U.S. patent application number 16/810590 was filed with the patent office on 2021-05-13 for prostate-specific membrane antigen cars and methods of use thereof.
The applicant listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Anne Chew, Szu Hua Sharon Lin, Xiaojun Liu, Yangbing Zhao.
Application Number | 20210137980 16/810590 |
Document ID | / |
Family ID | 1000004858636 |
Filed Date | 2021-05-13 |
United States Patent
Application |
20210137980 |
Kind Code |
A1 |
Zhao; Yangbing ; et
al. |
May 13, 2021 |
PROSTATE-SPECIFIC MEMBRANE ANTIGEN CARS AND METHODS OF USE
THEREOF
Abstract
The present disclosure provides modified immune cells (e.g.,
modified T cells) comprising a chimeric antigen receptor (CAR)
having affinity for a prostate-specific membrane antigen (PSMA)
(e.g., human PSMA). The present disclosure provides modified immune
cells (e.g., modified T cells) comprising a CAR having affinity for
PSMA and a dominant negative receptor and/or a switch receptor. The
present disclosure provides modified immune cells (e.g., modified T
cells) comprising a CAR having affinity for PSMA and a dominant
negative receptor and/or a switch receptor, wherein the modified
cell is capable of expressing and secreting a bispecific
antibody.
Inventors: |
Zhao; Yangbing; (Lumberton,
NJ) ; Lin; Szu Hua Sharon; (Philadelphia, PA)
; Liu; Xiaojun; (Wallingford, PA) ; Chew;
Anne; (Cherry Hill, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
PHILADELPHIA |
PA |
US |
|
|
Family ID: |
1000004858636 |
Appl. No.: |
16/810590 |
Filed: |
March 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2019/020729 |
Mar 5, 2019 |
|
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16810590 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/03 20130101;
A61K 31/7076 20130101; C07K 16/3069 20130101; C07K 14/7051
20130101; C12N 5/0638 20130101; A61K 35/17 20130101; C07K 2317/622
20130101; C07K 14/70517 20130101; C07K 2317/569 20130101; C07K
2319/02 20130101; C07K 2317/55 20130101; C07K 2319/33 20130101;
A61K 31/675 20130101; C07K 2317/24 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12N 5/0783 20060101 C12N005/0783; C07K 16/30 20060101
C07K016/30; C07K 14/705 20060101 C07K014/705; C07K 14/725 20060101
C07K014/725; A61K 31/675 20060101 A61K031/675; A61K 31/7076
20060101 A61K031/7076 |
Claims
1. A modified immune cell or precursor cell thereof, comprising (a)
a chimeric antigen receptor (CAR) capable of binding prostate
specific membrane antigen (PSMA) comprising an antigen binding
domain, a transmembrane domain, and an intracellular domain,
wherein the antigen binding domain comprises: a heavy chain
variable region (VH) that comprises the consensus sequence of SEQ
ID NO:183; and a light chain variable region (VL) that comprises
the consensus sequence of SEQ ID NO:184; and (b) a dominant
negative receptor and/or switch receptor.
2. The modified cell of claim 1, wherein: (a) the VH comprises the
sequence of SEQ ID NO:191; and/or (b) the VL comprises the sequence
of SEQ ID NO:192.
3. (canceled)
4. The modified cell of claim 1, wherein: (a) the antigen binding
domain comprises an antibody or an antigen-binding fragment
thereof; and/or (b) the antigen binding domain comprises an
antibody or an antigen-binding fragment thereof, and wherein the
antigen-binding fragment is selected from the group consisting of a
Fab, a single-chain variable fragment (scFv), or a single-domain
antibody.
5. (canceled)
6. The modified cell of claim 1, wherein the transmembrane domain:
(a) comprises a transmembrane region derived from CD8; and/or (b)
comprises a transmembrane region derived from CD8 comprising the
amino acid sequence set forth in SEQ ID NO: 88; and/or (c) further
comprises a hinge region derived from CD8; and/or (d) further
comprises a hinge region derived from CD8 comprising the amino acid
sequence set forth in SEQ ID NO: 86.
7.-9. (canceled)
10. The modified cell of claim 6, wherein the intracellular domain
comprises: (a) a 4-1BB signaling domain and a CD3 zeta signaling
domain; and/or (b) an ICOS signaling domain and a CD3 zeta
signaling domain; and/or (c) a variant ICOS signaling domain and a
CD3 zeta signaling domain; and/or (d) a 4-1BB signaling domain
comprising the amino acid sequence set forth in SEQ ID NO: 92;
and/or (e) an ICOS signaling domain and a CD3 zeta signaling
domain, and wherein the ICOS signaling domain the amino acid
sequence set forth in SEQ ID NO: 203; and/or (f) a variant ICOS
signaling domain and a CD3 zeta signaling domain, wherein the
variant ICOS signaling domain comprises the amino acid sequence set
forth in SEQ 1D NO: 95; and/or (g) a CD3 zeta signaling domain
comprising the amino acid sequence set forth in SEQ ID NOs: 97 or
100.
11.-16. (canceled)
17. The modified cell of claim 1, wherein the dominant negative
receptor: (a) is a truncated variant of a wild-type protein
associated with a negative signal; and/or (b) a truncated variant
of a wild-type protein associated with a negative signal comprising
the amino acid sequence set forth in SEQ ID NO: 115.
18. (canceled)
19. The modified cell of claim 1, wherein the switch receptor: (a)
comprises a first domain, wherein the first domain is derived from
a first polypeptide that is associated with a negative signal; and
a second domain, wherein the second domain is derived from a second
polypeptide that is associated with a positive signal; and/or (b)
comprises a first domain, wherein the first domain is derived from
a first polypeptide that is associated with a negative signal,
wherein the first domain comprises at least a portion of the
extracellular domain of the first polypeptide that is associated
with a negative signal; and a second domain, wherein the second
domain is derived from a second polypeptide that is associated with
a positive signal, and wherein the second domain comprises at least
a portion of the intracellular domain of the second polypeptide
that is associated with a positive signal; and/or (c) further
comprises a switch receptor transmembrane domain, and optionally
wherein the transmembrane domain comprises the transmembrane domain
of the first polypeptide that is associated with a negative signal;
or the transmembrane domain of the second polypeptide that is
associated with a positive signal; and/or (d) comprises a first
domain, wherein the first domain is derived from a first
polypeptide that is associated with a negative signal; and a second
domain, wherein the second domain is derived from a second
polypeptide that is associated with a positive signal, wherein the
first polypeptide that is associated with a negative signal is
selected from the group consisting of CTLA4, PD-1, BTLA, TIM-3, and
a TGF.beta.R, and/or (e) comprises a first domain, wherein the
first domain is derived from a first polypeptide that is associated
with a negative signal; and a second domain, wherein the second
domain is derived from a. second polypeptide that is associated
with a positive signal, wherein the second polypeptide that is
associated with a positive signal is selected from the group
consisting of CD28, ICOS, 4-1BB, and a IL-12R; and/or (f) comprises
a first domain comprising at least a portion of the extracellular
domain of PD1; a switch receptor transmembrane domain comprising at
least a portion of the transmembrane domain of CD28; and a second
domain comprising at least a portion of the intracellular domain of
CD28; and/or (g) comprises the amino acid sequence set forth in SEQ
ID NO: 117; and/or (h) comprises a first domain comprising at least
a portion of the extracellular domain of PD1; a switch receptor
transmembrane domain comprising at least a portion of the
transmembrane domain of PD1; and a second domain comprising at
least a portion of the intracellular domain of CD28, and optionally
wherein the first domain comprises at least a portion of the
extracellular domain of PD1 comprises an alanine (A) to leucine (L)
substitution at amino acid position 132; and/or (i) comprises the
amino acid sequence set forth in SEQ ID NO: 119; and/or (j)
comprises the amino acid sequence set forth in SEQ ID NO: 121;
and/or (k) the switch receptor comprises a first domain comprising
at least a portion of the extracellular domain of PD1 comprising an
alanine (A) to leucine (L) substitution at amino acid position 132;
and a second domain comprising at least a portion of the
intracellular domain of CD28; and/or (l) comprises a first domain
comprising at least a portion of the extracellular domain of PD1
comprising an alanine (A) to leucine (L) substitution at amino acid
position 132; and a second domain comprising at least a portion of
the intracellular domain of 4-1BB; and/or (m) comprises the amino
acid sequence set forth in SEQ ID NO: 215; and/or (n) comprises: a
first domain comprising at least a portion of the extracellular
domain of TIM-3; and a second domain comprising at least a portion
of the intracellular domain of CD28; and/or (o) comprises the amino
acid sequence set forth in SEQ ID NO: 127; and/or (p) comprises a
first domain comprising at least a portion of the extracellular
domain of a TGF.beta.R; and a second domain comprising at least a
portion of the intracellular domain of ILI2R.alpha.1; and/or (q)
comprises the amino acid sequence set forth in SEQ ID NO: 123;
and/or (r) comprises a first domain comprising at least a portion
of the extracellular domain of a TGF.beta.R; and a second domain
comprising at least a portion of the intracellular domain of
IL12R.beta.1; and/or (s) comprises the amino acid sequence set
forth in SEQ ID NO: 125.
20.-40. (canceled)
41. A modified immune cell or precursor cell thereof, comprising:
(a) a chimeric antigen receptor (CAR) having affinity for a
prostate specific membrane antigen (PSMA) on a target cell, wherein
the CAR comprises a PSMA binding domain comprising a heavy chain
variable region (VH) that comprises the sequence of SEQ ID NO:191;
and a light chain variable region (VL) that comprises the sequence
of SEQ ID NO:192; and a dominant negative receptor comprising the
amino acid sequence set forth in SEQ ID NO: 115; or (b) a chimeric
antigen receptor (CAR) having affinity for a prostate specific
membrane antigen (PSMA) on a target cell, wherein the CAR comprises
a PSMA binding domain comprising a heavy chain variable region (VH)
that comprises the sequence of SEQ ID NO: 191; and a light chain
variable region (VL) that comprises the sequence of SEQ ID NO: 192;
and a switch receptor comprising the amino acid sequence set forth
in SEQ ID NO: 213 or 215; or (c) a chimeric antigen receptor (CAR)
having affinity for a prostate specific membrane antigen (PSMA) on
a target cell, wherein the CAR comprises a PSMA binding domain
comprising a heavy chain variable region (VH) that comprises the
sequence of SEQ ID NO: 191; and a light chain variable region (VL)
that comprises the sequence of SEQ ID NO: 192; and a switch
receptor comprising the amino acid sequence set forth in SEQ ID
NOs: 117 or 119; or (d) a chimeric antigen receptor (CAR) having
affinity for a prostate specific membrane antigen (PSMA) on a
target cell, wherein the CAR comprises a PSMA binding domain
comprising a heavy chain variable region (VH) that comprises the
sequence of SEQ ID NO: 191; and a light chain variable region (VL)
that comprises the sequence of SEQ ID NO: 192; and a switch
receptor comprising the amino acid sequence set forth in SEQ ID NO:
121; or (e) a chimeric antigen receptor (CAR) having affinity for a
prostate specific membrane antigen (PSMA) on a target cell, wherein
the CAR comprises a PSMA binding domain comprising a heavy chain
variable region (VH) that comprises the sequence of SEQ ID NO: 191;
and a light chain variable region (VL) that comprises the sequence
of SEQ ID NO: 192; and a switch receptor comprising the amino acid
sequence set forth in SEQ ID NO: 127; or (f) a chimeric antigen
receptor (CAR) having affinity for a prostate specific membrane
antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA
binding domain comprising a heavy chain variable region (VH) that
comprises the sequence of SEQ ID NO: 191; and a light chain
variable region (VL) that comprises the sequence of SEQ ID NO: 192;
and a switch receptor comprising the amino acid sequence set forth
in SEQ ID NO: 123; or (g) a chimeric antigen receptor (CAR) having
affinity for a prostate specific membrane antigen (PSMA) on a
target cell, wherein the CAR comprises a PSMA binding domain
comprising a heavy chain variable region (VH) that comprises the
sequence of SEQ ID NO: 191; and a light chain variable region (VL)
that comprises the sequence of SEQ ID NO: 192; and a switch
receptor comprising the amino acid sequence set forth in SEQ ID NO:
125.
42.-47. (canceled)
48. The modified cell of claim 1, wherein the modified cell is a
modified T cell.
49. The modified T cell of claim 48, wherein the modified T cell is
an autologous cell.
50. The modified T cell of claim 48, wherein the modified T cell is
an allogeneic cell.
51. The modified cell of claim 1, wherein the modified cell is a
cytotoxic T lymphocyte (CTL).
52. The modified cell of claim 1, wherein the modified cell is
derived from a human cell.
53. An isolated nucleic acid, comprising: (a) a first nucleic acid
sequence encoding a chimeric antigen receptor (CAR) capable of
binding prostate specific membrane antigen (PSMA) comprising an
antigen binding domain, a transmembrane domain, and an
intracellular domain, wherein the antigen binding domain comprises:
a heavy chain variable region (VH) that comprises the consensus
sequence of SEQ ID NO:183; and a light chain variable region (VL)
that comprises the consensus sequence of SEQ ID NO:184; and (b) a
second nucleic acid sequence encoding a dominant negative receptor
and/or a switch receptor.
54. The isolated nucleic acid of claim 53, wherein: (a) the VH
comprises the sequence of SEQ ID NO:191; and/or (b) the VL
comprises the sequence of SEQ ID NO:192; and/or (c) the first
nucleic acid sequence comprises the nucleic acid sequence set forth
in any one of SEQ ID NOs: 246, 248, 250, 252, 254, or 256; and/or
(d) the second nucleic acid sequence comprises the nucleic acid
sequence set forth in any one of SEQ ID NOs: 116, 118, 120, 122,
124, 126 128, 214, or 216; and/or (e) the first nucleic acid
sequence and the second nucleic acid sequence are separated by a
linker; and/or (f) the first nucleic acid sequence and the second
nucleic acid sequence are separated by a linker, wherein the linker
comprises a nucleic acid sequence encoding an internal ribosome
entry site (IRES); and/or (g) the first nucleic acid sequence and
the second nucleic acid sequence are separated by a linker, wherein
the linker comprises a nucleic acid sequence encoding a
self-cleaving peptide; and/or (h) the first nucleic acid sequence
and the second nucleic acid sequence are separated by a linker,
wherein the linker comprises a nucleic acid sequence encoding a
self-cleaving 2A peptide; and/or (i) the first nucleic acid
sequence and the second nucleic acid sequence are separated by a
linker, wherein the linker comprises a nucleic acid sequence
encoding a self-cleaving 2A peptide, wherein the 2A peptide is
selected from the group consisting of porcine teschovirus-1 2 A
(P2A), Thoseaasigna virus 2A (T2A), equine rhinitis A virus 2A
(E2A), and foot-and-mouth disease virus 2A (F2A); and/or (j) the
first nucleic acid sequence and the second nucleic acid sequence
are separated by a linker, wherein the linker comprises a nucleic
acid sequence encoding a self-cleaving 2A peptide, wherein the 2A
peptide is T2A; and/or (k) the first nucleic acid sequence and the
second nucleic acid sequence are separated by a linker, wherein the
linker comprises a nucleic acid sequence encoding a self-cleaving
2A peptide, wherein the 2A peptide is F2A; and/or (l) the isolated
nucleic acid comprises from 5' to 3' the first nucleic acid
sequence, the linker, and the second nucleic acid sequence; and/or
(m) the isolated nucleic acid comprises from 5' to 3' the second
nucleic acid sequence, the linker, and the first nucleic acid
sequence.
55.-66. (canceled)
67. An isolated nucleic acid, comprising: (a) a first nucleic acid
sequence encoding a chimeric antigen receptor (CAR) capable of
binding prostate specific membrane antigen (PSMA) comprising an
antigen binding domain, a transmembrane domain, and an
intracellular domain, wherein the antigen binding domain comprises:
a heavy chain variable region (VH) that comprises the sequence of
SEQ ID NO:191; and a light chain variable region (VL) that
comprises the sequence of SEQ ID NO:192; and a second nucleic acid
sequence encoding a dominant negative receptor and/or switch
receptor comprising the nucleic acid sequence set forth in any one
of SEQ ID NOs: 116 118, 120, 122, 124, 126, 128, 214, or 216; or
(b) a first nucleic acid sequence encoding a chimeric antigen
receptor (CAR) capable of binding prostate specific membrane
antigen (PSMA) comprising an antigen binding domain, a
transmembrane domain, and an intracellular domain, wherein the
antigen binding domain comprises: a heavy chain variable region
(VH) that comprises the sequence of SEQ ID NO:191; and a light
chain variable region (VL) that comprises the sequence of SEQ ID
NO:192; and a second nucleic acid sequence encoding a dominant
negative receptor and/or switch receptor comprising the nucleic
acid sequence set forth in SEQ ID NO: 116.
68. (canceled)
69. The isolated nucleic acid of claim 67, wherein: (a) the first
nucleic acid sequence and the second nucleic acid sequence is
separated by a linker comprising a nucleic acid sequence encoding
T2A; or (b) the first nucleic acid sequence and the second nucleic
acid sequence is separated by a linker comprising a nucleic acid
sequence encoding F2A.
70. (canceled)
71. An expression construct comprising the isolated nucleic acid of
claim 53.
72. The expression construct of claim 71, wherein: (a) the
expression construct is a viral vector selected from the group
consisting of a retroviral vector, a lentiviral vector, an
adenoviral vector, and an adeno-associated viral vector; and/or (b)
the expression construct is a lentiviral vector; and/or (c) the
expression construct is a lentiviral vector, and wherein the
lentiviral vector further comprises an EF-1 a promoter; and/or (d)
the expression construct is a lentiviral vector, and wherein the
lentiviral vector further comprises a rev response element (RRE);
and/or (e) the expression construct is a lentiviral vector, and
wherein the lentiviral vector further comprises a woodchuck
hepatitis virus posttranscriptional regulatory element (WPRE);
and/or (f) the expression construct is a lentiviral vector, and
wherein the lentiviral vector further comprises a cPPT sequence;
and/or (g) the expression construct is a lentiviral vector, and
wherein the lentiviral vector further comprises an EF-1 a promoter,
a rev response element (RRE), a woodchuck hepatitis virus
posttranscriptional regulatory element (WPRE), and a cPPT sequence;
and/or (h) the expression construct is a lentiviral vector, and
wherein the lentiviral vector is a self-inactivating lentiviral
vector.
73.-80. (canceled)
81. A method of treating cancer in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of a composition comprising the modified immune
cell of claim 1.
82. The method of claim 81, further comprising: (a) administering
to the subject a lymphodepleting chemotherapy, and/or (b)
administering to the subject a lymphodepleting chemotherapy,
wherein the lymphodepleting chemotherapy comprises administering to
the subject a therapeutically effective amount of cyclophosphamide
and/or fludarabine.
83. (canceled)
84. The method of claim 81, wherein the method is directed to
treating prostate cancer in a subject in need thereof, the method
comprising: administering to the subject a lymphodepleting
chemotherapy comprising administering to the subject a
therapeutically effective amount of cyclophosphamide; and
administering to the subject a modified T cell comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises an antigen binding domain, a transmembrane domain, and an
intracellular domain, wherein the antigen binding domain comprises:
a heavy chain variable region (VH) that comprises the sequence of
SEQ ID NO:191; and a light chain variable region (VL) that
comprises the sequence of SEQ ID NO:192; and a dominant negative
receptor comprising an amino acid sequence set forth in SEQ ID NO:
115.
85. The method of claim 81, wherein the method is directed to
treating metastatic castrate resistant prostate cancer in a subject
in need thereof, the method comprising: administering to the
subject a lymphodepleting chemotherapy comprising administering to
the subject a therapeutically effective amount of cyclophosphamide;
and administering to the subject a modified T cell comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises an antigen binding domain, a transmembrane domain, and an
intracellular domain, wherein the antigen binding domain comprises:
a heavy chain variable region (VH) that comprises the sequence of
SEQ ID NO:191; and a light chain variable region (VL) that
comprises the sequence of SEQ ID NO:192; and a dominant negative
receptor comprising an amino acid sequence set forth in SEQ NO:
115.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is entitled to priority under 35
U.S.C. .sctn. 365(a) to International Application
PCT/US2019/020729, filed Mar. 5, 2019, which is hereby incorporated
by reference in its entirety herein.
BACKGROUND OF THE INVENTION
[0002] Breaking the tolerance to self-antigens is a major challenge
in the application of immunotherapy to solid malignancies. Vaccine
strategies aimed at harnessing endogenous anti-tumor T cells are
limited by the T cell receptor (TCR) repertoire, which can be
deleted within the thymus as part of central tolerance or rendered
non-functional by post-thymic mechanisms of peripheral tolerance.
One strategy to overcome such obstacles is to produce genetically
engineered T cells redirected toward tumor antigens using a
chimeric antigen receptor (CAR) approach. CAR T cells use
genetically programmed, patient-derived lymphocytes transduced with
chimeric receptor genes in order to combine the antigen recognition
domains of a specific antibody with the signaling domains of a
TCR.
[0003] Prostate-specific membrane antigen (PSMA) is a
membrane-bound protein expressed on the cell surface and is
reported to be highly overexpressed in prostate cancer tissues.
PSMA expression is directly correlated with advancing tumor grade
and stage, and is believed to confer a selective growth advantage
to prostate cancer cells. As such, PSMA may be an ideal target for
immunotherapies for prostate cancer.
[0004] Another major challenge in cancer immunotherapy is the
hostile microenvironment in which the targeted tumor resides. For
example, immunosuppressive receptor ligands such as, PDL1 (CD274)
which binds to PD1 (CD279), are up-regulated and negatively
regulate T cell activity in the tumor microenvironment. In
addition, TGF-.beta., which is over-expressed in prostate tumor
cells, can act as an immunosuppressive molecule.
[0005] Thus, there is a need in the art for novel cancer
immunotherapies targeting PSMA. The present invention satisfies
this need.
SUMMARY OF THE INVENTION
[0006] The present invention is based on the finding that humanized
prostate-specific membrane antigen (PSMA) chimeric antigen receptor
(CAR) T cells exhibit potent anti-tumor activity. The present
invention is also based on the finding that PSMA-CAR T cells
comprising a dominant negative receptor and/or switch receptor
exhibit significantly enhanced anti-tumor activity.
[0007] Accordingly, in certain aspects, the instant disclosure
provides a modified immune cell or precursor cell thereof,
comprising: (a) a chimeric antigen receptor (CAR) capable of
binding prostate specific membrane antigen (PSMA) comprising an
antigen binding domain, a transmembrane domain, and an
intracellular domain, wherein the antigen binding domain comprises:
a heavy chain variable region (VH) that comprises the consensus
sequence of SEQ ID NO:183; and a light chain variable region (VL)
that comprises the consensus sequence of SEQ ID NO:184; and (b) a
dominant negative receptor and/or switch receptor.
[0008] In certain exemplary embodiments, the VH comprises the
sequence of SEQ ID NO:191. In certain exemplary embodiments, the VL
comprises the sequence of SEQ ID NO:192.
[0009] In certain exemplary embodiments, the antigen binding domain
comprises an antibody or an antigen-binding fragment thereof. In
certain exemplary embodiments, the the antigen-binding fragment is
selected from the group consisting of a Fab, a single-chain
variable fragment (scFv), or a single-domain antibody.
[0010] In certain exemplary embodiments, the transmembrane domain
comprises a transmembrane region derived from CD8. In certain
exemplary embodiments, the transmembrane region derived from CD8
comprises the amino acid sequence set forth in SEQ ID NO: 88. In
certain exemplary embodiments, the transmembrane domain further
comprises a hinge region derived from CD8. In certain exemplary
embodiments, the hinge region is derived from CD8 comprises the
amino acid sequence set forth in SEQ ID NO:86.
[0011] In certain exemplary embodiments, the intracellular domain
comprises a 4-1BB signaling domain and a CD3 zeta signaling domain.
In certain exemplary embodiments, the intracellular domain
comprises an ICOS signaling domain and a CD3 zeta signaling domain.
In certain exemplary embodiments, the intracellular domain
comprises a variant ICOS signaling domain and a CD3 zeta signaling
domain. In certain exemplary embodiments, the 4-1BB signaling
domain comprises the amino acid sequence set forth in SEQ ID NO:92.
In certain exemplary embodiments, the ICOS signaling domain
comprises the amino acid sequence set forth in SEQ ID NO:203. In
certain exemplary embodiments, the variant ICOS signaling domain
comprises the amino acid sequence set forth in SEQ II) NO:95. In
certain exemplary embodiments, the the CD3 zeta signaling domain
comprises the amino acid sequence set forth in SEQ ID NOs:97 or
100.
[0012] In certain exemplary embodiments, the dominant negative
receptor is a truncated variant of a wild-type protein associated
with a negative signal. In certain exemplary embodiments, the
truncated variant of a wild-type protein associated 115 a negative
signal comprises the amino acid sequence set forth in SEQ ID NO:
115.
[0013] In certain exemplary embodiments, the the switch receptor
comprises: a first domain, wherein the first domain is derived from
a first polypeptide that is associated with a negative signal; and
a second domain, wherein the second domain is derived from a second
polypeptide that is associated with a positive signal. In certain
exemplary embodiments, the the first domain comprises at least a
portion of the extracellular domain of the first polypeptide that
is associated with a negative signal, and wherein the second domain
comprises at least a portion of the intracellular domain of the
second polypeptide that is associated with a positive signal. In
certain exemplary embodiments, the switch receptor further
comprises a switch receptor transmembrane domain. In certain
exemplary embodiments, the switch receptor transmembrane domain
comprises: the transmembrane domain of the first polypeptide that
is associated with a negative signal; or the transmembrane domain
of the second polypeptide that is associated with a positive
signal.
[0014] In certain exemplary embodiments, the first polypeptide that
is associated with a negative signal is selected from the group
consisting of CTLA4, PD-1, BTLA, TIM-3, and a TGF.beta.R. In
certain exemplary embodiments, the second polypeptide that is
associated with a positive signal is selected from the group
consisting of CD28, ICOS, 4-1BB, and a IL-12R.
[0015] In certain exemplary embodiments, the switch receptor
comprises: a first domain comprising at least a portion of the
extracellular domain of PD1; a switch receptor transmembrane domain
comprising at least a portion of the transmembrane domain of CD28;
and a second domain comprising at least a portion of the
intracellular domain of CD28. In certain exemplary embodiments, the
switch receptor comprises the amino acid sequence set forth in SEQ
ID NO: 117.
[0016] In certain exemplary embodiments, the switch receptor
comprises: a first domain comprising at least a portion of the
extracellular domain of PD1; a switch receptor transmembrane domain
comprising at least a portion of the transmembrane domain of PD1;
and a second domain comprising at least a portion of the
intracellular domain of CD28. In certain exemplary embodiments, the
switch receptor comprises the amino acid sequence set forth in SEQ
ID NO: 119.
[0017] In certain exemplary embodiments, the first domain comprises
at least a portion of the extracellular domain of PD1 comprises an
alanine (A) to leucine (L) substitution at amino acid position 132.
In certain exemplary embodiments, the switch receptor comprises the
amino acid sequence set forth in SEQ ID NO: 121.
[0018] In certain exemplary embodiments, the switch receptor
comprises: a first domain comprising at least a portion of the
extracellular domain of PD1 comprising an alanine (A) to leucine
(L) substitution at amino acid position 132; and a second domain
comprising at least a portion of the intracellular domain of CD28.
In certain exemplary embodiments, the switch receptor comprises the
amino acid sequence set forth in SEQ ID NO: 121.
[0019] In certain exemplary embodiments, the switch receptor
comprises: a first domain comprising at least a portion of the
extracellular domain of PD1 comprising an alanine (A) to leucine
(L) substitution at amino acid position 132; and a second domain
comprising at least a portion of the intracellular domain of 4-1BB.
In certain exemplary embodiments, the switch receptor comprises the
amino acid sequence set forth in SEQ ID NO:215.
[0020] In certain exemplary embodiments, the switch receptor
comprises: a first domain comprising at least a portion of the
extracellular domain of TIM-3; and a second domain comprising at
least a portion of the intracellular domain of CD28. In certain
exemplary embodiments, the switch receptor comprises the amino acid
sequence set forth in SEQ ID NO: 127.
[0021] In certain exemplary embodiments, the switch receptor
comprises: a first domain comprising at least a portion of the
extracellular domain of a TGF.beta.R; and a second domain
comprising at least a portion of the intracellular domain of
IL12R.alpha.1. In certain exemplary embodiments, the switch
receptor comprises the amino acid sequence set forth in SEQ ID NO:
123.
[0022] In certain exemplary embodiments, the switch receptor
comprises: a first domain comprising at least a portion of the
extracellular domain of a TGF.beta.R; and a second domain
comprising at least a portion of the intracellular domain of
IL12R.beta.1. In certain exemplary embodiments, the switch receptor
comprises the amino acid sequence set forth in SEQ ID NO: 125.
[0023] In another aspect, the instant disclosure provides a
modified immune cell or precursor cell thereof, comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises a PSMA binding domain comprising a heavy chain variable
region (VH) that comprises the sequence of SEQ ID NO:191; and a
light chain variable region (VL) that comprises the sequence of SEQ
ID NO: 192; and a dominant negative receptor comprising the amino
acid sequence set forth in SEQ ID NO: 115.
[0024] In another aspect, the instant disclosure provides a
modified immune cell or precursor cell thereof, comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises a PSMA binding domain comprising a heavy chain variable
region (VH) that comprises the sequence of SEQ ID NO:191; and a
light chain variable region (VL) that comprises the sequence of SEQ
ID NO: 192; and a switch receptor comprising the amino acid
sequence set forth in SEQ ID NO:213 or 215.
[0025] In another aspect, the instant disclosure provides a
modified immune cell or precursor cell thereof, comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises a PSMA binding domain comprising a heavy chain variable
region (VH) that comprises the sequence of SEQ ID NO:191; and a
light chain variable region (VL) that comprises the sequence of SEQ
ID NO: 192; and a switch receptor comprising the amino acid
sequence set forth in SEQ ID NOs: 117 or 119.
[0026] In another aspect, the instant disclosure provides a
modified immune cell or precursor cell thereof, comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises a PSMA binding domain comprising a heavy chain variable
region (VH) that comprises the sequence of SEQ ID NO:191; and a
light chain variable region (VL) that comprises the sequence of SEQ
ID NO: 192; and a switch receptor comprising the amino acid
sequence set forth in SEQ ID NO: 121.
[0027] In another aspect, the instant disclosure provides a
modified immune cell or precursor cell thereof, comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises a PSMA binding domain comprising a heavy chain variable
region (VH) that comprises the sequence of SEQ ID NO:191; and a
light chain variable region (VL) that comprises the sequence of SEQ
ID NO: 192; and a switch receptor comprising the amino acid
sequence set forth in SEQ ID NO: 127.
[0028] In another aspect, the instant disclosure provides a
modified immune cell or precursor cell thereof, comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises a PSMA binding domain comprising a heavy chain variable
region (VH) that comprises the sequence of SEQ ID NO:191; and a
light chain variable region (VL) that comprises the sequence of SEQ
ID NO: 192; and a switch receptor comprising the amino acid
sequence set forth in SEQ ID NO: 123.
[0029] In another aspect, the instant disclosure provides a
modified immune cell or precursor cell thereof, comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises a PSMA binding domain comprising a heavy chain variable
region (VH) that comprises the sequence of SEQ ID NO:191; and a
light chain variable region (VL) that comprises the sequence of SEQ
ID NO: 192; and a switch receptor comprising the amino acid
sequence set forth in SEQ ID NO: 125.
[0030] In certain exemplary embodiments, the modified cell is a
modified T cell. In certain exemplary embodiments, the modified T
cell is an autologous cell. In certain exemplary embodiments, the
modified T cell is an allogeneic cell. In certain exemplary
embodiments, the modified cell is a cytotoxic T lymphocyte (CTL).
In certain exemplary embodiments, the modified cell is derived from
a human cell.
[0031] In another aspect, the instant disclosure provides an
isolated nucleic acid, comprising: (a) a first nucleic acid
sequence encoding a chimeric antigen receptor (CAR) capable of
binding prostate specific membrane antigen (PSMA) comprising an
antigen binding domain, a transmembrane domain, and an
intracellular domain, wherein the antigen binding domain comprises:
a heavy chain variable region (VH) that comprises the consensus
sequence of SEQ ID NO: 183; and a light chain variable region (VL)
that comprises the consensus sequence of SEQ ID NO: 184; and (b) a
second nucleic acid sequence encoding a dominant negative receptor
and/or a switch receptor.
[0032] In certain exemplary embodiments, the VH comprises the
sequence of SEQ ID NO:191. In certain exemplary embodiments, the VL
comprises the sequence of SEQ ID NO: 192.
[0033] In certain exemplary embodiments, the first nucleic acid
sequence comprises the nucleic acid sequence set forth in any one
of SEQ ID NOs: 246, 248, 250, 252, 254, or 256.
[0034] In certain exemplary embodiments, the second nucleic acid
sequence comprises the nucleic acid sequence set forth in any one
of SEQ ID NOs: 116, 118, 120, 122, 124, 126 128, 214, or 216.
[0035] In certain exemplary embodiments, the first nucleic acid
sequence and the second nucleic acid sequence are separated by a
linker. In certain exemplary embodiments, the linker comprises a
nucleic acid sequence encoding an internal ribosome entry site
(IRES). In certain exemplary embodiments, the linker comprises a
nucleic acid sequence encoding a self-cleaving peptide. In certain
exemplary embodiments, the self-cleaving peptide is a 2A peptide.
In certain exemplary embodiments, the 2A peptide is selected from
the group consisting of porcine teschovirus-1 2 A (P2A),
Thoseaasigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and
foot-and-mouth disease virus 2A (F2A). In certain exemplary
embodiments, the 2A peptide is T2A. In certain exemplary
embodiments, the 2A peptide is F2A.
[0036] In certain exemplary embodiments, the isolated nucleic acid
comprises from 5' to 3' the first nucleic acid sequence, the
linker, and the second nucleic acid sequence.
[0037] In certain exemplary embodiments, the isolated nucleic acid
comprises from 5' to 3' the second nucleic acid sequence, the
linker, and the first nucleic acid sequence.
[0038] In another aspect, the instant disclosure provides an
isolated nucleic acid, comprising: a first nucleic acid sequence
encoding a chimeric antigen receptor (CAR) capable of binding
prostate specific membrane antigen (PSMA) comprising an antigen
binding domain, a transmembrane domain, and an intracellular
domain, wherein the antigen binding domain comprises: a heavy chain
variable region (VH) that comprises the consensus sequence of SEQ
ID NO:191; and a light chain variable region (VL) that comprises
the consensus sequence of SEQ ID NO: 192; and a second nucleic acid
sequence encoding a dominant negative receptor and/or switch
receptor comprising the nucleic acid sequence set forth in any one
of SEQ ID NOs: 116, 118, 120, 122, 124, 126, 128, 214, or 216.
[0039] In another aspect, the instant disclosure provides an
isolated nucleic acid, comprising: a first nucleic acid sequence
encoding a chimeric antigen receptor (CAR) capable of binding
prostate specific membrane antigen (PSMA) comprising an antigen
binding domain, a transmembrane domain, and an intracellular
domain, wherein the antigen binding domain comprises: a heavy chain
variable region (VH) that comprises the consensus sequence of SEQ
ID NO:191; and a light chain variable region (VL) that comprises
the consensus sequence of SEQ ID NO: 192; and a second nucleic acid
sequence encoding a dominant negative receptor and/or switch
receptor comprising the nucleic acid sequence set forth in SEQ ID
NO: 116.
[0040] In certain exemplary embodiments, the first nucleic acid
sequence and the second nucleic acid sequence is separated by a
linker comprising a nucleic acid sequence encoding T2A. In certain
exemplary embodiments, the first nucleic acid sequence and the
second nucleic acid sequence is separated by a linker comprising a
nucleic acid sequence encoding F2A.
[0041] In another aspect, the instant disclosure provides an
expression construct comprising the isolated nucleic acid as
described in the foregoing.
[0042] In certain exemplary embodiments, the expression construct
is a viral vector selected from the group consisting of a
retroviral vector, a lentiviral vector, an adenoviral vector, and
an adeno-associated viral vector. In certain exemplary embodiments,
the expression construct is a lentiviral vector. In certain
exemplary embodiments, the lentiviral vector further comprises an
EF-1 a promoter. In certain exemplary embodiments, the lentiviral
vector further comprises a rev response element (RRE). In certain
exemplary embodiments, the lentiviral vector further comprises a
woodchuck hepatitis virus posttranscriptional regulatory element
(WPRE). In certain exemplary embodiments, the lentiviral vector
further comprises a cPPT sequence.
[0043] In certain exemplary embodiments, the lentiviral vector
further comprises an EF-1 a promoter, a rev response element (RRE),
a woodchuck hepatitis virus posttranscriptional regulatory element
(WPRE), and a cPPT sequence.
[0044] In certain exemplary embodiments, the lentiviral vector is a
self-inactivating lentiviral vector.
[0045] In another aspect, the instant disclosure provides a method
for generating the modified immune cell or precursor cell thereof
as described in the foregoing, comprising introducing into the
immune cell one or more of the nucleic acid as described in the
foregoing, or the expression construct as described in the
foregoing.
[0046] In another aspect, the instant disclosure provides a method
of treating cancer in a subject in need thereof, the method
comprising administering to the subject a therapeutically effective
amount of a composition comprising the modified immune cell as
described in the foregoing.
[0047] In certain exemplary embodiments, the method further
comprises administering to the subject a lymphodepleting
chemotherapy. In certain exemplary embodiments, the lymphodepleting
chemotherapy comprises administering to the subject a
therapeutically effective amount of cyclophosphamide and/or
fludarabine.
[0048] In another aspect, the instant disclosure provides a method
of treating prostate cancer in a subject in need thereof, the
method comprising: administering to the subject a lymphodepleting
chemotherapy comprising administering to the subject a
therapeutically effective amount of cyclophosphamide; and
administering to the subject a modified T cell comprising: a
chimeric antigen receptor (CAR) having affinity for a prostate
specific membrane antigen (PSMA) on a target cell, wherein the CAR
comprises an antigen binding domain, a transmembrane domain, and an
intracellular domain, wherein the antigen binding domain comprises:
a heavy chain variable region (VH) that comprises the consensus
sequence of SEQ ID NO:191; and a light chain variable region (VL)
that comprises the consensus sequence of SEQ ID NO: 192; and a
dominant negative receptor comprising an amino acid sequence set
forth in SEQ ID NO: 115.
[0049] In another aspect, the instant disclosure provides a method
of treating metastatic castrate resistant prostate cancer in a
subject in need thereof, the method comprising: administering to
the subject a lymphodepleting chemotherapy comprising administering
to the subject a therapeutically effective amount of
cyclophosphamide; and administering to the subject a modified T
cell comprising: a chimeric antigen receptor (CAR) having affinity
for a prostate specific membrane antigen (PSMA) on a target cell,
wherein the CAR comprises an antigen binding domain, a
transmembrane domain, and an intracellular domain, wherein the
antigen binding domain comprises: a heavy chain variable region
(VH) that comprises the consensus sequence of SEQ ID NO:191; and a
light chain variable region (VL) that comprises the consensus
sequence of SEQ ID NO: 192; and a dominant negative receptor
comprising an amino acid sequence set forth in SEQ ID NO: 115.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The foregoing and other features and advantages of the
present invention will be more fully understood from the following
detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings. It should be understood
that the present invention is not limited to the precise
arrangements and instrumentalities of the embodiments shown in the
drawings.
[0051] FIG. 1A illustrates results using purified IVT PSMA RNA CARs
and full length PSMA RNA resolved on an agarose gel.
[0052] FIG. 1B shows results using purified PSMA RNA CARs
electroporated into ND444 T cells and CAR expression examined by
Flow Cytometry. The mean fluorescence intensity is labeled below
the graph.
[0053] FIG. 1C illustrates PSMA expression. Purified full length
PSMA RNA were electroporated into Nalm6 or K562 cells (middle and
right panel). PSMA expression was examined by Flow Cytometry.
[0054] FIG. 1D illustrates results using combined PC3.PSMA single
cell clones. Limited dilution was performed with PC3.PSMA cells
(left panel), seven single colonies were isolated and pooled to be
a new cell line, PC3.PSMA.7SC (right panel). PSMA expression was
examined by Flow Cytometry.
[0055] FIG. 2A illustrates results using various PSMA RNA CARs
incubated with tumor cells and CD107a assays performed. The cells
were gated by CD3.
[0056] FIG. 2B illustrates results using various PSMA RNA CARs
incubated with tumor cells and Luciferase based CTL assays
performed. Results are reported as percent killing based on
luciferase activity in wells with only tumor in the absence of T
cells.
[0057] FIG. 2C shows results using various PSMA RNA CARs incubated
with tumor cells and ELISA assays performed. (IL-2, left panel;
IFN-.gamma., right panel).
[0058] FIG. 3A illustrates results using PSMA Lenti CARs
constructed and transduced into primary human T cells (MOI=3). CAR
expression was examined by Flow Cytometry on day 8.
[0059] FIG. 3B shows results using various PSMA Lenti CARs
incubated with or without tumor cells and CD107a assays performed.
The cells were gated by CD3. Results from day 12 are shown.
[0060] FIG. 3C shows results using various PSMA Lenti CARs
incubated with tumor cells and Luciferase based CTL assays
performed. Results are reported as percent killing based on
luciferase activity in wells with only tumor in the absence of T
cells. Results from day 12 are shown.
[0061] FIG. 3D illustrates results using various PSMA Lenti CARs
incubated with PC3 or PC3.PSMA cells and ELISA assays performed
(IL-2, left panel; IFN-.gamma., right panel). Results from day 12
are shown.
[0062] FIG. 4A illustrates results using switch receptors,
PD1*PTM.CD28 or PD1.CD28 linked to each human PSMA Lenti CARs via
F2A and transduced into primary human T cells. PD1 and CAR
expression were examined by Flow Cytometry on day 12.
[0063] FIG. 4B illustrates results using a dominant negative (dn)
transforming growth factor .beta. receptor II (TGFR.beta.II)
sequence linked to each human PSMA Lenti CAR via T2A.
Dn-TGFR.beta.II-PSMA CAR transduced T cells were analyzed by Flow
Cytometry on day 7.
[0064] FIG. 4C illustrates results using various amounts of
purified full length PDL1 RNA electroporated into PC3.PSMA cells
and PDL1 expression examined by Flow Cytometry on day 13.
[0065] FIG. 4D shows results using various PSMA Lenti CARs
incubated with PC3.PSMA or PDL1 electroporated PC3.PSMA cells and
CD107a assays performed. The cells were gated by CD3. Results from
day 14 are shown.
[0066] FIG. 4E shows results using various PSMA Lenti CARs
incubated with PC3.PSMA or PDL1 electroporated PC3.PSMA cells and
CD107a assays performed. The cells were gated by CD3. Results from
day 14 are shown.
[0067] FIG. 4F shows results using various PSMA Lenti CARs
incubated with PC3.PSMA or PDL1 electroporated PC3.PSMA cells and
CD107a assays performed. The cells were gated by CD3. Results from
day 14 are shown.
[0068] FIG. 4G shows results using various PSMA Lenti CARs
incubated with PC3.PSMA or PDL1 electroporated PC3.PSMA cells and
CD107a assays performed. The cells were gated by CD3. Results from
day 14 are shown.
[0069] FIG. 4H shows results using various PSMA Lenti CARs
incubated with PC3.PSMA cells and Luciferase based CTL assays
performed. Results are reported as percent killing based on
luciferase activity in wells with only tumor in the absence of T
cells.
[0070] FIG. 4I shows results using various PSMA Lenti CARs
incubated with PC3.PSMA or PDL1 electroporated PC3.PSMA cells and
ELISA assays performed. (IL-2, top panel; IFN-.gamma., bottom
panel).
[0071] FIG. 5A shows results using switch receptor PD1.CD28 linked
to each human PSMA Lenti CARs via F2A transduced into primary human
T cells. PD1 and CAR expression were examined by Flow
Cytometry.
[0072] FIG. 5B shows results using a dominant negative (dn)
TGFR.beta.II sequence linked to human 2A10 PSMA Lenti CARs via T2A.
CARs transduced T cells were analyzed by Flow Cytometry.
[0073] FIG. 5C shows results using various PSMA Lenti CARs
incubated with PC3.PSMA.7SC cells and CD107a assays performed. The
cells were gated by CD3.
[0074] FIG. 5D shows results using various PSMA Lenti CARs were
incubated with PDL1 electroporated PC3.PSMA.7SC cells and CD107a
assay was performed. The cells were gated by CD3.
[0075] FIG. 5E shows results using various PSMA Lenti CARs
incubated with tumor cells and Luciferase based CTL assays
performed. Results are reported as percent killing based on
luciferase activity in wells with only tumor in the absence of T
cells.
[0076] FIG. 5F shows results using various PSMA Lenti CARs
incubated with PC3, PC3.PSMA.7SC or PDL1 electroporated
PC3.PSMA.PDL1 cells and ELISA assays performed. (IL-2, top panel;
IFN-.gamma., bottom panel).
[0077] FIG. 5G shows results using quantitative PCR for PSMA
expression. The fold changes (delta delta CT) were normalized to
Nalm6.CBG cells. See Table 1 for the abbreviations. P FIG. 5H shows
results using various PSMA Lenti CARs incubated with tumor cells or
primary human cells and CD107a assays performed. The cells were
gated by CD3.
[0078] FIG. 5I shows quantitative data from the experiments shown
in FIG. 5H. HSAEpC: Human Small Airway Epithelial Cells. HPMEC:
Human Pulmonary Microvascular Endothelial Cells.
[0079] FIG. 5J shows results using various PSMA Lenti CARs
incubated with primary human cells and ELISA assays performed.
(IL-2, left panel; IFN-.gamma., right panel). HREpC: Human Renal
Epithelial Cells. HSAEpC: Human Small Airway Epithelial Cells.
HPMEC: Human Pulmonary Microvascular Endothelial Cells.
[0080] FIG. 5K shows results using 2.times.10.sup.6 PC3.PSMA.7SC
cells transduced with click beetle and injected into mice (i.v.).
27 days later, 2.times.10.sup.6 PSMA CAR-T positive transduced T
cells were injected to the tumor bearing mice (i.v.).
Bioluminescence imaging (BLI) was conducted at multiple time
points. Upper panel with a minimal average radiance of
5.times.10.sup.5; Lower panel with a minimal average radiance of
3.times.10.sup.5.
[0081] FIG. 5L illustrates quantitative average radiances of FIG.
5K.
[0082] FIG. 6 is a schematic representation of a dn-TGFR.beta.II
PSMA CAR construct and pTRPE construct map.
[0083] FIG. 7 shows flow cytometry examination of CAR expression in
T cells transduced with 2F5 PSMA CAR alone (2F5 ICOS), or
co-transduced 2F5 PSMA CAR together with various switch receptors,
as indicated.
[0084] FIG. 8 shows flow cytometry examination of PD1 and Tim3
expression of T cells transduced with 2F5 PSMA CAR alone (2F5
ICOS), or co-transduced 2F5 PSMA CAR together with various switch
receptors, as indicated.
[0085] FIG. 9 is a graph depicting CD107a expression in T cells
transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone
(41BB), or co-transduced 2F5 PSMA CAR together with various switch
receptors, as indicated. UTD means untransduced.
[0086] FIG. 10 is a graph depicting granzyme B expression in T
cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR
alone (41BB), or co-transduced 2F5 PSMA CAR together with various
switch receptors, as indicated. UTD means untransduced.
[0087] FIG. 11A is a graph depicting IL-2 secretion of T cells
transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone
(41BB), or co-transduced 2F5 PSMA CAR together with various switch
receptors, as indicated. NTD means untransduced.
[0088] FIG. 11B is a graph depicting IFNgamma secretion of T cells
transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone
(41BB), or co-transduced 2F5 PSMA CAR together with various switch
receptors, as indicated. UTD means untransduced.
[0089] FIG. 12A is a graph depicting the quantification of
bioluminescence obtained from imaging of NSG mice bearing
PC3-PSMA.CBG induced tumors treated with T cells transduced as
indicated.
[0090] FIG. 12B is a graph depicting the quantification of
bioluminescence obtained from imaging of NSG mice bearing
PC3-PSMA.CBG induced tumors treated with T cells transduced as
indicated.
[0091] FIG. 13 is a graph depicting tumor sizes of NSG mice bearing
PC3-PSMA.CBG induced tumors treated with T cells transduced as
indicated.
[0092] FIG. 14A is a graph depicting the quantification of
bioluminescence obtained from imaging of NSG mice bearing
PC3-PSMA.CBG induced tumors treated with T cells transduced as
indicated.
[0093] FIG. 14B is a graph depicting the quantification of
bioluminescence obtained from imaging of NSG mice bearing
PC3-PSMA.CBG induced tumors treated with T cells transduced as
indicated.
[0094] FIG. 14C is a graph depicting the quantification of
bioluminescence obtained from imaging of NSG mice bearing
PC3-PSMA.CBG induced tumors treated with T cells transduced as
indicated.
[0095] FIG. 14D is a graph depicting the quantification of
bioluminescence obtained from imaging of NSG mice bearing
PC3-PSMA.CBG induced tumors treated with T cells transduced as
indicated.
[0096] FIG. 14E is a graph depicting the quantification of
bioluminescence obtained from imaging of NSG mice bearing
PC3-PSMA.CBG induced tumors treated with T cells transduced as
indicated.
[0097] FIG. 14F is a graph depicting the quantification of
bioluminescence (left) and tumor size (right) obtained from imaging
of NSG mice bearing PC3-PSMA.CBG induced tumors treated with T
cells transduced as indicated.
[0098] FIG. 14G is a table listing from top to bottom, T cells
transduced as indicated, in order of tumor control capability.
ICOS.sup.YMNM is superior to WT ICOS. PD1*BB is better than
PD1*CD28 when with ICOSz or ICOSzYMNM. FIG. 15A is a graph showing
that CART-PSMA-TGF.beta.Rdn cells (dnTGFBR2-T2A-Pbbz) demonstrated
enhanced antigen-specific proliferation versus CART-PSMA (Pbbz)
over 42 days co-culture and repetitive stimulation with
PSMA-expressing tumor cells (arrows). CD19-BBz CART (19bbz) and
transduced T cells (mock) were used as controls.
[0099] FIG. 15B is a graph showing the average radiance detected in
tumor-bearing mice up to 27 days post T cell injection with
CART-PSMA-TGF.beta.Rdn cells (dnTGFBR2-T2A-Pbbz), CART-PSMA cells
(Pbbz), and untransduced cells used as control (mock).
[0100] FIG. 15C are photographs showing the location and systemic
burden of tumor with weekly Bioluminescence imaging (BLI)
assessment.
[0101] FIG. 16 illustrates the study schema used in a phase 1
clinical trial.
[0102] FIG. 17 is a graph showing the evaluation of CAR-T cellular
kinetics via qPCR of CART-PSMA-TGF.beta.Rdn DNA in subjects.
Subjects 32816-02, -04, and -05 are in Cohort 1, and subjects
32816-06, -07, and -08 are in Cohort 2.
[0103] FIG. 18A is a graph showing marked increases in inflammatory
cytokines (IL-6, IL-15, IL-2, IFNgamma) and ferritin, correlating
with a grade 3 cytokine release syndrome event in subject
32816-06.
[0104] FIG. 18B is a graph showing marked increases in inflammatory
cytokines (IL-6, IL-15, IL-2, IFNgamma) and ferritin, correlating
with a grade 3 cytokine release syndrome event in subject
32816-07.
[0105] FIG. 19 is a graph showing the prostate specific antigen
(PSA) response among Cohort 1 and Cohort 2 patients.
[0106] FIG. 20A is a graph showing the expression of
PSMA-TGF.beta.RDN CART (left y-axis in copies/ug of genomic DNA)
and the level of IL-6 (right y-axis in pg/ml) in subject 32816-07,
indicating cytokine release syndrome exhibited in subject one day
post-infusion.
[0107] FIG. 20B is a graph showing that cytokine release syndrome
management was accompanied by transient PSA decrease, as measured
by the level of C-reactive protein (CRP; left y-axis in mg/L) and
the level of serum ferritin (right y-axis in ng/L).
[0108] FIG. 21 is a graph showing the number of PSMA-positive
circulating tumor cells (CTCs) detected in each subject over
time.
[0109] FIG. 22A is a schematic showing two humanized J591 CARs,
dnTGF.hJ591VHVK.BBZ and dnTGF.hJ591VKVH.BBZ.
[0110] FIG. 22B shows flow cytometry examination of CAR and
dominant negative TGFR.beta.II expression in transduced cells as
indicated.
[0111] FIG. 22C shows flow cytometry examination of CD107a
expression in transduced cells as indicated.
[0112] FIG. 23A is a graph showing the killing efficiency of
transduced cells as indicated against PC3-PSMA cells in
coculture.
[0113] FIG. 23B are plots showing the production of various
cytokines as indicated, of the various transduced cells as
indicated in coculture with PC3-PSMA cells.
[0114] FIG. 24A is a schematic showing the various humanized J591
CARs as indicated.
[0115] FIG. 24B shows the surface expression of the various CARs as
indicated on transduced T cells as measured using flow
cytometry.
[0116] FIG. 24C shows flow cytometry examination of CD107a
expression in transduced cells as indicated, when transudced cells
were cocultured with PC3 or PC3-PSMA cells.
[0117] FIG. 24D is a graph showing the killing efficiency of
transduced cells as indicated against PC3-PSMA cells in
coculture.
[0118] FIG. 24E are plots showing the production of cytokines as
indicated, of the various transduced cells as indicated in
coculture with PC3-PSMA cells.
[0119] FIG. 25 shows the surface expression of the various CARs as
indicated on transduced T cells as measured using flow
cytometry.
[0120] FIG. 26A shows flow cytometry examination of dominant
negative TGFR.beta.II and PD1 expression in transduced cells as
indicated.
[0121] FIG. 26B shows flow cytometry examination of CD107a
expression in transduced cells as indicated.
[0122] FIG. 27A is a graph showing the killing efficiency of
transduced cells as indicated against PC3-PSMA cells in
coculture.
[0123] FIG. 27B is a graph showing the killing efficiency of
transduced cells as indicated against PC3 cells in coculture.
[0124] FIG. 28A are plots showing the production of IFN-gamma of
the various transduced cells as indicated in coculture with PC3 or
PC3-PSMA cells.
[0125] FIG. 28B are plots showing the production of IL-2 of the
various transduced cells as indicated in coculture with PC3 or
PC3-PSMA cells.
[0126] FIG. 29 are photographs showing the location and systemic
burden of tumor assessed using bioluminiscence imaging, of various
mice administered transduced cells as indicated, on days as
indicated.
[0127] FIG. 30A is a plot showing the quantification of average
flux detected from tumors in various mice administered transduced
cells as indicated, on days as indicated.
[0128] FIG. 30B is a plot showing the quantification of average
flux detected from tumors in various mice administered transduced
cells as indicated, on day 21.
DETAILED DESCRIPTION
[0129] The present invention provides compositions and methods for
modified immune cells, e.g., T cells and NK cells, or precursors
thereof, e.g., modified T cells, comprising a chimeric antigen
receptor (CAR). In some embodiments, the CAR comprises a
prostate-specific membrane antigen (PSMA) binding domain
(PSMA-CAR), and has affinity for PSMA on a target cell, e.g., a
prostate cancer cell. In some embodiments, the modified immune cell
comprises a PSMA-CAR comprising a murine PSMA binding domain. In
some embodiments, the modified immune cell comprises a PSMA-CAR
comprising a human PSMA binding domain. Also provided are methods
of producing such genetically engineered cells. In some
embodiments, the cells and compositions can be used in adoptive
cell therapy, e.g., adoptive tumor immunotherapy.
[0130] In some embodiments, the provided immune cells comprise
additional receptors, e.g., a dominant negative receptor and/or a
switch receptor, to enhance the efficacy of the immune cell in the
tumor microenvironment. Such cells are capable of altering or
reducing the effects of immunosuppressive signals in the tumor
microenvironment. The modified immune cells of the invention
counteract the upregulation and/or expression of inhibitor receptor
or ligands that can negatively control T cell activation and T cell
function. For example, expression of certain immune checkpoint
proteins, e.g., PD-1 or PD-L1, on T cells and/or in the tumor
microenvironment can reduce the potency and efficacy of adoptive T
cell therapy. For example, expression of TGF-3 on T cells and/or in
the tumor microenvironment can reduce the potency and efficacy of
adoptive T cell therapy. Such immunosuppressive signals may
otherwise impair certain desirable effector functions in the
context of adoptive cell therapy. Tumor cells and/or cells in the
tumor microenvironment often upregulate immunosuppressive proteins,
e.g., PD-L1, delivering an immunosuppressive signal. Such
immunosuppressive proteins may also be upregulated on T cells in
the tumor microenvironment, e.g., on tumor-infiltrating T cells,
which can occur following signaling through the antigen receptor or
certain other activating signals. Such events may contribute to
genetically engineered immune cells (e.g., PSMA targeting) T cells
acquiring an exhausted phenotype, such as when present in proximity
with other cells that express such protein, which in turn can lead
to reduced functionality. Thus, the modified immune cells of the
invention address the T cell exhaustion and/or the lack of T cell
persistence that is a barrier to the efficacy and therapeutic
outcomes of conventional adoptive cell therapies.
[0131] The present invention includes a PSMA CAR and its use in
treating cancer. In certain embodiments, the invention includes a
human PSMA CAR with a dominant negative receptor and/or a switch
receptor. One of the major obstacles for cancer immunotherapy is
the tumor microenvironment. Up-regulation of immunosuppressive
molecules, e.g., PD-1, negatively regulates T cell activity.
[0132] The present invention is based on the finding that T cells
comprising a PSMA-CAR and a dominant negative receptor and/or a
switch receptor are capable of bypassing the effect of
immunosuppressive molecules in the tumor microenvironment,
providing continued and potent anti-tumor activity.
[0133] It is to be understood that the methods described in this
disclosure are not limited to particular methods and experimental
conditions disclosed herein as such methods and conditions may
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0134] Furthermore, the experiments described herein, unless
otherwise indicated, use conventional molecular and cellular
biological and immunological techniques within the skill of the
art. Such techniques are well known to the skilled worker, and are
explained fully in the literature. See, e.g., Ausubel, et al., ed.,
Current Protocols in Molecular Biology, John Wiley & Sons,
Inc., NY, N.Y. (1987-2008), including all supplements, Molecular
Cloning: A Laboratory Manual (Fourth Edition) by M R Green and J.
Sambrook and Harlow et al., Antibodies: A Laboratory Manual,
Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor
(2013, 2nd edition).
A. Definitions
[0135] Unless otherwise defined, scientific and technical terms
used herein have the meanings that are commonly understood by those
of ordinary skill in the art. In the event of any latent ambiguity,
definitions provided herein take precedent over any dictionary or
extrinsic definition. Unless otherwise required by context,
singular terms shall include pluralities and plural terms shall
include the singular. The use of"or" means "and/or" unless stated
otherwise. The use of the term "including," as well as other forms,
such as "includes" and "included," is not limiting.
[0136] Generally, nomenclature used in connection with cell and
tissue culture, molecular biology, immunology, microbiology,
genetics and protein and nucleic acid chemistry and hybridization
described herein is well-known and commonly used in the art. The
methods and techniques provided herein are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification unless
otherwise indicated. Enzymatic reactions and purification
techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well-known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0137] That the disclosure may be more readily understood, select
terms are defined below.
[0138] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0139] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0140] "Activation," as used herein, refers to the state of a T
cell that has been sufficiently stimulated to induce detectable
cellular proliferation. Activation can also be associated with
induced cytokine production, and detectable effector functions. The
term "activated T cells" refers to, among other things, T cells
that are undergoing cell division.
[0141] As used herein, to "alleviate" a disease means reducing the
severity of one or more symptoms of the disease.
[0142] The term "antibody," as used herein, refers to an
immunoglobulin molecule which specifically binds with an antigen.
Antibodies can be intact immunoglobulins derived from natural
sources or from recombinant sources and can be immunoreactive
portions of intact immunoglobulins (e.g., a binding fragment of an
antibody). Antibodies are typically tetramers of immunoglobulin
molecules. The antibodies in the present invention may exist in a
variety of forms including, for example, polyclonal antibodies,
monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain
antibodies (scFv) and humanized antibodies (Harlow et al., 1999,
In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A
Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science
242:423-426).
[0143] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab')2, and Fv
fragments, linear antibodies, scFv antibodies, and multispecific
antibodies formed from antibody fragments.
[0144] An "antibody heavy chain," as used herein, refers to the
larger of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations.
[0145] An "antibody light chain," as used herein, refers to the
smaller of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations.
.alpha. and .beta. light chains refer to the two major antibody
light chain isotypes.
[0146] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0147] The term "antigen" or "Ag" as used herein is defined as a
molecule that provokes an immune response. This immune response may
involve either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen.
[0148] Furthermore, antigens can be derived from recombinant or
genomic DNA. A skilled artisan will understand that any DNA, which
comprises a nucleotide sequences or a partial nucleotide sequence
encoding a protein that elicits an immune response therefore
encodes an "antigen" as that term is used herein. Furthermore, one
skilled in the art will understand that an antigen need not be
encoded solely by a full length nucleotide sequence of a gene. It
is readily apparent that the present invention includes, but is not
limited to, the use of partial nucleotide sequences of more than
one gene and that these nucleotide sequences are arranged in
various combinations to elicit the desired immune response.
Moreover, a skilled artisan will understand that an antigen need
not be encoded by a "gene" at all. It is readily apparent that an
antigen can be generated synthesized or can be derived from a
biological sample. Such a biological sample can include, but is not
limited to a tissue sample, a tumor sample, a cell or a biological
fluid.
[0149] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to which it is later
to be re-introduced into the individual. "Allogeneic" refers to any
material derived from a different animal of the same species.
"Xenogeneic" refers to any material derived from an animal of a
different species.
[0150] The term "chimeric antigen receptor" or "CAR," as used
herein refers to an artificial T cell receptor that is engineered
to be expressed on an immune cell and specifically bind an antigen.
CARs may be used as a therapy with adoptive cell transfer. T cells
are removed from a patient and modified so that they express the
receptors specific to an antigen or particular form of an antigen.
In some embodiments, the CARs have specificity to a selected
target, e.g., cells expressing a prostate-specific membrane
antigen. CARs may also comprise an intracellular activation domain,
a transmembrane domain and an extracellular domain comprising a
tumor associated antigen binding region.
[0151] "Co-stimulatory ligand," as the term is used herein,
includes a molecule on an antigen presenting cell (e.g., an
artificial APC (aAPC), dendritic cell, B cell, and the like) that
specifically binds a cognate co-stimulatory molecule on a T cell,
thereby providing a signal which, in addition to the primary signal
provided by, for instance, binding of a TCR/CD3 complex with an MHC
molecule loaded with peptide, mediates a T cell response,
including, but not limited to, proliferation, activation,
differentiation, and the like. A co-stimulatory ligand can include,
but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2,
4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L),
intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83,
HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3,
ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor
and a ligand that specifically binds with B7-H3. A co-stimulatory
ligand also encompasses, inter alia, an antibody that specifically
binds with a co-stimulatory molecule present on a T cell, such as,
but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,
ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,
LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with
CD83.
[0152] A "co-stimulatory molecule" refers to the cognate binding
partner on a T cell that specifically binds with a co-stimulatory
ligand, thereby mediating a co-stimulatory response by the T cell,
such as, but not limited to, proliferation. Co-stimulatory
molecules include, but are not limited to an MHC class I molecule,
BTLA and a Toll ligand receptor.
[0153] A "co-stimulatory signal", as used herein, refers to a
signal, which in combination with a primary signal, such as TCR/CD3
ligation, leads to T cell proliferation and/or upregulation or
downregulation of key molecules.
[0154] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in
which the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0155] The term "downregulation" as used herein refers to the
decrease or elimination of gene expression of one or more
genes.
[0156] "Effective amount" or "therapeutically effective amount" are
used interchangeably herein, and refer to an amount of a compound,
formulation, material, or composition, as described herein
effective to achieve a particular biological result or provides a
therapeutic or prophylactic benefit. Such results may include, but
are not limited to an amount that when administered to a mammal,
causes a detectable level of immune suppression or tolerance
compared to the immune response detected in the absence of the
composition of the invention. The immune response can be readily
assessed by a plethora of art-recognized methods. The skilled
artisan would understand that the amount of the composition
administered herein varies and can be readily determined based on a
number of factors such as the disease or condition being treated,
the age and health and physical condition of the mammal being
treated, the severity of the disease, the particular compound being
administered, and the like.
[0157] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0158] As used herein "endogenous" refers to any material from or
produced inside an organism, cell, tissue or system.
[0159] The term "epitope" as used herein is defined as a small
chemical molecule on an antigen that can elicit an immune response,
inducing B and/or T cell responses. An antigen can have one or more
epitopes. Most antigens have many epitopes; i.e., they are
multivalent. In general, an epitope is roughly about 10 amino acids
and/or sugars in size. Preferably, the epitope is about 4-18 amino
acids, more preferably about 5-16 amino acids, and even more most
preferably 6-14 amino acids, more preferably about 7-12, and most
preferably about 8-10 amino acids. One skilled in the art
understands that generally the overall three-dimensional structure,
rather than the specific linear sequence of the molecule, is the
main criterion of antigenic specificity and therefore distinguishes
one epitope from another. Based on the present disclosure, a
peptide of the present invention can be an epitope.
[0160] As used herein, the term "exogenous" refers to any material
introduced from or produced outside an organism, cell, tissue or
system.
[0161] The term "expand" as used herein refers to increasing in
number, as in an increase in the number of T cells. In one
embodiment, the T cells that are expanded ex vivo increase in
number relative to the number originally present in the culture. In
another embodiment, the T cells that are expanded ex vivo increase
in number relative to other cell types in the culture. The term "ex
vivo," as used herein, refers to cells that have been removed from
a living organism, (e.g., a human) and propagated outside the
organism (e.g., in a culture dish, test tube, or bioreactor).
[0162] The term "expression" as used herein is defined as the
transcription and/or translation of a particular nucleotide
sequence driven by its promoter.
[0163] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(e.g., naked or contained in liposomes) and viruses (e.g., Sendai
viruses, lentiviruses, retroviruses, adenoviruses, and
adeno-associated viruses) that incorporate the recombinant
polynucleotide.
[0164] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which
residues from a complementary-determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies can comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework
sequences. These modifications are made to further refine and
optimize antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody may also comprise
at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. For further details, see
Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature,
332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596,
1992. "Fully human" refers to an immunoglobulin, such as an
antibody, or binding fragment thereof, where the whole molecule is
of human origin or consists of an amino acid sequence identical to
a human form of the antibody.
[0165] The term "immunoglobulin" or "Ig," as used herein is defined
as a class of proteins, which function as antibodies. The five
members included in this class of proteins are IgA, IgG, IgM, IgD,
and IgE. IgA is the primary antibody that is present in body
secretions, such as saliva, tears, breast milk, gastrointestinal
secretions and mucus secretions of the respiratory and
genitourinary tracts. IgG is the most common circulating antibody.
IgM is the main immunoglobulin produced in the primary immune
response in most subjects. It is the most efficient immunoglobulin
in agglutination, complement fixation, and other antibody
responses, and is important in defense against bacteria and
viruses. IgD is the immunoglobulin that has no known antibody
function, but may serve as an antigen receptor. IgE is the
immunoglobulin that mediates immediate hypersensitivity by causing
release of mediators from mast cells and basophils upon exposure to
allergen.
[0166] "Identity" as used herein refers to the subunit sequence
identity between two polymeric molecules particularly between two
amino acid molecules, such as, between two polypeptide molecules.
When two amino acid sequences have the same residues at the same
positions; e.g., if a position in each of two polypeptide molecules
is occupied by an arginine, then they are identical at that
position. The identity or extent to which two amino acid sequences
have the same residues at the same positions in an alignment is
often expressed as a percentage. The identity between two amino
acid sequences is a direct function of the number of matching or
identical positions; e.g., if half (e.g., five positions in a
polymer ten amino acids in length) of the positions in two
sequences are identical, the two sequences are 50% identical; if
90% of the positions (e.g., 9 of 10), are matched or identical, the
two amino acids sequences are 90% identical.
[0167] The term "immune response" as used herein is defined as a
cellular response to an antigen that occurs when lymphocytes
identify antigenic molecules as foreign and induce the formation of
antibodies and/or activate lymphocytes to remove the antigen.
[0168] The term "immunosuppressive" is used herein to refer to
reducing overall immune response.
[0169] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a peptide naturally present in a
living animal is not "isolated," but the same nucleic acid or
peptide partially or completely separated from the coexisting
materials of its natural state is "isolated." An isolated nucleic
acid or protein can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a host
cell.
[0170] A "lentivirus" as used herein refers to a genus of the
Retroviridae family. Lentiviruses are unique among the retroviruses
in being able to infect non-dividing cells; they can deliver a
significant amount of genetic information into the DNA of the host
cell, so they are one of the most efficient methods of a gene
delivery vector. HIV, SIV, and FIV are all examples of
lentiviruses. Vectors derived from lentiviruses offer the means to
achieve significant levels of gene transfer in vivo.
[0171] By the term "modified" as used herein, is meant a changed
state or structure of a molecule or cell of the invention.
Molecules may be modified in many ways, including chemically,
structurally, and functionally. Cells may be modified through the
introduction of nucleic acids.
[0172] By the term "modulating," as used herein, is meant mediating
a detectable increase or decrease in the level of a response in a
subject compared with the level of a response in the subject in the
absence of a treatment or compound, and/or compared with the level
of a response in an otherwise identical but untreated subject. The
term encompasses perturbing and/or affecting a native signal or
response thereby mediating a beneficial therapeutic response in a
subject, preferably, a human.
[0173] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0174] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or an RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0175] The term "operably linked" or "operatively linked" refers to
functional linkage between a regulatory sequence and a heterologous
nucleic acid sequence resulting in expression of the latter. For
example, a first nucleic acid sequence is operably linked with a
second nucleic acid sequence when the first nucleic acid sequence
is placed in a functional relationship with the second nucleic acid
sequence. For instance, a promoter is operably linked to a coding
sequence if the promoter affects the transcription or expression of
the coding sequence.
[0176] The term "polynucleotide" as used herein is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, nucleic acids and polynucleotides as used herein
are interchangeable. One skilled in the art has the general
knowledge that nucleic acids are polynucleotides, which can be
hydrolyzed into the monomeric "nucleotides." The monomeric
nucleotides can be hydrolyzed into nucleosides. As used herein
polynucleotides include, but are not limited to, all nucleic acid
sequences which are obtained by any means available in the art,
including, without limitation, recombinant means, i.e., the cloning
of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR, and the like,
and by synthetic means.
[0177] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptides, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0178] By the term "specifically binds," as used herein with
respect to an antibody, is meant an antibody which recognizes a
specific antigen, but does not substantially recognize or bind
other molecules in a sample. For example, an antibody that
specifically binds to an antigen from one species may also bind to
that antigen from one or more species. But, such cross-species
reactivity does not itself alter the classification of an antibody
as specific. In another example, an antibody that specifically
binds to an antigen may also bind to different allelic forms of the
antigen. However, such cross reactivity does not itself alter the
classification of an antibody as specific. In some instances, the
terms "specific binding" or "specifically binding," can be used in
reference to the interaction of an antibody, a protein, or a
peptide with a second chemical species, to mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0179] By the term "stimulation," is meant a primary response
induced by binding of a stimulatory molecule (e.g., a TCR/CD3
complex) with its cognate ligand thereby mediating a signal
transduction event, such as, but not limited to, signal
transduction via the TCR/CD3 complex. Stimulation can mediate
altered expression of certain molecules, such as downregulation of
TGF-beta, and/or reorganization of cytoskeletal structures, and the
like.
[0180] A "stimulatory molecule," as the term is used herein, means
a molecule on a T cell that specifically binds with a cognate
stimulatory ligand present on an antigen presenting cell.
[0181] A "stimulatory ligand," as used herein, means a ligand that
when present on an antigen presenting cell (e.g., an aAPC, a
dendritic cell, a B-cell, and the like) can specifically bind with
a cognate binding partner (referred to herein as a "stimulatory
molecule") on a T cell, thereby mediating a primary response by the
T cell, including, but not limited to, activation, initiation of an
immune response, proliferation, and the like. Stimulatory ligands
are well-known in the art and encompass, inter alia, an MHC Class I
molecule loaded with a peptide, an anti-CD3 antibody, a
superagonist anti-CD28 antibody, and a superagonist anti-CD2
antibody.
[0182] The term "subject" is intended to include living organisms
in which an immune response can be elicited (e.g., mammals). A
"subject" or "patient," as used herein, may be a human or non-human
mammal. Non-human mammals include, for example, livestock and pets,
such as ovine, bovine, porcine, canine, feline and murine mammals.
Preferably, the subject is human.
[0183] A "target site" or "target sequence" refers to a genomic
nucleic acid sequence that defines a portion of a nucleic acid to
which a binding molecule may specifically bind under conditions
sufficient for binding to occur.
[0184] The term "therapeutic" as used herein means a treatment
and/or prophylaxis. A therapeutic effect is obtained by
suppression, remission, or eradication of a disease state.
[0185] "Transplant" refers to a biocompatible lattice or a donor
tissue, organ or cell, to be transplanted. An example of a
transplant may include but is not limited to skin cells or tissue,
bone marrow, and solid organs such as heart, pancreas, kidney, lung
and liver. A transplant can also refer to any material that is to
be administered to a host. For example, a transplant can refer to a
nucleic acid or a protein.
[0186] The term "transfected" or "transformed" or "transduced" as
used herein refers to a process by which exogenous nucleic acid is
transferred or introduced into the host cell. A "transfected" or
"transformed" or "transduced" cell is one which has been
transfected, transformed or transduced with exogenous nucleic acid.
The cell includes the primary subject cell and its progeny.
[0187] To "treat" a disease as the term is used herein, means to
reduce the frequency or severity of at least one sign or symptom of
a disease or disorder experienced by a subject.
[0188] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. The term should also
be construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for
example, polylysine compounds, liposomes, and the like. Examples of
viral vectors include, but are not limited to, Sendai viral
vectors, adenoviral vectors, adeno-associated virus vectors,
retroviral vectors, lentiviral vectors, and the like.
[0189] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
B. Chimeric Antigen Receptors
[0190] The present invention provides compositions and methods for
modified immune cells or precursors thereof, e.g., modified T
cells, comprising a chimeric antigen receptor (CAR). Thus, in some
embodiments, the immune cell has been genetically modified to
express the CAR. CARs of the present invention comprise an antigen
binding domain, a transmembrane domain, a hinge domain, and an
intracellular signaling domain.
[0191] The antigen binding domain may be operably linked to another
domain of the CAR, such as the transmembrane domain or the
intracellular domain, both described elsewhere herein, for
expression in the cell. In one embodiment, a first nucleic acid
sequence encoding the antigen binding domain is operably linked to
a second nucleic acid encoding a transmembrane domain, and further
operably linked to a third a nucleic acid sequence encoding an
intracellular domain.
[0192] The antigen binding domains described herein can be combined
with any of the transmembrane domains described herein, any of the
intracellular domains or cytoplasmic domains described herein, or
any of the other domains described herein that may be included in a
CAR of the present invention. A subject CAR of the present
invention may also include a spacer domain as described herein. In
some embodiments, each of the antigen binding domain, transmembrane
domain, and intracellular domain is separated by a linker.
Antigen Binding Domain
[0193] The antigen binding domain of a CAR is an extracellular
region of the CAR for binding to a specific target antigen
including proteins, carbohydrates, and glycolipids. In some
embodiments, the CAR comprises affinity to a target antigen on a
target cell. The target antigen may include any type of protein, or
epitope thereof, associated with the target cell. For example, the
CAR may comprise affinity to a target antigen on a target cell that
indicates a particular disease state of the target cell.
[0194] In an exemplary embodiment, the target cell antigen is a
prostate-specific membrane antigen (PSMA). PSMA is a membrane-bound
protein expressed on the cell surface and is reported to be highly
overexpressed in prostate cancer tissues. PSMA expression is
directly correlated with advancing tumor grade and stage, and is
believed to confer a selective growth advantage to prostate cancer
cells. As such, an exemplary CAR of the present disclosure has
affinity for PSMA on a target cell.
[0195] As described herein, a CAR of the present disclosure having
affinity for a specific target antigen on a target cell may
comprise a target-specific binding domain. In some embodiments, the
target-specific binding domain is a murine target-specific binding
domain, e.g., the target-specific binding domain is of murine
origin. In some embodiments, the target-specific binding domain is
a human target-specific binding domain, e.g., the target-specific
binding domain is of human origin. In an exemplary embodiment, a
CAR of the present disclosure having affinity for PSMA on a target
cell may comprise a PSMA binding domain. In some embodiments, the
PSMA binding domain is a murine PSMA binding domain, e.g., the PSMA
binding domain is of murine origin. In some embodiments, the PSMA
binding domain is a human PSMA binding domain, e.g., the PSMA
binding domain is of human origin.
[0196] In some embodiments, a CAR of the present disclosure may
have affinity for one or more target antigens on one or more target
cells. In some embodiments, a CAR may have affinity for one or more
target antigens on a target cell. In such embodiments, the CAR is a
bispecific CAR, or a multispecific CAR. In some embodiments, the
CAR comprises one or more target-specific binding domains that
confer affinity for one or more target antigens. In some
embodiments, the CAR comprises one or more target-specific binding
domains that confer affinity for the same target antigen. For
example, a CAR comprising one or more target-specific binding
domains having affinity for the same target antigen could bind
distinct epitopes of the target antigen. When a plurality of
target-specific binding domains is present in a CAR, the binding
domains may be arranged in tandem and may be separated by linker
peptides. For example, in a CAR comprising two target-specific
binding domains, the binding domains are connected to each other
covalently on a single polypeptide chain, through an oligo- or
polypeptide linker, an Fc hinge region, or a membrane hinge
region.
[0197] In some embodiments, the antigen binding domain is selected
from the group consisting of an antibody, an antigen binding
fragment (Fab), and a single-chain variable fragment (scFv). In
some embodiments, a PSMA binding domain of the present invention is
selected from the group consisting of a PSMA-specific antibody, a
PSMA-specific Fab, and a PSMA-specific scFv. In one embodiment, a
PSMA binding domain is a PSMA-specific antibody. In one embodiment,
a PSMA binding domain is a PSMA-specific Fab. In one embodiment, a
PSMA binding domain is a PSMA-specific scFv.
[0198] The antigen binding domain can include any domain that binds
to the antigen and may include, but is not limited to, a monoclonal
antibody, a polyclonal antibody, a synthetic antibody, a human
antibody, a humanized antibody, a non-human antibody, and any
fragment thereof. In some embodiments, the antigen binding domain
portion comprises a mammalian antibody or a fragment thereof. The
choice of antigen binding domain may depend upon the type and
number of antigens that are present on the surface of a target
cell.
[0199] As used herein, the term "single-chain variable fragment" or
"scFv" is a fusion protein of the variable regions of the heavy
(VH) and light chains (VL) of an immunoglobulin (e.g., mouse or
human) covalently linked to form a VH::VL heterodimer.
[0200] The heavy (VH) and light chains (VL) are either joined
directly or joined by a peptide-encoding linker, which connects the
N-terminus of the VH with the C-terminus of the VL, or the
C-terminus of the VH with the N-terminus of the VL. In some
embodiments, the antigen binding domain (e.g., PSMA binding domain)
comprises an scFv having the configuration from N-terminus to
C-terminus, VH-linker-VL. In some embodiments, the antigen binding
domain (e.g., PSMA binding domain) comprises an scFv having the
configuration from N-terminus to C-terminus, VL-linker-VH. Those of
skill in the art would be able to select the appropriate
configuration for use in the present invention.
[0201] The linker is usually rich in glycine for flexibility, as
well as serine or threonine for solubility. The linker can link the
heavy chain variable region and the light chain variable region of
the extracellular antigen-binding domain. Non-limiting examples of
linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917
(2008) and WO 2014/087010, the contents of which are hereby
incorporated by reference in their entireties. Various linker
sequences are known in the art, including, without limitation,
glycine serine (GS) linkers such as (GS).sub.n, (GSGGS).sub.n (SEQ
ID NO: 1), (GGGS).sub.n (SEQ ID NO:2), and (GGGGS).sub.n(SEQ ID
NO:3), where n represents an integer of at least 1. Exemplary
linker sequences can comprise amino acid sequences including,
without limitation, GGSG (SEQ ID NO:4), GGSGG (SEQ ID NO:5), GSGSG
(SEQ ID NO:6), GSGGG (SEQ ID NO:7), GGGSG (SEQ ID NO:8), GSSSG (SEQ
ID NO:9), GGGGS (SEQ ID NO:10), GGGGSGGGGSGGGGS (SEQ ID NO: 11) and
the like. Those of skill in the art would be able to select the
appropriate linker sequence for use in the present invention. In
one embodiment, an antigen binding domain (e.g., PSMA binding
domain) of the present invention comprises a heavy chain variable
region (VH) and a light chain variable region (VL), wherein the VH
and VL is separated by the linker sequence having the amino acid
sequence GGGGSGGGGSGGGGS (SEQ ID NO: 11), which may be encoded by
the nucleic acid sequence
GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQ ID NO:12).
[0202] Despite removal of the constant regions and the introduction
of a linker, scFv proteins retain the specificity of the original
immunoglobulin. Single chain Fv polypeptide antibodies can be
expressed from a nucleic acid comprising VH- and VL-encoding
sequences as described by Huston, et al. (Proc. Nat. Acad. Sci.
USA, 85:5879-5883, 1988). See, also, U.S. Pat. Nos. 5,091,513,
5,132,405 and 4,956,778; and U.S. Patent Publication Nos.
20050196754 and 20050196754. Antagonistic scFvs having inhibitory
activity have been described (see, e.g., Zhao et al., Hyrbidoma
(Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia
Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85;
Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J
Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology
1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40).
Agonistic scFvs having stimulatory activity have been described
(see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie
et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev
Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003
1638(3):257-66).
[0203] As used herein, "Fab" refers to a fragment of an antibody
structure that binds to an antigen but is monovalent and does not
have a Fc portion, for example, an antibody digested by the enzyme
papain yields two Fab fragments and an Fc fragment (e.g., a heavy
(H) chain constant region; Fc region that does not bind to an
antigen).
[0204] As used herein, "F(ab')2" refers to an antibody fragment
generated by pepsin digestion of whole IgG antibodies, wherein this
fragment has two antigen binding (ab) (bivalent) regions, wherein
each (ab') region comprises two separate amino acid chains, a part
of a H chain and a light (L) chain linked by an S--S bond for
binding an antigen and where the remaining H chain portions are
linked together. A "F(ab')2" fragment can be split into two
individual Fab' fragments.
[0205] In some embodiments, the antigen binding domain may be
derived from the same species in which the CAR will ultimately be
used. For example, for use in humans, the antigen binding domain of
the CAR may comprise a human antibody as described elsewhere
herein, or a fragment thereof.
[0206] In an exemplary embodiment, a PSMA-CAR of the present
invention comprises a PSMA binding domain, e.g., PSMA-specific
scFv.
[0207] (a) Murine PSMA Binding Domains and Variants Thereof
[0208] In certain embodiments, a PSMA-CAR of the present invention
comprises a murine PSMA binding domain or variant thereof.
[0209] In certain embodiments, a PSMA-CAR of the present invention
comprises a PSMA binding domain of a non-human PSMA antibody (e.g.,
a mouse or rat PSMA antibody), or a variant thereof. As is well
known in the art, a murine or other non-human antibody may be
raised by immunizing the non-human (e.g., a mouse) with human PSMA
or a fragment thereof.
[0210] In one embodiment, the PSMA binding domain is a murine J591
PSMA binding domain that is comprised in the amino acid sequence
set forth below:
TABLE-US-00001 (SEQ ID NO: 14)
MALPVTALLLPLALLLHAARPGSDIVMTQSHKFMSTSVGDRVSIICKAS
QDVGTAVDWYQQKPGQSPKWYWASTRHTGVPDRFTGSGSGTDFTLTITN
VQSEDLADYFCQQYNSYPLTFGAGTMLDLKGGGGSGGGGSSGGGSEVQL
QQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINP
NNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNF
DYWGQGTTLTVSS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00002 (SEQ ID NO: 15)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC
ACGCCGCCAGACCTGGATCTGACATTGTGATGACCCAGTCTCACAAATT
CATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGT
CAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAAT
CTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCC
TGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATT
ACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATA
ACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGG
AGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTG
CAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGC
GGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCA
CTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATC
AACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGG
CCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCG
GAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGG
AACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGC.
[0211] Tolerable variations of the murine J591 PSMA binding domain
will be known to those of skill in the art, while maintaining
binding to PSMA. For example, in some embodiments, the PSMA binding
domain is a murine J591 PSMA binding domain comprising an amino
acid sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the murine J591
PSMA binding domain amino acid sequence that is comprised in SEQ ID
NO: 14. In one embodiment, the PSMA binding domain is a murine J591
PSMA binding domain that is comprised in the amino acid sequence
set forth in SEQ ID NO:14.
[0212] In some embodiments, the PSMA binding domain is a murine
J591 PSMA binding domain encoded by a nucleic acid sequence that
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the murine J591 PSMA binding
domain coding sequence comprised in SEQ ID NO:15. In one
embodiment, the PSMA binding domain is a murine J591 PSMA binding
domain encoded by the coding sequence comprised in the nucleic acid
sequence set forth in SEQ ID NO:15.
[0213] In an exemplary embodiment, a PSMA-CAR of the present
invention comprises a PSMA binding domain, e.g., PSMA-specific
scFv. In one embodiment, the PSMA binding domain is a murine J591
PSMA binding domain comprising the amino acid sequence set forth
below:
TABLE-US-00003 (SEQ ID NO: 13)
DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIY
WASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTF
GAGTMLDLKGGGGSGGGGSSGGGSEVQLQQSGPELVKPGTSVRISCKTS
GYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDK
SSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00004 (SEQ ID NO:180)
GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAG
ACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGT
AGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTAT
TGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTG
GATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGA
CTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTC
GGTGCTGGGACCATGCTGGACCTGAAAGGAGGCGGAGGATCTGGCGGCG
GAGGAAGTTCTGGCGGAGGCAGCGAGGTGCAGCTGCAGCAGAGCGGACC
CGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGC
GGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACG
GCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCAC
CACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAG
AGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACA
GCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCA
GGGCACAACCCTGACAGTGTCTAGC.
[0214] Tolerable variations of the murine J591 PSMA binding domain
will be known to those of skill in the art, while maintaining
binding to human PSMA. For example, in some embodiments, the PSMA
binding domain is a murine J591 PSMA binding domain comprising an
amino acid sequence that has at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
amino acid sequence set forth in in SEQ ID NO:13. In one
embodiment, the PSMA binding domain is a murine J591 PSMA binding
domain comprising the amino acid sequence set forth in SEQ ID NO:
13.
[0215] In some embodiments, the PSMA binding domain is a murine
J591 PSMA binding domain encoded by a nucleic acid sequence that
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the nucleic acid sequence set
forth in SEQ ID NO: 180. In one embodiment, the PSMA binding domain
is a murine J591 PSMA binding domain encoded by the nucleic acid
sequence set forth in SEQ ID NO:180.
[0216] In one embodiment, the murine J591 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00005 (SEQ ID NO:16)
DIVIVITQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKWY
WASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTF GAGTMLDLK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00006 (SEQ ID NO:17)
GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAG
ACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGT
AGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTAT
TGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTG
GATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGA
CTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTC
GGTGCTGGGACCATGCTGGACCTGAAA.
[0217] Tolerable variations of the light chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the murine J591 PSMA binding domain comprises a light
chain variable region comprising an amino acid sequence that has at
least 60%/a, at least 65%, at least 70%/a, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the amino acid sequence set forth in
SEQ ID NO: 16. In one embodiment, the murine J591 PSMA binding
domain comprises a light chain variable region comprising the amino
acid sequence set forth in SEQ ID NO:16.
[0218] In some embodiments, the murine J591 PSMA binding domain
comprises a light chain variable region encoded by a nucleic acid
sequence that has at least 60%/a, at least 65%, at least 70/%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:17. In one embodiment, the murine
J591 PSMA binding domain comprises a light chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:
17.
[0219] In one embodiment, the murine J591 PSMA binding domain
comprises the light chain variable region described in NCBI GenBank
sequence database ID: CCA78125.1, comprising the amino acid
sequence set forth below:
TABLE-US-00007 (SEQ ID NO:181)
DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIY
WASTRHTGVPDRFTGSGSGTDFTLAITNVQSEDLADYFCQQYNSYPLTF GAGTKLEIKR.
[0220] Tolerable variations of the light chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the murine J591 PSMA binding domain comprises a light
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:181. In one embodiment, the murine J591 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:181. The light chain variable
region of the murine J591 PSMA binding domain comprises three light
chain complementarity-determining regions (CDRs). As used herein, a
"complementarity-determining region" or "CDR" refers to a region of
the variable chain of an antigen binding molecule that binds to a
specific antigen. Accordingly, a murine J591 PSMA binding domain
may comprise a light chain variable region that comprises a CDR1
represented by the amino acid sequence KASQDVGTAVD (SEQ ID NO: 18);
a CDR2 represented by the amino acid sequence WASTRHT (SEQ ID NO:
19); and a CDR3 represented by the amino acid sequence QQYNSYPLT
(SEQ ID NO:20). Tolerable variations to the CDRs of the light chain
will be known to those of skill in the art, while maintaining its
contribution to the binding of PSMA. For example, a murine J591
PSMA binding domain may comprise a light chain variable region
comprising a CDR1 that comprises an amino acid sequence that has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the CDR1 amino acid sequence set
forth in SEQ ID NO: 18. For example, a murine J591 PSMA binding
domain may comprise a light chain variable region comprising a CDR2
that comprises an amino acid sequence that has at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the CDR2 amino acid sequence set forth in SEQ
ID NO: 19. For example, a murine J591 PSMA binding domain may
comprise a light chain variable region comprising a CDR3 that
comprises an amino acid sequence that has at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the CDR3 amino acid sequence set forth in SEQ ID NO:20.
In one embodiment, the murine J591 PSMA binding domain comprises a
light chain variable region comprising the three aforementioned
light chain variable region CDRs.
[0221] In one embodiment, the murine J591 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00008 (SEQ ID NO:21)
EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTMWVKQSHGKSLEWIGN
INPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAG
WNFDYWGQGTTLTVSS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00009 (SEQ ID NO:22)
GAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAA
GCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACAC
CATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGC
AATATCAACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGG
ACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGA
ACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCC
GGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTA GC.
[0222] Tolerable variations of the heavy chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the murine J591 PSMA binding domain comprises a heavy
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:21. In one embodiment, the murine J591 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:21.
[0223] In some embodiments, the murine J591 PSMA binding domain
comprises a heavy chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:22. In one embodiment, the murine
J591 PSMA binding domain comprises a heavy chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:22.
[0224] In one embodiment, the murine J591 PSMA binding domain
comprises the heavy chain variable region described in NCBI GenBank
sequence database ID: CCA78124.1, comprising the amino acid
sequence set forth below:
TABLE-US-00010 (SEQ ID NO:182)
EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTMWVKQSHGKSLEWIGN
INPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAG
WNFDYWGQGTTLTVSS.
[0225] Tolerable variations of the heavy chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of PSMA. For example, in some
embodiments, the murine J591 PSMA binding domain comprises a heavy
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO: 182. In one embodiment, the murine J591 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 182.
[0226] The heavy chain variable region of the murine J591 PSMA
binding domain comprises three heavy chain
complementarity-determining regions (CDRs). Accordingly, a murine
J591 PSMA binding domain may comprise a heavy chain variable region
that comprises a CDR1 represented by the amino acid sequence
GYTFTEYTIH (SEQ ID NO:23); a CDR2 represented by the amino acid
sequence NINPNNGGTTYNQKFED (SEQ ID NO:24); and a CDR3 represented
by the amino acid sequence GWNFDY (SEQ ID NO:25). Tolerable
variations to the CDRs of the heavy chain will be known to those of
skill in the art, while maintaining its contribution to the binding
of human PSMA. For example, a murine J591 PSMA binding domain may
comprise a heavy chain variable region comprising a CDR1 that
comprises an amino acid sequence that has at least 85%, at least
86%, at least 87%, at least 88%, at least 89%/a, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the CDR1 amino acid sequence set forth in SEQ ID NO:23.
For example, a murine J591 PSMA binding domain may comprise a heavy
chain variable region comprising a CDR2 that comprises an amino
acid sequence that has at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%/a, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the CDR2
amino acid sequence set forth in SEQ ID NO:24. For example, a
murine J591 PSMA binding domain may comprise a heavy chain variable
region comprising a CDR3 that comprises an amino acid sequence that
has at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%/a, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the CDR3 amino acid
sequence set forth in SEQ ID NO:25. In one embodiment, the murine
J591 PSMA binding domain comprises a heavy chain variable region
comprising the three aforementioned heavy chain variable region
CDRs.
[0227] In one embodiment, the PSMA binding domain is a murine J591
PSMA binding domain comprising an amino acid sequence that
comprises at least 60%/a, at least 65%, at least 70%/a, at least
75%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the amino acid
sequences set forth in SEQ ID NOs:16 and 21.
[0228] In one embodiment, the PSMA binding domain is a murine J591
PSMA binding domain comprising an amino acid sequence that
comprises at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% sequence identity to the amino acid sequences set
forth in SEQ ID NOs: 181 and 182
[0229] (b) Humanized PSMA Binding Domains
[0230] In certain embodiments, a PSMA-CAR of the present invention
comprises a humanized variant of a PSMA binding domain of a
non-human PSMA antibody, or a variant or fragment thereof. In
certain exemplary embodiments, the PSMA CAR comprises a humanized
variant of the murine J591 antibody which binds human PSMA. Methods
for humanizing murine antibodies are well known in the art.
[0231] In one embodiment, the PSMA binding domain is a humanized
PSMA-specific binding domain. In certain embodiments, the PSMA
binding domain is a humanized J591 PSMA binding domain. In certain
embodiments, the PSMA binding domain comprises any of the heavy and
light chain variable regions disclosed in PCT Publication Nos.
WO2017212250A1 and WO2018033749A1, the disclosures of which are
hereby incorporated herein by reference in their entirety. For
example, a PSMA binding domain of the present invention can
comprise an scFv comprising any of the heavy and light chain
variable regions disclosed therein. Accordingly, a PSMA-CAR of the
present invention comprises a humanized variant of the murine J591
antibody which binds human PSMA, as disclosed in WO2017212250A1 and
WO2018033749A1.
[0232] In certain embodiments, a PSMA binding domain of the present
invention can comprise a heavy chain variable region and a light
chain variable region of any of those set forth in Table 19:
TABLE-US-00011 TABLE 19 Humanized PSMA binding heavy and light
chain variable sequences Heavy Chain Variable Region Sequences
Light Chain Variable Region Sequences VH Consensus Sequence VL
Consensus Sequence SEQ ID NO:183 SEQ ID NO:184
EVQLVQSGX.sub.1EX2KKPGASVKVSCKX.sub.3
DIX.sub.1MTQSPSX.sub.2LSASVGDRVTITCKASQDV
SGYTFTEYTIHWVX.sub.4QAX.sub.5GKGLEWIG GTAVDWYQQKPGQAPKLLIYWASTRHTG
NINPNX.sub.6GGTTYNQKFEDRX.sub.7TX.sub.8TVD
VPDRFX.sub.3GSGSGTDFTLTISRLQX.sub.4EDFAX.sub.5Y
KSTSTAYMELSSLRSEDTAVYYCAAG X.sub.6CQQYNSYPLTFGQGTX.sub.7VDIK
WNFDYWGQGTTVTVSS wherein: wherein: X.sub.1 is Q or V; X.sub.1 is A
or P; X.sub.2 is T or F; X.sub.2 is V or L; X.sub.3 iS S or T;
X.sub.3 is A or T; X.sub.4 is P or S; X.sub.4 is R or K; X.sub.5 is
V or D; X.sub.5 is P or H; X.sub.6 is Y or F; and X.sub.6 is N or
Q; X.sub.7 is K or M. X.sub.7 iS V or A; and X.sub.8 is I or L. SEQ
ID NO:185 SEQ ID NO:186 EVQLVQSGPELKKPGASVKVSCKTSG
DIVMTQSPSFLSASVGDRVTITCKASQDVG YTFTEYTIHWVKQAHGKGLEWIGNIN
TAVDWYQQKPGQAPKLLIYWASTRHTGV PNNGGTTYNQKFEDRATLTVDKSTST
PDRFTGSGSGTDFTLTISRLQSEDFADYFCQ AYMELSSLRSEDTAVYYCAAGWNFD
QYNSYPLTFGQGTMVDIK YWGQGTTVTVS S SEQ ID NO:187 SEQ ID NO:188
EVQLVQSGAEVKKPGASVKVSCKTSG DIVMTQSPSTLSASVGDRVTITCKASQDVG
YTFTEYTIHWVKQAPGKGLEWIGNIN TAVDWYQQKPGQAPKLLIYWASTRHTGV
PNNGGTTYNQKFEDRATITVDKSTST PDRFTGSGSGTDFTLTISRLQSEDFADYFCQ
AYMELSSLRSEDTAVYYCAAGWNFD QYNSYPLTFGQGTKVDIK YWGQGTTVTVSS SEQ ID
NO:189 SEQ ID NO:190 EVQLVQSGAEVKKPGASVKVSCKTSG
DIVMTQSPSTLSASVGDRVTITCKASQDVG YTFTEYTIHWVRQAPGKGLEWIGNIN
TAVDWYQQKPGQAPKLLIYWASTRHTGV PNNGGTTYNQKFEDRATITVDKSTST
PDRFSGSGSGTDFTLTISRLQPEDFADYYCQ AYMELSSLRSEDTAVYYCAAGWNFD
QYNSYPLTFGQGTKVDIK YWGQGTTVTVSS SEQ ID NO:191 SEQ ID NO:192
EVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSTLSASVGDRVTITCKASQDVG
GYTFTEYTIHWVRQAPGKGLEWIGNI TAVDWYQQKPGQAPKLLIYWASTRHTGV
NPNNGGTTYNQKFEDRVTITVDKSTS PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQ
TAYMELSSLRSEDTAVYYCAAGWNF QYNSYPLTFGQGTKVDIK DYWGQGTTVTVSS SEQ ID
NO:193 EVQLVQSGAEVKKPGASVKVSCKAS GYTFTEYTIHWVRQAPGKGLEWIGNI
NPNQGGTTYNQKFEDRVTITVDKSTS TAYMELSSLRSEDTAVYYCAAGWNF DYWGQGTTVTVSS
VH Consensus Sequence VL Consensus Sequence SEQ ID NO:194 SEQ ID
NO:195 EVQLVQSGX.sub.1EX.sub.2KKPGASVKVSCKX.sub.3
DIX.sub.1MTQSPSX.sub.2LSASVGDRVTITCKASQDV
SGYTFTEYTIHWVX.sub.4QAX.sub.5GKGLEWIG GTAVDWYQQKPGQAPKLLIYWASTRHTG
NINPNX.sub.6GGTTYNQKFEDRX.sub.7TX.sub.8TVD
VPDRFX.sub.3GSGSGTDFTLTISRLQX.sub.4EDFAX.sub.5Y
KSTSTAYMELSSX.sub.9RSEDTAVYYCAX.sub.10
X.sub.6CQQX.sub.7X.sub.8X.sub.9X.sub.10X.sub.11LTFGQGTX.sub.12VDIK
X.sub.11X.sub.12X.sub.13X.sub.14DYWGQGTTVTVSS wherein: wherein:
X.sub.1 is Q or V; X.sub.1 is A or P; X.sub.2 is T or F; X.sub.2 is
V or L; X.sub.3 iS S or T; X.sub.3 is A or T; X.sub.4 is P or S;
X.sub.4 is R or K; X.sub.5 is V or D; X.sub.5 is P or H; X.sub.6 is
Y or F; X.sub.6 is N or Q; X.sub.7-X.sub.11 is FTRYP or YNAYS; and
X.sub.7 is V or A; X.sub.12 is K or M. X.sub.8 is I or L; X.sub.9
is L or P; and X.sub.10-X.sub.14 is AYWLF, GGWTF, or GAWTM. SEQ ID
NO:196 SEQ ID NO:197 EVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSTLSASVGDRVTITCKASQDVG GYTFTEYTIHWVRQAPGKGLEWIGNI
TAVDWYQQKPGQAPKLLIYWASTRHTGV NPNNGGTTYNQKFEDRVTITVDKSTS
PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQ TAYMELSSLRSEDTAVYYCAAYWLF
QYNSYPLTFGQGTKVDIK DYWGQGTTVTVSS SEQ ID NO:198 SEQ ID NO:199
EVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSTLSASVGDRVTITCKASQDVG
GYTFTEYTIHWVRQAPGKGLEWIGNI TAVDWYQQKPGQAPKLLIYWASTRHTGV
NPNNGGTTYNQKFEDRVTITVDKSTS PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQ
TAYMELSSLRSEDTAVYYCAGGWTF QFTRYPLTFGQGTKVDIK DYWGQGTTVTVSS SEQ ID
NO:200 SEQ ID NO:201 EVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSTLSASVGDRVTITCKASQDVG GYTFTEYTIHWVRQAPGKGLEWIGNI
TAVDWYQQKPGQAPKLLIYWASTRHTGV NPNNGGTTYNQKFEDRVTITVDKSTS
PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQ TAYMELSSLRSEDTAVYYCAGAWTM
QYNAYSLTFGQGTKVDIK DYWGQGTTVTVSS SEQ ID NO:202
EVQLVQSGAEVKKPGASVKVSCKAS GYTFTEYTIHWVRQAPGKGLEWIGNI
NPNNGGTTYNQKFEDRVTITVDKSTS TAYMELSSPRSEDTAVYYCAAGWNF
DYWGQGTTVTVSS
[0233] In certain embodiments, a PSMA-CAR of the present disclosure
comprises a humanized PSMA-specific binding domain. In one
embodiment, the PSMA binding domain is a humanized J591("huJ591")
binding domain comprising the amino acid sequence set forth
below:
TABLE-US-00012 (SEQ ID NO:237)
EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIG
NINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAA
GWNFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIQMTQSPSTLSASVGD
RVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSG
SGTDFTLTISRLQPEDFAVYYCQQYNSYPLTFGQGTKVDIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00013 (SEQ ID NO:238)
Gaggtccagctggtgcagtctggagctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggatacacattcactgaatacac
catccactgggtgaggcaggcccctggaaagggccttgagtggattgga
aacattaatcctaacaatggtggtactacctacaaccagaagttcgagg
acagagtcacaatcactgtagacaagtccaccagcacagcctacatgga
gctcagcagcctgagatctgaggatactgcagtctattactgtgcagct
ggttggaactttgactactggggccaaggcaccacggtcaccgtctcct
caggaggcggaggatctggcggcggaggaagttctggcggaggcagcga
cattcagatgacccagtctcccagcaccctgtccgcatcagtaggagac
agggtcaccatcacttgcaaggccagtcaggatgtgggtactgctgtag
actggtatcaacagaaaccagggcaagctcctaaactactgatttactg
ggcatccacccggcacactggagtccctgatcgcttcagcggcagtgga
tctgggacagatttcactctcaccatcagcagactgcagcctgaagact
ttgcagtttattactgtcagcaatataacagctatcctctcacgttcgg
ccaggggaccaaggtggatatcaaa.
In certain embodiments, the PSMA binding domain is a huJ591 binding
domain comprising the amino acid sequence set forth below:
TABLE-US-00014 (SEQ ID NO:239)
DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIY
WASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTF
GQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVKKPGASVKVSCKAS
GYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDK
STSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00015 (SEQ ID NO: 240)
Gacattcagatgacccagtctcccagcaccctgtccgcatcagtaggag
acagggtcaccatcacttgcaaggccagtcaggatgtgggtactgctgt
agactggtatcaacagaaaccagggcaagctectaaactactgatttac
tgggcatccacccggcacactggagtccctgatcgcttcagcggcagtg
gatctgggacagatttcactctcaccatcagcagactgcagcctgaaga
ctttgcagtttattactgtcagcaatataacagctatcctctcacgttc
ggccaggggaccaaggtggatatcaaaggaggcggaggatctggcggcg
gaggaagttctggcggaggcagcgaggtccagctggtgcagtctggagc
tgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttct
ggatacacattcactgaatacaccatccactgggtgaggcaggcccctg
gaaagggccttgagtggattggaaacattaatcctaacaatggtggtac
tacctacaaccagaagttcgaggacagagtcacaatcactgtagacaag
tccaccagcacagcctacatggagctcagcagcctgagatctgaggata
ctgcagtctattactgtgcagctggttggaactttgactactggggcca
aggcaccacggtcaccgtctcctca.
In certain embodiments, the PSMA binding domain is a huJ591 binding
domain comprising the amino acid sequence set forth below:
TABLE-US-00016 (SEQ ID NO: 241)
DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAPKLLIY
WASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPLTF
GQGTKVDIKEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQA
PGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSE
DTAVYYCAAGWNFDYWGQGTTVTVSS.
[0234] Tolerable variations of the huJ591 PSMA binding domain will
be known to those of skill in the art, while maintaining binding to
human PSMA. For example, in some embodiments, the PSMA binding
domain is a huJ591 PSMA binding domain comprising an amino acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO: 237, SEQ ID NO: 239, or SEQ ID NO:
241. In one embodiment, the PSMA binding domain is a huJ591 PSMA
binding domain comprising the amino acid sequence set forth in SEQ
ID NO: 237, SEQ ID NO: 239, or SEQ ID NO: 241.
[0235] In some embodiments, the PSMA binding domain is a huJ591
PSMA binding domain encoded by a nucleic acid sequence that has at
least 60%/a, at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the nucleic acid sequence set forth
in SEQ ID NO: 238 or SEQ ID NO: 240. In one embodiment, the PSMA
binding domain is a huJ591 PSMA binding domain encoded by the
nucleic acid sequence set forth in SEQ ID NO: 238 or SEQ ID NO:
240.
[0236] In one embodiment, the huJ591 PSMA binding domain comprises
a heavy chain variable region comprising the amino acid sequence
set forth in SEQ ID NO: 191, which may be encoded by the nucleic
acid sequence set forth below:
TABLE-US-00017 (SEQ ID NO: 242)
GAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCT
CAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACAC
CATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGA
AACATTAATCCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGG
ACAGAGTCACAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGA
GCTCAGCAGCCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCT
GGTTGGAACTTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCT CA.
[0237] Tolerable variations of the heavy chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the huJ591 PSMA binding domain comprises a heavy chain
variable region comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 191. In one embodiment, the huJ591 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 191.
[0238] In some embodiments, the huJ591 PSMA binding domain
comprises a heavy chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO: 242. In one embodiment, the huJ591
PSMA binding domain comprises a heavy chain variable region encoded
by the nucleic acid sequence set forth in SEQ ID NO: 242.
[0239] The heavy chain variable region of the huJ591 PSMA binding
domain comprises three heavy chain complementarity-determining
regions (CDRs). Accordingly, a huJ591 PSMA binding domain may
comprise a heavy chain variable region that comprises a CDR1
represented by the amino acid sequence EYTIH (SEQ ID NO: 243); a
CDR2 represented by the amino acid sequence NINPNNGGTTYNQKFED (SEQ
ID NO: 24); and a CDR3 represented by the amino acid sequence
GWNFDY (SEQ ID NO: 25). Tolerable variations to the CDRs of the
heavy chain will be known to those of skill in the art, while
maintaining its contribution to the binding of PSMA. For example, a
huJ591 PSMA binding domain may comprise a heavy chain variable
region comprising a CDR1 that comprises an amino acid sequence that
has at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% sequence identity to the CDR1 amino acid sequence
set forth in SEQ ID NO: 243. For example, a huJ591 PSMA binding
domain may comprise a heavy chain variable region comprising a CDR2
that comprises an amino acid sequence that has at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the CDR2 amino acid sequence set forth in SEQ
ID NO: 24. For example, a huJ591 PSMA binding domain may comprise a
heavy chain variable region comprising a CDR3 that comprises an
amino acid sequence that has at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
CDR3 amino acid sequence set forth in SEQ ID NO: 25. In one
embodiment, the huJ591 PSMA binding domain comprises a heavy chain
variable region comprising the three aforementioned heavy chain
variable region CDRs.
[0240] In one embodiment, the huJ591 PSMA binding domain comprises
a light chain variable region comprising the amino acid sequence
set forth in SEQ ID NO: 192, which may be encoded by the nucleic
acid sequence set forth below:
TABLE-US-00018 (SEQ ID NO: 244)
GACATTCAGATGACCCAGTCTCCCAGCACCCTGTCCGCATCAGTAGGAG
ACAGGGTCACCATCACTTGCAAGGCCAGTCAGGATGTGGGTACTGCTGT
AGACTGGTATCAACAGAAACCAGGGCAAGCTCCTAAACTACTGATTTAC
TGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCAGCGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCAGACTGCAGCCTGAAGA
CTTTGCAGTTTATTACTGTCAGCAATATAACAGCTATCCTCTCACGTTC
GGCCAGGGGACCAAGGTGGATATCAAA.
[0241] Tolerable variations of the light chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the huJ591 PSMA binding domain comprises a light chain
variable region comprising an amino acid sequence that has at least
60%/a, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 192. In one embodiment, the huJ591 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 192.
[0242] In some embodiments, the huJ591 PSMA binding domain
comprises a light chain variable region encoded by a nucleic acid
sequence that has at least 60%/a, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO: 244. In one embodiment, the huJ591
PSMA binding domain comprises a light chain variable region encoded
by the nucleic acid sequence set forth in SEQ ID NO: 244.
[0243] The light chain variable region of the huJ591 PSMA binding
domain comprises three light chain complementarity-determining
regions (CDRs). Accordingly, a huJ591 PSMA binding domain may
comprise a light chain variable region that comprises a CDR1
represented by the amino acid sequence KASQDVGTAVD (SEQ ID NO: 18);
a CDR2 represented by the amino acid sequence WASTRHT (SEQ ID NO:
19); and a CDR3 represented by the amino acid sequence QQYNSYPLT
(SEQ ID NO: 20). Tolerable variations to the CDRs of the light
chain will be known to those of skill in the art, while maintaining
its contribution to the binding of PSMA. For example, a huJ591 PSMA
binding domain may comprise a light chain variable region
comprising a CDR1 that comprises an amino acid sequence that has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the CDR1 amino acid sequence set
forth in SEQ ID NO: 18. For example, a huJ591 PSMA binding domain
may comprise a light chain variable region comprising a CDR2 that
comprises an amino acid sequence that has at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the CDR2 amino acid sequence set forth in SEQ ID NO:
19. For example, a huJ591 PSMA binding domain may comprise a light
chain variable region comprising a CDR3 that comprises an amino
acid sequence that has at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% sequence identity to the CDR3 amino
acid sequence set forth in SEQ ID NO: 20. In one embodiment, the
huJ591 PSMA binding domain comprises a light chain variable region
comprising the three aforementioned light chain variable region
CDRs.
[0244] (c) Human PSMA Binding Domains
[0245] In certain embodiments, a PSMA-CAR of the present invention
comprises a PSMA binding domain of a human PSMA antibody, or a
variant thereof. In one embodiment, the PSMA binding domain is a
human 1C3 PSMA binding domain comprising the amino acid sequence
set forth below:
TABLE-US-00019 (SEQ ID NO: 26)
MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFT
FSSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKN
TLYLQMNSLRAEDTAVYYCARAVPWGSRYYYYGMDVWGQGTTVTVSSGG
GGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWY
QQKSGKAPKLLIFDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT
YYCQQFNSYPLTFGGGTKVEIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00020 (SEQ ID NO: 27)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGT
CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACC
TTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGC
TGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGT
ATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGG
TATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGC
GGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGT
TGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTAT
CAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCA
GTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGAC
AGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACT
TATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGA
CCAAGGTGGAGATCAAA.
[0246] Tolerable variations of the human 1C3 PSMA binding domain
will be known to those of skill in the art, while maintaining
binding to human PSMA. For example, in some embodiments, the PSMA
binding domain is a human 1C3 PSMA binding domain comprising an
amino acid sequence that has at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
amino acid sequence set forth in SEQ ID NO:26. In one embodiment,
the PSMA binding domain is a human 1C3 PSMA binding domain
comprising the amino acid sequence set forth in SEQ ID NO:26.
[0247] In some embodiments, the PSMA binding domain is a human 1C3
PSMA binding domain encoded by a nucleic acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO:27. In one embodiment, the PSMA binding domain is a human 1C3
PSMA binding domain encoded by the nucleic acid sequence set forth
in SEQ ID NO:27.
[0248] In one embodiment, the human 1C3 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00021 (SEQ ID NO: 28)
PQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWV
AVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA
RAVPWGSRYYYYGMDVWGQGTTVTVSS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00022 (SEQ ID NO: 29)
CCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTA
TGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTG
GCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGA
AGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCT
GCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCG
AGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCT
GGGGCCAAGGGACCACGGTCACCGTCTCCTCA.
[0249] Tolerable variations of the heavy chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the human 1C3 PSMA binding domain comprises a heavy
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:28. In one embodiment, the human 1C3 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:28.
[0250] In some embodiments, the human 1C3 PSMA binding domain
comprises a heavy chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:29. In one embodiment, the human
1C3 PSMA binding domain comprises a heavy chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:29.
[0251] The heavy chain variable region of the human 1C3 PSMA
binding domain comprises three heavy chain
complementarity-determining regions (CDRs). Accordingly, a human
1C3 PSMA binding domain may comprise a heavy chain variable region
that comprises a CDR1 represented by the amino acid sequence SYAMH
(SEQ ID NO:30); a CDR2 represented by the amino acid sequence
VISYDGNNKYYADSVKG (SEQ ID NO:31); and a CDR3 represented by the
amino acid sequence AVPWGSRYYYYGMDV (SEQ ID NO:32). Tolerable
variations to the CDRs of the heavy chain will be known to those of
skill in the art, while maintaining its contribution to the binding
of PSMA. For example, a human 1C3 PSMA binding domain may comprise
a heavy chain variable region comprising a CDR1 that comprises an
amino acid sequence that has at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
CDR1 amino acid sequence set forth in SEQ ID NO:30. For example, a
human 1C3 PSMA binding domain may comprise a heavy chain variable
region comprising a CDR2 that comprises an amino acid sequence that
has at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% sequence identity to the CDR2 amino acid sequence
set forth in SEQ ID NO:31. For example, a human 1C3 PSMA binding
domain may comprise a heavy chain variable region comprising a CDR3
that comprises an amino acid sequence that has at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the CDR3 amino acid sequence set forth in SEQ
ID NO:32. In one embodiment, the human 1C3 PSMA binding domain
comprises a heavy chain variable region comprising the three
aforementioned heavy chain variable region CDRs.
[0252] In one embodiment, the human 1C3 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00023 (SEQ ID NO: 33)
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKSGKAPKLLIF
DASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTF GGGTKVEIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00024 (SEQ ID NO: 34)
GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTT
AGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTT
GATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGA
TTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA.
[0253] Tolerable variations of the light chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the human 1C3 PSMA binding domain comprises a light
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:33. In one embodiment, the human 1C3 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:33.
[0254] In some embodiments, the human 1C3 PSMA binding domain
comprises a light chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:34. In one embodiment, the human
1C3 PSMA binding domain comprises a light chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:34.
[0255] The light chain variable region of the human 1C3 PSMA
binding domain comprises three light chain
complementarity-determining regions (CDRs). Accordingly, a human
1C3 PSMA binding domain may comprise a light chain variable region
that comprises a CDR1 represented by the amino acid sequence
RASQGISSALA (SEQ ID NO:35); a CDR2 represented by the amino acid
sequence DASSLES (SEQ ID NO:36); and a CDR3 represented by the
amino acid sequence QQFNSYPLT (SEQ ID NO:37). Tolerable variations
to the CDRs of the light chain will be known to those of skill in
the art, while maintaining its contribution to the binding of PSMA.
For example, a human 1C3 PSMA binding domain may comprise a light
chain variable region comprising a CDR1 that comprises an amino
acid sequence that has at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% sequence identity to the CDR1 amino
acid sequence set forth in SEQ ID NO:35. For example, a human 1C3
PSMA binding domain may comprise a light chain variable region
comprising a CDR2 that comprises an amino acid sequence that has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the CDR2 amino acid sequence set
forth in SEQ ID NO:36. For example, a human 1C3 PSMA binding domain
may comprise a light chain variable region comprising a CDR3 that
comprises an amino acid sequence that has at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the CDR3 amino acid sequence set forth in SEQ ID NO:37.
In one embodiment, the human 1C3 PSMA binding domain comprises a
light chain variable region comprising the three aforementioned
light chain variable region CDRs.
[0256] In one embodiment, the PSMA binding domain is a human 2A10
PSMA binding domain comprising the amino acid sequence set forth
below:
TABLE-US-00025 (SEQ ID NO: 38)
MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYS
FTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS
TAYLQWSSLKASDTAMYYCARQTGFLWSSDLWGRGTLVTVSSGGGGSGG
GGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPG
KAPKLLIYDASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQQ
FNSYPLTFGGGTKVEIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00026 (SEQ ID NO: 39)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGC
TTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGG
CCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGT
GGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC
AAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGG
AAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGG
TCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCAC
CATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAG
TTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA AA.
[0257] Tolerable variations of the human 2A10 PSMA binding domain
will be known to those of skill in the art, while maintaining
binding to human PSMA. For example, in some embodiments, the PSMA
binding domain is a human 2A10 PSMA binding domain comprising an
amino acid sequence that has at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
amino acid sequence set forth in SEQ ID NO:38. In one embodiment,
the PSMA binding domain is a human 2A10 PSMA binding domain
comprising the amino acid sequence set forth in SEQ ID NO:38.
[0258] In some embodiments, the PSMA binding domain is a human 2A10
PSMA binding domain encoded by a nucleic acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO:39. In one embodiment, the PSMA binding domain is a human
2A10 PSMA binding domain encoded by the nucleic acid sequence set
forth in SEQ ID NO:39.
[0259] In one embodiment, the human 2A10 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00027 (SEQ ID NO: 40)
PEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWM
GITYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCA
RQTGFLWSSDLWGRGTLVTVSS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00028 (SEQ ID NO: 41)
CCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGG
AGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAA
CTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATG
GGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCC
AAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCT
GCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCG
AGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCC
TGGTCACTGTCTCCTCA.
[0260] Tolerable variations of the heavy chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the human 2A10 PSMA binding domain comprises a heavy
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:40. In one embodiment, the human 2A10 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:40.
[0261] In some embodiments, the human 2A10 PSMA binding domain
comprises a heavy chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:41. In one embodiment, the human
2A10 PSMA binding domain comprises a heavy chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:41.
[0262] The heavy chain variable region of the human 2A10 PSMA
binding domain comprises three heavy chain
complementarity-determining regions (CDRs). Accordingly, a human
2A10 PSMA binding domain may comprise a heavy chain variable region
that comprises a CDR1 represented by the amino acid sequence SNWIG
(SEQ ID NO:42); a CDR2 represented by the amino acid sequence
IIYPGDSDTRYSPSFQG (SEQ ID NO:43); and a CDR3 represented by the
amino acid sequence QTGFLWSSDL (SEQ ID NO:44). Tolerable variations
to the CDRs of the heavy chain will be known to those of skill in
the art, while maintaining its contribution to the binding of human
PSMA. For example, a human 2A10 PSMA binding domain may comprise a
heavy chain variable region comprising a CDR1 that comprises an
amino acid sequence that has at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
CDR1 amino acid sequence set forth in SEQ ID NO:42. For example, a
human 2A10 PSMA binding domain may comprise a heavy chain variable
region comprising a CDR2 that comprises an amino acid sequence that
has at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%/a, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the CDR2 amino acid
sequence set forth in SEQ ID NO:43. For example, a human 2A10 PSMA
binding domain may comprise a heavy chain variable region
comprising a CDR3 that comprises an amino acid sequence that has at
least 85%, at least 86%, at least 87%, at least 88%, at least
89%/a, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the CDR3 amino acid sequence set
forth in SEQ ID NO:44. In one embodiment, the human 2A10 PSMA
binding domain comprises a heavy chain variable region comprising
the three aforementioned heavy chain variable region CDRs.
[0263] In one embodiment, the human 2A10 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00029 (SEQ ID NO: 45)
AIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIY
DASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQQFNSYPLTF GGGTKVEIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00030 (SEQ ID NO:46)
GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTT
AGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTAT
GATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATG
GATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGA
TTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA.
[0264] Tolerable variations of the light chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the human 2A10 PSMA binding domain comprises a light
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:45. In one embodiment, the human 2A10 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:45.
[0265] In some embodiments, the human 2A10 PSMA binding domain
comprises a light chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:46. In one embodiment, the human
2A10 PSMA binding domain comprises a light chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:46.
[0266] The light chain variable region of the human 2A10 PSMA
binding domain comprises three light chain
complementarity-determining regions (CDRs). Accordingly, a human
2A10 PSMA binding domain may comprise a light chain variable region
that comprises a CDR1 represented by the amino acid sequence
CRASQDISSAL (SEQ ID NO:47); a CDR2 represented by the amino acid
sequence YDASSLES (SEQ ID NO:48); and a CDR3 represented by the
amino acid sequence CQQFNSYPLT (SEQ ID NO:49). Tolerable variations
to the CDRs of the light chain will be known to those of skill in
the art, while maintaining its contribution to the binding of PSMA.
For example, a human 2A10 PSMA binding domain may comprise a light
chain variable region comprising a CDR1 that comprises an amino
acid sequence that has at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% sequence identity to the CDR1 amino
acid sequence set forth in SEQ ID NO:47. For example, a human 2A10
PSMA binding domain may comprise a light chain variable region
comprising a CDR2 that comprises an amino acid sequence that has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the CDR2 amino acid sequence set
forth in SEQ ID NO:48. For example, a human 2A10 PSMA binding
domain may comprise a light chain variable region comprising a CDR3
that comprises an amino acid sequence that has at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the CDR3 amino acid sequence set forth in SEQ
ID NO:49. In one embodiment, the human 2A10 PSMA binding domain
comprises a light chain variable region comprising the three
aforementioned light chain variable region CDRs.
[0267] In one embodiment, the PSMA binding domain is a human 2F5
PSMA binding domain comprising the amino acid sequence set forth
below:
TABLE-US-00031 (SEQ ID NO:50)
MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYS
FTSNWIGWVRQMPGKGLEWMGITYPGDSDTRYSPSFQGQVTISADKSIS
TAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGG
GGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPG
KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
FNSYPLTFGGGTKVEIKIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00032 (SEQ ID NO:51)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGT
TTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGG
CCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGT
GGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC
AAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGG
AAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGG
TCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCAC
CATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAG
TTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA AAATCAAA.
[0268] Tolerable variations of the human 2F5 PSMA binding domain
will be known to those of skill in the art, while maintaining
binding to human PSMA. For example, in some embodiments, the PSMA
binding domain is a human 2F5 PSMA binding domain comprising an
amino acid sequence that has at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
amino acid sequence set forth in SEQ ID NO:50. In one embodiment,
the PSMA binding domain is a human 2F5 PSMA binding domain
comprising the amino acid sequence set forth in SEQ ID NO:50.
[0269] In some embodiments, the PSMA binding domain is a human 2F5
PSMA binding domain encoded by a nucleic acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO:51. In one embodiment, the PSMA binding domain is a human 2F5
PSMA binding domain encoded by the nucleic acid sequence set forth
in SEQ ID NO:51.
[0270] In one embodiment, the human 2F5 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00033 (SEQ ID NO:52)
PEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWM
GITYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCA
RQTGFLWSFDLWGRGTLVTVSS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00034 (SEQ ID NO:53)
CCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGG
AGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAA
CTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATG
GGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCC
AAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCT
GCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCG
AGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCC
TGGTCACTGTCTCCTCA.
[0271] Tolerable variations of the heavy chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the human 2F5 PSMA binding domain comprises a heavy
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:52. In one embodiment, the human 2F5 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:52.
[0272] In some embodiments, the human 2F5 PSMA binding domain
comprises a heavy chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:53. In one embodiment, the human
2F5 PSMA binding domain comprises a heavy chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:53.
[0273] The heavy chain variable region of the human 2F5 PSMA
binding domain comprises three heavy chain
complementarity-determining regions (CDRs). Accordingly, a human
2F5 PSMA binding domain may comprise a heavy chain variable region
that comprises a CDR1 represented by the amino acid sequence SNWIG
(SEQ ID NO:54); a CDR2 represented by the amino acid sequence
IIYPGDSDTRYSPSFQG (SEQ ID NO:55); and a CDR3 represented by the
amino acid sequence QTGFLWSFDL (SEQ ID NO:56). Tolerable variations
to the CDRs of the heavy chain will be known to those of skill in
the art, while maintaining its contribution to the binding of PSMA.
For example, a human 2F5 PSMA binding domain may comprise a heavy
chain variable region comprising a CDR1 that comprises an amino
acid sequence that has at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% sequence identity to the CDR1 amino
acid sequence set forth in SEQ ID NO:54. For example, a human 2F5
PSMA binding domain may comprise a heavy chain variable region
comprising a CDR2 that comprises an amino acid sequence that has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the CDR2 amino acid sequence set
forth in SEQ ID NO:55. For example, a human 2F5 PSMA binding domain
may comprise a heavy chain variable region comprising a CDR3 that
comprises an amino acid sequence that has at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the CDR3 amino acid sequence set forth in SEQ ID NO:56.
In one embodiment, the human 2F5 PSMA binding domain comprises a
heavy chain variable region comprising the three aforementioned
heavy chain variable region CDRs.
[0274] In one embodiment, the human 2F5 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00035 (SEQ ID NO:57)
AIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIY
DASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTF GGGTKVEIKIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00036 (SEQ ID NO:58)
GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTT
AGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTAT
GATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGA
TTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAAATCAAA.
[0275] Tolerable variations of the light chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the human 2F5 PSMA binding domain comprises a light
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%/a, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the amino acid sequence set forth in
SEQ ID NO:57. In one embodiment, the human 2F5 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:57.
[0276] In some embodiments, the human 2F5 PSMA binding domain
comprises a light chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:58. In one embodiment, the human
2F5 PSMA binding domain comprises a light chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:58.
[0277] The light chain variable region of the human 2F5 PSMA
binding domain comprises three light chain
complementarity-determining regions (CDRs). Accordingly, a human
2F5 PSMA binding domain may comprise a light chain variable region
that comprises a CDR1 represented by the amino acid sequence
RASQDISSALA (SEQ ID NO:59); a CDR2 represented by the amino acid
sequence DASSLES (SEQ ID NO:60); and a CDR3 represented by the
amino acid sequence QQFNSYPLT (SEQ ID NO:61). Tolerable variations
to the CDRs of the light chain will be known to those of skill in
the art, while maintaining its contribution to the binding of PSMA.
For example, a human 2F5 PSMA binding domain may comprise a light
chain variable region comprising a CDR1 that comprises an amino
acid sequence that has at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% sequence identity to the CDR1 amino
acid sequence set forth in SEQ ID NO:59. For example, a human 2F5
PSMA binding domain may comprise a light chain variable region
comprising a CDR2 that comprises an amino acid sequence that has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the CDR2 amino acid sequence set
forth in SEQ ID NO:60. For example, a human 2F5 PSMA binding domain
may comprise a light chain variable region comprising a CDR3 that
comprises an amino acid sequence that has at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the CDR3 amino acid sequence set forth in SEQ ID NO:61.
In one embodiment, the human 2F5 PSMA binding domain comprises a
light chain variable region comprising the three aforementioned
light chain variable region CDRs.
[0278] In one embodiment, the PSMA binding domain is a human 2C6
PSMA binding domain comprising the amino acid sequence set forth
below:
TABLE-US-00037 (SEQ ID NO:62)
MALPVTALLLPLALLLHAARPEVQLVQSGSEVKKPGESLKISCKGSGYS
FTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS
TAYLQWSSLKASDTAMYYCASPGYTSSWTSFDYWGQGTLVTVSSGGGGS
GGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK
PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC
QQRSNWPLFTFGPGTKVDIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00038 (SEQ ID NO:63)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGC
TTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCG
GGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGT
CTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG
CAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAA
CCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCA
CTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCAC
TCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT
CAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAG TGGATATCAAA.
[0279] Tolerable variations of the human 2C6 PSMA binding domain
will be known to those of skill in the art, while maintaining
binding to human PSMA. For example, in some embodiments, the PSMA
binding domain is a human 2C6 PSMA binding domain comprising an
amino acid sequence that has at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
amino acid sequence set forth in SEQ ID NO:62. In one embodiment,
the PSMA binding domain is a human 2C6 PSMA binding domain
comprising the amino acid sequence set forth in SEQ ID NO:62.
[0280] In some embodiments, the PSMA binding domain is a human 2C6
PSMA binding domain encoded by a nucleic acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO:63. In one embodiment, the PSMA binding domain is a human 2C6
PSMA binding domain encoded by the nucleic acid sequence set forth
in SEQ ID NO:63.
[0281] In one embodiment, the human 2C6 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00039 (SEQ ID NO:64)
PEVQLVQSGSEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWM
GITYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCA
SPGYTSSWTSFDYWGQGTLVTVSS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00040 (SEQ ID NO:65)
CCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGG
AGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTA
CTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATG
GGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCC
AAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCT
GCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCG
AGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCA.
[0282] Tolerable variations of the heavy chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the human 2C6 PSMA binding domain comprises a heavy
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:64. In one embodiment, the human 2C6 PSMA binding domain
comprises a heavy chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:64.
[0283] In some embodiments, the human 2C6 PSMA binding domain
comprises a heavy chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:65. In one embodiment, the human
2C6 PSMA binding domain comprises a heavy chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:65.
[0284] The heavy chain variable region of the human 2C6 PSMA
binding domain comprises three heavy chain
complementarity-determining regions (CDRs). Accordingly, a human
2C6 PSMA binding domain may comprise a heavy chain variable region
that comprises a CDR1 represented by the amino acid sequence TNYWI
(SEQ ID NO:66); a CDR2 represented by the amino acid sequence
GIIYPGDSDTRYSPSFQG (SEQ ID NO:67); and a CDR3 represented by the
amino acid sequence SPGYTSSWTS (SEQ ID NO:68). Tolerable variations
to the CDRs of the heavy chain will be known to those of skill in
the art, while maintaining its contribution to the binding of PSMA.
For example, a human 2C6 PSMA binding domain may comprise a heavy
chain variable region comprising a CDR1 that comprises an amino
acid sequence that has at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% sequence identity to the CDR1 amino
acid sequence set forth in SEQ ID NO:66. For example, a human 2C6
PSMA binding domain may comprise a heavy chain variable region
comprising a CDR2 that comprises an amino acid sequence that has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the CDR2 amino acid sequence set
forth in SEQ ID NO:67. For example, a human 2C6 PSMA binding domain
may comprise a heavy chain variable region comprising a CDR3 that
comprises an amino acid sequence that has at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the CDR3 amino acid sequence set forth in SEQ ID NO:68.
In one embodiment, the human 2C6 PSMA binding domain comprises a
heavy chain variable region comprising the three aforementioned
heavy chain variable region CDRs.
[0285] In one embodiment, the human 2C6 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth below:
TABLE-US-00041 (SEQ ID NO:69)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLFT FGPGTKVDIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00042 (SEQ ID NO:70)
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT
AGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACT
TTCGGCCCTGGGACCAAAGTGGATATCAAA.
[0286] Tolerable variations of the light chain variable region will
be known to those of skill in the art, while maintaining its
contribution to the binding of human PSMA. For example, in some
embodiments, the human 2C6 PSMA binding domain comprises a light
chain variable region comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:69. In one embodiment, the human 2C6 PSMA binding domain
comprises a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO:69.
[0287] In some embodiments, the human 2C6 PSMA binding domain
comprises a light chain variable region encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:70. In one embodiment, the human
2C6 PSMA binding domain comprises a light chain variable region
encoded by the nucleic acid sequence set forth in SEQ ID NO:70.
[0288] The light chain variable region of the human 2C6 PSMA
binding domain comprises three light chain
complementarity-determining regions (CDRs). Accordingly, a human
2C6 PSMA binding domain may comprise a light chain variable region
that comprises a CDR1 represented by the amino acid sequence
CRASQSVSSYL (SEQ ID NO:71); a CDR2 represented by the amino acid
sequence YDASNRAT (SEQ ID NO:72); and a CDR3 represented by the
amino acid sequence CQQRSNWPLFT (SEQ ID NO:73). Tolerable
variations to the CDRs of the light chain will be known to those of
skill in the art, while maintaining its contribution to the binding
of PSMA. For example, a human 2C6 PSMA binding domain may comprise
a light chain variable region comprising a CDR1 that comprises an
amino acid sequence that has at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
CDR1 amino acid sequence set forth in SEQ ID NO:71. For example, a
human 2C6 PSMA binding domain may comprise a light chain variable
region comprising a CDR2 that comprises an amino acid sequence that
has at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% sequence identity to the CDR2 amino acid sequence
set forth in SEQ ID NO:72. For example, a human 2C6 PSMA binding
domain may comprise a light chain variable region comprising a CDR3
that comprises an amino acid sequence that has at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the CDR3 amino acid sequence set forth in SEQ
ID NO:73. In one embodiment, the human 2C6 PSMA binding domain
comprises a light chain variable region comprising the three
aforementioned light chain variable region CDRs.
Transmembrane Domain
[0289] CARs (e.g., PSMA-CARs) of the present invention comprise may
comprise a transmembrane domain that connects the antigen binding
domain of the CAR to the intracellular domain of the CAR. The
transmembrane domain of a subject CAR is a region that is capable
of spanning the plasma membrane of a cell (e.g., an immune cell or
precursor thereof). The transmembrane domain is for insertion into
a cell membrane, e.g., a eukaryotic cell membrane. In some
embodiments, the transmembrane domain is interposed between the
antigen binding domain and the intracellular domain of a CAR.
[0290] In some embodiments, the transmembrane domain is naturally
associated with one or more of the domains in the CAR. In some
embodiments, the transmembrane domain can be selected or modified
by one or more amino acid substitutions to avoid binding of such
domains to the transmembrane domains of the same or different
surface membrane proteins, to minimize interactions with other
members of the receptor complex.
[0291] The transmembrane domain may be derived either from a
natural or a synthetic source. Where the source is natural, the
domain may be derived from any membrane-bound or transmembrane
protein, e.g., a Type I transmembrane protein. Where the source is
synthetic, the transmembrane domain may be any artificial sequence
that facilitates insertion of the CAR into a cell membrane, e.g.,
an artificial hydrophobic sequence. Examples of the transmembrane
domain of particular use in this invention include, without
limitation, transmembrane domains derived from (i.e. comprise at
least the transmembrane region(s) of) the alpha, beta or zeta chain
of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7,
CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40),
CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR), TLR2,
TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments,
the transmembrane domain may be synthetic, in which case it will
comprise predominantly hydrophobic residues such as leucine and
valine. Preferably a triplet of phenylalanine, tryptophan and
valine will be found at each end of a synthetic transmembrane
domain.
[0292] The transmembrane domains described herein can be combined
with any of the antigen binding domains described herein, any of
the intracellular domains described herein, or any of the other
domains described herein that may be included in a subject CAR.
[0293] In some embodiments, the transmembrane domain further
comprises a hinge region. A subject CAR of the present invention
may also include an hinge region. The hinge region of the CAR is a
hydrophilic region which is located between the antigen binding
domain and the transmembrane domain. In some embodiments, this
domain facilitates proper protein folding for the CAR. The hinge
region is an optional component for the CAR. The hinge region may
include a domain selected from Fc fragments of antibodies, hinge
regions of antibodies, CH2 regions of antibodies, CH3 regions of
antibodies, artificial hinge sequences or combinations thereof.
Examples of hinge regions include, without limitation, a CD8a
hinge, artificial hinges made of polypeptides which may be as small
as, three glycines (Gly), as well as CH1 and CH3 domains of IgGs
(such as human IgG4).
[0294] In some embodiments, a subject CAR of the present disclosure
includes a hinge region that connects the antigen binding domain
with the transmembrane domain, which, in turn, connects to the
intracellular domain. The hinge region is preferably capable of
supporting the antigen binding domain to recognize and bind to the
target antigen on the target cells (see, e.g., Hudecek et al.,
Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments,
the hinge region is a flexible domain, thus allowing the antigen
binding domain to have a structure to optimally recognize the
specific structure and density of the target antigens on a cell
such as tumor cell (Hudecek et al., supra). The flexibility of the
hinge region permits the hinge region to adopt many different
conformations.
[0295] In some embodiments, the hinge region is an immunoglobulin
heavy chain hinge region. In some embodiments, the hinge region is
a hinge region polypeptide derived from a receptor (e.g., a
CD8-derived hinge region).
[0296] The hinge region can have a length of from about 4 amino
acids to about 50 amino acids, e.g., from about 4 aa to about 10
aa, from about 10 aa to about 15 aa, from about 15 aa to about 20
aa, from about 20 aa to about 25 aa, from about 25 aa to about 30
aa, from about 30 aa to about 40 aa, or from about 40 aa to about
50 aa.
[0297] Suitable hinge regions can be readily selected and can be of
any of a number of suitable lengths, such as from 1 amino acid
(e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino
acids, from 3 amino acids to 12 amino acids, including 4 amino
acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino
acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can
be 1, 2, 3, 4, 5, 6, or 7 amino acids.
[0298] For example, hinge regions include glycine polymers
(G).sub.n, glycine-serine polymers (including, for example,
(GS).sub.n, (GSGGS).sub.n (SEQ ID NO: 1) and (GGGS).sub.n (SEQ ID
NO:2), where n is an integer of at least one), glycine-alanine
polymers, alanine-serine polymers, and other flexible linkers known
in the art. Glycine and glycine-serine polymers can be used; both
Gly and Ser are relatively unstructured, and therefore can serve as
a neutral tether between components. Glycine polymers can be used;
glycine accesses significantly more phi-psi space than even
alanine, and is much less restricted than residues with longer side
chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2:
73-142). Exemplary hinge regions can comprise amino acid sequences
including, but not limited to, GGSG (SEQ ID NO:4), GGSGG (SEQ ID
NO:5), GSGSG (SEQ ID NO:6), GSGGG (SEQ ID NO:7), GGGSG (SEQ ID
NO:8), GSSSG (SEQ ID NO:9), and the like.
[0299] In some embodiments, the hinge region is an immunoglobulin
heavy chain hinge region. Immunoglobulin hinge region amino acid
sequences are known in the art; see, e.g., Tan et al., Proc. Natl.
Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids
Res. (1986) 14(4): 1779-1789. As non-limiting examples, an
immunoglobulin hinge region can include one of the following amino
acid sequences: DKTHT (SEQ ID NO:74); CPPC (SEQ ID NO:75);
CPEPKSCDTPPPCPR (SEQ ID NO:76) (see, e.g., Glaser et al., J. Biol.
Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO:77);
KSCDKTHTCP (SEQ ID NO:78); KCCVDCP (SEQ ID NO:79); KYGPPCP (SEQ ID
NO:80); EPKSCDKTHTCPPCP (SEQ ID NO:81) (human IgG1 hinge);
ERKCCVECPPCP (SEQ ID NO:82) (human IgG2 hinge); ELKTPLGDTTHTCPRCP
(SEQ ID NO:83) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:84)
(human IgG4 hinge); and the like.
[0300] The hinge region can comprise an amino acid sequence of a
human IgG1, IgG2, IgG3, or IgG4, hinge region. In one embodiment,
the hinge region can include one or more amino acid substitutions
and/or insertions and/or deletions compared to a wild-type
(naturally-occurring) hinge region. For example, His229 of human
IgG1 hinge can be substituted with Tyr, so that the hinge region
comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO:85); see, e.g.,
Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one
embodiment, the hinge region can comprise an amino acid sequence
derived from human CD8, or a variant thereof.
[0301] The transmembrane domain may be combined with any hinge
region and/or may comprise one or more transmembrane domains
described herein. In one embodiment, the transmembrane domain
comprises a CD8 transmembrane domain. In one embodiment, the
transmembrane domain comprises a CD8 hinge region and a CD8
transmembrane domain. In some embodiments, a subject CAR comprises
a CD8 hinge region having the amino acid sequence set forth
below:
TABLE-US-00043 (SEQ ID NO:86)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00044 (SEQ ID NO:87)
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGT
CGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGG
CGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT.
[0302] Tolerable variations of the transmembrane domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises a transmembrane domain comprising a CD8
hinge region comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:86. In one embodiment, the CAR comprises a transmembrane domain
comprising a CD8 hinge region comprising the amino acid sequence
set forth in SEQ ID NO:86.
[0303] In some embodiments, a subject CAR of the present invention
comprises a transmembrane domain comprising a CD8 hinge region
encoded by a nucleic acid sequence that has at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the nucleic acid sequence set forth in SEQ ID NO:87. In
one embodiment, the CAR comprises a transmembrane domain comprising
a CD8 hinge region encoded by the nucleic acid sequence set forth
in SEQ ID NO:87.
[0304] In some embodiments, a subject CAR comprises a CD8
transmembrane domain having the amino acid sequence set forth
below:
TABLE-US-00045 (SEQ ID NO:88) IYIWAPLAGTCGVLLLSLVITLYC.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00046 (SEQ ID NO:89)
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGT
CACTGGTTATCACCCTTTACTGC.
[0305] Tolerable variations of the transmembrane domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises a transmembrane domain comprising a CD8
transmembrane domain comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:88. In one embodiment, the CAR comprises a transmembrane
domain comprising a CD8 transmembrane domain comprising the amino
acid sequence set forth in SEQ ID NO:88.
[0306] In some embodiments, a subject CAR of the present invention
comprises a transmembrane domain comprising a CD8 transmembrane
domain encoded by a nucleic acid sequence that has at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the nucleic acid sequence set forth in SEQ ID NO:89. In
one embodiment, the CAR comprises a transmembrane domain comprising
a CD8 transmembrane domain encoded by the acid sequence set forth
in SEQ ID NO:89.
[0307] In some embodiments, the transmembrane domain comprises a
CD8 hinge region and a CD8 transmembrane domain, having the amino
acid sequence set forth below:
TABLE-US-00047 (SEQ ID NO:90)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW
APLAGTCGVLLLSLVITLYC.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00048 (SEQ ID NO:91)
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGT
CGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGG
CGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGG
GCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCA CCCTTTACTGC.
[0308] Tolerable variations of the transmembrane domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises a transmembrane domain comprising a CD8
hinge region and a CD8 transmembrane domain, comprising an amino
acid sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%/a, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO:90. In one embodiment, the CAR
comprises a transmembrane domain comprising a CD8 hinge region and
a CD8 transmembrane domain, comprising the amino acid sequence set
forth in SEQ ID NO:90.
[0309] In some embodiments, a subject CAR of the present invention
comprises a transmembrane domain comprising a CD8 hinge region and
a CD8 transmembrane domain, encoded by a nucleic acid sequence that
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the nucleic acid sequence set
forth in SEQ ID NO:91. In one embodiment, the CAR comprises a
transmembrane domain comprising a CD8 hinge region and a CD8
transmembrane domain, encoded by the nucleic acid sequence set
forth in SEQ ID NO:91.
[0310] Between the extracellular domain and the transmembrane
domain of the CAR, or between the intracellular domain and the
transmembrane domain of the CAR, there may be incorporated a spacer
domain. As used herein, the term "spacer domain" generally means
any oligo- or polypeptide that functions to link the transmembrane
domain to, either the extracellular domain or, the intracellular
domain in the polypeptide chain. A spacer domain may comprise up to
300 amino acids, e.g., 10 to 100 amino acids, or 25 to 50 amino
acids. In some embodiments, the spacer domain may be a short oligo-
or polypeptide linker, e.g., between 2 and 10 amino acids in
length. For example, glycine-serine doublet provides a particularly
suitable linker between the transmembrane domain and the
intracellular signaling domain of the subject CAR.
Intracellular Signaling Domain
[0311] A subject CAR of the present invention also includes an
intracellular signaling domain. The terms "intracellular signaling
domain" and "intracellular domain" are used interchangeably herein.
The intracellular signaling domain of the CAR is responsible for
activation of at least one of the effector functions of the cell in
which the CAR is expressed (e.g., immune cell). The intracellular
signaling domain transduces the effector function signal and
directs the cell (e.g., immune cell) to perform its specialized
function, e.g., harming and/or destroying a target cell.
[0312] Examples of an intracellular domain for use in the invention
include, but are not limited to, the cytoplasmic portion of a
surface receptor, co-stimulatory molecule, and any molecule that
acts in concert to initiate signal transduction in the T cell, as
well as any derivative or variant of these elements and any
synthetic sequence that has the same functional capability.
[0313] Examples of the intracellular signaling domain include,
without limitation, the .zeta. chain of the T cell receptor complex
or any of its homologs, e.g., .eta. chain, FcsRI.gamma. and .beta.
chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta
chain, CD3 polypeptides (.DELTA., .delta. and .epsilon.), syk
family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine
kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T
cell transduction, such as CD2, CD5 and CD28. In one embodiment,
the intracellular signaling domain may be human CD3 zeta chain,
FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an
immunoreceptor tyrosine-based activation motif (ITAM) bearing
cytoplasmic receptors, and combinations thereof.
[0314] In one embodiment, the intracellular signaling domain of the
CAR includes any portion of one or more co-stimulatory molecules,
such as at least one signaling domain from CD3, CD8, CD27, CD28,
ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic
sequence thereof that has the same functional capability, and any
combination thereof.
[0315] Other examples of the intracellular domain include a
fragment or domain from one or more molecules or receptors
including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta,
CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib),
CD79a, CD79b, Fcgamma R11a, DAPl0, DAP 12, T cell receptor (TCR),
CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS,
a KIR family protein, lymphocyte function-associated antigen-1
(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically
binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7,
NKp80 (KLRF), CD127, CD 160, CD19, CD4, CD8alpha, CD8beta, IL2R
beta, IL2R gamma, IL7R alpha, ITGA4, VLA, CD49a, ITGA4, IA4, CD49D,
ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD 11 a,
LFA-1, ITGAM, CD lib, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18,
LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244,
2B4), CD84, CD 96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160
(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM
(SLAMF, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT,
GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like
receptor 1 (TLR), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,
other co-stimulatory molecules described herein, any derivative,
variant, or fragment thereof, any synthetic sequence of a
co-stimulatory molecule that has the same functional capability,
and any combination thereof.
[0316] Additional examples of intracellular domains include,
without limitation, intracellular signaling domains of several
types of various other immune signaling receptors, including, but
not limited to, first, second, and third generation T cell
signaling proteins including CD3, B7 family costimulatory, and
Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see,
e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653).
Additionally, intracellular signaling domains may include signaling
domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman,
Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30
(B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5):
2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol.
(2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAPl0, and
CD3z.
[0317] Intracellular signaling domains suitable for use in a
subject CAR of the present invention include any desired signaling
domain that provides a distinct and detectable signal (e.g.,
increased production of one or more cytokines by the cell; change
in transcription of a target gene; change in activity of a protein;
change in cell behavior, e.g., cell death; cellular proliferation;
cellular differentiation; cell survival; modulation of cellular
signaling responses; etc.) in response to activation of the CAR
(i.e., activated by antigen and dimerizing agent). In some
embodiments, the intracellular signaling domain includes at least
one (e.g., one, two, three, four, five, six, etc.) ITAM motifs as
described below. In some embodiments, the intracellular signaling
domain includes DAP10/CD28 type signaling chains. In some
embodiments, the intracellular signaling domain is not covalently
attached to the membrane bound CAR, but is instead diffused in the
cytoplasm.
[0318] Intracellular signaling domains suitable for use in a
subject CAR of the present invention include immunoreceptor
tyrosine-based activation motif (ITAM)-containing intracellular
signaling polypeptides. In some embodiments, an ITAM motif is
repeated twice in an intracellular signaling domain, where the
first and second instances of the ITAM motif are separated from one
another by 6 to 8 amino acids. In one embodiment, the intracellular
signaling domain of a subject CAR comprises 3 ITAM motifs.
[0319] In some embodiments, intracellular signaling domains
includes the signaling domains of human immunoglobulin receptors
that contain immunoreceptor tyrosine based activation motifs
(ITAMs) such as, but not limited to, FcgammaRI, FcgammaRIIA,
FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis et al., Front.
Immunol. (2014) 5:254).
[0320] A suitable intracellular signaling domain can be an ITAM
motif-containing portion that is derived from a polypeptide that
contains an ITAM motif. For example, a suitable intracellular
signaling domain can be an ITAM motif-containing domain from any
ITAM motif-containing protein. Thus, a suitable intracellular
signaling domain need not contain the entire sequence of the entire
protein from which it is derived. Examples of suitable ITAM
motif-containing polypeptides include, but are not limited to:
DAPl2, FCERIG (Fc epsilon receptor I gamma chain), CD3D (CD3
delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and
CD79A (antigen receptor complex-associated protein alpha
chain).
[0321] In one embodiment, the intracellular signaling domain is
derived from DAP12 (also known as TYROBP; TYRO protein tyrosine
kinase binding protein; KARAP; PLOSL; DNAX-activation protein 12;
KAR-associated protein; TYRO protein tyrosine kinase-binding
protein; killer activating receptor associated protein;
killer-activating receptor-associated protein; etc.). In one
embodiment, the intracellular signaling domain is derived from
FCERIG (also known as FCRG; Fc epsilon receptor I gamma chain; Fc
receptor gamma-chain; fc-epsilon RI-gamma; fcRgamma; fceR1 gamma;
high affinity immunoglobulin epsilon receptor subunit gamma;
immunoglobulin E receptor, high affinity, gamma chain; etc.). In
one embodiment, the intracellular signaling domain is derived from
T-cell surface glycoprotein CD3 delta chain (also known as CD3D;
CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d
antigen, delta polypeptide (TiT3 complex); OKT3, delta chain;
T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3
delta chain; etc.). In one embodiment, the intracellular signaling
domain is derived from T-cell surface glycoprotein CD3 epsilon
chain (also known as CD3e, T-cell surface antigen T3/Leu-4 epsilon
chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783,
CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular
signaling domain is derived from T-cell surface glycoprotein CD3
gamma chain (also known as CD3G, T-cell receptor T3 gamma chain,
CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one
embodiment, the intracellular signaling domain is derived from
T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z,
T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z,
TCRZ, etc.). In one embodiment, the intracellular signaling domain
is derived from CD79A (also known as B-cell antigen receptor
complex-associated protein alpha chain; CD79a antigen
(immunoglobulin-associated alpha); MB-1 membrane glycoprotein;
ig-alpha; membrane-bound immunoglobulin-associated protein; surface
IgM-associated protein; etc.). In one embodiment, an intracellular
signaling domain suitable for use in an FN3 CAR of the present
disclosure includes a DAP10/CD28 type signaling chain. In one
embodiment, an intracellular signaling domain suitable for use in
an FN3 CAR of the present disclosure includes a ZAP70 polypeptide.
In some embodiments, the intracellular signaling domain includes a
cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3
gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d.
In one embodiment, the intracellular signaling domain in the CAR
includes a cytoplasmic signaling domain of human CD3 zeta.
[0322] While usually the entire intracellular signaling domain can
be employed, in many cases it is not necessary to use the entire
chain. To the extent that a truncated portion of the intracellular
signaling domain is used, such truncated portion may be used in
place of the intact chain as long as it transduces the effector
function signal. The intracellular signaling domain includes any
truncated portion of the intracellular signaling domain sufficient
to transduce the effector function signal.
[0323] The intracellular signaling domains described herein can be
combined with any of the antigen binding domains described herein,
any of the transmembrane domains described herein, or any of the
other domains described herein that may be included in the CAR.
[0324] In one embodiment, the intracellular domain of a subject CAR
comprises a 4-1BB domain comprising the amino acid sequence set
forth below:
TABLE-US-00049 (SEQ ID NO:92)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00050 (SEQ ID NO:93)
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA
GACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCC
AGAAGAAGAAGAAGGAGGATGTGAACTG.
or the nucleic acid sequence set forth below:
TABLE-US-00051 (SEQ ID NO:94)
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA
GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCC
AGAAGAAGAAGAAGGAGGATGTGAACTG.
[0325] Tolerable variations of the intracellular domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises an intracellular domain comprising a
4-1BB domain comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:92. In one embodiment, the CAR comprises an intracellular domain
comprising a 4-1BB domain comprising the amino acid sequence set
forth in SEQ ID NO:92.
[0326] In some embodiments, a subject CAR of the present invention
comprises an intracellular domain comprising a 4-1BB domain encoded
by a nucleic acid sequence that has at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
nucleic acid sequence set forth in SEQ ID NOs:93 or 94. In one
embodiment, the CAR comprises an intracellular domain comprising a
4-1BB domain encoded by the nucleic acid sequence set forth in SEQ
ID NOs:93 or 94.
[0327] In one embodiment, the intracellular domain of a subject CAR
comprises an ICOS domain comprising the amino acid sequence set
forth below:
TABLE-US-00052 (SEQ ID NO:203)
TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00053 (SEQ ID NO:204)
ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACA
TGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGT GACCCTA.
[0328] Tolerable variations of the intracellular domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises an intracellular domain comprising an
ICOS domain comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:203. In one embodiment, the CAR comprises an intracellular
domain comprising an ICOS domain comprising the amino acid sequence
set forth in SEQ ID NO:203.
[0329] In some embodiments, a subject CAR of the present invention
comprises an intracellular domain comprising an ICOS domain encoded
by a nucleic acid sequence that has at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
nucleic acid sequence set forth in SEQ ID NO:204. In one
embodiment, the CAR comprises an intracellular domain comprising an
ICOS domain encoded by the nucleic acid sequence set forth in SEQ
ID NO:204.
[0330] In one embodiment, the intracellular domain of a subject CAR
comprises a variant ICOS domain comprising the amino acid sequence
set forth below: TKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTL (SEQ ID
NO:95), which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00054 (SEQ ID NO:96)
ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACA
TGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGT GACCCTA.
[0331] The variant ICOS domain is also referred to herein as
ICOS(YMNM).
[0332] Tolerable variations of the intracellular domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises an intracellular domain comprising an
ICOS domain comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:95. In one embodiment, the CAR comprises an intracellular domain
comprising an ICOS domain comprising the amino acid sequence set
forth in SEQ ID NO:95.
[0333] In some embodiments, a subject CAR of the present invention
comprises an intracellular domain comprising an ICOS domain encoded
by a nucleic acid sequence that has at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
nucleic acid sequence set forth in SEQ ID NO:96. In one embodiment,
the CAR comprises an intracellular domain comprising an ICOS domain
encoded by the nucleic acid sequence set forth in SEQ ID NO:96.
[0334] In one embodiment, the intracellular domain of a subject CAR
comprises a CD3 zeta domain comprising the amino acid sequence set
forth below:
TABLE-US-00055 (SEQ ID NO:97)
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK
DTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00056 (SEQ ID NO:98)
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCC
AGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA
TGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG
AGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA
AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG
GGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG
GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
or the nucleic acid sequence set forth below:
TABLE-US-00057 (SEQ ID NO:99)
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCC
AGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA
CGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG
AGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATA
AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG
GGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG
GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0335] Tolerable variations of the intracellular domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises an intracellular domain comprising a
CD3 zeta domain comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:97. In one embodiment, a subject CAR of the present invention
comprises an intracellular domain comprising a CD3 zeta domain
comprising the amino acid sequence set forth in SEQ ID NO:97.
[0336] In some embodiments, a subject CAR of the present invention
comprises an intracellular domain comprising a CD3 zeta domain
encoded by a nucleic acid sequence that has at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the nucleic acid sequence set forth in SEQ ID NOs:98 or
99. In one embodiment, a subject CAR of the present invention
comprises an intracellular domain comprising a CD3 zeta domain
encoded by the nucleic acid sequence set forth in SEQ ID NOs:98 or
99 A CD3 zeta domain may comprise an amino acid sequence set forth
below:
TABLE-US-00058 (SEQ ID NO:100)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK
DTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00059 (SEQ ID NO:101)
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCC
AGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA
TGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG
AGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA
AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG
GGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG
GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0337] Tolerable variations of the intracellular domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises an intracellular domain comprising a
CD3 zeta domain comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 100. In one embodiment, a subject CAR of the present invention
comprises an intracellular domain comprising a CD3 zeta domain
comprising the amino acid sequence set forth in SEQ ID NO: 100.
[0338] In some embodiments, a subject CAR of the present invention
comprises an intracellular domain comprising a CD3 zeta domain
encoded by a nucleic acid sequence that has at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the nucleic acid sequence set forth in SEQ ID NO: 101.
In one embodiment, a subject CAR of the present invention comprises
an intracellular domain comprising a CD3 zeta domain encoded by the
nucleic acid sequence set forth in SEQ ID NO:101.
[0339] In one embodiment, the CAR comprises an intracellular domain
comprising a CD3 zeta domain comprising the amino acid sequence set
forth in SEQ ID NOs:97 or 100.
[0340] In one exemplary embodiment, the intracellular domain of a
subject CAR comprises a 4-1BB domain and a CD3 zeta domain,
comprising the amino acid sequence set forth below:
TABLE-US-00060 (SEQ ID NO:102)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE
GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00061 (SEQ ID NO:103)
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA
GACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCC
AGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGC
GCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGC
TCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGG
CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA
GGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTG
AGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCT
TTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCAC
ATGCAGGCCCTGCCCCCTCGC.
or the nucleic acid sequence set forth below:
TABLE-US-00062 (SEQ ID NO:104)
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA
GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCC
AGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGC
GCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGC
TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGG
CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA
GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTG
AGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCT
TTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCAC
ATGCAGGCCCTGCCCCCTCGC.
[0341] Tolerable variations of the intracellular domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises an intracellular domain comprising a
4-1BB domain and a CD3 zeta domain, comprising an amino acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO: 102. In one embodiment, the CAR
comprises an intracellular domain comprising a 4-1BB domain and a
CD3 zeta domain, comprising the amino acid sequence set forth in
SEQ ID NO: 102.
[0342] In some embodiments, a subject CAR of the present invention
comprises an intracellular domain comprising a 4-1BB domain and a
CD3 zeta domain, encoded by a nucleic acid sequence that has at
least 60%/a, at least 65%, at least 70%, at least 75%, at least
80%/a, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the nucleic acid sequence set forth
in SEQ ID NOs:103 or 104. In one embodiment, the CAR comprises an
intracellular domain comprising a 4-1BB domain and a CD3 zeta
domain, encoded by the nucleic acid sequence set forth in SEQ ID
NOs:103 or 104.
[0343] In one exemplary embodiment, the intracellular domain of a
subject CAR comprises an ICOS domain and a CD3 zeta domain,
comprising the amino acid sequence set forth below:
TABLE-US-00063 (SEQ ID NO:205)
TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQ
QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL
QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00064 (SEQ ID NO:206)
ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACA
TGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGT
GACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG
CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG
AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG
AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCG
AGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC
AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGC.
[0344] Tolerable variations of the intracellular domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises an intracellular domain comprising an
ICOS domain and a CD3 zeta domain, comprising an amino acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO:205. In one embodiment, the CAR
comprises an intracellular domain comprising an ICOS domain and a
CD3 zeta domain, comprising the amino acid sequence set forth in
SEQ ID NO:205.
[0345] In some embodiments, a subject CAR of the present invention
comprises an intracellular domain comprising an ICOS domain and a
CD3 zeta domain, encoded by a nucleic acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO:206. In one embodiment, the CAR comprises an intracellular
domain comprising an ICOS domain and a CD3 zeta domain, encoded by
the nucleic acid sequence set forth in SEQ ID NO:206.
[0346] In one exemplary embodiment, the intracellular domain of a
subject CAR comprises a variant ICOS domain and a CD3 zeta domain,
comprising the amino acid sequence set forth below:
TABLE-US-00065 (SEQ ID NO:207)
TKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQ
QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL
QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00066 (SEQ ID NO:208)
ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACA
TGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGT
GACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG
CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG
AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG
AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCG
AGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC
AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGC.
[0347] Tolerable variations of the intracellular domain will be
known to those of skill in the art, while maintaining its intended
function. For example, in some embodiments, a subject CAR of the
present invention comprises an intracellular domain comprising a
variant ICOS domain and a CD3 zeta domain, comprising an amino acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO:207. In one embodiment, the CAR
comprises an intracellular domain comprising a variant ICOS domain
and a CD3 zeta domain, comprising the amino acid sequence set forth
in SEQ ID NO:207.
[0348] In some embodiments, a subject CAR of the present invention
comprises an intracellular domain comprising a variant ICOS domain
and a CD3 zeta domain, encoded by a nucleic acid sequence that has
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the nucleic acid sequence set forth
in SEQ ID NO:208. In one embodiment, the CAR comprises an
intracellular domain comprising a variant ICOS domain and a CD3
zeta domain, encoded by the nucleic acid sequence set forth in SEQ
ID NO:208.
CAR Sequences
[0349] A subject CAR of the present invention may be selected from
the group consisting of a J591 murine PSMA-CAR, a humanized J591
PSMA-CAR, a 1C3 human PSMA-CAR, a 2A10 human PSMA-CAR, a 2F5 human
PSMA-CAR, and a 2C6 human PSMA-CAR.
[0350] In one embodiment, a subject CAR of the present invention is
a J591 murine PSMA-CAR. In one embodiment, the J591 murine PSMA-CAR
comprises the amino acid sequence set forth below:
TABLE-US-00067 (SEQ ID NO:105)
MALPVTALLLPLALLLHAARPGSDIVMTQSHKFMSTSVGDRVSIICKAS
QDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTI
TNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKGGGGSGGGGSSGGGSEV
QLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNI
NPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGW
NFDYWGQGTTLTVSSASSGTTTPAPRPPTPAPTIASQPLSLRPEACRPA
AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI
FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQ
NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK
MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00068 (SEQ ID NO:106)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC
ACGCCGCCAGACCTGGATCTGACATTGTGATGACCCAGTCTCACAAATT
CATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGT
CAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAAT
CTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCC
TGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATT
ACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATA
ACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGG
AGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTG
CAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGC
GGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCA
CTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATC
AACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGG
CCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCG
GAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGG
AACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGCGCTA
GCTCCGGAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCAC
CATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCG
GCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCT
ACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACT
GGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATA
TTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACG
GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAG
AGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAG
AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACG
TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAG
AAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAG
ATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG
GCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGA
CACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0351] In one embodiment, a subject CAR of the present invention is
a humanized PSMA-CAR, e.g., a humanized J591 PSMA-CAR. In such an
embodiment, the humanized PSMA-CAR comprises any of the heavy and
light chain variable regions disclosed in PCT Publication Nos.
WO2017212250A1 and WO2018033749A1. For example, a humanized
PSMA-CAR of the present invention can comprise an scFv comprising
any of the heavy and light chain variable regions disclosed
therein, see, e.g., sequences set forth in Table 19 of the present
disclosure.
[0352] In one embodiment, a subject CAR of the present invention is
a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR comprises
the amino acid sequence set forth below:
TABLE-US-00069 (SEQ ID NO:245)
MALPVTALLLPLALLLHAARPGEVQLVQSGAEVKKPGASVKVSCKASGY
TFTEYTIHWVRQAPGKGLEWIGNINPNNGGTTYNQKFEDRVTITVDKST
STAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGGGS
SGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQKPGQAP
KLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNS
YPLTFGQGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV
HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYN
ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00070 (SEQ ID NO:246)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC
ACGCCGCCAGACCTGGAGAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGT
GAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATAC
ACATTCACTGAATACACCATCCACTGGGTGAGGCAGGCCCCTGGAAAGG
GCCTTGAGTGGATTGGAAACATTAATCCTAACAATGGTGGTACTACCTA
CAACCAGAAGTTCGAGGACAGAGTCACAATCACTGTAGACAAGTCCACC
AGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGATACTGCAG
TCTATTACTGTGCAGCTGGTTGGAACTTTGACTACTGGGGCCAAGGCAC
CACGGTCACCGTCTCCTCAGGAGGCGGAGGATCTGGCGGCGGAGGAAGT
TCTGGCGGAGGCAGCGACATTCAGATGACCCAGTCTCCCAGCACCCTGT
CCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAGGA
TGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTCCT
AAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATC
GCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG
ACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACAGC
TATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAACCACGA
CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC
CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTG
CACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCT
TGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTA
CTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTT
ATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGAT
TTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAG
GAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAAC
GAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGAC
GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCA
GGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC
AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACG
GCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT
TCACATGCAGGCCCTGCCCCCTCGC.
[0353] In one embodiment, a subject CAR of the present invention is
a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR comprises
the amino acid sequence set forth below:
TABLE-US-00071 (SEQ ID NO:247)
MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCKASQ
DVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTIS
RLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQ
LVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNIN
PNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWN
FDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV
HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYN
ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00072 (SEQ ID NO:248)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC
ACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCT
GTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAG
GATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTC
CTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGA
TCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACA
GCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGG
CGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAG
CTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG
TCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTG
GGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAAT
CCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCA
CAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAG
CCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAAC
TTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGA
CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC
CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTG
CACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCT
TGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTA
CTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTT
ATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGAT
TTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAG GAGCGCAGACG.
TABLE-US-00073 (SEQ ID NO: 248)
CCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT
AGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGAC
CCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGT
ACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGG
GATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAG
GGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGG
CCCTGCCCCCTCGC.
[0354] In one embodiment, a subject CAR of the present invention is
a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR comprises
the amino acid sequence set forth below:
TABLE-US-00074 (SEQ ID NO: 249)
MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCKASQ
DVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTIS
RLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQ
LVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNIN
PNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWN
FDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV
HTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGE
YMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCS
CRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD
KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG
HDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00075 (SEQ ID NO: 250)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC
ACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCT
GTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAG
GATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTC
CTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGA
TCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACA
GCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGG
CGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAG
CTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG
TCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTG
GGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAAT
CCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCA
CAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAG
CCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAAC
TTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGA
CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC
CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTG
CACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGAT
GTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTG
GCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAA
TACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAG
ATGTGACCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACA
ACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGC
TGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGT
TCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCT
CTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGAC
AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGA
ACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGA
GGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG
CACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG
ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0355] In one embodiment, a subject CAR of the present invention is
a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR comprises
the amino acid sequence set forth below:
TABLE-US-00076 (SEQ ID NO: 251)
MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCKASQ
DVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTIS
RLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQ
LVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNIN
PNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWN
FDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV
HTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGE
YMNMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCS
SRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD
KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG
HDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00077 (SEQ ID NO: 252)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC
ACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCT
GTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAG
GATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTC
CTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGA
TCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACA
GCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGG
CGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAG
CTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG
TCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTG
GGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAAT
CCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCA
CAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAG
CCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAAC
TTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGA
CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC
CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTG
CACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGAT
GTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTG
GCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAA
TACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAG
ATGTGACCCTAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACA
ACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGC
TGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGT
TCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCT
CTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGAC
AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGA
ACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGG
GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC
ACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGA
CGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0356] In one embodiment, a subject CAR of the present invention is
a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR comprises
the amino acid sequence set forth below:
TABLE-US-00078 (SEQ ID NO: 253)
MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCKASQ
DVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTIS
RLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQ
LVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNIN
PNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWN
FDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV
HTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGE
YMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG
ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00079 (SEQ ID NO: 254)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC
ACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCT
GTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAG
GATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTC
CTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGA
TCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACA
GCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGG
CGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAG
CTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG
TCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTG
GGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAAT
CCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCA
CAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAG
CCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAAC
TTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGA
CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC
CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTG
CACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGAT
GTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTG
GCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAA
TACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAG
ATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA
CAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA
GAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG
GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACT
GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC
GAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTA
CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC TCGC.
[0357] In one embodiment, a subject CAR of the present invention is
a huJ591 PSMA-CAR. In one embodiment, the huJ591 PSMA-CAR comprises
the amino acid sequence set forth below:
TABLE-US-00080 (SEQ ID NO: 255)
MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVTITCKASQ
DVGTAVDWYQQKPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTIS
RLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQ
LVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNIN
PNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAAGWN
FDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV
HTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGE
YMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG
ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00081 (SEQ ID NO: 256)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC
ACGCCGCCAGACCTGGAGACATTCAGATGACCCAGTCTCCCAGCACCCT
GTCCGCATCAGTAGGAGACAGGGTCACCATCACTTGCAAGGCCAGTCAG
GATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGGCAAGCTC
CTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGA
TCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGACTGCAGCCTGAAGACTTTGCAGTTTATTACTGTCAGCAATATAACA
GCTATCCTCTCACGTTCGGCCAGGGGACCAAGGTGGATATCAAAGGAGG
CGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTCCAG
CTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG
TCTCCTGCAAGGCTTCTGGATACACATTCACTGAATACACCATCCACTG
GGTGAGGCAGGCCCCTGGAAAGGGCCTTGAGTGGATTGGAAACATTAAT
CCTAACAATGGTGGTACTACCTACAACCAGAAGTTCGAGGACAGAGTCA
CAATCACTGTAGACAAGTCCACCAGCACAGCCTACATGGAGCTCAGCAG
CCTGAGATCTGAGGATACTGCAGTCTATTACTGTGCAGCTGGTTGGAAC
TTTGACTACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAACCACGA
CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCC
CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTG
CACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGAT
GTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTG
GCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAA
TACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAG
ATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA
CAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA
GAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG
GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACT
GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC
GAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTA
CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCC TCGC.
[0358] In one embodiment, a subject CAR of the present invention is
a 1C3 human PSMA-CAR. In one embodiment, the 1C3 human PSMA-CAR
comprises the amino acid sequence set forth below:
TABLE-US-00082 (SEQ ID NO: 107)
MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFT
FSSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKN
TLYLQMNSLRAEDTAVYYCARAVPWGSRYYYYGMDVWGQGTTVTVSSGG
GGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWY
QQKSGKAPKLLIFDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT
YYCQQFNSYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEAC
RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYK
QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00083 (SEQ ID NO: 108)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGT
CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACC
TTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGC
TGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGT
ATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGG
TATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGC
GGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGT
TGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTAT
CAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCA
GTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGAC
AGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACT
TATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGA
CCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACC
GGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGC
CGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCT
GTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCT
CCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTC
CTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAG
AGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATG
TGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAG
CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG
AGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG
AAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAG
AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGC
GCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGC
CACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0359] In one embodiment, a subject CAR of the present invention is
a 2A10 human PSMA-CAR. In one embodiment, the 2A10 human PSMA-CAR
comprises the amino acid sequence set forth below:
TABLE-US-00084 (SEQ ID NO: 109)
MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYS
FTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS
TAYLQWSSLKASDTAMYYCARQTGFLWSSDLWGRGTLVTVSSGGGGSGG
GGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPG
KAPKLLIYDASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQQ
FNSYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG
GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFK
QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQ
LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA
EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00085 (SEQ ID NO: 110)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGC
TTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGG
CCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGT
GGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC
AAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGG
AAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGG
TCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCAC
CATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAG
TTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGC
GTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGG
GGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCT
GGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTAT
CACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA
CAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTA
GCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAA
GTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAG
CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGG
ACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAA
GAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCG
GAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG
GGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTA
CGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0360] In one embodiment, a subject CAR of the present invention is
a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR
comprises a 4-1BB domain and a CD3 zeta domain comprising the amino
acid sequence set forth below:
TABLE-US-00086 (SEQ ID NO: 111)
MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYS
FTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS
TAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGG
GGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPG
KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
FNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPA
AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI
FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQ
NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK
MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00087 (SEQ ID NO: 112)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGT
TTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGG
CCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGT
GGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC
AAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGG
AAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGG
TCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCAC
CATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAG
TTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCAC
CATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCG
GCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCT
ACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACT
GGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATA
TTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACG
GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAG
AGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAG
AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACG
TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAG
AAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAG
ATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG
GCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGA
CACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0361] In one embodiment, a subject CAR of the present invention is
a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR
comprises an ICOS domain and a CD3 zeta domain comprising the amino
acid sequence set forth below:
TABLE-US-00088 (SEQ ID NO: 209)
MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYS
FTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS
TAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGG
GGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPG
KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
FNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPA
AGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVH
DPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00089 (SEQ ID NO: 210)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGT
TTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGG
CCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGT
GGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC
AAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGG
AAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGG
TCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCAC
CATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAG
TTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCAC
CATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCG
GCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCT
GGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATG
CATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCAC
GACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAA
AATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGC
AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC
AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC
GGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGA
AGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT
GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC
TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCA
CATGCAGGCCCTGCCCCCTCGC.
[0362] In one embodiment, a subject CAR of the present invention is
a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR
comprises a variant ICOS domain and a CD3 zeta domain comprising
the amino acid sequence set forth below:
TABLE-US-00090 (SEQ ID NO:211)
MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYS
FTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS
TAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGG
GGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPG
KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
FNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPA
AGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVH
DPNGEYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00091 (SEQ ID NO:212)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGT
TTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGG
CCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGT
GGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC
AAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGG
AAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGG
TCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCAC
CATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAG
TTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCAC
CATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCG
GCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCT
GGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATG
CATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCAC
GACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAA
AATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGC
AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC
AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC
GGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGA
AGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT
GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC
TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCA
CATGCAGGCCCTGCCCCCTCGC.
[0363] In one embodiment, a subject CAR of the present invention is
a 2C6 human PSMA-CAR. In one embodiment, the 2C6 human PSMA-CAR
comprises the amino acid sequence set forth below:
TABLE-US-00092 (SEQ ID NO:113)
MALPVTALLLPLALLLHAARPEVQLVQSGSEVKKPGESLKISCKGSGYS
FTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSIS
TAYLQWSSLKASDTAMYYCASPGYTSSWTSFDYWGQGTLVTVSSGGGGS
GGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK
PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC
QQRSNWPLFTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRP
AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY
IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQG
QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD
KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00093 (SEQ ID NO:114)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGC
TTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCG
GGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGT
CTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG
CAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAA
CCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCA
CTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCAC
TCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT
CAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAG
TGGATATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCC
CACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCA
GCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATA
TCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTC
ACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTAT
ATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG
ACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACT
GAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGC
CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACG
ACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCC
GAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGAT
AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA
GGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAA
GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0364] Tolerable variations of the sequences of the subject CARs
will be known to those of skill in the art, while maintaining its
function.
[0365] For example, in some embodiments, a subject CAR of the
present invention is a J591 murine PSMA-CAR comprising an amino
acid sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO: 105. In one embodiment, the CAR is
a J591 murine PSMA-CAR comprising the amino acid sequence set forth
in SEQ ID NO:105.
[0366] For example, in some embodiments, a subject CAR of the
present invention is a humanized J591 PSMA-CAR. A humanized J591
PSMA-CAR comprises a humanized J591 PSMA binding domain comprising
a heavy and light chain variable region selected from any of the
heavy and light chain variable region sequences set forth in Table
19. In some embodiments, the humanized J591 PSMA-CAR comprises a
4-1BB domain and a CD3zeta domain.
[0367] For example, in some embodiments, a subject CAR of the
present invention is a huJ591 PSMA-CAR comprising an amino acid
sequence that has at least 60%, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO: 245, 247, 249, 251, 253, or 255.
In one embodiment, the CAR is a huJ591 PSMA-CAR comprising the
amino acid sequence set forth in SEQ ID NO: 245, 247, 249, 251,
253, or 255.
[0368] For example, in some embodiments, a subject CAR of the
present invention is a 1C3 human PSMA-CAR comprising an amino acid
sequence that has at least 60%/a, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO: 107. In one embodiment, the CAR is
a 1C3 human PSMA-CAR comprising the amino acid sequence set forth
in SEQ ID NO:107.
[0369] For example, in some embodiments, a subject CAR of the
present invention is a 2A10 human PSMA-CAR comprising an amino acid
sequence that has at least 60%/a, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the amino acid
sequence set forth in SEQ ID NO: 109. In one embodiment, the CAR is
a 2A10 human PSMA-CAR comprising the amino acid sequence set forth
in SEQ ID NO:109.
[0370] For example, in some embodiments, a subject CAR of the
present invention is a 2F5 human PSMA-CAR. In one embodiment, the
CAR is a 2F5 human PSMA-CAR that comprises a 4-1BB domain and a
CD3zeta domain comprising an amino acid sequence that has at least
60%/a, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 111. In one embodiment, the CAR is a 2F5 human PSMA-CAR that
comprises a 4-1BB domain and a CD3zeta domain comprising the amino
acid sequence set forth in SEQ ID NO:111. In one embodiment, the
CAR is a 2F5 human PSMA-CAR that comprises an ICOS domain and a
CD3zeta domain comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO:209. In one embodiment, the CAR is a 2F5 human PSMA-CAR that
comprises an ICOS domain and a CD3zeta domain comprising the amino
acid sequence set forth in SEQ ID NO:209. In one embodiment, the
CAR is a 2F5 human PSMA-CAR that comprises a variant ICOS domain
and a CD3zeta domain comprising an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the amino acid sequence set forth in SEQ
ID NO:211. In one embodiment, the CAR is a 2F5 human PSMA-CAR that
comprises a variant ICOS domain and a CD3zeta domain comprising the
amino acid sequence set forth in SEQ ID NO:211. For example, in
some embodiments, a subject CAR of the present invention is a 2C6
human PSMA-CAR comprising an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 113. In one embodiment, the CAR is a 2C6 human PSMA-CAR
comprising the amino acid sequence set forth in SEQ ID NO:113.
[0371] In some embodiments, a subject CAR of the present invention
is a J591 murine PSMA-CAR encoded by a nucleic acid sequence that
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the nucleic acid sequence set
forth in SEQ ID NO: 106. In one embodiment, the CAR is a J591
murine PSMA-CAR encoded by the nucleic acid sequence set forth in
SEQ ID NO: 106.
[0372] For example, in some embodiments, a subject CAR of the
present invention is a huJ591 PSMA-CAR encoded by a nucleic acid
sequence that has at least 60%, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO: 246, 248, 250, 252, 254, or 256.
In one embodiment, the CAR is a huJ591 PSMA-CAR encoded by the
nucleic acid sequence set forth in SEQ ID NO: 246, 248, 250, 252,
254, or 256.
[0373] For example, in some embodiments, a subject CAR of the
present invention is a 1C3 human PSMA-CAR encoded by a nucleic acid
sequence that has at least 60%/a, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:108. In one embodiment, the CAR is
a 1C3 human PSMA-CAR encoded by the nucleic acid sequence set forth
in SEQ ID NO: 108. For example, in some embodiments, a subject CAR
of the present invention is a 2A10 human PSMA-CAR encoded by a
nucleic acid sequence that has at least 60%/a, at least 65%, at
least 70%, at least 75%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
nucleic acid sequence set forth in SEQ ID NO:110. In one
embodiment, the CAR is a 2A10 human PSMA-CAR encoded by the nucleic
acid sequence set forth in SEQ ID NO: 110. For example, in some
embodiments, a subject CAR of the present invention is a 2F5 human
PSMA-CAR. In one embodiment, the CAR is a 2F5 human PSMA-CAR that
comprises a 4-1BB domain and a CD3zeta domain, encoded by a nucleic
acid sequence that has at least 60%/a, at least 65%, at least 70%,
at least 75%, at least 80%, at least 81%, at least 82%, at least
83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% sequence identity to the nucleic
acid sequence set forth in SEQ ID NO: 112. In one embodiment, the
CAR is a 2F5 human PSMA-CAR that comprises a 4-1BB domain and a
CD3zeta domain, encoded by the nucleic acid sequence set forth in
SEQ ID NO: 112. In one embodiment, the CAR is a 2F5 human PSMA-CAR
that comprises an ICOS domain and a CD3zeta domain, encoded by a
nucleic acid sequence that has at least 60%/a, at least 65%, at
least 70%, at least 75%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% sequence identity to the
nucleic acid sequence set forth in SEQ ID NO:210. In one
embodiment, the CAR is a 2F5 human PSMA-CAR that comprises an ICOS
domain and a CD3zeta domain, encoded by the nucleic acid sequence
set forth in SEQ ID NO:210. In one embodiment, the CAR is a 2F5
human PSMA-CAR that comprises a variant ICOS domain and a CD3zeta
domain, encoded by a nucleic acid sequence that has at least 60%/a,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the nucleic acid sequence set forth in SEQ ID
NO:212. In one embodiment, the CAR is a 2F5 human PSMA-CAR that
comprises a variant ICOS domain and a CD3zeta domain, encoded by
the nucleic acid sequence set forth in SEQ ID NO:212. For example,
in some embodiments, a subject CAR of the present invention is a
2C6 human PSMA-CAR encoded by a nucleic acid sequence that has at
least 60%/a, at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the nucleic acid sequence set forth
in SEQ ID NO: 114. In one embodiment, the CAR is a 2C6 human
PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID
NO: 114.
[0374] In certain embodiments, a subject CAR of the present
invention may comprise any one of the amino acid sequences
corresponding to SEQ ID NOs: 209, 211, 227-236, 245, 247, 249, 251,
253, or 255.
TABLE-US-00094 TABLE 20 Exemplary CAR sequences SEQ ID NO: CAR
Sequence 227 PD1-CD28- MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFS
2F5-ICOSz PALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT
DKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRAR
RNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVP
TAHPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTV
AFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA
PPRDFAAYRSVKQTLNFDLLKLAGDVESNPGPMALPVT
ALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCK
GSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYS
PSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQ
TGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQ
LTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPG
KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRP
PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDF
WLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGE
YMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQN
QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR 228
PD1*CD28- MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFS 2F5-ICOSz
PALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT
DKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRAR
RNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVP
TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAV
IRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA
AYRSVKQTLNFDLLKLAGDVESNPGPMALPVTALLLPL
ALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSF
TSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ
VTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWS
FDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPS
SLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLL
IYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT
YYCQQFNSYPLTFGGGTKVEIKIYYCQQFNSYPLTFGG
GTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRP
AAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILIC
WLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLR
VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR
GRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM
KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 229 PD1*BB-
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFS 2F5-ICOSz
PALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT
DKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRAR
RNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVP
TAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVI
TLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP
EEEEGGCELVKQTLNFDLLKLAGDVESNPGPMALPVTA
LLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKG
SGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSP
ASFQGQVTISADKSISTYLQWNSLKASDTAMYYCARQT
GFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQL
TQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGK
APKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRPP
TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFW
LPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEY
MFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQ
LYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQE
GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR 230
TIM3-CD28 MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPC -2F5-ICOSz
FYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVN
YWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQI
PGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRML
TTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLAN
DLRDSGATIRFWVLVVVGGVLACYSLLVTVAFIIFWVR
SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY
RSVKQTLNFDLLKLAGDVESNPGPMALPVTALLLPLAL
LLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTS
NWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVT
ISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFD
LWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSL
SASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIY
DASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY
CQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIA
SQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAA
FVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVN
TAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNL
GRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR 231 PD1*BB-
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFS TIM3-CD28
PALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT -2F5-ICOSz
DKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRAR
RNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVP
TAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVI
KTLYCKKRGRKLLYIFKQPFMRPVQTTQEEDGCSCRFP
EEEEGGCELVKQTLNFDLLKLAGDVESNPGPMFSHLPF
DCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAP
GNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYW
LNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDE
KFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGP
AETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGA
VTIRFWVLVVVGGVLACYSLLVTAFIIFWVRSKRSRLL
HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSVKQTL
NFDLLKLAGDVESNPGPMALPVTALLLPLALLLHAARP
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVR
QMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSI
STAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTL
VTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDR
VTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLES
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSY
PLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLR
PEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCIL
GCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRL
TDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD
VLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEA
YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR 232 PD1-CD28-
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFS 2F5-ICOSz
PALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT YMNM
DKLAAFPEDRSQPGQDCRERVTQLPNGRDEHMSVVRAR
RNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVP
TARPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTV
AFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA
PPRDFAAYRSVKQTLNFDLLKLAGDVESNPGPMALPVT
AALLLPLALLLHARPEVQLVQSGAEVKKPGESLKISCK
GSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYS
PSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQ
TGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQ
LTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPG
KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRP
PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDF
WLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGE
YMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQN
QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR 233
PD1*CD28- MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFS 2F5-ICOSz
PALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT YMNM
DKLAAFPEDRSQPGQDCRERVTQLPNGRDEHMSVVRAR
RNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVP
TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAV
IRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA
AYRSVKQTLNFDLLKLAGDVESNPGPMALPVTALLLPL
ALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSF
TSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQ
VTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWS
FDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPS
SLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLL
IYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT
YYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPT
IASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGC
AAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMNMRA
VNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNE
LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR 234 PD1*BB-
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFS 2F5-ICOSz
PALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT
DKYMNMLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSV
VRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERR
AEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLL
SLVITLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGSC
RFPEEEEGGCELVKQTLNFDLLKLAGDVESNPGPMALP
VTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKIS
CKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTR
YSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCA
RQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSA
IQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQK
PGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISS
LQPEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAP
RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC
DFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPN
GEYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQG
QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKN
PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR 235
TIM3-CD28 MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPC -2F5-ICOSz
FYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVN YMNM
YWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQI
PGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRML
TTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLAN
DLRDSGATIRFWVLVVVGGVLACYSLLVTVAFIIFWVR
SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY
RSVKQTLNFDLLKLAGDVESNPGPMALPVTALLLPLAL
LLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTS
NWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVT
ISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFD
LWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSL
SASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIY
DASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY
CQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIA
SQPSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAF
MNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQ
VVVCILGCILICWLTKKKYSSSVHDPNGEYLYNELNLG
RREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR 236 PD1*BB-
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFS TIM3-CD28
PALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQT -2F5-ICOSz
DKLAAFPEDRSQPGQDCRERVTQLPNGRDEHMSVVRAR
RNDYMNMSGTYLCGAISLAPKLQIKESLRAELRVTERR
AEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLL
SLVITLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGCS
CRFPEEEEGGCELVKQTLNFDLLKLAGDVESNPGPMFS
HLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYT
PAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWT
SRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGI
MNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTR
GHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLR
DSGATIRFWVLVVVGGVLACYSLLVTVAFBFWVRSKRS
RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSVK
QTLNFDLLKLAGDVESNPGPMALPVTALLLPLALLLHA
ARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIG
WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISAD
KSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGR
GTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASV
GDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASS
LESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQF
NSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPL
SLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVV
CILGCILICWLTKKKYSSSVHDPNGEYMNMRAVNTAKK
SRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRRE
EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM
AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR 245 hJ591VHVK
MALPVTALLLPLALLLHAARPGEVQLVQSGAEVKKPGA .BBZ
SVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPN
NGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTA
VYYCAAGWNFDYWGQGTTVTVSSGGGGSGGGGSSGGGS
DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVDWYQQ
KPGQAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTIS
RLQPEDFAVYYCQQYNSYPLTFGQGTKVDIKTTTPAPR
PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP
FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA
PAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR
247 hJ591VKVH MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVG
.BBZ DRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTR
HTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYN
SYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSG
AEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLE
WIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELS
SLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPR
PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP
FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA
PAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK
PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR
249 hJ591VKVH MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVG .ICOSBBZ
DRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTR
HTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYN
SYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSG
AEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLE
WIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELS
SLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPR
PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNG
EYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMR
PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR
KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR 251
hJ591VKVH MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVG .ICOSBBZY
DRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTR MNM
HTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYN
SYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSG
AEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLE
WIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELS
SLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPR
PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNG
EYMNMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMR
PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR
KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR 253
hJ591VKVH MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVG .ICOSZ
DRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTR
HTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYN
SYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSG
AEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLE
WIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELS
SLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPR
PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNG
EYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQ
NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR 255
hJ591VKVH MALPVTALLLPLALLLHAARPGDIQMTQSPSTLSASVG .ICOSZYMN
DRVTITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTR M
HTGVPDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYN
SYPLTFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSG
AEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLE
WIGNINPNNGGTTYNQKFEDRVTITVDKSTSTAYMELS
SLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPR
PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNG
EYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQ
NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQVALPPR 257
dnTGF.hJ59 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIV 1VHVK.BB
TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI Z
CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE
DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYR
VNRQQKLSSSGRSGGGEGRGSLLTCGDVEENPGPMALP
VTALLLPLALLLHAARPGEVQLVQSGAEVKKPGASVKV
SCKASGYTFTEYTIHWVRQAPGKGLEWIGNINPNNGGT
TYNQKFEDRVTITVDKSTSTAYMELSSLRSEDTAVYYC
VAAGWNFDYWGQGTTVTSSGGGGSGGGGSSGGGSDIQM
CTQSPSTLSASVGDRVTITKASQDVGTAVDWYQQKPGQ
APKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISRLQP
EDFAVYYCQQYNSYPLTFGQGTKVDIKTTTPAPRPPTP
APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW
APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP
VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYK
QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK
NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR 259
dnTGF.hJ59 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIV 1VKVH.BB
TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI Z
CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE
DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYR
VNRQQKLSSSGRSGGGEGRGSLLTCGDVEENPGPMALP
VTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVT
ITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGV
PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPL
TFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVK
KPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGN
INPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRS
EDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTP
APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW
APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP
VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYK
QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK
NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR 261
dnTGF.hJ59 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIV 1VKVH.ICO
TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI SZYMNM
CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE
DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE
YNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYR
VNRQQKLSSSGRSGGGEGRGSLLTCGDVEENPGPMALP
VTALLLPLALLLHAARPGDIQMTQSPSTLSASVGDRVT
ITCKASQDVGTAVDWYQQKPGQAPKLLIYWASTRHTGV
PDRFSGSGSGTDFTLTISRLQPEDFAVYYCQQYNSYPL
TFGQGTKVDIKGGGGSGGGGSSGGGSEVQLVQSGAEVK
KPGASVKVSCKASGYTFTEYTIHWVRQAPGKGLEWIGN
INPNNGGTTYNQKFEDRVTITVDKSTSTAYMELSSLRS
EDTAVYYCAAGWNFDYWGQGTTVTVSSTTTPAPRPPTP
APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLP
IGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMN
MRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQNQLY
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY
NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR
[0375] Accordingly, the present invention provides a modified
immune cell or precursor cell thereof, e.g., a modified T cell,
comprising a chimeric antigen receptor (CAR) having affinity for a
prostate-specific membrane antigen (PSMA) on a target cell (e.g., a
prostate cancer cell). In some embodiments, the CAR comprises a
PSMA binding domain. In some embodiments, the CAR comprises a
murine PSMA binding domain. In one embodiment, the CAR comprises a
J591 murine PSMA binding domain. In one embodiment, the CAR
comprises a humanized J591 PSMA binding domain. In some
embodiments, the CAR comprises a human PSMA binding domain. In some
embodiments, the CAR comprises a human PSMA binding domain selected
from the group consisting of a 1C3, a 2A10, a 2F5, and a 2C6 human
PSMA binding domain.
[0376] Accordingly, a subject CAR of the present invention
comprises a PSMA binding domain and a transmembrane domain. In one
embodiment, the CAR comprises a PSMA binding domain and a
transmembrane domain, wherein the transmembrane domain comprises a
CD8 hinge region. In one embodiment, the CAR comprises a PSMA
binding domain and a transmembrane domain, wherein the
transmembrane domain comprises a CD8 transmembrane domain. In one
embodiment, the CAR comprises a PSMA binding domain and a
transmembrane domain, wherein the transmembrane domain comprises a
CD8 hinge region and a CD8 transmembrane domain.
[0377] Accordingly, a subject CAR of the present invention
comprises a PSMA binding domain, a transmembrane domain, and an
intracellular domain. In one embodiment, the CAR comprises a PSMA
binding domain, a transmembrane domain, and an intracellular
domain, wherein the intracellular domain comprises a 4-1BB domain.
In one embodiment, the CAR comprises a PSMA binding domain, a
transmembrane domain, and an intracellular domain, wherein the
intracellular domain comprises a CD3 zeta domain. In one
embodiment, the CAR comprises a PSMA binding domain, a
transmembrane domain, and an intracellular domain, wherein the
intracellular domain comprises a 4-1BB domain and a CD3 zeta
domain.
C. Dominant Negative Receptors and Switch Receptors
[0378] The present invention provides compositions and methods for
modified immune cells or precursors thereof, e.g., modified T
cells, comprising a dominant negative receptor and/or a switch
receptor. Thus, in some embodiments, the immune cell has been
genetically modified to express the dominant negative receptor
and/or switch receptor. As used herein, the term "dominant negative
receptor" refers to a molecule designed to reduce the effect of a
negative signal transduction molecule, e.g., the effect of a
negative signal transduction molecule on a modified immune cell of
the present invention. A dominant negative receptor of the present
invention may bind a negative signal transduction molecule, e.g.,
TGF-3 or PD-1, by virtue of an extracellular domain associated with
the negative signal, and reduce the effect of the negative signal
transduction molecule. Such dominant negative receptors are
described herein. For example, a modified immune cell comprising a
dominant negative receptor may bind a negative signal transduction
molecule in the microenvironment of the modified immune cell, and
reduce the effect the negative signal transduction molecule may
have on the modified immune cell.
[0379] A switch receptor of the present invention may be designed
to, in addition to reducing the effects of a negative signal
transduction molecule, to convert the negative signal into a
positive signal, by virtue of comprising an intracellular domain
associated with the positive signal. Switch receptors designed to
convert a negative signal into a positive signal are described
herein. Accordingly, switch receptors comprise an extracellular
domain associated with a negative signal and/or an intracellular
domain associated with a positive signal.
[0380] Tumor cells generate an immunosuppressive microenvironment
that serves to protect them from immune recognition and
elimination. This immunosuppressive microenvironment can limit the
effectiveness of immunosuppressive therapies such as CAR-T cell
therapy. The secreted cytokine Transforming Growth Factor .beta.
(TGF.beta.) directly inhibits the function of cytotoxic T cells and
additionally induces regulatory T cell formation to further
suppress immune responses. T cell immunosuppression due to
TGF.beta. in the context of prostate cancers has been previously
demonstrated (Donkor et al., 2011; Shalapour et al., 2015). To
reduce the immunosuppressive effects of TGF.beta., immune cells can
be modified to express a dominant negative receptor that is a
dominant negative receptor for TGF-.beta..
[0381] In some embodiments, the dominant negative receptor is a
truncated variant of a wild-type protein associated with a negative
signal. In some embodiments, the dominant negative receptor is a
dominant negative receptor for TGF-.beta.. Accordingly, in some
embodiments, the dominant negative receptor for TGF-3 is a
truncated variant of a wild-type TGF-3 receptor. In some
embodiments, the dominant negative receptor is a truncated dominant
negative variant of the TGF-.beta. receptor type II
(TGF.beta.RII-DN). In one embodiment, the TGF.beta.RII-DN comprises
the amino acid sequence set forth below:
TABLE-US-00095 (SEQ ID NO :115)
MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQ
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE
TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI
IFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQK LSSSG.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00096 (SEQ ID NO :116)
ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGT
GGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAA
TAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAA
CTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAAT
CCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGA
AGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAG
ACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAG
ATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGA
GACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATC
ATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCA
TATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCAT
ATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAG
CTGAGTTCATCCGGA.
[0382] Tolerable variations of the sequence of TGF.beta.RII-DN will
be known to those of skill in the art, while maintaining its
intended function. For example, in some embodiments, a dominant
negative receptor of the present invention is TGF.beta.RII-DN
comprising an amino acid sequence that has at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the amino acid sequence set forth in SEQ ID NO: 115 In
one embodiment, the dominant negative receptor is TGF.beta.RII-DN
comprising the amino acid sequence set forth in SEQ ID NO: 115.
[0383] In some embodiments, a dominant negative receptor of the
present invention is TGF.beta.RII-DN encoded by a nucleic acid
sequence that has at least 60%/a, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO: 116. In one embodiment, the
dominant negative receptor is TGF.beta.RII-DN encoded by the
nucleic acid sequence set forth in SEQ ID NO: 116.
[0384] In one embodiment, a switch receptor suitable for use in the
present invention is a PD1-CTM-CD28 receptor. The PD1-CTM-CD28
receptor converts a negative PD1 signal into a positive CD28 signal
when expressed in a cell. The PD1-CTM-CD28 receptor comprises a
variant of the PD1 extracellular domain, a CD28 transmembrane
domain, and a CD28 cytoplasmic domain. In one embodiment, the
PD1-CTM-CD28 receptor comprises an amino acid sequence set forth
below:
TABLE-US-00097 (SEQ ID NO :117)
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN
ATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVT
QLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTER
RAEVPTAHPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTVAFIIFW
VRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00098 (SEQ ID NO :118)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG
GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCC.
[0385] Tolerable variations of the PD1-CTM-CD28 receptor will be
known to those of skill in the art, while maintaining its intended
biological activity (e.g., converting a negative PD1 signal into a
positive CD28 signal when expressed in a cell). Accordingly, a
PD1-CTM-CD28 receptor of the present invention may comprise an
amino acid sequence that has at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
PD1-CTM-CD28 receptor amino acid sequence set forth in SEQ ID NO:
117. Accordingly, a PD1-CTM-CD28 receptor of the present invention
may be encoded by a nucleic acid comprising a nucleic acid sequence
that has at least 60%/a, at least 65%, at least 70%, at least 75%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%/a, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the PD1-CTM-CD28
receptor nucleic acid sequence set forth in SEQ ID NO: 118.
[0386] In one embodiment, a switch receptor suitable for use in the
present invention is a PD1-PTM-CD28 receptor. The PD1-PTM-CD28
receptor converts a negative PD1 signal into a positive CD28 signal
when expressed in a cell. The PD1-PTM-CD28 receptor comprises a
variant of the PD1 extracellular domain, a PD1 transmembrane
domain, and a CD28 cytoplasmic domain. In one embodiment, the
PD1-PTM-CD28 receptor comprises an amino acid sequence set forth
below:
TABLE-US-00099 (SEQ ID NO :119)
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN
ATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVT
QLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTER
RAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVIRSKRS
RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00100 (SEQ ID NO:120)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC CTATCGCTCC.
[0387] Tolerable variations of the PD1-PTM-CD28 receptor will be
known to those of skill in the art, while maintaining its intended
biological activity (e.g., converting a negative PD1 signal into a
positive CD28 signal when expressed in a cell). Accordingly, a
PD1-PTM-CD28 receptor of the present invention may comprise an
amino acid sequence that has at least 60%/a, at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
PD1-PTM-CD28 receptor amino acid sequence set forth in SEQ ID NO:
119. Accordingly, a PD1-PTM-CD28 receptor of the present invention
may be encoded by a nucleic acid comprising a nucleic acid sequence
that has at least 60%/a, at least 65%, at least 70%, at least 75%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%/a, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the PD1-PTM-CD28
receptor nucleic acid sequence set forth in SEQ ID NO: 120.
[0388] In one embodiment, a switch receptor suitable for use in the
present invention is a PD1.sup.A132L-PTM-CD28 receptor. The
PD1.sup.A132L-PTM-CD28 receptor converts a negative PD1 signal into
a positive CD28 signal when expressed in a cell. A point mutation
at amino acid position 132, substituting alanine with leucine
(A132L), of PD1 was found to increase its affinity with PD-L1 by
two fold (see, e.g., Zhang et al., Immunity (2004) 20(3), 337-347).
The PD1.sup.A132L-PTM-CD28 receptor comprises a variant of the PD1
extracellular domain that has an amino acid substitution at
position 132 (A132L), a PD1 transmembrane domain, and a CD28
cytoplasmic domain. In one embodiment, the PD1.sup.A132L-PTM-CD28
receptor comprises an amino acid sequence set forth below:
TABLE-US-00101 (SEQ ID NO:121)
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN
ATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVT
QLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTER
RAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVIRSKRS
RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00102 (SEQ ID NO:122)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC CTATCGC.
[0389] Tolerable variations of the PD1.sup.A132L-PTM-CD28 receptor
will be known to those of skill in the art, while maintaining its
intended biological activity (e.g., converting a negative PD1
signal into a positive CD28 signal when expressed in a cell).
Accordingly, a PD1.sup.A132L-PTM-CD28 receptor of the present
invention may comprise an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the PD1.sup.A132L-PTM-CD28 receptor amino acid
sequence set forth in SEQ ID NO: 121. Accordingly, a
PD1.sup.A132L-PTM-CD28 receptor of the present invention may be
encoded by a nucleic acid comprising a nucleic acid sequence that
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the PD1.sup.A132L-PTM-CD28
receptor nucleic acid sequence set forth in SEQ ID NO: 122. In one
embodiment, a switch receptor suitable for use in the present
invention is a PD1-4-1BB receptor. The PD1-4-1BB receptor (also
referred to herein as PD1-BB) converts a negative PD1 signal into a
positive 4-1BB signal when expressed in a cell. In one embodiment,
the PD1-4-1BB receptor comprises an amino acid sequence set forth
below:
TABLE-US-00103 (SEQ ID NO:213)
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN
ATFTCSFSNTSESFVLNWYRIVISPSNQTDKLAAFPEDRSQPGQDCRFR
VTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVT
ERRAEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVITLYC
KKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00104 (SEQ ID NO:214)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGAC
TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAA
CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC
CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA
AGAAGAAGAAGGAGGATGTGAACTG.
[0390] Tolerable variations of the PD1-4-1BB receptor will be known
to those of skill in the art, while maintaining its intended
biological activity (e.g., converting a negative PD1 signal into a
positive 4-1BB signal when expressed in a cell). Accordingly, a
PD1-4-1BB receptor of the present invention may comprise an amino
acid sequence that has at least 60%/a, at least 65%, at least
70%/a, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
PD1-4-1BB receptor amino acid sequence set forth in SEQ ID NO:213.
Accordingly, a PD1-4-1BB receptor of the present invention may be
encoded by a nucleic acid comprising a nucleic acid sequence that
has at least 60%/a, at least 65%, at least 70%/a, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%/a, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the PD1-4-1BB receptor nucleic
acid sequence set forth in SEQ ID NO:214.
[0391] In one embodiment, a switch receptor suitable for use in the
present invention is a PD1.sup.A132L-4-1BB receptor. The
PD1.sup.A132L-4-1BB receptor (also referred to herein as PD1*BB)
converts a negative PD1 signal into a positive 4-1BB signal when
expressed in a cell. In one embodiment, the PD1.sup.A132L-4-1BB
receptor comprises an amino acid sequence set forth below:
TABLE-US-00105 (SEQ ID NO:215)
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN
ATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVT
QLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTER
RAEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVITLYCKK
RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00106 (SEQ ID NO:216)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGAC
TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAA
CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC
CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA
AGAAGAAGAAGGAGGATGTGAACTG.
[0392] Tolerable variations of the PD1.sup.A132L-4-1BB receptor
will be known to those of skill in the art, while maintaining its
intended biological activity (e.g., converting a negative PD1
signal into a positive 4-1BB signal when expressed in a cell).
Accordingly, a PD1.sup.A132L-4-1BB receptor of the present
invention may comprise an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the PD1.sup.A132L-4-1BB receptor amino acid
sequence set forth in SEQ ID NO:215. Accordingly, a
PD1.sup.A132L-4-1BB receptor of the present invention may be
encoded by a nucleic acid comprising a nucleic acid sequence that
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the PD1.sup.A132L-4-1BB receptor
nucleic acid sequence set forth in SEQ ID NO:216.
[0393] In one embodiment, a switch receptor suitable for use in the
present invention is a TGF.beta.R-IL12R.beta.1 receptor. The
TGF.beta.R-IL12R.beta.1 receptor converts a negative TGF-3 signal
into a positive IL-12 signal when expressed in a cell. In one
embodiment, the TGF.beta.R-IL12R.beta.1 receptor comprises an amino
acid sequence set forth below:
TABLE-US-00107 (SEQ ID NO:123)
MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCV
TDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTT
YCCNQDHCNKIELPTTVKSSPGLGPVELAAVIAGPVCFVCISLMLMVYI
RAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVE
MSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00108 (SEQ ID NO:124)
ATGGAGGCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCCTCGTGC
TGGCGGCGGCGGCGGCGGCGGCGGCGGCGCTGCTCCCGGGGGCGACGGC
GTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTG
ACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTA
TACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAG
GCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACA
TATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTG
TAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGC
TGGACCAGTGTGCTTCGTCTGCATCTCACTCATGTTGATGGTCTATATC
AGGGCCGCACGGCACCTGTGCCCGCCGCTGCCCACACCCTGTGCCAGCT
CCGCCATTGAGTTCCCTGGAGGGAAGGAGACTTGGCAGTGGATCAACCC
AGTGGACTTCCAGGAAGAGGCATCCCTGCAGGAGGCCCTGGTGGTAGAG
ATGTCCTGGGACAAAGGCGAGAGGACTGAGCCTCTCGAGAAGACAGAGC
TACCTGAGGGTGCCCCTGAGCTGGCCCTGGATACAGAGTTGTCCTTGGA
GGATGGAGACAGGTGCAAGGCCAAGATG.
[0394] Tolerable variations of the TGF.beta.R-IL12R.beta.1 receptor
will be known to those of skill in the art, while maintaining its
intended biological activity (e.g., converting a negative
TGF-.beta. signal into a positive IL-12 signal when expressed in a
cell). Accordingly, a TGF.beta.R-IL12R.beta.1 receptor of the
present invention may comprise an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the TGF.beta.R-IL12R.beta.1 receptor amino
acid sequence set forth in SEQ ID NO:123. Accordingly, a
TGF.beta.R-IL12R.beta.1 receptor of the present invention may be
encoded by a nucleic acid comprising a nucleic acid sequence that
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the TGF.beta.R-IL12R.beta.1
receptor nucleic acid sequence set forth in SEQ ID NO: 124.
[0395] In one embodiment, a switch receptor suitable for use in the
present invention is a TGF.beta.R-IL12R.beta.2 receptor. The
TGF.beta.R-IL12R.beta.2 receptor converts a negative TGF-3 signal
into a positive IL-12 signal when expressed in a cell. In one
embodiment, the TGF.beta.R-IL12R32 receptor comprises an amino acid
sequence set forth below:
TABLE-US-00109 (SEQ ID NO :125)
MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQ
LCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE
TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI
IFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYQQKVFVLLA
ALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEP
LVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPP
RALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLP
SNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLD QLKMRCDSLML.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00110 (SEQ ID NO:126)
ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGT
GGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAA
TAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAA
CTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAAT
CCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGA
AGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAG
ACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAG
ATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGA
GACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATC
ATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCA
TATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCAT
ATCTGTCATCATCATCTTCTACCAGCAAAAGGTGTTTGTTCTCCTAGCA
GCCCTCAGACCTCAGTGGTGTAGCAGAGAAATTCCAGATCCAGCAAATA
GCACTTGCGCTAAGAAATATCCCATTGCAGAGGAGAAGACACAGCTGCC
CTTGGACAGGCTCCTGATAGACTGGCCCACGCCTGAAGATCCTGAACCG
CTGGTCATCAGTGAAGTCCTTCATCAAGTGACCCCAGTTTTCAGACATC
CCCCCTGCTCCAACTGGCCACAAAGGGAAAAAGGAATCCAAGGTCATCA
GGCCTCTGAGAAAGACATGATGCACAGTGCCTCAAGCCCACCACCTCCA
AGAGCTCTCCAAGCTGAGAGCAGACAACTGGTGGATCTGTACAAGGTGC
TGGAGAGCAGGGGCTCCGACCCAAAGCCAGAAAACCCAGCCTGTCCCTG
GACGGTGCTCCCAGCAGGTGACCTTCCCACCCATGATGGCTACTTACCC
TCCAACATAGATGACCTCCCCTCACATGAGGCACCTCTCGCTGACTCTC
TGGAAGAACTGGAGCCTCAGCACATCTCCCTTTCTGTTTTCCCCTCAAG
TTCTCTTCACCCACTCACCTTCTCCTGTGGTGATAAGCTGACTCTGGAT
CAGTTAAAGATGAGGTGTGACTCCCTCATGCTC.
[0396] Tolerable variations of the TGF.beta.R-IL12R.beta.2 receptor
will be known to those of skill in the art, while maintaining its
intended biological activity (e.g., converting a negative
TGF-.beta. signal into a positive IL-12 signal when expressed in a
cell). Accordingly, a TGF.beta.R-IL12R32 receptor of the present
invention may comprise an amino acid sequence that has at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the TGF.beta.R-IL12R32 receptor amino acid
sequence set forth in SEQ ID NO:125. Accordingly, a
TGF.beta.R-IL12R32 receptor of the present invention may be encoded
by a nucleic acid comprising a nucleic acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the TGF.beta.R-IL12R.beta.2 receptor
nucleic acid sequence set forth in SEQ ID NO:126.
[0397] In one embodiment, a switch receptor suitable for use in the
present invention is a TIM3-CD28 receptor. The TIM3-CD28 receptor
converts a negative TIM-3 signal into a positive CD28 signal when
expressed in a cell. In one embodiment, the TIM3-CD28 receptor
comprises an amino acid sequence set forth below:
TABLE-US-00111 (SEQ ID NO:127)
MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLV
PVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIE
NVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAA
FPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSG
ATIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR
RPGPTRKHYQPYAPPRDFAAYRS.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00112 (SEQ ID NO:128)
ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTAC
TACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAA
TGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTG
CCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACG
TGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATA
CTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAG
AATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCC
CAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACC
AGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCC
TTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGA
CACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGC
CAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGA
GCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT
GCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG
TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG
ACTTCGCAGCCTATCGCTCC.
[0398] Tolerable variations of the TIM3-CD28 receptor will be known
to those of skill in the art, while maintaining its intended
biological activity (e.g., converting a negative TIM-3 signal into
a positive CD28 signal when expressed in a cell). Accordingly, a
TIM3-CD28 receptor of the present invention may comprise an amino
acid sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the TIM3-CD28
receptor amino acid sequence set forth in SEQ ID NO: 127.
Accordingly, a TIM3-CD28 receptor of the present invention may be
encoded by a nucleic acid comprising a nucleic acid sequence that
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the TIM3-CD28 receptor nucleic
acid sequence set forth in SEQ ID NO:128.
[0399] Other suitable dominant negative receptors and switch
receptors for use in the present invention are described in PCT
Publication No. WO2013019615A2, the disclosure of which is
incorporated herein by reference.
D. Bispecific Antibodies
[0400] The present invention provides compositions and methods for
modified immune cells or precursors thereof, e.g., modified T
cells, comprising a nucleic acid encoding a bispecific antibody.
Thus, in some embodiments, the immune cell has been genetically
modified to express the bispecific antibody. A "bispecific
antibody," as used herein, refers to an antibody having binding
specificities for at least two different antigenic epitopes. In one
embodiment, the epitopes are from the same antigen. In another
embodiment, the epitopes are from two different antigens. Methods
for making bispecific antibodies are known in the art. For example,
bispecific antibodies can be produced recombinantly using the
co-expression of two immunoglobulin heavy chain/light chain pairs.
See, e.g., Milstein et al. (1983) Nature 305: 537-39.
Alternatively, bispecific antibodies can be prepared using chemical
linkage. See, e.g., Brennan et al. (1985) Science 229:81.
Bispecific antibodies include bispecific antibody fragments. See,
e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A.
90:6444-48, Gruber et al. (1994) J. Immunol. 152:5368.
[0401] In certain embodiments, the modified cell of the present
invention comprises a CAR having affinity for a prostate specific
membrane antigen (PSMA) on a target cell and a bispecific antibody.
In certain embodiments, the modified cell of the present invention
secretes a bispecific antibody.
[0402] In one embodiment, the bispecific antibody comprises a first
antigen binding domain that binds to a first antigen and a second
antigen binding domain that binds to a second antigen. In some
embodiments, the bispecific antibody comprises an antigen binding
domain comprising a first and a second single chain variable
fragment (scFv) molecules. In one embodiment, the first and a
second antigen binding domains bind an antigen on a target cell and
an antigen on an activating T cell.
[0403] In one embodiment, the bispecific antibody comprises
specificity to at least one antigen on an activating T cell. The
activating T cell antigen includes antigens found on the surface of
a T cell that can activate another cell. The activating T cell
antigen may bind a co-stimulatory molecule. A costimulatory
molecule is a cell surface molecule, other than an antigen receptor
or their ligands, that is required for an efficient response of
lymphocytes to an antigen. Examples of the activating T cell
antigen can include but are not limited to CD3, CD4, CD8, T cell
receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1,
ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,
LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or
any fragment thereof. In some embodiments, the bispecific antibody
comprises specificity to the T cell antigen CD28.
[0404] Other costimulatory elements are also within the scope of
the invention. In these examples, the bispecific antibody
recognizes a T cell antigen and may be referred to as a Bispecific
T Cell Engager (BiTE). However, the present invention is not
limited by the use of any particular bispecific antibody. Rather,
any bispecific antibody or BiTE can be used. The bispecific
antibody or BiTE molecule may also be expressed as a soluble
protein with specificity for at least one target cell associated
antigen.
[0405] In one embodiment, the bispecific antibody comprises more
than one antigen binding domain. In this embodiment, at least one
antigen binding domain includes a synthetic antibody, human
antibody, a humanized antibody, single chain variable fragment,
single domain antibody, an antigen binding fragment thereof, and
any combination thereof. Techniques for making human and humanized
antibodies are described elsewhere herein.
[0406] In some embodiments, the bispecific antibody comprises more
than one antigen binding domain, wherein at least one antigen
binding domain binds to a negative signal transduction molecule
(e.g., a negative signal transduction molecule that may be found in
the microenvironment of the cell secreting the bispecific antibody)
or an interacting partner thereof (e.g., receptor). In some
embodiments, at least one antigen binding domain of the bispecific
antibody binds to TGF-.beta. or an interacting partner thereof
(e.g., receptor). In some embodiments, at least one antigen binding
domain of the bispecific antibody binds to PD-1 or an interacting
partner thereof. In one embodiment, at least one antigen binding
domain of the bispecific antibody binds to TGF-.beta.R. In another
embodiment, at least one antigen binding domain of the bispecific
antibody binds to PD-L1.
[0407] In some embodiments, the bispecific antibody comprises at
least one antigen binding domain that binds to a molecule on a T
cell and activates the T cell. For example, a bispecific antibody
of the present disclosure may comprise a superagonistic anti-CD28
binding domain as described in U.S. Pat. No. 7,585,960, contents of
which are incorporated herein in its entirety.
[0408] In some embodiments, the bispecific antibody comprises at
least one antigen binding domain that binds PD-L1. For example, a
bispecific antibody of the present disclosure may comprise, without
limitation, a PD-L1 binding domain derived from 10A5, 13G4, or 1B12
as described in PCT Publication No. WO2007005874A2, contents of
which are incorporated herein in its entirety. In some embodiments,
the bispecific antibody comprises at least one antigen binding
domain that binds a TGF-.beta. receptor, e.g., TGF.beta.RII. For
example, a bispecific antibody of the present disclosure may
comprise, without limitation, a TGF.beta.RII binding domain derived
from TGF1 or TGF.beta. as described in U.S. Pat. No. 8,147,834,
contents of which are incorporated herein in its entirety.
[0409] Accordingly, in one embodiment, a bispecific antibody of the
present disclosure comprises at least one antigen binding domain
that binds PD-L1 or TGF.beta.RII, and an antigen binding domain
that binds CD28.
[0410] In some embodiments, the target cell antigen may be the same
antigen that a T cell receptor binds to or may be a different
antigen. The target cell antigen includes any tumor associated
antigen (TAA) or viral, bacterial and parasitic antigen, or any
fragment thereof. The target cell antigen may include any type of
ligand that defines the target cell. For example, the target cell
antigen may be chosen to recognize a ligand that acts as a cell
marker on target cells associated with a particular disease state.
Thus, cell markers may act as ligands for the antigen binding
domain in the bispecific antibody, including those associated with
viral, bacterial and parasitic infections, autoimmune disease and
cancer cells.
[0411] In some embodiments, the target cell antigen is the same
antigen as the activating T cell antigen including, but not limited
to, CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB
(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte
function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C,
B7-H3, a ligand that specifically binds with CD83, and fragments
thereof. In one aspect, the invention includes a nucleic acid
encoding a bispecific antibody comprising bispecificity for an
antigen on a target cell and an antigen on an activating T cell,
wherein the T cell transiently secretes the bispecific antibody.
Techniques for engineering and expressing bispecific antibodies
include, but are not limited to, recombinant co-expression of two
immunoglobulin heavy chain-light chain pairs having different
specificities (see, e.g., Milstein and Cuello, Nature 305: 537
(1983), WO 93/08829, and Traunecker et al, EMBO J. 10: 3655
(1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No.
5,731,168). Multi-specific antibodies may also be made by
engineering electrostatic steering effects for making antibody
Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or
more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980,
and Brennan et al, Science 229:81 (1985)); using leucine zippers to
produce bispecific antibodies (see, e.g., Kostelny et al, J.
Immunol. 148(5): 1547-1553 (1992)); using "diabody" technology for
making bispecific antibody fragments (see, e.g., Hollinger et al,
Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using
single-chain Fv (scFv) dimers (see, e.g. Gruber et al, J. Immunol,
152:5368 (1994)); and preparing trispecific antibodies as
described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).
Engineered antibodies with three or more functional antigen binding
sites, including "Octopus antibodies," are also included herein
(see, e.g. US 2006/0025576A1). Bispecific antibodies can be
constructed by linking two different antibodies, or portions
thereof. For example, a bispecific antibody can comprise Fab,
F(ab')2, Fab', scFv, and sdAb from two different antibodies.
[0412] A bispecific antibody of the present invention includes a
bispecific antibody having affinity for PD-L1 and CD28. In one
embodiment, a 13G4-1211 PD-L1/CD28 bispecific antibody of the
present invention comprises an amino acid sequence set forth
below:
TABLE-US-00113 (SEQ ID NO:129)
MGWSCIILFLVATATGVHSAIQLTQSPSSLSASVGDRVTITCRASQGIS
SALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQFNSYPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPG
RSLRLSCAASGITFDDYGMHWVRQAPGKGLEWVSGISWNRGRIEYADSV
KGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGRFRYFDWFLDYWGQ
GTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHW
VRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSR
LRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGS
GGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAP
KLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQT
YPYTFGGGTKVEI.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00114 (SEQ ID NO:130)
ATGGGGTGGTCGTGTATCATCCTGTTCCTGGTCGCGACAGCAACCGGCG
TGCATTCGGCCATACAGCTGACCCAGAGCCCCTCCTCCCTCTCCGCTTC
CGTGGGGGACCGCGTGACAATCACGTGCCGCGCCAGCCAGGGAATCTCC
TCGGCCCTCGCCTGGTACCAGCAGAAACCCGGGAAGGCTCCCAAGCTGC
TCATCTACGATGCCTCCTCGCTTGAGTCGGGCGTGCCATCCAGGTTCTC
CGGATCCGGGTCCGGAACCGACTTTACACTCACGATTTCCTCTCTGCAG
CCCGAGGACTTCGCCACATACTACTGTCAGCAGTTCAACTCCTACCCAT
TCACCTTCGGCCCGGGCACCAAGGTGGACATCAAGTCTGGCGGGGGAGG
CTCCGAAGTCCAGCTCGTGGAATCCGGGGGCGGTCTCGTGCAGCCAGGC
CGGAGTCTGCGCCTGTCTTGCGCTGCCTCGGGGATCACTTTCGACGACT
ACGGCATGCATTGGGTTCGCCAGGCCCCAGGGAAGGGGTTGGAGTGGGT
CAGTGGCATTTCATGGAACAGGGGGCGCATCGAATACGCCGACTCCGTT
AAGGGCAGATTCACCATCTCGCGCGATAACGCCAAAAACAGTCTCTACC
TCCAGATGAACTCGCTTCGAGCAGAGGATACTGCCCTGTACTATTGCGC
GAAGGGACGCTTCCGCTACTTTGACTGGTTTCTGGACTACTGGGGCCAG
GGGACACTGGTGACGGTGTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGC
TGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGT
GTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGG
GTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACC
CAGGGAACGTCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAAC
CCTGACCGTGGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGC
CTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACG
GCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGT
GTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCA
GGCGGCGTGATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCT
CCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAA
TATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCC
AAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCC
GCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTC
GCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACC
TACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGAT.
[0413] Tolerable variations of the 13G4-1211 PD-L1/CD28 bispecific
antibody will be known to those of skill in the art, while
maintaining its intended biological activity (e.g., binding to
PD-L1 and CD28). Accordingly, a 13G4-1211 PD-L1/CD28 bispecific
antibody of the present invention may comprise an amino acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the 13G4-1211
PD-L1/CD28 bispecific antibody amino acid sequence set forth in SEQ
ID NO:129. Accordingly, a 13G4-1211 PD-L1/CD28 bispecific antibody
of the present invention may be encoded by a nucleic acid
comprising a nucleic acid sequence that has at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the 13G4-1211 PD-L1/CD28 bispecific antibody nucleic
acid sequence set forth in SEQ ID NO: 130.
[0414] A bispecific antibody of the present invention includes a
bispecific antibody having affinity for PD-L1 and CD28. In one
embodiment, a 10A5-1412 PD-L1/CD28 bispecific antibody of the
present invention comprises an amino acid sequence set forth
below:
TABLE-US-00115 (SEQ ID NO:131)
MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQGIS
SWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQYNSYPYTFGQGTKLEIKSGGGGSQVQLVQSGAEVKKPG
ASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKF
QGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTL
VTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYTHWVRQ
APGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRS
DDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGV
MDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLL
IYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPY TFGGGTKVEI.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00116 (SEQ ID NO:132)
ATGGGCTGGAGTTGCATCATTCTCTTCCTCGTGGCGACCGCAACAGGGG
TGCACTCCGACATCCAGATGACCCAGTCCCCGAGTTCCCTGTCTGCTTC
CGTGGGAGATCGCGTGACTATCACCTGCCGGGCTTCCCAGGGCATCTCT
TCCTGGCTGGCGTGGTACCAGCAGAAACCAGAAAAGGCTCCTAAGTCCC
TGATCTACGCAGCTTCGTCCCTCCAATCCGGCGTCCCCTCTCGCTTCTC
CGGCTCCGGATCCGGCACCGACTTCACGCTGACAATCTCGAGTTTGCAG
CCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACTCCTACCCTT
ACACCTTCGGCCAGGGCACAAAGCTCGAAATCAAGTCGGGGGGGGGCGG
GTCGCAGGTCCAGCTGGTGCAGTCCGGCGCCGAAGTCAAGAAGCCCGGA
GCAAGTGTGAAAGTGTCGTGCAAGGCAAGTGGGTATACCTTCACCTCAT
ACGACGTACACTGGGTGCGCCAGGCGCCCGGTCAGCGCCTTGAGTGGAT
GGGCTGGCTCCACGCCGACACCGGCATTACCAAGTTCTCTCAGAAGTTC
CAGGGAAGAGTGACCATAACACGCGACACCAGTGCTTCCACAGCTTACA
TGGAACTTTCGAGTCTGAGATCCGAGGACACAGCCGTGTATTACTGTGC
CCGTGAGCGCATCCAGCTGTGGTTCGACTACTGGGGGCAGGGCACCCTC
GTGACGGTGTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGCTGGTGCAGT
CCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGTGTCATGCAA
GGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGGGTGCGCCAA
GCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACCCAGGGAACG
TCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAACCCTGACCGT
GGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGCCTGCGCTCT
GATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACGGCCTGGACT
GGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGTGTCCAGTGT
GGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCAGGCGGCGTG
ATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCTCCGCTTCCG
TCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAATATCTACGT
GTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCCAAGCTGCTT
ATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCCGCTTCTCCG
GATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTCGCTGCAGCC
AGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACCTACCCATAT
ACTTTCGGCGGCGGGACCAAGGTGGAGAT.
[0415] Tolerable variations of the 10A5-1412 PD-L1/CD28 bispecific
antibody will be known to those of skill in the art, while
maintaining its intended biological activity (e.g., binding to
PD-L1 and CD28). Accordingly, a 10A5-1412 PD-L1/CD28 bispecific
antibody of the present invention may comprise an amino acid
sequence that has at least 60%/a, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the 10A5-1412
PD-L1/CD28 bispecific antibody amino acid sequence set forth in SEQ
ID NO:131. Accordingly, a 10A5-1412 PD-L1/CD28 bispecific antibody
of the present invention may be encoded by a nucleic acid
comprising a nucleic acid sequence that has at least 60%/a, at
least 65%, at least 70%, at least 75%, at least 80%/a, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the 10A5-1412 PD-L1/CD28 bispecific antibody
nucleic acid sequence set forth in SEQ ID NO: 132.
[0416] A bispecific antibody of the present invention includes a
bispecific antibody having affinity for PD-L1 and CD28. In one
embodiment, a 1B12-1412 PD-L1/CD28 bispecific antibody of the
present invention comprises an amino acid sequence set forth
below:
TABLE-US-00117 (SEQ ID NO:133)
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVS
SYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLE
PEDFAVYYCQQRSNWPTFGQGTKVEIKSGGGGSQVQLVQSGAEVKKPGS
SVKVSCKTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAHYAQKFQ
GRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWG
QGTVTVSSGGSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSY
YTHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYM
ELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGG
SGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKP
GKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ
QGQTYPYTFGGGTKVEI.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00118 (SEQ ID NO:134)
ATGGGCTGGAGTTGCATCATCCTCTTTCTAGTCGCCACGGCCACCGGCG
TACACTCAGAGATCGTGCTGACACAGTCGCCTGCGACGCTGTCGCTCAG
TCCAGGGGAGCGCGCTACTCTCTCCTGCCGCGCGTCGCAGAGCGTGTCG
TCCTACTTGGCCTGGTACCAGCAGAAGCCTGGCCAGGCTCCGCGCCTGC
TGATATACGACGCCTCGAACAGAGCCACGGGCATCCCCGCCCGTTTTAG
TGGCTCCGGGTCGGGGACCGACTTCACTCTGACAATCTCATCCCTCGAG
CCCGAGGATTTCGCCGTGTACTACTGTCAGCAGCGCTCGAATTGGCCAA
CCTTCGGGCAGGGGACGAAAGTTGAGATCAAAAGCGGCGGCGGGGGCAG
CCAGGTCCAGCTCGTCCAGTCTGGCGCCGAGGTCAAAAAGCCGGGCTCT
TCGGTCAAGGTCTCCTGCAAGACTTCCGGCGACACCTTCTCCTCCTATG
CTATCTCCTGGGTGCGGCAGGCCCCGGGGCAGGGCCTGGAGTGGATGGG
AGGCATCATCCCAATCTTTGGGAGGGCCCACTACGCCCAGAAGTTCCAG
GGACGCGTGACAATCACCGCAGACGAGTCCACATCCACTGCCTACATGG
AGTTGTCCTCGCTCCGGTCGGAGGATACTGCCGTGTACTTCTGCGCCCG
GAAGTTCCACTTCGTGTCAGGCTCCCCCTTCGGGATGGACGTGTGGGGA
CAAGGAACCGTGACGGTGTCGTCGGGGGGCTCGTCGGGGGGCGGGGGGA
GTCAGGTGCAGCTGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGC
TTCCGTCAAGGTGTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTAT
TACATCCACTGGGTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCG
GGTGCATTTACCCAGGGAACGTCAACACAAACTACAACGAGAAGTTCAA
GGATCGGGCAACCCTGACCGTGGACACATCCATCTCTACCGCCTACATG
GAGCTGTCACGCCTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCA
GGAGTCACTACGGCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAAC
CACCGTGACGGTGTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGG
TCCGGAGGCTCAGGCGGCGTGATGGATGACATTCAGATGACCCAGAGTC
CCTCCTCCCTCTCCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCA
CGCCTCACAGAATATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCC
GGCAAGGCCCCCAAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGG
GAGTCCCTTCCCGCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACT
CACAATCTCGTCGCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAG
CAGGGCCAGACCTACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGA T.
[0417] Tolerable variations of the 1B12-1412 PD-L1/CD28 bispecific
antibody will be known to those of skill in the art, while
maintaining its intended biological activity (e.g., binding to
PD-L1 and CD28). Accordingly, a 1B12-1412 PD-L1/CD28 bispecific
antibody of the present invention may comprise an amino acid
sequence that has at least 60%/a, at least 65%, at least 70%/a, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the 1B12-1412
PD-L1/CD28 bispecific antibody amino acid sequence set forth in SEQ
ID NO:133. Accordingly, a 1B12-1412 PD-L1/CD28 bispecific antibody
of the present invention may be encoded by a nucleic acid
comprising a nucleic acid sequence that has at least 60%/a, at
least 65%, at least 70%, at least 75%, at least 80%/a, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the 1B12-1412 PD-L1/CD28 bispecific antibody
nucleic acid sequence set forth in SEQ ID NO: 134.
[0418] A bispecific antibody of the present invention includes a
bispecific antibody having affinity for TGF-.beta. receptor type II
(TGF.beta.RII) and CD28. In one embodiment, a TGF.beta.R-1-1412
TGF.beta.RII/CD28 bispecific antibody of the present invention
comprises an amino acid sequence set forth below:
TABLE-US-00119 (SEQ ID NO:135)
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVR
SYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLE
PEDFAVYYCQQRSNWPPTFGQGTKVEIKSGGGGSQLQVQESGPGLVKPS
ETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPS
LKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWGQ
GTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYTHW
VRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSR
LRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGS
GGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAP
KLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQT
YPYTFGGGTKVEIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00120 (SEQ ID NO:136)
ATGGGTTGGTCCTGCATCATCCTGTTTCTCGTGGCCACCGCCACCGGCG
TGCACTCCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTC
TCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTCGC
AGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC
TCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAG
TGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAG
CCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTC
CGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAAGTGGAGGGGGCGG
TTCACAGCTGCAGGTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCG
GAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAACA
GTTATTTCTCCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGACTGGA
GTGGATTGGGAGTTTCTATTATGGTGAAAAAACCTACTACAACCCGTCC
CTCAAGAGCCGAGCCACCATATCCATTGACACGTCCAAGAGCCAGTTCT
CCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTG
TCCGAGAGGGCCTACTATGATTCGGGGAGTTATAGACTCCTGGGGCCAG
GGAACCCTGGTGACGGTGTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGC
TGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGT
GTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGG
GTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACC
CAGGGAACGTCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAAC
CCTGACCGTGGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGC
CTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACG
GCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGT
GTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCA
GGCGGCGTGATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCT
CCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAA
TATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCC
AAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCC
GCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTC
GCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACC
TACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGATTAAG.
[0419] Tolerable variations of the TGF.beta.R-1-1412
TGF.beta.RII/CD28 bispecific antibody will be known to those of
skill in the art, while maintaining its intended biological
activity (e.g., binding to TGF.beta.RII and CD28). Accordingly, a
TGF.beta.R-1-1412 TGF.beta.RII/CD28 bispecific antibody of the
present invention may comprise an amino acid sequence that has at
least 60%/a, at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% sequence identity to the TGF.beta.R-1-1412
TGF.beta.RII/CD28 bispecific antibody amino acid sequence set forth
in SEQ ID NO:135. Accordingly, a TGF.beta.R-1-1412
TGF.beta.RII/CD28 bispecific antibody of the present invention may
be encoded by a nucleic acid comprising a nucleic acid sequence
that has at least 60%/a, at least 65%, at least 70%/a, at least
75%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the TGF.beta.R-1-1412
TGF.beta.RII/CD28 bispecific antibody nucleic acid sequence set
forth in SEQ ID NO:136.
[0420] A bispecific antibody of the present invention includes a
bispecific antibody having affinity for TGF-.beta. receptor type II
(TGF.beta.RII) and CD28. In one embodiment, a TGF.beta.R-3-1412
TGF.beta.RII/CD28 bispecific antibody of the present invention
comprises an amino acid sequence set forth below:
TABLE-US-00121 (SEQ ID NO:137)
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVR
SFLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLE
PEDFAVYYCQQRSNWPPTFGQGTKVEIKSGGGGSQLQLQESGPGLVKPS
ETLSLTCTVSGGSISSSYSWGWIRQPPGKGLEWIGSFYYSGITYYSPSL
KSRIIISEDTSKNQFSLKLSSVTAADTAVYYCASGFTMIRGALDYWGQG
TLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYTHWV
RQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRL
RSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSG
GVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK
LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTY
PYTFGGGTKVEIK.
which may be encoded by the nucleic acid sequence set forth
below:
TABLE-US-00122 (SEQ ID NO:138)
ATGGGTTGGTCCTGCATCATCCTGTTTCTCGTGGCCACCGCCACCGGCG
TGCACTCCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTC
TCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGA
AGTTTCTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC
TCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAG
TGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAG
CCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTC
CGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAAGTGGAGGGGGCGG
TTCACAGCTACAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCG
GAGACCCTATCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTA
GTAGTTACTCCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGCCTGGA
GTGGATTGGGAGTTTCTATTACAGTGGGATCACCTACTACAGCCCGTCC
CTCAAGAGTCGAATTATCATATCCGAAGACACGTCCAAGAACCAGTTCT
CCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCTGTGTATTACTG
TGCGAGCGGGTTTACTATGATTCGGGGAGCCCTTGACTACTGGGGCCAG
GGAACCCTGGTGACGGTGTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGC
TGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGT
GTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGG
GTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACC
CAGGGAACGTCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAAC
CCTGACCGTGGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGC
CTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACG
GCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGT
GTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCA
GGCGGCGTGATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCT
CCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAA
TATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCC
AAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCC
GCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTC
GCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACC
TACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGATTAAG.
[0421] Tolerable variations of the TGF.beta.R-3-1412
TGF.beta.RII/CD28 bispecific antibody will be known to those of
skill in the art, while maintaining its intended biological
activity (e.g., binding to TGF.beta.RII and CD28). Accordingly, a
TGF.beta.R-3-1412 TGF.beta.RII/CD28 bispecific antibody of the
present invention may comprise an amino acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the TGF.beta.R-3-1412 TGF.beta.RII/CD28
bispecific antibody amino acid sequence set forth in SEQ ID NO:137.
Accordingly, a TGF.beta.R-3-1412 TGF.beta.RII/CD28 bispecific
antibody of the present invention may be encoded by a nucleic acid
comprising a nucleic acid sequence that has at least 60%, at least
65%, at least 70%/a, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the TGF.beta.R-3-1412 TGF.beta.RII/CD28 bispecific
antibody nucleic acid sequence set forth in SEQ ID NO:138.
[0422] Other suitable bispecific antibodies for use in the present
invention are described in PCT Publication No. WO2016122738A1, the
disclosure of which is incorporated herein by reference.
E. Nucleic Acids and Expression Vectors
[0423] The present invention provides a nucleic acid encoding a CAR
and/or a dominant negative receptor and/or a switch receptor. In
one embodiment, a nucleic acid of the present disclosure comprises
a nucleic acid sequence encoding a subject CAR of the present
invention (e.g., PSMA-CAR). In one embodiment, a nucleic acid of
the present disclosure comprises a nucleic acid sequence encoding a
dominant negative receptor and/or a switch receptor (e.g., a
PD1-PTM-CD28 receptor).
[0424] In some embodiments, a nucleic acid of the present
disclosure provides for the production of a CAR and/or dominant
negative receptor and/or a switch receptor as described herein,
e.g., in a mammalian cell. In some embodiments, a nucleic acid of
the present disclosure provides for amplification of the CAR and/or
dominant negative receptor and/or a switch receptor-encoding
nucleic acid.
[0425] As described herein, a subject CAR comprises an antigen
binding domain, a transmembrane domain, and an intracellular
domain. Accordingly, the present disclosure provides a nucleic acid
encoding an antigen binding domain, a transmembrane domain, and an
intracellular domain of a subject CAR. As described herein, various
dominant negative receptors and switch receptors are provided.
Accordingly, the present invention provides a nucleic acid encoding
a dominant negative receptor and/or a switch receptor.
[0426] In some embodiments, the nucleic acid encoding a CAR is
separate from the nucleic acid encoding a dominant negative
receptor and/or a switch receptor. In an exemplary embodiment, the
nucleic acid encoding a CAR, and the nucleic acid encoding a
dominant negative receptor and/or a switch receptor, resides within
the same nucleic acid.
[0427] In some embodiments, a nucleic acid of the present invention
comprises a nucleic acid comprising a CAR coding sequence and a
dominant negative receptor and/or a switch receptor coding
sequence. In some embodiments, a nucleic acid of the present
invention comprises a nucleic acid comprising a CAR coding sequence
and a dominant negative receptor and/or a switch receptor coding
sequence that is separated by a linker. A linker for use in the
present invention (e.g., in the context of linking a CAR coding
sequence and a dominant negative receptor and/or a switch receptor
coding sequence) allows for multiple proteins to be encoded by the
same nucleic acid sequence (e.g., a multicistronic or bicistronic
sequence), which are translated as a polyprotein that is
dissociated into separate protein components. For example, a linker
for use in a nucleic acid of the present disclosure comprising a
CAR coding sequence and a dominant negative receptor and/or a
switch receptor coding sequence, allows for the CAR and dominant
negative receptor and/or switch receptor to be translated as a
polyprotein that is dissociated into separate CAR and dominant
negative receptor and/or switch receptor components.
[0428] In some embodiments, the linker comprises a nucleic acid
sequence that encodes for an internal ribosome entry site (IRES).
As used herein, "an internal ribosome entry site" or "IRES" refers
to an element that promotes direct internal ribosome entry to the
initiation codon, such as ATG, of a protein coding region, thereby
leading to cap-independent translation of the gene. Various
internal ribosome entry sites are known to those of skill in the
art, including, without limitation, IRES obtainable from viral or
cellular mRNA sources, e.g., immunogloublin heavy-chain binding
protein (BiP); vascular endothelial growth factor (VEGF);
fibroblast growth factor 2; insulin-like growth factor;
translational initiation factor eIF4G; yeast transcription factors
TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus,
rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV),
and Moloney murine leukemia virus (MoMLV). Those of skill in the
art would be able to select the appropriate IRES for use in the
present invention.
[0429] In some embodiments, the linker comprises a nucleic acid
sequence that encodes for a self-cleaving peptide. As used herein,
a "self-cleaving peptide" or "2A peptide" refers to an oligopeptide
that allow multiple proteins to be encoded as polyproteins, which
dissociate into component proteins upon translation. Use of the
term "self-cleaving" is not intended to imply a proteolytic
cleavage reaction. Various self-cleaving or 2A peptides are known
to those of skill in the art, including, without limitation, those
found in members of the Picornaviridae virus family, e.g.,
foot-and-mouth disease virus (FMDV), equine rhinitis A virus
(ERAVO, Thosea asigna virus (TaV), and porcine tescho virus-1
(PTV-1); and carioviruses such as Theilovirus and
encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV,
PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and
"T2A," respectively. Those of skill in the art would be able to
select the appropriate self-cleaving peptide for use in the present
invention.
[0430] In some embodiments, a nucleic acid of the present
disclosure comprises a nucleic acid sequence comprising a CAR
coding sequence and a dominant negative receptor and/or a switch
receptor coding sequence that is separated by a linker comprising a
T2A peptide sequence. In some embodiments, the T2A peptide sequence
comprises the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID
NO:139), which may be encoded by the nucleic acid sequence
GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO:
140). In some embodiments, the linker comprising a T2A peptide
sequence may further comprise a spacer sequence as described
herein. For example, the linker comprising a T2A peptide sequence
may further comprise a spacer sequence comprising the amino acid
sequence SGRSGGG (SEQ ID NO: 141), which may be encoded by the
nucleic acid sequence TCCGGAAGATCTGGCGGCGGA (SEQ ID NO: 142).
[0431] In some embodiments, a nucleic acid of the present
disclosure comprises a nucleic acid sequence comprising a CAR
coding sequence and a dominant negative receptor and/or a switch
receptor coding sequence that is separated by a linker comprising a
F2A peptide sequence. In some embodiments, the F2A peptide sequence
comprises the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID
NO: 143), which may be encoded by the nucleic acid sequence
TABLE-US-00123 (SEQ ID NO:144)
GTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGG
AGTCCAACCCAGGGCCG.
[0432] In some embodiments, a linker further comprises a nucleic
acid sequence that encodes a furin cleavage site. Furin is a
ubiquitously expressed protease that resides in the trans-golgi and
processes protein precursors before their secretion. Furin cleaves
at the COOH-- terminus of its consensus recognition sequence.
Various furin consensus recognition sequences (or "furin cleavage
sites") are known to those of skill in the art, including, without
limitation, Arg-X-Lys-Arg (SEQ ID NO: 145) or Arg-X-Arg-Arg (SEQ ID
NO: 146), and Arg-X-X-Arg (SEQ ID NO: 147), such as an
Arg-Gln-Lys-Arg (SEQ ID NO: 148), where X is any naturally
occurring amino acid. Another example of a furin cleavage site is
X1-Arg-X2-X3-Arg (SEQ ID NO: 149), where X1 is Lys or Arg, X2 is
any naturally occurring amino acid, and X3 is Lys or Arg. Those of
skill in the art would be able to select the appropriate Furin
cleavage site for use in the present invention.
[0433] In some embodiments, the linker comprises a nucleic acid
sequence encoding a combination of a Furin cleavage site and a 2A
peptide. Examples include, without limitation, a linker comprising
a nucleic acid sequence encoding Furin and F2A, a linker comprising
a nucleic acid sequence encoding Furin and E2A, a linker comprising
a nucleic acid sequence encoding Furin and P2A, a linker comprising
a nucleic acid sequence encoding Furin and T2A. Those of skill in
the art would be able to select the appropriate combination for use
in the present invention. In such embodiments, the linker may
further comprise a spacer sequence between the Furin and 2A
peptide. Various spacer sequences are known in the art, including,
without limitation, glycine serine (GS) spacers such as (GS)n,
(GSGGS)n (SEQ ID NO: 1) and (GGGS)n (SEQ ID NO:2), where n
represents an integer of at least 1. Exemplary spacer sequences can
comprise amino acid sequences including, without limitation, GGSG
(SEQ ID NO:4), GGSGG (SEQ ID NO:5), GSGSG (SEQ ID NO:6), GSGGG (SEQ
ID NO:7), GGGSG (SEQ ID NO:8), GSSSG (SEQ ID NO:9), and the like.
Those of skill in the art would be able to select the appropriate
spacer sequence for use in the present invention.
[0434] In some embodiments, a nucleic acid of the present
disclosure comprises a nucleic acid sequence comprising a CAR
coding sequence and a dominant negative receptor and/or a switch
receptor coding sequence that is separated by a Furin-(G4S)2-T2A
(F-GS2-T2A) linker. The F-GS2-T2A linker may be encoded by the
nucleic acid sequence
TABLE-US-00124 (SEQ ID NO:150)
CGTGCGAAGAGGGGCGGCGGGGGCTCCGGCGGGGGAGGCAGTGAGGGCC
GCGGCTCCCTGCTGACCTGCGGAGATGTAGAAGAGAACCCAGGCCCC.
and may comprise the amino acid sequence
RAKRGGGGSGGGGSEGRGSLLTCGDVEENPGP (SEQ ID NO:151). Those of skill in
the art would appreciate that linkers of the present invention may
include tolerable sequence variations.
[0435] In some embodiments, the present invention provides a
nucleic acid comprising a nucleic acid sequence encoding a dominant
negative receptor and/or a switch receptor as described herein. In
some embodiments, a nucleic acid comprises a nucleic acid sequence
encoding a dominant negative receptor and/or a switch receptor and
a nucleic acid sequence encoding a CAR as described herein (e.g., a
PSMA-CAR). In one embodiment, the nucleic acid sequence encoding
the dominant negative receptor and/or the switch receptor and the
nucleic acid sequence encoding the CAR resides on separate nucleic
acids. In one embodiment, the nucleic acid sequence encoding the
dominant negative receptor and/or the switch receptor and the
nucleic acid sequence encoding the CAR resides within the same
nucleic acid. In such an embodiment, the nucleic acid sequence
encoding the dominant negative receptor and/or the switch receptor
and the nucleic acid sequence encoding the CAR is separated by a
linker as described herein.
[0436] For example, a nucleic acid of the present disclosure may
comprise a nucleic acid sequence encoding a dominant receptor, a
linker, and a nucleic acid sequence encoding a CAR. In one
embodiment, the linker comprises a nucleic acid sequence encoding a
2A peptide (e.g., T2A). In an exemplary embodiment, a nucleic acid
of the present disclosure may comprise a nucleic acid sequence
encoding a dominant negative receptor and/or a switch receptor and
a nucleic acid sequence encoding a CAR separated by a linker
sequence comprising a nucleic acid sequence encoding T2A.
[0437] Accordingly, in one embodiment, a nucleic acid of the
present disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a dominant negative receptor and/or a switch receptor, a
nucleic acid sequence encoding a linker, and a nucleic acid
sequence encoding a CAR. In one embodiment, a nucleic acid of the
present disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a CAR, a nucleic acid sequence encoding a linker, and a
nucleic acid sequence encoding a dominant negative receptor and/or
a switch receptor.
[0438] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a dominant negative receptor and/or a switch receptor, a
nucleic acid sequence encoding a linker comprising T2A, and a
nucleic acid sequence encoding a CAR. In one embodiment, the
dominant negative receptor is TGF.beta.RII-DN. In one embodiment,
the CAR is a murine J591 PSMA-CAR. Accordingly, in an exemplary
embodiment, a nucleic acid of the present invention comprises from
5' to 3': a nucleic acid sequence encoding TGF.beta.RII-DN, a
nucleic acid sequence encoding a linker comprising T2A, and a
nucleic acid sequence encoding a murine J591 PSMA-CAR. In one
embodiment, the nucleic acid comprising from 5' to 3': a nucleic
acid sequence encoding TGF.beta.RII-DN, a nucleic acid sequence
encoding a linker comprising T2A, and a nucleic acid sequence
encoding a murine J591 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00125 (SEQ ID NO:152)
ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGT
GGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAA
TAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAA
CTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAAT
CCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGA
AGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAG
ACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAG
ATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGA
GACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATC
ATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCA
TATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCAT
ATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAG
CTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTC
TAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGC
CCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCC
GCCAGACCTGGATCTGACATTGTGATGACCCAGTCTCACAAATTCATGT
CCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGA
TGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCT
AAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATC
GCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAA
CGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGC
TATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGGAGGCG
GAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTGCAGCT
GCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATC
AGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGG
TCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCC
CAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGGCCACC
CTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCC
TGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTT
CGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGCGCTAGCTCC
GGAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCG
CGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG
GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATC
TGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA
TCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAA
ACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGT
AGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGA
AGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCA
GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTG
GACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA
AGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGC
GGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAG
GGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT
ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0439] In one embodiment, the CAR is a humanized J591 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence
encoding a humanized J591 PSMA-CAR. In one embodiment, a nucleic
acid of the present disclosure comprises from 5' to 3': a nucleic
acid encoding a humanized PSMA-CAR, a nucleic acid encoding a
linker comprising a 2A peptide (e.g., T2A), and a nucleic acid
encoding a dominant negative receptor and/or a switch receptor.
[0440] In one embodiment, the CAR is a humanized J591 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising T2A, and a nucleic acid sequence encoding a humanized
J591 PSMA-CAR. In one embodiment, the nucleic acid comprising from
5' to 3': a nucleic acid sequence encoding TGF.beta.RII-DN, a
nucleic acid sequence encoding a linker comprising T2A, and a
nucleic acid sequence encoding a humanized J591 PSMA-CAR.
[0441] The humanized PSMA-CAR can comprise any of the heavy and
light chain variable regions disclosed in PCT Publication Nos.
WO2017212250A1 and WO2018033749A1. For example, the humanized
PSMA-CAR of the present invention can comprise an scFv comprising
any of the heavy and light chain variable regions disclosed
therein. In some embodiments, the humanized J591 PSMA-CAR comprises
a humanized J591 PSMA binding domain comprising a heavy and light
chain variable region selected from any of the heavy and light
chain variable region sequences set forth in Table 19.
[0442] In some embodiments, a nucleic acid of the present invention
comprising from 5' to 3': a nucleic acid sequence encoding
TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence
encoding a humanized J591 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00126 (SEQ ID NO:258)
atgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcctgt
ggacgcgtatcgccagcacgatcccaccgcacgttcagaagteggttaa
taacgacatgatagtcactgacaacaacggtgcagtcaagtttccacaa
ctgtgtaaatifigtgatgtgagattttccacctgtgacaaccagaaat
cctgcatgagcaactgcagcatcacctccatctgtgagaagccacagga
agtctgtgtggctgtatggagaaagaatgacgagaacataacactagag
acagtttgccatgaccccaagctcccctaccatgactttattctggaag
atgctgcttctccaaagtgcattatgaaggaaaaaaaaaagcctggtga
gactttcttcatgtgttcctgtagctctgatgagtgcaatgacaacatc
atcttctcagaagaatataacaccagcaatcctgacttgttgctagtca
tatttcaagtgacaggcatcagcctcctgccaccactgggagttgccat
atctgtcatcatcatcttctactgctaccgcgttaaccggcagcagaag
ctgagttcatccggaagatctggcggcggagagggcagaggaagtcttc
taacatgcggtgacgtggaggagaatcccggccctatggccctgcctgt
gacagccctgctgctgcctctggctctgctgctgcacgccgccagacct
ggagaggtccagctggtgcagtctggagctgaggtgaagaagcctgggg
cctcagtgaaggtctcctgcaaggcttctggatacacattcactgaata
caccatccactgggtgaggcaggcccctggaaagggccttgagtggatt
ggaaacattaatcctaacaatggtggtactacctacaaccagaagttcg
aggacagagtcacaatcactgtagacaagtccaccagcacagcctacat
ggagctcagcagcctgagatctgaggatactgcagtctattactgtgca
gctggttggaactttgactactggggccaaggcaccacggtcaccgtct
cctcaggaggcggaggatctggcggcggaggaagttctggcggaggcag
cgacattcagatgacccagtctcccagcaccctgtccgcatcagtagga
gacagggtcaccatcacttgcaaggccagtcaggatgtgggtactgctg
tagactggtatcaacagaaaccagggcaagctectaaactactgattta
ctgggcatccacccggcacactggagtccctgatcgcttcageggcagt
ggatctgggacagatttcactctcaccatcagcagactgcagcctgaag
actttgcagtttattactgtcagcaatataacagctatcctctcacgtt
cggccaggggaccaaggtggatatcaaaaccacgacgccagcgccgcga
ccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcc
cagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggct
ggacttcgcctgtgatatctacatctgggcgccettggccgggacttgt
ggggtecttctcctgtcactggttatcaccctttactgcaaacggggca
gaaagaaactcctgtatatattcaaacaaccatttatgagaccagtaca
aactactcaagaggaagacggctgtagctgccgatttccagaagaagaa
gaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgccc
ccgcgtacaagcagggccagaaccagctctataacgagctcaatctagg
acgaagagaggagtacgacgttttggacaagagacgtggccgggaccct
gagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtaca
acgaactgcagaaagataagatggcggaggcctacagtgagattgggat
gaaaggcgagcgccggaggggcaaggggcacgacggcctttaccagggt
ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccc tgccccctcgc.
[0443] In some embodiments, a nucleic acid of the present invention
comprising from 5' to 3': a nucleic acid sequence encoding
TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence
encoding a humanized J591 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00127 (SEQ ID NO:260)
atgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcctgt
ggacgcgtatcgccagcacgatcccaccgcacgttcagaagteggttaa
taacgacatgatagtcactgacaacaacggtgcagtcaagtttccacaa
ctgtgtaaatifigtgatgtgagattttccacctgtgacaaccagaaat
cctgcatgagcaactgcagcatcacctccatctgtgagaagccacagga
agtctgtgtggctgtatggagaaagaatgacgagaacataacactagag
acagtttgccatgaccccaagctcccctaccatgactttattctggaag
atgctgcttctccaaagtgcattatgaaggaaaaaaaaaagcctggtga
gactttcttcatgtgttcctgtagctctgatgagtgcaatgacaacatc
atcttctcagaagaatataacaccagcaatcctgacttgttgctagtca
tatttcaagtgacaggcatcagcctcctgccaccactgggagttgccat
atctgtcatcatcatcttctactgctaccgcgttaaccggcagcagaag
ctgagttcatccggaagatctggcggcggagagggcagaggaagtcttc
taacatgcggtgacgtggaggagaatcccggccctatggccctgcctgt
gacagccctgctgctgcctctggctctgctgctgcacgccgccagacct
ggagacattcagatgacccagtctcccagcaccctgtccgcatcagtag
gagacagggtcaccatcacttgcaaggccagtcaggatgtgggtactgc
tgtagactggtatcaacagaaaccagggcaagctcctaaactactgatt
tactgggcatccacccggcacactggagtccctgatcgcttcagcggca
gtggatctgggacagatttcactctcaccatcagcagactgcagcctga
agactttgcagtttattactgtcagcaatataacagctatcctctcacg
ttcggccaggggaccaaggtggatatcaaaggaggcggaggatctggcg
gcggaggaagttctggcggaggcagcgaggtccagctggtgcagtctgg
agctgaggtgaagaagcctggggcctcagtgaaggtctectgcaagget
tctggatacacattcactgaatacaccatccactgggtgaggcaggccc
ctggaaagggccttgagtggattggaaacattaatcctaacaatggtgg
tactacctacaaccagaagttcgaggacagagtcacaatcactgtagac
aagtccaccagcacagcctacatggagctcagcagcctgagatctgagg
atactgcagtctattactgtgcagctggttggaactttgactactgggg
ccaaggcaccacggtcaccgtctcctcaaccacgacgccagcgccgcga
ccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcc
cagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggct
ggacttcgcctgtgatatctacatctgggcgccettggccgggacttgt
ggggtecttctcctgtcactggttatcaccctttactgcaaacggggca
gaaagaaactectgtatatattcaaacaaccatttatgagaccagtaca
aactactcaagaggaagacggctgtagctgccgatttccagaagaagaa
gaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgccc
ccgcgtacaagcagggccagaaccagctctataacgagctcaatctagg
acgaagagaggagtacgacgttttggacaagagacgtggccgggaccct
gagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtaca
acgaactgcagaaagataagatggcggaggcctacagtgagattgggat
gaaaggcgagcgccggaggggcaaggggcacgacggcctttaccagggt
ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccc tgccccctcgc.
[0444] In some embodiments, a nucleic acid of the present invention
comprising from 5' to 3': a nucleic acid sequence encoding
TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence
encoding a humanized J591 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00128 (SEQ ID NO:262)
atgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcctgt
ggacgcgtatcgccagcacgatcccaccgcacgttcagaagteggttaa
taacgacatgatagtcactgacaacaacggtgcagtcaagtttccacaa
ctgtgtaaatifigtgatgtgagattttccacctgtgacaaccagaaat
cctgcatgagcaactgcagcatcacctccatctgtgagaagccacagga
agtctgtgtggctgtatggagaaagaatgacgagaacataacactagag
acagtttgccatgaccccaagctcccctaccatgactttattctggaag
atgctgcttctccaaagtgcattatgaaggaaaaaaaaaagcctggtga
gactttcttcatgtgttcctgtagctctgatgagtgcaatgacaacatc
atcttctcagaagaatataacaccagcaatcctgacttgttgctagtca
tatttcaagtgacaggcatcagcctcctgccaccactgggagttgccat
atctgtcatcatcatcttctactgctaccgcgttaaccggcagcagaag
ctgagttcatccggaagatctggcggcggagagggcagaggaagtcttc
taacatgcggtgacgtggaggagaatcccggccctatggccctgcctgt
gacagccctgctgctgcctctggctctgctgctgcacgccgccagacct
ggagacattcagatgacccagtctcccagcaccctgtccgcatcagtag
gagacagggtcaccatcacttgcaaggccagtcaggatgtgggtactgc
tgtagactggtatcaacagaaaccagggcaagctcctaaactactgatt
tactgggcatccacccggcacactggagtccctgatcgcttcagcggca
gtggatctgggacagatttcactctcaccatcagcagactgcagcctga
agactttgcagtttattactgtcagcaatataacagctatcctctcacg
ttcggccaggggaccaaggtggatatcaaaggaggcggaggatctggcg
gcggaggaagttctggcggaggcagcgaggtccagctggtgcagtctgg
agctgaggtgaagaagcctggggcctcagtgaaggtctectgcaaggat
ctggatacacattcactgaatacaccatccactgggtgaggcaggcccc
tggaaagggccttgagtggattggaaacattaatcctaacaatggtggt
actacctacaaccagaagttcgaggacagagtcacaatcactgtagaca
agtccaccagcacagcctacatggagctcagcagcctgagatctgagga
tactgcagtctattactgtgcagctggttggaactttgactactggggc
caaggcaccacggtcaccgtctcctcaaccacgacgccagcgccgcgac
caccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgccc
agaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctg
gacttcgcctgtgatttctggttacccataggatgtgcagccifigttg
tagtctgcattttgggatgcatacttatttgttggcttacaaaaaagaa
gtattcatccagtgtgcacgaccctaacggtgaatacatgaacatgaga
gcagtgaacacagccaaaaaatccagactcacagatgtgaccctaagag
tgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaa
ccagctctataacgagctcaatctaggacgaagagaggagtacgacgtt
ttggacaagagacgtggccgggaccctgagatggggggaaagccgagaa
ggaagaaccctcaggaaggcctgtacaacgaactgcagaaagataagat
ggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggc
aaggggcacgacggcctttaccagggtctcagtacagccaccaaggaca
cctacgacgcccttcacatgcaggccctgccccctcgc.
[0445] In one embodiment, the CAR is a human 1C3 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising T2A, and a nucleic acid sequence encoding a human 1C3
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding TGF.beta.RII-DN, a nucleic
acid sequence encoding a linker comprising T2A, and a nucleic acid
sequence encoding a human 1C3 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00129 (SEQ ID NO:153)
ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGT
GGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAA
TAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAA
CTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAAT
CCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGA
AGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAG
ACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAG
ATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGA
GACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATC
ATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCA
TATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCAT
ATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAG
CTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTC
TAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGC
CTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCC
GCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGC
CTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAG
TAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACT
CCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCT
GTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTAC
TGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGG
ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGG
CTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACC
CAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA
CTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCA
GAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTG
GAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATT
TCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTA
CTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAG
GTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGC
CCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCC
AGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGT
CACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTA
TATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA
GACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAAC
TGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGG
CCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTAC
GACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGC
CGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGA
TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG
AGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCA
AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0446] In one embodiment, the CAR is a human 2A10 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising T2A, and a nucleic acid sequence encoding a human 2A10
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding TGF.beta.RII-DN, a nucleic
acid sequence encoding a linker comprising T2A, and a nucleic acid
sequence encoding a human 2A10 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00130 (SEQ ID NO:154)
ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGT
GGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAA
TAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAA
CTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAAT
CCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGA
AGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAG
ACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAG
ATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGA
GACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATC
ATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCA
TATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCAT
ATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAG
CTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTC
TAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGC
CTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCC
GCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGC
CCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTAC
CAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAG
TGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGT
CCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGC
CTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTAC
TGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTG
GCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGG
GTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCC
CTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTC
AGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGC
TCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCA
TCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCA
ACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAA
TAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACC
ACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGC
AGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGC
AGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCG
CCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCC
TTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACC
ATTTATGAGACCAGTACAAACTACTAAGAGGAAGACGGCTGTAGCTGCC
GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAG
CAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTAT
AACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGA
GACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCC
TCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCC
TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG
ACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC
CCTTCACATGCAGGCCCTGCCCCCTCGC.
[0447] In one embodiment, the CAR is a human 2F5 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising T2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding TGF.beta.RII-DN, a nucleic
acid sequence encoding a linker comprising T2A, and a nucleic acid
sequence encoding a human 2F5 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00131 (SEQ ID NO:155)
ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGT
GGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAA
TAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAA
CTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAAT
CCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGA
AGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAG
ACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAG
ATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGA
GACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATC
ATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCA
TATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCAT
ATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAG
CTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTC
TAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGC
CTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCC
GCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGC
CCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTAC
CAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAG
TGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGT
CCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGC
CTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTAC
TGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTG
GCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGG
GTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCC
CTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTC
AGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGC
TCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCA
TCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCA
GCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAA
TAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATC
AAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCG
CGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG
GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATC
TGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA
TCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAA
ACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGT
AGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGA
AGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCA
GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTG
GACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA
AGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGC
GGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAG
GGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT
ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0448] In one embodiment, the CAR is a human 2C6 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN, a nucleic acid sequence encoding a linker
comprising T2A, and a nucleic acid sequence encoding a human 2C6
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding TGF.beta.RII-DN, a nucleic
acid sequence encoding a linker comprising T2A, and a nucleic acid
sequence encoding a human 2C6 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00132 (SEQ ID NO:156)
ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGT
GGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAA
TAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAA
CTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAAT
CCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGA
AGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAG
ACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAG
ATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGA
GACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATC
ATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCA
TATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCAT
ATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAG
CTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTC
TAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGC
CTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCC
GCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGC
CCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTAC
CAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAG
TGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGT
CCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGC
CTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTAC
TGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGG
TGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCA
GCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGG
CCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGG
CCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGC
ATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCA
CCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCA
GCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGAT
ATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCA
TCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGC
GGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTAC
ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG
TTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATT
CAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGC
TGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAG
TGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAA
CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTT
TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA
GGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGAT
GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGC
AAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACA
CCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0449] Tolerable variations of the nucleic acid sequence encoding
for TGF.beta.RII-DN and a PSMA-CAR will be known to those of skill
in the art. For example, in some embodiments, the nucleic acid
sequence has at least 60%, at least 65%, at least 70%, at least
75%, at least 80%/a, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in any one of SEQ ID NOs: 152-156, 258, 260, or
262. In one embodiment, the nucleic acid sequence encoding for
TGF.beta.RII-DN and murine J591 PSMA-CAR comprises the nucleic acid
sequence set forth in SEQ ID NO: 152. In one embodiment, the
nucleic acid sequence encoding for TGF.beta.RII-DN and human 1C3
PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID
NO:153. In one embodiment, the nucleic acid sequence encoding for
TGF.beta.RII-DN and human 2A10 PSMA-CAR comprises the nucleic acid
sequence set forth in SEQ ID NO: 154. In one embodiment, the
nucleic acid sequence encoding for TGF.beta.RII-DN and human 2F5
PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID
NO: 155. In one embodiment, the nucleic acid sequence encoding for
TGF.beta.RII-DN and human 2C6 PSMA-CAR comprises the nucleic acid
sequence set forth in SEQ ID NO: 156. In one embodiment, the
nucleic acid sequence encoding for TGF.beta.RII-DN and humanized
J591 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ
ID NO: 258, 260, or 262.
[0450] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1-CTM-CD28.
[0451] In one embodiment, the CAR is a humanized J591 CAR (huJ591
PSMA-CAR). Accordingly, in an exemplary embodiment, a nucleic acid
of the present invention comprises from 5' to 3': a nucleic acid
sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a
linker (e.g., a 2A linker), and a nucleic acid sequence encoding a
huJ591 PSMA-CAR. In some embodiments, a nucleic acid of the present
invention comprises from 5' to 3' a nucleic acid sequence encoding
PD1-CTM-CD28 having an amino acid sequence set forth in SEQ ID NO:
117, a nucleic acid sequence encoding a linker (e.g., a 2A linker),
and a nucleic acid sequence encoding a huJ591 PSMA-CAR having an
amino acid sequence set forth in SEQ ID NO: 245 or 247.
[0452] In one embodiment, the CAR is a human 1C3 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 1C3
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid
sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 1C3 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00133 (SEQ ID NO:157)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG
GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTT
GACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGG
CCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC
CGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAG
CCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCA
GTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGA
GTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGAC
TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGC
TGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA
CTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATG
GACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTG
GCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGAC
CCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC
ACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGC
AGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTT
GGAAAGTGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATT
TCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTA
CTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAG
GTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGC
CCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCC
AGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGT
CACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTA
TATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA
GATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAAC
TGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGG
CCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTAC
GATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGC
CGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA
TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG
AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA
AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0453] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1-CTM-CD28. In one
embodiment, the CAR is a human 2A10 PSMA-CAR. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2A10 PSMA-CAR. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid
sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2A10 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00134 (SEQ ID NO:158)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG
GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTT
GACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGG
CCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC
CGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAG
CCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTA
CCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGA
GTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCG
TCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCG
CCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTA
CTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGT
GGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTG
GGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTC
CCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT
CAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAG
CTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCC
ATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATC
AACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTA
ATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAAC
CACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCG
CAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG
CAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGC
GCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACC
CTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAAC
CATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG
CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTC
AGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCT
ATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAA
GAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC
CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGG
CCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA
CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC
GCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0454] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1-CTM-CD28. In one
embodiment, the CAR is a human 2F5 PSMA-CAR. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid
sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2F5 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00135 (SEQ ID NO:159)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG
GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTT
GACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGG
CCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC
CGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAG
CCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTA
CCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGA
GTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCG
TCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCG
CCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTA
CTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGT
GGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTG
GGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTC
CCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT
CAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAG
CTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCC
ATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC
AGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTA
ATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAAT
CAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATC
GCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGG
GGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACAT
CTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTT
ATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCA
AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTG
TAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTG
AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACC
AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT
GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGG
AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGG
CGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA
GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC
TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0455] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1-CTM-CD28. In one
embodiment, the CAR is a human 2C6 PSMA-CAR. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid
sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2C6 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00136 (SEQ ID NO:160)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG
GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTT
GACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGG
CCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC
CGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAG
CCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTA
CCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGA
GTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCG
TCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCG
CCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTA
CTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGG
GGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCG
GTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCC
AGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG
GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG
GCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGG
CATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTC
ACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGC
AGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGA
TATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACC
ATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGG
CGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTA
CATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTG
GTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATAT
TCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGG
CTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGA
GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGA
ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT
TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGA
AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGA
TGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG
CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGAC
ACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0456] Tolerable variations of the nucleic acid sequence encoding
PD1-CTM-CD28 and a PSMA-CAR will be known to those of skill in the
art. For example, in some embodiments, the nucleic acid sequence
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%/a, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% sequence identity to the nucleic acid sequence
set forth in any one of SEQ ID NOs:157-160. In one embodiment, the
nucleic acid sequence encoding for PD1-CTM-CD28 and human 1C3
PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID
NO: 157. In one embodiment, the nucleic acid sequence encoding for
PD1-CTM-CD28 and human 2A10 PSMA-CAR comprises the nucleic acid
sequence set forth in SEQ ID NO: 158. In one embodiment, the
nucleic acid sequence encoding for PD1-CTM-CD28 and human 2F5
PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID
NO: 159. In one embodiment, the nucleic acid sequence encoding for
PD1-CTM-CD28 and human 2C6 PSMA-CAR comprises the nucleic acid
sequence set forth in SEQ ID NO:160.
[0457] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1.sup.A132L-PTM-CD28.
In one embodiment, the CAR is a human 1C3 PSMA-CAR. Accordingly, in
an exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1.sup.A132L-PTM-CD28, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 1C3
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding PD1.sup.A132L-PTM-CD28, a
nucleic acid sequence encoding a linker comprising F2A, and a
nucleic acid sequence encoding a human 1C3 PSMA-CAR, comprises the
nucleic acid sequence set forth below:
TABLE-US-00137 (SEQ ID NO:161)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC
CTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCG
GGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCT
TGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCA
ACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA
CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACT
GGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATC
ATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTC
ACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACA
GCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCC
CTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGG
ACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGT
CGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCT
GTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG
GGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTC
CTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATC
AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACA
GTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCAC
GACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG
CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAG
TGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCC
CTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTT
TACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCAT
TTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCG
ATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGC
AGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATA
ACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAG
ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCT
CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCT
ACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA
TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC
CTTCACATGCAGGCCCTGCCCCCTCGC.
[0458] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is
PD1.sup.A132L-PTM-CD28.
[0459] In one embodiment, the CAR is a humanized J591 CAR (huJ591
PSMA-CAR). Accordingly, in an exemplary embodiment, a nucleic acid
of the present invention comprises from 5' to 3': a nucleic acid
sequence encoding PD1.sup.A132L-PTM-CD28, a nucleic acid sequence
encoding a linker (e.g., a 2A linker), and a nucleic acid sequence
encoding a huJ591 PSMA-CAR. In some embodiments, a nucleic acid of
the present invention comprises from 5' to 3' a nucleic acid
sequence encoding PD1.sup.A132L-PTM-CD28 having an amino acid
sequence set forth in SEQ ID NO: 121, a nucleic acid sequence
encoding a linker (e.g., a 2A linker), and a nucleic acid sequence
encoding a huJ591 PSMA-CAR having an amino acid sequence set forth
in SEQ ID NO: 245 or 247.
[0460] In one embodiment, the CAR is a human 2A10 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding PD1.sup.A132L-PTM-CD28, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a human
2A10 PSMA-CAR. In one embodiment, the nucleic acid comprising from
5' to 3': a nucleic acid sequence encoding PD1.sup.A32L-PTM-CD28, a
nucleic acid sequence encoding a linker comprising F2A, and a
nucleic acid sequence encoding a human 2A10 PSMA-CAR, comprises the
nucleic acid sequence set forth below:
TABLE-US-00138 (SEQ ID NO:162)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC
CTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCG
GGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCT
TGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCA
GCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG
ATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCT
GGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTA
TCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTC
ACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCA
GCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGG
TTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTC
TCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGAT
CTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCT
TTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCT
ATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTA
TGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAA
GATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTT
TCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCG
ACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGC
CCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGC
TGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTG
TGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGC
AGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTAC
AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGA
AGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCC
CCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG
GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC
TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC
AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA
TGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG
TCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCC CTGCCCCCTCGC.
[0461] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1.sup.A132L-PTM-CD28.
In one embodiment, the CAR is a human 2F5 PSMA-CAR. Accordingly, in
an exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1.sup.A132L-PTM-CD28, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding PD1.sup.A132L-PTM-CD28, a
nucleic acid sequence encoding a linker comprising F2A, and a
nucleic acid sequence encoding a human 2F5 PSMA-CAR, comprises the
nucleic acid sequence set forth below:
TABLE-US-00139 (SEQ ID NO:163)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC
CTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCG
GGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCT
TGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCA
GCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG
ATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCT
GGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTA
TCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTC
ACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACA
GCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGG
TTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTC
TCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGAT
CTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCT
TTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCT
ATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAG
TGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAA
GATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTT
TCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGC
GCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCC
CTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGA
GGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGG
GACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAA
CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC
CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA
AGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCA
GACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCA
ATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCG
GGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC
CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGA
TTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA
CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG
CAGGCCCTGCCCCCTCGC.
[0462] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1.sup.A132L-PTM-CD28.
In one embodiment, the CAR is a human 2C6 PSMA-CAR. Accordingly, in
an exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1.sup.A132L-PTM-CD28, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 2C6
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding PD1.sup.A132L-PTM-CD28, a
nucleic acid sequence encoding a linker comprising F2A, and a
nucleic acid sequence encoding a human 2C6 PSMA-CAR, comprises the
nucleic acid sequence set forth below:
TABLE-US-00140 (SEQ ID NO:164)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC
CTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCG
GGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCT
TGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCA
GCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG
ATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCT
GGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTA
TCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTC
ACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCA
GCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTA
TACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTC
ACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCG
GCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTC
TCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGC
AGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCC
TCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAG
TGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAG
CCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCC
TATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAACCACGACGCC
AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG
TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACA
CGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGC
CGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA
GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCC
AGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGC
GCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGC
TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGG
CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA
GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTG
AGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCT
TTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCAC
ATGCAGGCCCTGCCCCCTCGC.
[0463] Tolerable variations of the nucleic acid sequence encoding
PD1.sup.A132L-PTM-CD28 and a PSMA-CAR will be known to those of
skill in the art. For example, in some embodiments, the nucleic
acid sequence has at least 60%/a, at least 65%, at least 70%, at
least 75%, at least 80%/a, at least 81%, at least 82%, at least
83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% sequence identity to the nucleic
acid sequence set forth in any one of SEQ ID NOs:161-164. In one
embodiment, the nucleic acid sequence encoding for
PD1.sup.A132L-PTM-CD28 and human 1C3 PSMA-CAR comprises the nucleic
acid sequence set forth in SEQ ID NO: 161. In one embodiment, the
nucleic acid sequence encoding for PD1.sup.A132L-PTM-CD28 and human
2A10 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ
ID NO: 162. In one embodiment, the nucleic acid sequence encoding
for PD1.sup.A132L-PTM-CD28 and human 2F5 PSMA-CAR comprises the
nucleic acid sequence set forth in SEQ ID NO: 163. In one
embodiment, the nucleic acid sequence encoding for
PD1.sup.A132L-PTM-CD28 and human 2C6 PSMA-CAR comprises the nucleic
acid sequence set forth in SEQ ID NO:164.
[0464] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is TIM3-CD28. In one
embodiment, the CAR is a human 1C3 PSMA-CAR. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
TIM3-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 1C3 PSMA-CAR. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence
encoding a linker comprising F2A, and a nucleic acid sequence
encoding a human 1C3 PSMA-CAR, comprises the nucleic acid sequence
set forth below:
TABLE-US-00141 (SEQ ID NO:165)
ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTAC
TACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAA
TGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTG
CCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACG
TGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATA
CTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAG
AATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCC
CAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACC
AGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCC
TTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGA
CACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGC
CAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGA
GCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT
GCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG
TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG
ACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCT
CAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCA
GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGC
CGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTAT
GCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGG
CAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAA
GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTG
CAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGA
GAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTG
GGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGC
GGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTC
CATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCG
GGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCA
GGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTG
GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCT
CACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAA
CAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGA
TCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCAT
CGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCG
GGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACA
TCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGT
TATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTC
AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCT
GTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGT
GAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAAC
CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTT
TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG
GAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATG
GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA
AGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC
CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0465] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is TIM3-CD28.
[0466] In one embodiment, the CAR is a humanized J591 CAR (huJ591
PSMA-CAR). Accordingly, in an exemplary embodiment, a nucleic acid
of the present invention comprises from 5' to 3': a nucleic acid
sequence encoding TIM3-CD28, a nucleic acid sequence encoding a
linker (e.g., a 2A linker), and a nucleic acid sequence encoding a
huJ591 PSMA-CAR. In some embodiments, a nucleic acid of the present
invention comprises from 5' to 3' a nucleic acid sequence encoding
TIM3-CD28 having an amino acid sequence set forth in SEQ ID NO:
127, a nucleic acid sequence encoding a linker (e.g., a 2A linker),
and a nucleic acid sequence encoding a huJ591 PSMA-CAR having an
amino acid sequence set forth in SEQ ID NO: 245 or 247.
[0467] In one embodiment, the CAR is a human 2A10 PSMA-CAR.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding TIM3-CD28, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 2A10
PSMA-CAR. In one embodiment, the nucleic acid comprising from 5' to
3': a nucleic acid sequence encoding TIM3-CD28, a nucleic acid
sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2A10 PSMA-CAR, comprises the nucleic acid
sequence set forth below:
TABLE-US-00142 (SEQ ID NO:166)
ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTAC
TACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAA
TGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTG
CCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACG
TGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATA
CTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAG
AATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCC
CAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACC
AGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCC
TTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGA
CACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGC
CAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGA
GCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT
GCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG
TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG
ACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCT
CAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCA
GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGC
CGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGA
GTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAAC
TGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGG
GGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCA
AGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG
CAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGA
GGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCT
GGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGT
GGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTG
CATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACAT
TAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGT
TCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCT
GCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTAC
CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGC
CAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCT
GTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC
ACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGG
CCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTG
CAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATG
AGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC
CAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAG
CGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAG
CTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG
GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGA
AGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT
GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC
TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCA
CATGCAGGCCCTGCCCCCTCGC.
[0468] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is TIM3-CD28. In one
embodiment, the CAR is a human 2F5 PSMA-CAR. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
TIM3-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence
encoding a linker comprising F2A, and a nucleic acid sequence
encoding a human 2F5 PSMA-CAR, comprises the nucleic acid sequence
set forth below:
TABLE-US-00143 (SEQ ID NO:167)
ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTAC
TACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAA
TGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTG
CCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACG
TGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATA
CTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAG
AATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCC
CAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACC
AGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCC
TTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGA
CACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGC
CAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGA
GCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT
GCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG
TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG
ACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCT
CAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCA
GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGC
CGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGA
GTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAAC
TGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGG
GGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCA
AGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG
CAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGA
GACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCT
GGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGT
GGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTG
CATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACAT
TAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAG
CTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGT
TCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCT
GCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTAC
CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCA
CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCA
GCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA
GTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGC
CCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCT
TTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCA
TTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCC
GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAG
CAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTAT
AACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGA
GACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCC
TCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCC
TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG
ACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC
CCTTCACATGCAGGCCCTGCCCCCTCGC.
[0469] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is TIM3-CD28. In one
embodiment, the CAR is a human 2C6 PSMA-CAR. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
TIM3-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2C6 PSMA-CAR. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence
encoding a linker comprising F2A, and a nucleic acid sequence
encoding a human 2C6 PSMA-CAR, comprises the nucleic acid sequence
set forth below:
TABLE-US-00144 (SEQ ID NO:168)
ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTAC
TACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAA
TGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTG
CCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACG
TGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATA
CTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAG
AATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCC
CAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACC
AGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCC
TTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGA
CACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGC
CAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGA
GCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT
GCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG
TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG
ACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCT
CAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCA
GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGC
CGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGA
GTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTAC
TGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGG
GGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCA
AGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTG
CAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGA
GTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGG
AACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGG
TCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCC
TGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCA
GAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCT
CCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAG
CCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGC
AACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAA
CCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTC
GCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGC
GCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGG
CGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCAC
CCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAA
CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCT
GCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTT
CAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTC
TATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACA
AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAA
CCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAG
GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC
ACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGA
CGCCCTTCACATGCAGGCCCTGCCCCCTCGC.
[0470] Tolerable variations of the nucleic acid sequence encoding
TIM3-CD28 and a PSMA-CAR will be known to those of skill in the
art. For example, in some embodiments, the nucleic acid sequence
has at least 60%/a, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%/a, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the nucleic acid sequence set
forth in any one of SEQ ID NOs: 165-168. In one embodiment, the
nucleic acid sequence encoding for TIM3-CD28 and human 1C3 PSMA-CAR
comprises the nucleic acid sequence set forth in SEQ ID NO: 165. In
one embodiment, the nucleic acid sequence encoding for TIM3-CD28
and human 2A10 PSMA-CAR comprises the nucleic acid sequence set
forth in SEQ ID NO: 166. In one embodiment, the nucleic acid
sequence encoding for TIM3-CD28 and human 2F5 PSMA-CAR comprises
the nucleic acid sequence set forth in SEQ ID NO: 167. In one
embodiment, the nucleic acid sequence encoding for TIM3-CD28 and
human 2C6 PSMA-CAR comprises the nucleic acid sequence set forth in
SEQ ID NO: 168.
[0471] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1-CTM-CD28.
[0472] In one embodiment, the CAR is a human 2F5 PSMA-CAR
comprising an ICOS domain and a CD3zeta domain. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR
comprising an ICOS domain and a CD3zeta domain. In one embodiment,
the nucleic acid comprising from 5' to 3': a nucleic acid sequence
encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR comprising an ICOS domain and a CD3zeta domain, comprises
the nucleic acid sequence set forth below:
TABLE-US-00145 (SEQ ID NO:217)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG
GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTT
GACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGG
CCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC
CGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAG
CCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTA
CCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGA
GTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCG
TCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCG
CCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTA
CTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGT
GGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTG
GGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTC
CCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT
CAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAG
CTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCC
ATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC
AGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTA
ATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAAT
CAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATC
GCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGG
GGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTT
ACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATA
CTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACC
CTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATC
CAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGAC
GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC
TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGA
CCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGC
CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGA
TTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA
CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG
CAGGCCCTGCCCCCTCGC.
[0473] Tolerable variations of the nucleic acid sequence encoding
PD1-CTM-CD28 and a human 2F5 PSMA-CAR comprising an ICOS domain and
a CD3zeta domain will be known to those of skill in the art. For
example, in some embodiments, the nucleic acid sequence has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO:217. In one embodiment, the nucleic acid sequence encoding
for PD1-CTM-CD28 and human 2F5 PSMA-CAR comprising an ICOS domain
and a CD3zeta domain comprises the nucleic acid sequence set forth
in SEQ ID NO:217.
[0474] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1-CTM-CD28. In one
embodiment, the CAR is a human 2F5 PSMA-CAR comprising a variant
ICOS domain and a CD3zeta domain. Accordingly, in an exemplary
embodiment, a nucleic acid of the present invention comprises from
5' to 3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic
acid sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2F5 PSMA-CAR comprising a variant ICOS
domain and a CD3zeta domain. In one embodiment, the nucleic acid
comprising from 5' to 3': a nucleic acid sequence encoding
PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR
comprising a variant ICOS domain and a CD3zeta domain, comprises
the nucleic acid sequence set forth below:
TABLE-US-00146 (SEQ ID NO:218)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG
GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTT
GACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGG
CCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGC
CGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAG
CCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTA
CCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGA
GTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCG
TCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCG
CCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTA
CTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGT
GGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTG
GGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTC
CCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT
CAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAG
CTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCC
ATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC
AGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTA
ATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAAT
CAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATC
GCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGG
GGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTT
ACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATA
CTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACC
CTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATC
CAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGAC
GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC
TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGA
CCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGC
CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGA
TTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA
CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG
CAGGCCCTGCCCCCTCGC.
[0475] Tolerable variations of the nucleic acid sequence encoding
PD1-CTM-CD28 and a human 2F5 PSMA-CAR comprising a variant ICOS
domain and a CD3zeta domain will be known to those of skill in the
art. For example, in some embodiments, the nucleic acid sequence
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the nucleic acid sequence set
forth in SEQ ID NO:218. In one embodiment, the nucleic acid
sequence encoding for PD1-CTM-CD28 and human 2F5 PSMA-CAR
comprising a variant ICOS domain and a CD3zeta domain comprises the
nucleic acid sequence set forth in SEQ ID NO:218.
[0476] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1.sup.A132L-PTM-CD28.
In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising an
ICOS domain and a CD3zeta domain. Accordingly, in an exemplary
embodiment, a nucleic acid of the present invention comprises from
5' to 3': a nucleic acid sequence encoding PD1.sup.A132L-PTM-CD28,
a nucleic acid sequence encoding a linker comprising F2A, and a
nucleic acid sequence encoding a human 2F5 PSMA-CAR comprising an
ICOS domain and a CD3zeta domain. In one embodiment, the nucleic
acid comprising from 5' to 3': a nucleic acid sequence encoding
PD1.sup.A132L-PTM-CD28, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR comprising an ICOS domain and a CD3zeta domain, comprises
the nucleic acid sequence set forth below:
TABLE-US-00147 (SEQ ID NO:219)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC
CTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCG
GGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCT
TGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCA
GCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG
ATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCT
GGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTA
TCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTC
ACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACA
GCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGG
TTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTC
TCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGAT
CTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCT
TTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCT
ATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAG
TGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAA
GATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTT
TCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGC
GCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCC
CTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGA
GGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGC
CTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACA
AAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGT
TCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGAC
CCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG
GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGT
ACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAA
GCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG
AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGC
GCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGC
CACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC .
[0477] Tolerable variations of the nucleic acid sequence encoding
PD1.sup.A132L-PTM-CD28 and a human 2F5 PSMA-CAR comprising an ICOS
domain and a CD3zeta domain will be known to those of skill in the
art. For example, in some embodiments, the nucleic acid sequence
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the nucleic acid sequence set
forth in SEQ ID NO:219. In one embodiment, the nucleic acid
sequence encoding for PD1.sup.A132L-PTM-CD28and human 2F5 PSMA-CAR
comprising an ICOS domain and a CD3zeta domain comprises the
nucleic acid sequence set forth in SEQ ID NO:219.
[0478] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1.sup.A132L-PTM-CD28.
In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising a
variant ICOS domain and a CD3zeta domain. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1.sup.A132L-PTM-CD28, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR comprising a variant ICOS domain and a CD3zeta domain. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding PD1.sup.A132L-PTM-CD28, a nucleic
acid sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2F5 PSMA-CAR comprising a variant ICOS
domain and a CD3zeta domain, comprises the nucleic acid sequence
set forth below:
TABLE-US-00148 (SEQ ID NO:220)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC
CTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCG
GGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCT
TGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCA
GCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG
ATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCT
GGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTA
TCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTC
ACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACA
GCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGG
TTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTC
TCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGAT
CTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGT
AGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGT
GCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGA
TCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGG
CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
GAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCA
CTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCC
AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG
TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACA
CGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGC
AGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTT
ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACA
TGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGT
GACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG
CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG
AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG
AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCG
AGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC
AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGC.
[0479] Tolerable variations of the nucleic acid sequence encoding
PD1.sup.A132L-PTM-CD28and a human 2F5 PSMA-CAR comprising a variant
ICOS domain and a CD3zeta domain will be known to those of skill in
the art. For example, in some embodiments, the nucleic acid
sequence has at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:220. In one embodiment, the nucleic
acid sequence encoding for PD1.sup.A132L-PTM-CD28and human 2F5
PSMA-CAR comprising a variant ICOS domain and a CD3zeta domain
comprises the nucleic acid sequence set forth in SEQ ID NO:220.
[0480] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1.sup.A132L-4-1BB. In
one embodiment, the CAR is a human 2F5 PSMA-CAR comprising an ICOS
domain and a CD3zeta domain. Accordingly, in an exemplary
embodiment, a nucleic acid of the present invention comprises from
5' to 3': a nucleic acid sequence encoding PD1.sup.A132L-4-1BB, a
nucleic acid sequence encoding a linker comprising F2A, and a
nucleic acid sequence encoding a human 2F5 PSMA-CAR comprising an
ICOS domain and a CD3zeta domain. In one embodiment, the nucleic
acid comprising from 5' to 3': a nucleic acid sequence encoding
PD1.sup.A132L-4-1BB, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR comprising an ICOS domain and a CD3zeta domain, comprises
the nucleic acid sequence set forth below:
TABLE-US-00149 (SEQ ID NO:221)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGAC
TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAA
CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC
CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA
AGAAGAAGAAGGAGGATGTGAACTGGTGAAACAGACTTTGAATTTTGAC
CTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCT
TACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGC
CAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCC
GGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCA
GCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTG
GATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCC
TTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCT
ACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTG
TGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGC
ACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGT
CGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCT
GTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG
GACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTC
CTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATC
AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATA
GTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAA
AACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCG
TCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGG
GCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACC
CATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTT
ATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTA
ACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAG
ACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCC
CCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG
GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC
TGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTG
TACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTG
GGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCA
GGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG
GCCCTGCCCCCTCGC.
[0481] Tolerable variations of the nucleic acid sequence encoding
PD1.sup.A132L-4-1BB and a human 2F5 PSMA-CAR comprising an ICOS
domain and a CD3zeta domain will be known to those of skill in the
art. For example, in some embodiments, the nucleic acid sequence
has at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% sequence identity to the nucleic acid sequence set
forth in SEQ ID NO:221. In one embodiment, the nucleic acid
sequence encoding for PD1.sup.A132L-4-1BB and human 2F5 PSMA-CAR
comprising an ICOS domain and a CD3zeta domain comprises the
nucleic acid sequence set forth in SEQ ID NO:221.
[0482] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is PD1.sup.A132L-4-1BB. In
one embodiment, the CAR is a human 2F5 PSMA-CAR comprising a
variant ICOS domain and a CD3zeta domain. Accordingly, in an
exemplary embodiment, a nucleic acid of the present invention
comprises from 5' to 3': a nucleic acid sequence encoding
PD1.sup.A132L-4-1BB, a nucleic acid sequence encoding a linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR comprising a variant ICOS domain and a CD3zeta domain. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding PD1.sup.A132L-4-1BB, a nucleic acid
sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2F5 PSMA-CAR comprising a variant ICOS
domain and a CD3zeta domain, comprises the nucleic acid sequence
set forth below:
TABLE-US-00150 (SEQ ID NO:222)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGAC
TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAA
CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC
CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA
AGAAGAAGAAGGAGGATGTGAACTGGTGAAACAGACTTTGAATTTTGAC
CTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCT
TACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGC
CAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCC
GGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCA
GCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTG
GATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCC
TTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCT
ACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTG
TGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGC
ACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGT
CGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCT
GTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG
GACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTC
CTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATC
AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATA
GTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAA
AACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCG
TCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGG
GCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACC
CATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTT
ATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTA
ACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAG
ACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCC
CCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG
GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC
TGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTG
TACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTG
GGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCA
GGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG
GCCCTGCCCCCTCGC.
[0483] Tolerable variations of the nucleic acid sequence encoding
PD1.sup.A132L-4-1BB and a human 2F5 PSMA-CAR comprising a variant
ICOS domain and a CD3zeta domain will be known to those of skill in
the art. For example, in some embodiments, the nucleic acid
sequence has at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:222. In one embodiment, the nucleic
acid sequence encoding for PD1.sup.A132L-4-1BB and human 2F5
PSMA-CAR comprising a variant ICOS domain and a CD3zeta domain
comprises the nucleic acid sequence set forth in SEQ ID NO:222.
[0484] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is TIM3-CD28. In one
embodiment, the CAR is a human 2F5 PSMA-CAR comprising an ICOS
domain and a CD3zeta domain. Accordingly, in an exemplary
embodiment, a nucleic acid of the present invention comprises from
5' to 3': a nucleic acid sequence encoding TIM3-CD28, a nucleic
acid sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2F5 PSMA-CAR comprising an ICOS domain
and a CD3zeta domain. In one embodiment, the nucleic acid
comprising from 5' to 3': a nucleic acid sequence encoding
TIM3-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR
comprising an ICOS domain and a CD3zeta domain, comprises the
nucleic acid sequence set forth below:
TABLE-US-00151 (SEQ ID NO:223)
ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTAC
TACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAA
TGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTG
CCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACG
TGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATA
CTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAG
AATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCC
CAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACC
AGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCC
TTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGA
CACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGC
CAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGA
GCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT
GCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG
TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG
ACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCT
CAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCA
GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGC
CGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGA
GTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAAC
TGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGG
GGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCA
AGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG
CAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGA
GACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCT
GGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGT
GGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTG
CATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACAT
TAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAG
CTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGT
TCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCT
GCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTAC
CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCA
CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCA
GCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA
GTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAG
GATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTG
TTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGT
GAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCA
CAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGC
GTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGA
AGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA
TGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA
TGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATG
AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTC
TCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCT GCCCCCTCGC.
[0485] Tolerable variations of the nucleic acid sequence encoding
TIM3-CD28and a human 2F5 PSMA-CAR comprising an ICOS domain and a
CD3zeta domain will be known to those of skill in the art. For
example, in some embodiments, the nucleic acid sequence has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO:223. In one embodiment, the nucleic acid sequence encoding
for TIM3-CD28and human 2F5 PSMA-CAR comprising an ICOS domain and a
CD3zeta domain comprises the nucleic acid sequence set forth in SEQ
ID NO:223.
[0486] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a switch receptor, a nucleic acid sequence encoding a
linker comprising F2A, and a nucleic acid sequence encoding a CAR.
In one embodiment, the switch receptor is TIM3-CD28. In one
embodiment, the CAR is a human 2F5 PSMA-CAR comprising a variant
ICOS domain and a CD3zeta domain. Accordingly, in an exemplary
embodiment, a nucleic acid of the present invention comprises from
5' to 3': a nucleic acid sequence encoding TIM3-CD28, a nucleic
acid sequence encoding a linker comprising F2A, and a nucleic acid
sequence encoding a human 2F5 PSMA-CAR comprising a variant ICOS
domain and a CD3zeta domain. In one embodiment, the nucleic acid
comprising from 5' to 3': a nucleic acid sequence encoding
TIM3-CD28, a nucleic acid sequence encoding a linker comprising
F2A, and a nucleic acid sequence encoding a human 2F5 PSMA-CAR
comprising a variant ICOS domain and a CD3zeta domain, comprises
the nucleic acid sequence set forth below:
TABLE-US-00152 (SEQ ID NO:224)
ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTAC
TACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAA
TGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTG
CCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACG
TGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATA
CTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAG
AATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCC
CAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACC
AGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCC
TTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGA
CACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGC
CAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGA
GCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT
GCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAG
TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG
ACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCT
CAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCA
GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGC
CGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGA
GTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAAC
TGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGG
GGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCA
AGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG
CAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGA
GACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCT
GGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGT
GGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTG
CATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACAT
TAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAG
CTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGT
TCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCT
GCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTAC
CCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCA
CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCA
GCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCA
GTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAG
GATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTG
TTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGT
GAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCA
CAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGC
GTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGA
AGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA
TGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA
TGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATG
AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTC
TCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCT GCCCCCTCGC.
[0487] Tolerable variations of the nucleic acid sequence encoding
TIM3-CD28and a human 2F5 PSMA-CAR comprising a variant ICOS domain
and a CD3zeta domain will be known to those of skill in the art.
For example, in some embodiments, the nucleic acid sequence has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO:224. In one embodiment, the nucleic acid sequence encoding
for TIM3-CD28and human 2F5 PSMA-CAR comprising a variant ICOS
domain and a CD3zeta domain comprises the nucleic acid sequence set
forth in SEQ ID NO:224.
[0488] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a first switch receptor, a nucleic acid sequence encoding
a first linker comprising F2A, a nucleic acid sequence encoding a
second switch receptor, a nucleic acid encoding a second linker
comprising F2A, and a nucleic acid sequence encoding a CAR. In one
embodiment, the first switch receptor is TIM3-CD28, and the second
switch receptor is PD1.sup.A132L-4-1BB. In one embodiment, the
first switch receptor is PD1.sup.A132L-4-1BB, and the second switch
receptor is TIM3-CD28. In one embodiment, the CAR is a human 2F5
PSMA-CAR comprising an ICOS domain and a CD3zeta domain. In one
embodiment, the first and second linkers are the same. In one
embodiment, the first and second linkers are different.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding PD1.sup.A132L-4-1BB, a nucleic acid sequence encoding a
first linker comprising F2A, a nucleic acid sequence encoding
TIM3-CD28, a nucleic acid sequence encoding a second linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR comprising an ICOS domain and a CD3zeta domain. In one
embodiment, the nucleic acid comprising from 5' to 3': a nucleic
acid sequence encoding PD1.sup.A132L-4-1BB, a nucleic acid sequence
encoding a first linker comprising F2A, a nucleic acid sequence
encoding TIM3-CD28, a nucleic acid encoding a second linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR comprising an ICOS domain and a CD3zeta domain, comprises
the nucleic acid sequence set forth below:
TABLE-US-00153 (SEQ ID NO:225)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGAC
TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAA
CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC
CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA
AGAAGAAGAAGGAGGATGTGAACTGGTGAAGCAGACGTTGAACTTCGAT
TTGCTCAAACTTGCCGGTGACGTGGAATCCAATCCGGGGCCGATGTTTT
CACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTAC
AAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTAT
CTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCT
GCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCT
CAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTA
AATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGA
CTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCAT
AATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAG
GTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAA
GGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGG
GAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAG
TTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCA
TCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAG
CTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGG
AGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCG
GGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGC
AGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTG
GCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCG
CCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGT
GCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTG
AAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCG
GCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCAT
CTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAG
GTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
ACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAAC
TGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACT
GTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCG
GATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGT
AGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGT
GCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGA
TCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGG
CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
GAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCA
CTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCC
AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG
TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACA
CGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGC
AGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTT
ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACA
TGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGT
GACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG
CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG
AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG
AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCG
AGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC
AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGC.
[0489] Tolerable variations of the nucleic acid sequence encoding
PD1.sup.A132L-4-1BB, TIM3-CD28, and a human 2F5 PSMA-CAR comprising
an ICOS domain and a CD3zeta domain will be known to those of skill
in the art. For example, in some embodiments, the nucleic acid
sequence has at least 60%/a, at least 65%, at least 70%, at least
75%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%/a, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:225. In one embodiment, the nucleic
acid sequence encoding for PD1.sup.A132L-4-1BB, TIM3-CD28, and a
human 2F5 PSMA-CAR comprising an ICOS domain and a CD3zeta domain
comprises the nucleic acid sequence set forth in SEQ ID NO:225.
[0490] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a first switch receptor, a nucleic acid sequence encoding
a first linker comprising F2A, a nucleic acid sequence encoding a
second switch receptor, a nucleic acid encoding a second linker
comprising F2A, and a nucleic acid sequence encoding a CAR. In one
embodiment, the first switch receptor is TIM3-CD28, and the second
switch receptor is PD1.sup.A132L-4-1BB. In one embodiment, the
first switch receptor is PD1.sup.A132L-4-1BB, and the second switch
receptor is TIM3-CD28. In one embodiment, the CAR is a human 2F5
PSMA-CAR comprising a variant ICOS domain and a CD3zeta domain. In
one embodiment, the first and second linkers are the same. In one
embodiment, the first and second linkers are different.
Accordingly, in an exemplary embodiment, a nucleic acid of the
present invention comprises from 5' to 3': a nucleic acid sequence
encoding PD1.sup.A132L-4-1BB, a nucleic acid sequence encoding a
first linker comprising F2A, a nucleic acid sequence encoding
TIM3-CD28, a nucleic acid sequence encoding a second linker
comprising F2A, and a nucleic acid sequence encoding a human 2F5
PSMA-CAR comprising a variant ICOS domain and a CD3zeta domain. In
one embodiment, the nucleic acid comprising from 5' to 3': a
nucleic acid sequence encoding PD1.sup.A132L-4-1BB, a nucleic acid
sequence encoding a first linker comprising F2A, a nucleic acid
sequence encoding TIM3-CD28, a nucleic acid encoding a second
linker comprising F2A, and a nucleic acid sequence encoding a human
2F5 PSMA-CAR comprising a variant ICOS domain and a CD3zeta domain,
comprises the nucleic acid sequence set forth below:
TABLE-US-00154 (SEQ ID NO:226)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGAC
TTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAA
CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC
CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA
AGAAGAAGAAGGAGGATGTGAACTGGTGAAGCAGACGTTGAACTTCGAT
TTGCTCAAACTTGCCGGTGACGTGGAATCCAATCCGGGGCCGATGTTTT
CACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTAC
AAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTAT
CTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCT
GCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCT
CAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTA
AATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGA
CTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCAT
AATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAG
GTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAA
GGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGG
GAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAG
TTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCA
TCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAG
CTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGG
AGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCG
GGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGC
AGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTG
GCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCG
CCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGT
GCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTG
AAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCG
GCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCAT
CTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAG
GTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
ACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAAC
TGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACT
GTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCG
GATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGT
AGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGT
GCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGA
TCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGG
CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
GAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCA
CTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCC
AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG
TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACA
CGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGC
AGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTT
ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACA
TGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGT
GACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG
CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG
AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG
AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCG
AGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC
AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGC.
[0491] Tolerable variations of the nucleic acid sequence encoding
PD1.sup.A132L-4-1BB, TIM3-CD28, and a human 2F5 PSMA-CAR comprising
a variant ICOS domain and a CD3zeta domain will be known to those
of skill in the art. For example, in some embodiments, the nucleic
acid sequence has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% sequence identity to the nucleic acid
sequence set forth in SEQ ID NO:226. In one embodiment, the nucleic
acid sequence encoding for PD1.sup.A132L-4-1BB, TIM3-CD28, and a
human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3zeta
domain comprises the nucleic acid sequence set forth in SEQ ID
NO:226.
[0492] In some embodiments, the present invention provides a
nucleic acid comprising a nucleic acid sequence encoding a dominant
negative receptor and a switch receptor as described herein. In
some embodiments, a nucleic acid comprises a nucleic acid sequence
encoding a dominant negative receptor and a switch receptor and a
nucleic acid sequence encoding a CAR as described herein (e.g., a
PSMA-CAR). In one embodiment, the nucleic acid sequence encoding
the dominant negative receptor and the switch receptor and the
nucleic acid sequence encoding the CAR resides on separate nucleic
acids. In one embodiment, the nucleic acid sequence encoding the
dominant negative receptor and the switch receptor and the nucleic
acid sequence encoding the CAR resides within the same nucleic
acid. In such an embodiment, the nucleic acid sequences encoding
the dominant negative receptor and the switch receptor and the
nucleic acid sequence encoding the CAR are separated by a linker as
described herein.
[0493] The nucleic acid of the present disclosure may comprise a 5'
to 3' orientation of the nucleic acid sequence encoding the
dominant negative receptor, the nucleic acid sequence encoding the
switch receptor, and the nucleic acid sequence encoding the CAR, in
any order. Those of skill in the art will readily be able to
determine the optimal 5' to 3' orientation of the nucleic acid
sequence encoding the dominant negative receptor, the nucleic acid
sequence encoding the switch receptor, and the nucleic acid
sequence encoding the CAR.
[0494] In some embodiments, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding a dominant negative receptor, a nucleic acid sequence
encoding a first linker, a nucleic acid sequence encoding a switch
receptor, a nucleic acid encoding a second linker, and a nucleic
acid sequence encoding a CAR.
[0495] In one embodiment, the dominant negative receptor is
TGF.beta.RII-DN (for example, having the amino acid sequence set
forth in SEQ ID NO: 115), and the switch receptor is PD1-CTM-CD28
(for example, having the amino acid sequence set forth in SEQ ID
NO: 117). In one embodiment, the dominant negative receptor is
TGF.beta.RII-DN (for example, having the amino acid sequence set
forth in SEQ ID NO: 115), and the switch receptor is PD1-PTM-CD28
(for example, having the amino acid sequence set forth in SEQ ID
NO: 119). In one embodiment, the dominant negative receptor is
TGF.beta.RII-DN (for example, having the amino acid sequence set
forth in SEQ ID NO: 115), and the switch receptor is
PD1.sup.A132L-PTM-CD28 (for example, having the amino acid sequence
set forth in SEQ ID NO: 121). In one embodiment, the dominant
negative receptor is TGF.beta.RII-DN (for example, having the amino
acid sequence set forth in SEQ ID NO: 115), and the switch receptor
is PD1-4-1BB (for example, having the amino acid sequence set forth
in SEQ ID NO: 213). In one embodiment, the dominant negative
receptor is TGF.beta.RII-DN (for example, having the amino acid
sequence set forth in SEQ ID NO: 115), and the switch receptor is
PD1.sup.A132L-4-1BB (for example, having the amino acid sequence
set forth in SEQ ID NO: 215). In one embodiment, the dominant
negative receptor is TGF.beta.RII-DN (for example, having the amino
acid sequence set forth in SEQ ID NO: 115), and the switch receptor
is TGF.beta.R-IL12R.beta.1 (for example, having the amino acid
sequence set forth in SEQ ID NO: 123). In one embodiment, the
dominant negative receptor is TGF.beta.RII-DN (for example, having
the amino acid sequence set forth in SEQ ID NO: 115), and the
switch receptor is TGF.beta.R-IL12R32 (for example, having the
amino acid sequence set forth in SEQ ID NO: 125). In one
embodiment, the dominant negative receptor is TGF.beta.RII-DN (for
example, having the amino acid sequence set forth in SEQ ID NO:
115), and the switch receptor is TIM3-CD28 (for example, having the
amino acid sequence set forth in SEQ ID NO: 127).
[0496] In an exemplary embodiment, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN (for example, having the amino acid
sequence set forth in SEQ ID NO: 115), a nucleic acid sequence
encoding a first linker, a nucleic acid sequence encoding
PD1-CTM-CD28 (for example, having the amino acid sequence set forth
in SEQ ID NO: 117), a nucleic acid encoding a second linker, and a
nucleic acid sequence encoding a J591 murine PSMA-CAR (for example,
having the amino acid sequence set forth in SEQ ID NO: 105). In an
exemplary embodiment, a nucleic acid of the present disclosure
comprises from 5' to 3': a nucleic acid sequence encoding
TGF.beta.RII-DN (for example, having the amino acid sequence set
forth in SEQ ID NO: 115), a nucleic acid sequence encoding a first
linker, a nucleic acid sequence encoding PD1-PTM-CD28 (for example,
having the amino acid sequence set forth in SEQ ID NO: 119), a
nucleic acid encoding a second linker, and a nucleic acid sequence
encoding a J591 murine PSMA-CAR (for example, having the amino acid
sequence set forth in SEQ ID NO: 105).
[0497] In an exemplary embodiment, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN (for example, having the amino acid
sequence set forth in SEQ ID NO: 115), a nucleic acid sequence
encoding a first linker, a nucleic acid sequence encoding
PD1-CTM-CD28 (for example, having the amino acid sequence set forth
in SEQ ID NO: 117), a nucleic acid encoding a second linker, and a
nucleic acid sequence encoding a huJ591 PSMA-CAR (for example,
having the amino acid sequence set forth in SEQ ID NO: 245, 247,
249, 251, 253, or 255). In an exemplary embodiment, a nucleic acid
of the present disclosure comprises from 5' to 3': a nucleic acid
sequence encoding TGF.beta.RII-DN (for example, having the amino
acid sequence set forth in SEQ ID NO: 115), a nucleic acid sequence
encoding a first linker, a nucleic acid sequence encoding
PD1-PTM-CD28 (for example, having the amino acid sequence set forth
in SEQ ID NO: 119), a nucleic acid encoding a second linker, and a
nucleic acid sequence encoding a huJ591 PSMA-CAR (for example,
having the amino acid sequence set forth in SEQ ID NO: 245, 247,
249, 251, 253, or 255).
[0498] In an exemplary embodiment, a nucleic acid of the present
disclosure comprises from 5' to 3': a nucleic acid sequence
encoding TGF.beta.RII-DN (for example, having the amino acid
sequence set forth in SEQ ID NO: 115), a nucleic acid sequence
encoding a first linker, a nucleic acid sequence encoding
PD1-CTM-CD28 (for example, having the amino acid sequence set forth
in SEQ ID NO: 117), a nucleic acid encoding a second linker, and a
nucleic acid sequence encoding a huJ591 PSMA-CAR (for example,
having the amino acid sequence set forth in SEQ ID NO: 245). In an
exemplary embodiment, a nucleic acid of the present disclosure
comprises from 5' to 3': a nucleic acid sequence encoding
TGF.beta.RII-DN (for example, having the amino acid sequence set
forth in SEQ ID NO: 115), a nucleic acid sequence encoding a first
linker, a nucleic acid sequence encoding PD1-PTM-CD28 (for example,
having the amino acid sequence set forth in SEQ ID NO: 119), a
nucleic acid encoding a second linker, and a nucleic acid sequence
encoding a huJ591 PSMA-CAR (for example, having the amino acid
sequence set forth in SEQ ID NO: 247).
[0499] In some embodiments, a nucleic acid of the present
disclosure may be operably linked to a transcriptional control
element, e.g., a promoter, and enhancer, etc. Suitable promoter and
enhancer elements are known to those of skill in the art.
[0500] For expression in a bacterial cell, suitable promoters
include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P
and trc. For expression in a eukaryotic cell, suitable promoters
include, but are not limited to, light and/or heavy chain
immunoglobulin gene promoter and enhancer elements; cytomegalovirus
immediate early promoter; herpes simplex virus thymidine kinase
promoter; early and late SV40 promoters; promoter present in long
terminal repeats from a retrovirus; mouse metallothionein-I
promoter; and various art-known tissue specific promoters. Suitable
reversible promoters, including reversible inducible promoters are
known in the art. Such reversible promoters may be isolated and
derived from many organisms, e.g., eukaryotes and prokaryotes.
Modification of reversible promoters derived from a first organism
for use in a second organism, e.g., a first prokaryote and a second
a eukaryote, a first eukaryote and a second a prokaryote, etc., is
well known in the art.
[0501] Such reversible promoters, and systems based on such
reversible promoters but also comprising additional control
proteins, include, but are not limited to, alcohol regulated
promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter,
promoters responsive to alcohol transactivator proteins (AlcR),
etc.), tetracycline regulated promoters, (e.g., promoter systems
including TetActivators, TetON, TetOFF, etc.), steroid regulated
promoters (e.g., rat glucocorticoid receptor promoter systems,
human estrogen receptor promoter systems, retinoid promoter
systems, thyroid promoter systems, ecdysone promoter systems,
mifepristone promoter systems, etc.), metal regulated promoters
(e.g., metallothionein promoter systems, etc.),
pathogenesis-related regulated promoters (e.g., salicylic acid
regulated promoters, ethylene regulated promoters, benzothiadiazole
regulated promoters, etc.), temperature regulated promoters (e.g.,
heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat
shock promoter, etc.), light regulated promoters, synthetic
inducible promoters, and the like.
[0502] In some embodiments, the promoter is a CD8 cell-specific
promoter, a CD4 cell-specific promoter, a neutrophil-specific
promoter, or an NK-specific promoter. For example, a CD4 gene
promoter can be used; see, e.g., Salmon et al. Proc. Natl. Acad.
Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.
As another example, a CD8 gene promoter can be used. NK
cell-specific expression can be achieved by use of an NcrI (p46)
promoter; see, e.g., Eckelhart et al. Blood (2011) 117:1565.
[0503] For expression in a yeast cell, a suitable promoter is a
constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an
ENO promoter, a PYK1 promoter and the like; or a regulatable
promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2
promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a
MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter,
an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1
promoter, a TRP 1 promoter, a URA3 promoter, a LEU2 promoter, an
ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia).
Selection of the appropriate vector and promoter is well within the
level of ordinary skill in the art. Suitable promoters for use in
prokaryotic host cells include, but are not limited to, a
bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac
operon promoter; a hybrid promoter, e.g., a lac/tac hybrid
promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac
promoter; a trc promoter; a tac promoter, and the like; an araBAD
promoter; in vivo regulated promoters, such as an ssaG promoter or
a related promoter (see, e.g., U.S. Patent Publication No.
20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol.
(1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci.
USA (1992) 89(21): 10079-83), a nirB promoter (Harborne et al. Mol.
Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan et
al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine
(2004) 22:3243-3255; and Chatfield et al., Biotechnol. (1992)
10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter
(see, e.g., GenBank Accession Nos. AX798980, AX798961, and
AX798183); a stationary phase promoter, e.g., a dps promoter, an
spy promoter, and the like; a promoter derived from the
pathogenicity island SPI-2 (see, e.g., WO96/17951); an actA
promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002)
70:1087-1096); an rpsM promoter (see, e.g., Valdivia and Falkow
Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g., Hillen,
W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds),
Topics in Molecular and Structural Biology, Protein-Nucleic Acid
Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6
promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984)
12:7035); and the like. Suitable strong promoters for use in
prokaryotes such as Escherichia coli include, but are not limited
to Trc, Tac, T5, T7, and PLambda. Non-limiting examples of
operators for use in bacterial host cells include a lactose
promoter operator (LacI repressor protein changes conformation when
contacted with lactose, thereby preventing the Lad repressor
protein from binding to the operator), a tryptophan promoter
operator (when complexed with tryptophan, TrpR repressor protein
has a conformation that binds the operator; in the absence of
tryptophan, the TrpR repressor protein has a conformation that does
not bind to the operator), and a tac promoter operator (see, e.g.,
deBoer et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25).
[0504] Other examples of suitable promoters include the immediate
early cytomegalovirus (CMV) promoter sequence. This promoter
sequence is a strong constitutive promoter sequence capable of
driving high levels of expression of any polynucleotide sequence
operatively linked thereto. Other constitutive promoter sequences
may also be used, including, but not limited to a simian virus 40
(SV40) early promoter, a mouse mammary tumor virus (MMTV) or human
immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a
MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr
virus immediate early promoter, a Rous sarcoma virus promoter, the
EF-1 alpha promoter, as well as human gene promoters such as, but
not limited to, an actin promoter, a myosin promoter, a hemoglobin
promoter, and a creatine kinase promoter. Further, the invention
should not be limited to the use of constitutive promoters.
Inducible promoters are also contemplated as part of the invention.
The use of an inducible promoter provides a molecular switch
capable of turning on expression of the polynucleotide sequence
which it is operatively linked when such expression is desired, or
turning off the expression when expression is not desired. Examples
of inducible promoters include, but are not limited to a
metallothionine promoter, a glucocorticoid promoter, a progesterone
promoter, and a tetracycline promoter.
[0505] In some embodiments, the locus or construct or transgene
containing the suitable promoter is irreversibly switched through
the induction of an inducible system. Suitable systems for
induction of an irreversible switch are well known in the art,
e.g., induction of an irreversible switch may make use of a
Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et
al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of
which is incorporated herein by reference). Any suitable
combination of recombinase, endonuclease, ligase, recombination
sites, etc. known to the art may be used in generating an
irreversibly switchable promoter. Methods, mechanisms, and
requirements for performing site-specific recombination, described
elsewhere herein, find use in generating irreversibly switched
promoters and are well known in the art, see, e.g., Grindley et al.
Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular
Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.),
the disclosures of which are incorporated herein by reference.
[0506] In some embodiments, a nucleic acid of the present
disclosure further comprises a nucleic acid sequence encoding a
TCR/CAR inducible expression cassette. In one embodiment, the
TCR/CAR inducible expression cassette is for the production of a
transgenic polypeptide product that is released upon TCR/CAR
signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol.
Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015)
7(5): 535-544. In some embodiments, a nucleic acid of the present
disclosure further comprises a nucleic acid sequence encoding a
cytokine operably linked to a T-cell activation responsive
promoter. In some embodiments, the cytokine operably linked to a
T-cell activation responsive promoter is present on a separate
nucleic acid sequence. In one embodiment, the cytokine is
IL-12.
[0507] A nucleic acid of the present disclosure may be present
within an expression vector and/or a cloning vector. An expression
vector can include a selectable marker, an origin of replication,
and other features that provide for replication and/or maintenance
of the vector.
[0508] Suitable expression vectors include, e.g., plasmids, viral
vectors, and the like. Large numbers of suitable vectors and
promoters are known to those of skill in the art; many are
commercially available for generating a subject recombinant
construct. The following vectors are provided by way of example,
and should not be construed in anyway as limiting: Bacterial: pBs,
phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a,
pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A,
pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3,
pBPV, pMSG and pSVL (Pharmacia).
[0509] Expression vectors generally have convenient restriction
sites located near the promoter sequence to provide for the
insertion of nucleic acid sequences encoding heterologous proteins.
A selectable marker operative in the expression host may be
present. Suitable expression vectors include, but are not limited
to, viral vectors (e.g. viral vectors based on vaccinia virus;
poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol.
Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6:
515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92:
7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO
94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO
95/00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene
Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA
(1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci.
(1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690,
Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al.,
Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239,
Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al.,
Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad.
Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human
immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl.
Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol.
(1999) 73: 7812-7816); a retroviral vector (e.g., Murine Leukemia
Virus, spleen necrosis virus, and vectors derived from retroviruses
such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis
virus, human immunodeficiency virus, myeloproliferative sarcoma
virus, and mammary tumor virus); and the like.
[0510] Additional expression vectors suitable for use are, e.g.,
without limitation, a lentivirus vector, a gamma retrovirus vector,
a foamy virus vector, an adeno-associated virus vector, an
adenovirus vector, a pox virus vector, a herpes virus vector, an
engineered hybrid virus vector, a transposon mediated vector, and
the like. Viral vector technology is well known in the art and is
described, for example, in Sambrook et al., 2012, Molecular
Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor
Press, NY), and in other virology and molecular biology manuals.
Viruses, which are useful as vectors include, but are not limited
to, retroviruses, adenoviruses, adeno-associated viruses, herpes
viruses, and lentiviruses.
[0511] In general, a suitable vector contains an origin of
replication functional in at least one organism, a promoter
sequence, convenient restriction endonuclease sites, and one or
more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S.
Pat. No. 6,326,193).
[0512] In some embodiments, an expression vector (e.g., a
lentiviral vector) may be used to introduce the TCR/CAR and/or the
dominant negative receptor and/or switch receptor into an immune
cell or precursor thereof (e.g., a T cell). Accordingly, an
expression vector (e.g., a lentiviral vector) of the present
invention may comprise a nucleic acid encoding for a TCR/CAR and/or
the dominant negative receptor and/or switch receptor. In some
embodiments, the expression vector (e.g., lentiviral vector) will
comprise additional elements that will aid in the functional
expression of the TCR/CAR and/or the dominant negative receptor
and/or switch receptor encoded therein. In some embodiments, an
expression vector comprising a nucleic acid encoding for a TCR/CAR
and/or the dominant negative receptor and/or switch receptor
further comprises a mammalian promoter. In one embodiment, the
vector further comprises an elongation-factor-1-alpha promoter
(EF-1.alpha. promoter). Use of an EF-1.alpha. promoter may increase
the efficiency in expression of downstream transgenes (e.g., a
TCR/CAR and/or the dominant negative receptor and/or switch
receptor encoding nucleic acid sequence). Physiologic promoters
(e.g., an EF-1.alpha. promoter) may be less likely to induce
integration mediated genotoxicity, and may abrogate the ability of
the retroviral vector to transform stem cells. Other physiological
promoters suitable for use in a vector (e.g., lentiviral vector)
are known to those of skill in the art and may be incorporated into
a vector of the present invention. In some embodiments, the vector
(e.g., lentiviral vector) further comprises a non-requisite cis
acting sequence that may improve titers and gene expression. One
non-limiting example of a non-requisite cis acting sequence is the
central polypurine tract and central termination sequence
(cPPT/CTS) which is important for efficient reverse transcription
and nuclear import. Other non-requisite cis acting sequences are
known to those of skill in the art and may be incorporated into a
vector (e.g., lentiviral vector) of the present invention. In some
embodiments, the vector further comprises a posttranscriptional
regulatory element. Posttranscriptional regulatory elements may
improve RNA translation, improve transgene expression and stabilize
RNA transcripts. One example of a posttranscriptional regulatory
element is the woodchuck hepatitis virus posttranscriptional
regulatory element (WPRE). Accordingly, in some embodiments a
vector for the present invention further comprises a WPRE sequence.
Various posttranscriptional regulator elements are known to those
of skill in the art and may be incorporated into a vector (e.g.,
lentiviral vector) of the present invention. A vector of the
present invention may further comprise additional elements such as
a rev response element (RRE) for RNA transport, packaging
sequences, and 5' and 3' long terminal repeats (LTRs). The term
"long terminal repeat" or "LTR" refers to domains of base pairs
located at the ends of retroviral DNAs which comprise U3, R and U5
regions. LTRs generally provide functions required for the
expression of retroviral genes (e.g., promotion, initiation and
polyadenylation of gene transcripts) and to viral replication. In
one embodiment, a vector (e.g., lentiviral vector) of the present
invention includes a 3' U3 deleted LTR. Accordingly, a vector
(e.g., lentiviral vector) of the present invention may comprise any
combination of the elements described herein to enhance the
efficiency of functional expression of transgenes. For example, a
vector (e.g., lentiviral vector) of the present invention may
comprise a WPRE sequence, cPPT sequence, RRE sequence, 5'LTR, 3' U3
deleted LTR' in addition to a nucleic acid encoding for a TCR/CAR
and/or the dominant negative receptor and/or switch receptor.
[0513] Vectors of the present invention may be self-inactivating
vectors. As used herein, the term "self-inactivating vector" refers
to vectors in which the 3' LTR enhancer promoter region (U3 region)
has been modified (e.g., by deletion or substitution). A
self-inactivating vector may prevent viral transcription beyond the
first round of viral replication. Consequently, a self-inactivating
vector may be capable of infecting and then integrating into a host
genome (e.g., a mammalian genome) only once, and cannot be passed
further.
[0514] Accordingly, self-inactivating vectors may greatly reduce
the risk of creating a replication-competent virus.
[0515] In some embodiments, a nucleic acid of the present invention
may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro
synthesis of RNA are known to those of skill in the art; any known
method can be used to synthesize RNA comprising a sequence encoding
a TCR/CAR and/or the dominant negative receptor and/or switch
receptor of the present disclosure. Methods for introducing RNA
into a host cell are known in the art. See, e.g., Zhao et al.
Cancer Res. (2010) 15: 9053. Introducing RNA comprising a
nucleotide sequence encoding a TCR/CAR and/or the dominant negative
receptor and/or switch receptor of the present disclosure into a
host cell can be carried out in vitro or ex vivo or in vivo. For
example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte,
etc.) can be electroporated in vitro or ex vivo with RNA comprising
a nucleotide sequence encoding a TCR/CAR and/or the dominant
negative receptor and/or switch receptor of the present
disclosure.
[0516] In order to assess the expression of a polypeptide or
portions thereof, the expression vector to be introduced into a
cell may also contain either a selectable marker gene or a reporter
gene, or both, to facilitate identification and selection of
expressing cells from the population of cells sought to be
transfected or infected through viral vectors. In some embodiments,
the selectable marker may be carried on a separate piece of DNA and
used in a co-transfection procedure. Both selectable markers and
reporter genes may be flanked with appropriate regulatory sequences
to enable expression in the host cells. Useful selectable markers
include, without limitation, antibiotic-resistance genes.
[0517] Reporter genes are used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. In general, a reporter gene is a gene that is
not present in or expressed by the recipient organism or tissue and
that encodes a polypeptide whose expression is manifested by some
easily detectable property, e.g., enzymatic activity.
[0518] Expression of the reporter gene is assessed at a suitable
time after the DNA has been introduced into the recipient cells.
Suitable reporter genes may include, without limitation, genes
encoding luciferase, beta-galactosidase, chloramphenicol acetyl
transferase, secreted alkaline phosphatase, or the green
fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters
479: 79-82).
F. Modified Immune Cells
[0519] The present invention provides a modified immune cell or
precursor cell thereof (e.g., a T cell), comprising a CAR and/or a
dominant negative receptor and/or a switch receptor. Accordingly,
such modified cells possess the specificity directed by the CAR
that is expressed therein. For example, a modified cell of the
present invention comprising a PSMA-CAR possesses specificity for
PSMA on a target cell.
[0520] In some embodiments, a modified cell of the present
invention comprises a CAR. In one embodiment, a modified cell of
the present invention comprises a CAR having affinity for a
prostate-specific membrane antigen (PSMA) on a target cell. In some
embodiments, a modified cell of the present invention comprises a
dominant negative receptor and/or a switch receptor. In one
embodiment, a modified cell of the present invention comprises a
dominant negative receptor capable of reducing the effect of a
negative signal transduction molecule in the microenvironment. In
one embodiment, a modified cell of the present invention comprises
a switch receptor capable of reducing the effect of a negative
signal transduction molecule in the microenvironment, and
converting the negative signal into a positive signal within the
modified cell. In some embodiments, a modified cell of the present
invention comprises a CAR and a dominant negative receptor and/or a
switch receptor. In one embodiment, a modified cell of the present
invention comprises a CAR having affinity for PSMA on a target
cell, and a dominant negative receptor and/or a switch receptor.
Modified cells comprising a dominant negative receptor and/or a
switch receptor of the present invention are able to engage
negative signal transduction molecules (e.g., inhibitory ligands)
in the microenvironment by virtue of their respective extracellular
domains. In some embodiments, a modified cell of the present
invention comprising a dominant negative receptor is capable of
reducing the effect of a negative signal transduction molecule in
the microenvironment, wherein the dominant negative receptor
comprises an extracellular domain associated with the negative
signal. In some embodiments, a modified cell of the present
invention comprising a switch receptor is capable of converting the
effect of a negative signal transduction molecule in the
microenvironment into a positive signal, wherein the switch
receptor comprises an extracellular domain associated with the
negative signal and an intracellular domain associated with the
positive signal.
[0521] In an exemplary embodiment, a modified cell of the present
invention comprises a dominant negative receptor that is capable of
reducing the effect of a negative signal transduction molecule. In
one embodiment, a modified cell of the present invention comprises
TGF.beta.RII-DN.
[0522] In an exemplary embodiment, a modified cell of the present
invention comprises a switch receptor that is capable of converting
the effect of a negative signal transduction molecule into a
positive (e.g., activating) signal within the modified cell. In one
embodiment, a modified cell of the present invention comprises
PD1-CTM-CD28. In one embodiment, a modified cell of the present
invention comprises PD1.sup.A132L-PTM-CD28. In one embodiment, a
modified cell of the present invention comprises TIM3-CD28.
[0523] In an exemplary embodiment, a modified cell of the present
invention comprises a PSMA-CAR and a dominant negative receptor
that is capable of reducing the effect of a negative signal
transduction molecule. In one embodiment, a modified cell of the
present invention comprises a murine J591 PSMA-CAR and
TGF.beta.RII-DN. In one embodiment, a modified cell of the present
invention comprises a humanized J591 PSMA-CAR and TGF.beta.RII-DN.
In one embodiment, a modified cell of the present invention
comprises a human 1C3 PSMA-CAR and TGF.beta.RII-DN. In one
embodiment, a modified cell of the present invention comprises a
human 2A10 PSMA-CAR and TGF.beta.RII-DN. In one embodiment, a
modified cell of the present invention comprises a human 2F5
PSMA-CAR and TGF.beta.RII-DN. In one embodiment, a modified cell of
the present invention comprises a human 2C6 PSMA-CAR and
TGF.beta.RII-DN. Such modified cells (e.g., modified T cells) in
addition to having affinity for PSMA on a target cell, are capable
of reducing inhibitory TGF-.beta. signals from the microenvironment
they reside in.
[0524] In an exemplary embodiment, a modified cell of the present
invention comprises a PSMA-CAR and a switch receptor that is
capable of converting the inhibitory effect of a negative signal
transduction molecule into a positive signal within the modified
cell. In one embodiment, a modified cell of the present invention
comprises a murine J591 PSMA-CAR and PD1-CTM-CD28. In one
embodiment, a modified cell of the present invention comprises a
humanized J591 PSMA-CAR (huJ591 PSMA-CAR) and PD1-CTM-CD28. In one
embodiment, a modified cell of the present invention comprises a
human 1C3 PSMA-CAR and PD1-CTM-CD28. In one embodiment, a modified
cell of the present invention comprises a human 2A10 PSMA-CAR and
PD1-CTM-CD28. In one embodiment, a modified cell of the present
invention comprises a human 2F5 PSMA-CAR and PD1-CTM-CD28. In one
embodiment, a modified cell of the present invention comprises a
human 2C6 PSMA-CAR and PD1-CTM-CD28. In one embodiment, a modified
cell of the present invention comprises a murine J591 PSMA-CAR and
PD1-PTM-CD28. In one embodiment, a modified cell of the present
invention comprises a humanized J591 PSMA-CAR (huJ591 PSMA-CAR) and
PD1-PTM-CD28. In one embodiment, a modified cell of the present
invention comprises a human 1C3 PSMA-CAR and PD1-PTM-CD28. In one
embodiment, a modified cell of the present invention comprises a
human 2A10 PSMA-CAR and PD1-PTM-CD28. In one embodiment, a modified
cell of the present invention comprises a human 2F5 PSMA-CAR and
PD1-PTM-CD28. In one embodiment, a modified cell of the present
invention comprises a human 2C6 PSMA-CAR and PD1-PTM-CD28. In one
embodiment, a modified cell of the present invention comprises a
murine J591 PSMA-CAR and PD1.sup.A132L-PTM-CD28. In one embodiment,
a modified cell of the present invention comprises a humanized J591
PSMA-CAR (huJ591 PSMA-CAR) and PD1.sup.A132L-PTM-CD28. In one
embodiment, a modified cell of the present invention comprises a
human 1C3 PSMA-CAR and PD1.sup.A132L-PTM-CD28. In one embodiment, a
modified cell of the present invention comprises a human 2A10
PSMA-CAR and PD1.sup.A132L-PTM-CD28. In one embodiment, a modified
cell of the present invention comprises a human 2F5 PSMA-CAR and
PD1.sup.A132L-PTM-CD28. In one embodiment, a modified cell of the
present invention comprises a human 2C6 PSMA-CAR and
PD1.sup.A132L-PTM-CD28. In one embodiment, a modified cell of the
present invention comprises a murine J591 PSMA-CAR and TIM3-CD28.
In one embodiment, a modified cell of the present invention
comprises a humanized J591 PSMA-CAR (huJ591 PSMA-CAR) and
TIM3-CD28. In one embodiment, a modified cell of the present
invention comprises a human 1C3 PSMA-CAR and TIM3-CD28. In one
embodiment, a modified cell of the present invention comprises a
human 2A10 PSMA-CAR and TIM3-CD28. In one embodiment, a modified
cell of the present invention comprises a human 2F5 PSMA-CAR and
TIM3-CD28. In one embodiment, a modified cell of the present
invention comprises a human 2C6 PSMA-CAR and TIM3-CD28. In one
embodiment, a modified cell of the present invention comprises a
murine J591 PSMA-CAR and PD1-4-1BB. In one embodiment, a modified
cell of the present invention comprises a humanized J591 PSMA-CAR
(huJ591 PSMA-CAR) and PD1-4-1BB. In one embodiment, a modified cell
of the present invention comprises a human 1C3 PSMA-CAR and
PD1-4-1BB. In one embodiment, a modified cell of the present
invention comprises a human 2A10 PSMA-CAR and PD1-4-1BB. In one
embodiment, a modified cell of the present invention comprises a
human 2F5 PSMA-CAR and PD1-4-1BB. In one embodiment, a modified
cell of the present invention comprises a human 2C6 PSMA-CAR and
PD1-4-1BB. In one embodiment, a modified cell of the present
invention comprises a murine J591 PSMA-CAR and PD1.sup.A132L-4-1BB.
In one embodiment, a modified cell of the present invention
comprises a humanized J591 PSMA-CAR (huJ591 PSMA-CAR) and
PD1.sup.A132L-4-1BB. In one embodiment, a modified cell of the
present invention comprises a human 1C3 PSMA-CAR and
PD1.sup.A132L-4-1BB. In one embodiment, a modified cell of the
present invention comprises a human 2A10 PSMA-CAR and
PD1.sup.A132L-4-1BB. In one embodiment, a modified cell of the
present invention comprises a human 2F5 PSMA-CAR and
PD1.sup.A132L-4-1BB. In one embodiment, a modified cell of the
present invention comprises a human 2C6 PSMA-CAR and
PD1.sup.A132L-4-1BB. In one embodiment, a modified cell of the
present invention comprises a murine J591 PSMA-CAR and
TGF.beta.R-IL12R.beta.1. In one embodiment, a modified cell of the
present invention comprises a humanized J591 PSMA-CAR (huJ591
PSMA-CAR) and TGF.beta.R-IL12R.beta.1. In one embodiment, a
modified cell of the present invention comprises a human 1C3
PSMA-CAR and TGF.beta.R-IL12R.beta.1. In one embodiment, a modified
cell of the present invention comprises a human 2A10 PSMA-CAR and
TGF.beta.R-IL12R.beta.1. In one embodiment, a modified cell of the
present invention comprises a human 2F5 PSMA-CAR and
TGF.beta.R-IL12R.beta.1. In one embodiment, a modified cell of the
present invention comprises a human 2C6 PSMA-CAR and
TGF.beta.R-IL12R.beta.1. In one embodiment, a modified cell of the
present invention comprises a murine J591 PSMA-CAR and
TGF.beta.R-IL12.beta.2. In one embodiment, a modified cell of the
present invention comprises a humanized J591 PSMA-CAR (huJ591
PSMA-CAR) and TGF.beta.R-IL12R.beta.2. In one embodiment, a
modified cell of the present invention comprises a human 1C3
PSMA-CAR and TGF.beta.R-IL12R.beta.2. In one embodiment, a modified
cell of the present invention comprises a human 2A10 PSMA-CAR and
TGF.beta.R-IL12R.beta.2. In one embodiment, a modified cell of the
present invention comprises a human 2F5 PSMA-CAR and
TGF.beta.R-IL12R.beta.2. In one embodiment, a modified cell of the
present invention comprises a human 2C6 PSMA-CAR and
TGF.beta.R-IL12R.beta.2. Such modified cells (e.g., modified T
cells) in addition to having affinity for PSMA on a target cell,
are capable of converting inhibitory PD-1 or TGF.beta. signals from
the microenvironment into a positive (e.g., activating) signal
within the modified cell. Such modified cells (e.g., modified T
cells) in addition to having affinity for PSMA on a target cell,
are capable of converting inhibitory PD-1 or TIM-3 signals from the
microenvironment into a positive (e.g., activating) CD28 signal
within the modified cell.
[0525] In an exemplary embodiment, a modified cell of the present
invention comprises a murine J591 PSMA-CAR, TGF.beta.RII-DN, and
PD1-CTM-CD28. In an exemplary embodiment, a modified cell of the
present invention comprises a murine J591 PSMA-CAR,
TGF.beta.RII-DN, and PD1-PTM-CD28.
[0526] In an exemplary embodiment, a modified cell of the present
invention comprises a humanized J591 PSMA-CAR (huJ591 PSMA-CAR),
TGF.beta.RII-DN, and PD1-CTM-CD28. In an exemplary embodiment, a
modified cell of the present invention comprises a humanized J591
PSMA-CAR (huJ591 PSMA-CAR), TGF.beta.RII-DN, and PD1-PTM-CD28.
[0527] In an exemplary embodiment, a modified cell of the present
invention comprises a nucleic acid encoding a bispecific antibody.
In one embodiment, such modified cells can secrete the bispecific
antibody outside of the modified cell. In one embodiment, a
modified cell of the present invention comprises a nucleic acid
encoding a bispecific antibody, wherein the bispecific antibody
comprises more than one antigen binding domain, wherein at least
one antigen binding domain binds to a negative signal transduction
molecule (e.g., a negative signal transduction molecule found in
the microenvironment of the modified cell), and at least one
antigen binding domain binds a co-stimulatory molecule on the
surface of the modified cell. In one embodiment, a modified cell of
the present invention comprises a nucleic acid encoding a 13G4-1211
PD-L1/CD28 bispecific antibody as described herein. In one
embodiment, a modified cell of the present invention comprises a
nucleic acid encoding a 10A5-1412 PD-L1/CD28 bispecific antibody as
described herein. In one embodiment, a modified cell of the present
invention comprises a nucleic acid encoding a 1B12-1412 PD-L1/CD28
bispecific antibody as described herein. In one embodiment, a
modified cell of the present invention comprises a nucleic acid
encoding a TGF.beta.R-1-1412 TGF.beta.RII/CD28 bispecific antibody
as described herein. In one embodiment, a modified cell of the
present invention comprises a nucleic acid encoding a
TGF.beta.R-3-1412 TGF.beta.RII/CD28 bispecific antibody as
described herein.
[0528] In an exemplary embodiment, a modified cell of the present
invention comprises a PSMA-CAR, a dominant negative receptor and/or
a switch receptor, and may further comprise a nucleic acid encoding
a bispecific antibody. Such modified cells (e.g., modified T cells)
in addition to having affinity for PSMA on a target cell, are
capable of reducing inhibitory signals from the microenvironment
they reside in, and secreting the bispecific antibody into the
microenvironment they reside in. In such cells, the activity of the
bispecific antibody may further increase the activation of the
modified cell (e.g., modified T cell). In one embodiment, a
modified cell of the present invention comprises a PSMA-CAR
selected from the group consisting of a murine J591 PSMA-CAR, a
humanized J591 PSMA-CAR (huJ591 PSMA-CAR), a human 1C3 PSMA-CAR, a
human 2A10 PSMA-CAR, a human 2F5 PSMA-CAR, and a human 2C6
PSMA-CAR; TGF.beta.RII-DN; and expresses and secretes a bispecific
antibody selected from the group consisting of a 13G4-1211
PD-L1/CD28 bispecific antibody, a 10A5-1412 PD-L1/CD28 bispecific
antibody, a 1B12-1412 PD-L1/CD28 bispecific antibody, a
TGF.beta.R-1-1412 TGF.beta.RII/CD28 bispecific antibody, and a
TGF.beta.R-3-1412 TGF.beta.RII/CD28 bispecific antibody.
[0529] In an exemplary embodiment, a modified cell of the present
invention comprises a PSMA-CAR, a switch receptor, and may further
comprise a nucleic acid encoding a bispecific antibody. Such
modified cells (e.g., modified T cells) in addition to having
affinity for PSMA on a target cell, are capable of converting
inhibitory signals from the microenvironment they reside in into a
positive (e.g., activating) signal within the modified cell, and
secreting the bispecific antibody into the microenvironment they
reside in. In such cells, the activity of the bispecific antibody
may further increase the activation of the modified cell (e.g.,
modified T cell). In one embodiment, a modified cell of the present
invention comprises a PSMA-CAR selected from the group consisting
of a murine J591 PSMA-CAR, a humanized J591 PSMA-CAR (huJ591
PSMA-CAR), a human 1C3 PSMA-CAR, a human 2A10 PSMA-CAR, a human 2F5
PSMA-CAR, and a human 2C6 PSMA-CAR; a switch receptor selected from
the group consisting of a PD1-CTM-CD28 switch receptor, a
PD1A132L-PTM-CD28 switch receptor, and a TIM3-CD28 switch receptor;
and expresses and secretes a bispecific antibody selected from the
group consisting of a 13G4-1211 PD-L1/CD28 bispecific antibody, a
10A5-1412 PD-L1/CD28 bispecific antibody, a 1B12-1412 PD-L1/CD28
bispecific antibody, a TGF.beta.R-1-1412 TGF.beta.RII/CD28
bispecific antibody, and a TGF.beta.R-3-1412 TGF.beta.RII/CD28
bispecific antibody.
[0530] Any modified cell comprising a PSMA-CAR of the present
invention, a dominant negative receptor and/or a switch receptor of
the present invention, and/or expresses and secretes a bispecific
antibody of the present invention is envisioned, and can readily be
understood and made by a person of skill in the art in view of the
disclosure herein.
G. Methods of Producing Modified Immune Cells
[0531] The present invention provides methods for producing or
generating a modified immune cell or precursor thereof (e.g., a T
cell) of the invention for tumor immunotherapy, e.g., adoptive
immunotherapy. The cells generally are engineered by introducing
one or more nucleic acids encoding a subject CAR, dominant negative
receptor and/or switch receptor, and/or bispecific antibody, and/or
combinations thereof.
[0532] In some embodiments, one or more nucleic acids encoding the
subject CAR, dominant negative receptor and/or switch receptor,
and/or bispecific antibody is introduced into a cell by an
expression vector. Expression vectors comprising a nucleic acid
sequence encoding a subject CAR, dominant negative receptor and/or
switch receptor, and/or bispecific antibody, and/or combinations
thereof, of the present invention are provided herein. Suitable
expression vectors include lentivirus vectors, gamma retrovirus
vectors, foamy virus vectors, adeno associated virus (AAV) vectors,
adenovirus vectors, engineered hybrid viruses, naked DNA, including
but not limited to transposon mediated vectors, such as Sleeping
Beauty, Piggybak, and Integrases such as Phi31. Some other suitable
expression vectors include Herpes simplex virus (HSV) and
retrovirus expression vectors.
[0533] Adenovirus expression vectors are based on adenoviruses,
which have a low capacity for integration into genomic DNA but a
high efficiency for transfecting host cells. Adenovirus expression
vectors contain adenovirus sequences sufficient to: (a) support
packaging of the expression vector and (b) to ultimately express
the subject CAR, dominant negative receptor and/or switch receptor,
and/or bispecific antibody, and/or combinations thereof, in the
host cell. In some embodiments, the adenovirus genome is a 36 kb,
linear, double stranded DNA, where a foreign DNA sequence (e.g., a
nucleic acid encoding a subject CAR, dominant negative receptor
and/or switch receptor, and/or bispecific antibody, and/or
combinations thereof) may be inserted to substitute large pieces of
adenoviral DNA in order to make the expression vector of the
present invention (see, e.g., Danthinne and Imperiale, Gene Therapy
(2000) 7(20): 1707-1714).
[0534] Another expression vector is based on an adeno associated
virus, which takes advantage of the adenovirus coupled systems.
This AAV expression vector has a high frequency of integration into
the host genome. It can infect non-dividing cells, thus making it
useful for delivery of genes into mammalian cells, for example, in
tissue cultures or in vivo. The AAV vector has a broad host range
for infectivity. Details concerning the generation and use of AAV
vectors are described in U.S. Pat. Nos. 5,139,941 and
4,797,368.
[0535] Retrovirus expression vectors are capable of integrating
into the host genome, delivering a large amount of foreign genetic
material, infecting a broad spectrum of species and cell types and
being packaged in special cell lines. The retrovirus vector is
constructed by inserting a nucleic acid (e.g., a nucleic acid
encoding a subject CAR, dominant negative receptor and/or switch
receptor, and/or bispecific antibody, and/or combinations thereof)
into the viral genome at certain locations to produce a virus that
is replication defective. Though the retrovirus vectors are able to
infect a broad variety of cell types, integration and stable
expression of the subject CAR, dominant negative receptor and/or
switch receptor, and/or bispecific antibody, and/or combinations
thereof, requires the division of host cells.
[0536] Lentivirus vectors are derived from lentiviruses, which are
complex retroviruses that, in addition to the common retroviral
genes gag, pol, and env, contain other genes with regulatory or
structural function (see, e.g., U.S. Pat. Nos. 6,013,516 and
5,994,136). Some examples of lentiviruses include the Human
Immunodeficiency Viruses (HIV-1, HIV-2) and the Simian
Immunodeficiency Virus (SIV). Lentivirus vectors have been
generated by multiply attenuating the HIV virulence genes, for
example, the genes env, vif, vpr, vpu and nef are deleted making
the vector biologically safe. Lentivirus vectors are capable of
infecting non-dividing cells and can be used for both in vivo and
ex vivo gene transfer and expression, e.g., of a nucleic acid
encoding a subject CAR, dominant negative receptor and/or switch
receptor, and/or bispecific antibody, and/or combinations thereof
(see, e.g., U.S. Pat. No. 5,994,136).
[0537] Expression vectors including a nucleic acid of the present
disclosure can be introduced into a host cell by any means known to
persons skilled in the art. The expression vectors may include
viral sequences for transfection, if desired. Alternatively, the
expression vectors may be introduced by fusion, electroporation,
biolistics, transfection, lipofection, or the like. The host cell
may be grown and expanded in culture before introduction of the
expression vectors, followed by the appropriate treatment for
introduction and integration of the vectors. The host cells are
then expanded and may be screened by virtue of a marker present in
the vectors. Various markers that may be used are known in the art,
and may include hprt, neomycin resistance, thymidine kinase,
hygromycin resistance, etc. As used herein, the terms "cell," "cell
line," and "cell culture" may be used interchangeably. In some
embodiments, the host cell an immune cell or precursor thereof,
e.g., a T cell, an NK cell, or an NKT cell.
[0538] The present invention also provides genetically engineered
cells which include and stably express a subject CAR, dominant
negative receptor and/or switch receptor, and/or bispecific
antibody, and/or combinations thereof, of the present disclosure.
In some embodiments, the genetically engineered cells are
genetically engineered T-lymphocytes (T cells), naive T cells (TN),
memory T cells (for example, central memory T cells (TCM), effector
memory cells (TEM)), natural killer cells (NK cells), and
macrophages capable of giving rise to therapeutically relevant
progeny. In one embodiment, the genetically engineered cells are
autologous cells.
[0539] Modified cells (e.g., comprising a subject CAR, dominant
negative receptor and/or switch receptor, and/or expresses and
secretes a bispecific antibody, and/or combinations thereof) may be
produced by stably transfecting host cells with an expression
vector including a nucleic acid of the present disclosure.
Additional methods to generate a modified cell of the present
disclosure include, without limitation, chemical transformation
methods (e.g., using calcium phosphate, dendrimers, liposomes
and/or cationic polymers), non-chemical transformation methods
(e.g., electroporation, optical transformation, gene
electrotransfer and/or hydrodynamic delivery) and/or particle-based
methods (e.g., impalefection, using a gene gun and/or
magnetofection). Transfected cells expressing a subject CAR,
dominant negative receptor and/or switch receptor, and/or
bispecific antibody, and/or combinations thereof, of the present
disclosure may be expanded ex vivo.
[0540] Physical methods for introducing an expression vector into
host cells include calcium phosphate precipitation, lipofection,
particle bombardment, microinjection, electroporation, and the
like. Methods for producing cells including vectors and/or
exogenous nucleic acids are well-known in the art. See, e.g.,
Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, New York. Chemical methods for
introducing an expression vector into a host cell include colloidal
dispersion systems, such as macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes.
[0541] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP")
can be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Choi") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform may be used as the only
solvent since it is more readily evaporated than methanol.
"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed
lipid bilayers or aggregates. Liposomes can be characterized as
having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology
5: 505-10). Compositions that have different structures in solution
than the normal vesicular structure are also encompassed. For
example, the lipids may assume a micellar structure or merely exist
as non-uniform aggregates of lipid molecules. Also contemplated are
lipofectamine-nucleic acid complexes.
[0542] Regardless of the method used to introduce exogenous nucleic
acids into a host cell or otherwise expose a cell to the inhibitor
of the present invention, in order to confirm the presence of the
nucleic acids in the host cell, a variety of assays may be
performed. Such assays include, for example, molecular biology
assays well known to those of skill in the art, such as Southern
and Northern blotting, RT-PCR and PCR biochemistry assays, such as
detecting the presence or absence of a particular peptide, e.g., by
immunological means (ELISAs and Western blots) or by assays
described herein to identify agents falling within the scope of the
invention.
[0543] In one embodiment, the nucleic acids introduced into the
host cell are RNA. In another embodiment, the RNA is mRNA that
comprises in vitro transcribed RNA or synthetic RNA. The RNA may be
produced by in vitro transcription using a polymerase chain
reaction (PCR)-generated template. DNA of interest from any source
can be directly converted by PCR into a template for in vitro mRNA
synthesis using appropriate primers and RNA polymerase. The source
of the DNA may be, for example, genomic DNA, plasmid DNA, phage
DNA, cDNA, synthetic DNA sequence or any other appropriate source
of DNA.
[0544] PCR may be used to generate a template for in vitro
transcription of mRNA which is then introduced into cells. Methods
for performing PCR are well known in the art. Primers for use in
PCR are designed to have regions that are substantially
complementary to regions of the DNA to be used as a template for
the PCR. "Substantially complementary," as used herein, refers to
sequences of nucleotides where a majority or all of the bases in
the primer sequence are complementary. Substantially complementary
sequences are able to anneal or hybridize with the intended DNA
target under annealing conditions used for PCR. The primers can be
designed to be substantially complementary to any portion of the
DNA template. For example, the primers can be designed to amplify
the portion of a gene that is normally transcribed in cells (the
open reading frame), including 5' and 3' UTRs. The primers may also
be designed to amplify a portion of a gene that encodes a
particular domain of interest. In one embodiment, the primers are
designed to amplify the coding region of a human cDNA, including
all or portions of the 5' and 3' UTRs. Primers useful for PCR are
generated by synthetic methods that are well known in the art.
"Forward primers" are primers that contain a region of nucleotides
that are substantially complementary to nucleotides on the DNA
template that are upstream of the DNA sequence that is to be
amplified. "Upstream" is used herein to refer to a location 5, to
the DNA sequence to be amplified relative to the coding strand.
"Reverse primers" are primers that contain a region of nucleotides
that are substantially complementary to a double-stranded DNA
template that are downstream of the DNA sequence that is to be
amplified. "Downstream" is used herein to refer to a location 3' to
the DNA sequence to be amplified relative to the coding strand.
[0545] Chemical structures that have the ability to promote
stability and/or translation efficiency of the RNA may also be
used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the
5' UTR is between zero and 3000 nucleotides in length. The length
of 5' and 3' UTR sequences to be added to the coding region can be
altered by different methods, including, but not limited to,
designing primers for PCR that anneal to different regions of the
UTRs. Using this approach, one of ordinary skill in the art can
modify the 5' and 3' UTR lengths required to achieve optimal
translation efficiency following transfection of the transcribed
RNA.
[0546] The 5' and 3' UTRs can be the naturally occurring,
endogenous 5' and 3' UTRs for the gene of interest. Alternatively,
UTR sequences that are not endogenous to the gene of interest can
be added by incorporating the UTR sequences into the forward and
reverse primers or by any other modifications of the template. The
use of UTR sequences that are not endogenous to the gene of
interest can be useful for modifying the stability and/or
translation efficiency of the RNA. For example, it is known that
AU-rich elements in 3' UTR sequences can decrease the stability of
mRNA. Therefore, 3' UTRs can be selected or designed to increase
the stability of the transcribed RNA based on properties of UTRs
that are well known in the art.
[0547] In one embodiment, the 5' UTR can contain the Kozak sequence
of the endogenous gene. Alternatively, when a 5' UTR that is not
endogenous to the gene of interest is being added by PCR as
described above, a consensus Kozak sequence can be redesigned by
adding the 5' UTR sequence. Kozak sequences can increase the
efficiency of translation of some RNA transcripts, but does not
appear to be required for all RNAs to enable efficient translation.
The requirement for Kozak sequences for many mRNAs is known in the
art. In other embodiments the 5' UTR can be derived from an RNA
virus whose RNA genome is stable in cells. In other embodiments
various nucleotide analogues can be used in the 3' or 5' UTR to
impede exonuclease degradation of the mRNA.
[0548] To enable synthesis of RNA from a DNA template without the
need for gene cloning, a promoter of transcription should be
attached to the DNA template upstream of the sequence to be
transcribed. When a sequence that functions as a promoter for an
RNA polymerase is added to the 5' end of the forward primer, the
RNA polymerase promoter becomes incorporated into the PCR product
upstream of the open reading frame that is to be transcribed. In
one embodiment, the promoter is a T7 polymerase promoter, as
described elsewhere herein. Other useful promoters include, but are
not limited to, T3 and SP6 RNA polymerase promoters. Consensus
nucleotide sequences for T7, T3 and SP6 promoters are known in the
art.
[0549] In one embodiment, the mRNA has both a cap on the 5' end and
a 3' poly(A) tail which determine ribosome binding, initiation of
translation and stability mRNA in the cell. On a circular DNA
template, for instance, plasmid DNA, RNA polymerase produces a long
concatameric product which is not suitable for expression in
eukaryotic cells. The transcription of plasmid DNA linearized at
the end of the 3' UTR results in normal sized mRNA which is not
effective in eukaryotic transfection even if it is polyadenylated
after transcription.
[0550] On a linear DNA template, phage T7 RNA polymerase can extend
the 3' end of the transcript beyond the last base of the template
(Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985);
Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65
(2003).
[0551] The polyA/T segment of the transcriptional DNA template can
be produced during PCR by using a reverse primer containing a polyT
tail, such as 100T tail (size can be 50-5000 T), or after PCR by
any other method, including, but not limited to, DNA ligation or in
vitro recombination. Poly(A) tails also provide stability to RNAs
and reduce their degradation. Generally, the length of a poly(A)
tail positively correlates with the stability of the transcribed
RNA. In one embodiment, the poly(A) tail is between 100 and 5000
adenosines.
[0552] Poly(A) tails of RNAs can be further extended following in
vitro transcription with the use of a poly(A) polymerase, such as
E. coli polyA polymerase (E-PAP). In one embodiment, increasing the
length of a poly(A) tail from 100 nucleotides to between 300 and
400 nucleotides results in about a two-fold increase in the
translation efficiency of the RNA. Additionally, the attachment of
different chemical groups to the 3' end can increase mRNA
stability. Such attachment can contain modified/artificial
nucleotides, aptamers and other compounds. For example, ATP analogs
can be incorporated into the poly(A) tail using poly(A) polymerase.
ATP analogs can further increase the stability of the RNA.
[0553] 5' caps also provide stability to RNA molecules. In a
preferred embodiment, RNAs produced by the methods disclosed herein
include a 5' cap. The 5' cap is provided using techniques known in
the art and described herein (Cougot, et al., Trends in Biochem.
Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001);
Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966
(2005)).
[0554] In some embodiments, the RNA is electroporated into the
cells, such as in vitro transcribed RNA. Any solutes suitable for
cell electroporation, which can contain factors facilitating
cellular permeability and viability such as sugars, peptides,
lipids, proteins, antioxidants, and surfactants can be
included.
[0555] In some embodiments, a nucleic acid encoding a subject CAR,
dominant negative receptor and/or switch receptor, and/or
bispecific antibody, and/or combinations thereof, of the present
disclosure will be RNA, e.g., in vitro synthesized RNA. Methods for
in vitro synthesis of RNA are known in the art; any known method
can be used to synthesize RNA comprising a sequence encoding a
subject CAR, dominant negative receptor and/or switch receptor,
and/or bispecific antibody, and/or combinations thereof. Methods
for introducing RNA into a host cell are known in the art. See,
e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA
comprising a nucleotide sequence encoding a subject CAR, dominant
negative receptor and/or switch receptor, and/or bispecific
antibody, and/or combinations thereof, into a host cell can be
carried out in vitro or ex vivo or in vivo. For example, a host
cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be
electroporated in vitro or ex vivo with RNA comprising a nucleotide
sequence encoding a subject CAR, dominant negative receptor and/or
switch receptor, and/or bispecific antibody, and/or combinations
thereof.
[0556] The disclosed methods can be applied to the modulation of T
cell activity in basic research and therapy, in the fields of
cancer, stem cells, acute and chronic infections, and autoimmune
diseases, including the assessment of the ability of the
genetically modified T cell to kill a target cancer cell.
[0557] The methods also provide the ability to control the level of
expression over a wide range by changing, for example, the promoter
or the amount of input RNA, making it possible to individually
regulate the expression level. Furthermore, the PCR-based technique
of mRNA production greatly facilitates the design of the mRNAs with
different structures and combination of their domains.
[0558] One advantage of RNA transfection methods of the invention
is that RNA transfection is essentially transient and a
vector-free. A RNA transgene can be delivered to a lymphocyte and
expressed therein following a brief in vitro cell activation, as a
minimal expressing cassette without the need for any additional
viral sequences. Under these conditions, integration of the
transgene into the host cell genome is unlikely. Cloning of cells
is not necessary because of the efficiency of transfection of the
RNA and its ability to uniformly modify the entire lymphocyte
population.
[0559] Genetic modification of T cells with in vitro-transcribed
RNA (IVT-RNA) makes use of two different strategies both of which
have been successively tested in various animal models. Cells are
transfected with in vitro-transcribed RNA by means of lipofection
or electroporation. It is desirable to stabilize IVT-RNA using
various modifications in order to achieve prolonged expression of
transferred IVT-RNA.
[0560] Some IVT vectors are known in the literature which are
utilized in a standardized manner as template for in vitro
transcription and which have been genetically modified in such a
way that stabilized RNA transcripts are produced. Currently
protocols used in the art are based on a plasmid vector with the
following structure: a 5' RNA polymerase promoter enabling RNA
transcription, followed by a gene of interest which is flanked
either 3' and/or 5' by untranslated regions (UTR), and a 3'
polyadenyl cassette containing 50-70 A nucleotides. Prior to in
vitro transcription, the circular plasmid is linearized downstream
of the polyadenyl cassette by type II restriction enzymes
(recognition sequence corresponds to cleavage site). The polyadenyl
cassette thus corresponds to the later poly(A) sequence in the
transcript. As a result of this procedure, some nucleotides remain
as part of the enzyme cleavage site after linearization and extend
or mask the poly(A) sequence at the 3' end. It is not clear,
whether this nonphysiological overhang affects the amount of
protein produced intracellularly from such a construct.
[0561] In another aspect, the RNA construct is delivered into the
cells by electroporation. See, e.g., the formulations and
methodology of electroporation of nucleic acid constructs into
mammalian cells as taught in US 2004/0014645, US 2005/0052630A1, US
2005/0070841A1, US 2004/0059285A1, US 2004/0092907A1. The various
parameters including electric field strength required for
electroporation of any known cell type are generally known in the
relevant research literature as well as numerous patents and
applications in the field. See e.g., U.S. Pat. Nos. 6,678,556,
7,171,264, and 7,173,116. Apparatus for therapeutic application of
electroporation are available commercially, e.g., the MedPulser.TM.
DNA Electroporation Therapy System (Inovio/Genetronics, San Diego,
Calif.), and are described in patents such as U.S. Pat. Nos.
6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and
6,233,482; electroporation may also be used for transfection of
cells in vitro as described e.g. in US20070128708A1.
Electroporation may also be utilized to deliver nucleic acids into
cells in vitro. Accordingly, electroporation-mediated
administration into cells of nucleic acids including expression
constructs utilizing any of the many available devices and
electroporation systems known to those of skill in the art presents
an exciting new means for delivering an RNA of interest to a target
cell.
[0562] In some embodiments, the immune cells (e.g. T cells) can be
incubated or cultivated prior to, during and/or subsequent to
introducing the nucleic acid molecule encoding the subject CAR,
dominant negative receptor and/or switch receptor, and/or
bispecific antibody, and/or combinations thereof. In some
embodiments, the cells (e.g. T cells) can be incubated or
cultivated prior to, during or subsequent to the introduction of
the nucleic acid molecule encoding the subject CAR, dominant
negative receptor and/or switch receptor, and/or bispecific
antibody, and/or combinations thereof, such as prior to, during or
subsequent to the transduction of the cells with a viral vector
(e.g. lentiviral vector) encoding the subject CAR, dominant
negative receptor and/or switch receptor, and/or bispecific
antibody, and/or combinations thereof. In some embodiments, the
method includes activating or stimulating cells with a stimulating
or activating agent (e.g. anti-CD3/anti-CD28 antibodies) prior to
introducing the nucleic acid molecule encoding the subject CAR,
dominant negative receptor and/or switch receptor, and/or
bispecific antibody, and/or combinations thereof.
[0563] In some embodiments, where the nucleic acid sequences
encoding the subject CAR, dominant negative receptor and/or switch
receptor, and/or bispecific antibody, and/or combinations thereof,
of the present invention reside on one or more separate nucleic
acid sequences, the order of introducing each of the one or more
nucleic acid sequences may vary.
[0564] For example, a nucleic acid sequence encoding a subject CAR
and dominant negative receptor and/or switch receptor may first be
introduced into the host cell, followed by introduction of a
nucleic acid sequence encoding a subject bispecific antibody. For
example, a nucleic acid sequence encoding a subject bispecific
antibody may first be introduced into the host cell, followed by
introduction of a nucleic acid sequence encoding a subject CAR and
dominant negative receptor and/or switch receptor. In some
embodiments, each of the one or more nucleic acid sequences are
introduced into the host cell simultaneously. Those of skill in the
art will be able to determine the order in which each of the one or
more nucleic acid sequences are introduced into the host cell.
H. Sources of Immune Cells
[0565] Prior to expansion, a source of immune cells is obtained
from a subject for ex vivo manipulation. Sources of target cells
for ex vivo manipulation may also include, e.g., autologous or
heterologous donor blood, cord blood, or bone marrow. For example,
the source of immune cells may be from the subject to be treated
with the modified immune cells of the invention, e.g., the
subject's blood, the subject's cord blood, or the subject's bone
marrow. Non-limiting examples of subjects include humans, dogs,
cats, mice, rats, and transgenic species thereof. Preferably, the
subject is a human.
[0566] Immune cells can be obtained from a number of sources,
including blood, peripheral blood mononuclear cells, bone marrow,
lymph node tissue, spleen tissue, umbilical cord, lymph, or
lymphoid organs. Immune cells are cells of the immune system, such
as cells of the innate or adaptive immunity, e.g., myeloid or
lymphoid cells, including lymphocytes, typically T cells and/or NK
cells. Other exemplary cells include stem cells, such as
multipotent and pluripotent stem cells, including induced
pluripotent stem cells (iPSCs). In some aspects, the cells are
human cells. With reference to the subject to be treated, the cells
may be allogeneic and/or autologous. The cells typically are
primary cells, such as those isolated directly from a subject
and/or isolated from a subject and frozen.
[0567] In certain embodiments, the immune cell is a T cell, e.g., a
CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or
effector memory T cell), a CD4+ T cell, a natural killer T cell
(NKT cells), a regulatory T cell (Treg), a stem cell memory T cell,
a lymphoid progenitor cell a hematopoietic stem cell, a natural
killer cell (NK cell) or a dendritic cell. In some embodiments, the
cells are monocytes or granulocytes, e.g., myeloid cells,
macrophages, neutrophils, dendritic cells, mast cells, eosinophils,
and/or basophils. In an embodiment, the target cell is an induced
pluripotent stem (iPS) cell or a cell derived from an iPS cell,
e.g., an iPS cell generated from a subject, manipulated to alter
(e.g., induce a mutation in) or manipulate the expression of one or
more target genes, and differentiated into, e.g., a T cell, e.g., a
CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or
effector memory T cell), a CD4+ T cell, a stem cell memory T cell,
a lymphoid progenitor cell or a hematopoietic stem cell.
[0568] In some embodiments, the cells include one or more subsets
of T cells or other cell types, such as whole T cell populations,
CD4+ cells, CD8+ cells, and subpopulations thereof, such as those
defined by function, activation state, maturity, potential for
differentiation, expansion, recirculation, localization, and/or
persistence capacities, antigen-specificity, type of antigen
receptor, presence in a particular organ or compartment, marker or
cytokine secretion profile, and/or degree of differentiation. Among
the sub-types and subpopulations of T cells and/or of CD4+ and/or
of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF),
memory T cells and sub-types thereof, such as stem cell memory T
(TSCM), central memory T (TCM), effector memory T (TEM), or
terminally differentiated effector memory T cells,
tumor-infiltrating lymphocytes (TIL), immature T cells, mature T
cells, helper T cells, cytotoxic T cells, mucosa-associated
invariant T (MAIT) cells, naturally occurring and adaptive
regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2
cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular
helper T cells, alpha/beta T cells, and delta/gamma T cells. In
certain embodiments, any number of T cell lines available in the
art, may be used.
[0569] In some embodiments, the methods include isolating immune
cells from the subject, preparing, processing, culturing, and/or
engineering them. In some embodiments, preparation of the
engineered cells includes one or more culture and/or preparation
steps. The cells for engineering as described may be isolated from
a sample, such as a biological sample, e.g., one obtained from or
derived from a subject. In some embodiments, the subject from which
the cell is isolated is one having the disease or condition or in
need of a cell therapy or to which cell therapy will be
administered. The subject in some embodiments is a human in need of
a particular therapeutic intervention, such as the adoptive cell
therapy for which cells are being isolated, processed, and/or
engineered. Accordingly, the cells in some embodiments are primary
cells, e.g., primary human cells. The samples include tissue,
fluid, and other samples taken directly from the subject, as well
as samples resulting from one or more processing steps, such as
separation, centrifugation, genetic engineering (e.g. transduction
with viral vector), washing, and/or incubation. The biological
sample can be a sample obtained directly from a biological source
or a sample that is processed. Biological samples include, but are
not limited to, body fluids, such as blood, plasma, serum,
cerebrospinal fluid, synovial fluid, urine and sweat, tissue and
organ samples, including processed samples derived therefrom.
[0570] In some aspects, the sample from which the cells are derived
or isolated is blood or a blood-derived sample, or is or is derived
from an apheresis or leukapheresis product. Exemplary samples
include whole blood, peripheral blood mononuclear cells (PBMCs),
leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia,
lymphoma, lymph node, gut associated lymphoid tissue, mucosa
associated lymphoid tissue, spleen, other lymphoid tissues, liver,
lung, stomach, intestine, colon, kidney, pancreas, breast, bone,
prostate, cervix, testes, ovaries, tonsil, or other organ, and/or
cells derived therefrom. Samples include, in the context of cell
therapy, e.g., adoptive cell therapy, samples from autologous and
allogeneic sources.
[0571] In some embodiments, the cells are derived from cell lines,
e.g., T cell lines. The cells in some embodiments are obtained from
a xenogeneic source, for example, from mouse, rat, non-human
primate, and pig. In some embodiments, isolation of the cells
includes one or more preparation and/or non-affinity based cell
separation steps. In some examples, cells are washed, centrifuged,
and/or incubated in the presence of one or more reagents, for
example, to remove unwanted components, enrich for desired
components, lyse or remove cells sensitive to particular reagents.
In some examples, cells are separated based on one or more
property, such as density, adherent properties, size, sensitivity
and/or resistance to particular components.
[0572] In some examples, cells from the circulating blood of a
subject are obtained, e.g., by apheresis or leukapheresis. The
samples, in some aspects, contain lymphocytes, including T cells,
monocytes, granulocytes, B cells, other nucleated white blood
cells, red blood cells, and/or platelets, and in some aspects
contains cells other than red blood cells and platelets. In some
embodiments, the blood cells collected from the subject are washed,
e.g., to remove the plasma fraction and to place the cells in an
appropriate buffer or media for subsequent processing steps. In
some embodiments, the cells are washed with phosphate buffered
saline (PBS). In some aspects, a washing step is accomplished by
tangential flow filtration (TFF) according to the manufacturer's
instructions. In some embodiments, the cells are resuspended in a
variety of biocompatible buffers after washing. In certain
embodiments, components of a blood cell sample are removed and the
cells directly resuspended in culture media. In some embodiments,
the methods include density-based cell separation methods, such as
the preparation of white blood cells from peripheral blood by
lysing the red blood cells and centrifugation through a Percoll or
Ficoll gradient.
[0573] In one embodiment, immune are obtained cells from the
circulating blood of an individual are obtained by apheresis or
leukapheresis. The apheresis product typically contains
lymphocytes, including T cells, monocytes, granulocytes, B cells,
other nucleated white blood cells, red blood cells, and platelets.
The cells collected by apheresis may be washed to remove the plasma
fraction and to place the cells in an appropriate buffer or media,
such as phosphate buffered saline (PBS) or wash solution lacks
calcium and may lack magnesium or may lack many if not all divalent
cations, for subsequent processing steps. After washing, the cells
may be resuspended in a variety of biocompatible buffers, such as,
for example, Ca-free, Mg-free PBS. Alternatively, the undesirable
components of the apheresis sample may be removed and the cells
directly resuspended in culture media.
[0574] In some embodiments, the isolation methods include the
separation of different cell types based on the expression or
presence in the cell of one or more specific molecules, such as
surface markers, e.g., surface proteins, intracellular markers, or
nucleic acid. In some embodiments, any known method for separation
based on such markers may be used. In some embodiments, the
separation is affinity- or immunoaffinity-based separation. For
example, the isolation in some aspects includes separation of cells
and cell populations based on the cells' expression or expression
level of one or more markers, typically cell surface markers, for
example, by incubation with an antibody or binding partner that
specifically binds to such markers, followed generally by washing
steps and separation of cells having bound the antibody or binding
partner, from those cells having not bound to the antibody or
binding partner.
[0575] Such separation steps can be based on positive selection, in
which the cells having bound the reagents are retained for further
use, and/or negative selection, in which the cells having not bound
to the antibody or binding partner are retained. In some examples,
both fractions are retained for further use. In some aspects,
negative selection can be particularly useful where no antibody is
available that specifically identifies a cell type in a
heterogeneous population, such that separation is best carried out
based on markers expressed by cells other than the desired
population. The separation need not result in 100% enrichment or
removal of a particular cell population or cells expressing a
particular marker. For example, positive selection of or enrichment
for cells of a particular type, such as those expressing a marker,
refers to increasing the number or percentage of such cells, but
need not result in a complete absence of cells not expressing the
marker. Likewise, negative selection, removal, or depletion of
cells of a particular type, such as those expressing a marker,
refers to decreasing the number or percentage of such cells, but
need not result in a complete removal of all such cells.
[0576] In some examples, multiple rounds of separation steps are
carried out, where the positively or negatively selected fraction
from one step is subjected to another separation step, such as a
subsequent positive or negative selection. In some examples, a
single separation step can deplete cells expressing multiple
markers simultaneously, such as by incubating cells with a
plurality of antibodies or binding partners, each specific for a
marker targeted for negative selection. Likewise, multiple cell
types can simultaneously be positively selected by incubating cells
with a plurality of antibodies or binding partners expressed on the
various cell types.
[0577] In some embodiments, one or more of the T cell populations
is enriched for or depleted of cells that are positive for
(marker+) or express high levels (marker.sup.high) of one or more
particular markers, such as surface markers, or that are negative
for (marker.sup.-) or express relatively low levels
(marker.sup.low) of one or more markers. For example, in some
aspects, specific subpopulations of T cells, such as cells positive
or expressing high levels of one or more surface markers, e.g.,
CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or
CD45RO+ T cells, are isolated by positive or negative selection
techniques. In some cases, such markers are those that are absent
or expressed at relatively low levels on certain populations of T
cells (such as non-memory cells) but are present or expressed at
relatively higher levels on certain other populations of T cells
(such as memory cells). In one embodiment, the cells (such as the
CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for
(i.e., positively selected for) cells that are positive or
expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44,
CD 127, and/or CD62L and/or depleted of (e.g., negatively selected
for) cells that are positive for or express high surface levels of
CD45RA. In some embodiments, cells are enriched for or depleted of
cells positive or expressing high surface levels of CD 122, CD95,
CD25, CD27, and/or IL7-Ra (CD 127). In some examples, CD8+ T cells
are enriched for cells positive for CD45RO (or negative for CD45RA)
and for CD62L. For example, CD3+, CD28+ T cells can be positively
selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS@
M-450 CD3/CD28 T Cell Expander).
[0578] In some embodiments, T cells are separated from a PBMC
sample by negative selection of markers expressed on non-T cells,
such as B cells, monocytes, or other white blood cells, such as
CD14. In some aspects, a CD4+ or CD8+ selection step is used to
separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+
populations can be further sorted into sub-populations by positive
or negative selection for markers expressed or expressed to a
relatively higher degree on one or more naive, memory, and/or
effector T cell subpopulations. In some embodiments, CD8+ cells are
further enriched for or depleted of naive, central memory, effector
memory, and/or central memory stem cells, such as by positive or
negative selection based on surface antigens associated with the
respective subpopulation. In some embodiments, enrichment for
central memory T (TCM) cells is carried out to increase efficacy,
such as to improve long-term survival, expansion, and/or
engraftment following administration, which in some aspects is
particularly robust in such sub-populations. In some embodiments,
combining TCM-enriched CD8+ T cells and CD4+ T cells further
enhances efficacy.
[0579] In embodiments, memory T cells are present in both CD62L+
and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can
be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+
fractions, such as using anti-CD8 and anti-CD62L antibodies. In
some embodiments, a CD4+ T cell population and a CD8+ T cell
sub-population, e.g., a sub-population enriched for central memory
(TCM) cells. In some embodiments, the enrichment for central memory
T (TCM) cells is based on positive or high surface expression of
CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it
is based on negative selection for cells expressing or highly
expressing CD45RA and/or granzyme B. In some aspects, isolation of
a CD8+ population enriched for TCM cells is carried out by
depletion of cells expressing CD4, CD 14, CD45RA, and positive
selection or enrichment for cells expressing CD62L. In one aspect,
enrichment for central memory T (TCM) cells is carried out starting
with a negative fraction of cells selected based on CD4 expression,
which is subjected to a negative selection based on expression of
CD 14 and CD45RA, and a positive selection based on CD62L. Such
selections in some aspects are carried out simultaneously and in
other aspects are carried out sequentially, in either order. In
some aspects, the same CD4 expression-based selection step used in
preparing the CD8+ cell population or subpopulation, also is used
to generate the CD4+ cell population or sub-population, such that
both the positive and negative fractions from the CD4-based
separation are retained and used in subsequent steps of the
methods, optionally following one or more further positive or
negative selection steps.
[0580] CD4+ T helper cells are sorted into naive, central memory,
and effector cells by identifying cell populations that have cell
surface antigens. CD4+ lymphocytes can be obtained by standard
methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-,
CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory
CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector
CD4+ cells are CD62L- and CD45RO. In one example, to enrich for
CD4+ cells by negative selection, a monoclonal antibody cocktail
typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR,
and CD8. In some embodiments, the antibody or binding partner is
bound to a solid support or matrix, such as a magnetic bead or
paramagnetic bead, to allow for separation of cells for positive
and/or negative selection.
[0581] In some embodiments, the cells are incubated and/or cultured
prior to or in connection with genetic engineering. The incubation
steps can include culture, cultivation, stimulation, activation,
and/or propagation. In some embodiments, the compositions or cells
are incubated in the presence of stimulating conditions or a
stimulatory agent. Such conditions include those designed to induce
proliferation, expansion, activation, and/or survival of cells in
the population, to mimic antigen exposure, and/or to prime the
cells for genetic engineering, such as for the introduction of a
recombinant antigen receptor. The conditions can include one or
more of particular media, temperature, oxygen content, carbon
dioxide content, time, agents, e.g., nutrients, amino acids,
antibiotics, ions, and/or stimulatory factors, such as cytokines,
chemokines, antigens, binding partners, fusion proteins,
recombinant soluble receptors, and any other agents designed to
activate the cells. In some embodiments, the stimulating conditions
or agents include one or more agent, e.g., ligand, which is capable
of activating an intracellular signaling domain of a TCR complex.
In some aspects, the agent turns on or initiates TCR/CD3
intracellular signaling cascade in a T cell. Such agents can
include antibodies, such as those specific for a TCR component
and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for
example, bound to solid support such as a bead, and/or one or more
cytokines. Optionally, the expansion method may further comprise
the step of adding anti-CD3 and/or anti CD28 antibody to the
culture medium (e.g., at a concentration of at least about 0.5
ng/ml). In some embodiments, the stimulating agents include IL-2
and/or IL-15, for example, an IL-2 concentration of at least about
10 units/mL.
[0582] In another embodiment, T cells are isolated from peripheral
blood by lysing the red blood cells and depleting the monocytes,
for example, by centrifugation through a PERCOLL.TM. gradient.
Alternatively, T cells can be isolated from an umbilical cord. In
any event, a specific subpopulation of T cells can be further
isolated by positive or negative selection techniques.
[0583] The cord blood mononuclear cells so isolated can be depleted
of cells expressing certain antigens, including, but not limited
to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can
be accomplished using an isolated antibody, a biological sample
comprising an antibody, such as ascites, an antibody bound to a
physical support, and a cell bound antibody.
[0584] Enrichment of a T cell population by negative selection can
be accomplished using a combination of antibodies directed to
surface markers unique to the negatively selected cells. A
preferred method is cell sorting and/or selection via negative
magnetic immunoadherence or flow cytometry that uses a cocktail of
monoclonal antibodies directed to cell surface markers present on
the cells negatively selected. For example, to enrich for CD4.sup.+
cells by negative selection, a monoclonal antibody cocktail
typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR,
and CD8.
[0585] For isolation of a desired population of cells by positive
or negative selection, the concentration of cells and surface
(e.g., particles such as beads) can be varied. In certain
embodiments, it may be desirable to significantly decrease the
volume in which beads and cells are mixed together (i.e., increase
the concentration of cells), to ensure maximum contact of cells and
beads. For example, in one embodiment, a concentration of 2 billion
cells/ml is used. In one embodiment, a concentration of 1 billion
cells/ml is used. In a further embodiment, greater than 100 million
cells/ml is used. In a further embodiment, a concentration of cells
of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
In yet another embodiment, a concentration of cells from 75, 80,
85, 90, 95, or 100 million cells/ml is used. In further
embodiments, concentrations of 125 or 150 million cells/ml can be
used. Using high concentrations can result in increased cell yield,
cell activation, and cell expansion.
[0586] T cells can also be frozen after the washing step, which
does not require the monocyte-removal step. While not wishing to be
bound by theory, the freeze and subsequent thaw step provides a
more uniform product by removing granulocytes and to some extent
monocytes in the cell population. After the washing step that
removes plasma and platelets, the cells may be suspended in a
freezing solution. While many freezing solutions and parameters are
known in the art and will be useful in this context, in a
non-limiting example, one method involves using PBS containing 20%
DMSO and 8% human serum albumin, or other suitable cell freezing
media. The cells are then frozen to -80.degree. C. at a rate of
1.degree. C. per minute and stored in the vapor phase of a liquid
nitrogen storage tank. Other methods of controlled freezing may be
used as well as uncontrolled freezing immediately at -20.degree. C.
or in liquid nitrogen.
[0587] In one embodiment, the population of T cells is comprised
within cells such as peripheral blood mononuclear cells, cord blood
cells, a purified population of T cells, and a T cell line. In
another embodiment, peripheral blood mononuclear cells comprise the
population of T cells. In yet another embodiment, purified T cells
comprise the population of T cells.
[0588] In certain embodiments, T regulatory cells (Tregs) can be
isolated from a sample. The sample can include, but is not limited
to, umbilical cord blood or peripheral blood. In certain
embodiments, the Tregs are isolated by flow-cytometry sorting. The
sample can be enriched for Tregs prior to isolation by any means
known in the art. The isolated Tregs can be cryopreserved, and/or
expanded prior to use. Methods for isolating Tregs are described in
U.S. Pat. Nos. 7,754,482, 8,722,400, and 9,555,105, and U.S. patent
application Ser. No. 13/639,927, contents of which are incorporated
herein in their entirety.
I. Expansion of Immune Cells
[0589] Whether prior to or after modification of cells to express a
subject CAR, dominant negative receptor, and/or switch receptor,
and/or bispecific antibody, and/or combinations thereof, the cells
can be activated and expanded in number using methods as described,
for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680;
6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318;
7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514;
6,867,041; and U.S. Publication No. 20060121005. For example, the T
cells of the invention may be expanded by contact with a surface
having attached thereto an agent that stimulates a CD3/TCR complex
associated signal and a ligand that stimulates a co-stimulatory
molecule on the surface of the T cells. In particular, T cell
populations may be stimulated by contact with an anti-CD3 antibody,
or an antigen-binding fragment thereof, or an anti-CD2 antibody
immobilized on a surface, or by contact with a protein kinase C
activator (e.g., bryostatin) in conjunction with a calcium
ionophore. For co-stimulation of an accessory molecule on the
surface of the T cells, a ligand that binds the accessory molecule
is used. For example, T cells can be contacted with an anti-CD3
antibody and an anti-CD28 antibody, under conditions appropriate
for stimulating proliferation of the T cells. Examples of an
anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon,
France) and these can be used in the invention, as can other
methods and reagents known in the art (see, e.g., ten Berge et al.,
Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp.
Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol.
Methods (1999) 227(1-2): 53-63).
[0590] Expanding T cells by the methods disclosed herein can be
multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60
fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400
fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold,
2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold,
8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold,
10,000,000 fold, or greater, and any and all whole or partial
integers therebetween. In one embodiment, the T cells expand in the
range of about 20 fold to about 50 fold.
[0591] Following culturing, the T cells can be incubated in cell
medium in a culture apparatus for a period of time or until the
cells reach confluency or high cell density for optimal passage
before passing the cells to another culture apparatus. The
culturing apparatus can be of any culture apparatus commonly used
for culturing cells in vitro. Preferably, the level of confluence
is 70% or greater before passing the cells to another culture
apparatus. More preferably, the level of confluence is 90% or
greater. A period of time can be any time suitable for the culture
of cells in vitro. The T cell medium may be replaced during the
culture of the T cells at any time. Preferably, the T cell medium
is replaced about every 2 to 3 days. The T cells are then harvested
from the culture apparatus whereupon the T cells can be used
immediately or cryopreserved to be stored for use at a later time.
In one embodiment, the invention includes cryopreserving the
expanded T cells. The cryopreserved T cells are thawed prior to
introducing nucleic acids into the T cell.
[0592] In another embodiment, the method comprises isolating T
cells and expanding the T cells. In another embodiment, the
invention further comprises cryopreserving the T cells prior to
expansion. In yet another embodiment, the cryopreserved T cells are
thawed for electroporation with the RNA encoding the chimeric
membrane protein.
[0593] Another procedure for ex vivo expansion cells is described
in U.S. Pat. No. 5,199,942 (incorporated herein by reference).
Expansion, such as described in U.S. Pat. No. 5,199,942 can be an
alternative or in addition to other methods of expansion described
herein. Briefly, ex vivo culture and expansion of T cells comprises
the addition to the cellular growth factors, such as those
described in U.S. Pat. No. 5,199,942, or other factors, such as
flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding
the T cells comprises culturing the T cells with a factor selected
from the group consisting of flt3-L, IL-1, IL-3 and c-kit
ligand.
[0594] The culturing step as described herein (contact with agents
as described herein or after electroporation) can be very short,
for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
The culturing step as described further herein (contact with agents
as described herein) can be longer, for example 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or more days.
[0595] Various terms are used to describe cells in culture. Cell
culture refers generally to cells taken from a living organism and
grown under controlled condition. A primary cell culture is a
culture of cells, tissues or organs taken directly from an organism
and before the first subculture. Cells are expanded in culture when
they are placed in a growth medium under conditions that facilitate
cell growth and/or division, resulting in a larger population of
the cells. When cells are expanded in culture, the rate of cell
proliferation is typically measured by the amount of time required
for the cells to double in number, otherwise known as the doubling
time.
[0596] Each round of subculturing is referred to as a passage. When
cells are subcultured, they are referred to as having been
passaged. A specific population of cells, or a cell line, is
sometimes referred to or characterized by the number of times it
has been passaged. For example, a cultured cell population that has
been passaged ten times may be referred to as a P10 culture. The
primary culture, i.e., the first culture following the isolation of
cells from tissue, is designated P0. Following the first
subculture, the cells are described as a secondary culture (P1 or
passage 1). After the second subculture, the cells become a
tertiary culture (P2 or passage 2), and so on. It will be
understood by those of skill in the art that there may be many
population doublings during the period of passaging; therefore the
number of population doublings of a culture is greater than the
passage number. The expansion of cells (i.e., the number of
population doublings) during the period between passaging depends
on many factors, including but is not limited to the seeding
density, substrate, medium, and time between passaging.
[0597] In one embodiment, the cells may be cultured for several
hours (about 3 hours) to about 14 days or any hourly integer value
in between. Conditions appropriate for T cell culture include an
appropriate media (e.g., Minimal Essential Media or RPMI Media 1640
or, X-vivo 15, (Lonza)) that may contain factors necessary for
proliferation and viability, including serum (e.g., fetal bovine or
human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7,
GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-.alpha.. or any
other additives for the growth of cells known to the skilled
artisan. Other additives for the growth of cells include, but are
not limited to, surfactant, plasmanate, and reducing agents such as
N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI
1640, AIM-V, DMEM, MEM, .alpha.-MEM, F-12, X-Vivo 15, and X-Vivo
20, Optimizer, with added amino acids, sodium pyruvate, and
vitamins, either serum-free or supplemented with an appropriate
amount of serum (or plasma) or a defined set of hormones, and/or an
amount of cytokine(s) sufficient for the growth and expansion of T
cells. Antibiotics, e.g., penicillin and streptomycin, are included
only in experimental cultures, not in cultures of cells that are to
be infused into a subject. The target cells are maintained under
conditions necessary to support growth, for example, an appropriate
temperature (e.g., 37.degree. C.) and atmosphere (e.g., air plus 5%
CO.sub.2).
[0598] The medium used to culture the T cells may include an agent
that can co-stimulate the T cells. For example, an agent that can
stimulate CD3 is an antibody to CD3, and an agent that can
stimulate CD28 is an antibody to CD28. A cell isolated by the
methods disclosed herein can be expanded approximately 10 fold, 20
fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90
fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold,
700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000
fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000
fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In
one embodiment, the T cells expand in the range of about 20 fold to
about 50 fold, or more. In one embodiment, human T regulatory cells
are expanded via anti-CD3 antibody coated KT64.86 artificial
antigen presenting cells (aAPCs). In one embodiment, human T
regulatory cells are expanded via anti-CD3 antibody coated K562
artificial antigen presenting cells (aAPCs). Methods for expanding
and activating T cells can be found in U.S. Pat. Nos. 7,754,482,
8,722,400, and 9,555,105, contents of which are incorporated herein
in their entirety.
[0599] In one embodiment, the method of expanding the T cells can
further comprise isolating the expanded T cells for further
applications. In another embodiment, the method of expanding can
further comprise a subsequent electroporation of the expanded T
cells followed by culturing. The subsequent electroporation may
include introducing a nucleic acid encoding an agent, such as
transducing the expanded T cells, transfecting the expanded T
cells, or electroporating the expanded T cells with a nucleic acid,
into the expanded population of T cells, wherein the agent further
stimulates the T cell. The agent may stimulate the T cells, such as
by stimulating further expansion, effector function, or another T
cell function.
J. Methods of Treatment
[0600] The modified cells (e.g., T cells) described herein may be
included in a composition for immunotherapy. The composition may
include a pharmaceutical composition and further include a
pharmaceutically acceptable carrier. A therapeutically effective
amount of the pharmaceutical composition comprising the modified T
cells may be administered.
[0601] In one aspect, the invention includes a method for adoptive
cell transfer therapy comprising administering to a subject in need
thereof a modified T cell of the present invention. In another
aspect, the invention includes a method of treating a disease or
condition in a subject comprising administering to a subject in
need thereof a population of modified T cells.
[0602] Also included is a method of treating a disease or condition
in a subject in need thereof comprising administering to the
subject a modified cell (e.g., modified T cell) of the present
invention. In one embodiment, the method of treating a disease or
condition in a subject in need thereof comprises administering to
the subject a modified cell (e.g., a modified T cell) comprising a
subject CAR, dominant negative receptor and/or switch receptor,
and/or a bispecific antibody, and/or combinations thereof. In one
embodiment, the method of treating a disease or condition in a
subject in need thereof comprises administering to the subject a
modified cell (e.g., a modified T cell) comprising a subject CAR
(e.g., a CAR having affinity for PSMA on a target cell) and a
dominant negative receptor and/or switch receptor. In one
embodiment, the method of treating a disease or condition in a
subject in need thereof comprises administering to the subject a
modified cell (e.g., a modified T cell) comprising a subject CAR
(e.g., a CAR having affinity for PSMA on a target cell), a dominant
negative receptor and/or switch receptor, and wherein the modified
cell is capable of expressing and secreting a bispecific
antibody.
[0603] Methods for administration of immune cells for adoptive cell
therapy are known and may be used in connection with the provided
methods and compositions. For example, adoptive T cell therapy
methods are described, e.g., in U.S. Patent Application Publication
No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to
Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See,
e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933;
Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9;
Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments,
the cell therapy, e.g., adoptive T cell therapy is carried out by
autologous transfer, in which the cells are isolated and/or
otherwise prepared from the subject who is to receive the cell
therapy, or from a sample derived from such a subject. Thus, in
some aspects, the cells are derived from a subject, e.g., patient,
in need of a treatment and the cells, following isolation and
processing are administered to the same subject.
[0604] In some embodiments, the cell therapy, e.g., adoptive T cell
therapy, is carried out by allogeneic transfer, in which the cells
are isolated and/or otherwise prepared from a subject other than a
subject who is to receive or who ultimately receives the cell
therapy, e.g., a first subject. In such embodiments, the cells then
are administered to a different subject, e.g., a second subject, of
the same species. In some embodiments, the first and second
subjects are genetically identical. In some embodiments, the first
and second subjects are genetically similar. In some embodiments,
the second subject expresses the same HLA class or supertype as the
first subject.
[0605] In some embodiments, the subject has been treated with a
therapeutic agent targeting the disease or condition, e.g., the
tumor, prior to administration of the cells or composition
containing the cells. In some aspects, the subject is refractory or
non-responsive to the other therapeutic agent. In some embodiments,
the subject has persistent or relapsed disease, e.g., following
treatment with another therapeutic intervention, including
chemotherapy, radiation, and/or hematopoietic stem cell
transplantation (HSCT), e.g., allogenic HSCT. In some embodiments,
the administration effectively treats the subject despite the
subject having become resistant to another therapy.
[0606] In some embodiments, the subject is responsive to the other
therapeutic agent, and treatment with the therapeutic agent reduces
disease burden. In some aspects, the subject is initially
responsive to the therapeutic agent, but exhibits a relapse of the
disease or condition over time. In some embodiments, the subject
has not relapsed. In some such embodiments, the subject is
determined to be at risk for relapse, such as at a high risk of
relapse, and thus the cells are administered prophylactically,
e.g., to reduce the likelihood of or prevent relapse. In some
aspects, the subject has not received prior treatment with another
therapeutic agent.
[0607] In some embodiments, the subject has persistent or relapsed
disease, e.g., following treatment with another therapeutic
intervention, including chemotherapy, radiation, and/or
hematopoietic stem cell transplantation (HSCT), e.g., allogenic
HSCT. In some embodiments, the administration effectively treats
the subject despite the subject having become resistant to another
therapy.
[0608] The modified immune cells of the present invention can be
administered to an animal, preferably a mammal, even more
preferably a human, to treat a cancer. In addition, the cells of
the present invention can be used for the treatment of any
condition related to a cancer, especially a cell-mediated immune
response against a tumor cell(s), where it is desirable to treat or
alleviate the disease. The types of cancers to be treated with the
modified cells or pharmaceutical compositions of the invention
include, carcinoma, blastoma, and sarcoma, and certain leukemia or
lymphoid malignancies, benign and malignant tumors, and
malignancies e.g., sarcomas, carcinomas, and melanomas. Other
exemplary cancers include but are not limited breast cancer,
prostate cancer, ovarian cancer, cervical cancer, skin cancer,
pancreatic cancer, colorectal cancer, renal cancer, liver cancer,
brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and
the like. The cancers may be non-solid tumors (such as
hematological tumors) or solid tumors. Adult tumors/cancers and
pediatric tumors/cancers are also included.
[0609] Solid tumors are abnormal masses of tissue that usually do
not contain cysts or liquid areas. Solid tumors can be benign or
malignant. Different types of solid tumors are named for the type
of cells that form them (such as sarcomas, carcinomas, and
lymphomas). Examples of solid tumors, such as sarcomas and
carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, lymphoid malignancy, pancreatic cancer, breast
cancer, lung cancers, ovarian cancer, prostate cancer,
hepatocellular carcinoma, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid
carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,
cervical cancer, testicular tumor, seminoma, bladder carcinoma,
melanoma, and CNS tumors (such as a glioma (such as brainstem
glioma and mixed gliomas), glioblastoma (also known as glioblastoma
multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma,
Schwannoma craniopharyogioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,
neuroblastoma, retinoblastoma and brain metastases).
[0610] Carcinomas that can be amenable to therapy by a method
disclosed herein include, but are not limited to, esophageal
carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form
of skin cancer), squamous cell carcinoma (various tissues), bladder
carcinoma, including transitional cell carcinoma (a malignant
neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma,
colorectal carcinoma, gastric carcinoma, lung carcinoma, including
small cell carcinoma and non-small cell carcinoma of the lung,
adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma,
breast carcinoma, ovarian carcinoma, prostate carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma,
medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ
or bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma,
testicular carcinoma, osteogenic carcinoma, epithelial carcinoma,
and nasopharyngeal carcinoma.
[0611] Sarcomas that can be amenable to therapy by a method
disclosed herein include, but are not limited to, fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic
sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma,
and other soft tissue sarcomas.
[0612] Prostate adenocarcinoma is an extremely common and lethal
disease. Prostate cancer is the most common malignancy among men.
Prostate cancer is the second-leading cause of cancer-related
deaths among men, accounting for an estimated 10% of annual male
cancer deaths. PSMA is highly expressed in malignant prostate
tissue, with low-levels of expression in some normal human tissues.
Under normal physiologic conditions, PSMA is expressed in the
prostate gland (secretory acinar epithelium), kidney (proximal
tubules), nervous system glia (astrocytes and Schwann cells), and
the small intestine (jejunal brush border). PSMA is much more
highly expressed in prostate epithelium and is significantly
upregulated in malignant prostate tissues. PSMA expression in
normal cells has been found to be 100-fold to 1000-fold less than
in prostate carcinoma cells. PSMA expression increases
significantly during the transformation from benign prostatic
hyperplasia to prostatic adenocarcinoma. PSMA expression has been
found to be directly correlated with the histologic grade of
malignant prostate tissue and increases with more advanced disease
(i.e. highest PSMA expression found in prostate cancer metastases
in lymph node and bone).
[0613] In one embodiment, the methods of the invention are useful
for treating prostate cancer, for example advanced
castrate-resistant prostate cancer. It should be readily understood
by one of ordinary skill in the art that any type of cancer wherein
the PSMA tumor antigen is expressed, can be treated using the
methods of the present invention. For example, neovascular
expression of PSMA was found in non-small cell lung cancer, see,
e.g., PLoS One. 2017 Oct. 27; 12(10). Accordingly, the methods of
the invention may also be useful for treating non-small cell lung
cancer (NSCLC).
[0614] In certain exemplary embodiments, the modified immune cells
of the invention are used to treat prostate cancer. In one
embodiment, a method of the present disclosure is used to treat
castrate-resistant prostate cancer. In one embodiment, a method of
the present disclosure is used to treat advanced castrate-resistant
prostate cancer. In one embodiment, a method of the present
disclosure is used to treat metastatic castrate-resistant prostate
cancer. In one embodiment, a method of the present disclosure is
used to treat metastatic castrate-resistant prostate cancer,
wherein the patient with metastatic castrate-resistant prostate
cancer has .gtoreq.10% tumor cells expressing PSMA. In one
embodiment, a method of the present disclosure is used to treat
castrate-resistant prostate adenocarcinoma, wherein the patient has
castrate levels of testosterone (e.g., <50 ng/mL) with or
without the use of androgen deprivation therapy.
[0615] In certain embodiments, the subject is provided a secondary
treatment. Secondary treatments include but are not limited to
chemotherapy, radiation, surgery, and medications.
[0616] Cells of the invention can be administered in dosages and
routes and at times to be determined in appropriate pre-clinical
and clinical experimentation and trials. Cell compositions may be
administered multiple times at dosages within these ranges.
Administration of the cells of the invention may be combined with
other methods useful to treat the desired disease or condition as
determined by those of skill in the art.
[0617] The cells of the invention to be administered may be
autologous, with respect to the subject undergoing therapy.
[0618] The administration of the cells of the invention may be
carried out in any convenient manner known to those of skill in the
art. The cells of the present invention may be administered to a
subject by aerosol inhalation, injection, ingestion, transfusion,
implantation or transplantation. The compositions described herein
may be administered to a patient transarterially, subcutaneously,
intradermally, intratumorally, intranodally, intramedullary,
intramuscularly, by intravenous (i.v.) injection, or
intraperitoneally. In other instances, the cells of the invention
are injected directly into a site of inflammation in the subject, a
local disease site in the subject, alymph node, an organ, a tumor,
and the like.
[0619] In some embodiments, the cells are administered at a desired
dosage, which in some aspects includes a desired dose or number of
cells or cell type(s) and/or a desired ratio of cell types. Thus,
the dosage of cells in some embodiments is based on a total number
of cells (or number per kg body weight) and a desired ratio of the
individual populations or sub-types, such as the CD4+ to CD8+
ratio. In some embodiments, the dosage of cells is based on a
desired total number (or number per kg of body weight) of cells in
the individual populations or of individual cell types. In some
embodiments, the dosage is based on a combination of such features,
such as a desired number of total cells, desired ratio, and desired
total number of cells in the individual populations.
[0620] In some embodiments, the populations or sub-types of cells,
such as CD8.sup.+ and CD4.sup.+ T cells, are administered at or
within a tolerated difference of a desired dose of total cells,
such as a desired dose of T cells. In some aspects, the desired
dose is a desired number of cells or a desired number of cells per
unit of body weight of the subject to whom the cells are
administered, e.g., cells/kg. In some aspects, the desired dose is
at or above a minimum number of cells or minimum number of cells
per unit of body weight. In some aspects, among the total cells,
administered at the desired dose, the individual populations or
sub-types are present at or near a desired output ratio (such as
CD4.sup.+ to CD8.sup.+ ratio), e.g., within a certain tolerated
difference or error of such a ratio.
[0621] In some embodiments, the cells are administered at or within
a tolerated difference of a desired dose of one or more of the
individual populations or sub-types of cells, such as a desired
dose of CD4+ cells and/or a desired dose of CD8+ cells. In some
aspects, the desired dose is a desired number of cells of the
sub-type or population, or a desired number of such cells per unit
of body weight of the subject to whom the cells are administered,
e.g., cells/kg. In some aspects, the desired dose is at or above a
minimum number of cells of the population or subtype, or minimum
number of cells of the population or sub-type per unit of body
weight. Thus, in some embodiments, the dosage is based on a desired
fixed dose of total cells and a desired ratio, and/or based on a
desired fixed dose of one or more, e.g., each, of the individual
sub-types or sub-populations. Thus, in some embodiments, the dosage
is based on a desired fixed or minimum dose of T cells and a
desired ratio of CD4.sup.+ to CD8.sup.+ cells, and/or is based on a
desired fixed or minimum dose of CD4.sup.+ and/or CD8.sup.+
cells.
[0622] In certain embodiments, the cells, or individual populations
of sub-types of cells, are administered to the subject at a range
of about one million to about 100 billion cells, such as, e.g., 1
million to about 50 billion cells (e.g., about 5 million cells,
about 25 million cells, about 500 million cells, about 1 billion
cells, about 5 billion cells, about 20 billion cells, about 30
billion cells, about 40 billion cells, or a range defined by any
two of the foregoing values), such as about 10 million to about 100
billion cells (e.g., about 20 million cells, about 30 million
cells, about 40 million cells, about 60 million cells, about 70
million cells, about 80 million cells, about 90 million cells,
about 10 billion cells, about 25 billion cells, about 50 billion
cells, about 75 billion cells, about 90 billion cells, or a range
defined by any two of the foregoing values), and in some cases
about 100 million cells to about 50 billion cells (e.g., about 120
million cells, about 250 million cells, about 350 million cells,
about 450 million cells, about 650 million cells, about 800 million
cells, about 900 million cells, about 3 billion cells, about 30
billion cells, about 45 billion cells) or any value in between
these ranges.
[0623] In some embodiments, the dose of total cells and/or dose of
individual sub-populations of cells is within a range of between at
or about 1.times.10.sup.5 cells/kg to about 1.times.10.sup.11
cells/kg, 10.sup.4, and at or about 10.sup.11 cells/kilograms (kg)
body weight, such as between 10.sup.5 and 10.sup.6 cells/kg body
weight, for example, at or about 1.times.10.sup.5 cells/kg,
1.5.times.10.sup.5 cells/kg, 2.times.10.sup.5 cells/kg, or
1.times.10.sup.6 cells/kg body weight. For example, in some
embodiments, the cells are administered at, or within a certain
range of error of, between at or about 10.sup.4 and at or about
10.sup.9 T cells/kilograms (kg) body weight, such as between
10.sup.5 and 10.sup.6 T cells/kg body weight, for example, at or
about 1.times.10.sup.5 T cells/kg, 1.5.times.10.sup.5 T cells/kg,
2.times.10.sup.5 T cells/kg, or 1.times.10.sup.6 T cells/kg body
weight. In other exemplary embodiments, a suitable dosage range of
modified cells for use in a method of the present disclosure
includes, without limitation, from about 1.times.10.sup.5 cells/kg
to about 1.times.10.sup.6 cells/kg, from about 1.times.10.sup.6
cells/kg to about 1.times.10.sup.7 cells/kg, from about
1.times.10.sup.7 cells/kg about 1.times.10.sup.8 cells/kg, from
about 1.times.10.sup.8 cells/kg about 1.times.10.sup.9 cells/kg,
from about 1.times.10.sup.9 cells/kg about 1.times.10.sup.10
cells/kg, from about 1.times.10.sup.10 cells/kg about
1.times.10.sup.11 cells/kg. In an exemplary embodiment, a suitable
dosage for use in a method of the present disclosure is about
1.times.10s cells/kg. In an exemplary embodiment, a suitable dosage
for use in a method of the present disclosure is about
1.times.10.sup.7 cells/kg. In other embodiments, a suitable dosage
is from about 1.times.10.sup.7 total cells to about
5.times.10.sup.7 total cells. In some embodiments, a suitable
dosage is from about 1.times.10.sup.8 total cells to about
5.times.10.sup.8 total cells. In some embodiments, a suitable
dosage is from about 1.4.times.10.sup.7 total cells to about
1.1.times.10.sup.9 total cells. In an exemplary embodiment, a
suitable dosage for use in a method of the present disclosure is
about 7.times.10.sup.9 total cells. In an exemplary embodiment, a
suitable dosage is from about 1.times.10.sup.7 total cells to about
3.times.10.sup.7 total cells.
[0624] In some embodiments, the dose of total cells and/or dose of
individual sub-populations of cells is within a range of between at
or about 1.times.10.sup.5 cells/m.sup.2 to about 1.times.10.sup.11
cells/m.sup.2. In an exemplary embodiment, the dose of total cells
and/or dose of individual sub-populations of cells is within a
range of between at or about 1.times.10.sup.7/m.sup.2 to at or
about 3.times.10.sup.7/m.sup.2. In an exemplary embodiment, the
dose of total cells and/or dose of individual sub-populations of
cells is within a range of between at or about
1.times.10.sup.8/m.sup.2 to at or about 3.times.10.sup.8/m.sup.2.
In some embodiments, the dose of total cells and/or dose of
individual sub-populations of cells is the maximum tolerated dose
by a given patient.
[0625] In some embodiments, the cells are administered at or within
a certain range of error of between at or about 10.sup.4 and at or
about 10.sup.9 CD4.sup.+ and/or CD8.sup.+ cells/kilograms (kg) body
weight, such as between 10.sup.5 and 10.sup.6 CD4.sup.+ and/or CD8+
cells/kg body weight, for example, at or about 1.times.10.sup.5
CD4.sup.+ and/or CD8.sup.+ cells/kg, 1.5.times.10.sup.5 CD4.sup.+
and/or CD8.sup.+ cells/kg, 2.times.10.sup.5 CD4.sup.+ and/or
CD8.sup.+ cells/kg, or 1.times.10.sup.6 CD4.sup.+ and/or CD8.sup.+
cells/kg body weight. In some embodiments, the cells are
administered at or within a certain range of error of, greater
than, and/or at least about 1.times.10.sup.6, about
2.5.times.10.sup.6, about 5.times.10.sup.6, about
7.5.times.10.sup.6, or about 9.times.10.sup.6 CD4.sup.+ cells,
and/or at least about 1.times.10.sup.6, about 2.5.times.10.sup.6,
about 5.times.10.sup.6, about 7.5.times.10.sup.6, or about
9.times.10.sup.6 CD8+ cells, and/or at least about
1.times.10.sup.6, about 2.5.times.10.sup.6, about 5.times.10.sup.6,
about 7.5.times.10.sup.6, or about 9.times.10.sup.6 T cells. In
some embodiments, the cells are administered at or within a certain
range of error of between about 10.sup.8 and 10.sup.12 or between
about 10.sup.10 and 10.sup.11 T cells, between about 10.sup.8 and
10.sup.12 or between about 10.sup.10 and 10.sup.11 CD4.sup.+ cells,
and/or between about 10.sup.8 and 10.sup.12 or between about
10.sup.10 and 10.sup.11 CD8.sup.+ cells.
[0626] In some embodiments, the cells are administered at or within
a tolerated range of a desired output ratio of multiple cell
populations or sub-types, such as CD4+ and CD8+ cells or sub-types.
In some aspects, the desired ratio can be a specific ratio or can
be a range of ratios, for example, in some embodiments, the desired
ratio (e.g., ratio of CD4.sup.+ to CD8.sup.+ cells) is between at
or about 5:1 and at or about 5:1 (or greater than about 1:5 and
less than about 5:1), or between at or about 1:3 and at or about
3:1 (or greater than about 1:3 and less than about 3:1), such as
between at or about 2:1 and at or about 1:5 (or greater than about
1:5 and less than about 2:1, such as at or about 5:1, 4.5:1, 4:1,
3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,
1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,
1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In
some aspects, the tolerated difference is within about 1%, about
2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%/a,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of
the desired ratio, including any value in between these ranges.
[0627] In some embodiments, a dose of modified cells is
administered to a subject in need thereof, in a single dose or
multiple doses. In some embodiments, a dose of modified cells is
administered in multiple doses, e.g., once a week or every 7 days,
once every 2 weeks or every 14 days, once every 3 weeks or every 21
days, once every 4 weeks or every 28 days. In an exemplary
embodiment, a single dose of modified cells is administered to a
subject in need thereof. In an exemplary embodiment, a single dose
of modified cells is administered to a subject in need thereof by
rapid intravenous infusion.
[0628] For the prevention or treatment of disease, the appropriate
dosage may depend on the type of disease to be treated, the type of
cells or recombinant receptors, the severity and course of the
disease, whether the cells are administered for preventive or
therapeutic purposes, previous therapy, the subject's clinical
history and response to the cells, and the discretion of the
attending physician. The compositions and cells are in some
embodiments suitably administered to the subject at one time or
over a series of treatments.
[0629] In some embodiments, the cells are administered as part of a
combination treatment, such as simultaneously with or sequentially
with, in any order, another therapeutic intervention, such as an
antibody or engineered cell or receptor or agent, such as a
cytotoxic or therapeutic agent. The cells in some embodiments are
co-administered with one or more additional therapeutic agents or
in connection with another therapeutic intervention, either
simultaneously or sequentially in any order. In some contexts, the
cells are co-administered with another therapy sufficiently close
in time such that the cell populations enhance the effect of one or
more additional therapeutic agents, or vice versa. In some
embodiments, the cells are administered prior to the one or more
additional therapeutic agents. In some embodiments, the cells are
administered after the one or more additional therapeutic agents.
In some embodiments, the one or more additional agents includes a
cytokine, such as IL-2, for example, to enhance persistence. In
some embodiments, the methods comprise administration of a
chemotherapeutic agent.
[0630] Following administration of the cells, the biological
activity of the engineered cell populations in some embodiments is
measured, e.g., by any of a number of known methods. Parameters to
assess include specific binding of an engineered or natural T cell
or other immune cell to antigen, in vivo, e.g., by imaging, or ex
vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the
ability of the engineered cells to destroy target cells can be
measured using any suitable method known in the art, such as
cytotoxicity assays described in, for example, Kochenderfer et al.,
J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J.
Immunological Methods, 285(1): 25-40 (2004). In certain
embodiments, the biological activity of the cells is measured by
assaying expression and/or secretion of one or more cytokines, such
as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological
activity is measured by assessing clinical outcome, such as
reduction in tumor burden or load.
[0631] In some embodiments, a specific dosage regimen of the
present disclosure includes a lymphodepletion step prior to the
administration of the modified T cells. In an exemplary embodiment,
the lymphodepletion step includes administration of
cyclophosphamide and/or fludarabine.
[0632] In some embodiments, the lymphodepletion step includes
administration of cyclophosphamide at a dose of between about 200
mg/m.sup.2/day and about 2000 mg/m.sup.2/day (e.g., 200
mg/m.sup.2/day, 300 mg/m.sup.2/day, or 500 mg/m.sup.2/day). In an
exemplary embodiment, the dose of cyclophosphamide is about 300
mg/m.sup.2/day. In some embodiments, the lymphodepletion step
includes administration of fludarabine at a dose of between about
20 mg/m.sup.2/day and about 900 mg/m.sup.2/day (e.g., 20
mg/m.sup.2/day, 25 mg/m.sup.2/day, 30 mg/m.sup.2/day, or 60
mg/m.sup.2/day). In an exemplary embodiment, the dose of
fludarabine is about 30 mg/m.sup.2/day.
[0633] In some embodiment, the lymphodepletion step includes
administration of cyclophosphamide at a dose of between about 200
mg/m.sup.2/day and about 2000 mg/m.sup.2/day (e.g., 200
mg/m.sup.2/day, 300 mg/m.sup.2/day, or 500 mg/m.sup.2/day), and
fludarabine at a dose of between about 20 mg/m.sup.2/day and about
900 mg/m.sup.2/day (e.g., 20 mg/m.sup.2/day, 25 mg/m.sup.2/day, 30
mg/m.sup.2/day, or 60 mg/m.sup.2/day). In an exemplary embodiment,
the lymphodepletion step includes administration of
cyclophosphamide at a dose of about 300 mg/m.sup.2/day, and
fludarabine at a dose of about 30 mg/m.sup.2/day.
[0634] In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy prior to the administration of the
modified T cells. In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including at or about 500 mg/m.sup.2
to at or about 1 g/m.sup.2 of cyclophosphamide by intravenous
infusion. In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including at or about 500 mg/m.sup.2
to at or about 1 g/m.sup.2 of cyclophosphamide by intravenous
infusion about 3 days (+1 day) prior to administration of the
modified T cells. In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including at or about 500 mg/m.sup.2
to at or about 1 g/m.sup.2 of cyclophosphamide by intravenous
infusion up to 4 days prior to administration of the modified T
cells. In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including at or about 500 mg/m.sup.2
to at or about 1 g/m.sup.2 of cyclophosphamide by intravenous
infusion 4 days prior to administration of the modified T cells. In
an exemplary embodiment, for a subject having castrate-resistant
prostate cancer, the subject receives lymphodepleting chemotherapy
including at or about 500 mg/m.sup.2 to at or about 1 g/m.sup.2 of
cyclophosphamide by intravenous infusion 3 days prior to
administration of the modified T cells. In an exemplary embodiment,
for a subject having castrate-resistant prostate cancer, the
subject receives lymphodepleting chemotherapy including at or about
500 mg/m.sup.2 to at or about 1 g/m.sup.2 of cyclophosphamide by
intravenous infusion 2 days prior to administration of the modified
T cells.
[0635] In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including 300 mg/m.sup.2 of
cyclophosphamide by intravenous infusion 3 days prior to
administration of the modified T cells. In an exemplary embodiment,
for a subject having castrate-resistant prostate cancer, the
subject receives lymphodepleting chemotherapy including 300
mg/m.sup.2 of cyclophosphamide by intravenous infusion for 3 days
prior to administration of the modified T cells.
[0636] In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including fludarabine at a dose of
between about 20 mg/m.sup.2/day and about 900 mg/m.sup.2/day (e.g.,
20 mg/m.sup.2/day, 25 mg/m.sup.2/day, 30 mg/m.sup.2/day, or 60
mg/m.sup.2/day). In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including fludarabine at a dose of 30
mg/m.sup.2 for 3 days.
[0637] In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including cyclophosphamide at a dose
of between about 200 mg/m.sup.2/day and about 2000 mg/m.sup.2/day
(e.g., 200 mg/m.sup.2/day, 300 mg/m.sup.2/day, or 500
mg/m.sup.2/day), and fludarabine at a dose of between about 20
mg/m.sup.2/day and about 900 mg/m.sup.2/day (e.g., 20
mg/m.sup.2/day, 25 mg/m.sup.2/day, 30 mg/m.sup.2/day, or 60
mg/m.sup.2/day). In an exemplary embodiment, for a subject having
castrate-resistant prostate cancer, the subject receives
lymphodepleting chemotherapy including cyclophosphamide at a dose
of about 300 mg/m.sup.2/day, and fludarabine at a dose of 30
mg/m.sup.2 for 3 days.
[0638] Cells of the invention can be administered in dosages and
routes and at times to be determined in appropriate pre-clinical
and clinical experimentation and trials. Cell compositions may be
administered multiple times at dosages within these ranges.
Administration of the cells of the invention may be combined with
other methods useful to treat the desired disease or condition as
determined by those of skill in the art.
[0639] It is known in the art that one of the adverse effects
following infusion of CAR T cells is the onset of immune
activation, known as cytokine release syndrome (CRS). CRS is immune
activation resulting in elevated inflammatory cytokines. Clinical
and laboratory measures range from mild CRS (constitutional
symptoms and/or grade-2 organ toxicity) to severe CRS (sCRS; grade
.gtoreq.3 organ toxicity, aggressive clinical intervention, and/or
potentially life threatening). Clinical features include: high
fever, malaise, fatigue, myalgia, nausea, anorexia,
tachycardia/hypotension, capillary leak, cardiac dysfunction, renal
impairment, hepatic failure, and disseminated intravascular
coagulation. Dramatic elevations of cytokines including
interferon-gamma, granulocyte macrophage colony-stimulating factor,
IL-10, and IL-6 have been shown following CAR T-cell infusion. The
presence of CRS generally correlates with expansion and progressive
immune activation of adoptively transferred cells. It has been
demonstrated that the degree of CRS severity is dictated by disease
burden at the time of infusion as patients with high tumor burden
experience a more sCRS.
[0640] Accordingly, the invention provides for, following the
diagnosis of CRS, appropriate CRS management strategies to mitigate
the physiological symptoms of uncontrolled inflammation without
dampening the antitumor efficacy of the engineered cells (e.g., CAR
T cells). CRS management strategies are known in the art. For
example, systemic corticosteroids may be administered to rapidly
reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising
initial antitumor response.
[0641] In some embodiments, an anti-IL-6R antibody may be
administered. An example of an anti-IL-6R antibody is the Food and
Drug Administration-approved monoclonal antibody tocilizumab, also
known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab
is a humanized monoclonal antibody against the interleukin-6
receptor (IL-6R). Administration of tocilizumab has demonstrated
near-immediate reversal of CRS.
[0642] CRS is generally managed based on the severity of the
observed syndrome and interventions are tailored as such. CRS
management decisions may be based upon clinical signs and symptoms
and response to interventions, not solely on laboratory values
alone.
[0643] Mild to moderate cases generally are treated with symptom
management with fluid therapy, non-steroidal anti-inflammatory drug
(NSAID) and antihistamines as needed for adequate symptom relief.
More severe cases include patients with any degree of hemodynamic
instability; with any hemodynamic instability, the administration
of tocilizumab is recommended. The first-line management of CRS may
be tocilizumab, in some embodiments, at the labeled dose of 8 mg/kg
IV over 60 minutes (not to exceed 800 mg/dose); tocilizumab can be
repeated Q8 hours. If suboptimal response to the first dose of
tocilizumab, additional doses of tocilizumab may be considered.
Tocilizumab can be administered alone or in combination with
corticosteroid therapy. Patients with continued or progressive CRS
symptoms, inadequate clinical improvement in 12-18 hours or poor
response to tocilizumab, may be treated with high-dose
corticosteroid therapy, generally hydrocortisone 100 mg IV or
methylprednisolone 1-2 mg/kg. In patients with more severe
hemodynamic instability or more severe respiratory symptoms,
patients may be administered high-dose corticosteroid therapy early
in the course of the CRS. CRS management guidance may be based on
published standards (Lee et al. (2019) Biol Blood Marrow
Transplant, doi.org/10.1016/j.bbmt.2018.12.758; Neelapu et al.
(2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer
Discov, 6(6):664-679).
[0644] Features consistent with Macrophage Activation Syndrome
(MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been
observed in patients treated with CAR-T therapy (Henter, 2007),
coincident with clinical manifestations of the CRS. MAS appears to
be a reaction to immune activation that occurs from the CRS, and
should therefore be considered a manifestation of CRS. MAS is
similar to HLH (also a reaction to immune stimulation). The
clinical syndrome of MAS is characterized by high grade
non-remitting fever, cytopenias affecting at least two of three
lineages, and hepatosplenomegaly. It is associated with high serum
ferritin, soluble interleukin-2 receptor, and triglycerides, and a
decrease of circulating natural killer (NK) activity.
[0645] In some embodiments, the methods of the invention involve
selecting and treating a subject having failed at least one prior
course of standard of cancer therapy. For example, a suitable
subject may have had a confirmed diagnosis of relapsed prostate
cancer. In some embodiments, the methods of the invention involve
selecting and treating a subject having had at least one prior
course of standard of cancer therapy. For example, a suitable
subject may have had prior therapy with at least one standard 17a
lyase inhibitor or second-generation anti-androgen therapy for the
treatment of metastatic castrate resistant prostate cancer.
[0646] In an exemplary embodiment, a suitable subject is a subject
having metastatic castrate resistant prostate cancer. In an
exemplary embodiment, a suitable subject is a subject having
metastatic castrate resistant prostate cancer having .gtoreq.10%
tumor cells expressing PSMA as demonstrated by immunohistochemistry
analysis on fresh tissue.
[0647] In some embodiments, a suitable subject is a subject that
has radiographic evidence of osseous metastatic disease and/or
measurable, non-osseous metastatic disease (nodal or visceral).
[0648] In some embodiments, a suitable subject is a subject that
has an ECOG performance status of 0-1.
[0649] In some embodiments, a suitable subject is a subject that
has adequate organ function, as defined by: serum creatinine
.ltoreq.1.5 mg/dl or creatinine clearance .gtoreq.60 cc/min; and/or
serum total bilirubin <1.5.times.ULN; serum
ALT/AST<2.times.ULN.
[0650] In some embodiments, a suitable subject is a subject that
has adequate hematologic reserve as defined by: Hgb>10 g/dl;
PLT>100 k/ul; and/or ANC>1.5 k/ul.
[0651] In some embodiments, a suitable subject is a subject that is
not transfusion dependent.
[0652] In some embodiments, a suitable subject is a subject that
has evidence of progressive castrate resistant prostate
adenocarcinoma, as defined by: castrate levels of testosterone
(<50 ng/ml) with or without the use of androgen deprivation
therapy; and/or evidence of one of the following measures of
progressive disease: soft tissue progression by RECIST 1.1
criteria, osseous disease progression with 2 or more new lesions on
bone scan (as per PCWG2 criteria), increase in serum PSA of at
least 25% and an absolute increase of 2 ng/ml or more from nadir
(as per PCWG2 criteria).
[0653] In some embodiments, a suitable subject has had previous
treatment with at least one second-generation androgen signaling
inhibitor. In some embodiments, a suitable subject has had previous
treatment with abiraterone. In some embodiments, a suitable subject
has had previous treatment with enzalutamide.
[0654] In some embodiments, a suitable subject has .gtoreq.10%
tumor cells expressing PSMA by immunohistochemistry (IHC) on a
metastatic tissue biopsy.
[0655] In some embodiments, a suitable subject has radiographic
evidence for metastatic disease (osseous or nodal/visceral).
[0656] In some embodiments, a suitable subject has .ltoreq.4 lines
of therapy for metastatic CRPC.
K. Pharmaceutical Compositions and Formulations
[0657] Also provided are populations of immune cells of the
invention, compositions containing such cells and/or enriched for
such cells, such as in which cells expressing the recombinant
receptor make up at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the
composition or cells of a certain type such as T cells or CD8+ or
CD4+ cells. Among the compositions are pharmaceutical compositions
and formulations for administration, such as for adoptive cell
therapy. Also provided are therapeutic methods for administering
the cells and compositions to subjects, e.g., patients.
[0658] Also provided are compositions including the cells for
administration, including pharmaceutical compositions and
formulations, such as unit dose form compositions including the
number of cells for administration in a given dose or fraction
thereof. The pharmaceutical compositions and formulations generally
include one or more optional pharmaceutically acceptable carrier or
excipient. In some embodiments, the composition includes at least
one additional therapeutic agent.
[0659] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be administered.
A "pharmaceutically acceptable carrier" refers to an ingredient in
a pharmaceutical formulation, other than an active ingredient,
which is nontoxic to a subject. A pharmaceutically acceptable
carrier includes, but is not limited to, a buffer, excipient,
stabilizer, or preservative. In some aspects, the choice of carrier
is determined in part by the particular cell and/or by the method
of administration.
[0660] Accordingly, there are a variety of suitable formulations.
For example, the pharmaceutical composition can contain
preservatives. Suitable preservatives may include, for example,
methylparaben, propylparaben, sodium benzoate, and benzalkonium
chloride. In some aspects, a mixture of two or more preservatives
is used. The preservative or mixtures thereof are typically present
in an amount of about 0.0001% to about 2% by weight of the total
composition. Carriers are described, e.g., by Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Pharmaceutically acceptable carriers are generally nontoxic to
recipients at the dosages and concentrations employed, and include,
but are not limited to: buffers such as phosphate, citrate, and
other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride;
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as polyethylene glycol (PEG).
[0661] Buffering agents in some aspects are included in the
compositions. Suitable buffering agents include, for example,
citric acid, sodium citrate, phosphoric acid, potassium phosphate,
and various other acids and salts. In some aspects, a mixture of
two or more buffering agents is used. The buffering agent or
mixtures thereof are typically present in an amount of about 0.001%
to about 4% by weight of the total composition. Methods for
preparing administrable pharmaceutical compositions are known.
Exemplary methods are described in more detail in, for example,
Remington: The Science and Practice of Pharmacy, Lippincott
Williams & Wilkins; 21st ed. (May 1, 2005).
[0662] The formulations can include aqueous solutions. The
formulation or composition may also contain more than one active
ingredient useful for the particular indication, disease, or
condition being treated with the cells, preferably those with
activities complementary to the cells, where the respective
activities do not adversely affect one another. Such active
ingredients are suitably present in combination in amounts that are
effective for the purpose intended. Thus, in some embodiments, the
pharmaceutical composition further includes other pharmaceutically
active agents or drugs, such as chemotherapeutic agents, e.g.,
asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,
doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,
paclitaxel, rituximab, vinblastine, and/or vincristine. The
pharmaceutical composition in some embodiments contains the cells
in amounts effective to treat or prevent the disease or condition,
such as a therapeutically effective or prophylactically effective
amount. Therapeutic or prophylactic efficacy in some embodiments is
monitored by periodic assessment of treated subjects. The desired
dosage can be delivered by a single bolus administration of the
cells, by multiple bolus administrations of the cells, or by
continuous infusion administration of the cells.
[0663] Formulations include those for oral, intravenous,
intraperitoneal, subcutaneous, pulmonary, transdermal,
intramuscular, intranasal, buccal, sublingual, or suppository
administration. In some embodiments, the cell populations are
administered parenterally. The term "parenteral," as used herein,
includes intravenous, intramuscular, subcutaneous, rectal, vaginal,
and intraperitoneal administration. In some embodiments, the cells
are administered to the subject using peripheral systemic delivery
by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid
preparations, e.g., isotonic aqueous solutions, suspensions,
emulsions, dispersions, or viscous compositions, which may in some
aspects be buffered to a selected pH. Liquid preparations are
normally easier to prepare than gels, other viscous compositions,
and solid compositions. Additionally, liquid compositions are
somewhat more convenient to administer, especially by injection.
Viscous compositions, on the other hand, can be formulated within
the appropriate viscosity range to provide longer contact periods
with specific tissues. Liquid or viscous compositions can comprise
carriers, which can be a solvent or dispersing medium containing,
for example, water, saline, phosphate buffered saline, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol)
and suitable mixtures thereof.
[0664] Sterile injectable solutions can be prepared by
incorporating the cells in a solvent, such as in admixture with a
suitable carrier, diluent, or excipient such as sterile water,
physiological saline, glucose, dextrose, or the like. The
compositions can contain auxiliary substances such as wetting,
dispersing, or emulsifying agents (e.g., methylcellulose), pH
buffering agents, gelling or viscosity enhancing additives,
preservatives, flavoring agents, and/or colors, depending upon the
route of administration and the preparation desired. Standard texts
may in some aspects be consulted to prepare suitable
preparations.
[0665] Various additives which enhance the stability and sterility
of the compositions, including antimicrobial preservatives,
antioxidants, chelating agents, and buffers, can be added.
Prevention of the action of microorganisms can be ensured by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the
injectable pharmaceutical form can be brought about by the use of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0666] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0667] The contents of the articles, patents, and patent
applications, and all other documents and electronically available
information mentioned or cited herein, are hereby incorporated by
reference in their entirety to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference. Applicants reserve the right to
physically incorporate into this application any and all materials
and information from any such articles, patents, patent
applications, or other physical and electronic documents.
[0668] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. It will be readily apparent
to those skilled in the art that other suitable modifications and
adaptations of the methods described herein may be made using
suitable equivalents without departing from the scope of the
embodiments disclosed herein. In addition, many modifications may
be made to adapt a particular situation, material, composition of
matter, process, process step or steps, to the objective, spirit
and scope of the present invention. All such modifications are
intended to be within the scope of the claims appended hereto.
Having now described certain embodiments in detail, the same will
be more clearly understood by reference to the following examples,
which are included for purposes of illustration only and are not
intended to be limiting.
EXAMPLES
[0669] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only, and the invention is not limited to these
Examples, but rather encompasses all variations that are evident as
a result of the teachings provided herein.
[0670] The materials and methods employed in these experiments are
now described.
[0671] RNA CAR Construct Design: Four human scFvs specifically
targeting human PSMA, 1C3, 2A10, 2C6 and 2F5, were synthesized from
IDT as gBlocks. CARs with 4-1BB-zeta (BBZ) were assembled by
overlapping PCR and cloned into the RNA in vitro transcription
vector pD-A. The pD-A vector was optimized for T cell transfection,
CAR expression and RNA production. The four human PSMA CARs and one
mouse PSMA CAR (J591) were linearized by SpeI digestion prior to
RNA IVT. The T7 mScript Standard mRNA Production System
(Cellscript, Inc., Madison, Wis.) was utilized to generate
capped/tailed IVT RNA. The IVT RNA was purified by RNeasy Mini Kit
(Qiagen, Inc., Valencia, Calif.). Purified RNA was eluted in
RNase-free water at 1-2 mg/mL and stored at -80.degree. C. until
use. RNA integrity was confirmed by 260/280 absorbance and visually
on an Agarose gel.
[0672] Lenti CAR Construct Design: All PSMA CARs were subcloned
into pTRPE Lenti vectors. Switch receptor: PD1.CD28-F2A (SW),
PD1.sup.A132L-PTM.CD28-F2A (SW*) and a dominant negative
TGFR.beta.II sequence, dnTGFR.beta.II-T2A (dn), were then subcloned
into each Lenti vector followed by human PSMA scFv.
Examples of sequences comprised by a Lenti vector are as
follows:
TABLE-US-00155 1C3 (SEQ ID NO: 169)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGT
CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACC
TTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGC
TGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGT
ATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGG
TATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGC
GGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGT
TGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTAT
CAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCA
GTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGAC
AGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACT
TATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGA
CCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCG. 2A10 (SEQ ID NO:170)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGC
TTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGG
CCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGT
GGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC
AAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGG
AAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGG
TCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCAC
CATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAG
TTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AAACCACGACGCCAGCGCCGCG. 2F5 (SEQ ID NO. 171)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGT
TTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGG
CCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGT
GGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGC
AAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGG
AAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGG
TCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCAC
CATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAG
TTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCA
AAATCAAAACCACGACGCCAGCGCCGCG. 2C6 (SEQ ID NO. 172)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC
ACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAA
AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGC
TTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCC
TGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAG
CCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGC
ACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGT
ATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCG
GGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGT
CTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTG
CAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAA
CCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCA
CTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCAC
TCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT
CAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAG
TGGATATCAAAACCACGACGCCAGCGCCGCG. PD1.CD28-F2A (SW) (SEQ ID NO:173)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGG
GTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA
CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTT
GACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCG.
PD1.sup.A132L-PTMCD28-F2A (SW*) (SEQ ID NO:174)
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAAC
TGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAA
CCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAAC
GCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAA
ACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT
CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACA
CAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGC
GCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAA
GCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGA
AGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCG
GCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAG
CCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGC
CCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC
CTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCG
GGAGACGTGGAGTCCAACCCAGGGCCG. dnTGFR.beta.II-T2A (dn) (SEQ ID NO.
175) ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGT
GGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAA
TAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAA
CTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAAT
CCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGA
AGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAG
ACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAG
ATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGA
GACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATC
ATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCA
TATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCAT
ATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAG
CTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTC
TAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACC.
[0673] Transduction protocol: Bulk T cells (CD4 and CD8) obtained
from the Human Immunology Core were diluted to 10.sup.6 cells/mL,
and stimulated with CD3/28 beads (T cell expanders, Invitrogen) at
a cell:bead ratio of 1:3. Transductions of packaged lentiviral
vectors were performed on day 1 post-stimulation using a MOI of
3:1, and allowed to expand in a 37.degree. C./5% CO.sub.2
incubator.
[0674] Transduction efficacy: The CAR transduction efficacy was
evaluated by flow cytometry using Biotin-SP-AffiniPure Goat
Anti-Mouse IgG (Cat #: 115-065-072, Jackson ImmunoResearch Labs) or
Biotin-SP-AffiniPure Rabbit Anti-Human IgG (Cat #: 309-065-082,
Jackson ImmunoResearch Labs) followed by Streptavidin APC (Cat #:
17-4317-82, eBioscience) or Streptavidin PE (Cat #: 554061, BD
Pharmingen). APC anti-human CD279 (PD-1) antibody (Cat #: 329908,
BioLegend) and Human TGF-beta RII APC-conjugated Antibody (Catalog
# FAB241A, R&D systems) were used to examine the switch
receptor or dominant negative TGFR.beta.II portion.
[0675] T cell expansion: Cells were fed and split every 2 days
starting at day 3 post stimulation. T cells were de-beaded at day 4
and frozen at day 10 for later use.
[0676] RNA electroporation: Resting T cells were electroporated
with 10 or 20 .mu.g IVT PSMA RNA CARs using BTX830 at 500 V and 700
.mu.s. Nalm6.CBG or K562 cells were electroporated with 5 .mu.g or
15 .mu.g PSMA IVT RNA using BTX830 at 300 V and 500 .mu.s. PC3.PSMA
cells were electroporated with 0.5 .mu.g, 2 .mu.g or 5 .mu.g PDL1
IVT RNA using BTX830 at 300 V and 500 .mu.s. Following
electroporation, the cells were immediately placed in pre-warmed
culture media at 37.degree. C. and 5% CO.sub.2. 18 hr later, PSMA
or PDL1 electroporated tumor cells were stained by APC anti-human
PSMA (FOLH1) antibody (Cat #: 342507, BioLegend) or APC anti-human
CD274 (PDL-1 or B7-H1) antibody (Cat #: 17-5983-42, BD Biosciences)
and analyzed by Flow Cytometry.
[0677] Cell counting: At various time-points during the
expansion-resting cycles, cells were gently mixed and a 40 .mu.L
aliquot of cells was collected from known culture volume and placed
into accuvettes (Beckman Coulter) with 20 mL Isoton II Diluent
Buffer for counting using a Coulter Multisizer 3 (Beckman Coulter)
in accordance with the CCI laboratory SOP. These assays determined
cell concentration, total cell numbers, growth rates, and cell
volumes and were used to calculate dilution volumes and determine
when cells were rested for freezing.
[0678] Quantitative-PCR: Primary cells or tumor cell lines were
lysed and passed through QIA shredder (Cat #79656). Total RNA was
extracted by RNeasy Mini kit (Cat #74104) according to the
manufacturer's protocol. Reverse transcription (Cat #: 11904-018,
Invitrogen) was performed to obtain cDNA. cDNA was subjected to
quantitative PCR with primers specific for PSMA:
TABLE-US-00156 (F primer: AGGAAGTCTCAAAGTGCCCT (SEQ ID NO:176), R
primer: GAACAACAGCTGCTCCACTC (SEQ ID NO:177)) or GAPDH: (F primer:
GCTACACTGAGCACCAGGTGGTCTC (SEQ ID NO:178), R primer:
CCCAGCAGTGAGGGTCTCTCTCTTC (SEQ ID NO:179)).
[0679] ELISA for IL-2 and IFN-.gamma.: The T cells or target cells
were washed and suspended in R10 medium at 1.times.10.sup.6
cells/mL. Approximately 0.1 mL of each cell line was added to a
well of a 96-well plate (Corning) and incubated at 37.degree. C.
for 18 to 20 hours. The supernatant was harvested and subjected to
ELISA.
[0680] CD107a Assay: An E:T ratio of 1:2 (5.times.10.sup.4
effectors: 1.times.10.sup.5 targets) of cells were prepared in 100
.mu.L of R10 medium and plated in a 96 well plate. 10 .mu.L of
phycoerythrin-labeled anti-CD107a Ab was added and the plate was
incubated at 37.degree. C. for 1 hour. Golgi Stop (2 ul Golgi Stop
in 3 ml R10 medium, 10 ul/well; BD Biosciences, 51-2092KZ) was
added and the plate was incubated for another 2.5 hours. Then 2
.mu.L FITC-anti-CD8 (Cat #: 551347, BD Pharmingen) and 2 uL
APC-anti-CD3 (Cat #: 555342, BD Pharmingen) was added and incubated
at 37.degree. C. for 30 min. After incubation, the samples were
washed with FACS buffer and analyzed by flow cytometry.
[0681] Luciferase based CTL assay: Nalm6-CBG, PC3-CBG, PC3.PSMA-CBG
tumor cells were resuspended at 1.times.10.sup.5 cells/mL in R10
medium and incubated with different ratios of T cells (e.g. 10:1,
5:1, 2.5 etc.) for 18 hr at 37.degree. C. Equal volume of substrate
was added and the luminescence was immediately determined. Results
are reported as percent killing based on luciferase activity in
wells with only tumor in the absence of T cells (%
killing=100-((RLU from well with effector and target cell
co-culture)/(RLU from well with target cells).times.100)).
[0682] PC3.PSMA tumor model: 2E6 PC3.PSMA.7SC cells transduced with
click beetle were injected to the mice (i.v.), and 28 days later,
2E6 PSMA CAR-T positive transduced T cells were injected to the
tumor bearing mice (i.v.). Bioluminescence imaging (BLI) was
conducted at multiple time points.
[0683] The results of the experiments are now described.
Example 1: Human RNA PSMA CARs have Equivalent Anti-Tumor Activity
as Mouse RNA PSMA CAR, J591
[0684] Four human RNA CARs targeting PSMA were constructed using
one of four scFv sequences, 1C3 (SEQ ID NO:169), 2A10 (SEQ ID
NO:170), 2C6 (SEQ ID NO:172) and 2F5 (SEQ ID NO: 171), (from U.S.
patent application, US 2009/0297438 A1, incorporated by reference
herein in its entirety). ScFvs were linked to a CD8 transmembrane
domain and 4-1BB and CD3 zeta intracellular signaling domains.
Purified RNA was visualized on an Agarose gel (FIG. 1A) and
electroporated into resting human primary T cells. All CARs had
nearly 100% CAR expression under the condition tested. 10 ug of IVT
RNA CARs, whether it was human or mouse PSMA CAR, reached maximal
mean fluorescence intensity (MFI) for CAR expression; thus, 10 ug
IVT RNA was used for further experiments (FIG. 1B). CAR expression
varied among different human CARs, the highest MFI for CAR
expression being 1C3.BBZ. The MFI for mouse J591 CAR expression was
4 fold higher than that of 1C3.BBZ; however, since the species of
antibody origins differ, 10 ug mouse J591 RNA CAR was also used for
further experiments.
[0685] Full length PSMA was cloned into a PD-A vector for optimal
RNA expression. Purified RNA was visualized on an Agarose gel (FIG.
1A), electroporated into Nalm6.CBG or K562 tumor cells and the
expression of PSMA was analyzed by flow cytometry (FIG. 1C). 15 ug
and 5 ug PSMA RNA was used for further experiments for Nalm6.CBG
and K562 tumor cells respectively. PC3.PSMA.PSCA.CBG tumor cells
were constructed previously with diminishing PSMA expression
(37.5%) (FIG. 1C). Limited dilutions were performed for
PC3.PSMA.CBG tumor cell line and 7 single cell clones were isolated
and combined to be a new cell line, PC3.PSMA.7SC.CBG (FIG. 1D).
[0686] Nalm6.CBG or K562 electroporated with PSMA RNA, PC3 or
PC3.PSMA.PSCA.CBG tumor cells were co-cultured with various PSMA
RNA CARs. CD107a assays, Luciferase based CTL assays and ELISA
assays were performed to determine the functionality of the four
new human CARs. All four human PSMA CARs had equivalent
de-granulation activity as mouse PSMA CAR, J591, when co-cultured
with PSMA positive cells (FIG. 2A). However, three out of four
human PSMA CARs exerted higher non-specific activation toward PC3
cells than J591 CAR (FIG. 2A). All four human CARs had comparable
cytotoxicity and anti-tumor activity toward PSMA positive cells
compared with the J591 CAR (FIG. 2B and FIG. 2C). Cytokine
production was PSMA target specific for 1C3.BBZ, 2A10.BBZ and
2F5.BBZ CARs (FIG. 2C).
Example 2: Human Lenti PSMA CARs Specifically Target PSMA Positive
Cells
[0687] The four human PSMA CARs were subcloned into pTRPE Lenti
vector. Primary human T cells transduced with human PSMA CARs had
different CAR expression levels: 40% for 1C3.BBZ, 66% for 2A10.BBZ,
50% for 2C6.BBZ and 61% for 2F5.BBZ (FIG. 3A). A dominant negative
TGFR.beta.II sequence was linked to the mouse J591.BBZ CAR via a
T2A sequence (dnTGFR.beta.II-J591.BBZ). Nalm6.CBG electroporated
with PSMA RNA, PC3 or PC3.PSMA.CBG cells were co-cultured with
various PSMA Lenti CARs. CD107a assays, Luciferase based CTL assays
and ELISA assays were performed to determine the functionality of
the four new human PSMA Lenti CARs, and compared with mouse J591
CAR. 1C3.BBZ, 2A10.BBZ and 2F5.BBZ exerted similar anti-tumor
activity in the de-granulation assay and Luciferase based Killing
assay as dnTGFR.beta.II-J591 (FIG. 3B and FIG. 3C). In terms of
cytokine production, all four human PSMA Lenti CARs elicited
specific IL-2 and INF-.gamma. production but the amount varied
among the human CARs (FIG. 3D).
Example 3: Construction of Switch Receptor or Dominant
Negative-TGFR.beta.II Linked--Human Lenti CARs
[0688] A switch receptor (PD1.CD28), comprising a truncated
extracellular domain of PD1 and the transmembrane and cytoplasmic
signaling domains of CD28 was designed and linked to each human
PSMA CAR via a T2A sequence. A point mutation at the 132 (99 for
mouse) position from Alanine to Leucine on PD 1 increases its
affinity with PDL 1 by two fold (Zhang et al. Immunity 20, 337-347,
2004). Thus, the second version of switch receptor,
"PD1.sup.A32LPTM.CD28" (with truncated extracellular and
transmembrane domains of PD1 and cytoplasmic signaling domain of
CD28) was linked to each human PSMA CAR. The dominant negative
TGFR.beta.II sequence was subcloned into each human PSMA CARs as
well. Flow cytometry was performed to examine the transduction
efficiency of each switch receptor-CAR (FIG. 4A) and dnTGFR.beta.II
CAR (FIG. 4B). A distinct CAR/PD1 double positive population was
observed for all switch receptor-CAR transduced T cells. The
transduction efficiency was similar between the human PSMA Lenti
CARs and their two switch receptor counterparts (FIG. 4A). Each 2C6
CAR had the lowest transduction efficiency. There was no separate
dnTGFR.beta.II population but a clear shift was observed for each
dnTGFR.beta.II-linked human PSMA CAR (FIG. 4B).
[0689] To examine the functionality of the switch receptors,
various amounts of PDL1 RNA, 0.5 ug, 2 ug, and 5 ug, were
electroporated into PC3.PSMA cells (FIG. 4C). The PC3.PSMA cells
electroporated with 5 ug PDL1 RNA were co-cultured with each PSMA
CAR. dnTGFR.beta.II-J591.BBZ was normalized to a transduction
efficiency of 50% prior to the co-cultured experiment. Three out of
four human PSMA CARs and their switch receptors or dnTGFR.beta.II
counterparts showed comparable de-granulation activity with
dnTGFR.beta.II-J591.BBZ CAR when co-cultured with PSMA positive
cells (FIG. 4D, FIG. 4E, and FIG. 4F). 2C6.BBZ CAR and its relevant
counterparts demonstrated a lower degranulation activity which
might be due to lower transduction efficiency (FIG. 4G). All four
human PSMA CARs and their switch receptor or dnTGFR.beta.II
counterparts showed similar cytotoxicity toward PC3.PSMA cells
(FIG. 4H). All human PSMA CARs elicited comparable amounts of
cytokine with dnTGFR.beta.II-J591.BBZ CAR (FIG. 4I). Each switch
receptor CAR secreted almost two fold higher IL-2 compared with
their non-switch receptor or dnTGFR.beta.II CAR counterparts when
co-cultured with PDL1 electroporated PC3.PSMA cells (FIG. 4I).
Example 4: PC3.PSMA.7SC Xenograft Model
[0690] The following six human PSMA CARs were selected for use in
PC3.PSMA.7SC mouse xenograft model: 1C3.BBZ, PD1CD28.1C3.BBZ,
2A10.BBZ, PD1CD28.2A10.BBZ, dnTGFR.beta.II-2A10.BBZ and
dnTGFR.beta.II-J591.BBZ. CAR expression was tested by flow
cytometry (FIG. 5A and FIG. 5B). Mouse J591.BBZ Lenti CAR was
included in the functional test. All the PSMA CARs tested showed
similar degranulation activity in the CD107a assay (FIG. 5C and
FIG. 5D) and comparable killing activity in the Luciferase based
CTL assay, with 2A10.BBZ being the lowest and
dnTGFR.beta.II-J591.BBZ being the highest (FIG. 5E). Each switch
receptor CAR secreted almost two fold higher IL-2 comparing with
their non-switch receptor or dnTGFR.beta.II CAR counterparts when
co-cultured with PDL1 electroporated PC3.PSMA.7SC cells (FIG.
5F).
[0691] To ensure the safety of using the above-mentioned human PSMA
CARs, a panel of primary human cells (Table 1) was tested for PSMA
expression by quantitative PCR (FIG. 5G). Normalized by Nalm6.CBG,
all primary cells tested had various PSMA expression levels even
for PC3 cells which only had limited reactivity toward PSMA RNA
CARs (FIG. 2A) but not PSMA Lenti CARs (FIG. 5H) (PSMA expression
is 1800 fold higher for PC3.PSMA than PC3 cells). HREpC, HSAEpC and
HPMEC human primary were co-cultured with above-mentioned CARs.
CD107a and ELISA assays were performed. All PSMA CARs tested had
minimal detectable de-granulation activity when co-culturing with
HPMEC (FIG. 5H and FIG. 5I). PD1CD28.1C3.BBZ elicited increased
IL-2, compared with 1C3.BBZ when co-cultured with HPMEC (FIG. 5J).
Even though the level of cytokine elicited by primary human cells,
specifically, HPMEC is negligible when compared to that by
PC3.PSMA.7SC (FIG. 5J as compared to FIG. 5F).
TABLE-US-00157 TABLE 1 Primary human cells tested for PSMA
expression HRCEpC Human Renal Cortical Epithelial Cells Hn2 Primary
human Neuron hNP1 Human Neuronal progenitors hMSC-BM Human
Mesenchymal Stem Cells from Bone Marrow HPASMC Human Pulmonary
Artery Smooth Muscle Cells HCM Human cardiac myocytes HOB Human
Osteoblasts HAoSMC Human Aortic Smooth Muscle Cells HREpC Human
Renal Epithelial Cells HPAEC Human Pulmonary Artery Endothelial
Cells Kera Kerotinocyte HSAEpC Human Small Airway Epithelial Cells
HPMEC Human Pulmonary Microvascular Endothelial Cells
[0692] The in vivo NSG mouse experiment was designed for 7 groups
(five mice per group) to test the six above-mentioned PSMA CARs
plus a non-transduced control group. 2E6 PC3.PSMA.7SC cells
transduced with click beetle green were injected in the mice (i.v.)
and 28 days later, 2E6 CAR positive transduced T cells were
injected into the tumor bearing mice (i.v.). Bioluminescence
imaging (BLI) was conducted at different time points: day 27, 34,
42, 49 post tumor injection (FIG. 5K and FIG. 5L). Without being
bound by any theory, the results of this experiment indicated that
all the PSMA CARs tested had comparable anti-tumor activity as
dnTGFR.beta.II.J591.BBZ.
[0693] FIG. 6 shows the different domains of a dnTGFRII-T2A
PSMA-CAR construct, and a pTRPE dnTGFRII-T2A PSMA CAR vector
map.
Example 5: In Vivo Tumor Control by PSMA CAR-T Cells
[0694] Transduction protocol: Bulk T cells (CD4 and CD8) obtained
from the Human Immunology Core were diluted to 10.sup.6 cells/ml,
and stimulated with CD3/28 beads (T cell expanders, Invitrogen) at
a cell:bead ratio of 1:3. Transductions of packaged lentiviral
vectors were performed on day 1 post-stimulation using a MOI of
3:1, and allowed to expand in a 37.degree. C./5% CO.sub.2
incubator.
[0695] Transduction efficacy: The transduction efficacy was
evaluated by flow cytometry using the PE anti-human TCR V38
antibody (Cat #: 348104, BioLegend) and APC anti-human CD279 (PD-1)
antibody (Cat #: 329908, BioLegend).
[0696] T cell expansion: Cells were fed and split every 2 days
starting day 3 post stimulation. T cells were de-beaded at day 3 or
day 4 and frozen at day 12 for later use.
[0697] Cell counting: At various time-points during the
expansion-resting cycles, cells were gently mixed and a 40 .mu.l
aliquot of cells was collected from known culture volume and placed
into accuvettes (Beckman Coulter) with 20 ml Isoton II Diluent
Buffer for counting using a Coulter Multisizer 3 (Beckman Coulter)
in accordance with the CCI laboratory SOP. These assays determined
cell concentration, total cell numbers, growth rates, and cell
volumes and were used to calculate dilution volumes and determine
when cells were rested for freezing.
[0698] ELISA for IL-2 and IFN.gamma.: The T cells were washed and
suspended in R10 medium at 1.times.10.sup.6 cells/ml. Approximately
0.1 ml of each cell line was added to a well of a 96-well plate
(Corning) and incubated at 37.degree. C. for 18 to 20 hours. The
supernatant was harvested and subjected to ELISA.
[0699] CD107a assay: The cells were plated at an effector:target
(E:T) cell ratio of 1:1 (10.sup.5 effectors: 10.sup.5 targets) in
160 .mu.l of R/10 medium in a 96-well plate. An anti-CD107a
antibody was added and incubated with the cells for 1 hour at
37.degree. C. before Golgi Stop was added and incubated for an
additional 2.5 hours. The anti-CD8 and anti-CD3 antibodies were
added and incubated at 37.degree. C. for 30 min. After incubation,
the samples were washed once and subjected to flow cytometry with a
BD Accuri C6. The data were analyzed with the FlowJo software.
[0700] PC3-PMSA tumor models: 1E6 PC3-PMSA-CBG were injected to the
mice subcutaneously (s.c.), and 21 days later, lentiviral
transduced T cells were injected to the tumor bearing mice
intravenously (i.v.). Bioluminescence imaging (BLI) and tumor
measurements were conducted at multiple time points.
[0701] Results: The sequences set forth in Table 2 were generated
and tested for their ability to control tumors in vivo.
TABLE-US-00158 TABLE 2 PSMA CAR in combination with various switch
receptor sequences SEQ FIG. ID NO: Ref. Sequence CAR Switch
Description 111 B 2F5BBZ 2F5BBZ N/A comprises a 2F5 scFv, a 4-1BB
or costimulatory domain, and a 112 CD3 zeta intracellular signaling
domain 159 C PD1CD2 2F5BBZ PD1-CD28 comprises a PD1-CD28 switch
8.2F5BBZ and a 2F5BBZ PSMA CAR 163 D PD1*CD 2F5BBZ PD1.sup.A132L-
comprises a PD1.sup.A132L-CD28 28.2F5BBZ CD28 switch and a 2F5BBZ
PSMA CAR 209 E 2F5ICOSz 2F5ICOSz N/A comprises a 2F5 scFv, an ICOS
or costimulatory domain, and a CD3 210 zeta intracellular signaling
domain 211 F 2F5ICOSz 2F5ICOSz N/A comprises a 2F5 scFv, a variant
or YMNM YMNM ICOS costimulatory domain 212 comprising a YMNM motif,
and a CD3 zeta intracellular signaling domain 217 G PD1CD2 2F5ICOSz
PD1-CD28 comprises a PD1-CD28 switch or 8.2F5ICOSz and a 2F5ICOSz
PSMA CAR 227 218 H PD1CD2 2F5ICOSz PD1-CD28 comprises a PD1-CD28
switch or 8.2F5ICOSz YMNM and a 2F5ICOSzYMNM PSMA 232 YMNM CAR 219
I PD1*CD 2F5ICOSz PD1.sup.A132L- comprises a PD1.sup.A132L-CD28 or
28.2F5ICOSz CD28 switch and a 2F5ICOSz PSMA 233 CAR 220 J PD1*CD
2F5ICOSz PD1.sup.A132L- comprises a PD1.sup.A132L-CD28 28.2F5ICOSz
YMNM CD28 switch and a 2F5ICOSzYMNM YMNM PSMA CAR 221 K PD1*BB.
2F5ICOSz PD1.sup.A132L- comprises a PD1.sup.A132L-41BB or 2F5ICOSz
41BB switch and a 2F5ICOSz PSMA 229 CAR 222 L PD1*BB. 2F5ICOSz
PD1.sup.A132L- comprises a PD1.sup.A132L-41BB or 2F5ICOSz YMNM 41BB
switch and a 2F5ICOSzYMNM 234 YMNM PSMA CAR 223 M TIM3CD 2F5ICOSz
TIM3-CD28 comprises a TIM3-CD28 switch 28.2F5ICOSz and a 2F5ICOSz
PSMA CAR 224 N TIM3CD 2F5ICOSz TIM3-CD28 comprises a TIM3-CD28
switch or 28.2F5ICOSz YMNM and a 2F5ICOSzYMNM PSMA 235 YMNM CAR 225
O PD1*BB. 2F5ICOSz PD1.sup.A132L- comprises a PD1.sup.A132L-41BB
TIM3CD 41BB; switch, a TIM3-CD28 switch, 28.2F5ICOSz and TIM3- and
a 2F5ICOSz PSMA CAR CD28 226 P PD1*BB. 2F5ICOSz PD1.sup.A132L-
comprises a PD1.sup.A132L-41BB or TIM3CD YMNM 41BB; switch, a
TIM3-CD28 switch, 236 28.2F5ICOSz and TIM3- and a 2F5ICOSzYMNM YMNM
CD28 PSMA CAR
[0702] PSMA CARs with either ICOS or ICOS.YMNM signaling domain and
combination of CAR+PD1 (or Tim3) switch receptors were constructed
and cloned into a lentiviral vector (see, Table 2 for sequences).
The CAR expression levels in transduced T cells were comparable for
most of the CAR constructs (FIG. 7), and the switch receptors were
expressed properly (FIG. 8). When stimulated with PSMA positive
cell lines PC3.PSMA or PC3.PSMA.PD-L1 and examined for CD107a
upregulation, PSMA CAR with ICOS.YMNM signaling domain (ICOS ymnm)
showed significantly higher CD107a expression compared to wild type
ICOS (ICOS) or 4-1BB (41BB) CARs (FIG. 9). GranzymeB expression for
both ICOS and ICOS.YMNM PSMA CARs were similar to 4-1BB PSMA CAR
(FIG. 10). Cytokine production (IL-2 and IFN-gamma) of the PD1
switch receptors co-transduced with PSMA CARs comprising ICOS,
ICOS.YMNM, or 4-1BB were significantly higher when stimulated with
PD-L1 expressing PC3.PSMA cells (FIGS. 11A and 11B).
[0703] FIG. 12 and shows the quantification of bioluminescence
imaging of NSG mice bearing PC3-PSMA.CBG induced tumors treated
with T cells transduced with the CARs as indicated, up to 86 days
(FIG. 12A), and up to 151 days (FIG. 12B). FIG. 13 shows the tumor
size of NSG mice bearing PC3-PSMA.CBG induced tumors treated with T
cells transduced with the CARs as indicated, up to 164 days. As
shown, both ICOS (2F5.ICOSz) and ICOS.YMNM PSMA CARs
(2F5.ICOSzYMNM) showed worse tumor control than the 4-1BB PSMA CAR
(2F5.BBZ). The PD1-CD28 switch receptor improved the PSMA CAR with
4-1BB costimulatory domain (PD1.CD28.2F5.BBZ), but not for ICOS
(PD1CD28.2F5ICOSz) or ICOS.YMNM (PD1CD28.2F5ICOSzYMNM) PSMA CARs.
Both PD1.CD28.2F5.BBZ and PD1CD28.2F5ICOSzYMNM showed inferior
tumor control compared to 4-1BB PMSA CAR. When these ICOS based
CARs were co-delivered to T cells with a high affinity PD1 switch
receptor with 4-1BB signaling domain (PD1*BB), the tumor can be
controlled as efficiently as 4-1BB PMSA CAR. As shown in FIGS.
14A-14F, T cells co-delivered with ICOS.YMNM CAR and PD1 switch
receptor with 4-1BB signaling domain (PD1*BB.2F5ICOSzYMNM)
eliminated tumors. FIG. 14G provides a list of the T cells in order
of tumor control capabilities.
Example 7: PSMA CAR-T Cells to Co-Express Bispecific Antibodies for
PD1 to CD28 Switch or TGF Beta Receptor II to CD28 Switch
[0704] Five bispecific antibodies using scFvs that could bind PD-L1
(10A5, 13G4 and 1B12, see, e.g., PCT Publication No.
WO2007005874A2) or TGF beta receptor II (aTGFbRII-1 and aTGFbRII-3
(TGFb1 and TGFb3, see, e.g., U.S. Pat. No. 8,147,834) and an
anti-CD28 scFv (1412, see, e.g., U.S. Pat. No. 7,585,960) were
designed and the genes were synthesized by PCR. Sequence verified
DNA was cloned into PGEM.64A based RNA in vitro transcription
vector to generate pGEM.aTGFbR-1-1412 and pGEM.aTGFbR-3-1412. See,
e.g., PCT Publication No. WO2016122738A1.
[0705] PSMA CAR-T cells are generated that co-express a bispecific
antibody selected from the above described.
Example 8: Manufacture and Administration of Clinical
CART-PSMA-TGF.beta.RDN Autologous T Cells
[0706] CART-PSMA-TGF.beta.RDN investigational cell product
manufacturing, final formulation, testing, and labeling were
performed as described below, according to The Clinical Cell and
Vaccine Production Facility's (CVPF) standard operating protocols
(SOPs). The CVPF is a unit within the Division of Transfusion
Medicine and Therapeutic Pathology in the Department of Pathology
and Laboratory Medicine at the University of Pennsylvania. Within
the Division, in addition to the CVPF and the apheresis collection
facility, there is a separate hematopoietic stem cell processing
laboratory that is responsible for bone marrow and peripheral blood
stem cell products primarily dedicated to support the clinical
hematopoietic stem cell transplantation service. The CVPF is a
registered HCT Facility and accredited by the Foundation for the
Accreditation of Cellular Therapy (FACT).
[0707] Dynabeads CD3/C28 CTS.TM. (formerly named ClinExVivo) beads
were used for T cell activations and expansions.
[0708] CART-PSMA-TGF.beta.RDN investigational product manufacturing
was initiated from a leukapheresis product. Based on the
constitution of the leukaphereis product, as assessed by Beckman
Coulter Multisizer and BD FACS Calibur devices, the following
occured: depletion of monocytes via counterflow centrifugal
elutriation on the TerumoBCT Elutra, which employs a single use
closed system disposable set, washing step using a semi-automated,
closed-system device Haemonetics CellSaver 5, and/or Ficoll
separation of the buffy fraction of the PBMCs. On day 0, the
CART-PSMATGF.beta.RDN manufacturing process was initiated with
activation of T lymphocytes with the Dynabeads CD3/CD28 CTS beads.
The PSMA-TGFbRIIDN CAR LV vector was added on Day 1 at the total
final MOI. Vector transduction occured between days 1 and 3. On day
3, the cells were washed and media was replaced. Cultures were
allowed to continue expansion in the GE Wave Bioreactor System. On
the final day of the culture, cells were harvested and concentrated
using the Cell Saver Prior to harvest, the cell product was placed
on the Baxter MaxSep for removal of the Dynabeads CD3/CD28 CTS
beads. Following bead removal, the cell expansion was washed using
the Haemonetics Cell Saver 5 to remove residual vector, viral
particles, and cell debris. CART-PSMA-TGF.beta.RDN cells were
resuspended in cryopreservation media containing 31.25%
Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 1% Dextran 40 and 5%
Dextrose, 5% Human Serum Albumin, and 7.5% DMSO. Cells were frozen
in Cryostore Ethylene-Vinyl Acetate (EVA) (OriGen Biomedical) or
equivalent clear bags using a controlled-rate freezer.
[0709] Cryopreserved CART-PSMA-TGF.beta.RDN: Each infusion bag
contained .about.10-50 mL of cells. Cryopreserved cells were also
retained in small aliquots in identical cell concentrations to the
infusion dose and used as sentinel vials for performing a viability
and endotoxin test prior to infusions, and for stability
testing.
[0710] Leukapheresis collection and cell separation/enrichment:
Autologous peripheral blood lymphocytes were obtained via
leukapheresis collection at the Apheresis Unit at the Hospital of
the University of Pennsylvania (HUP). Cryopreserved historical
apheresis products collected from the patient prior to study entry
may be usable for CART-PSMA-TGF.beta.RDN cell manufacturing. If
used, the sample must have been collected at an appropriately
certified apheresis center and the product must have met adequate
mononuclear cell yields.
[0711] Approximately 10-15 L of blood was processed on the COBE
Spectra Apheresis System or equivalent system to obtain a
population of approximately 5.times.10.sup.9 white blood cells. In
addition to the screening testing requirements provided in the
protocol, blood from all apheresis donors underwent infectious
disease testing performed by the American Red Cross National
Testing Laboratory.
[0712] The apheresis product was transported in an insulated
container to the CVPF, and temperature was logged upon receipt.
Samples were removed for bacterial and fungal cultures, real-time
phenotyping by flow cytometry, and research and correlative study
purposes. Apheresis products were cryopreserved or processed by
elutriation. After Elutriation or cell washing, the cell number was
determined on the Coulter Multisizer M3/M4 and viability by trypan
blue dye exclusion assay. Elutriated products were cryopreserved or
proceeded to further processing. Cryopreserved apheresis or
elutriated products were thawed and washed prior to culture to
remove cryopreservation medium.
[0713] These products were then processed via either 1) washing and
seeding of elutriated lymphocytes, 2) positive selection with
CD3/CD28 beads, or 3) Ficoll based gradient separation for further
T-cell selection.
[0714] Culture Initiation and Expansion: Enriched lymphocytes were
stimulated with Dynabeads CD3/CD28 CTS in static tissue culture
flasks at an approximate range of 8.times.10.sup.5-1.times.10.sup.6
cells in XVIVO-15 media supplemented with 5% human AB Serum, 2 mM
L-GlutaMAX, 20 mM Hepes, 1 mM Sodium Pyruvate, 1% MEM Vitamin
Essential Mixture, 10 mM N-Acetylcysteine, and 100 IU/ml IL-2
(Modified X-VIVO 15 Media). Beads were added at a 3:1 bead to cell
ratio. On day 5 of culture, if an acceptable cell number was
achieved, cells were transferred to the WAVE 2/10EH Bioreactor for
expansion to the appropriate cell number allowing for harvest,
electroporation, sampling and final formulation. On the final day
of the culture, cells were harvested and concentrated using the
Cell Saver Wash system. Prior to harvest, the cell product was
placed on the Baxter MaxSep for removal of the anti-CD3/CD28
magnetic microbeads. Post-harvest, the expanded T cells were
resuspended at 2.times.10.sup.6 cells per mL of X-VIVO media
supplemented with 5% Human AB Serum. Cells were placed in a
37.degree. C. incubator overnight.
[0715] CART-PSMA-TGFBRDN Dose Formulation: The dose formulation
started at a dose of 1-3.times.10.sup.7/m.sup.2 CART-PSMA-TGFBRDN
cells for one cohort, and a dose of 1-3.times.10.sup.8/m.sup.2 for
other cohorts. Dosing was based on anti-PSMA CAR expression. The
total dose was formulated as a single dose.
[0716] Final Formulation: Post-incubation, after all release
testing samples and archives were removed, the cells were
resuspended in infusible cryopreservation media containing 31.25%
PlasmaLyte-A, 31.25% Dextrose (5%) in NaCl (0.45%), 7.5% DMSO, 5%
Human Serum Albumin, and 1% Low Molecular Weight Dextran (LMD).
[0717] Product Administration:
[0718] Cell thawing: The cells were thawed at the CVPF or at the
bedside using a water bath or comparable device maintained at
36.degree. C. to 38.degree. C. by trained personnel. There should
be no frozen clumps left in the container by the time it is
connected to the I.V. tube. If the CART-PSMA-TGF.beta.RDN cell
product appeared to have a damaged or leaking bag, or be
compromised, it was not infused, and was returned to the CVPF.
[0719] Administration: The infusion took place in an isolated room
in the CTRC or elsewhere in the Hospital of the University of
Pennsylvania, using precautions for immunosuppressed patients.
Prior to the infusion, two individuals independently verified the
information on the infusion product label in the presence of the
subject and confirmed that the information is correctly matched to
the participant. Cells were infused within approximately 30 minutes
after thaw. The CART cells were infused intravenously into an 18
gauge intravenous catheter, either through a peripheral vein
(preferred) or central vein. Macrodrip intravenous tubing was used
to infuse the CART cells by gravity (i.e., without an infusion
pump) at a rate of approximately .about.10 mL/minute through a
latex free-Y-type blood set with a 3-way stopcock. A leukoreduction
filter was not used for the infusion of the CART cell product.
Emergency medical equipment (i.e., emergency trolley) was available
during the infusion in case the subject had an allergic response,
or severe hypotensive crisis, or any other reaction to the
infusion. Vital signs (temperature, respiration rate, pulse, blood
pressure, and oxygen saturation by pulse oximetry) were measured
prior, and after the infusion. If the subject's vital signs were
not satisfactory and stable, vital signs were continually monitored
at a minimum of every hour or as clinically indicated until stable.
The subject was discharged after the physician managing their care
has determined the subject was in satisfactory condition.
Example 9: CART-PSMA-TGFBRDN Clinical Trial Design
[0720] This protocol tested the safety of 2 dose-levels of
CART-PSMA-TGF.beta.RDN cells administered intravenously alone or
after lymphodepletion with a moderate dose of cyclophosphamide
administered three days prior to CART-PSMA-TGF.beta.RDN cells. The
dose escalation followed a 3+3 design. CART-PSMA-TGF.beta.RDN cells
were permanently modified to be directed to the PSMA protein with
an anti-PSMA CAR fused to the signaling domains of 4-1BB and TCR.
The study population included patients with castrate resistant
prostate cancer with radiographic evidence of lymph node, visceral,
or osseous metastases. All patients must have progressed after
therapy with at least one standard 17a lyase inhibitor or
second-generation anti-androgen therapy.
[0721] The date the first patient was dosed was Aug. 31, 2017.
[0722] As part of informed consent, subjects were asked for
permission to test their tumor for PSMA as one of the eligibility
criteria. Evaluation of PSMA expression on a fresh tumor biopsy was
preferred; however, if a biopsy was not feasible or clinically
appropriate, then archived tissue from a recent metastatic tissue
biopsy was used to determine eligibility if obtained within prior
90 days.
[0723] Patients with confirmed .gtoreq.10% of tumor cells with PSMA
expression and who meet all other inclusion criteria were eligible
to participate.
[0724] Cohort 1 subjects (N=3 or 6) received a single dose of
1-3.times.10.sup.7/m.sup.2 lentivirally transduced
CART-PSMA-TGF.beta.RDN cells on day 0 without any conditioning
chemotherapeutic regimen. If the number of manufactured CAR T cells
did not meet the pre-specified minimum infused dose of
1.times.10.sup.7/m.sup.2 cells, then the dose was not administered,
and the subject was replaced in the study. If 1 DLT/3 subjects
occurs, the study enrolls an additional 3 subjects at this dose
level. If 0 DLT/3 subjects or 1 DLT/6 subjects occurs, the study
advances to Cohort 2. If 2 DLT/3 subjects occurs at dose of
1-3.times.10.sup.7/m.sup.2 cells, then enrollment in this Cohort is
stopped and the dose is de-escalated by 10-fold to
1-3.times.10.sup.6 cells/m.sup.2 (Cohort -1). In this situation, up
to 6 subjects are enrolled in Cohort -1.
[0725] Cohort 2 subjects (N=3 or 6) received a single dose of
1-3.times.10.sup.8/m.sup.2 lentivirally transduced
CART-PSMA-TGF.beta.RDN cells on day 0 without any conditioning
chemotherapeutic regimen. If the number of manufactured CAR T cells
did not meet the protocol-specified minimum of
1.times.10.sup.8/m.sup.2 cells, but does meet the minimum dose
requirement of at least 1.times.10.sup.7/m.sup.2 cells, then the
subject receives the dose and was not included in the DLT
assessment for Cohort 2. This subject would be replaced for DLT
assessment at this dose. If, the number of manufactured CAR T cells
did not meet the pre-specified minimum infused dose as outlined for
Cohort 1, then no dose was administered, and the subject was
replaced in the study. If 1 DLT/3 subjects occurs, the study
enrolls an additional 3 subjects at this dose level. If 0 DLT/3
subjects or 1 DLT/6 subjects occur, the study advances to Cohort 3.
If 2 DLT/3 subjects occur, then the study stops and declares
maximum tolerated dose (MTD).
[0726] Cohorts 1 and 2 served to identify the MTD of
CART-PSMA-TGF.beta.RDN cells. The MTD is defined as the highest
dose at which 0/3 or 1/6 DLTs occur.
[0727] Cohort 3 subjects (N=3 or 6) received a single infusion at
the MTD of lentivirally transduced CART-PSMA-TGF.beta.RDN cells on
day 0, following a single dose of 1.0 gram/m.sup.2 of
cyclophosphamide administered up to 4 days prior to the CAR T cells
(day -3+1 day). If 0 DLT/3 subjects occur, the study enrolls an
additional 3 patients to confirm tolerability. If 1 DLT/3 subject
occurs, the study enrolls an additional 3 subjects at this dose
level. If two of the initial three subjects experience a DLT, three
additional patients are accrued with a dose-reduction in the
lymphodepleting chemotherapy to 500 mg/m.sup.2 administered up to 4
days prior to the CAR T cells (day -3.+-.1 day).
[0728] Subjects were enrolled serially. Infusions were staggered to
allow assessment of DLTs for cohort progression, expansion, or dose
de-escalation. The infusions for the first 2 subjects in each
cohort were staggered by 28 days; the second subject was not
infused until 28 days after the infusion of the first subject. The
2nd and 3rd subjects in each cohort were infused and followed in
parallel but only after the 1st subject in that cohort completed
the day 28 visit without DLT.
[0729] DLT was defined as any new grade 3 or greater adverse event
at least possibly related to the T cell regimen that occured within
28 days of T cell infusion. If 1 DLT occurs in the first 3 subjects
treated at a dose level, the study enrolls an additional 3 subjects
at that dose level. If 2 DLT/3 subjects occur, then the study stops
and declares maximum tolerated dose, except for Cohort 1, where a
10-fold dose de-escalation occurs. If 0 DLT/3 subjects or 1 DLT/6
subjects at a dose level, the study advances to the next Cohort.
For cohort 3, if two of the initial three subjects experience a
DLT, three additional patients are accrued with a dose-reduction in
the lymphodepleting chemotherapy to 500 mg/m.sup.2 administered up
to 4 days prior to the CAR T cells (day -3.+-.1 day). Otherwise, if
0-1 DLT/3 subjects occur in cohort 3, the study enrolls an
additional 3 patients to confirm tolerability.
[0730] Subjects were followed up for safety assessments and
research assessments. Subjects returned for study follow-up on Days
1, 3, 7, 10, 14, 21, and 28 for safety assessments. On Day 28
(.+-.5), disease staging was performed with a CT
chest/abdomen/pelvis, bone scan, and serum PSA. The reasons for
this early imaging assessment at day 28 were to assess for systemic
inflammation effects and to monitor disease status at the time of
the expected homing of CART-PSMA-TGF.beta.RDN cells. Repeat disease
assessments (including imaging) were performed at Months 3 and 6
and as standard of care thereafter. If a subject had relevant
imaging data (CT abd/pelvis, MRI abd/pelvis, bone scan) within 4
weeks of Month 3 and/or 6 performed as part of their standard of
care, this was not repeated at Month 3 and/or 6.
[0731] Adverse event reporting began at the time of consent and
continues until the subject is off-study. While on study, subjects
were continually reassessed for evidence of acute and cumulative
toxicity. Upon discontinuation from the primary follow-up phase,
subjects enter long-term follow-up for up to 5 years from their
CART-PSMA-TGF.beta.RDN infusion. During long-term follow-up,
subjects are monitored for delayed adverse events that may be
associated with the administration of the CART-PSMA-TGF.beta.RDN
cells.
[0732] Peripheral blood samples were obtained at defined time
points to monitor for measures of safety and efficacy. Additional
blood and tissue samples (e.g. fluids, tissue biopsy) that were
obtained for clinical indications may also be sent for research
analysis. At any time that tissue or body fluids were obtained (for
example, drainage of pleural fluid or ascites fluid), fluid samples
that would otherwise be discarded were used instead for research
purposes. These studies include, but were not limited to,
CART-PSMA-TGF.beta.RDN cell persistence by Q-PCR and inflammation
marker assessment with a Luminex-based cytokine and chemokine
panel.
[0733] In case of unexpected AEs, additional blood and tissues were
collected for research analysis, focused at evaluating the
potential causality of the unexpected event with the infused
CART-PSMA-TGF.beta.RDN cells. The additional samples collected for
research did not exceed 3 tablespoons of blood twice in one week,
and one tissue sample collection procedure for per month.
[0734] Inclusion Criteria: [0735] 1. Metastatic castrate resistant
prostate cancer [0736] 2. .gtoreq.10% tumor cells expressing PSMA
as demonstrated by immunohistochemistry analysis on biopsied
tissue. [0737] 3. Radiographic evidence of osseous metastatic
disease and/or measurable, non-osseous metastatic disease (nodal or
visceral) [0738] 4. Patients .gtoreq.18 years of age [0739] 5. ECOG
performance status of 0-1 [0740] 6. Adequate organ function, as
defined by: [0741] a. Serum creatinine .ltoreq.1.5 mg/dl or
creatinine clearance .gtoreq.60 cc/min [0742] b. Serum total
bilirubin <1.5.times.ULN [0743] c. Serum ALT/AST<2.times.ULN
[0744] 7. Adequate hematologic reserve within 4 weeks of study
enrollment as defined by: [0745] a. Hgb>10 g/dl [0746] b.
PLT>100 k/ul [0747] c. ANC>1.5 k/ul [0748] Note: Subjects
must not be transfusion dependent [0749] 8. Evidence of progressive
castrate resistant prostate adenocarcinoma, as defined by: [0750]
a. Castrate levels of testosterone (<50 ng/ml) with or without
the use of androgen-deprivation therapy AND [0751] b. Evidence of
one of the following measures of progressive disease in the 12
weeks preceding study enrollment: [0752] i. soft tissue progression
by RECIST 1.1 criteria [0753] ii. osseous disease progression with
2 or more new lesions on bone scan (as per PCWG2 criteria) [0754]
iii. increase in serum PSA of at least 25% and an absolute increase
of 2 ng/ml or more from nadir (as per PCWG2 criteria) [0755] 9.
Prior therapy with at least one standard 17a lyase inhibitor or
second-generation anti-androgen therapy for the treatment of
metastatic castrate resistant prostate cancer [0756] 10. Provides
written informed consent [0757] 11. Subjects of reproductive
potential must agree to use acceptable birth control methods.
[0758] Exclusion Criteria: [0759] 1. Prior treatment with an
immune-based therapy for the treatment of prostate cancer,
including cancer vaccine therapies (such as SipuleucelT, PROSTVAC),
immune checkpoint inhibitors, radium-223 and immunoconjugate
therapies [0760] 2. History of an active non-curative non-prostate
primary malignancy within the prior 5 years [0761] 3. Subjects who
require the chronic use of systemic corticosteroid therapy [0762]
4. Subjects who have received >3 prior therapies for the
treatment of castrate resistant prostate cancer (excluding
luteinizing hormone-releasing hormone agonists or antagonists, or
first generation anti-androgen therapies). This includes subjects
who received Taxotere in non-castrate resistant setting. [0763] 5.
Subjects with Class III/IV cardiovascular disability according to
the New York Heart Association Classification (see Attachment 2)
[0764] 6. Subjects with symptomatic vertebral metastases affecting
spinal cord function (as determined by clinical history, physical
exam, or MRI imaging) [0765] 7. History of active autoimmune
disease requiring immunosuppressive therapy [0766] 8. Patients with
ongoing or active infection. [0767] 9. History of allergy or
hypersensitivity to study product excipients (human serum albumin,
DMSO, and Dextran 40) [0768] 10. Active hepatitis B, hepatitis C or
HIV infection.
Example 10: Phase 1 Clinical Safety Data
[0769] A total of six subjects have been infused and two subjects
remain on study as of Jul. 25, 2018. Three subjects were infused in
Cohort 1 and three subjects were infused in Cohort 2. Thus, Cohort
2 was filled. In contract to Cohort 1, all three subjects infused
in Cohort 2 experienced cytokine release syndrome (CRS): two
subjects had grade 3 CRS and one subject had grade 1 CRS, all of
which developed within 12 hours CAR T cell infusion. These
toxicities were managed per protocol/institutional guidelines and
resolved. Thus, Cohort 2 was completed without a DLT.
[0770] The study Site Initiation Visit was held on Wednesday, Feb.
22, 2017 and the study was activated on Mar. 8, 2017. As of Jul.
25, 2018, the clinical site consented 8 subjects. Of the 8 subjects
consented there was 1 screen failure, 1 subject withdrew prior to
treatment, and 6 subjects were infused.
[0771] Table 3 shows a summary of the demographics of screened
subjects (N=8).
TABLE-US-00159 TABLE 3 Demographics of screened subjects Screen
Reason for Reason Sex Age at Fail Screen Infused for End Subject ID
Cohort (F/M) Consent Race (Y/N) Fail (Y/N) of Study 1 32816-01 N/A
M 66 Caucasian Y Excluded N Screen based on Failure prior immune
therapy 2 32816-02 1 M 55 Caucasian N N/A Y Death; Neutropenic
Sepsis* 3 32816-03 N/A M 67 Caucasian N N/A N Subject Withdrew
Consent 4 32816-04 1 M 50 Caucasian N N/A Y Death; Disease
Progression* 5 32816-05 1 M 71 Caucasian N N/A Y Disease
Progression 6 32816-06 2 M 72 Caucasian N N/A Y Disease Progression
7 32816-07 2 M 73 Caucasian N N/A Y Active on Study 8 32816-08 2 M
64 Caucasian N Y Active on Study N/A = not applicable *Death
occurred during long term follow up. Therefore, this event did not
qualify as a PDAE and determined unrelated to the IP.
[0772] Table 4 shows a summary of the current protocol status for
infused subjects (N=6).
TABLE-US-00160 TABLE 4 Current protocol status for infused subjects
Last Study Related Serious Visit/Date of Protocol Adverse Adverse
Adverse Last Visit in Off-Study Date/ Deviation Events Events
Events Study Subject ID Primary Study Reason (Y/N) (Y/N) (Y/N)
(Y/N) Status 1 32816-02 Day 28/ Nov. 16, 2017/Death N Y Y Y Off-
Sep. 29, 2017 (Neutropenic Sepsis) Study 2 32816-04 Month 3/ May
19, 2018/Death N Y N N Off- Feb. 15, 2018 (Disease Progression)
Study 3 32816-05 Month 6/ Jun. 13, 2018/Disease N Y Y Y Off- May
22, 2018 Progression Study 4 32816-06 Day 28/ Jul. 17, 2018/Disease
Y Y Y Y Off- Jul. 13, 2018 Progression Study 5 32816-07 Month 2/
N/A N Y Y Y On- Jul. 9, 2018 Study 6 32816-08 Day 28/ N/A Y Y Y Y
On- Jul. 25, 2018 Study
[0773] Table 5 shows a summary of deviations or exceptions for
infused subjects (N=6).
TABLE-US-00161 TABLE 5 Deviations or exceptions for infused
subjects Date Protocol Exception Description Status of Exception or
Deviation of Exception Exception Subject ID or Deviation Identified
or Deviation or Deviation 32816-02 Exception Apr. 24, 2017 Subject
had repeate Sponsor approved; screening biopsy as approved by all
initial biopsied local regulatory material was review communities
determined to be insufficient for PSMA expression analysis as it
contained fat, marrow tissue 32816-04 No Deviations or Exceptions
to report 32816-05 No Deviations or Exceptions to report 32816-06
Deviation Jun. 12, 2018 Subject was infused Sponsor on Jun. 11,
2018; the acknowledgment vital signs source communicated to
documentation was the site Aug. 7, lost during the 2018; did not
subject's transfer to require real time the ICU and therefore
reporting as it did there is no record not affect subject of the
subject's safety. Corrective protocol-required and preventative
pre- and post-infusion action plan being vital signs implemented.
32816-07 No Deviations or Exceptions to report 32816-08 Deviation
Mar. 29, 2018 The study pathologist Sponsor approved; performed
PSMA IRB approved; did expression testing on not require real a
specimen collected time reporting as as standard of care it did not
affect prior to the patient subject safety. signing the pre-
Corrective and screening informed preventative action consent form
plan implemented.
[0774] Table 6 shows a summary of infusion dates and doses among
infused subjects (N=6).
TABLE-US-00162 TABLE 6 PSMA-TGF.beta.RDN infusion dates and dose
summary among infused subjects Cells Infused Transduction
Efficiency Total CART- CART- Met Met Target PSMA- PSMA- Target %
scFv % scFv Total Cell TGF.beta.RDN TGF.beta.RDN Dose Flow Flow
Subject ID Cohort Infusion Date Dose Cell Dose Cell Dose/m.sup.2
(Y/N) (%) (Y/N) 1 32816-02 1 Aug. 31, 2017 9.25 .times. 10.sup.7
5.61 .times. 10.sup.7 3 .times. 10.sup.7/m.sup.2 Y 60.5 Y 2
32816-04 1 Nov. 13, 2017 1.20 .times. 10.sup.8 7.56 .times.
10.sup.7 3 .times. 10.sup.7/m.sup.2 Y 62.9 Y 3 32816-05 1 Nov. 20,
2017 7.66 .times. 10.sup.7 5.58 .times. 10.sup.7 3 .times.
10.sup.7/m.sup.2 Y 72.7 Y 4 32816-06 2 Jun. 11, 2018 1.05 .times.
10.sup.9 7.29 .times. 10.sup.8 3 .times. 10.sup.8/m.sup.2 Y 69.7 Y
5 32816-07 2 May 7, 2018 1.18 .times. 10.sup.9 6.60 .times.
10.sup.8 3 .times. 10.sup.8/m.sup.2 Y 56.1 Y 6 32816-08 2 Jun. 27,
2018 1.13 .times. 10.sup.9 6.36 .times. 10.sup.8 3 .times.
10.sup.8/m.sup.2 Y 56.4 Y
[0775] Table 7 is a summary of disease response for infused
subjects (N=6).
TABLE-US-00163 TABLE 7 Disease response for infused subjects
Overall Tumor Response Response Subject ID Cohort Criteria Day 28
Month 2 Month 3 Month 6 1 32816-02 1 RECIST 1.1 NE N/A N/A N/A Bone
Scan New N/A N/A N/A Lesions 2 32816-04 1 RECIST 1.1 NE Not PD N/A
Assessed Bone Scan No New Not New N/A Lesions Assessed Lesions 3
32816-05 1 RECIST 1.1 SD Not SD PD Assessed Bone Scan New Not New
No New Lesions Assessed Lesions Lesions 4 32816-06 2 RECIST 1.1 PD
N/A N/A N/A Bone Scan Not N/A N/A N/A Assessed 5 32816-07 2 RECIST
1.1 SD Not Pending Pending Assessed Bone Scan No New Not Pending
Pending Lesions Assessed 6 32816-08 2 RECIST 1.1 PD Pending Pending
Pending Bone Scan New Pending Pending Pending Lesions NE = Not
Evaluable PD = Progressive Disease SD = Stable Disease Pending =
Subject has not yet reached this time point Not Assessed = An
assessment was not done at this time point N/A = Not
applicable/Subject discontinued primary follow-up prior to this
time point
[0776] Table 8 is a summary of serum PSA levels for infused
subjects (N=6).
TABLE-US-00164 TABLE 8 Serum PSA levels for infused patients (data
provided in ng/mL) Pre-Infusion Subject ID Cohort Screening Safety
Day 28 Month 2 Month 3 Month 6 Unscheduled 1 32816-02 1 163.80
237.80 167.40 317.10 N/A N/A (Day + 20): 162.20 2 32816-04 1 9.35
7.60 10.45 19.79 38.11 N/A -- 3 32816-05 1 17.83 10.47 14.01 11.75
18.77 47.31 -- 4 32816-06 2 14.26 41.75 132.20 N/A N/A N/A -- 5
32816-07 2 219.30 324.30 340.50 372.50 Pending Pending (Day + 10:
286.80 (Day + 14): 285.40 (Day + 21): 341.60 6 32816-08 2 70.56
134.50 197.10 Pending Pending Pending -- N/A = not applicable =
Subject entered LTFU on Nov. 1, 2017; Month 2 PSA was drawn on Nov.
2, 2017 -- = no unscheduled data for this subject Pending = subject
has not yet reached this time point
[0777] Table 9 is a summary showing PSMA-TGF.beta.RDN cell marking
in the peripheral blood by qPCR for infused subjects (N=6).
TABLE-US-00165 TABLE 9 PSMA-TGF.beta.RDN cell marking in the
peripheral blood by qPCR for infused subjects (data provided in
copies/microgram genomic DNA) Pre- Infusion Day 0 Day 0 Subject ID
Cohort Safety pre post Day 1 Day 3 Day 7 Day 10 32816-02 1 ND ND
85.15 4.23 101.82 343.70 1412.81 32816-04 1 ND ND 151.6 ND 25.90
417.31 3351.64 32816-05 1 ND ND 46.99 5.95 9.23 ND 33.38 32816-06 2
ND ND 394.94 16.87 201.73 3099.79 1084.13 32816-07 2 ND ND 530.57
16.27 89.39 457.40 151.45 32816-08 2 ND ND 422.92 63.32 96.76
253.85 217.28 Subject ID Day 14 Day 21 Day 28 Month 2 Month 3 Month
6 32816-02 110.46 11.46 8.04 N/A N/A N/A 32816-04 216.02 27.03 ND
ND ND N/A 32816-05 43.61 7.85 14.50 ND ND ND 32816-06 218.64 Not
26.96 N/A N/A N/A Collected 32816-07 84.31 43.40 14.50 Not Yet
Pending Pending Resulted 32816-08 114.46 72.06 Not Yet Pending
Pending Pending Resulted ND = not detected N/A = not applicable -
subject discontinued primary follow-up priot to this visit Pending
= subject has not yet reached this time point Not Collected =
Research samples were not collected for analysis Not Yet Resulted =
Sample has not yet been tested
[0778] Table 10 is a summary showing PSMA-TGF.beta.RDN cell marking
in other tissues by qPCR for infused subjects (N=6).
TABLE-US-00166 TABLE 10 PSMA-TGF.beta.RDN cell marking in other
tissues by qPCR for infused subjects (data provided in
copies/microgram genomic DNA) Time Subject ID Cohort Point Sample
Type Results 32816-02 1 Day Tumor (1A FFPE 122.32 10 tissue curls)
Tumor (2A FFPE 57.99 tissue culrs) Day Other (BMBMX ND 21 core
cells) Other (Marrow) 27.12 Month Other (BMBMX ND 2 core cells)
32816-04 1 Day Tumor (HS17- 133.36 10 37343-1A Right Iliac) Tumor
(HS17- 211.16 37343-1B Right Iliac) 32816-05 1 Day Tumor 758.51 10
(Retroperitoneal lymph node) 32816-06 2 Day Tumor BX curls 98.24 10
32816-07 2 Day Tumor BX curls ND 10 32816-08 2 No other tissue data
at this time FFPE = Formalin-fixed, paraffin embedded BMBMX = Bone
marrow biopsy BX = Biopsy ND = Not detected
[0779] Table 11 is a summary showing percent PSMA positive tumor
cells for enrolled subjects as determined by immunohistochemistry
(N=7).
TABLE-US-00167 TABLE 11 Percent PSMA positive tumor cells for
enrolled subjects Sample Subject ID Cohort Timepoint Type Location
Results (%) 1 32816-02 1 Screening Fresh Right iliac bone ND,
biopsy insufficient Screening Fresh Bladder 100 Day 10 Fresh
Bladder 100 2 32816-03 NA Screening Fresh Right external 100 iliac
lymph node 3 32816-04 1 Screening Archived Iliac bone 30 Day 10
Fresh Left iliac bone 75 4 32816-05 1 Screening Fresh Left 100
retroperitoneal lymph node Day 10 Fresh Left 100 retroperitoneal
lymph node 5 32816-06 2 Screening Fresh Para aortic lymph 25 node
Day 10 Fresh Para aortic lymph 100 node 6 32816-07 2 Screening
Fresh Posterior vertebra 100 Day 10 Fresh L1 vertebra 80 7 32816-08
2 Screening Fresh Bladder 100 Day 10 Fresh Primary 70-80 NA = Not
assigned ND = Not detected
Example 11: Phase 1 Clinical Trial of
PSMA-Directed/TGF.beta.-Insensitive CAR-T Cells in Metastatic
Castration-Resistant Prostate Cancer
[0780] Background:
[0781] Adoptive immunotherapy with CAR-T cells has transformative
potential for the treatment of cancer. A primary challenge to the
success of these therapies in prostate cancer is the
immunosuppressive microenvironment, including high levels of
TGF.beta., encountered by re-directed T cells upon tumor
infiltration. Importantly, these immunosuppressive functions of
TGF.beta. can be inhibited in T cells using a dominant negative
TGF.beta. receptor (TGF.beta.Rdn), thereby enhancing antitumor
immunity. In in vivo disseminated tumor models, co-expression of
TGF.beta.Rdn on PSMA-directed CAR-T cells led to increased T cell
proliferation, enhanced cytokine secretion, long-term persistence,
and greater induction of tumor eradication. Mechanisms of adaptive
tumor resistance are unknown.
[0782] FIG. 15 shows the efficacy of CART-PSMA-TGF.beta.Rdn cells
in in vivo disseminated tumor models. FIG. 15A is a graph showing
that CART-PSMA-TGF.beta.Rdn cells demonstrated enhanced
antigen-specific proliferation versus CART-PSMA over 42 days
co-culture and repetitive stimulation with PSMA-expressing tumor
cells. FIG. 15B is a graph showing that in vivo,
CART-PSMA-TGF.beta.Rdn cells demonstrated significantly increased
tumor reduction compared to CART-PSMA, as measured by BLI imaging
weekly to assess tumor burden. FIG. 15C are photographs showing the
location and systemic burden of tumor with weekly BLI assessment.
Abbreviations used in FIG. 15: Pbbz=CAR-T PSMA;
dnTGFBR2-T2A-Pbbz=CART-PSMA-TGF.beta.Rdn; 19bbz=anti-CD 19 CAR.
[0783] Study Design:
[0784] Study Overview: A first-in-human phase 1 clinical trial was
initiated to evaluate the safety and preliminary efficacy of
lentivirally-transduced PSMA-directed/TGF.beta.-insensitive CAR-T
cells (CART-PSMA-TGF.beta.Rdn) in men with treatment-refractory
metastatic castrate resistant prostate cancer (CRPC) (NCT03089203).
In preliminary dose-escalation cohorts, patients received a single
dose of 1-3.times.10.sup.7/m.sup.2 (Cohort 1) or
1-3.times.10.sup.8/m.sup.2 (Cohort 2) CART-PSMA-TGF.beta.Rdn cells
without lymphodepleting chemotherapy in a 3+3 design. In Cohort 3,
patients receive the maximum tolerated dose (MTD) of
CART-PSMA-TGF.beta.Rdn cells following lymphodepleting chemotherapy
with Cyclophosphamide 300 mg/m.sup.2 and Fludarabine 30 mg/m.sup.2
for 3 days. All treated patients underwent metastatic tumor
biopsies at baseline, as well as on day +10 following the CAR-T
cell infusion.
[0785] Key Eligibility Criteria: Metastatic CRPC, with previous
treatment with at least one second-generation androgen signaling
inhibitor (abiraterone or enzalutamide); .gtoreq.10% tumor cells
expressing PSMA by IHC on metastatic tissue biopsy; radiographic
evidence for metastatic disease (osseous or nodal/visceral);
.ltoreq.4 lines of therapy for metastatic CRPC.
[0786] Study Schema: FIG. 16 shows the study schema used in this
clinical trial.
[0787] Correlative Analyses: Quantitative PCR of
CART-PSMA-TGF.beta.Rdn DNA was performed at serial timepoints to
evaluate for CAR-T expansion and persistence in peripheral blood
and trafficking to target tissues. Bioactivity of
CART-PSMA-TGF.beta.Rdn cells in peripheral blood was evaluated
through Luminex analyses of immune and inflammatory factors.
Circulating tumor material was collected at serial time points and
correlated with clinical response.
[0788] Study Status and Preliminary Findings: Six patients received
CART-PSMA-TGF.beta.Rdn cell infusions at the specified dose levels
(Cohort 1, N=3; Cohort 2, N=3). All CART-PSMA-TGF.beta.Rdn infusion
products met target transduction efficiency. No dose limiting
toxicitities were observed in preliminary dose escalation.
[0789] Evaluation of CAR-T cellular kinetics via qPCR of
CART-PSMA-TGF.beta.Rdn DNA demonstrated peripheral blood T cell
expansion (FIG. 17), as well as tumor tissue trafficking in
post-treatment tumor biopsies (Table 17).
TABLE-US-00168 TABLE 17 CART-PSMA-TGF.beta.RDN cell trafficking:
qPCR detection in tissue biopsy samples in infused subjects Time
Subject ID Cohort Point Sample Type Results* 32816-02 1 Day Bladder
(FFPE 122.32 10 tissue curls) Bladder (FFPE 57.99 tissue curls) Day
Bone marrow ND 21 biopsy core Bone marrow 27.12 Month Bone marrow
ND 2 biopsy core 32816-04 1 Day Bone (FFPE 133.36 10 tissue curls)
Bone (FFPE 211.16 tissue curls) 32816-05 1 Day Lymph node 758.51 10
(FFPE tissue) 32816-06 2 Day Lymph node 98.24 10 (FFPE tissue)
32816-07 2 Day Bone (FFPE ND 10 tissue curls) 32816-08 2 Data
analysis pending *copies/ug gDNA FFPE = formalin-fixed, paraffin
embedded ND = not detected
[0790] In Cohort 2, two patients developed anticipated Grade 3
cytokine release syndrome (CRS), which is a critical marker of
biologic activity with CAR-T therapy, and one patient developed
Grade 3 CAR-T neurotoxicity requiring corticosteroids.
[0791] Marked increases in inflammatory cytokines (IL-6, IL-15,
IL-2, IFNgamma) and ferritin correlated with all Grade 3 CRS events
(Subject 32816-06: FIG. 18A; and subject 32816-07: FIG. 18B). All
CRS events rapidly resolved with tocilizumab (anti-IL6R)
rescue.
[0792] Cohort 3 enrollment (MTD with lymphodepleting chemotherapy)
began in September 2018.
Example 12: Cohorts 1 and 2 Observations and Case Study
[0793] FIG. 19 shows a graph of prostate specific antigen (PSA)
response among Cohort 1 and Cohort 2 patients.
[0794] Subject 32816-07: 74 year old with metastatic castration
resistant prostate cancer (mCRPC; initial diagnosis: May 2014).
Fever to 103F several hours post-PSMA-TGF.beta.RDN CART infusion
(no lymphodepletion) was observed. Hypotension was observed
approximately 6 hours post-PSMA-TGF.beta.RDN CART at 83/44 mmHg
nadir. Hypotension was managed with crystalloid infusion (no
pharmacologic management required during ICU admission) and
tocilizumab with resolution by the following day after
PSMA-TGF.beta.RDN CART infusion.
[0795] Cytokine release syndrome (CRS) was observed in patient
32816-07 following PSMA-TGF.beta.RDN CART-infusion (FIG. 20A). In
FIG. 20A, the left y-axis indicates the level of PSMA-TGF.beta.RDN
CART in peripheral blood in copies/ug of genomic DNA (32816-07),
and the right y-axis indicates the level of IL-6 in pg/ml (IL6).
CRS was accompanied by transient PSA decrease (FIG. 20B). In FIG.
20B, the left y-axis indicates the serum level of C-reactive
protein (CRP) in mg/L and the right y-axis indicates the serum
level of ferritin in ng/L.
[0796] PSMA Positive CTC Observations in Cohorts 1 and 2: Table 18
shows a summary of the number of PSMA-positive circulating tumor
cells (CTCs) detected in each subject across various time points,
the data of which is graphed in FIG. 21.
TABLE-US-00169 TABLE 18 PSMA-positive CTCs in Cohorts 1 and 2 Week
-8 Day 10 Day 28 Month 3 Screening Post-Infusion Post-Infusion
Post-Infusion Total # PSMA + Total # PSMA + Total # PSMA + Total #
PSMA + Cohort Subject ID CTC CTCs (%) CTC CTCs (%) CTC CTCs (%) CTC
CTCs (%) 1 32816-02 248 144 421 241 230 117 788 409 (58.1%) (57.2%)
(50.9%) (51.9%) 32816-03 3 3 Off study (100.0%) 32816-04 3 2 0 0 15
11 3 1 (66.7%) (73.3%) (33.3%) 32816-05 1 0 1 0 0 0 3 3 (0.0%)
(0.0%) (100.0%) 2 32816-06 12 12 3 0 0 0 0 Off (100.0%) study
32816-07 13 -- 3 2 0 0 0 0 32816-08 3 -- Pending
Example 13: Humanized PSMA Chimeric Antigen Receptor
[0797] Chimeric antigen receptors (CARs) were generated using a
fully humanized antibody derived from the anti-PSMA monoclonal
antibody, J591.
Materials and Methods
[0798] Cell lines and primary human T lymphocyte cultures: PC3 is a
prostate cancer tumor line (ATCC; Cat #: CRL-1435).
Prostate-specific membrane antigen (PSMA) was lentivirally
transduced into PC3 to produce PC3-PSMA. CBG was lentivirally
transduced into PC3 and PC3-PSMA to produce PC3-CBG and
PC3-PSMA-CBG.
[0799] Primary human CD4 and CD8 T cells were isolated from healthy
volunteer donors following leukapheresis by negative selection
using RosetteSep Kits (Stem Cell Technologies). All specimens were
collected under a University Institutional Review Board-approved
protocol, and written informed consent was obtained from each
donor. Mixed primary human CD4 and CD8 T cells (1:1) were
stimulated with anti-CD3/CD28 Dynabeads (Life Technologies).
[0800] CAR constructs and lentiviral transduction: CAR scFv domains
against PSMA were synthesized and/or amplified by PCR, linked to
CD8 transmembrane domain and 4-1BB or ICOS and CD3 zeta
intracellular signaling domains, and subcloned into pTRPE
lentiviral vectors. T cells were transduced with lentiviral vectors
at an MOI of 5.
[0801] Flow cytometry: Flow cytometry was used to determine the
transduction efficiency of transduced cells following staining with
biotin-labeled polyclonal anti-mouse F(ab)2 antibody (Jackson
Immunoresearch). The following antibodies were used in flow
cytometry experiments: PE conjugated Streptavidin. Data acquisition
was performed on FACSCalibur (BC Biosciences) and analyzed via
FlowJo.
[0802] CD107a assay: Cells were plated at an E:T of 1:2
(1.times.10.sup.5 effectors: 2.times.10.sup.5) in 160 .mu.L of R10
medium in a 96-well plate. Of note, 20 .mu.L of
phycoerythrin-labeled anti-CD107a Ab was added and the plate was
incubated at 37.degree. C. for 1 hour before adding Golgi Stop (2
mL Golgi Stop in 3 mL R10 medium, 20 mL/well; BD Biosciences,
51-2092 KZ) and incubating for another 2.5 hours. Then 5 mL
FITC-anti-CD8 and 5 mL streptavidin-allophycocyanin (APC)-anti-CD3
were added and incubated at 37.degree. C. for 30 minutes. After
incubation, the samples were washed with FACS buffer and analyzed
by flow cytometry.
[0803] Enzyme-linked immunosorbent assay (ELISA): Target cells were
washed and suspended at 1.times.10.sup.6 cells/mL in R10 medium. Of
note, 100 .mu.L each target cell type were added in triplicate to a
96-well round bottom plate (Corning). Effector T cells were washed
and resuspended at 1.times.10.sup.6 cells/mL in R10 cells and then
100 .mu.L of T cells were combined with target cells in the
indicated wells. The plates were incubated at 37C for 18 to 24
hours. After the incubation, supernatant was harvested and
subjected to an ELISA assay (eBioscience).
[0804] Luciferase-based cytolytic T-cell (CTL) assay: Click beetle
green luciferase (CBG)-T2A-eGFP was lentivirally transduced into
PC3 and PC3-PSMA tumor cells and sorted for GFP expression. Tumor
cells were incubated with different ratios of T cells 8 hours at
37.degree. C. Of note, 100 mL of the mixture was transferred to a
96-well white luminometer plate, 100 mL of substrate was added, and
the luminescence was immediately determined. Results are reported
as percent killing based on luciferase activity in wells with
tumor, but no T cells. (% killing=100-((RLU from well with effector
and target cell coculture)/(RLU from well with target
cells).times.100).
Results
[0805] In FIGS. 22A-22C, FIGS. 23A-23B, and FIGS. 24A-24E,
dnTGF-hJ591VHVK.BBZ represents transduced T cells comprising a
dominant negative TGFR.beta.II sequence linked to the humanized
J591 (hJ591) BBZ CAR via a T2A sequence, wherein the hJ591 CAR
comprises a 4-1BB costimulatory domain and CD3zeta signaling
domain, and wherein the hJ591 binding sequence comprises from 5' to
3' the hJ591 heavy chain variable sequence and the hJ591 light
chain variable sequence (VHVK); dnTGF-hJ591VKVH.BBZ represents
transduced T cells comprising a dominant negative TGFR.beta.II
sequence linked to the humanized J591 (hJ591) BBZ CAR via a T2A
sequence, wherein the hJ591 CAR comprises a 4-1BB costimulatory
domain and CD3zeta signaling domain, and wherein the hJ591 binding
sequence comprises from 5' to 3' the hJ591 light chain variable
sequence and the hJ591 heavy chain variable sequence (VKVH);
dnTGF-mJ591.BBZ represents transduced T cells comprising a dominant
negative TGFR.beta.II sequence linked to the murine J591 (mJ591)
BBZ CAR via a T2A sequence (also referred to herein as
dnTGFR.beta.II.J591.BBZ).
[0806] Two humanized J591 CARs, dnTGF.hJ591VHVK.BBZ and
dnTGF.hJ591VKVH.BBZ, were constructed and cloned into a lentiviral
vector (FIG. 22A). FIG. 22B shows the transgene expression of
lentivirus transduced T cells that express the PSMA CAR staining
with human recombinant PSMA-Fc protein (PMSA) or anti-hIgG, or
TGF-b receptor II (TGFbRII), as measured by flow cytometry. CAR-T
cells expressing the indicated PMSA CAR constructs were cocultured
with PC3-PMSA for 4 hrs and the percentage of CD107a expression was
quantified by using flow cytometry, showing that the humanized J591
CARs confer T cell functionality (FIG. 22C).
[0807] CAR-T cells expressing the indicated PMSA CAR constructs
were tested for the lytic activity using PC3-PMSA cells as target,
showing T cells comprising hJ591 CARs specifically kill target
cells (FIG. 23A). Cytokine production was analyzed by Luminex,
showing that the VKVH hJ591 CAR was superior to the VHVK hJ591 CAR
(FIG. 23B).
[0808] Humanized J591 (hJ591) CARs were tested with different
costimulatory domains. FIG. 24A shows a schematic of the hJ591 CAR
vectors used for studies encoding the PSMA-targeted CARs, which
differ in the transmembrane and intracellular domains. FIG. 24B
shows the surface expression of the hJ591-CARs on lentivirus
transduced CAR T cells. CAR T cells were cocultured with either PC3
or PC3-PSMA cells for 4 hours and the percentage CD107a expression
was quantified (FIG. 24C). Specific lysis of PC3-PSMA by hJ591-CARs
18 hours post coculture was measured (FIG. 24D). FIG. 24E shows
cytokine production as measured by ELISA of the supernatants of CAR
T cells after 24 hours of coculture with PC3-PSMA cells
(E:T=1:1).
[0809] Several constructs were generated and cloned into a
lentiviral vector for in vitro and in vivo testing. The results are
shown in FIGS. 25-30. In FIGS. 25-30, UTD represents untranduced
cells; mJ591.BBZ represents transduced T cells comprising
comprising a murine J591 BBZ CAR, wherein the mJ591 CAR comprises a
4-1BB costimulatory domain and CD3zeta signaling domain;
dnTGF.beta.R-mJ591.BBZ represents transduced T cells comprising
comprising a dominant negative TGFR.beta.II sequence linked to the
murine J591 BBZ CAR via a T2A sequence, wherein the mJ591 CAR
comprises a 4-1BB costimulatory domain and CD3zeta signaling
domain; PD1.CD28-mJ591-hJ591KH.BBZ represents
dnTGF.beta.R-mJ591.BBZ wherein the cells additionally comprise a
PD1-CD28 switch receptor; hJ591KH.BBZ represents transduced T cells
comprising comprising a humanized J591 (hJ591) BBZ CAR, wherein the
hJ591 CAR comprises a 4-1BB costimulatory domain and CD3zeta
signaling domain, and wherein the hJ591 binding sequence comprises
from 5' to 3' the hJ591 light chain variable sequence and the hJ591
heavy chain variable sequence (VKVH); dnTGF.beta.R-hJ591KH.BBZ
represents transduced T cells comprising comprising a dominant
negative TGFR.beta.II sequence linked to the humanized J591 (hJ591)
BBZ CAR via a T2A sequence, wherein the hJ591 CAR comprises a 4-1BB
costimulatory domain and CD3zeta signaling domain, and wherein the
hJ591 binding sequence comprises from 5' to 3' the hJ591 light
chain variable sequence and the hJ591 heavy chain variable sequence
(VKVH); and PD1.CD28-dnTGF.beta.R-hJ591KH.BBZ represents
dnTGF.beta.R-hJ591KH.BBZ wherein the cells additionally comprise a
PD1-CD28 switch receptor.
[0810] CAR expression in the transduced cells as indicated was
tested by flow cytometry (FIG. 25). CAR T cells were cocultured
with either PC3 or PC3-PSMA cells for 4 hours and the percentage
dominant negative TGFR.beta.II and PD1 expression was quantified
(FIG. 26A). CAR T cells were cocultured with either PC3 or PC3-PSMA
cells for 4 hours and the percentage CD107a expression was
quantified (FIG. 26B). CAR-T cells expressing the indicated PMSA
CAR constructs were tested for the lytic activity using PC3-PMSA
cells as target, showing T cells comprising the CARs as indicated
specifically kill target cells (FIG. 27A). In a control experiment,
CAR-T cells expressing the indicated PMSA CAR constructs were
tested for the lytic activity using PC3 cells as target (FIG. 27B).
Production of IFN-gamma and IL-2 by cells as indicated was analyzed
by Luminex (FIGS. 28A and 28B, respectively). As shown in FIG.
28A-28B, cytokine production was only mediated in the presence of
PC3-PSMA cells and not in the presence of PC3 cells.
[0811] In vivo tumor killing efficacy of the various cells were
assessed. The various transduced cells as indicated were
administered to a mouse PSMA tumor model, and the bioluminescence
of tumors were assessed on days 1, 7, 12, and 21
post-administration (FIG. 29). FIG. 30A shows the quantification of
average flux detected from tumors in the mice administered
transduced cells as indicated, on days 1, 7, 12, and 21
post-administration. FIG. 30B shows the quantification of average
flux detected from tumors in the mice administered transduced cells
as indicated, on day 21 post-administration.
[0812] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
Sequence CWU 1
1
26215PRTArtificial SequencelinkerREPEAT(1)..(5)repeat n times,
where n represents an integer of at least 1 1Gly Ser Gly Gly Ser1
524PRTArtificial SequencelinkerREPEAT(1)..(4)repeat n times, where
n represents an integer of at least 1 2Gly Gly Gly
Ser135PRTArtificial SequencelinkerREPEAT(1)..(5)repeat n times,
where n represents an integer of at least 1 3Gly Gly Gly Gly Ser1
544PRTArtificial Sequencelinker 4Gly Gly Ser Gly155PRTArtificial
Sequencelinker 5Gly Gly Ser Gly Gly1 565PRTArtificial
Sequencelinker 6Gly Ser Gly Ser Gly1 575PRTArtificial
Sequencelinker 7Gly Ser Gly Gly Gly1 585PRTArtificial
Sequencelinker 8Gly Gly Gly Ser Gly1 595PRTArtificial
Sequencelinker 9Gly Ser Ser Ser Gly1 5105PRTArtificial
Sequencelinker 10Gly Gly Gly Gly Ser1 51115PRTArtificial
Sequencelinker 11Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 10 151245DNAArtificial Sequencelinker 12ggtggcggtg
gctcgggcgg tggtgggtcg ggtggcggcg gatct 4513237PRTMus musculus 13Asp
Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10
15Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala
20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn
Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr
Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Met Leu Asp Leu
Lys Gly Gly Gly Gly Ser 100 105 110Gly Gly Gly Gly Ser Ser Gly Gly
Gly Ser Glu Val Gln Leu Gln Gln 115 120 125Ser Gly Pro Glu Leu Val
Lys Pro Gly Thr Ser Val Arg Ile Ser Cys 130 135 140Lys Thr Ser Gly
Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp Val Lys145 150 155 160Gln
Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asn Ile Asn Pro Asn 165 170
175Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu Asp Lys Ala Thr Leu
180 185 190Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg
Ser Leu 195 200 205Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ala
Gly Trp Asn Phe 210 215 220Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
Val Ser Ser225 230 23514260PRTMus musculus 14Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Gly Ser Asp Ile Val Met Thr Gln Ser His Lys 20 25 30Phe Met Ser
Thr Ser Val Gly Asp Arg Val Ser Ile Ile Cys Lys Ala 35 40 45Ser Gln
Asp Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly 50 55 60Gln
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly65 70 75
80Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
85 90 95Thr Ile Thr Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys
Gln 100 105 110Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr
Met Leu Asp 115 120 125Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ser Gly Gly Gly 130 135 140Ser Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys Pro Gly145 150 155 160Thr Ser Val Arg Ile Ser
Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu 165 170 175Tyr Thr Ile His
Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp 180 185 190Ile Gly
Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys 195 200
205Phe Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
210 215 220Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr225 230 235 240Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu 245 250 255Thr Val Ser Ser 26015780DNAMus
musculus 15atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca
cgccgccaga 60cctggatctg acattgtgat gacccagtct cacaaattca tgtccacatc
agtaggagac 120agggtcagca tcatctgtaa ggccagtcaa gatgtgggta
ctgctgtaga ctggtatcaa 180cagaaaccag gacaatctcc taaactactg
atttattggg catccactcg gcacactgga 240gtccctgatc gcttcacagg
cagtggatct gggacagact tcactctcac cattactaac 300gttcagtctg
aagacttggc agattatttc tgtcagcaat ataacagcta tcctctcacg
360ttcggtgctg ggaccatgct ggacctgaaa ggaggcggag gatctggcgg
cggaggaagt 420tctggcggag gcagcgaggt gcagctgcag cagagcggac
ccgagctcgt gaagcctgga 480acaagcgtgc ggatcagctg caagaccagc
ggctacacct tcaccgagta caccatccac 540tgggtcaagc agtcccacgg
caagagcctg gagtggatcg gcaatatcaa ccccaacaac 600ggcggcacca
cctacaacca gaagttcgag gacaaggcca ccctgaccgt ggacaagagc
660agcagcaccg cctacatgga actgcggagc ctgaccagcg aggacagcgc
cgtgtactat 720tgtgccgccg gttggaactt cgactactgg ggccagggca
caaccctgac agtgtctagc 78016107PRTMus musculus 16Asp Ile Val Met Thr
Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser
Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser65 70 75
80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys 100
10517321DNAMus musculus 17gacattgtga tgacccagtc tcacaaattc
atgtccacat cagtaggaga cagggtcagc 60atcatctgta aggccagtca agatgtgggt
actgctgtag actggtatca acagaaacca 120ggacaatctc ctaaactact
gatttattgg gcatccactc ggcacactgg agtccctgat 180cgcttcacag
gcagtggatc tgggacagac ttcactctca ccattactaa cgttcagtct
240gaagacttgg cagattattt ctgtcagcaa tataacagct atcctctcac
gttcggtgct 300gggaccatgc tggacctgaa a 3211811PRTMus musculus 18Lys
Ala Ser Gln Asp Val Gly Thr Ala Val Asp1 5 10197PRTMus musculus
19Trp Ala Ser Thr Arg His Thr1 5209PRTMus musculus 20Gln Gln Tyr
Asn Ser Tyr Pro Leu Thr1 521115PRTMus musculus 21Glu Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10 15Ser Val Arg
Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile
His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly
Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55
60Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Leu Thr 100 105 110Val Ser Ser 11522345DNAMus musculus 22gaggtgcagc
tgcagcagag cggacccgag ctcgtgaagc ctggaacaag cgtgcggatc 60agctgcaaga
ccagcggcta caccttcacc gagtacacca tccactgggt caagcagtcc
120cacggcaaga gcctggagtg gatcggcaat atcaacccca acaacggcgg
caccacctac 180aaccagaagt tcgaggacaa ggccaccctg accgtggaca
agagcagcag caccgcctac 240atggaactgc ggagcctgac cagcgaggac
agcgccgtgt actattgtgc cgccggttgg 300aacttcgact actggggcca
gggcacaacc ctgacagtgt ctagc 3452310PRTMus musculus 23Gly Tyr Thr
Phe Thr Glu Tyr Thr Ile His1 5 102417PRTMus musculus 24Asn Ile Asn
Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu1 5 10
15Asp256PRTMus musculus 25Gly Trp Asn Phe Asp Tyr1 526267PRTHomo
sapiens 26Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val 20 25 30Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe 35 40 45Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys 50 55 60Gly Leu Glu Trp Val Ala Val Ile Ser Tyr
Asp Gly Asn Asn Lys Tyr65 70 75 80Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95Lys Asn Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110Ala Val Tyr Tyr Cys
Ala Arg Ala Val Pro Trp Gly Ser Arg Tyr Tyr 115 120 125Tyr Tyr Gly
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150
155 160Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly 165 170 175Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Ser Ser Ala 180 185 190Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala
Pro Lys Leu Leu Ile 195 200 205Phe Asp Ala Ser Ser Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly 210 215 220Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro225 230 235 240Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 245 250 255Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 260 26527801DNAHomo sapiens
27atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
60ccgcaggtgc aactggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga
120ctctcctgtg cagcctctgg attcaccttc agtagctatg ctatgcactg
ggtccgccag 180gctccaggca aggggctgga gtgggtggca gttatatcat
atgatggaaa caataaatac 240tacgcagact ccgtgaaggg ccgattcacc
atctccagag acaattccaa gaacacgctg 300tatctgcaaa tgaacagcct
gagagctgag gacacggctg tgtattactg tgcgagagcc 360gtcccctggg
gatcgaggta ctactactac ggtatggacg tctggggcca agggaccacg
420gtcaccgtct cctcaggtgg cggtggctcg ggcggtggtg ggtcgggtgg
cggcggatct 480gccatccagt tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 540atcacttgcc gggcaagtca gggcattagc
agtgctttag cctggtatca gcagaaatca 600gggaaagctc ctaagctcct
gatctttgat gcctccagtt tggaaagtgg ggtcccatca 660aggttcagcg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
720gaagattttg caacttatta ctgtcaacag tttaacagtt atcctctcac
tttcggcgga 780gggaccaagg tggagatcaa a 80128125PRTHomo sapiens 28Pro
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly1 5 10
15Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
20 25 30Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp 35 40 45Val Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala
Asp Ser 50 55 60Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu65 70 75 80Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Ala Val Pro Trp Gly Ser Arg
Tyr Tyr Tyr Tyr Gly Met 100 105 110Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 12529375DNAHomo sapiens 29ccgcaggtgc
aactggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga 60ctctcctgtg
cagcctctgg attcaccttc agtagctatg ctatgcactg ggtccgccag
120gctccaggca aggggctgga gtgggtggca gttatatcat atgatggaaa
caataaatac 180tacgcagact ccgtgaaggg ccgattcacc atctccagag
acaattccaa gaacacgctg 240tatctgcaaa tgaacagcct gagagctgag
gacacggctg tgtattactg tgcgagagcc 300gtcccctggg gatcgaggta
ctactactac ggtatggacg tctggggcca agggaccacg 360gtcaccgtct cctca
375305PRTHomo sapiens 30Ser Tyr Ala Met His1 53117PRTHomo sapiens
31Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys1
5 10 15Gly3215PRTHomo sapiens 32Ala Val Pro Trp Gly Ser Arg Tyr Tyr
Tyr Tyr Gly Met Asp Val1 5 10 1533107PRTHomo sapiens 33Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Phe Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser
Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10534321DNAHomo sapiens 34gccatccagt tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattagc
agtgctttag cctggtatca gcagaaatca 120gggaaagctc ctaagctcct
gatctttgat gcctccagtt tggaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagattttg caacttatta ctgtcaacag tttaacagtt atcctctcac
tttcggcgga 300gggaccaagg tggagatcaa a 3213511PRTHomo sapiens 35Arg
Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala1 5 10367PRTHomo sapiens
36Asp Ala Ser Ser Leu Glu Ser1 5379PRTHomo sapiens 37Gln Gln Phe
Asn Ser Tyr Pro Leu Thr1 538262PRTHomo sapiens 38Met Ala Leu Pro
Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala
Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30Lys Lys
Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr 35 40 45Ser
Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys 50 55
60Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg65
70 75 80Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys
Ser 85 90 95Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser
Asp Thr 100 105 110Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu
Trp Ser Ser Asp 115 120 125Leu Trp Gly Arg Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Ala Ile Gln Leu Thr145 150 155 160Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 165 170 175Thr Cys Arg
Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln 180 185 190Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser 195 200
205Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Tyr Gly Ser Gly Thr
210 215 220Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe
Ala Thr225 230 235 240Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu
Thr Phe Gly Gly Gly 245 250 255Thr Lys Val Glu Ile Lys
26039786DNAHomo sapiens 39atggccttac cagtgaccgc cttgctcctg
ccgctggcct tgctgctcca cgccgccagg 60ccggaggtgc agctggtgca gtctggagca
gaggtgaaaa agcccgggga gtctctgaag 120atctcctgta agggttctgg
atacagcttt accagtaact ggatcggctg ggtgcgccag 180atgcccggga
aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga
240tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat
cagcaccgcc 300tacctgcagt ggagcagcct gaaggcctcg gacaccgcca
tgtattactg tgcgaggcaa 360actggtttcc tctggtcctc cgatctctgg
ggccgtggca ccctggtcac tgtctcctca 420ggtggcggtg gctcgggcgg
tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480cagtctccat
cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca
540agtcaggaca ttagcagtgc tttagcctgg tatcaacaga aaccagggaa
agctcctaag 600ctcctgatct atgatgcctc cagtttggaa agtggggtcc
catcaaggtt cagcggctat 660ggatctggga cagatttcac tctcaccatc
aacagcctgc agcctgaaga ttttgcaact 720tattactgtc aacagtttaa
tagttacccg ctcactttcg gcggagggac
caaggtggag 780atcaaa 78640120PRTHomo sapiens 40Pro Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly1 5 10 15Glu Ser Leu Lys
Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser 20 25 30Asn Trp Ile
Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp 35 40 45Met Gly
Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser 50 55 60Phe
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala65 70 75
80Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr
85 90 95Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Ser Asp Leu Trp Gly
Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12041360DNAHomo
sapiens 41ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga
gtctctgaag 60atctcctgta agggttctgg atacagcttt accagtaact ggatcggctg
ggtgcgccag 120atgcccggga aaggcctgga gtggatgggg atcatctatc
ctggtgactc tgataccaga 180tacagcccgt ccttccaagg ccaggtcacc
atctcagccg acaagtccat cagcaccgcc 240tacctgcagt ggagcagcct
gaaggcctcg gacaccgcca tgtattactg tgcgaggcaa 300actggtttcc
tctggtcctc cgatctctgg ggccgtggca ccctggtcac tgtctcctca
360425PRTHomo sapiens 42Ser Asn Trp Ile Gly1 54317PRTHomo sapiens
43Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln1
5 10 15Gly4410PRTHomo sapiens 44Gln Thr Gly Phe Leu Trp Ser Ser Asp
Leu1 5 1045107PRTHomo sapiens 45Ala Ile Gln Leu Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu
Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Tyr Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10546321DNAHomo sapiens
46gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggcaagtca ggacattagc agtgctttag cctggtatca acagaaacca
120gggaaagctc ctaagctcct gatctatgat gcctccagtt tggaaagtgg
ggtcccatca 180aggttcagcg gctatggatc tgggacagat ttcactctca
ccatcaacag cctgcagcct 240gaagattttg caacttatta ctgtcaacag
tttaatagtt acccgctcac tttcggcgga 300gggaccaagg tggagatcaa a
3214711PRTHomo sapiens 47Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala
Leu1 5 10488PRTHomo sapiens 48Tyr Asp Ala Ser Ser Leu Glu Ser1
54910PRTHomo sapiens 49Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr1 5
1050264PRTHomo sapiens 50Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val 20 25 30Lys Lys Pro Gly Glu Ser Leu Lys
Ile Ser Cys Lys Gly Ser Gly Tyr 35 40 45Ser Phe Thr Ser Asn Trp Ile
Gly Trp Val Arg Gln Met Pro Gly Lys 50 55 60Gly Leu Glu Trp Met Gly
Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg65 70 75 80Tyr Ser Pro Ser
Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser 85 90 95Ile Ser Thr
Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr 100 105 110Ala
Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp 115 120
125Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln
Leu Thr145 150 155 160Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile 165 170 175Thr Cys Arg Ala Ser Gln Asp Ile Ser
Ser Ala Leu Ala Trp Tyr Gln 180 185 190Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr Asp Ala Ser Ser 195 200 205Leu Glu Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 210 215 220Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr225 230 235
240Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly
245 250 255Thr Lys Val Glu Ile Lys Ile Lys 26051792DNAHomo sapiens
51atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
60ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag
120atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg
ggtgcgccag 180atgcccggga aaggcctgga gtggatgggg atcatctatc
ctggtgactc tgataccaga 240tacagcccgt ccttccaagg ccaggtcacc
atctcagccg acaagtccat cagcaccgcc 300tacctgcagt ggaacagcct
gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 360actggtttcc
tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca
420ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat
ccagttgacc 480cagtctccat cctccctgtc tgcatctgta ggagacagag
tcaccatcac ttgccgggca 540agtcaggaca ttagcagtgc tttagcctgg
tatcagcaga aaccggggaa agctcctaag 600ctcctgatct atgatgcctc
cagtttggaa agtggggtcc catcaaggtt cagcggcagt 660ggatctggga
cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact
720tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac
caaggtggag 780atcaaaatca aa 79252120PRTHomo sapiens 52Pro Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly1 5 10 15Glu Ser
Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser 20 25 30Asn
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp 35 40
45Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser
50 55 60Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr
Ala65 70 75 80Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala
Met Tyr Tyr 85 90 95Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp
Leu Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12053360DNAHomo sapiens 53ccggaggtgc agctggtgca gtctggagca
gaggtgaaaa agcccgggga gtctctgaag 60atctcctgta agggttctgg atacagtttt
accagcaact ggatcggctg ggtgcgccag 120atgcccggga aaggcctgga
gtggatgggg atcatctatc ctggtgactc tgataccaga 180tacagcccgt
ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc
240tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg
tgcgagacaa 300actggtttcc tctggtcctt cgatctctgg ggccgtggca
ccctggtcac tgtctcctca 360545PRTHomo sapiens 54Ser Asn Trp Ile Gly1
55517PRTHomo sapiens 55Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr
Ser Pro Ser Phe Gln1 5 10 15Gly5610PRTHomo sapiens 56Gln Thr Gly
Phe Leu Trp Ser Phe Asp Leu1 5 1057109PRTHomo sapiens 57Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser
Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ile
Lys 100 10558327DNAHomo sapiens 58gccatccagt tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattagc
agtgctttag cctggtatca gcagaaaccg 120gggaaagctc ctaagctcct
gatctatgat gcctccagtt tggaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagattttg caacttatta ctgtcaacag tttaatagtt acccgctcac
tttcggcgga 300gggaccaagg tggagatcaa aatcaaa 3275911PRTHomo sapiens
59Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala1 5 10607PRTHomo
sapiens 60Asp Ala Ser Ser Leu Glu Ser1 5619PRTHomo sapiens 61Gln
Gln Phe Asn Ser Tyr Pro Leu Thr1 562265PRTHomo sapiens 62Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His
Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ser Glu Val 20 25
30Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr
35 40 45Ser Phe Thr Asn Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly
Lys 50 55 60Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp
Thr Arg65 70 75 80Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser
Ala Asp Lys Ser 85 90 95Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr 100 105 110Ala Met Tyr Tyr Cys Ala Ser Pro Gly
Tyr Thr Ser Ser Trp Thr Ser 115 120 125Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val145 150 155 160Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala 165 170
175Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp
180 185 190Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
Asp Ala 195 200 205Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly Ser Gly Ser 210 215 220Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro Glu Asp Phe225 230 235 240Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Leu Phe Thr Phe 245 250 255Gly Pro Gly Thr Lys
Val Asp Ile Lys 260 26563795DNAHomo sapiens 63atggccttac cagtgaccgc
cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggaggtgc agctggtgca
gtctggatca gaggtgaaaa agcccgggga gtctctgaag 120atctcctgta
agggttctgg atacagcttt accaactact ggatcggctg ggtgcgccag
180atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc
tgataccaga 240tacagcccgt ccttccaagg ccaggtcacc atctcagccg
acaagtccat cagcaccgcc 300tatctgcagt ggagcagcct gaaggcctcg
gacaccgcca tgtattactg tgcgagtccc 360gggtatacca gcagttggac
ttcttttgac tactggggcc agggaaccct ggtcaccgtc 420tcctcaggtg
gcggtggctc gggcggtggt gggtcgggtg gcggcggatc tgaaattgtg
480ttgacacagt ctccagccac cctgtctttg tctccagggg aaagagccac
cctctcctgc 540agggccagtc agagtgttag cagctactta gcctggtacc
aacagaaacc tggccaggct 600cccaggctcc tcatctatga tgcatccaac
agggccactg gcatcccagc caggttcagt 660ggcagtgggt ctgggacaga
cttcactctc accatcagca gcctagagcc tgaagatttt 720gcagtttatt
actgtcagca gcgtagcaac tggcccctat tcactttcgg ccctgggacc
780aaagtggata tcaaa 79564122PRTHomo sapiens 64Pro Glu Val Gln Leu
Val Gln Ser Gly Ser Glu Val Lys Lys Pro Gly1 5 10 15Glu Ser Leu Lys
Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn 20 25 30Tyr Trp Ile
Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp 35 40 45Met Gly
Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser 50 55 60Phe
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala65 70 75
80Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr
85 90 95Cys Ala Ser Pro Gly Tyr Thr Ser Ser Trp Thr Ser Phe Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12065366DNAHomo sapiens 65ccggaggtgc agctggtgca gtctggatca
gaggtgaaaa agcccgggga gtctctgaag 60atctcctgta agggttctgg atacagcttt
accaactact ggatcggctg ggtgcgccag 120atgcccggga aaggcctgga
gtggatgggg atcatctatc ctggtgactc tgataccaga 180tacagcccgt
ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc
240tatctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg
tgcgagtccc 300gggtatacca gcagttggac ttcttttgac tactggggcc
agggaaccct ggtcaccgtc 360tcctca 366664PRTHomo sapiens 66Thr Asn Tyr
Trp16718PRTHomo sapiens 67Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr
Arg Tyr Ser Pro Ser Phe1 5 10 15Gln Gly6810PRTHomo sapiens 68Ser
Pro Gly Tyr Thr Ser Ser Trp Thr Ser1 5 1069108PRTHomo sapiens 69Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp Pro Leu 85 90 95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys 100 10570324DNAHomo sapiens 70gaaattgtgt tgacacagtc
tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc agctacttag cctggtacca acagaaacct 120ggccaggctc
ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc
180aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag
cctagagcct 240gaagattttg cagtttatta ctgtcagcag cgtagcaact
ggcccctatt cactttcggc 300cctgggacca aagtggatat caaa 3247111PRTHomo
sapiens 71Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu1 5
10728PRTHomo sapiens 72Tyr Asp Ala Ser Asn Arg Ala Thr1
57311PRTHomo sapiens 73Cys Gln Gln Arg Ser Asn Trp Pro Leu Phe Thr1
5 10745PRTArtificial Sequencehinge 74Asp Lys Thr His Thr1
5754PRTArtificial Sequencehinge 75Cys Pro Pro Cys17615PRTArtificial
Sequencehinge 76Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys
Pro Arg1 5 10 157712PRTArtificial Sequencehinge 77Glu Leu Lys Thr
Pro Leu Gly Asp Thr Thr His Thr1 5 107810PRTArtificial
Sequencehinge 78Lys Ser Cys Asp Lys Thr His Thr Cys Pro1 5
10797PRTArtificial Sequencehinge 79Lys Cys Cys Val Asp Cys Pro1
5807PRTArtificial Sequencehinge 80Lys Tyr Gly Pro Pro Cys Pro1
58115PRTArtificial Sequencehinge 81Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro1 5 10 158212PRTArtificial Sequencehinge
82Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro1 5
108317PRTArtificial Sequencehinge 83Glu Leu Lys Thr Pro Leu Gly Asp
Thr Thr His Thr Cys Pro Arg Cys1 5 10 15Pro8412PRTArtificial
Sequencehinge 84Ser Pro Asn Met Val Pro His Ala His His Ala Gln1 5
108515PRTArtificial Sequencehinge 85Glu Pro Lys Ser Cys Asp Lys Thr
Tyr Thr Cys Pro Pro Cys Pro1 5 10 158645PRTArtificial SequenceCD8
hinge 86Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
Ala1 5 10 15Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
Ala Gly 20 25 30Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 4587135DNAArtificial SequenceCD8 hinge 87accacgacgc
cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60tccctgcgcc
cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg
120gacttcgcct gtgat 1358824PRTArtificial SequenceCD8 transmembrane
domain 88Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
Leu Leu1 5 10 15Ser Leu Val Ile Thr Leu Tyr Cys 208972DNAArtificial
SequenceCD8 transmembrane domain 89atctacatct gggcgccctt ggccgggact
tgtggggtcc ttctcctgtc actggttatc 60accctttact
gc 729069PRTArtificial SequenceCD8 hinge and CD8 transmembrane
domain 90Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
Ile Ala1 5 10 15Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
Ala Ala Gly 20 25 30Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
Asp Ile Tyr Ile 35 40 45Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
Leu Leu Ser Leu Val 50 55 60Ile Thr Leu Tyr Cys6591207DNAArtificial
SequenceCD8 hinge and CD8 transmembrane domain 91accacgacgc
cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60tccctgcgcc
cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg
120gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg
ggtccttctc 180ctgtcactgg ttatcaccct ttactgc 2079242PRTArtificial
Sequence4-1BB 92Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
Pro Phe Met1 5 10 15Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
Ser Cys Arg Phe 20 25 30Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35
4093126DNAArtificial Sequence4-1BB 93aaacggggca gaaagaaact
cctgtatata ttcaaacaac catttatgag accagtacaa 60actactcaag aggaagacgg
ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg
12694126DNAArtificial Sequence4-1BB 94aaacggggca gaaagaaact
cctgtatata ttcaaacaac catttatgag accagtacaa 60actactcaag aggaagatgg
ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg
1269535PRTArtificial SequenceICOS(YMNM) 95Thr Lys Lys Lys Tyr Ser
Ser Ser Val His Asp Pro Asn Gly Glu Tyr1 5 10 15Met Asn Met Arg Ala
Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp 20 25 30Val Thr Leu
3596105DNAArtificial SequenceICOS(YMNM) 96acaaaaaaga agtattcatc
cagtgtgcac gaccctaacg gtgaatacat gaacatgaga 60gcagtgaaca cagccaaaaa
atccagactc acagatgtga cccta 10597112PRTArtificial SequenceCD3 zeta
domain 97Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala
Leu His Met Gln Ala Leu Pro Pro Arg 100 105 11098336DNAArtificial
SequenceCD3 zeta domain 98agagtgaagt tcagcaggag cgcagacgcc
cccgcgtaca agcagggcca gaaccagctc 60tataacgagc tcaatctagg acgaagagag
gagtacgatg ttttggacaa gagacgtggc 120cgggaccctg agatgggggg
aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180gaactgcaga
aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc
240cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac
caaggacacc 300tacgacgccc ttcacatgca ggccctgccc cctcgc
33699336DNAArtificial SequenceCD3 zeta domain 99agagtgaagt
tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60tataacgagc
tcaatctagg acgaagagag gagtacgacg ttttggacaa gagacgtggc
120cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg
cctgtacaac 180gaactgcaga aagataagat ggcggaggcc tacagtgaga
ttgggatgaa aggcgagcgc 240cggaggggca aggggcacga cggcctttac
cagggtctca gtacagccac caaggacacc 300tacgacgccc ttcacatgca
ggccctgccc cctcgc 336100112PRTArtificial SequenceCD3 zeta domain
100Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly1
5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His
Met Gln Ala Leu Pro Pro Arg 100 105 110101336DNAArtificial
SequenceCD3 zeta domain 101agagtgaagt tcagcaggag cgcagacgcc
cccgcgtacc agcagggcca gaaccagctc 60tataacgagc tcaatctagg acgaagagag
gagtacgatg ttttggacaa gagacgtggc 120cgggaccctg agatgggggg
aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180gaactgcaga
aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc
240cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac
caaggacacc 300tacgacgccc ttcacatgca ggccctgccc cctcgc
336102154PRTArtificial Sequence4-1BB domain and CD3 zeta domain
102Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1
5 10 15Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
Phe 20 25 30Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
Ser Arg 35 40 45Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
Leu Tyr Asn 50 55 60Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
Leu Asp Lys Arg65 70 75 80Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
Pro Arg Arg Lys Asn Pro 85 90 95Gln Glu Gly Leu Tyr Asn Glu Leu Gln
Lys Asp Lys Met Ala Glu Ala 100 105 110Tyr Ser Glu Ile Gly Met Lys
Gly Glu Arg Arg Arg Gly Lys Gly His 115 120 125Asp Gly Leu Tyr Gln
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 130 135 140Ala Leu His
Met Gln Ala Leu Pro Pro Arg145 150103462DNAArtificial Sequence4-1BB
domain and CD3 zeta domain 103aaacggggca gaaagaaact cctgtatata
ttcaaacaac catttatgag accagtacaa 60actactcaag aggaagacgg ctgtagctgc
cgatttccag aagaagaaga aggaggatgt 120gaactgagag tgaagttcag
caggagcgca gacgcccccg cgtacaagca gggccagaac 180cagctctata
acgagctcaa tctaggacga agagaggagt acgacgtttt ggacaagaga
240cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca
ggaaggcctg 300tacaacgaac tgcagaaaga taagatggcg gaggcctaca
gtgagattgg gatgaaaggc 360gagcgccgga ggggcaaggg gcacgacggc
ctttaccagg gtctcagtac agccaccaag 420gacacctacg acgcccttca
catgcaggcc ctgccccctc gc 462104462DNAArtificial Sequence4-1BB
domain and CD3 zeta domain 104aaacggggca gaaagaaact cctgtatata
ttcaaacaac catttatgag accagtacaa 60actactcaag aggaagatgg ctgtagctgc
cgatttccag aagaagaaga aggaggatgt 120gaactgagag tgaagttcag
caggagcgca gacgcccccg cgtacaagca gggccagaac 180cagctctata
acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga
240cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca
ggaaggcctg 300tacaatgaac tgcagaaaga taagatggcg gaggcctaca
gtgagattgg gatgaaaggc 360gagcgccgga ggggcaaggg gcacgatggc
ctttaccagg gtctcagtac agccaccaag 420gacacctacg acgcccttca
catgcaggcc ctgccccctc gc 462105487PRTArtificial SequenceJ591 murine
PSMA-CAR 105Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
Leu Leu1 5 10 15His Ala Ala Arg Pro Gly Ser Asp Ile Val Met Thr Gln
Ser His Lys 20 25 30Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile
Ile Cys Lys Ala 35 40 45Ser Gln Asp Val Gly Thr Ala Val Asp Trp Tyr
Gln Gln Lys Pro Gly 50 55 60Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala
Ser Thr Arg His Thr Gly65 70 75 80Val Pro Asp Arg Phe Thr Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu 85 90 95Thr Ile Thr Asn Val Gln Ser
Glu Asp Leu Ala Asp Tyr Phe Cys Gln 100 105 110Gln Tyr Asn Ser Tyr
Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp 115 120 125Leu Lys Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly 130 135 140Ser
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly145 150
155 160Thr Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr
Glu 165 170 175Tyr Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser
Leu Glu Trp 180 185 190Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr
Thr Tyr Asn Gln Lys 195 200 205Phe Glu Asp Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Ser Thr Ala 210 215 220Tyr Met Glu Leu Arg Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr225 230 235 240Cys Ala Ala Gly
Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 245 250 255Thr Val
Ser Ser Ala Ser Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro 260 265
270Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
Gly Leu 290 295 300Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu
Ala Gly Thr Cys305 310 315 320Gly Val Leu Leu Leu Ser Leu Val Ile
Thr Leu Tyr Cys Lys Arg Gly 325 330 335Arg Lys Lys Leu Leu Tyr Ile
Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350Gln Thr Thr Gln Glu
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365Glu Glu Gly
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380Ala
Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn385 390
395 400Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg 405 410 415Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
Gln Glu Gly 420 425 430Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
Glu Ala Tyr Ser Glu 435 440 445Ile Gly Met Lys Gly Glu Arg Arg Arg
Gly Lys Gly His Asp Gly Leu 450 455 460Tyr Gln Gly Leu Ser Thr Ala
Thr Lys Asp Thr Tyr Asp Ala Leu His465 470 475 480Met Gln Ala Leu
Pro Pro Arg 4851061461DNAArtificial SequenceJ591 murine PSMA-CAR
106atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca
cgccgccaga 60cctggatctg acattgtgat gacccagtct cacaaattca tgtccacatc
agtaggagac 120agggtcagca tcatctgtaa ggccagtcaa gatgtgggta
ctgctgtaga ctggtatcaa 180cagaaaccag gacaatctcc taaactactg
atttattggg catccactcg gcacactgga 240gtccctgatc gcttcacagg
cagtggatct gggacagact tcactctcac cattactaac 300gttcagtctg
aagacttggc agattatttc tgtcagcaat ataacagcta tcctctcacg
360ttcggtgctg ggaccatgct ggacctgaaa ggaggcggag gatctggcgg
cggaggaagt 420tctggcggag gcagcgaggt gcagctgcag cagagcggac
ccgagctcgt gaagcctgga 480acaagcgtgc ggatcagctg caagaccagc
ggctacacct tcaccgagta caccatccac 540tgggtcaagc agtcccacgg
caagagcctg gagtggatcg gcaatatcaa ccccaacaac 600ggcggcacca
cctacaacca gaagttcgag gacaaggcca ccctgaccgt ggacaagagc
660agcagcaccg cctacatgga actgcggagc ctgaccagcg aggacagcgc
cgtgtactat 720tgtgccgccg gttggaactt cgactactgg ggccagggca
caaccctgac agtgtctagc 780gctagctccg gaaccacgac gccagcgccg
cgaccaccaa caccggcgcc caccatcgcg 840tcgcagcccc tgtccctgcg
cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900acgagggggc
tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt
960ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag
aaagaaactc 1020ctgtatatat tcaaacaacc atttatgaga ccagtacaaa
ctactcaaga ggaagacggc 1080tgtagctgcc gatttccaga agaagaagaa
ggaggatgtg aactgagagt gaagttcagc 1140aggagcgcag acgcccccgc
gtacaagcag ggccagaacc agctctataa cgagctcaat 1200ctaggacgaa
gagaggagta cgacgttttg gacaagagac gtggccggga ccctgagatg
1260gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaacgaact
gcagaaagat 1320aagatggcgg aggcctacag tgagattggg atgaaaggcg
agcgccggag gggcaagggg 1380cacgacggcc tttaccaggg tctcagtaca
gccaccaagg acacctacga cgcccttcac 1440atgcaggccc tgccccctcg c
1461107490PRTArtificial Sequence1C3 human PSMA-CAR 107Met Ala Leu
Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala
Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val 20 25 30Val
Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40
45Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
50 55 60Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys
Tyr65 70 75 80Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser 85 90 95Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Ala Val Pro
Trp Gly Ser Arg Tyr Tyr 115 120 125Tyr Tyr Gly Met Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150 155 160Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 165 170 175Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 180 185
190Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile
195 200 205Phe Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 210 215 220Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro225 230 235 240Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Phe Asn Ser Tyr Pro Leu 245 250 255Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Thr Thr Thr Pro Ala 260 265 270Pro Arg Pro Pro Thr
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285Leu Arg Pro
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300Arg
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala305 310
315 320Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
Cys 325 330 335Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
Pro Phe Met 340 345 350Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
Cys Ser Cys Arg Phe 355 360 365Pro Glu Glu Glu Glu Gly Gly Cys Glu
Leu Arg Val Lys Phe Ser Arg 370 375 380Ser Ala Asp Ala Pro Ala Tyr
Lys Gln Gly Gln Asn Gln Leu Tyr Asn385 390 395 400Glu Leu Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 405 410 415Arg Gly
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 420 425
430Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
435 440 445Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His 450 455 460Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp465 470 475 480Ala Leu His Met Gln Ala Leu Pro Pro
Arg 485 4901081470DNAArtificial Sequence1C3 human PSMA-CAR
108atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca
cgccgccagg 60ccgcaggtgc aactggtgga gtctggggga ggcgtggtcc agcctgggag
gtccctgaga 120ctctcctgtg cagcctctgg attcaccttc agtagctatg
ctatgcactg ggtccgccag 180gctccaggca aggggctgga gtgggtggca
gttatatcat atgatggaaa caataaatac 240tacgcagact ccgtgaaggg
ccgattcacc atctccagag acaattccaa gaacacgctg 300tatctgcaaa
tgaacagcct gagagctgag gacacggctg tgtattactg tgcgagagcc
360gtcccctggg gatcgaggta ctactactac ggtatggacg tctggggcca
agggaccacg 420gtcaccgtct cctcaggtgg cggtggctcg ggcggtggtg
ggtcgggtgg cggcggatct 480gccatccagt tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 540atcacttgcc gggcaagtca
gggcattagc agtgctttag cctggtatca gcagaaatca 600gggaaagctc
ctaagctcct gatctttgat gcctccagtt tggaaagtgg ggtcccatca
660aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 720gaagattttg caacttatta ctgtcaacag tttaacagtt
atcctctcac tttcggcgga 780gggaccaagg tggagatcaa aaccacgacg
ccagcgccgc gaccaccaac
accggcgccc 840accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt
gccggccagc ggcggggggc 900gcagtgcaca cgagggggct ggacttcgcc
tgtgatatct acatctgggc gcccttggcc 960gggacttgtg gggtccttct
cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020aagaaactcc
tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag
1080gaagacggct gtagctgccg atttccagaa gaagaagaag gaggatgtga
actgagagtg 1140aagttcagca ggagcgcaga cgcccccgcg tacaagcagg
gccagaacca gctctataac 1200gagctcaatc taggacgaag agaggagtac
gacgttttgg acaagagacg tggccgggac 1260cctgagatgg ggggaaagcc
gagaaggaag aaccctcagg aaggcctgta caacgaactg 1320cagaaagata
agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg
1380ggcaaggggc acgacggcct ttaccagggt ctcagtacag ccaccaagga
cacctacgac 1440gcccttcaca tgcaggccct gccccctcgc
1470109485PRTArtificial Sequence2A10 human PSMA-CAR 109Met Ala Leu
Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala
Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30Lys
Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr 35 40
45Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys
50 55 60Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr
Arg65 70 75 80Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala
Asp Lys Ser 85 90 95Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys
Ala Ser Asp Thr 100 105 110Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly
Phe Leu Trp Ser Ser Asp 115 120 125Leu Trp Gly Arg Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr145 150 155 160Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 165 170 175Thr
Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln 180 185
190Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser
195 200 205Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Tyr Gly Ser
Gly Thr 210 215 220Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu
Asp Phe Ala Thr225 230 235 240Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr
Pro Leu Thr Phe Gly Gly Gly 245 250 255Thr Lys Val Glu Ile Lys Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270Pro Ala Pro Thr Ile
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285Cys Arg Pro
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300Ala
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val305 310
315 320Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
Lys 325 330 335Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
Val Gln Thr 340 345 350Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
Pro Glu Glu Glu Glu 355 360 365Gly Gly Cys Glu Leu Arg Val Lys Phe
Ser Arg Ser Ala Asp Ala Pro 370 375 380Ala Tyr Lys Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly385 390 395 400Arg Arg Glu Glu
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415Glu Met
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425
430Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
435 440 445Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
Tyr Gln 450 455 460Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
Leu His Met Gln465 470 475 480Ala Leu Pro Pro Arg
4851101455DNAArtificial Sequence2A10 human PSMA-CAR 110atggccttac
cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggaggtgc
agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag
120atctcctgta agggttctgg atacagcttt accagtaact ggatcggctg
ggtgcgccag 180atgcccggga aaggcctgga gtggatgggg atcatctatc
ctggtgactc tgataccaga 240tacagcccgt ccttccaagg ccaggtcacc
atctcagccg acaagtccat cagcaccgcc 300tacctgcagt ggagcagcct
gaaggcctcg gacaccgcca tgtattactg tgcgaggcaa 360actggtttcc
tctggtcctc cgatctctgg ggccgtggca ccctggtcac tgtctcctca
420ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat
ccagttgacc 480cagtctccat cctccctgtc tgcatctgta ggagacagag
tcaccatcac ttgccgggca 540agtcaggaca ttagcagtgc tttagcctgg
tatcaacaga aaccagggaa agctcctaag 600ctcctgatct atgatgcctc
cagtttggaa agtggggtcc catcaaggtt cagcggctat 660ggatctggga
cagatttcac tctcaccatc aacagcctgc agcctgaaga ttttgcaact
720tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac
caaggtggag 780atcaaaacca cgacgccagc gccgcgacca ccaacaccgg
cgcccaccat cgcgtcgcag 840cccctgtccc tgcgcccaga ggcgtgccgg
ccagcggcgg ggggcgcagt gcacacgagg 900gggctggact tcgcctgtga
tatctacatc tgggcgccct tggccgggac ttgtggggtc 960cttctcctgt
cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat
1020atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga
cggctgtagc 1080tgccgatttc cagaagaaga agaaggagga tgtgaactga
gagtgaagtt cagcaggagc 1140gcagacgccc ccgcgtacaa gcagggccag
aaccagctct ataacgagct caatctagga 1200cgaagagagg agtacgacgt
tttggacaag agacgtggcc gggaccctga gatgggggga 1260aagccgagaa
ggaagaaccc tcaggaaggc ctgtacaacg aactgcagaa agataagatg
1320gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa
ggggcacgac 1380ggcctttacc agggtctcag tacagccacc aaggacacct
acgacgccct tcacatgcag 1440gccctgcccc ctcgc 1455111487PRTArtificial
Sequence2F5 human PSMA-CAR 111Met Ala Leu Pro Val Thr Ala Leu Leu
Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val 20 25 30Lys Lys Pro Gly Glu Ser Leu
Lys Ile Ser Cys Lys Gly Ser Gly Tyr 35 40 45Ser Phe Thr Ser Asn Trp
Ile Gly Trp Val Arg Gln Met Pro Gly Lys 50 55 60Gly Leu Glu Trp Met
Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg65 70 75 80Tyr Ser Pro
Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser 85 90 95Ile Ser
Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr 100 105
110Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp
115 120 125Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
Ile Gln Leu Thr145 150 155 160Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile 165 170 175Thr Cys Arg Ala Ser Gln Asp
Ile Ser Ser Ala Leu Ala Trp Tyr Gln 180 185 190Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser 195 200 205Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 210 215 220Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr225 230
235 240Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly
Gly 245 250 255Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala
Pro Arg Pro 260 265 270Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
Leu Ser Leu Arg Pro 275 280 285Glu Ala Cys Arg Pro Ala Ala Gly Gly
Ala Val His Thr Arg Gly Leu 290 295 300Asp Phe Ala Cys Asp Ile Tyr
Ile Trp Ala Pro Leu Ala Gly Thr Cys305 310 315 320Gly Val Leu Leu
Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335Arg Lys
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345
350Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
Ala Asp 370 375 380Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr
Asn Glu Leu Asn385 390 395 400Leu Gly Arg Arg Glu Glu Tyr Asp Val
Leu Asp Lys Arg Arg Gly Arg 405 410 415Asp Pro Glu Met Gly Gly Lys
Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430Leu Tyr Asn Glu Leu
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445Ile Gly Met
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460Tyr
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His465 470
475 480Met Gln Ala Leu Pro Pro Arg 4851121461DNAArtificial
Sequence2F5 human PSMA-CAR 112atggccttac cagtgaccgc cttgctcctg
ccgctggcct tgctgctcca cgccgccagg 60ccggaggtgc agctggtgca gtctggagca
gaggtgaaaa agcccgggga gtctctgaag 120atctcctgta agggttctgg
atacagtttt accagcaact ggatcggctg ggtgcgccag 180atgcccggga
aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga
240tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat
cagcaccgcc 300tacctgcagt ggaacagcct gaaggcctcg gacaccgcca
tgtattactg tgcgagacaa 360actggtttcc tctggtcctt cgatctctgg
ggccgtggca ccctggtcac tgtctcctca 420ggtggcggtg gctcgggcgg
tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480cagtctccat
cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca
540agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa
agctcctaag 600ctcctgatct atgatgcctc cagtttggaa agtggggtcc
catcaaggtt cagcggcagt 660ggatctggga cagatttcac tctcaccatc
agcagcctgc agcctgaaga ttttgcaact 720tattactgtc aacagtttaa
tagttacccg ctcactttcg gcggagggac caaggtggag 780atcaaaatca
aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg
840tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg
cgcagtgcac 900acgagggggc tggacttcgc ctgtgatatc tacatctggg
cgcccttggc cgggacttgt 960ggggtccttc tcctgtcact ggttatcacc
ctttactgca aacggggcag aaagaaactc 1020ctgtatatat tcaaacaacc
atttatgaga ccagtacaaa ctactcaaga ggaagacggc 1080tgtagctgcc
gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc
1140aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa
cgagctcaat 1200ctaggacgaa gagaggagta cgacgttttg gacaagagac
gtggccggga ccctgagatg 1260gggggaaagc cgagaaggaa gaaccctcag
gaaggcctgt acaacgaact gcagaaagat 1320aagatggcgg aggcctacag
tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380cacgacggcc
tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac
1440atgcaggccc tgccccctcg c 1461113488PRTArtificial Sequence2C6
human PSMA-CAR 113Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu
Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Val Gln Leu Val Gln
Ser Gly Ser Glu Val 20 25 30Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser
Cys Lys Gly Ser Gly Tyr 35 40 45Ser Phe Thr Asn Tyr Trp Ile Gly Trp
Val Arg Gln Met Pro Gly Lys 50 55 60Gly Leu Glu Trp Met Gly Ile Ile
Tyr Pro Gly Asp Ser Asp Thr Arg65 70 75 80Tyr Ser Pro Ser Phe Gln
Gly Gln Val Thr Ile Ser Ala Asp Lys Ser 85 90 95Ile Ser Thr Ala Tyr
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr 100 105 110Ala Met Tyr
Tyr Cys Ala Ser Pro Gly Tyr Thr Ser Ser Trp Thr Ser 115 120 125Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 130 135
140Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile
Val145 150 155 160Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
Gly Glu Arg Ala 165 170 175Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Tyr Leu Ala Trp 180 185 190Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile Tyr Asp Ala 195 200 205Ser Asn Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser 210 215 220Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe225 230 235 240Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu Phe Thr Phe 245 250
255Gly Pro Gly Thr Lys Val Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg
260 265 270Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
Leu Arg 275 280 285Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
His Thr Arg Gly 290 295 300Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
Ala Pro Leu Ala Gly Thr305 310 315 320Cys Gly Val Leu Leu Leu Ser
Leu Val Ile Thr Leu Tyr Cys Lys Arg 325 330 335Gly Arg Lys Lys Leu
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 340 345 350Val Gln Thr
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 355 360 365Glu
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 370 375
380Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu
Leu385 390 395 400Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
Lys Arg Arg Gly 405 410 415Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
Arg Lys Asn Pro Gln Glu 420 425 430Gly Leu Tyr Asn Glu Leu Gln Lys
Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445Glu Ile Gly Met Lys Gly
Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460Leu Tyr Gln Gly
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu465 470 475 480His
Met Gln Ala Leu Pro Pro Arg 4851141464DNAArtificial Sequence2C6
human PSMA-CAR 114atggccttac cagtgaccgc cttgctcctg ccgctggcct
tgctgctcca cgccgccagg 60ccggaggtgc agctggtgca gtctggatca gaggtgaaaa
agcccgggga gtctctgaag 120atctcctgta agggttctgg atacagcttt
accaactact ggatcggctg ggtgcgccag 180atgcccggga aaggcctgga
gtggatgggg atcatctatc ctggtgactc tgataccaga 240tacagcccgt
ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc
300tatctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg
tgcgagtccc 360gggtatacca gcagttggac ttcttttgac tactggggcc
agggaaccct ggtcaccgtc 420tcctcaggtg gcggtggctc gggcggtggt
gggtcgggtg gcggcggatc tgaaattgtg 480ttgacacagt ctccagccac
cctgtctttg tctccagggg aaagagccac cctctcctgc 540agggccagtc
agagtgttag cagctactta gcctggtacc aacagaaacc tggccaggct
600cccaggctcc tcatctatga tgcatccaac agggccactg gcatcccagc
caggttcagt 660ggcagtgggt ctgggacaga cttcactctc accatcagca
gcctagagcc tgaagatttt 720gcagtttatt actgtcagca gcgtagcaac
tggcccctat tcactttcgg ccctgggacc 780aaagtggata tcaaaaccac
gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840gcgtcgcagc
ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg
900cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt
ggccgggact 960tgtggggtcc ttctcctgtc actggttatc accctttact
gcaaacgggg cagaaagaaa 1020ctcctgtata tattcaaaca accatttatg
agaccagtac aaactactca agaggaagac 1080ggctgtagct gccgatttcc
agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140agcaggagcg
cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc
1200aatctaggac gaagagagga gtacgacgtt ttggacaaga gacgtggccg
ggaccctgag 1260atggggggaa agccgagaag gaagaaccct caggaaggcc
tgtacaacga actgcagaaa 1320gataagatgg cggaggccta cagtgagatt
gggatgaaag gcgagcgccg gaggggcaag 1380gggcacgacg gcctttacca
gggtctcagt acagccacca aggacaccta cgacgccctt 1440cacatgcagg
ccctgccccc tcgc 1464115201PRTArtificial SequenceTGFBRII-DN 115Met
Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu1 5 10
15Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val
20 25 30Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe
Pro 35 40 45Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp
Asn Gln 50 55 60Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys
Glu Lys Pro65 70 75 80Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn
Asp Glu Asn Ile Thr 85 90 95Leu Glu Thr Val Cys His Asp Pro Lys Leu
Pro Tyr His Asp Phe Ile 100 105 110Leu
Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120
125Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
130 135 140Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro
Asp Leu145 150 155 160Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser
Leu Leu Pro Pro Leu 165 170 175Gly Val Ala Ile Ser Val Ile Ile Ile
Phe Tyr Cys Tyr Arg Val Asn 180 185 190Arg Gln Gln Lys Leu Ser Ser
Ser Gly 195 200116603DNAArtificial SequenceTGFBRII-DN 116atgggtcggg
ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60gccagcacga
tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac
120aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag
attttccacc 180tgtgacaacc agaaatcctg catgagcaac tgcagcatca
cctccatctg tgagaagcca 240caggaagtct gtgtggctgt atggagaaag
aatgacgaga acataacact agagacagtt 300tgccatgacc ccaagctccc
ctaccatgac tttattctgg aagatgctgc ttctccaaag 360tgcattatga
aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct
420gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa
tcctgacttg 480ttgctagtca tatttcaagt gacaggcatc agcctcctgc
caccactggg agttgccata 540tctgtcatca tcatcttcta ctgctaccgc
gttaaccggc agcagaagct gagttcatcc 600gga 603117238PRTArtificial
SequencePD1-CTM-CD28 switch receptor 117Met Gln Ile Pro Gln Ala Pro
Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro Gly
Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr Phe
Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala Thr Phe
Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu Asn Trp
Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75 80Ala
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90
95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile
Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala
Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr Ala
His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe Gln
Thr Leu Val Phe Trp Val Leu Val Val 165 170 175Val Gly Gly Val Leu
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 180 185 190Ile Ile Phe
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp 195 200 205Tyr
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 210 215
220Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser225 230
235118714DNAArtificial SequencePD1-CTM-CD28 switch receptor
118atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
ttttgggtgc tggtggtggt tggtggagtc 540ctggcttgct atagcttgct
agtaacagtg gcctttatta ttttctgggt gaggagtaag 600aggagcaggc
tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc
660cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctcc
714119232PRTArtificial SequencePD1-PTM-CD28 switch receptor 119Met
Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10
15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly
Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser
Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp
Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln
Asp Cys Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe
His Met Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr
Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Leu Gln Ile
Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg
Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro145 150 155 160Arg
Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170
175Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Arg
180 185 190Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
Thr Pro 195 200 205Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
Tyr Ala Pro Pro 210 215 220Arg Asp Phe Ala Ala Tyr Arg Ser225
230120696DNAArtificial SequencePD1-PTM-CD28 switch receptor
120atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
gttggtgtcg tgggcggcct gctgggcagc 540ctggtgctgc tagtctgggt
cctggccgtc atcaggagta agaggagcag gctcctgcac 600agtgactaca
tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc
660tatgccccac cacgcgactt cgcagcctat cgctcc 696121232PRTArtificial
SequencePD1A132L-PTM-CD28 switch receptor 121Met Gln Ile Pro Gln
Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg
Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro
Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala
Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75
80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val
Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala
Ile Ser Leu 115 120 125Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr
Ala His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe
Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175Leu Leu Gly Ser
Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Arg 180 185 190Ser Lys
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 195 200
205Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
210 215 220Arg Asp Phe Ala Ala Tyr Arg Ser225
230122693DNAArtificial SequencePD1A132L-PTM-CD28 switch receptor
122atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
gttggtgtcg tgggcggcct gctgggcagc 540ctggtgctgc tagtctgggt
cctggccgtc atcaggagta agaggagcag gctcctgcac 600agtgactaca
tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc
660tatgccccac cacgcgactt cgcagcctat cgc 693123238PRTArtificial
SequenceTGFBR-IL12RB1 receptor 123Met Glu Ala Ala Val Ala Ala Pro
Arg Pro Arg Leu Leu Leu Leu Val1 5 10 15Leu Ala Ala Ala Ala Ala Ala
Ala Ala Ala Leu Leu Pro Gly Ala Thr 20 25 30Ala Leu Gln Cys Phe Cys
His Leu Cys Thr Lys Asp Asn Phe Thr Cys 35 40 45Val Thr Asp Gly Leu
Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys 50 55 60Val Ile His Asn
Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg65 70 75 80Asp Arg
Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr 85 90 95Thr
Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro 100 105
110Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Ala Ala
115 120 125Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser Leu Met
Leu Met 130 135 140Val Tyr Ile Arg Ala Ala Arg His Leu Cys Pro Pro
Leu Pro Thr Pro145 150 155 160Cys Ala Ser Ser Ala Ile Glu Phe Pro
Gly Gly Lys Glu Thr Trp Gln 165 170 175Trp Ile Asn Pro Val Asp Phe
Gln Glu Glu Ala Ser Leu Gln Glu Ala 180 185 190Leu Val Val Glu Met
Ser Trp Asp Lys Gly Glu Arg Thr Glu Pro Leu 195 200 205Glu Lys Thr
Glu Leu Pro Glu Gly Ala Pro Glu Leu Ala Leu Asp Thr 210 215 220Glu
Leu Ser Leu Glu Asp Gly Asp Arg Cys Lys Ala Lys Met225 230
235124714DNAArtificial SequenceTGFBR-IL12RB1 receptor 124atggaggcgg
cggtcgctgc tccgcgtccc cggctgctcc tcctcgtgct ggcggcggcg 60gcggcggcgg
cggcggcgct gctcccgggg gcgacggcgt tacagtgttt ctgccacctc
120tgtacaaaag acaattttac ttgtgtgaca gatgggctct gctttgtctc
tgtcacagag 180accacagaca aagttataca caacagcatg tgtatagctg
aaattgactt aattcctcga 240gataggccgt ttgtatgtgc accctcttca
aaaactgggt ctgtgactac aacatattgc 300tgcaatcagg accattgcaa
taaaatagaa cttccaacta ctgtaaagtc atcacctggc 360cttggtcctg
tggaactggc agctgtcatt gctggaccag tgtgcttcgt ctgcatctca
420ctcatgttga tggtctatat cagggccgca cggcacctgt gcccgccgct
gcccacaccc 480tgtgccagct ccgccattga gttccctgga gggaaggaga
cttggcagtg gatcaaccca 540gtggacttcc aggaagaggc atccctgcag
gaggccctgg tggtagagat gtcctgggac 600aaaggcgaga ggactgagcc
tctcgagaag acagagctac ctgagggtgc ccctgagctg 660gccctggata
cagagttgtc cttggaggat ggagacaggt gcaaggccaa gatg
714125403PRTArtificial SequenceTGFBR-IL12RB2 receptor 125Met Gly
Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu1 5 10 15Trp
Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25
30Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro
35 40 45Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
Gln 50 55 60Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu
Lys Pro65 70 75 80Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp
Glu Asn Ile Thr 85 90 95Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
Tyr His Asp Phe Ile 100 105 110Leu Glu Asp Ala Ala Ser Pro Lys Cys
Ile Met Lys Glu Lys Lys Lys 115 120 125Pro Gly Glu Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140Asp Asn Ile Ile Phe
Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu145 150 155 160Leu Leu
Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170
175Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Gln Gln Lys Val Phe
180 185 190Val Leu Leu Ala Ala Leu Arg Pro Gln Trp Cys Ser Arg Glu
Ile Pro 195 200 205Asp Pro Ala Asn Ser Thr Cys Ala Lys Lys Tyr Pro
Ile Ala Glu Glu 210 215 220Lys Thr Gln Leu Pro Leu Asp Arg Leu Leu
Ile Asp Trp Pro Thr Pro225 230 235 240Glu Asp Pro Glu Pro Leu Val
Ile Ser Glu Val Leu His Gln Val Thr 245 250 255Pro Val Phe Arg His
Pro Pro Cys Ser Asn Trp Pro Gln Arg Glu Lys 260 265 270Gly Ile Gln
Gly His Gln Ala Ser Glu Lys Asp Met Met His Ser Ala 275 280 285Ser
Ser Pro Pro Pro Pro Arg Ala Leu Gln Ala Glu Ser Arg Gln Leu 290 295
300Val Asp Leu Tyr Lys Val Leu Glu Ser Arg Gly Ser Asp Pro Lys
Pro305 310 315 320Glu Asn Pro Ala Cys Pro Trp Thr Val Leu Pro Ala
Gly Asp Leu Pro 325 330 335Thr His Asp Gly Tyr Leu Pro Ser Asn Ile
Asp Asp Leu Pro Ser His 340 345 350Glu Ala Pro Leu Ala Asp Ser Leu
Glu Glu Leu Glu Pro Gln His Ile 355 360 365Ser Leu Ser Val Phe Pro
Ser Ser Ser Leu His Pro Leu Thr Phe Ser 370 375 380Cys Gly Asp Lys
Leu Thr Leu Asp Gln Leu Lys Met Arg Cys Asp Ser385 390 395 400Leu
Met Leu1261209DNAArtificial SequenceTGFBR-IL12RB2 receptor
126atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg
gacgcgtatc 60gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat
agtcactgac 120aacaacggtg cagtcaagtt tccacaactg tgtaaatttt
gtgatgtgag attttccacc 180tgtgacaacc agaaatcctg catgagcaac
tgcagcatca cctccatctg tgagaagcca 240caggaagtct gtgtggctgt
atggagaaag aatgacgaga acataacact agagacagtt 300tgccatgacc
ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag
360tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc
ctgtagctct 420gatgagtgca atgacaacat catcttctca gaagaatata
acaccagcaa tcctgacttg 480ttgctagtca tatttcaagt gacaggcatc
agcctcctgc caccactggg agttgccata 540tctgtcatca tcatcttcta
ccagcaaaag gtgtttgttc tcctagcagc cctcagacct 600cagtggtgta
gcagagaaat tccagatcca gcaaatagca cttgcgctaa gaaatatccc
660attgcagagg agaagacaca gctgcccttg gacaggctcc tgatagactg
gcccacgcct 720gaagatcctg aaccgctggt catcagtgaa gtccttcatc
aagtgacccc agttttcaga 780catcccccct gctccaactg gccacaaagg
gaaaaaggaa tccaaggtca tcaggcctct 840gagaaagaca tgatgcacag
tgcctcaagc ccaccacctc caagagctct ccaagctgag 900agcagacaac
tggtggatct gtacaaggtg ctggagagca ggggctccga cccaaagcca
960gaaaacccag cctgtccctg gacggtgctc ccagcaggtg accttcccac
ccatgatggc 1020tacttaccct ccaacataga tgacctcccc tcacatgagg
cacctctcgc tgactctctg 1080gaagaactgg agcctcagca catctccctt
tctgttttcc cctcaagttc tcttcaccca 1140ctcaccttct cctgtggtga
taagctgact ctggatcagt taaagatgag gtgtgactcc 1200ctcatgctc
1209127268PRTArtificial SequenceTIM3-CD28 receptor 127Met Phe Ser
His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu
Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln 20 25 30Asn
Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu 35 40
45Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly
50 55 60Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr
Ser65 70 75 80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val
Ser Leu Thr 85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr
Cys Cys Arg Ile 100 105 110Gln Ile Pro Gly Ile Met Asn Asp Glu Lys
Phe Asn Leu Lys Leu Val 115 120 125Ile Lys Pro Ala Lys Val Thr Pro
Ala Pro Thr Arg Gln Arg Asp Phe 130 135 140Thr Ala Ala Phe Pro Arg
Met Leu Thr Thr Arg Gly His Gly Pro Ala145 150 155 160Glu Thr Gln
Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile
165 170 175Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn
Asp Leu 180 185 190Arg Asp Ser Gly Ala Thr Ile Arg Phe Trp Val Leu
Val Val Val Gly 195 200 205Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
Thr Val Ala Phe Ile Ile 210 215 220Phe Trp Val Arg Ser Lys Arg Ser
Arg Leu Leu His Ser Asp Tyr Met225 230 235 240Asn Met Thr Pro Arg
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 245 250 255Tyr Ala Pro
Pro Arg Asp Phe Ala Ala Tyr Arg Ser 260 265128804DNAArtificial
SequenceTIM3-CD28 receptor 128atgttttcac atcttccctt tgactgtgtc
ctgctgctgc tgctgctact acttacaagg 60tcctcagaag tggaatacag agcggaggtc
ggtcagaatg cctatctgcc ctgcttctac 120accccagccg ccccagggaa
cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180tttgaatgtg
gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc
240agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat
agagaatgtg 300actctagcag acagtgggat ctactgctgc cgaatccaaa
tcccaggcat aatgaatgat 360gaaaaattta acctgaagtt ggtcatcaaa
ccagccaagg tcacccctgc accgactcgg 420cagagagact tcactgcagc
ctttccaagg atgcttacca ccaggggaca tggcccagca 480gagacacaga
cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc
540aatgagttac gggactctag gttggccaat gacttacggg actccggagc
aaccatcaga 600ttttgggtgc tggtggtggt tggtggagtc ctggcttgct
atagcttact agtaacagtg 660gcctttatta ttttctgggt gaggagtaag
aggagcaggc tcctgcacag tgactacatg 720aacatgactc cccgccgccc
cgggcccacc cgcaagcatt accagcccta tgccccacca 780cgcgacttcg
cagcctatcg ctcc 804129503PRTArtificial Sequence13G4-1211 PD-L1/CD28
bispecific antibody 129Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ala Ile Gln Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala 20 25 30Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile 35 40 45Ser Ser Ala Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys 50 55 60Leu Leu Ile Tyr Asp Ala Ser
Ser Leu Glu Ser Gly Val Pro Ser Arg65 70 75 80Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser 100 105 110Tyr Pro
Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Gly 115 120
125Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
130 135 140Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Ile Thr145 150 155 160Phe Asp Asp Tyr Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly 165 170 175Leu Glu Trp Val Ser Gly Ile Ser Trp
Asn Arg Gly Arg Ile Glu Tyr 180 185 190Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys 195 200 205Asn Ser Leu Tyr Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 210 215 220Leu Tyr Tyr
Cys Ala Lys Gly Arg Phe Arg Tyr Phe Asp Trp Phe Leu225 230 235
240Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
245 250 255Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro 260 265 270Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr 275 280 285Ser Tyr Tyr Ile His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu 290 295 300Trp Ile Gly Cys Ile Tyr Pro Gly
Asn Val Asn Thr Asn Tyr Asn Glu305 310 315 320Lys Phe Lys Asp Arg
Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr 325 330 335Ala Tyr Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr 340 345 350Phe
Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp 355 360
365Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Glu Gly Gly Ser Gly
370 375 380Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Met Asp Asp
Ile Gln385 390 395 400Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val 405 410 415Thr Ile Thr Cys His Ala Ser Gln Asn
Ile Tyr Val Trp Leu Asn Trp 420 425 430Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Lys Ala 435 440 445Ser Asn Leu His Thr
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 450 455 460Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe465 470 475
480Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly
485 490 495Gly Gly Thr Lys Val Glu Ile 5001301508DNAArtificial
Sequence13G4-1211 PD-L1/CD28 bispecific antibody 130atggggtggt
cgtgtatcat cctgttcctg gtcgcgacag caaccggcgt gcattcggcc 60atacagctga
cccagagccc ctcctccctc tccgcttccg tgggggaccg cgtgacaatc
120acgtgccgcg ccagccaggg aatctcctcg gccctcgcct ggtaccagca
gaaacccggg 180aaggctccca agctgctcat ctacgatgcc tcctcgcttg
agtcgggcgt gccatccagg 240ttctccggat ccgggtccgg aaccgacttt
acactcacga tttcctctct gcagcccgag 300gacttcgcca catactactg
tcagcagttc aactcctacc cattcacctt cggcccgggc 360accaaggtgg
acatcaagtc tggcggggga ggctccgaag tccagctcgt ggaatccggg
420ggcggtctcg tgcagccagg ccggagtctg cgcctgtctt gcgctgcctc
ggggatcact 480ttcgacgact acggcatgca ttgggttcgc caggccccag
ggaaggggtt ggagtgggtc 540agtggcattt catggaacag ggggcgcatc
gaatacgccg actccgttaa gggcagattc 600accatctcgc gcgataacgc
caaaaacagt ctctacctcc agatgaactc gcttcgagca 660gaggatactg
ccctgtacta ttgcgcgaag ggacgcttcc gctactttga ctggtttctg
720gactactggg gccaggggac actggtgacg gtgtcgtcgg ggggcggggg
gagtcaggtg 780cagctggtgc agtccggagc cgaggtaaag aagccaggcg
cttccgtcaa ggtgtcatgc 840aaggcctcag gctacacctt cacaagctat
tacatccact gggtgcgcca agctcccggt 900cagggcttgg agtggatcgg
gtgcatttac ccagggaacg tcaacacaaa ctacaacgag 960aagttcaagg
atcgggcaac cctgaccgtg gacacatcca tctctaccgc ctacatggag
1020ctgtcacgcc tgcgctctga tgacaccgca gtgtacttct gtaccaggag
tcactacggc 1080ctggactgga actttgatgt ctggggccag ggaaccaccg
tgacggtgtc cagtgtggag 1140ggcggtagtg gcggctctgg tgggtccgga
ggctcaggcg gcgtgatgga tgacattcag 1200atgacccaga gtccctcctc
cctctccgct tccgtcggag accgcgtgac catcacttgt 1260cacgcctcac
agaatatcta cgtgtggctg aactggtacc aacagaagcc cggcaaggcc
1320cccaagctgc ttatctataa agcgtccaac ctccacacgg gagtcccttc
ccgcttctcc 1380ggatccggca gtgggacgga cttcacactc acaatctcgt
cgctgcagcc agaggacttt 1440gcgacgtact actgccagca gggccagacc
tacccatata ctttcggcgg cgggaccaag 1500gtggagat
1508131500PRTArtificial Sequence10A5-1412 PD-L1/CD28 bispecific
antibody 131Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala 20 25 30Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile 35 40 45Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys 50 55 60Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg65 70 75 80Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 100 105 110Tyr Pro Tyr Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Gly 115 120 125Gly Gly Gly
Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 130 135 140Lys
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr145 150
155 160Phe Thr Ser Tyr Asp Val His Trp Val Arg Gln Ala Pro Gly Gln
Arg 165 170 175Leu Glu Trp Met Gly Trp Leu His Ala Asp Thr Gly Ile
Thr Lys Phe 180 185 190Ser Gln Lys Phe Gln Gly Arg Val Thr Ile Thr
Arg Asp Thr Ser Ala 195 200 205Ser Thr Ala Tyr Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala 210 215 220Val Tyr Tyr Cys Ala Arg Glu
Arg Ile Gln Leu Trp Phe Asp Tyr Trp225 230 235 240Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln 245 250 255Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser 260 265
270Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr
275 280 285Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile Gly 290 295 300Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn
Glu Lys Phe Lys305 310 315 320Asp Arg Ala Thr Leu Thr Val Asp Thr
Ser Ile Ser Thr Ala Tyr Met 325 330 335Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr Phe Cys Thr 340 345 350Arg Ser His Tyr Gly
Leu Asp Trp Asn Phe Asp Val Trp Gly Gln Gly 355 360 365Thr Thr Val
Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly 370 375 380Gly
Ser Gly Gly Ser Gly Gly Val Met Asp Asp Ile Gln Met Thr Gln385 390
395 400Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
Thr 405 410 415Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp
Tyr Gln Gln 420 425 430Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Lys Ala Ser Asn Leu 435 440 445His Thr Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp 450 455 460Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr465 470 475 480Tyr Cys Gln Gln
Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr 485 490 495Lys Val
Glu Ile 5001321499DNAArtificial Sequence10A5-1412 PD-L1/CD28
bispecific antibody 132atgggctgga gttgcatcat tctcttcctc gtggcgaccg
caacaggggt gcactccgac 60atccagatga cccagtcccc gagttccctg tctgcttccg
tgggagatcg cgtgactatc 120acctgccggg cttcccaggg catctcttcc
tggctggcgt ggtaccagca gaaaccagaa 180aaggctccta agtccctgat
ctacgcagct tcgtccctcc aatccggcgt cccctctcgc 240ttctccggct
ccggatccgg caccgacttc acgctgacaa tctcgagttt gcagcccgag
300gacttcgcca cctactactg ccagcagtac aactcctacc cttacacctt
cggccagggc 360acaaagctcg aaatcaagtc gggggggggc gggtcgcagg
tccagctggt gcagtccggc 420gccgaagtca agaagcccgg agcaagtgtg
aaagtgtcgt gcaaggcaag tgggtatacc 480ttcacctcat acgacgtaca
ctgggtgcgc caggcgcccg gtcagcgcct tgagtggatg 540ggctggctcc
acgccgacac cggcattacc aagttctctc agaagttcca gggaagagtg
600accataacac gcgacaccag tgcttccaca gcttacatgg aactttcgag
tctgagatcc 660gaggacacag ccgtgtatta ctgtgcccgt gagcgcatcc
agctgtggtt cgactactgg 720gggcagggca ccctcgtgac ggtgtcgtcg
gggggcgggg ggagtcaggt gcagctggtg 780cagtccggag ccgaggtaaa
gaagccaggc gcttccgtca aggtgtcatg caaggcctca 840ggctacacct
tcacaagcta ttacatccac tgggtgcgcc aagctcccgg tcagggcttg
900gagtggatcg ggtgcattta cccagggaac gtcaacacaa actacaacga
gaagttcaag 960gatcgggcaa ccctgaccgt ggacacatcc atctctaccg
cctacatgga gctgtcacgc 1020ctgcgctctg atgacaccgc agtgtacttc
tgtaccagga gtcactacgg cctggactgg 1080aactttgatg tctggggcca
gggaaccacc gtgacggtgt ccagtgtgga gggcggtagt 1140ggcggctctg
gtgggtccgg aggctcaggc ggcgtgatgg atgacattca gatgacccag
1200agtccctcct ccctctccgc ttccgtcgga gaccgcgtga ccatcacttg
tcacgcctca 1260cagaatatct acgtgtggct gaactggtac caacagaagc
ccggcaaggc ccccaagctg 1320cttatctata aagcgtccaa cctccacacg
ggagtccctt cccgcttctc cggatccggc 1380agtgggacgg acttcacact
cacaatctcg tcgctgcagc cagaggactt tgcgacgtac 1440tactgccagc
agggccagac ctacccatat actttcggcg gcgggaccaa ggtggagat
1499133507PRTArtificial Sequence1B12-1412 PD-L1/CD28 bispecific
antibody 133Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu 20 25 30Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val 35 40 45Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg 50 55 60Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala Arg65 70 75 80Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn 100 105 110Trp Pro Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Ser Gly Gly 115 120 125Gly Gly Ser
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 130 135 140Pro
Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe145 150
155 160Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu 165 170 175Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Arg Ala
His Tyr Ala 180 185 190Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala
Asp Glu Ser Thr Ser 195 200 205Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val 210 215 220Tyr Phe Cys Ala Arg Lys Phe
His Phe Val Ser Gly Ser Pro Phe Gly225 230 235 240Met Asp Val Trp
Gly Gln Gly Thr Val Thr Val Ser Ser Gly Gly Ser 245 250 255Ser Gly
Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 260 265
270Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
275 280 285Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Val Arg Gln Ala
Pro Gly 290 295 300Gln Gly Leu Glu Trp Ile Gly Cys Ile Tyr Pro Gly
Asn Val Asn Thr305 310 315 320Asn Tyr Asn Glu Lys Phe Lys Asp Arg
Ala Thr Leu Thr Val Asp Thr 325 330 335Ser Ile Ser Thr Ala Tyr Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp 340 345 350Thr Ala Val Tyr Phe
Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn 355 360 365Phe Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Glu 370 375 380Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Met385 390
395 400Asp Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val 405 410 415Gly Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn
Ile Tyr Val 420 425 430Trp Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu 435 440 445Ile Tyr Lys Ala Ser Asn Leu His Thr
Gly Val Pro Ser Arg Phe Ser 450 455 460Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln465 470 475 480Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro 485 490 495Tyr Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile 500 5051341520DNAArtificial
Sequence1B12-1412 PD-L1/CD28 bispecific antibody 134atgggctgga
gttgcatcat cctctttcta gtcgccacgg ccaccggcgt acactcagag 60atcgtgctga
cacagtcgcc tgcgacgctg tcgctcagtc caggggagcg cgctactctc
120tcctgccgcg cgtcgcagag cgtgtcgtcc tacttggcct ggtaccagca
gaagcctggc 180caggctccgc gcctgctgat atacgacgcc tcgaacagag
ccacgggcat ccccgcccgt 240tttagtggct ccgggtcggg gaccgacttc
actctgacaa tctcatccct cgagcccgag 300gatttcgccg tgtactactg
tcagcagcgc tcgaattggc caaccttcgg gcaggggacg 360aaagttgaga
tcaaaagcgg cggcgggggc agccaggtcc agctcgtcca gtctggcgcc
420gaggtcaaaa agccgggctc ttcggtcaag gtctcctgca agacttccgg
cgacaccttc 480tcctcctatg ctatctcctg ggtgcggcag gccccggggc
agggcctgga gtggatggga 540ggcatcatcc caatctttgg gagggcccac
tacgcccaga agttccaggg acgcgtgaca 600atcaccgcag acgagtccac
atccactgcc tacatggagt tgtcctcgct ccggtcggag 660gatactgccg
tgtacttctg cgcccggaag ttccacttcg tgtcaggctc ccccttcggg
720atggacgtgt ggggacaagg aaccgtgacg gtgtcgtcgg ggggctcgtc
ggggggcggg 780gggagtcagg tgcagctggt gcagtccgga gccgaggtaa
agaagccagg cgcttccgtc 840aaggtgtcat gcaaggcctc aggctacacc
ttcacaagct attacatcca ctgggtgcgc 900caagctcccg gtcagggctt
ggagtggatc gggtgcattt acccagggaa
cgtcaacaca 960aactacaacg agaagttcaa ggatcgggca accctgaccg
tggacacatc catctctacc 1020gcctacatgg agctgtcacg cctgcgctct
gatgacaccg cagtgtactt ctgtaccagg 1080agtcactacg gcctggactg
gaactttgat gtctggggcc agggaaccac cgtgacggtg 1140tccagtgtgg
agggcggtag tggcggctct ggtgggtccg gaggctcagg cggcgtgatg
1200gatgacattc agatgaccca gagtccctcc tccctctccg cttccgtcgg
agaccgcgtg 1260accatcactt gtcacgcctc acagaatatc tacgtgtggc
tgaactggta ccaacagaag 1320cccggcaagg cccccaagct gcttatctat
aaagcgtcca acctccacac gggagtccct 1380tcccgcttct ccggatccgg
cagtgggacg gacttcacac tcacaatctc gtcgctgcag 1440ccagaggact
ttgcgacgta ctactgccag cagggccaga cctacccata tactttcggc
1500ggcgggacca aggtggagat 1520135504PRTArtificial
SequenceTGFBR-1-1412 TGFBRII/CD28 bispecific antibody 135Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu 20 25
30Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
35 40 45Arg Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg 50 55 60Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala Arg65 70 75 80Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser 85 90 95Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn 100 105 110Trp Pro Pro Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Ser Gly 115 120 125Gly Gly Gly Ser Gln Leu Gln
Val Gln Glu Ser Gly Pro Gly Leu Val 130 135 140Lys Pro Ser Glu Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser145 150 155 160Ile Ser
Asn Ser Tyr Phe Ser Trp Gly Trp Ile Arg Gln Pro Pro Gly 165 170
175Lys Gly Leu Glu Trp Ile Gly Ser Phe Tyr Tyr Gly Glu Lys Thr Tyr
180 185 190Tyr Asn Pro Ser Leu Lys Ser Arg Ala Thr Ile Ser Ile Asp
Thr Ser 195 200 205Lys Ser Gln Phe Ser Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr 210 215 220Ala Val Tyr Tyr Cys Pro Arg Gly Pro Thr
Met Ile Arg Gly Val Ile225 230 235 240Asp Ser Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly 245 250 255Gly Ser Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 260 265 270Gly Ala Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 275 280 285Ser
Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 290 295
300Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn
Glu305 310 315 320Lys Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Thr
Ser Ile Ser Thr 325 330 335Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr 340 345 350Phe Cys Thr Arg Ser His Tyr Gly
Leu Asp Trp Asn Phe Asp Val Trp 355 360 365Gly Gln Gly Thr Thr Val
Thr Val Ser Ser Val Glu Gly Gly Ser Gly 370 375 380Gly Ser Gly Gly
Ser Gly Gly Ser Gly Gly Val Met Asp Asp Ile Gln385 390 395 400Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 405 410
415Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp
420 425 430Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Lys Ala 435 440 445Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser 450 455 460Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe465 470 475 480Ala Thr Tyr Tyr Cys Gln Gln
Gly Gln Thr Tyr Pro Tyr Thr Phe Gly 485 490 495Gly Gly Thr Lys Val
Glu Ile Lys 5001361512DNAArtificial SequenceTGFBR-1-1412
TGFBRII/CD28 bispecific antibody 136atgggttggt cctgcatcat
cctgtttctc gtggccaccg ccaccggcgt gcactccgaa 60attgtgttga cacagtctcc
agccaccctg tctttgtctc caggggaaag agccaccctc 120tcctgcaggg
ccagtcagag tgttcgcagc tacttagcct ggtaccaaca gaaacctggc
180caggctccca ggctcctcat ctatgatgca tccaacaggg ccactggcat
cccagccagg 240ttcagtggca gtgggtctgg gacagacttc actctcacca
tcagcagcct agagcctgaa 300gattttgcag tttattactg tcagcagcgt
agcaactggc ctccgacgtt cggccaaggg 360accaaggtgg aaatcaaaag
tggagggggc ggttcacagc tgcaggtgca ggagtcgggc 420ccaggactgg
tgaagccttc ggagaccctg tccctcacct gcactgtctc tggtggctcc
480atcagcaaca gttatttctc ctggggctgg atccgccagc ccccagggaa
gggactggag 540tggattggga gtttctatta tggtgaaaaa acctactaca
acccgtccct caagagccga 600gccaccatat ccattgacac gtccaagagc
cagttctccc tgaagctgag ctctgtgacc 660gccgcagaca cggctgtgta
ttactgtccg agagggccta ctatgattcg gggagttata 720gactcctggg
gccagggaac cctggtgacg gtgtcgtcgg ggggcggggg gagtcaggtg
780cagctggtgc agtccggagc cgaggtaaag aagccaggcg cttccgtcaa
ggtgtcatgc 840aaggcctcag gctacacctt cacaagctat tacatccact
gggtgcgcca agctcccggt 900cagggcttgg agtggatcgg gtgcatttac
ccagggaacg tcaacacaaa ctacaacgag 960aagttcaagg atcgggcaac
cctgaccgtg gacacatcca tctctaccgc ctacatggag 1020ctgtcacgcc
tgcgctctga tgacaccgca gtgtacttct gtaccaggag tcactacggc
1080ctggactgga actttgatgt ctggggccag ggaaccaccg tgacggtgtc
cagtgtggag 1140ggcggtagtg gcggctctgg tgggtccgga ggctcaggcg
gcgtgatgga tgacattcag 1200atgacccaga gtccctcctc cctctccgct
tccgtcggag accgcgtgac catcacttgt 1260cacgcctcac agaatatcta
cgtgtggctg aactggtacc aacagaagcc cggcaaggcc 1320cccaagctgc
ttatctataa agcgtccaac ctccacacgg gagtcccttc ccgcttctcc
1380ggatccggca gtgggacgga cttcacactc acaatctcgt cgctgcagcc
agaggacttt 1440gcgacgtact actgccagca gggccagacc tacccatata
ctttcggcgg cgggaccaag 1500gtggagatta ag 1512137504PRTArtificial
SequenceTGFBR-3-1412 TGFBRII/CD28 bispecific antibody 137Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val
His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu 20 25
30Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
35 40 45Arg Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg 50 55 60Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala Arg65 70 75 80Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser 85 90 95Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn 100 105 110Trp Pro Pro Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Ser Gly 115 120 125Gly Gly Gly Ser Gln Leu Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val 130 135 140Lys Pro Ser Glu Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser145 150 155 160Ile Ser
Ser Ser Ser Tyr Ser Trp Gly Trp Ile Arg Gln Pro Pro Gly 165 170
175Lys Gly Leu Glu Trp Ile Gly Ser Phe Tyr Tyr Ser Gly Ile Thr Tyr
180 185 190Tyr Ser Pro Ser Leu Lys Ser Arg Ile Ile Ile Ser Glu Asp
Thr Ser 195 200 205Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr 210 215 220Ala Val Tyr Tyr Cys Ala Ser Gly Phe Thr
Met Ile Arg Gly Ala Leu225 230 235 240Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly 245 250 255Gly Ser Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 260 265 270Gly Ala Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 275 280 285Ser
Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 290 295
300Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn
Glu305 310 315 320Lys Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Thr
Ser Ile Ser Thr 325 330 335Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr 340 345 350Phe Cys Thr Arg Ser His Tyr Gly
Leu Asp Trp Asn Phe Asp Val Trp 355 360 365Gly Gln Gly Thr Thr Val
Thr Val Ser Ser Val Glu Gly Gly Ser Gly 370 375 380Gly Ser Gly Gly
Ser Gly Gly Ser Gly Gly Val Met Asp Asp Ile Gln385 390 395 400Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 405 410
415Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp
420 425 430Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Lys Ala 435 440 445Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser 450 455 460Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe465 470 475 480Ala Thr Tyr Tyr Cys Gln Gln
Gly Gln Thr Tyr Pro Tyr Thr Phe Gly 485 490 495Gly Gly Thr Lys Val
Glu Ile Lys 5001381512DNAArtificial SequenceTGFBR-3-1412
TGFBRII/CD28 bispecific antibody 138atgggttggt cctgcatcat
cctgtttctc gtggccaccg ccaccggcgt gcactccgaa 60attgtgttga cacagtctcc
agccaccctg tctttgtctc caggggaaag agccaccctc 120tcctgcaggg
ccagtcagag tgttagaagt ttcttagcct ggtaccaaca gaaacctggc
180caggctccca ggctcctcat ctatgatgca tccaacaggg ccactggcat
cccagccagg 240ttcagtggca gtgggtctgg gacagacttc actctcacca
tcagcagcct agagcctgaa 300gattttgcag tttattactg tcagcagcgt
agcaactggc ctccgacgtt cggccaaggg 360accaaggtgg aaatcaaaag
tggagggggc ggttcacagc tacagctgca ggagtcgggc 420ccaggactgg
tgaagccttc ggagacccta tccctcacct gcactgtctc tggtggctcc
480atcagcagta gtagttactc ctggggctgg atccgccagc ccccagggaa
gggcctggag 540tggattggga gtttctatta cagtgggatc acctactaca
gcccgtccct caagagtcga 600attatcatat ccgaagacac gtccaagaac
cagttctccc tgaagctgag ttctgtgacc 660gccgcagaca cggctgtgta
ttactgtgcg agcgggttta ctatgattcg gggagccctt 720gactactggg
gccagggaac cctggtgacg gtgtcgtcgg ggggcggggg gagtcaggtg
780cagctggtgc agtccggagc cgaggtaaag aagccaggcg cttccgtcaa
ggtgtcatgc 840aaggcctcag gctacacctt cacaagctat tacatccact
gggtgcgcca agctcccggt 900cagggcttgg agtggatcgg gtgcatttac
ccagggaacg tcaacacaaa ctacaacgag 960aagttcaagg atcgggcaac
cctgaccgtg gacacatcca tctctaccgc ctacatggag 1020ctgtcacgcc
tgcgctctga tgacaccgca gtgtacttct gtaccaggag tcactacggc
1080ctggactgga actttgatgt ctggggccag ggaaccaccg tgacggtgtc
cagtgtggag 1140ggcggtagtg gcggctctgg tgggtccgga ggctcaggcg
gcgtgatgga tgacattcag 1200atgacccaga gtccctcctc cctctccgct
tccgtcggag accgcgtgac catcacttgt 1260cacgcctcac agaatatcta
cgtgtggctg aactggtacc aacagaagcc cggcaaggcc 1320cccaagctgc
ttatctataa agcgtccaac ctccacacgg gagtcccttc ccgcttctcc
1380ggatccggca gtgggacgga cttcacactc acaatctcgt cgctgcagcc
agaggacttt 1440gcgacgtact actgccagca gggccagacc tacccatata
ctttcggcgg cgggaccaag 1500gtggagatta ag 151213918PRTArtificial
SequenceT2A 139Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
Glu Asn Pro1 5 10 15Gly Pro14054DNAArtificial SequenceT2A
140gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct
541417PRTArtificial Sequencespacer 141Ser Gly Arg Ser Gly Gly Gly1
514221DNAArtificial Sequencespacer 142tccggaagat ctggcggcgg a
2114322PRTArtificial SequenceF2A 143Val Lys Gln Thr Leu Asn Phe Asp
Leu Leu Lys Leu Ala Gly Asp Val1 5 10 15Glu Ser Asn Pro Gly Pro
2014466DNAArtificial SequenceF2A 144gtgaaacaga ctttgaattt
tgaccttctc aagttggcgg gagacgtgga gtccaaccca 60gggccg
661454PRTArtificial Sequencefurin cleavage
sitemisc_feature(2)..(2)Xaa can be any naturally occurring amino
acid 145Arg Xaa Lys Arg11464PRTArtificial Sequencefurin cleavage
sitemisc_feature(2)..(2)Xaa can be any naturally occurring amino
acid 146Arg Xaa Arg Arg11474PRTArtificial Sequencefurin cleavage
sitemisc_feature(2)..(3)Xaa can be any naturally occurring amino
acid 147Arg Xaa Xaa Arg11484PRTArtificial Sequencefurin cleavage
site 148Arg Gln Lys Arg11495PRTArtificial Sequencefurin cleavage
sitemisc_feature(1)..(1)Xaa can be any naturally occurring amino
acidmisc_feature(3)..(4)Xaa can be any naturally occurring amino
acid 149Xaa Arg Xaa Xaa Arg1 515096DNAArtificial SequenceF-GS2-T2A
linker 150cgtgcgaaga ggggcggcgg gggctccggc gggggaggca gtgagggccg
cggctccctg 60ctgacctgcg gagatgtaga agagaaccca ggcccc
9615132PRTArtificial SequenceF-GS2-T2A linker 151Arg Ala Lys Arg
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gly1 5 10 15Arg Gly Ser
Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25
301522142DNAArtificial SequenceTGFbRDN-PSMA-CAR 152atgggtcggg
ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60gccagcacga
tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac
120aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag
attttccacc 180tgtgacaacc agaaatcctg catgagcaac tgcagcatca
cctccatctg tgagaagcca 240caggaagtct gtgtggctgt atggagaaag
aatgacgaga acataacact agagacagtt 300tgccatgacc ccaagctccc
ctaccatgac tttattctgg aagatgctgc ttctccaaag 360tgcattatga
aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct
420gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa
tcctgacttg 480ttgctagtca tatttcaagt gacaggcatc agcctcctgc
caccactggg agttgccata 540tctgtcatca tcatcttcta ctgctaccgc
gttaaccggc agcagaagct gagttcatcc 600ggaagatctg gcggcggaga
gggcagagga agtcttctaa catgcggtga cgtggaggag 660aatcccggcc
ctagagccac catggccctg cctgtgacag ccctgctgct gcctctggct
720ctgctgctgc acgccgccag acctggatct gacattgtga tgacccagtc
tcacaaattc 780atgtccacat cagtaggaga cagggtcagc atcatctgta
aggccagtca agatgtgggt 840actgctgtag actggtatca acagaaacca
ggacaatctc ctaaactact gatttattgg 900gcatccactc ggcacactgg
agtccctgat cgcttcacag gcagtggatc tgggacagac 960ttcactctca
ccattactaa cgttcagtct gaagacttgg cagattattt ctgtcagcaa
1020tataacagct atcctctcac gttcggtgct gggaccatgc tggacctgaa
aggaggcgga 1080ggatctggcg gcggaggaag ttctggcgga ggcagcgagg
tgcagctgca gcagagcgga 1140cccgagctcg tgaagcctgg aacaagcgtg
cggatcagct gcaagaccag cggctacacc 1200ttcaccgagt acaccatcca
ctgggtcaag cagtcccacg gcaagagcct ggagtggatc 1260ggcaatatca
accccaacaa cggcggcacc acctacaacc agaagttcga ggacaaggcc
1320accctgaccg tggacaagag cagcagcacc gcctacatgg aactgcggag
cctgaccagc 1380gaggacagcg ccgtgtacta ttgtgccgcc ggttggaact
tcgactactg gggccagggc 1440acaaccctga cagtgtctag cgctagctcc
ggaaccacga cgccagcgcc gcgaccacca 1500acaccggcgc ccaccatcgc
gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 1560gcggcggggg
gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg
1620gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac
cctttactgc 1680aaacggggca gaaagaaact cctgtatata ttcaaacaac
catttatgag accagtacaa 1740actactcaag aggaagacgg ctgtagctgc
cgatttccag aagaagaaga aggaggatgt 1800gaactgagag tgaagttcag
caggagcgca gacgcccccg cgtacaagca gggccagaac 1860cagctctata
acgagctcaa tctaggacga agagaggagt acgacgtttt ggacaagaga
1920cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca
ggaaggcctg 1980tacaacgaac tgcagaaaga taagatggcg gaggcctaca
gtgagattgg gatgaaaggc 2040gagcgccgga ggggcaaggg gcacgacggc
ctttaccagg gtctcagtac agccaccaag 2100gacacctacg acgcccttca
catgcaggcc ctgccccctc gc 21421532151DNAArtificial
SequenceTGFbRDN-1C3PSMA-CAR 153atgggtcggg ggctgctcag gggcctgtgg
ccgctgcaca tcgtcctgtg gacgcgtatc 60gccagcacga tcccaccgca cgttcagaag
tcggttaata acgacatgat agtcactgac 120aacaacggtg cagtcaagtt
tccacaactg tgtaaatttt gtgatgtgag attttccacc 180tgtgacaacc
agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca
240caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact
agagacagtt 300tgccatgacc ccaagctccc ctaccatgac tttattctgg
aagatgctgc ttctccaaag 360tgcattatga aggaaaaaaa aaagcctggt
gagactttct tcatgtgttc ctgtagctct 420gatgagtgca atgacaacat
catcttctca gaagaatata acaccagcaa tcctgacttg 480ttgctagtca
tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata
540tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct
gagttcatcc 600ggaagatctg gcggcggaga gggcagagga agtcttctaa
catgcggtga cgtggaggag 660aatcccggcc ctagagccac catggcctta
ccagtgaccg ccttgctcct gccgctggcc 720ttgctgctcc acgccgccag
gccgcaggtg caactggtgg agtctggggg aggcgtggtc 780cagcctggga
ggtccctgag actctcctgt gcagcctctg gattcacctt cagtagctat
840gctatgcact gggtccgcca ggctccaggc aaggggctgg agtgggtggc
agttatatca 900tatgatggaa acaataaata ctacgcagac tccgtgaagg
gccgattcac catctccaga 960gacaattcca
agaacacgct gtatctgcaa atgaacagcc tgagagctga ggacacggct
1020gtgtattact gtgcgagagc cgtcccctgg ggatcgaggt actactacta
cggtatggac 1080gtctggggcc aagggaccac ggtcaccgtc tcctcaggtg
gcggtggctc gggcggtggt 1140gggtcgggtg gcggcggatc tgccatccag
ttgacccagt ctccatcctc cctgtctgca 1200tctgtaggag acagagtcac
catcacttgc cgggcaagtc agggcattag cagtgcttta 1260gcctggtatc
agcagaaatc agggaaagct cctaagctcc tgatctttga tgcctccagt
1320ttggaaagtg gggtcccatc aaggttcagc ggcagtggat ctgggacaga
tttcactctc 1380accatcagca gcctgcagcc tgaagatttt gcaacttatt
actgtcaaca gtttaacagt 1440tatcctctca ctttcggcgg agggaccaag
gtggagatca aaaccacgac gccagcgccg 1500cgaccaccaa caccggcgcc
caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 1560tgccggccag
cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc
1620tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact
ggttatcacc 1680ctttactgca aacggggcag aaagaaactc ctgtatatat
tcaaacaacc atttatgaga 1740ccagtacaaa ctactcaaga ggaagacggc
tgtagctgcc gatttccaga agaagaagaa 1800ggaggatgtg aactgagagt
gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1860ggccagaacc
agctctataa cgagctcaat ctaggacgaa gagaggagta cgacgttttg
1920gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa
gaaccctcag 1980gaaggcctgt acaacgaact gcagaaagat aagatggcgg
aggcctacag tgagattggg 2040atgaaaggcg agcgccggag gggcaagggg
cacgacggcc tttaccaggg tctcagtaca 2100gccaccaagg acacctacga
cgcccttcac atgcaggccc tgccccctcg c 21511542136DNAArtificial
SequenceTGFbRDN-2A10PSMA-CAR 154atgggtcggg ggctgctcag gggcctgtgg
ccgctgcaca tcgtcctgtg gacgcgtatc 60gccagcacga tcccaccgca cgttcagaag
tcggttaata acgacatgat agtcactgac 120aacaacggtg cagtcaagtt
tccacaactg tgtaaatttt gtgatgtgag attttccacc 180tgtgacaacc
agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca
240caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact
agagacagtt 300tgccatgacc ccaagctccc ctaccatgac tttattctgg
aagatgctgc ttctccaaag 360tgcattatga aggaaaaaaa aaagcctggt
gagactttct tcatgtgttc ctgtagctct 420gatgagtgca atgacaacat
catcttctca gaagaatata acaccagcaa tcctgacttg 480ttgctagtca
tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata
540tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct
gagttcatcc 600ggaagatctg gcggcggaga gggcagagga agtcttctaa
catgcggtga cgtggaggag 660aatcccggcc ctagagccac catggcctta
ccagtgaccg ccttgctcct gccgctggcc 720ttgctgctcc acgccgccag
gccggaggtg cagctggtgc agtctggagc agaggtgaaa 780aagcccgggg
agtctctgaa gatctcctgt aagggttctg gatacagctt taccagtaac
840tggatcggct gggtgcgcca gatgcccggg aaaggcctgg agtggatggg
gatcatctat 900cctggtgact ctgataccag atacagcccg tccttccaag
gccaggtcac catctcagcc 960gacaagtcca tcagcaccgc ctacctgcag
tggagcagcc tgaaggcctc ggacaccgcc 1020atgtattact gtgcgaggca
aactggtttc ctctggtcct ccgatctctg gggccgtggc 1080accctggtca
ctgtctcctc aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc
1140ggatctgcca tccagttgac ccagtctcca tcctccctgt ctgcatctgt
aggagacaga 1200gtcaccatca cttgccgggc aagtcaggac attagcagtg
ctttagcctg gtatcaacag 1260aaaccaggga aagctcctaa gctcctgatc
tatgatgcct ccagtttgga aagtggggtc 1320ccatcaaggt tcagcggcta
tggatctggg acagatttca ctctcaccat caacagcctg 1380cagcctgaag
attttgcaac ttattactgt caacagttta atagttaccc gctcactttc
1440ggcggaggga ccaaggtgga gatcaaaacc acgacgccag cgccgcgacc
accaacaccg 1500gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag
aggcgtgccg gccagcggcg 1560gggggcgcag tgcacacgag ggggctggac
ttcgcctgtg atatctacat ctgggcgccc 1620ttggccggga cttgtggggt
ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1680ggcagaaaga
aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact
1740caagaggaag acggctgtag ctgccgattt ccagaagaag aagaaggagg
atgtgaactg 1800agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca
agcagggcca gaaccagctc 1860tataacgagc tcaatctagg acgaagagag
gagtacgacg ttttggacaa gagacgtggc 1920cgggaccctg agatgggggg
aaagccgaga aggaagaacc ctcaggaagg cctgtacaac 1980gaactgcaga
aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc
2040cggaggggca aggggcacga cggcctttac cagggtctca gtacagccac
caaggacacc 2100tacgacgccc ttcacatgca ggccctgccc cctcgc
21361552142DNAArtificial SequenceTGFbRDN-2F5PSMA-CAR 155atgggtcggg
ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60gccagcacga
tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac
120aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag
attttccacc 180tgtgacaacc agaaatcctg catgagcaac tgcagcatca
cctccatctg tgagaagcca 240caggaagtct gtgtggctgt atggagaaag
aatgacgaga acataacact agagacagtt 300tgccatgacc ccaagctccc
ctaccatgac tttattctgg aagatgctgc ttctccaaag 360tgcattatga
aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct
420gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa
tcctgacttg 480ttgctagtca tatttcaagt gacaggcatc agcctcctgc
caccactggg agttgccata 540tctgtcatca tcatcttcta ctgctaccgc
gttaaccggc agcagaagct gagttcatcc 600ggaagatctg gcggcggaga
gggcagagga agtcttctaa catgcggtga cgtggaggag 660aatcccggcc
ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc
720ttgctgctcc acgccgccag gccggaggtg cagctggtgc agtctggagc
agaggtgaaa 780aagcccgggg agtctctgaa gatctcctgt aagggttctg
gatacagttt taccagcaac 840tggatcggct gggtgcgcca gatgcccggg
aaaggcctgg agtggatggg gatcatctat 900cctggtgact ctgataccag
atacagcccg tccttccaag gccaggtcac catctcagcc 960gacaagtcca
tcagcaccgc ctacctgcag tggaacagcc tgaaggcctc ggacaccgcc
1020atgtattact gtgcgagaca aactggtttc ctctggtcct tcgatctctg
gggccgtggc 1080accctggtca ctgtctcctc aggtggcggt ggctcgggcg
gtggtgggtc gggtggcggc 1140ggatctgcca tccagttgac ccagtctcca
tcctccctgt ctgcatctgt aggagacaga 1200gtcaccatca cttgccgggc
aagtcaggac attagcagtg ctttagcctg gtatcagcag 1260aaaccgggga
aagctcctaa gctcctgatc tatgatgcct ccagtttgga aagtggggtc
1320ccatcaaggt tcagcggcag tggatctggg acagatttca ctctcaccat
cagcagcctg 1380cagcctgaag attttgcaac ttattactgt caacagttta
atagttaccc gctcactttc 1440ggcggaggga ccaaggtgga gatcaaaatc
aaaaccacga cgccagcgcc gcgaccacca 1500acaccggcgc ccaccatcgc
gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 1560gcggcggggg
gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg
1620gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac
cctttactgc 1680aaacggggca gaaagaaact cctgtatata ttcaaacaac
catttatgag accagtacaa 1740actactcaag aggaagacgg ctgtagctgc
cgatttccag aagaagaaga aggaggatgt 1800gaactgagag tgaagttcag
caggagcgca gacgcccccg cgtacaagca gggccagaac 1860cagctctata
acgagctcaa tctaggacga agagaggagt acgacgtttt ggacaagaga
1920cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca
ggaaggcctg 1980tacaacgaac tgcagaaaga taagatggcg gaggcctaca
gtgagattgg gatgaaaggc 2040gagcgccgga ggggcaaggg gcacgacggc
ctttaccagg gtctcagtac agccaccaag 2100gacacctacg acgcccttca
catgcaggcc ctgccccctc gc 21421562145DNAArtificial
SequenceTGFbRDN-2C6PSMA-CAR 156atgggtcggg ggctgctcag gggcctgtgg
ccgctgcaca tcgtcctgtg gacgcgtatc 60gccagcacga tcccaccgca cgttcagaag
tcggttaata acgacatgat agtcactgac 120aacaacggtg cagtcaagtt
tccacaactg tgtaaatttt gtgatgtgag attttccacc 180tgtgacaacc
agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca
240caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact
agagacagtt 300tgccatgacc ccaagctccc ctaccatgac tttattctgg
aagatgctgc ttctccaaag 360tgcattatga aggaaaaaaa aaagcctggt
gagactttct tcatgtgttc ctgtagctct 420gatgagtgca atgacaacat
catcttctca gaagaatata acaccagcaa tcctgacttg 480ttgctagtca
tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata
540tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct
gagttcatcc 600ggaagatctg gcggcggaga gggcagagga agtcttctaa
catgcggtga cgtggaggag 660aatcccggcc ctagagccac catggcctta
ccagtgaccg ccttgctcct gccgctggcc 720ttgctgctcc acgccgccag
gccggaggtg cagctggtgc agtctggatc agaggtgaaa 780aagcccgggg
agtctctgaa gatctcctgt aagggttctg gatacagctt taccaactac
840tggatcggct gggtgcgcca gatgcccggg aaaggcctgg agtggatggg
gatcatctat 900cctggtgact ctgataccag atacagcccg tccttccaag
gccaggtcac catctcagcc 960gacaagtcca tcagcaccgc ctatctgcag
tggagcagcc tgaaggcctc ggacaccgcc 1020atgtattact gtgcgagtcc
cgggtatacc agcagttgga cttcttttga ctactggggc 1080cagggaaccc
tggtcaccgt ctcctcaggt ggcggtggct cgggcggtgg tgggtcgggt
1140ggcggcggat ctgaaattgt gttgacacag tctccagcca ccctgtcttt
gtctccaggg 1200gaaagagcca ccctctcctg cagggccagt cagagtgtta
gcagctactt agcctggtac 1260caacagaaac ctggccaggc tcccaggctc
ctcatctatg atgcatccaa cagggccact 1320ggcatcccag ccaggttcag
tggcagtggg tctgggacag acttcactct caccatcagc 1380agcctagagc
ctgaagattt tgcagtttat tactgtcagc agcgtagcaa ctggccccta
1440ttcactttcg gccctgggac caaagtggat atcaaaacca cgacgccagc
gccgcgacca 1500ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc
tgcgcccaga ggcgtgccgg 1560ccagcggcgg ggggcgcagt gcacacgagg
gggctggact tcgcctgtga tatctacatc 1620tgggcgccct tggccgggac
ttgtggggtc cttctcctgt cactggttat caccctttac 1680tgcaaacggg
gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta
1740caaactactc aagaggaaga cggctgtagc tgccgatttc cagaagaaga
agaaggagga 1800tgtgaactga gagtgaagtt cagcaggagc gcagacgccc
ccgcgtacaa gcagggccag 1860aaccagctct ataacgagct caatctagga
cgaagagagg agtacgacgt tttggacaag 1920agacgtggcc gggaccctga
gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1980ctgtacaacg
aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa
2040ggcgagcgcc ggaggggcaa ggggcacgac ggcctttacc agggtctcag
tacagccacc 2100aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc
21451572250DNAArtificial SequencePD1-CTM-CD28-1C3PSMA-CAR
157atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
ttttgggtgc tggtggtggt tggtggagtc 540ctggcttgct atagcttgct
agtaacagtg gcctttatta ttttctgggt gaggagtaag 600aggagcaggc
tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc
660cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg
ctccgtgaaa 720cagactttga attttgacct tctcaagttg gcgggagacg
tggagtccaa cccagggccg 780atggccttac cagtgaccgc cttgctcctg
ccgctggcct tgctgctcca cgccgccagg 840ccgcaggtgc aactggtgga
gtctggggga ggcgtggtcc agcctgggag gtccctgaga 900ctctcctgtg
cagcctctgg attcaccttc agtagctatg ctatgcactg ggtccgccag
960gctccaggca aggggctgga gtgggtggca gttatatcat atgatggaaa
caataaatac 1020tacgcagact ccgtgaaggg ccgattcacc atctccagag
acaattccaa gaacacgctg 1080tatctgcaaa tgaacagcct gagagctgag
gacacggctg tgtattactg tgcgagagcc 1140gtcccctggg gatcgaggta
ctactactac ggtatggacg tctggggcca agggaccacg 1200gtcaccgtct
cctcaggtgg cggtggctcg ggcggtggtg ggtcgggtgg cggcggatct
1260gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 1320atcacttgcc gggcaagtca gggcattagc agtgctttag
cctggtatca gcagaaatca 1380gggaaagctc ctaagctcct gatctttgat
gcctccagtt tggaaagtgg ggtcccatca 1440aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 1500gaagattttg
caacttatta ctgtcaacag tttaacagtt atcctctcac tttcggcgga
1560gggaccaagg tggagatcaa aaccacgacg ccagcgccgc gaccaccaac
accggcgccc 1620accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt
gccggccagc ggcggggggc 1680gcagtgcaca cgagggggct ggacttcgcc
tgtgatatct acatctgggc gcccttggcc 1740gggacttgtg gggtccttct
cctgtcactg gttatcaccc tttactgcaa acggggcaga 1800aagaaactcc
tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag
1860gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga
actgagagtg 1920aagttcagca ggagcgcaga cgcccccgcg tacaagcagg
gccagaacca gctctataac 1980gagctcaatc taggacgaag agaggagtac
gatgttttgg acaagagacg tggccgggac 2040cctgagatgg ggggaaagcc
gagaaggaag aaccctcagg aaggcctgta caatgaactg 2100cagaaagata
agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg
2160ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga
cacctacgac 2220gcccttcaca tgcaggccct gccccctcgc
22501582235DNAArtificial SequencePD1-CTM-CD28-2A10PSMA-CAR
158atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
ttttgggtgc tggtggtggt tggtggagtc 540ctggcttgct atagcttgct
agtaacagtg gcctttatta ttttctgggt gaggagtaag 600aggagcaggc
tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc
660cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg
ctccgtgaaa 720cagactttga attttgacct tctcaagttg gcgggagacg
tggagtccaa cccagggccg 780atggccttac cagtgaccgc cttgctcctg
ccgctggcct tgctgctcca cgccgccagg 840ccggaggtgc agctggtgca
gtctggagca gaggtgaaaa agcccgggga gtctctgaag 900atctcctgta
agggttctgg atacagcttt accagtaact ggatcggctg ggtgcgccag
960atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc
tgataccaga 1020tacagcccgt ccttccaagg ccaggtcacc atctcagccg
acaagtccat cagcaccgcc 1080tacctgcagt ggagcagcct gaaggcctcg
gacaccgcca tgtattactg tgcgaggcaa 1140actggtttcc tctggtcctc
cgatctctgg ggccgtggca ccctggtcac tgtctcctca 1200ggtggcggtg
gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc
1260cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac
ttgccgggca 1320agtcaggaca ttagcagtgc tttagcctgg tatcaacaga
aaccagggaa agctcctaag 1380ctcctgatct atgatgcctc cagtttggaa
agtggggtcc catcaaggtt cagcggctat 1440ggatctggga cagatttcac
tctcaccatc aacagcctgc agcctgaaga ttttgcaact 1500tattactgtc
aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag
1560atcaaaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat
cgcgtcgcag 1620cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg
ggggcgcagt gcacacgagg 1680gggctggact tcgcctgtga tatctacatc
tgggcgccct tggccgggac ttgtggggtc 1740cttctcctgt cactggttat
caccctttac tgcaaacggg gcagaaagaa actcctgtat 1800atattcaaac
aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc
1860tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt
cagcaggagc 1920gcagacgccc ccgcgtacaa gcagggccag aaccagctct
ataacgagct caatctagga 1980cgaagagagg agtacgatgt tttggacaag
agacgtggcc gggaccctga gatgggggga 2040aagccgagaa ggaagaaccc
tcaggaaggc ctgtacaatg aactgcagaa agataagatg 2100gcggaggcct
acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat
2160ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct
tcacatgcag 2220gccctgcccc ctcgc 22351592241DNAArtificial
SequencePD1-CTM-CD28-2F5PSMA-CAR 159atgcagatcc cacaggcgcc
ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60ccaggatggt tcttagactc
cccagacagg ccctggaacc cccccacctt ctccccagcc 120ctgctcgtgg
tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg
180gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga
caagctggcc 240gccttccccg aggaccgcag ccagcccggc caggactgcc
gcttccgtgt cacacaactg 300cccaacgggc gtgacttcca catgagcgtg
gtcagggccc ggcgcaatga cagcggcacc 360tacctctgtg gggccatctc
cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420gagctcaggg
tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc
480aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt
tggtggagtc 540ctggcttgct atagcttgct agtaacagtg gcctttatta
ttttctgggt gaggagtaag 600aggagcaggc tcctgcacag tgactacatg
aacatgactc cccgccgccc cgggcccacc 660cgcaagcatt accagcccta
tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720cagactttga
attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg
780atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca
cgccgccagg 840ccggaggtgc agctggtgca gtctggagca gaggtgaaaa
agcccgggga gtctctgaag 900atctcctgta agggttctgg atacagtttt
accagcaact ggatcggctg ggtgcgccag 960atgcccggga aaggcctgga
gtggatgggg atcatctatc ctggtgactc tgataccaga 1020tacagcccgt
ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc
1080tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg
tgcgagacaa 1140actggtttcc tctggtcctt cgatctctgg ggccgtggca
ccctggtcac tgtctcctca 1200ggtggcggtg gctcgggcgg tggtgggtcg
ggtggcggcg gatctgccat ccagttgacc 1260cagtctccat cctccctgtc
tgcatctgta ggagacagag tcaccatcac ttgccgggca 1320agtcaggaca
ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag
1380ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt
cagcggcagt 1440ggatctggga cagatttcac tctcaccatc agcagcctgc
agcctgaaga ttttgcaact 1500tattactgtc aacagtttaa tagttacccg
ctcactttcg gcggagggac caaggtggag 1560atcaaaatca aaaccacgac
gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 1620tcgcagcccc
tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac
1680acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc
cgggacttgt 1740ggggtccttc tcctgtcact ggttatcacc ctttactgca
aacggggcag aaagaaactc 1800ctgtatatat tcaaacaacc atttatgaga
ccagtacaaa ctactcaaga ggaagatggc 1860tgtagctgcc gatttccaga
agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1920aggagcgcag
acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat
1980ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga
ccctgagatg 2040gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt
acaatgaact gcagaaagat 2100aagatggcgg aggcctacag tgagattggg
atgaaaggcg agcgccggag gggcaagggg 2160cacgatggcc tttaccaggg
tctcagtaca gccaccaagg acacctacga cgcccttcac 2220atgcaggccc
tgccccctcg c 22411602244DNAArtificial
SequencePD1-CTM-CD28-2C6PSMA-CAR 160atgcagatcc cacaggcgcc
ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60ccaggatggt tcttagactc
cccagacagg ccctggaacc cccccacctt ctccccagcc 120ctgctcgtgg
tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg
180gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga
caagctggcc 240gccttccccg aggaccgcag ccagcccggc caggactgcc
gcttccgtgt cacacaactg 300cccaacgggc gtgacttcca catgagcgtg
gtcagggccc ggcgcaatga cagcggcacc 360tacctctgtg gggccatctc
cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420gagctcaggg
tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc
480aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt
tggtggagtc 540ctggcttgct atagcttgct agtaacagtg gcctttatta
ttttctgggt gaggagtaag 600aggagcaggc tcctgcacag tgactacatg
aacatgactc cccgccgccc cgggcccacc 660cgcaagcatt accagcccta
tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720cagactttga
attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg
780atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca
cgccgccagg 840ccggaggtgc agctggtgca gtctggatca gaggtgaaaa
agcccgggga gtctctgaag 900atctcctgta agggttctgg atacagcttt
accaactact ggatcggctg ggtgcgccag 960atgcccggga aaggcctgga
gtggatgggg atcatctatc ctggtgactc tgataccaga 1020tacagcccgt
ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc
1080tatctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg
tgcgagtccc 1140gggtatacca gcagttggac ttcttttgac tactggggcc
agggaaccct ggtcaccgtc 1200tcctcaggtg gcggtggctc gggcggtggt
gggtcgggtg gcggcggatc tgaaattgtg 1260ttgacacagt ctccagccac
cctgtctttg tctccagggg aaagagccac cctctcctgc 1320agggccagtc
agagtgttag cagctactta gcctggtacc aacagaaacc tggccaggct
1380cccaggctcc tcatctatga tgcatccaac agggccactg gcatcccagc
caggttcagt 1440ggcagtgggt ctgggacaga cttcactctc accatcagca
gcctagagcc tgaagatttt 1500gcagtttatt actgtcagca gcgtagcaac
tggcccctat tcactttcgg ccctgggacc 1560aaagtggata tcaaaaccac
gacgccagcg ccgcgaccac caacaccggc gcccaccatc 1620gcgtcgcagc
ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg
1680cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt
ggccgggact 1740tgtggggtcc ttctcctgtc actggttatc accctttact
gcaaacgggg cagaaagaaa 1800ctcctgtata tattcaaaca accatttatg
agaccagtac aaactactca agaggaagat 1860ggctgtagct gccgatttcc
agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1920agcaggagcg
cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc
1980aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg
ggaccctgag 2040atggggggaa agccgagaag gaagaaccct caggaaggcc
tgtacaatga actgcagaaa 2100gataagatgg cggaggccta cagtgagatt
gggatgaaag gcgagcgccg gaggggcaag 2160gggcacgatg gcctttacca
gggtctcagt acagccacca aggacaccta cgacgccctt 2220cacatgcagg
ccctgccccc tcgc 22441612232DNAArtificial
SequencePD1A132L-PTM-CD28-1C3PSMA-CAR 161atgcagatcc cacaggcgcc
ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60ccaggatggt tcttagactc
cccagacagg ccctggaacc cccccacctt ctccccagcc 120ctgctcgtgg
tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg
180gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga
caagctggcc 240gccttccccg aggaccgcag ccagcccggc caggactgcc
gcttccgtgt cacacaactg 300cccaacgggc gtgacttcca catgagcgtg
gtcagggccc ggcgcaatga cagcggcacc 360tacctctgtg gggccatctc
cctggccccc aagctgcaga tcaaagagag cctgcgggca 420gagctcaggg
tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc
480aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct
gctgggcagc 540ctggtgctgc tagtctgggt cctggccgtc atcaggagta
agaggagcag gctcctgcac 600agtgactaca tgaacatgac tccccgccgc
cccgggccca cccgcaagca ttaccagccc 660tatgccccac cacgcgactt
cgcagcctat cgctccgtga aacagacttt gaattttgac 720cttctcaagt
tggcgggaga cgtggagtcc aacccagggc cgatggcctt accagtgacc
780gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccgcaggt
gcaactggtg 840gagtctgggg gaggcgtggt ccagcctggg aggtccctga
gactctcctg tgcagcctct 900ggattcacct tcagtagcta tgctatgcac
tgggtccgcc aggctccagg caaggggctg 960gagtgggtgg cagttatatc
atatgatgga aacaataaat actacgcaga ctccgtgaag 1020ggccgattca
ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc
1080ctgagagctg aggacacggc tgtgtattac tgtgcgagag ccgtcccctg
gggatcgagg 1140tactactact acggtatgga cgtctggggc caagggacca
cggtcaccgt ctcctcaggt 1200ggcggtggct cgggcggtgg tgggtcgggt
ggcggcggat ctgccatcca gttgacccag 1260tctccatcct ccctgtctgc
atctgtagga gacagagtca ccatcacttg ccgggcaagt 1320cagggcatta
gcagtgcttt agcctggtat cagcagaaat cagggaaagc tcctaagctc
1380ctgatctttg atgcctccag tttggaaagt ggggtcccat caaggttcag
cggcagtgga 1440tctgggacag atttcactct caccatcagc agcctgcagc
ctgaagattt tgcaacttat 1500tactgtcaac agtttaacag ttatcctctc
actttcggcg gagggaccaa ggtggagatc 1560aaaaccacga cgccagcgcc
gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 1620ctgtccctgc
gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg
1680ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg
tggggtcctt 1740ctcctgtcac tggttatcac cctttactgc aaacggggca
gaaagaaact cctgtatata 1800ttcaaacaac catttatgag accagtacaa
actactcaag aggaagatgg ctgtagctgc 1860cgatttccag aagaagaaga
aggaggatgt gaactgagag tgaagttcag caggagcgca 1920gacgcccccg
cgtacaagca gggccagaac cagctctata acgagctcaa tctaggacga
1980agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat
ggggggaaag 2040ccgagaagga agaaccctca ggaaggcctg tacaatgaac
tgcagaaaga taagatggcg 2100gaggcctaca gtgagattgg gatgaaaggc
gagcgccgga ggggcaaggg gcacgatggc 2160ctttaccagg gtctcagtac
agccaccaag gacacctacg acgcccttca catgcaggcc 2220ctgccccctc gc
22321622217DNAArtificial SequencePD1A132L-PTM-CD28-2A10PSMA-CAR
162atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
gttggtgtcg tgggcggcct gctgggcagc 540ctggtgctgc tagtctgggt
cctggccgtc atcaggagta agaggagcag gctcctgcac 600agtgactaca
tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc
660tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt
gaattttgac 720cttctcaagt tggcgggaga cgtggagtcc aacccagggc
cgatggcctt accagtgacc 780gccttgctcc tgccgctggc cttgctgctc
cacgccgcca ggccggaggt gcagctggtg 840cagtctggag cagaggtgaa
aaagcccggg gagtctctga agatctcctg taagggttct 900ggatacagct
ttaccagtaa ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg
960gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc
gtccttccaa 1020ggccaggtca ccatctcagc cgacaagtcc atcagcaccg
cctacctgca gtggagcagc 1080ctgaaggcct cggacaccgc catgtattac
tgtgcgaggc aaactggttt cctctggtcc 1140tccgatctct ggggccgtgg
caccctggtc actgtctcct caggtggcgg tggctcgggc 1200ggtggtgggt
cgggtggcgg cggatctgcc atccagttga cccagtctcc atcctccctg
1260tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagga
cattagcagt 1320gctttagcct ggtatcaaca gaaaccaggg aaagctccta
agctcctgat ctatgatgcc 1380tccagtttgg aaagtggggt cccatcaagg
ttcagcggct atggatctgg gacagatttc 1440actctcacca tcaacagcct
gcagcctgaa gattttgcaa cttattactg tcaacagttt 1500aatagttacc
cgctcacttt cggcggaggg accaaggtgg agatcaaaac cacgacgcca
1560gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc
cctgcgccca 1620gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga
gggggctgga cttcgcctgt 1680gatatctaca tctgggcgcc cttggccggg
acttgtgggg tccttctcct gtcactggtt 1740atcacccttt actgcaaacg
gggcagaaag aaactcctgt atatattcaa acaaccattt 1800atgagaccag
tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa
1860gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc
ccccgcgtac 1920aagcagggcc agaaccagct ctataacgag ctcaatctag
gacgaagaga ggagtacgat 1980gttttggaca agagacgtgg ccgggaccct
gagatggggg gaaagccgag aaggaagaac 2040cctcaggaag gcctgtacaa
tgaactgcag aaagataaga tggcggaggc ctacagtgag 2100attgggatga
aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc
2160agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgc
22171632223DNAArtificial SequencePD1A132L-PTM-CD28-25FPSMA-CAR
163atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
gttggtgtcg tgggcggcct gctgggcagc 540ctggtgctgc tagtctgggt
cctggccgtc atcaggagta agaggagcag gctcctgcac 600agtgactaca
tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc
660tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt
gaattttgac 720cttctcaagt tggcgggaga cgtggagtcc aacccagggc
cgatggcctt accagtgacc 780gccttgctcc tgccgctggc cttgctgctc
cacgccgcca ggccggaggt gcagctggtg 840cagtctggag cagaggtgaa
aaagcccggg gagtctctga agatctcctg taagggttct 900ggatacagtt
ttaccagcaa ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg
960gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc
gtccttccaa 1020ggccaggtca ccatctcagc cgacaagtcc atcagcaccg
cctacctgca gtggaacagc 1080ctgaaggcct cggacaccgc catgtattac
tgtgcgagac aaactggttt cctctggtcc 1140ttcgatctct ggggccgtgg
caccctggtc actgtctcct caggtggcgg tggctcgggc 1200ggtggtgggt
cgggtggcgg cggatctgcc atccagttga cccagtctcc atcctccctg
1260tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagga
cattagcagt 1320gctttagcct ggtatcagca gaaaccgggg aaagctccta
agctcctgat ctatgatgcc 1380tccagtttgg aaagtggggt cccatcaagg
ttcagcggca gtggatctgg gacagatttc 1440actctcacca tcagcagcct
gcagcctgaa gattttgcaa cttattactg tcaacagttt 1500aatagttacc
cgctcacttt cggcggaggg accaaggtgg agatcaaaat caaaaccacg
1560acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc
cctgtccctg 1620cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc
acacgagggg gctggacttc 1680gcctgtgata tctacatctg ggcgcccttg
gccgggactt gtggggtcct tctcctgtca 1740ctggttatca ccctttactg
caaacggggc agaaagaaac tcctgtatat attcaaacaa 1800ccatttatga
gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca
1860gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc
agacgccccc 1920gcgtacaagc agggccagaa ccagctctat aacgagctca
atctaggacg aagagaggag 1980tacgatgttt tggacaagag acgtggccgg
gaccctgaga tggggggaaa gccgagaagg 2040aagaaccctc aggaaggcct
gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 2100agtgagattg
ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag
2160ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc
cctgccccct 2220cgc 22231642226DNAArtificial
SequencePD1A132L-PTM-CD28-2C6PSMA-CAR 164atgcagatcc cacaggcgcc
ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60ccaggatggt tcttagactc
cccagacagg ccctggaacc cccccacctt ctccccagcc 120ctgctcgtgg
tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg
180gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga
caagctggcc 240gccttccccg aggaccgcag ccagcccggc caggactgcc
gcttccgtgt cacacaactg 300cccaacgggc gtgacttcca catgagcgtg
gtcagggccc ggcgcaatga cagcggcacc 360tacctctgtg gggccatctc
cctggccccc aagctgcaga tcaaagagag cctgcgggca 420gagctcaggg
tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc
480aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct
gctgggcagc 540ctggtgctgc tagtctgggt cctggccgtc atcaggagta
agaggagcag gctcctgcac 600agtgactaca tgaacatgac tccccgccgc
cccgggccca cccgcaagca ttaccagccc 660tatgccccac cacgcgactt
cgcagcctat cgctccgtga aacagacttt gaattttgac 720cttctcaagt
tggcgggaga cgtggagtcc aacccagggc cgatggcctt accagtgacc
780gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccggaggt
gcagctggtg 840cagtctggat cagaggtgaa aaagcccggg gagtctctga
agatctcctg taagggttct 900ggatacagct ttaccaacta ctggatcggc
tgggtgcgcc agatgcccgg gaaaggcctg 960gagtggatgg ggatcatcta
tcctggtgac tctgatacca gatacagccc gtccttccaa 1020ggccaggtca
ccatctcagc cgacaagtcc atcagcaccg cctatctgca gtggagcagc
1080ctgaaggcct cggacaccgc catgtattac tgtgcgagtc ccgggtatac
cagcagttgg 1140acttcttttg actactgggg ccagggaacc ctggtcaccg
tctcctcagg tggcggtggc 1200tcgggcggtg gtgggtcggg tggcggcgga
tctgaaattg tgttgacaca gtctccagcc 1260accctgtctt tgtctccagg
ggaaagagcc accctctcct gcagggccag tcagagtgtt 1320agcagctact
tagcctggta ccaacagaaa cctggccagg ctcccaggct cctcatctat
1380gatgcatcca acagggccac tggcatccca gccaggttca gtggcagtgg
gtctgggaca 1440gacttcactc tcaccatcag cagcctagag cctgaagatt
ttgcagttta ttactgtcag 1500cagcgtagca actggcccct attcactttc
ggccctggga ccaaagtgga tatcaaaacc 1560acgacgccag cgccgcgacc
accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 1620ctgcgcccag
aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac
1680ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt
ccttctcctg 1740tcactggtta tcacccttta ctgcaaacgg ggcagaaaga
aactcctgta tatattcaaa 1800caaccattta tgagaccagt acaaactact
caagaggaag atggctgtag ctgccgattt 1860ccagaagaag aagaaggagg
atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 1920cccgcgtaca
agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag
1980gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg
aaagccgaga 2040aggaagaacc ctcaggaagg cctgtacaat gaactgcaga
aagataagat ggcggaggcc 2100tacagtgaga ttgggatgaa aggcgagcgc
cggaggggca aggggcacga tggcctttac 2160cagggtctca gtacagccac
caaggacacc tacgacgccc ttcacatgca ggccctgccc 2220cctcgc
22261652340DNAArtificial SequenceTIM3-CD28-1C3PSMA-CAR
165atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact
acttacaagg 60tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc
ctgcttctac 120accccagccg ccccagggaa cctcgtgccc gtctgctggg
gcaaaggagc ctgtcctgtg 180tttgaatgtg gcaacgtggt gctcaggact
gatgaaaggg atgtgaatta ttggacatcc 240agatactggc taaatgggga
tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300actctagcag
acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat
360gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc
accgactcgg 420cagagagact tcactgcagc ctttccaagg atgcttacca
ccaggggaca tggcccagca 480gagacacaga cactggggag cctccctgac
ataaatctaa cacaaatatc cacattggcc 540aatgagttac gggactctag
gttggccaat gacttacggg actccggagc aaccatcaga 600ttttgggtgc
tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg
660gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag
tgactacatg 720aacatgactc cccgccgccc cgggcccacc cgcaagcatt
accagcccta tgccccacca 780cgcgacttcg cagcctatcg ctccgtgaaa
cagactttga attttgacct tctcaagttg 840gcgggagacg tggagtccaa
cccagggccg atggccttac cagtgaccgc cttgctcctg 900ccgctggcct
tgctgctcca cgccgccagg ccgcaggtgc aactggtgga gtctggggga
960ggcgtggtcc agcctgggag gtccctgaga ctctcctgtg cagcctctgg
attcaccttc 1020agtagctatg ctatgcactg ggtccgccag gctccaggca
aggggctgga gtgggtggca 1080gttatatcat atgatggaaa caataaatac
tacgcagact ccgtgaaggg ccgattcacc 1140atctccagag acaattccaa
gaacacgctg tatctgcaaa tgaacagcct gagagctgag 1200gacacggctg
tgtattactg tgcgagagcc gtcccctggg gatcgaggta ctactactac
1260ggtatggacg tctggggcca agggaccacg gtcaccgtct cctcaggtgg
cggtggctcg 1320ggcggtggtg ggtcgggtgg cggcggatct gccatccagt
tgacccagtc tccatcctcc 1380ctgtctgcat ctgtaggaga cagagtcacc
atcacttgcc gggcaagtca gggcattagc 1440agtgctttag cctggtatca
gcagaaatca gggaaagctc ctaagctcct gatctttgat 1500gcctccagtt
tggaaagtgg ggtcccatca aggttcagcg gcagtggatc tgggacagat
1560ttcactctca ccatcagcag cctgcagcct gaagattttg caacttatta
ctgtcaacag 1620tttaacagtt atcctctcac tttcggcgga gggaccaagg
tggagatcaa aaccacgacg 1680ccagcgccgc gaccaccaac accggcgccc
accatcgcgt cgcagcccct gtccctgcgc 1740ccagaggcgt gccggccagc
ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 1800tgtgatatct
acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg
1860gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt
caaacaacca 1920tttatgagac cagtacaaac tactcaagag gaagacggct
gtagctgccg atttccagaa 1980gaagaagaag gaggatgtga actgagagtg
aagttcagca ggagcgcaga cgcccccgcg 2040tacaagcagg gccagaacca
gctctataac gagctcaatc taggacgaag agaggagtac 2100gacgttttgg
acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag
2160aaccctcagg aaggcctgta caacgaactg cagaaagata agatggcgga
ggcctacagt 2220gagattggga tgaaaggcga gcgccggagg ggcaaggggc
acgacggcct ttaccagggt 2280ctcagtacag ccaccaagga cacctacgac
gcccttcaca tgcaggccct gccccctcgc 23401662325DNAArtificial
SequenceTIM3-CD28-2A10PSMA-CAR 166atgttttcac atcttccctt tgactgtgtc
ctgctgctgc tgctgctact acttacaagg 60tcctcagaag tggaatacag agcggaggtc
ggtcagaatg cctatctgcc ctgcttctac 120accccagccg ccccagggaa
cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180tttgaatgtg
gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc
240agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat
agagaatgtg 300actctagcag acagtgggat ctactgctgc cgaatccaaa
tcccaggcat aatgaatgat 360gaaaaattta acctgaagtt ggtcatcaaa
ccagccaagg tcacccctgc accgactcgg 420cagagagact tcactgcagc
ctttccaagg atgcttacca ccaggggaca tggcccagca 480gagacacaga
cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc
540aatgagttac gggactctag gttggccaat gacttacggg actccggagc
aaccatcaga 600ttttgggtgc tggtggtggt tggtggagtc ctggcttgct
atagcttact agtaacagtg 660gcctttatta ttttctgggt gaggagtaag
aggagcaggc tcctgcacag tgactacatg 720aacatgactc cccgccgccc
cgggcccacc cgcaagcatt accagcccta tgccccacca 780cgcgacttcg
cagcctatcg ctccgtgaaa cagactttga attttgacct tctcaagttg
840gcgggagacg tggagtccaa cccagggccg atggccttac cagtgaccgc
cttgctcctg 900ccgctggcct tgctgctcca cgccgccagg ccggaggtgc
agctggtgca gtctggagca 960gaggtgaaaa agcccgggga gtctctgaag
atctcctgta agggttctgg atacagcttt 1020accagtaact ggatcggctg
ggtgcgccag atgcccggga aaggcctgga gtggatgggg 1080atcatctatc
ctggtgactc tgataccaga tacagcccgt ccttccaagg ccaggtcacc
1140atctcagccg
acaagtccat cagcaccgcc tacctgcagt ggagcagcct gaaggcctcg
1200gacaccgcca tgtattactg tgcgaggcaa actggtttcc tctggtcctc
cgatctctgg 1260ggccgtggca ccctggtcac tgtctcctca ggtggcggtg
gctcgggcgg tggtgggtcg 1320ggtggcggcg gatctgccat ccagttgacc
cagtctccat cctccctgtc tgcatctgta 1380ggagacagag tcaccatcac
ttgccgggca agtcaggaca ttagcagtgc tttagcctgg 1440tatcaacaga
aaccagggaa agctcctaag ctcctgatct atgatgcctc cagtttggaa
1500agtggggtcc catcaaggtt cagcggctat ggatctggga cagatttcac
tctcaccatc 1560aacagcctgc agcctgaaga ttttgcaact tattactgtc
aacagtttaa tagttacccg 1620ctcactttcg gcggagggac caaggtggag
atcaaaacca cgacgccagc gccgcgacca 1680ccaacaccgg cgcccaccat
cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 1740ccagcggcgg
ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc
1800tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat
caccctttac 1860tgcaaacggg gcagaaagaa actcctgtat atattcaaac
aaccatttat gagaccagta 1920caaactactc aagaggaaga tggctgtagc
tgccgatttc cagaagaaga agaaggagga 1980tgtgaactga gagtgaagtt
cagcaggagc gcagacgccc ccgcgtacaa gcagggccag 2040aaccagctct
ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag
2100agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc
tcaggaaggc 2160ctgtacaatg aactgcagaa agataagatg gcggaggcct
acagtgagat tgggatgaaa 2220ggcgagcgcc ggaggggcaa ggggcacgat
ggcctttacc agggtctcag tacagccacc 2280aaggacacct acgacgccct
tcacatgcag gccctgcccc ctcgc 23251672331DNAArtificial
SequenceTIM3-CD28-25FPSMA-CAR 167atgttttcac atcttccctt tgactgtgtc
ctgctgctgc tgctgctact acttacaagg 60tcctcagaag tggaatacag agcggaggtc
ggtcagaatg cctatctgcc ctgcttctac 120accccagccg ccccagggaa
cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180tttgaatgtg
gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc
240agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat
agagaatgtg 300actctagcag acagtgggat ctactgctgc cgaatccaaa
tcccaggcat aatgaatgat 360gaaaaattta acctgaagtt ggtcatcaaa
ccagccaagg tcacccctgc accgactcgg 420cagagagact tcactgcagc
ctttccaagg atgcttacca ccaggggaca tggcccagca 480gagacacaga
cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc
540aatgagttac gggactctag gttggccaat gacttacggg actccggagc
aaccatcaga 600ttttgggtgc tggtggtggt tggtggagtc ctggcttgct
atagcttact agtaacagtg 660gcctttatta ttttctgggt gaggagtaag
aggagcaggc tcctgcacag tgactacatg 720aacatgactc cccgccgccc
cgggcccacc cgcaagcatt accagcccta tgccccacca 780cgcgacttcg
cagcctatcg ctccgtgaaa cagactttga attttgacct tctcaagttg
840gcgggagacg tggagtccaa cccagggccg atggccttac cagtgaccgc
cttgctcctg 900ccgctggcct tgctgctcca cgccgccagg ccggaggtgc
agctggtgca gtctggagca 960gaggtgaaaa agcccgggga gtctctgaag
atctcctgta agggttctgg atacagtttt 1020accagcaact ggatcggctg
ggtgcgccag atgcccggga aaggcctgga gtggatgggg 1080atcatctatc
ctggtgactc tgataccaga tacagcccgt ccttccaagg ccaggtcacc
1140atctcagccg acaagtccat cagcaccgcc tacctgcagt ggaacagcct
gaaggcctcg 1200gacaccgcca tgtattactg tgcgagacaa actggtttcc
tctggtcctt cgatctctgg 1260ggccgtggca ccctggtcac tgtctcctca
ggtggcggtg gctcgggcgg tggtgggtcg 1320ggtggcggcg gatctgccat
ccagttgacc cagtctccat cctccctgtc tgcatctgta 1380ggagacagag
tcaccatcac ttgccgggca agtcaggaca ttagcagtgc tttagcctgg
1440tatcagcaga aaccggggaa agctcctaag ctcctgatct atgatgcctc
cagtttggaa 1500agtggggtcc catcaaggtt cagcggcagt ggatctggga
cagatttcac tctcaccatc 1560agcagcctgc agcctgaaga ttttgcaact
tattactgtc aacagtttaa tagttacccg 1620ctcactttcg gcggagggac
caaggtggag atcaaaatca aaaccacgac gccagcgccg 1680cgaccaccaa
caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg
1740tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc
ctgtgatatc 1800tacatctggg cgcccttggc cgggacttgt ggggtccttc
tcctgtcact ggttatcacc 1860ctttactgca aacggggcag aaagaaactc
ctgtatatat tcaaacaacc atttatgaga 1920ccagtacaaa ctactcaaga
ggaagacggc tgtagctgcc gatttccaga agaagaagaa 1980ggaggatgtg
aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag
2040ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta
cgacgttttg 2100gacaagagac gtggccggga ccctgagatg gggggaaagc
cgagaaggaa gaaccctcag 2160gaaggcctgt acaacgaact gcagaaagat
aagatggcgg aggcctacag tgagattggg 2220atgaaaggcg agcgccggag
gggcaagggg cacgacggcc tttaccaggg tctcagtaca 2280gccaccaagg
acacctacga cgcccttcac atgcaggccc tgccccctcg c
23311682334DNAArtificial SequenceTIM3-CD28-2C6PSMA-CAR
168atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact
acttacaagg 60tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc
ctgcttctac 120accccagccg ccccagggaa cctcgtgccc gtctgctggg
gcaaaggagc ctgtcctgtg 180tttgaatgtg gcaacgtggt gctcaggact
gatgaaaggg atgtgaatta ttggacatcc 240agatactggc taaatgggga
tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300actctagcag
acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat
360gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc
accgactcgg 420cagagagact tcactgcagc ctttccaagg atgcttacca
ccaggggaca tggcccagca 480gagacacaga cactggggag cctccctgac
ataaatctaa cacaaatatc cacattggcc 540aatgagttac gggactctag
gttggccaat gacttacggg actccggagc aaccatcaga 600ttttgggtgc
tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg
660gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag
tgactacatg 720aacatgactc cccgccgccc cgggcccacc cgcaagcatt
accagcccta tgccccacca 780cgcgacttcg cagcctatcg ctccgtgaaa
cagactttga attttgacct tctcaagttg 840gcgggagacg tggagtccaa
cccagggccg atggccttac cagtgaccgc cttgctcctg 900ccgctggcct
tgctgctcca cgccgccagg ccggaggtgc agctggtgca gtctggatca
960gaggtgaaaa agcccgggga gtctctgaag atctcctgta agggttctgg
atacagcttt 1020accaactact ggatcggctg ggtgcgccag atgcccggga
aaggcctgga gtggatgggg 1080atcatctatc ctggtgactc tgataccaga
tacagcccgt ccttccaagg ccaggtcacc 1140atctcagccg acaagtccat
cagcaccgcc tatctgcagt ggagcagcct gaaggcctcg 1200gacaccgcca
tgtattactg tgcgagtccc gggtatacca gcagttggac ttcttttgac
1260tactggggcc agggaaccct ggtcaccgtc tcctcaggtg gcggtggctc
gggcggtggt 1320gggtcgggtg gcggcggatc tgaaattgtg ttgacacagt
ctccagccac cctgtctttg 1380tctccagggg aaagagccac cctctcctgc
agggccagtc agagtgttag cagctactta 1440gcctggtacc aacagaaacc
tggccaggct cccaggctcc tcatctatga tgcatccaac 1500agggccactg
gcatcccagc caggttcagt ggcagtgggt ctgggacaga cttcactctc
1560accatcagca gcctagagcc tgaagatttt gcagtttatt actgtcagca
gcgtagcaac 1620tggcccctat tcactttcgg ccctgggacc aaagtggata
tcaaaaccac gacgccagcg 1680ccgcgaccac caacaccggc gcccaccatc
gcgtcgcagc ccctgtccct gcgcccagag 1740gcgtgccggc cagcggcggg
gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 1800atctacatct
gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc
1860accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca
accatttatg 1920agaccagtac aaactactca agaggaagac ggctgtagct
gccgatttcc agaagaagaa 1980gaaggaggat gtgaactgag agtgaagttc
agcaggagcg cagacgcccc cgcgtacaag 2040cagggccaga accagctcta
taacgagctc aatctaggac gaagagagga gtacgacgtt 2100ttggacaaga
gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct
2160caggaaggcc tgtacaacga actgcagaaa gataagatgg cggaggccta
cagtgagatt 2220gggatgaaag gcgagcgccg gaggggcaag gggcacgacg
gcctttacca gggtctcagt 2280acagccacca aggacaccta cgacgccctt
cacatgcagg ccctgccccc tcgc 2334169821DNAArtificial Sequence1C3
169atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca
cgccgccagg 60ccgcaggtgc aactggtgga gtctggggga ggcgtggtcc agcctgggag
gtccctgaga 120ctctcctgtg cagcctctgg attcaccttc agtagctatg
ctatgcactg ggtccgccag 180gctccaggca aggggctgga gtgggtggca
gttatatcat atgatggaaa caataaatac 240tacgcagact ccgtgaaggg
ccgattcacc atctccagag acaattccaa gaacacgctg 300tatctgcaaa
tgaacagcct gagagctgag gacacggctg tgtattactg tgcgagagcc
360gtcccctggg gatcgaggta ctactactac ggtatggacg tctggggcca
agggaccacg 420gtcaccgtct cctcaggtgg cggtggctcg ggcggtggtg
ggtcgggtgg cggcggatct 480gccatccagt tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 540atcacttgcc gggcaagtca
gggcattagc agtgctttag cctggtatca gcagaaatca 600gggaaagctc
ctaagctcct gatctttgat gcctccagtt tggaaagtgg ggtcccatca
660aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 720gaagattttg caacttatta ctgtcaacag tttaacagtt
atcctctcac tttcggcgga 780gggaccaagg tggagatcaa aaccacgacg
ccagcgccgc g 821170806DNAArtificial Sequence2A10 170atggccttac
cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggaggtgc
agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag
120atctcctgta agggttctgg atacagcttt accagtaact ggatcggctg
ggtgcgccag 180atgcccggga aaggcctgga gtggatgggg atcatctatc
ctggtgactc tgataccaga 240tacagcccgt ccttccaagg ccaggtcacc
atctcagccg acaagtccat cagcaccgcc 300tacctgcagt ggagcagcct
gaaggcctcg gacaccgcca tgtattactg tgcgaggcaa 360actggtttcc
tctggtcctc cgatctctgg ggccgtggca ccctggtcac tgtctcctca
420ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat
ccagttgacc 480cagtctccat cctccctgtc tgcatctgta ggagacagag
tcaccatcac ttgccgggca 540agtcaggaca ttagcagtgc tttagcctgg
tatcaacaga aaccagggaa agctcctaag 600ctcctgatct atgatgcctc
cagtttggaa agtggggtcc catcaaggtt cagcggctat 660ggatctggga
cagatttcac tctcaccatc aacagcctgc agcctgaaga ttttgcaact
720tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac
caaggtggag 780atcaaaacca cgacgccagc gccgcg 806171812DNAArtificial
Sequence2F5 171atggccttac cagtgaccgc cttgctcctg ccgctggcct
tgctgctcca cgccgccagg 60ccggaggtgc agctggtgca gtctggagca gaggtgaaaa
agcccgggga gtctctgaag 120atctcctgta agggttctgg atacagtttt
accagcaact ggatcggctg ggtgcgccag 180atgcccggga aaggcctgga
gtggatgggg atcatctatc ctggtgactc tgataccaga 240tacagcccgt
ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc
300tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg
tgcgagacaa 360actggtttcc tctggtcctt cgatctctgg ggccgtggca
ccctggtcac tgtctcctca 420ggtggcggtg gctcgggcgg tggtgggtcg
ggtggcggcg gatctgccat ccagttgacc 480cagtctccat cctccctgtc
tgcatctgta ggagacagag tcaccatcac ttgccgggca 540agtcaggaca
ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag
600ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt
cagcggcagt 660ggatctggga cagatttcac tctcaccatc agcagcctgc
agcctgaaga ttttgcaact 720tattactgtc aacagtttaa tagttacccg
ctcactttcg gcggagggac caaggtggag 780atcaaaatca aaaccacgac
gccagcgccg cg 812172815DNAArtificial Sequence2C6 172atggccttac
cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggaggtgc
agctggtgca gtctggatca gaggtgaaaa agcccgggga gtctctgaag
120atctcctgta agggttctgg atacagcttt accaactact ggatcggctg
ggtgcgccag 180atgcccggga aaggcctgga gtggatgggg atcatctatc
ctggtgactc tgataccaga 240tacagcccgt ccttccaagg ccaggtcacc
atctcagccg acaagtccat cagcaccgcc 300tatctgcagt ggagcagcct
gaaggcctcg gacaccgcca tgtattactg tgcgagtccc 360gggtatacca
gcagttggac ttcttttgac tactggggcc agggaaccct ggtcaccgtc
420tcctcaggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc
tgaaattgtg 480ttgacacagt ctccagccac cctgtctttg tctccagggg
aaagagccac cctctcctgc 540agggccagtc agagtgttag cagctactta
gcctggtacc aacagaaacc tggccaggct 600cccaggctcc tcatctatga
tgcatccaac agggccactg gcatcccagc caggttcagt 660ggcagtgggt
ctgggacaga cttcactctc accatcagca gcctagagcc tgaagatttt
720gcagtttatt actgtcagca gcgtagcaac tggcccctat tcactttcgg
ccctgggacc 780aaagtggata tcaaaaccac gacgccagcg ccgcg
815173780DNAArtificial SequencePD1.CD28-F2A 173atgcagatcc
cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60ccaggatggt
tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc
120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc
caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg agccccagca
accagacgga caagctggcc 240gccttccccg aggaccgcag ccagcccggc
caggactgcc gcttccgtgt cacacaactg 300cccaacgggc gtgacttcca
catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360tacctctgtg
gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca
420gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag
cccctcaccc 480aggccagccg gccagttcca aaccctggtg ttttgggtgc
tggtggtggt tggtggagtc 540ctggcttgct atagcttgct agtaacagtg
gcctttatta ttttctgggt gaggagtaag 600aggagcaggc tcctgcacag
tgactacatg aacatgactc cccgccgccc cgggcccacc 660cgcaagcatt
accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa
720cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa
cccagggccg 780174762DNAArtificial SequencePD1A132L-PTM.CD28-F2A
174atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
gttggtgtcg tgggcggcct gctgggcagc 540ctggtgctgc tagtctgggt
cctggccgtc atcaggagta agaggagcag gctcctgcac 600agtgactaca
tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc
660tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt
gaattttgac 720cttctcaagt tggcgggaga cgtggagtcc aacccagggc cg
762175681DNAArtificial SequencednTGFRBII-T2A 175atgggtcggg
ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60gccagcacga
tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac
120aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag
attttccacc 180tgtgacaacc agaaatcctg catgagcaac tgcagcatca
cctccatctg tgagaagcca 240caggaagtct gtgtggctgt atggagaaag
aatgacgaga acataacact agagacagtt 300tgccatgacc ccaagctccc
ctaccatgac tttattctgg aagatgctgc ttctccaaag 360tgcattatga
aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct
420gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa
tcctgacttg 480ttgctagtca tatttcaagt gacaggcatc agcctcctgc
caccactggg agttgccata 540tctgtcatca tcatcttcta ctgctaccgc
gttaaccggc agcagaagct gagttcatcc 600ggaagatctg gcggcggaga
gggcagagga agtcttctaa catgcggtga cgtggaggag 660aatcccggcc
ctagagccac c 68117620DNAArtificial Sequenceprimer 176aggaagtctc
aaagtgccct 2017720DNAArtificial Sequenceprimer 177gaacaacagc
tgctccactc 2017825DNAArtificial Sequenceprimer 178gctacactga
gcaccaggtg gtctc 2517925DNAArtificial Sequenceprimer 179cccagcagtg
agggtctctc tcttc 25180711DNAArtificial SequenceJ591 PSMA scFv
180gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga
cagggtcagc 60atcatctgta aggccagtca agatgtgggt actgctgtag actggtatca
acagaaacca 120ggacaatctc ctaaactact gatttattgg gcatccactc
ggcacactgg agtccctgat 180cgcttcacag gcagtggatc tgggacagac
ttcactctca ccattactaa cgttcagtct 240gaagacttgg cagattattt
ctgtcagcaa tataacagct atcctctcac gttcggtgct 300gggaccatgc
tggacctgaa aggaggcgga ggatctggcg gcggaggaag ttctggcgga
360ggcagcgagg tgcagctgca gcagagcgga cccgagctcg tgaagcctgg
aacaagcgtg 420cggatcagct gcaagaccag cggctacacc ttcaccgagt
acaccatcca ctgggtcaag 480cagtcccacg gcaagagcct ggagtggatc
ggcaatatca accccaacaa cggcggcacc 540acctacaacc agaagttcga
ggacaaggcc accctgaccg tggacaagag cagcagcacc 600gcctacatgg
aactgcggag cctgaccagc gaggacagcg ccgtgtacta ttgtgccgcc
660ggttggaact tcgactactg gggccagggc acaaccctga cagtgtctag c
711181108PRTArtificial SequenceJ591 VL 181Asp Ile Val Met Thr Gln
Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile
Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala
Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Ala Ile Thr Asn Val Gln Ser65 70 75
80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg 100
105182115PRTArtificial SequenceJ591 VH 182Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10 15Ser Val Arg Ile Ser
Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile His Trp
Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Asn Ile
Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60Glu Asp
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
Thr 100 105 110Val Ser Ser 115183115PRTArtificial SequenceHumanized
PSMA VH consensusmisc_feature(9)..(9)X is A or
Pmisc_feature(11)..(11)X is V or Lmisc_feature(24)..(24)X is A or
Tmisc_feature(38)..(38)X is R or Kmisc_feature(41)..(41)X is P or
Hmisc_feature(55)..(55)X is N or Qmisc_feature(68)..(68)X is V or
Amisc_feature(70)..(70)X is I or L 183Glu Val Gln Leu Val Gln Ser
Gly Xaa Glu Xaa Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Xaa Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val
Xaa Gln Ala Xaa Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile Asn
Pro Asn Xaa Gly Gly Thr Thr Tyr
Asn Gln Lys Phe 50 55 60Glu Asp Arg Xaa Thr Xaa Thr Val Asp Lys Ser
Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr
Trp Gly Gln Gly Thr Thr Val Thr 100 105 110Val Ser Ser
115184107PRTArtificial SequenceHumanized PSMA VL
consensusmisc_feature(3)..(3)X is Q or Vmisc_feature(10)..(10)X is
T or Fmisc_feature(63)..(63)X is S or Tmisc_feature(80)..(80)X is P
or Smisc_feature(85)..(85)X is V or Dmisc_feature(87)..(87)X is Y
or Fmisc_feature(103)..(103)X is K or M 184Asp Ile Xaa Met Thr Gln
Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala
Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Xaa Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Xaa65 70 75
80Glu Asp Phe Ala Xaa Tyr Xaa Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Xaa Val Asp Ile Lys 100
105185115PRTArtificial SequenceHumanized PSMA VH 185Glu Val Gln Leu
Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile
His Trp Val Lys Gln Ala His Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly
Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55
60Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Val Thr 100 105 110Val Ser Ser 115186107PRTArtificial
SequenceHumanized PSMA VL 186Asp Ile Val Met Thr Gln Ser Pro Ser
Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Ser65 70 75 80Glu Asp Phe
Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe
Gly Gln Gly Thr Met Val Asp Ile Lys 100 105187115PRTArtificial
SequenceHumanized PSMA VH 187Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Thr
Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val Lys Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile Asn Pro Asn
Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60Glu Asp Arg Ala Thr
Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105
110Val Ser Ser 115188107PRTArtificial SequenceHumanized PSMA VL
188Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr
Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Gln Ser65 70 75 80Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln
Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Asp
Ile Lys 100 105189115PRTArtificial SequenceHumanized PSMA VH 189Glu
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln
Lys Phe 50 55 60Glu Asp Arg Ala Thr Ile Thr Val Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Val Thr 100 105 110Val Ser Ser
115190107PRTArtificial SequenceHumanized PSMA VL 190Asp Ile Val Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro65
70 75 80Glu Asp Phe Ala Asp Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro
Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100
105191115PRTArtificial SequenceHumanized PSMA VH 191Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly
Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55
60Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Val Thr 100 105 110Val Ser Ser 115192107PRTArtificial
SequenceHumanized PSMA VL 192Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Asp Ile Lys 100 105193115PRTArtificial
SequenceHumanized PSMA VH 193Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile Asn Pro Asn
Gln Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60Glu Asp Arg Val Thr
Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105
110Val Ser Ser 115194115PRTArtificial SequenceHumanized PSMA VH
consensusmisc_feature(9)..(9)X is A or Pmisc_feature(11)..(11)X is
V or Lmisc_feature(24)..(24)X is A or Tmisc_feature(37)..(37)X is R
or Kmisc_feature(38)..(38)Xaa can be any naturally occurring amino
acidmisc_feature(40)..(40)X is P or Hmisc_feature(41)..(41)Xaa can
be any naturally occurring amino acidmisc_feature(54)..(54)X is N
or Qmisc_feature(55)..(55)Xaa can be any naturally occurring amino
acidmisc_feature(68)..(68)X is V or Amisc_feature(70)..(70)X is I
or Lmisc_feature(86)..(86)Xaa can be any naturally occurring amino
acidmisc_feature(98)..(102)is AYWLF, GGWTF, or GAWTM 194Glu Val Gln
Leu Val Gln Ser Gly Xaa Glu Xaa Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Xaa Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr
Ile His Trp Val Xaa Gln Ala Xaa Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Asn Ile Asn Pro Asn Xaa Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60Glu Asp Arg Xaa Thr Xaa Thr Val Asp Lys Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Xaa Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Xaa Xaa Xaa Xaa Xaa Asp Tyr Trp Gly Gln Gly
Thr Thr Val Thr 100 105 110Val Ser Ser 115195106PRTArtificial
SequenceHumanized PSMA VL consensusmisc_feature(3)..(3)X is Q or
Vmisc_feature(10)..(10)X is T or Fmisc_feature(63)..(63)X is S or
Tmisc_feature(80)..(80)X is P or Smisc_feature(85)..(85)X is V or
Dmisc_feature(87)..(87)X is Y or Fmisc_feature(91)..(95)is FTRYP or
YNAYSmisc_feature(103)..(103)X is K or M 195Asp Ile Xaa Met Thr Gln
Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala
Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Xaa Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Xaa65 70 75
80Glu Asp Phe Ala Xaa Tyr Xaa Cys Gln Gln Xaa Xaa Xaa Xaa Xaa Leu
85 90 95Thr Phe Gly Gln Gly Thr Val Asp Ile Lys 100
105196115PRTArtificial SequenceHumanized PSMA VH 196Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly
Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55
60Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Tyr Trp Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Val Thr 100 105 110Val Ser Ser 115197107PRTArtificial
SequenceHumanized PSMA VL 197Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Asp Ile Lys 100 105198115PRTArtificial
SequenceHumanized PSMA VH 198Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Asn Ile Asn Pro Asn
Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60Glu Asp Arg Val Thr
Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Gly
Gly Trp Thr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105
110Val Ser Ser 115199107PRTArtificial SequenceHumanized PSMA VL
199Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr
Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Phe Thr Arg Tyr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Asp
Ile Lys 100 105200115PRTArtificial SequenceHumanized PSMA VH 200Glu
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln
Lys Phe 50 55 60Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Gly Ala Trp Thr Met Asp Tyr Trp Gly
Gln Gly Thr Thr Val Thr 100 105 110Val Ser Ser
115201107PRTArtificial SequenceHumanized PSMA VL 201Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro65
70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ala Tyr Ser
Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100
105202115PRTArtificial SequenceHumanized PSMA VH 202Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly
Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55
60Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Pro Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Val Thr 100 105 110Val Ser Ser 11520335PRTArtificial SequenceICOS
ICD 203Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu
Tyr1 5 10 15Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg
Leu
Thr Asp 20 25 30Val Thr Leu 35204105DNAArtificial SequenceICOS ICD
204acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat
gttcatgaga 60gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta
105205148PRTArtificial SequenceICOS CD3zeta ICD 205Thr Lys Lys Lys
Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr1 5 10 15Met Phe Met
Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp 20 25 30Val Thr
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 35 40 45Gln
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 50 55
60Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met65
70 75 80Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn 85 90 95Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
Gly Met 100 105 110Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
Leu Tyr Gln Gly 115 120 125Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
Ala Leu His Met Gln Ala 130 135 140Leu Pro Pro
Arg145206444DNAArtificial SequenceICOS CD3zeta ICD 206acaaaaaaga
agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga 60gcagtgaaca
cagccaaaaa atccagactc acagatgtga ccctaagagt gaagttcagc
120aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa
cgagctcaat 180ctaggacgaa gagaggagta cgatgttttg gacaagagac
gtggccggga ccctgagatg 240gggggaaagc cgcagagaag gaagaaccct
caggaaggcc tgtacaatga actgcagaaa 300gataagatgg cggaggccta
cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 360gggcacgatg
gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt
420cacatgcagg ccctgccccc tcgc 444207148PRTArtificial
Sequencevariant ICOS CD3zeta ICD 207Thr Lys Lys Lys Tyr Ser Ser Ser
Val His Asp Pro Asn Gly Glu Tyr1 5 10 15Met Asn Met Arg Ala Val Asn
Thr Ala Lys Lys Ser Arg Leu Thr Asp 20 25 30Val Thr Leu Arg Val Lys
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 35 40 45Gln Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 50 55 60Glu Glu Tyr Asp
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met65 70 75 80Gly Gly
Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 85 90 95Glu
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 100 105
110Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
115 120 125Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
Gln Ala 130 135 140Leu Pro Pro Arg145208444DNAArtificial
Sequencevariant ICOS CD3zeta ICD 208acaaaaaaga agtattcatc
cagtgtgcac gaccctaacg gtgaatacat gaacatgaga 60gcagtgaaca cagccaaaaa
atccagactc acagatgtga ccctaagagt gaagttcagc 120aggagcgcag
acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat
180ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga
ccctgagatg 240gggggaaagc cgcagagaag gaagaaccct caggaaggcc
tgtacaatga actgcagaaa 300gataagatgg cggaggccta cagtgagatt
gggatgaaag gcgagcgccg gaggggcaag 360gggcacgatg gcctttacca
gggtctcagt acagccacca aggacaccta cgacgccctt 420cacatgcagg
ccctgccccc tcgc 444209481PRTArtificial Sequence2F5 human PSMA-CAR
ICOS CD3z 209Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val 20 25 30Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys
Lys Gly Ser Gly Tyr 35 40 45Ser Phe Thr Ser Asn Trp Ile Gly Trp Val
Arg Gln Met Pro Gly Lys 50 55 60Gly Leu Glu Trp Met Gly Ile Ile Tyr
Pro Gly Asp Ser Asp Thr Arg65 70 75 80Tyr Ser Pro Ser Phe Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser 85 90 95Ile Ser Thr Ala Tyr Leu
Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr 100 105 110Ala Met Tyr Tyr
Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp 115 120 125Leu Trp
Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 130 135
140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu
Thr145 150 155 160Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile 165 170 175Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser
Ala Leu Ala Trp Tyr Gln 180 185 190Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr Asp Ala Ser Ser 195 200 205Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 210 215 220Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr225 230 235 240Tyr
Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly 245 250
255Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
Arg Pro 275 280 285Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
Thr Arg Gly Leu 290 295 300Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile
Gly Cys Ala Ala Phe Val305 310 315 320Val Val Cys Ile Leu Gly Cys
Ile Leu Ile Cys Trp Leu Thr Lys Lys 325 330 335Lys Tyr Ser Ser Ser
Val His Asp Pro Asn Gly Glu Tyr Met Phe Met 340 345 350Arg Ala Val
Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu 355 360 365Arg
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 370 375
380Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
Tyr385 390 395 400Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
Met Gly Gly Lys 405 410 415Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly
Leu Tyr Asn Glu Leu Gln 420 425 430Lys Asp Lys Met Ala Glu Ala Tyr
Ser Glu Ile Gly Met Lys Gly Glu 435 440 445Arg Arg Arg Gly Lys Gly
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 450 455 460Ala Thr Lys Asp
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro465 470 475
480Arg2101443DNAArtificial Sequence2F5 human PSMA-CAR ICOS CD3z
210atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca
cgccgccagg 60ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga
gtctctgaag 120atctcctgta agggttctgg atacagtttt accagcaact
ggatcggctg ggtgcgccag 180atgcccggga aaggcctgga gtggatgggg
atcatctatc ctggtgactc tgataccaga 240tacagcccgt ccttccaagg
ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300tacctgcagt
ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa
360actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac
tgtctcctca 420ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg
gatctgccat ccagttgacc 480cagtctccat cctccctgtc tgcatctgta
ggagacagag tcaccatcac ttgccgggca 540agtcaggaca ttagcagtgc
tttagcctgg tatcagcaga aaccggggaa agctcctaag 600ctcctgatct
atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt
660ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga
ttttgcaact 720tattactgtc aacagtttaa tagttacccg ctcactttcg
gcggagggac caaggtggag 780atcaaaatca aaaccacgac gccagcgccg
cgaccaccaa caccggcgcc caccatcgcg 840tcgcagcccc tgtccctgcg
cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900acgagggggc
tggacttcgc ctgtgatttc tggttaccca taggatgtgc agcctttgtt
960gtagtctgca ttttgggatg catacttatt tgttggctta caaaaaagaa
gtattcatcc 1020agtgtgcacg accctaacgg tgaatacatg ttcatgagag
cagtgaacac agccaaaaaa 1080tccagactca cagatgtgac cctaagagtg
aagttcagca ggagcgcaga cgcccccgcg 1140taccagcagg gccagaacca
gctctataac gagctcaatc taggacgaag agaggagtac 1200gatgttttgg
acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg
1260aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc
ggaggcctac 1320agtgagattg ggatgaaagg cgagcgccgg aggggcaagg
ggcacgatgg cctttaccag 1380ggtctcagta cagccaccaa ggacacctac
gacgcccttc acatgcaggc cctgccccct 1440cgc 1443211481PRTArtificial
Sequence2F5 human PSMA-CAR varICOS CD3z 211Met Ala Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30Lys Lys Pro Gly
Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr 35 40 45Ser Phe Thr
Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys 50 55 60Gly Leu
Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg65 70 75
80Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
85 90 95Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp
Thr 100 105 110Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp
Ser Phe Asp 115 120 125Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Ala Ile Gln Leu Thr145 150 155 160Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Ile 165 170 175Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln 180 185 190Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser 195 200
205Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
210 215 220Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr225 230 235 240Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu
Thr Phe Gly Gly Gly 245 250 255Thr Lys Val Glu Ile Lys Ile Lys Thr
Thr Thr Pro Ala Pro Arg Pro 260 265 270Pro Thr Pro Ala Pro Thr Ile
Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285Glu Ala Cys Arg Pro
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300Asp Phe Ala
Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val305 310 315
320Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys
325 330 335Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met
Asn Met 340 345 350Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr
Asp Val Thr Leu 355 360 365Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
Pro Ala Tyr Gln Gln Gly 370 375 380Gln Asn Gln Leu Tyr Asn Glu Leu
Asn Leu Gly Arg Arg Glu Glu Tyr385 390 395 400Asp Val Leu Asp Lys
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 405 410 415Pro Gln Arg
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 420 425 430Lys
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 435 440
445Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
450 455 460Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
Pro Pro465 470 475 480Arg2121443DNAArtificial Sequence2F5 human
PSMA-CAR varICOS CD3z 212atggccttac cagtgaccgc cttgctcctg
ccgctggcct tgctgctcca cgccgccagg 60ccggaggtgc agctggtgca gtctggagca
gaggtgaaaa agcccgggga gtctctgaag 120atctcctgta agggttctgg
atacagtttt accagcaact ggatcggctg ggtgcgccag 180atgcccggga
aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga
240tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat
cagcaccgcc 300tacctgcagt ggaacagcct gaaggcctcg gacaccgcca
tgtattactg tgcgagacaa 360actggtttcc tctggtcctt cgatctctgg
ggccgtggca ccctggtcac tgtctcctca 420ggtggcggtg gctcgggcgg
tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480cagtctccat
cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca
540agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa
agctcctaag 600ctcctgatct atgatgcctc cagtttggaa agtggggtcc
catcaaggtt cagcggcagt 660ggatctggga cagatttcac tctcaccatc
agcagcctgc agcctgaaga ttttgcaact 720tattactgtc aacagtttaa
tagttacccg ctcactttcg gcggagggac caaggtggag 780atcaaaatca
aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg
840tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg
cgcagtgcac 900acgagggggc tggacttcgc ctgtgatttc tggttaccca
taggatgtgc agcctttgtt 960gtagtctgca ttttgggatg catacttatt
tgttggctta caaaaaagaa gtattcatcc 1020agtgtgcacg accctaacgg
tgaatacatg aacatgagag cagtgaacac agccaaaaaa 1080tccagactca
cagatgtgac cctaagagtg aagttcagca ggagcgcaga cgcccccgcg
1140taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag
agaggagtac 1200gatgttttgg acaagagacg tggccgggac cctgagatgg
ggggaaagcc gcagagaagg 1260aagaaccctc aggaaggcct gtacaatgaa
ctgcagaaag ataagatggc ggaggcctac 1320agtgagattg ggatgaaagg
cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1380ggtctcagta
cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1440cgc
1443213237PRTArtificial SequencePD1-4-1BB switch receptor 213Met
Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10
15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly
Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser
Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp
Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln
Asp Cys Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe
His Met Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr
Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile
Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg
Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro145 150 155 160Arg
Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro 165 170
175Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
180 185 190Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
Lys Gln 195 200 205Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
Asp Gly Cys Ser 210 215 220Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
Cys Glu Leu225 230 235214711DNAArtificial SequencePD1-4-1BB switch
receptor 214atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtt
atctacatct gggcgccctt ggccgggact 540tgtggggtcc ttctcctgtc
actggttatc accctttact gcaaaaaacg gggcagaaag 600aaactcctgt
atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa
660gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact g
711215237PRTArtificial SequencePD1A132L-4-1BB switch receptor
215Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1
5 10 15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro
Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu
Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu
Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr
Asp Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly
Gln Asp Cys Arg Phe Arg 85 90
95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile
Ser Leu 115 120 125Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala
Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr Ala
His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe Gln
Thr Leu Val Ile Tyr Ile Trp Ala Pro 165 170 175Leu Ala Gly Thr Cys
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 180 185 190Tyr Cys Lys
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 195 200 205Pro
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 210 215
220Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu225 230
235216711DNAArtificial SequencePD1A132L-4-1BB switch receptor
216atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtt
atctacatct gggcgccctt ggccgggact 540tgtggggtcc ttctcctgtc
actggttatc accctttact gcaaaaaacg gggcagaaag 600aaactcctgt
atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa
660gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact g
7112172223DNAArtificial SequencePD1-CD28-F2A-2F5PSMA-CAR ICOS CD3z
217atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
ttttgggtgc tggtggtggt tggtggagtc 540ctggcttgct atagcttgct
agtaacagtg gcctttatta ttttctgggt gaggagtaag 600aggagcaggc
tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc
660cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg
ctccgtgaaa 720cagactttga attttgacct tctcaagttg gcgggagacg
tggagtccaa cccagggccg 780atggccttac cagtgaccgc cttgctcctg
ccgctggcct tgctgctcca cgccgccagg 840ccggaggtgc agctggtgca
gtctggagca gaggtgaaaa agcccgggga gtctctgaag 900atctcctgta
agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag
960atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc
tgataccaga 1020tacagcccgt ccttccaagg ccaggtcacc atctcagccg
acaagtccat cagcaccgcc 1080tacctgcagt ggaacagcct gaaggcctcg
gacaccgcca tgtattactg tgcgagacaa 1140actggtttcc tctggtcctt
cgatctctgg ggccgtggca ccctggtcac tgtctcctca 1200ggtggcggtg
gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc
1260cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac
ttgccgggca 1320agtcaggaca ttagcagtgc tttagcctgg tatcagcaga
aaccggggaa agctcctaag 1380ctcctgatct atgatgcctc cagtttggaa
agtggggtcc catcaaggtt cagcggcagt 1440ggatctggga cagatttcac
tctcaccatc agcagcctgc agcctgaaga ttttgcaact 1500tattactgtc
aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag
1560atcaaaatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc
caccatcgcg 1620tcgcagcccc tgtccctgcg cccagaggcg tgccggccag
cggcgggggg cgcagtgcac 1680acgagggggc tggacttcgc ctgtgatttc
tggttaccca taggatgtgc agcctttgtt 1740gtagtctgca ttttgggatg
catacttatt tgttggctta caaaaaagaa gtattcatcc 1800agtgtgcacg
accctaacgg tgaatacatg ttcatgagag cagtgaacac agccaaaaaa
1860tccagactca cagatgtgac cctaagagtg aagttcagca ggagcgcaga
cgcccccgcg 1920taccagcagg gccagaacca gctctataac gagctcaatc
taggacgaag agaggagtac 1980gatgttttgg acaagagacg tggccgggac
cctgagatgg ggggaaagcc gcagagaagg 2040aagaaccctc aggaaggcct
gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 2100agtgagattg
ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag
2160ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc
cctgccccct 2220cgc 22232182223DNAArtificial
SequencePD1-CD28-2F5PSMA-CAR varICOS CD3z 218atgcagatcc cacaggcgcc
ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60ccaggatggt tcttagactc
cccagacagg ccctggaacc cccccacctt ctccccagcc 120ctgctcgtgg
tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg
180gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga
caagctggcc 240gccttccccg aggaccgcag ccagcccggc caggactgcc
gcttccgtgt cacacaactg 300cccaacgggc gtgacttcca catgagcgtg
gtcagggccc ggcgcaatga cagcggcacc 360tacctctgtg gggccatctc
cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420gagctcaggg
tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc
480aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt
tggtggagtc 540ctggcttgct atagcttgct agtaacagtg gcctttatta
ttttctgggt gaggagtaag 600aggagcaggc tcctgcacag tgactacatg
aacatgactc cccgccgccc cgggcccacc 660cgcaagcatt accagcccta
tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720cagactttga
attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg
780atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca
cgccgccagg 840ccggaggtgc agctggtgca gtctggagca gaggtgaaaa
agcccgggga gtctctgaag 900atctcctgta agggttctgg atacagtttt
accagcaact ggatcggctg ggtgcgccag 960atgcccggga aaggcctgga
gtggatgggg atcatctatc ctggtgactc tgataccaga 1020tacagcccgt
ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc
1080tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg
tgcgagacaa 1140actggtttcc tctggtcctt cgatctctgg ggccgtggca
ccctggtcac tgtctcctca 1200ggtggcggtg gctcgggcgg tggtgggtcg
ggtggcggcg gatctgccat ccagttgacc 1260cagtctccat cctccctgtc
tgcatctgta ggagacagag tcaccatcac ttgccgggca 1320agtcaggaca
ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag
1380ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt
cagcggcagt 1440ggatctggga cagatttcac tctcaccatc agcagcctgc
agcctgaaga ttttgcaact 1500tattactgtc aacagtttaa tagttacccg
ctcactttcg gcggagggac caaggtggag 1560atcaaaatca aaaccacgac
gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 1620tcgcagcccc
tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac
1680acgagggggc tggacttcgc ctgtgatttc tggttaccca taggatgtgc
agcctttgtt 1740gtagtctgca ttttgggatg catacttatt tgttggctta
caaaaaagaa gtattcatcc 1800agtgtgcacg accctaacgg tgaatacatg
aacatgagag cagtgaacac agccaaaaaa 1860tccagactca cagatgtgac
cctaagagtg aagttcagca ggagcgcaga cgcccccgcg 1920taccagcagg
gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac
1980gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc
gcagagaagg 2040aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag
ataagatggc ggaggcctac 2100agtgagattg ggatgaaagg cgagcgccgg
aggggcaagg ggcacgatgg cctttaccag 2160ggtctcagta cagccaccaa
ggacacctac gacgcccttc acatgcaggc cctgccccct 2220cgc
22232192205DNAArtificial SequencePD1A132L-CD28-2F5PSMA-CAR ICOS
CD3z 219atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
gttggtgtcg tgggcggcct gctgggcagc 540ctggtgctgc tagtctgggt
cctggccgtc atcaggagta agaggagcag gctcctgcac 600agtgactaca
tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc
660tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt
gaattttgac 720cttctcaagt tggcgggaga cgtggagtcc aacccagggc
cgatggcctt accagtgacc 780gccttgctcc tgccgctggc cttgctgctc
cacgccgcca ggccggaggt gcagctggtg 840cagtctggag cagaggtgaa
aaagcccggg gagtctctga agatctcctg taagggttct 900ggatacagtt
ttaccagcaa ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg
960gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc
gtccttccaa 1020ggccaggtca ccatctcagc cgacaagtcc atcagcaccg
cctacctgca gtggaacagc 1080ctgaaggcct cggacaccgc catgtattac
tgtgcgagac aaactggttt cctctggtcc 1140ttcgatctct ggggccgtgg
caccctggtc actgtctcct caggtggcgg tggctcgggc 1200ggtggtgggt
cgggtggcgg cggatctgcc atccagttga cccagtctcc atcctccctg
1260tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagga
cattagcagt 1320gctttagcct ggtatcagca gaaaccgggg aaagctccta
agctcctgat ctatgatgcc 1380tccagtttgg aaagtggggt cccatcaagg
ttcagcggca gtggatctgg gacagatttc 1440actctcacca tcagcagcct
gcagcctgaa gattttgcaa cttattactg tcaacagttt 1500aatagttacc
cgctcacttt cggcggaggg accaaggtgg agatcaaaat caaaaccacg
1560acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc
cctgtccctg 1620cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc
acacgagggg gctggacttc 1680gcctgtgatt tctggttacc cataggatgt
gcagcctttg ttgtagtctg cattttggga 1740tgcatactta tttgttggct
tacaaaaaag aagtattcat ccagtgtgca cgaccctaac 1800ggtgaataca
tgttcatgag agcagtgaac acagccaaaa aatccagact cacagatgtg
1860accctaagag tgaagttcag caggagcgca gacgcccccg cgtaccagca
gggccagaac 1920cagctctata acgagctcaa tctaggacga agagaggagt
acgatgtttt ggacaagaga 1980cgtggccggg accctgagat ggggggaaag
ccgcagagaa ggaagaaccc tcaggaaggc 2040ctgtacaatg aactgcagaa
agataagatg gcggaggcct acagtgagat tgggatgaaa 2100ggcgagcgcc
ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc
2160aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc
22052202205DNAArtificial SequencePD1A132L-CD28-2F5PSMA-CAR varICOS
CD3z 220atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtg
gttggtgtcg tgggcggcct gctgggcagc 540ctggtgctgc tagtctgggt
cctggccgtc atcaggagta agaggagcag gctcctgcac 600agtgactaca
tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc
660tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt
gaattttgac 720cttctcaagt tggcgggaga cgtggagtcc aacccagggc
cgatggcctt accagtgacc 780gccttgctcc tgccgctggc cttgctgctc
cacgccgcca ggccggaggt gcagctggtg 840cagtctggag cagaggtgaa
aaagcccggg gagtctctga agatctcctg taagggttct 900ggatacagtt
ttaccagcaa ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg
960gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc
gtccttccaa 1020ggccaggtca ccatctcagc cgacaagtcc atcagcaccg
cctacctgca gtggaacagc 1080ctgaaggcct cggacaccgc catgtattac
tgtgcgagac aaactggttt cctctggtcc 1140ttcgatctct ggggccgtgg
caccctggtc actgtctcct caggtggcgg tggctcgggc 1200ggtggtgggt
cgggtggcgg cggatctgcc atccagttga cccagtctcc atcctccctg
1260tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagga
cattagcagt 1320gctttagcct ggtatcagca gaaaccgggg aaagctccta
agctcctgat ctatgatgcc 1380tccagtttgg aaagtggggt cccatcaagg
ttcagcggca gtggatctgg gacagatttc 1440actctcacca tcagcagcct
gcagcctgaa gattttgcaa cttattactg tcaacagttt 1500aatagttacc
cgctcacttt cggcggaggg accaaggtgg agatcaaaat caaaaccacg
1560acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc
cctgtccctg 1620cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc
acacgagggg gctggacttc 1680gcctgtgatt tctggttacc cataggatgt
gcagcctttg ttgtagtctg cattttggga 1740tgcatactta tttgttggct
tacaaaaaag aagtattcat ccagtgtgca cgaccctaac 1800ggtgaataca
tgaacatgag agcagtgaac acagccaaaa aatccagact cacagatgtg
1860accctaagag tgaagttcag caggagcgca gacgcccccg cgtaccagca
gggccagaac 1920cagctctata acgagctcaa tctaggacga agagaggagt
acgatgtttt ggacaagaga 1980cgtggccggg accctgagat ggggggaaag
ccgcagagaa ggaagaaccc tcaggaaggc 2040ctgtacaatg aactgcagaa
agataagatg gcggaggcct acagtgagat tgggatgaaa 2100ggcgagcgcc
ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc
2160aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc
22052212220DNAArtificial SequencePD1A132L-41BB-2F5PSMA-CAR ICOS
CD3z 221atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtt
atctacatct gggcgccctt ggccgggact 540tgtggggtcc ttctcctgtc
actggttatc accctttact gcaaaaaacg gggcagaaag 600aaactcctgt
atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa
660gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact
ggtgaaacag 720actttgaatt ttgaccttct caagttggcg ggagacgtgg
agtccaaccc agggccgatg 780gccttaccag tgaccgcctt gctcctgccg
ctggccttgc tgctccacgc cgccaggccg 840gaggtgcagc tggtgcagtc
tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 900tcctgtaagg
gttctggata cagttttacc agcaactgga tcggctgggt gcgccagatg
960cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga
taccagatac 1020agcccgtcct tccaaggcca ggtcaccatc tcagccgaca
agtccatcag caccgcctac 1080ctgcagtgga acagcctgaa ggcctcggac
accgccatgt attactgtgc gagacaaact 1140ggtttcctct ggtccttcga
tctctggggc cgtggcaccc tggtcactgt ctcctcaggt 1200ggcggtggct
cgggcggtgg tgggtcgggt ggcggcggat ctgccatcca gttgacccag
1260tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg
ccgggcaagt 1320caggacatta gcagtgcttt agcctggtat cagcagaaac
cggggaaagc tcctaagctc 1380ctgatctatg atgcctccag tttggaaagt
ggggtcccat caaggttcag cggcagtgga 1440tctgggacag atttcactct
caccatcagc agcctgcagc ctgaagattt tgcaacttat 1500tactgtcaac
agtttaatag ttacccgctc actttcggcg gagggaccaa ggtggagatc
1560aaaatcaaaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac
catcgcgtcg 1620cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg
cggggggcgc agtgcacacg 1680agggggctgg acttcgcctg tgatttctgg
ttacccatag gatgtgcagc ctttgttgta 1740gtctgcattt tgggatgcat
acttatttgt tggcttacaa aaaagaagta ttcatccagt 1800gtgcacgacc
ctaacggtga atacatgttc atgagagcag tgaacacagc caaaaaatcc
1860agactcacag atgtgaccct aagagtgaag ttcagcagga gcgcagacgc
ccccgcgtac 1920cagcagggcc agaaccagct ctataacgag ctcaatctag
gacgaagaga ggagtacgat 1980gttttggaca agagacgtgg ccgggaccct
gagatggggg gaaagccgca gagaaggaag 2040aaccctcagg aaggcctgta
caatgaactg cagaaagata agatggcgga ggcctacagt 2100gagattggga
tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt
2160ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct
gccccctcgc 22202222220DNAArtificial
SequencePD1A132L-41BB-2F5PSMA-CAR varICOS CD3z 222atgcagatcc
cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60ccaggatggt
tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc
120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc
caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg agccccagca
accagacgga caagctggcc 240gccttccccg aggaccgcag ccagcccggc
caggactgcc gcttccgtgt cacacaactg 300cccaacgggc gtgacttcca
catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360tacctctgtg
gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca
420gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag
cccctcaccc 480aggccagccg gccagttcca aaccctggtt atctacatct
gggcgccctt ggccgggact 540tgtggggtcc ttctcctgtc actggttatc
accctttact gcaaaaaacg gggcagaaag 600aaactcctgt atatattcaa
acaaccattt atgagaccag tacaaactac tcaagaggaa 660gatggctgta
gctgccgatt tccagaagaa gaagaaggag gatgtgaact ggtgaaacag
720actttgaatt ttgaccttct caagttggcg ggagacgtgg agtccaaccc
agggccgatg 780gccttaccag tgaccgcctt gctcctgccg ctggccttgc
tgctccacgc cgccaggccg 840gaggtgcagc tggtgcagtc tggagcagag
gtgaaaaagc ccggggagtc tctgaagatc 900tcctgtaagg gttctggata
cagttttacc agcaactgga tcggctgggt gcgccagatg 960cccgggaaag
gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac
1020agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag
caccgcctac 1080ctgcagtgga acagcctgaa ggcctcggac accgccatgt
attactgtgc gagacaaact 1140ggtttcctct ggtccttcga tctctggggc
cgtggcaccc tggtcactgt ctcctcaggt 1200ggcggtggct cgggcggtgg
tgggtcgggt ggcggcggat ctgccatcca gttgacccag 1260tctccatcct
ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt
1320caggacatta gcagtgcttt agcctggtat cagcagaaac cggggaaagc
tcctaagctc 1380ctgatctatg atgcctccag tttggaaagt ggggtcccat
caaggttcag cggcagtgga 1440tctgggacag atttcactct caccatcagc
agcctgcagc ctgaagattt tgcaacttat 1500tactgtcaac agtttaatag
ttacccgctc actttcggcg gagggaccaa ggtggagatc 1560aaaatcaaaa
ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg
1620cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc
agtgcacacg 1680agggggctgg acttcgcctg tgatttctgg ttacccatag
gatgtgcagc ctttgttgta 1740gtctgcattt tgggatgcat acttatttgt
tggcttacaa aaaagaagta ttcatccagt 1800gtgcacgacc ctaacggtga
atacatgaac atgagagcag tgaacacagc caaaaaatcc
1860agactcacag atgtgaccct aagagtgaag ttcagcagga gcgcagacgc
ccccgcgtac 1920cagcagggcc agaaccagct ctataacgag ctcaatctag
gacgaagaga ggagtacgat 1980gttttggaca agagacgtgg ccgggaccct
gagatggggg gaaagccgca gagaaggaag 2040aaccctcagg aaggcctgta
caatgaactg cagaaagata agatggcgga ggcctacagt 2100gagattggga
tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt
2160ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct
gccccctcgc 22202232313DNAArtificial SequenceTIM3-CD28-2F5PSMA-CAR
ICOS CD3z 223atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact
acttacaagg 60tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc
ctgcttctac 120accccagccg ccccagggaa cctcgtgccc gtctgctggg
gcaaaggagc ctgtcctgtg 180tttgaatgtg gcaacgtggt gctcaggact
gatgaaaggg atgtgaatta ttggacatcc 240agatactggc taaatgggga
tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300actctagcag
acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat
360gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc
accgactcgg 420cagagagact tcactgcagc ctttccaagg atgcttacca
ccaggggaca tggcccagca 480gagacacaga cactggggag cctccctgac
ataaatctaa cacaaatatc cacattggcc 540aatgagttac gggactctag
gttggccaat gacttacggg actccggagc aaccatcaga 600ttttgggtgc
tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg
660gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag
tgactacatg 720aacatgactc cccgccgccc cgggcccacc cgcaagcatt
accagcccta tgccccacca 780cgcgacttcg cagcctatcg ctccgtgaaa
cagactttga attttgacct tctcaagttg 840gcgggagacg tggagtccaa
cccagggccg atggccttac cagtgaccgc cttgctcctg 900ccgctggcct
tgctgctcca cgccgccagg ccggaggtgc agctggtgca gtctggagca
960gaggtgaaaa agcccgggga gtctctgaag atctcctgta agggttctgg
atacagtttt 1020accagcaact ggatcggctg ggtgcgccag atgcccggga
aaggcctgga gtggatgggg 1080atcatctatc ctggtgactc tgataccaga
tacagcccgt ccttccaagg ccaggtcacc 1140atctcagccg acaagtccat
cagcaccgcc tacctgcagt ggaacagcct gaaggcctcg 1200gacaccgcca
tgtattactg tgcgagacaa actggtttcc tctggtcctt cgatctctgg
1260ggccgtggca ccctggtcac tgtctcctca ggtggcggtg gctcgggcgg
tggtgggtcg 1320ggtggcggcg gatctgccat ccagttgacc cagtctccat
cctccctgtc tgcatctgta 1380ggagacagag tcaccatcac ttgccgggca
agtcaggaca ttagcagtgc tttagcctgg 1440tatcagcaga aaccggggaa
agctcctaag ctcctgatct atgatgcctc cagtttggaa 1500agtggggtcc
catcaaggtt cagcggcagt ggatctggga cagatttcac tctcaccatc
1560agcagcctgc agcctgaaga ttttgcaact tattactgtc aacagtttaa
tagttacccg 1620ctcactttcg gcggagggac caaggtggag atcaaaatca
aaaccacgac gccagcgccg 1680cgaccaccaa caccggcgcc caccatcgcg
tcgcagcccc tgtccctgcg cccagaggcg 1740tgccggccag cggcgggggg
cgcagtgcac acgagggggc tggacttcgc ctgtgatttc 1800tggttaccca
taggatgtgc agcctttgtt gtagtctgca ttttgggatg catacttatt
1860tgttggctta caaaaaagaa gtattcatcc agtgtgcacg accctaacgg
tgaatacatg 1920ttcatgagag cagtgaacac agccaaaaaa tccagactca
cagatgtgac cctaagagtg 1980aagttcagca ggagcgcaga cgcccccgcg
taccagcagg gccagaacca gctctataac 2040gagctcaatc taggacgaag
agaggagtac gatgttttgg acaagagacg tggccgggac 2100cctgagatgg
ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa
2160ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg
cgagcgccgg 2220aggggcaagg ggcacgatgg cctttaccag ggtctcagta
cagccaccaa ggacacctac 2280gacgcccttc acatgcaggc cctgccccct cgc
23132242313DNAArtificial SequenceTIM3-CD28-2F5PSMA-CAR varICOS CD3z
224atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact
acttacaagg 60tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc
ctgcttctac 120accccagccg ccccagggaa cctcgtgccc gtctgctggg
gcaaaggagc ctgtcctgtg 180tttgaatgtg gcaacgtggt gctcaggact
gatgaaaggg atgtgaatta ttggacatcc 240agatactggc taaatgggga
tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300actctagcag
acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat
360gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc
accgactcgg 420cagagagact tcactgcagc ctttccaagg atgcttacca
ccaggggaca tggcccagca 480gagacacaga cactggggag cctccctgac
ataaatctaa cacaaatatc cacattggcc 540aatgagttac gggactctag
gttggccaat gacttacggg actccggagc aaccatcaga 600ttttgggtgc
tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg
660gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag
tgactacatg 720aacatgactc cccgccgccc cgggcccacc cgcaagcatt
accagcccta tgccccacca 780cgcgacttcg cagcctatcg ctccgtgaaa
cagactttga attttgacct tctcaagttg 840gcgggagacg tggagtccaa
cccagggccg atggccttac cagtgaccgc cttgctcctg 900ccgctggcct
tgctgctcca cgccgccagg ccggaggtgc agctggtgca gtctggagca
960gaggtgaaaa agcccgggga gtctctgaag atctcctgta agggttctgg
atacagtttt 1020accagcaact ggatcggctg ggtgcgccag atgcccggga
aaggcctgga gtggatgggg 1080atcatctatc ctggtgactc tgataccaga
tacagcccgt ccttccaagg ccaggtcacc 1140atctcagccg acaagtccat
cagcaccgcc tacctgcagt ggaacagcct gaaggcctcg 1200gacaccgcca
tgtattactg tgcgagacaa actggtttcc tctggtcctt cgatctctgg
1260ggccgtggca ccctggtcac tgtctcctca ggtggcggtg gctcgggcgg
tggtgggtcg 1320ggtggcggcg gatctgccat ccagttgacc cagtctccat
cctccctgtc tgcatctgta 1380ggagacagag tcaccatcac ttgccgggca
agtcaggaca ttagcagtgc tttagcctgg 1440tatcagcaga aaccggggaa
agctcctaag ctcctgatct atgatgcctc cagtttggaa 1500agtggggtcc
catcaaggtt cagcggcagt ggatctggga cagatttcac tctcaccatc
1560agcagcctgc agcctgaaga ttttgcaact tattactgtc aacagtttaa
tagttacccg 1620ctcactttcg gcggagggac caaggtggag atcaaaatca
aaaccacgac gccagcgccg 1680cgaccaccaa caccggcgcc caccatcgcg
tcgcagcccc tgtccctgcg cccagaggcg 1740tgccggccag cggcgggggg
cgcagtgcac acgagggggc tggacttcgc ctgtgatttc 1800tggttaccca
taggatgtgc agcctttgtt gtagtctgca ttttgggatg catacttatt
1860tgttggctta caaaaaagaa gtattcatcc agtgtgcacg accctaacgg
tgaatacatg 1920aacatgagag cagtgaacac agccaaaaaa tccagactca
cagatgtgac cctaagagtg 1980aagttcagca ggagcgcaga cgcccccgcg
taccagcagg gccagaacca gctctataac 2040gagctcaatc taggacgaag
agaggagtac gatgttttgg acaagagacg tggccgggac 2100cctgagatgg
ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa
2160ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg
cgagcgccgg 2220aggggcaagg ggcacgatgg cctttaccag ggtctcagta
cagccaccaa ggacacctac 2280gacgcccttc acatgcaggc cctgccccct cgc
23132253090DNAArtificial
SequencePD1A132L-4-1BB-TIM3-CD28-2F5PSMA-CAR ICOS CD3z
225atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtt
atctacatct gggcgccctt ggccgggact 540tgtggggtcc ttctcctgtc
actggttatc accctttact gcaaaaaacg gggcagaaag 600aaactcctgt
atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa
660gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact
ggtgaagcag 720acgttgaact tcgatttgct caaacttgcc ggtgacgtgg
aatccaatcc ggggccgatg 780ttttcacatc ttccctttga ctgtgtcctg
ctgctgctgc tgctactact tacaaggtcc 840tcagaagtgg aatacagagc
ggaggtcggt cagaatgcct atctgccctg cttctacacc 900ccagccgccc
cagggaacct cgtgcccgtc tgctggggca aaggagcctg tcctgtgttt
960gaatgtggca acgtggtgct caggactgat gaaagggatg tgaattattg
gacatccaga 1020tactggctaa atggggattt ccgcaaagga gatgtgtccc
tgaccataga gaatgtgact 1080ctagcagaca gtgggatcta ctgctgccga
atccaaatcc caggcataat gaatgatgaa 1140aaatttaacc tgaagttggt
catcaaacca gccaaggtca cccctgcacc gactcggcag 1200agagacttca
ctgcagcctt tccaaggatg cttaccacca ggggacatgg cccagcagag
1260acacagacac tggggagcct ccctgacata aatctaacac aaatatccac
attggccaat 1320gagttacggg actctaggtt ggccaatgac ttacgggact
ccggagcaac catcagattt 1380tgggtgctgg tggtggttgg tggagtcctg
gcttgctata gcttactagt aacagtggcc 1440tttattattt tctgggtgag
gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1500atgactcccc
gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc
1560gacttcgcag cctatcgctc cgtgaaacag actttgaatt ttgaccttct
caagttggcg 1620ggagacgtgg agtccaaccc agggccgatg gccttaccag
tgaccgcctt gctcctgccg 1680ctggccttgc tgctccacgc cgccaggccg
gaggtgcagc tggtgcagtc tggagcagag 1740gtgaaaaagc ccggggagtc
tctgaagatc tcctgtaagg gttctggata cagttttacc 1800agcaactgga
tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc
1860atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca
ggtcaccatc 1920tcagccgaca agtccatcag caccgcctac ctgcagtgga
acagcctgaa ggcctcggac 1980accgccatgt attactgtgc gagacaaact
ggtttcctct ggtccttcga tctctggggc 2040cgtggcaccc tggtcactgt
ctcctcaggt ggcggtggct cgggcggtgg tgggtcgggt 2100ggcggcggat
ctgccatcca gttgacccag tctccatcct ccctgtctgc atctgtagga
2160gacagagtca ccatcacttg ccgggcaagt caggacatta gcagtgcttt
agcctggtat 2220cagcagaaac cggggaaagc tcctaagctc ctgatctatg
atgcctccag tttggaaagt 2280ggggtcccat caaggttcag cggcagtgga
tctgggacag atttcactct caccatcagc 2340agcctgcagc ctgaagattt
tgcaacttat tactgtcaac agtttaatag ttacccgctc 2400actttcggcg
gagggaccaa ggtggagatc aaaatcaaaa ccacgacgcc agcgccgcga
2460ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc
agaggcgtgc 2520cggccagcgg cggggggcgc agtgcacacg agggggctgg
acttcgcctg tgatttctgg 2580ttacccatag gatgtgcagc ctttgttgta
gtctgcattt tgggatgcat acttatttgt 2640tggcttacaa aaaagaagta
ttcatccagt gtgcacgacc ctaacggtga atacatgttc 2700atgagagcag
tgaacacagc caaaaaatcc agactcacag atgtgaccct aagagtgaag
2760ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct
ctataacgag 2820ctcaatctag gacgaagaga ggagtacgat gttttggaca
agagacgtgg ccgggaccct 2880gagatggggg gaaagccgca gagaaggaag
aaccctcagg aaggcctgta caatgaactg 2940cagaaagata agatggcgga
ggcctacagt gagattggga tgaaaggcga gcgccggagg 3000ggcaaggggc
acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac
3060gcccttcaca tgcaggccct gccccctcgc 30902263090DNAArtificial
SequencePD1A132L-4-1BB-TIM3-CD28-2F5PSMA-CAR varICOS CD3z
226atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact
gggctggcgg 60ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 120ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 180gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 240gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 300cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc
360tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag
cctgcgggca 420gagctcaggg tgacagagag aagggcagaa gtgcccacag
cccaccccag cccctcaccc 480aggccagccg gccagttcca aaccctggtt
atctacatct gggcgccctt ggccgggact 540tgtggggtcc ttctcctgtc
actggttatc accctttact gcaaaaaacg gggcagaaag 600aaactcctgt
atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa
660gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact
ggtgaagcag 720acgttgaact tcgatttgct caaacttgcc ggtgacgtgg
aatccaatcc ggggccgatg 780ttttcacatc ttccctttga ctgtgtcctg
ctgctgctgc tgctactact tacaaggtcc 840tcagaagtgg aatacagagc
ggaggtcggt cagaatgcct atctgccctg cttctacacc 900ccagccgccc
cagggaacct cgtgcccgtc tgctggggca aaggagcctg tcctgtgttt
960gaatgtggca acgtggtgct caggactgat gaaagggatg tgaattattg
gacatccaga 1020tactggctaa atggggattt ccgcaaagga gatgtgtccc
tgaccataga gaatgtgact 1080ctagcagaca gtgggatcta ctgctgccga
atccaaatcc caggcataat gaatgatgaa 1140aaatttaacc tgaagttggt
catcaaacca gccaaggtca cccctgcacc gactcggcag 1200agagacttca
ctgcagcctt tccaaggatg cttaccacca ggggacatgg cccagcagag
1260acacagacac tggggagcct ccctgacata aatctaacac aaatatccac
attggccaat 1320gagttacggg actctaggtt ggccaatgac ttacgggact
ccggagcaac catcagattt 1380tgggtgctgg tggtggttgg tggagtcctg
gcttgctata gcttactagt aacagtggcc 1440tttattattt tctgggtgag
gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1500atgactcccc
gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc
1560gacttcgcag cctatcgctc cgtgaaacag actttgaatt ttgaccttct
caagttggcg 1620ggagacgtgg agtccaaccc agggccgatg gccttaccag
tgaccgcctt gctcctgccg 1680ctggccttgc tgctccacgc cgccaggccg
gaggtgcagc tggtgcagtc tggagcagag 1740gtgaaaaagc ccggggagtc
tctgaagatc tcctgtaagg gttctggata cagttttacc 1800agcaactgga
tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc
1860atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca
ggtcaccatc 1920tcagccgaca agtccatcag caccgcctac ctgcagtgga
acagcctgaa ggcctcggac 1980accgccatgt attactgtgc gagacaaact
ggtttcctct ggtccttcga tctctggggc 2040cgtggcaccc tggtcactgt
ctcctcaggt ggcggtggct cgggcggtgg tgggtcgggt 2100ggcggcggat
ctgccatcca gttgacccag tctccatcct ccctgtctgc atctgtagga
2160gacagagtca ccatcacttg ccgggcaagt caggacatta gcagtgcttt
agcctggtat 2220cagcagaaac cggggaaagc tcctaagctc ctgatctatg
atgcctccag tttggaaagt 2280ggggtcccat caaggttcag cggcagtgga
tctgggacag atttcactct caccatcagc 2340agcctgcagc ctgaagattt
tgcaacttat tactgtcaac agtttaatag ttacccgctc 2400actttcggcg
gagggaccaa ggtggagatc aaaatcaaaa ccacgacgcc agcgccgcga
2460ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc
agaggcgtgc 2520cggccagcgg cggggggcgc agtgcacacg agggggctgg
acttcgcctg tgatttctgg 2580ttacccatag gatgtgcagc ctttgttgta
gtctgcattt tgggatgcat acttatttgt 2640tggcttacaa aaaagaagta
ttcatccagt gtgcacgacc ctaacggtga atacatgaac 2700atgagagcag
tgaacacagc caaaaaatcc agactcacag atgtgaccct aagagtgaag
2760ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct
ctataacgag 2820ctcaatctag gacgaagaga ggagtacgat gttttggaca
agagacgtgg ccgggaccct 2880gagatggggg gaaagccgca gagaaggaag
aaccctcagg aaggcctgta caatgaactg 2940cagaaagata agatggcgga
ggcctacagt gagattggga tgaaaggcga gcgccggagg 3000ggcaaggggc
acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac
3060gcccttcaca tgcaggccct gccccctcgc 3090227741PRTArtificial
SequencePD1-CD28-2F5PSMA-CAR ICOS CD3z 227Met Gln Ile Pro Gln Ala
Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro
Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr
Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala Thr
Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu Asn
Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75
80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val
Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala
Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr
Ala His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe
Gln Thr Leu Val Phe Trp Val Leu Val Val 165 170 175Val Gly Gly Val
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 180 185 190Ile Ile
Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp 195 200
205Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
210 215 220Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
Val Lys225 230 235 240Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala
Gly Asp Val Glu Ser 245 250 255Asn Pro Gly Pro Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu 260 265 270Ala Leu Leu Leu His Ala Ala
Arg Pro Glu Val Gln Leu Val Gln Ser 275 280 285Gly Ala Glu Val Lys
Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys 290 295 300Gly Ser Gly
Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln305 310 315
320Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp
325 330 335Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr
Ile Ser 340 345 350Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp
Asn Ser Leu Lys 355 360 365Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala
Arg Gln Thr Gly Phe Leu 370 375 380Trp Ser Phe Asp Leu Trp Gly Arg
Gly Thr Leu Val Thr Val Ser Ser385 390 395 400Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 405 410 415Ile Gln Leu
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 420 425 430Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu 435 440
445Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
450 455 460Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser465 470 475 480Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu 485 490 495Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Phe Asn Ser Tyr Pro Leu Thr 500 505 510Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Ile Lys Thr Thr Thr Pro 515 520 525Ala Pro Arg Pro Pro
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 530 535 540Ser Leu Arg
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His545 550 555
560Thr Arg Gly Leu Asp Phe Ala
Cys Asp Phe Trp Leu Pro Ile Gly Cys 565 570 575Ala Ala Phe Val Val
Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp 580 585 590Leu Thr Lys
Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu 595 600 605Tyr
Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr 610 615
620Asp Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
Ala625 630 635 640Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
Asn Leu Gly Arg 645 650 655Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
Arg Gly Arg Asp Pro Glu 660 665 670Met Gly Gly Lys Pro Gln Arg Arg
Lys Asn Pro Gln Glu Gly Leu Tyr 675 680 685Asn Glu Leu Gln Lys Asp
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 690 695 700Met Lys Gly Glu
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln705 710 715 720Gly
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 725 730
735Ala Leu Pro Pro Arg 740228735PRTArtificial
SequencePD1-CD28-2F5PSMA-CAR varICOS CD3z 228Met Gln Ile Pro Gln
Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg
Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro
Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala
Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75
80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val
Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala
Ile Ser Leu 115 120 125Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr
Ala His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe
Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175Leu Leu Gly Ser
Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Arg 180 185 190Ser Lys
Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 195 200
205Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
210 215 220Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Gln Thr Leu Asn
Phe Asp225 230 235 240Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn
Pro Gly Pro Met Ala 245 250 255Leu Pro Val Thr Ala Leu Leu Leu Pro
Leu Ala Leu Leu Leu His Ala 260 265 270Ala Arg Pro Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 275 280 285Pro Gly Glu Ser Leu
Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe 290 295 300Thr Ser Asn
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu305 310 315
320Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser
325 330 335Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
Ile Ser 340 345 350Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser
Asp Thr Ala Met 355 360 365Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu
Trp Ser Phe Asp Leu Trp 370 375 380Gly Arg Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly385 390 395 400Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ala Ile Gln Leu Thr Gln Ser 405 410 415Pro Ser Ser
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 420 425 430Arg
Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln Gln Lys 435 440
445Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu
450 455 460Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe465 470 475 480Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr 485 490 495Cys Gln Gln Phe Asn Ser Tyr Pro Leu
Thr Phe Gly Gly Gly Thr Lys 500 505 510Val Glu Ile Lys Ile Lys Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr 515 520 525Pro Ala Pro Thr Ile
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 530 535 540Cys Arg Pro
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe545 550 555
560Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val
565 570 575Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys
Lys Tyr 580 585 590Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met
Phe Met Arg Ala 595 600 605Val Asn Thr Ala Lys Lys Ser Arg Leu Thr
Asp Val Thr Leu Arg Val 610 615 620Lys Phe Ser Arg Ser Ala Asp Ala
Pro Ala Tyr Gln Gln Gly Gln Asn625 630 635 640Gln Leu Tyr Asn Glu
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 645 650 655Leu Asp Lys
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln 660 665 670Arg
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 675 680
685Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
690 695 700Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
Ala Thr705 710 715 720Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
Leu Pro Pro Arg 725 730 735229740PRTArtificial
SequencePD1A132L-41BB-2F5PSMA-CAR ICOS CD3z 229Met Gln Ile Pro Gln
Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg
Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro
Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala
Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75
80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val
Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala
Ile Ser Leu 115 120 125Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr
Ala His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe
Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro 165 170 175Leu Ala Gly Thr
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 180 185 190Tyr Cys
Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 195 200
205Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
210 215 220Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Val
Lys Gln225 230 235 240Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
Asp Val Glu Ser Asn 245 250 255Pro Gly Pro Met Ala Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala 260 265 270Leu Leu Leu His Ala Ala Arg
Pro Glu Val Gln Leu Val Gln Ser Gly 275 280 285Ala Glu Val Lys Lys
Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly 290 295 300Ser Gly Tyr
Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met305 310 315
320Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser
325 330 335Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile
Ser Ala 340 345 350Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn
Ser Leu Lys Ala 355 360 365Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg
Gln Thr Gly Phe Leu Trp 370 375 380Ser Phe Asp Leu Trp Gly Arg Gly
Thr Leu Val Thr Val Ser Ser Gly385 390 395 400Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile 405 410 415Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 420 425 430Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala 435 440
445Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp
450 455 460Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly465 470 475 480Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp 485 490 495Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
Asn Ser Tyr Pro Leu Thr Phe 500 505 510Gly Gly Gly Thr Lys Val Glu
Ile Lys Ile Lys Thr Thr Thr Pro Ala 515 520 525Pro Arg Pro Pro Thr
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 530 535 540Leu Arg Pro
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr545 550 555
560Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala
565 570 575Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys
Trp Leu 580 585 590Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro
Asn Gly Glu Tyr 595 600 605Met Phe Met Arg Ala Val Asn Thr Ala Lys
Lys Ser Arg Leu Thr Asp 610 615 620Val Thr Leu Arg Val Lys Phe Ser
Arg Ser Ala Asp Ala Pro Ala Tyr625 630 635 640Gln Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 645 650 655Glu Glu Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 660 665 670Gly
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 675 680
685Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
690 695 700Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
Gln Gly705 710 715 720Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
Leu His Met Gln Ala 725 730 735Leu Pro Pro Arg
740230771PRTArtificial SequenceTIM3-CD28-2F5PSMA-CAR ICOS CD3z
230Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1
5 10 15Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly
Gln 20 25 30Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly
Asn Leu 35 40 45Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe
Glu Cys Gly 50 55 60Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn
Tyr Trp Thr Ser65 70 75 80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys
Gly Asp Val Ser Leu Thr 85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser
Gly Ile Tyr Cys Cys Arg Ile 100 105 110Gln Ile Pro Gly Ile Met Asn
Asp Glu Lys Phe Asn Leu Lys Leu Val 115 120 125Ile Lys Pro Ala Lys
Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe 130 135 140Thr Ala Ala
Phe Pro Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala145 150 155
160Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile
165 170 175Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn
Asp Leu 180 185 190Arg Asp Ser Gly Ala Thr Ile Arg Phe Trp Val Leu
Val Val Val Gly 195 200 205Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
Thr Val Ala Phe Ile Ile 210 215 220Phe Trp Val Arg Ser Lys Arg Ser
Arg Leu Leu His Ser Asp Tyr Met225 230 235 240Asn Met Thr Pro Arg
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 245 250 255Tyr Ala Pro
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Gln Thr 260 265 270Leu
Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro 275 280
285Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
290 295 300Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser
Gly Ala305 310 315 320Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile
Ser Cys Lys Gly Ser 325 330 335Gly Tyr Ser Phe Thr Ser Asn Trp Ile
Gly Trp Val Arg Gln Met Pro 340 345 350Gly Lys Gly Leu Glu Trp Met
Gly Ile Ile Tyr Pro Gly Asp Ser Asp 355 360 365Thr Arg Tyr Ser Pro
Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp 370 375 380Lys Ser Ile
Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser385 390 395
400Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser
405 410 415Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
Gly Gly 420 425 430Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala Ile Gln 435 440 445Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly Asp Arg Val 450 455 460Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Ser Ala Leu Ala Trp465 470 475 480Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala 485 490 495Ser Ser Leu
Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 500 505 510Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 515 520
525Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly
530 535 540Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro
Ala Pro545 550 555 560Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
Gln Pro Leu Ser Leu 565 570 575Arg Pro Glu Ala Cys Arg Pro Ala Ala
Gly Gly Ala Val His Thr Arg 580 585 590Gly Leu Asp Phe Ala Cys Asp
Phe Trp Leu Pro Ile Gly Cys Ala Ala 595 600 605Phe Val Val Val Cys
Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr 610 615 620Lys Lys Lys
Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met625 630 635
640Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val
645 650 655Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
Tyr Gln 660 665 670Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
Gly Arg Arg Glu 675 680 685Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg Asp Pro Glu Met Gly 690 695 700Gly Lys Pro Gln Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn Glu705 710 715 720Leu Gln Lys Asp Lys
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 725 730 735Gly Glu Arg
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 740 745 750Ser
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 755 760
765Pro Pro Arg 7702311030PRTArtificial
SequencePD1A132L-4-1BB-TIM3-CD28-2F5PSMA-CAR ICOS CD3z 231Met Gln
Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1
5 10 15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro
Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu
Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu
Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr
Asp Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly
Gln Asp Cys Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp
Phe His Met Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly
Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Leu Gln
Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg
Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro145 150 155
160Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro
165 170 175Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
Thr Leu 180 185 190Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr
Ile Phe Lys Gln 195 200 205Pro Phe Met Arg Pro Val Gln Thr Thr Gln
Glu Glu Asp Gly Cys Ser 210 215 220Cys Arg Phe Pro Glu Glu Glu Glu
Gly Gly Cys Glu Leu Val Lys Gln225 230 235 240Thr Leu Asn Phe Asp
Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 245 250 255Pro Gly Pro
Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu 260 265 270Leu
Leu Leu Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu 275 280
285Val Gly Gln Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro
290 295 300Gly Asn Leu Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro
Val Phe305 310 315 320Glu Cys Gly Asn Val Val Leu Arg Thr Asp Glu
Arg Asp Val Asn Tyr 325 330 335Trp Thr Ser Arg Tyr Trp Leu Asn Gly
Asp Phe Arg Lys Gly Asp Val 340 345 350Ser Leu Thr Ile Glu Asn Val
Thr Leu Ala Asp Ser Gly Ile Tyr Cys 355 360 365Cys Arg Ile Gln Ile
Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu 370 375 380Lys Leu Val
Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln385 390 395
400Arg Asp Phe Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly His
405 410 415Gly Pro Ala Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile
Asn Leu 420 425 430Thr Gln Ile Ser Thr Leu Ala Asn Glu Leu Arg Asp
Ser Arg Leu Ala 435 440 445Asn Asp Leu Arg Asp Ser Gly Ala Thr Ile
Arg Phe Trp Val Leu Val 450 455 460Val Val Gly Gly Val Leu Ala Cys
Tyr Ser Leu Leu Val Thr Val Ala465 470 475 480Phe Ile Ile Phe Trp
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser 485 490 495Asp Tyr Met
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 500 505 510Tyr
Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val 515 520
525Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu
530 535 540Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu
Leu Pro545 550 555 560Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu
Val Gln Leu Val Gln 565 570 575Ser Gly Ala Glu Val Lys Lys Pro Gly
Glu Ser Leu Lys Ile Ser Cys 580 585 590Lys Gly Ser Gly Tyr Ser Phe
Thr Ser Asn Trp Ile Gly Trp Val Arg 595 600 605Gln Met Pro Gly Lys
Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly 610 615 620Asp Ser Asp
Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile625 630 635
640Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu
645 650 655Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr
Gly Phe 660 665 670Leu Trp Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu
Val Thr Val Ser 675 680 685Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 690 695 700Ala Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly705 710 715 720Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 725 730 735Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 740 745 750Tyr
Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 755 760
765Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
770 775 780Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr
Pro Leu785 790 795 800Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Ile Lys Thr Thr Thr 805 810 815Pro Ala Pro Arg Pro Pro Thr Pro Ala
Pro Thr Ile Ala Ser Gln Pro 820 825 830Leu Ser Leu Arg Pro Glu Ala
Cys Arg Pro Ala Ala Gly Gly Ala Val 835 840 845His Thr Arg Gly Leu
Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly 850 855 860Cys Ala Ala
Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys865 870 875
880Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly
885 890 895Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser
Arg Leu 900 905 910Thr Asp Val Thr Leu Arg Val Lys Phe Ser Arg Ser
Ala Asp Ala Pro 915 920 925Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
Asn Glu Leu Asn Leu Gly 930 935 940Arg Arg Glu Glu Tyr Asp Val Leu
Asp Lys Arg Arg Gly Arg Asp Pro945 950 955 960Glu Met Gly Gly Lys
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu 965 970 975Tyr Asn Glu
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 980 985 990Gly
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 995
1000 1005Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
His 1010 1015 1020Met Gln Ala Leu Pro Pro Arg1025
1030232741PRTArtificial SequencePD1-CD28-2F5PSMA-CAR varICOS CD3z
232Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1
5 10 15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro
Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu
Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu
Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr
Asp Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly
Gln Asp Cys Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp
Phe His Met Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly
Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln
Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg
Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro145 150 155
160Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Phe Trp Val Leu Val Val
165 170 175Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
Ala Phe 180 185 190Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
Leu His Ser Asp 195 200 205Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
Pro Thr Arg Lys His Tyr 210 215 220Gln Pro Tyr Ala Pro Pro Arg Asp
Phe Ala Ala Tyr Arg Ser Val Lys225 230 235 240Gln Thr Leu Asn Phe
Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 245 250 255Asn Pro Gly
Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu 260 265 270Ala
Leu Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser 275 280
285Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys
290 295 300Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val
Arg Gln305 310 315 320Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile
Ile Tyr Pro Gly Asp 325 330 335Ser Asp Thr Arg Tyr Ser Pro Ser Phe
Gln Gly Gln Val Thr Ile Ser 340 345 350Ala Asp Lys Ser Ile Ser Thr
Ala Tyr Leu Gln Trp Asn Ser Leu Lys 355 360 365Ala Ser Asp Thr Ala
Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu 370 375 380Trp Ser Phe
Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser385 390 395
400Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
405 410 415Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp 420 425 430Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile
Ser Ser Ala Leu 435 440 445Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr 450 455 460Asp Ala Ser Ser Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser465 470 475 480Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 485 490 495Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr 500 505 510Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro 515 520
525Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
530 535 540Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
Val His545 550 555 560Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp
Leu Pro Ile Gly Cys 565 570 575Ala Ala Phe Val Val Val Cys Ile Leu
Gly Cys Ile Leu Ile Cys Trp 580 585 590Leu Thr Lys Lys Lys Tyr Ser
Ser Ser Val His Asp Pro Asn Gly Glu 595 600 605Tyr Met Asn Met Arg
Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr 610 615 620Asp Val Thr
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala625 630 635
640Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
645 650 655Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
Pro Glu 660 665 670Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln
Glu Gly Leu Tyr 675 680 685Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
Ala Tyr Ser Glu Ile Gly 690 695 700Met Lys Gly Glu Arg Arg Arg Gly
Lys Gly His Asp Gly Leu Tyr Gln705 710 715 720Gly Leu Ser Thr Ala
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 725 730 735Ala Leu Pro
Pro Arg 740233735PRTArtificial SequencePD1A132L-CD28-2F5PSMA-CAR
ICOS CD3z 233Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala
Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro
Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val
Val Thr Glu Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn
Thr Ser Glu Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser
Asn Gln Thr Asp Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser
Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn
Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110Ala Arg Arg Asn
Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro
Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135
140Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser
Pro145 150 155 160Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly
Val Val Gly Gly 165 170 175Leu Leu Gly Ser Leu Val Leu Leu Val Trp
Val Leu Ala Val Ile Arg 180 185 190Ser Lys Arg Ser Arg Leu Leu His
Ser Asp Tyr Met Asn Met Thr Pro 195 200 205Arg Arg Pro Gly Pro Thr
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 210 215 220Arg Asp Phe Ala
Ala Tyr Arg Ser Val Lys Gln Thr Leu Asn Phe Asp225 230 235 240Leu
Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Ala 245 250
255Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala
260 265 270Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys 275 280 285Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Ser Phe 290 295 300Thr Ser Asn Trp Ile Gly Trp Val Arg Gln
Met Pro Gly Lys Gly Leu305 310 315 320Glu Trp Met Gly Ile Ile Tyr
Pro Gly Asp Ser Asp Thr Arg Tyr Ser 325 330 335Pro Ser Phe Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser 340 345 350Thr Ala Tyr
Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met 355 360 365Tyr
Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp Leu Trp 370 375
380Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly385 390 395 400Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln
Leu Thr Gln Ser 405 410 415Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys 420 425 430Arg Ala Ser Gln Asp Ile Ser Ser
Ala Leu Ala Trp Tyr Gln Gln Lys 435 440 445Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu 450 455 460Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe465 470 475 480Thr
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 485 490
495Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys
500 505 510Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro
Pro Thr 515 520 525Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
Arg Pro Glu Ala 530 535 540Cys Arg Pro Ala Ala Gly Gly Ala Val His
Thr Arg Gly Leu Asp Phe545 550 555 560Ala Cys Asp Phe Trp Leu Pro
Ile Gly Cys Ala Ala Phe Val Val Val 565 570 575Cys Ile Leu Gly Cys
Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr 580 585 590Ser Ser Ser
Val His Asp Pro Asn Gly Glu Tyr Met Asn Met Arg Ala 595 600 605Val
Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu Arg Val 610 615
620Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
Asn625 630 635 640Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
Glu Tyr Asp Val 645 650 655Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
Met Gly Gly Lys Pro Gln 660 665 670Arg Arg Lys Asn Pro Gln Glu Gly
Leu Tyr Asn Glu Leu Gln Lys Asp 675 680 685Lys
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 690 695
700Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
Thr705 710 715 720Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
Pro Pro Arg 725 730 735234740PRTArtificial
SequencePD1A132L-41BB-2F5PSMA-CAR varICOS CD3z 234Met Gln Ile Pro
Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp
Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro
Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn
Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55
60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65
70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe
Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val
Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly
Ala Ile Ser Leu 115 120 125Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu
Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro
Thr Ala His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln
Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro 165 170 175Leu Ala Gly
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 180 185 190Tyr
Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 195 200
205Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
210 215 220Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Val
Lys Gln225 230 235 240Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
Asp Val Glu Ser Asn 245 250 255Pro Gly Pro Met Ala Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala 260 265 270Leu Leu Leu His Ala Ala Arg
Pro Glu Val Gln Leu Val Gln Ser Gly 275 280 285Ala Glu Val Lys Lys
Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly 290 295 300Ser Gly Tyr
Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met305 310 315
320Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser
325 330 335Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile
Ser Ala 340 345 350Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn
Ser Leu Lys Ala 355 360 365Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg
Gln Thr Gly Phe Leu Trp 370 375 380Ser Phe Asp Leu Trp Gly Arg Gly
Thr Leu Val Thr Val Ser Ser Gly385 390 395 400Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile 405 410 415Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 420 425 430Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala 435 440
445Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp
450 455 460Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly465 470 475 480Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp 485 490 495Phe Ala Thr Tyr Tyr Cys Gln Gln Phe
Asn Ser Tyr Pro Leu Thr Phe 500 505 510Gly Gly Gly Thr Lys Val Glu
Ile Lys Ile Lys Thr Thr Thr Pro Ala 515 520 525Pro Arg Pro Pro Thr
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 530 535 540Leu Arg Pro
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr545 550 555
560Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala
565 570 575Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys
Trp Leu 580 585 590Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro
Asn Gly Glu Tyr 595 600 605Met Asn Met Arg Ala Val Asn Thr Ala Lys
Lys Ser Arg Leu Thr Asp 610 615 620Val Thr Leu Arg Val Lys Phe Ser
Arg Ser Ala Asp Ala Pro Ala Tyr625 630 635 640Gln Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 645 650 655Glu Glu Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 660 665 670Gly
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 675 680
685Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
690 695 700Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
Gln Gly705 710 715 720Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
Leu His Met Gln Ala 725 730 735Leu Pro Pro Arg
740235771PRTArtificial SequenceTIM3-CD28-2F5PSMA-CAR varICOS CD3z
235Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1
5 10 15Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly
Gln 20 25 30Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly
Asn Leu 35 40 45Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe
Glu Cys Gly 50 55 60Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn
Tyr Trp Thr Ser65 70 75 80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys
Gly Asp Val Ser Leu Thr 85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser
Gly Ile Tyr Cys Cys Arg Ile 100 105 110Gln Ile Pro Gly Ile Met Asn
Asp Glu Lys Phe Asn Leu Lys Leu Val 115 120 125Ile Lys Pro Ala Lys
Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe 130 135 140Thr Ala Ala
Phe Pro Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala145 150 155
160Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile
165 170 175Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn
Asp Leu 180 185 190Arg Asp Ser Gly Ala Thr Ile Arg Phe Trp Val Leu
Val Val Val Gly 195 200 205Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
Thr Val Ala Phe Ile Ile 210 215 220Phe Trp Val Arg Ser Lys Arg Ser
Arg Leu Leu His Ser Asp Tyr Met225 230 235 240Asn Met Thr Pro Arg
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 245 250 255Tyr Ala Pro
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Gln Thr 260 265 270Leu
Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro 275 280
285Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
290 295 300Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser
Gly Ala305 310 315 320Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile
Ser Cys Lys Gly Ser 325 330 335Gly Tyr Ser Phe Thr Ser Asn Trp Ile
Gly Trp Val Arg Gln Met Pro 340 345 350Gly Lys Gly Leu Glu Trp Met
Gly Ile Ile Tyr Pro Gly Asp Ser Asp 355 360 365Thr Arg Tyr Ser Pro
Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp 370 375 380Lys Ser Ile
Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser385 390 395
400Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser
405 410 415Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
Gly Gly 420 425 430Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala Ile Gln 435 440 445Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly Asp Arg Val 450 455 460Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Ser Ala Leu Ala Trp465 470 475 480Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala 485 490 495Ser Ser Leu
Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 500 505 510Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 515 520
525Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly
530 535 540Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro
Ala Pro545 550 555 560Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
Gln Pro Leu Ser Leu 565 570 575Arg Pro Glu Ala Cys Arg Pro Ala Ala
Gly Gly Ala Val His Thr Arg 580 585 590Gly Leu Asp Phe Ala Cys Asp
Phe Trp Leu Pro Ile Gly Cys Ala Ala 595 600 605Phe Val Val Val Cys
Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr 610 615 620Lys Lys Lys
Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met625 630 635
640Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val
645 650 655Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
Tyr Gln 660 665 670Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
Gly Arg Arg Glu 675 680 685Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg Asp Pro Glu Met Gly 690 695 700Gly Lys Pro Gln Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn Glu705 710 715 720Leu Gln Lys Asp Lys
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 725 730 735Gly Glu Arg
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 740 745 750Ser
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 755 760
765Pro Pro Arg 7702361030PRTArtificial
SequencePD1A132L-4-1BB-TIM3-CD28-2F5PSMA-CAR varICOS CD3z 236Met
Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10
15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly
Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser
Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp
Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln
Asp Cys Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe
His Met Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr
Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Leu Gln Ile
Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg
Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro145 150 155 160Arg
Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro 165 170
175Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
180 185 190Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
Lys Gln 195 200 205Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
Asp Gly Cys Ser 210 215 220Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
Cys Glu Leu Val Lys Gln225 230 235 240Thr Leu Asn Phe Asp Leu Leu
Lys Leu Ala Gly Asp Val Glu Ser Asn 245 250 255Pro Gly Pro Met Phe
Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu 260 265 270Leu Leu Leu
Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu 275 280 285Val
Gly Gln Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro 290 295
300Gly Asn Leu Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val
Phe305 310 315 320Glu Cys Gly Asn Val Val Leu Arg Thr Asp Glu Arg
Asp Val Asn Tyr 325 330 335Trp Thr Ser Arg Tyr Trp Leu Asn Gly Asp
Phe Arg Lys Gly Asp Val 340 345 350Ser Leu Thr Ile Glu Asn Val Thr
Leu Ala Asp Ser Gly Ile Tyr Cys 355 360 365Cys Arg Ile Gln Ile Pro
Gly Ile Met Asn Asp Glu Lys Phe Asn Leu 370 375 380Lys Leu Val Ile
Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln385 390 395 400Arg
Asp Phe Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly His 405 410
415Gly Pro Ala Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu
420 425 430Thr Gln Ile Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg
Leu Ala 435 440 445Asn Asp Leu Arg Asp Ser Gly Ala Thr Ile Arg Phe
Trp Val Leu Val 450 455 460Val Val Gly Gly Val Leu Ala Cys Tyr Ser
Leu Leu Val Thr Val Ala465 470 475 480Phe Ile Ile Phe Trp Val Arg
Ser Lys Arg Ser Arg Leu Leu His Ser 485 490 495Asp Tyr Met Asn Met
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 500 505 510Tyr Gln Pro
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val 515 520 525Lys
Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu 530 535
540Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro545 550 555 560Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val
Gln Leu Val Gln 565 570 575Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
Ser Leu Lys Ile Ser Cys 580 585 590Lys Gly Ser Gly Tyr Ser Phe Thr
Ser Asn Trp Ile Gly Trp Val Arg 595 600 605Gln Met Pro Gly Lys Gly
Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly 610 615 620Asp Ser Asp Thr
Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile625 630 635 640Ser
Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu 645 650
655Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe
660 665 670Leu Trp Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr
Val Ser 675 680 685Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 690 695 700Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly705 710 715 720Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Ser Ala 725 730 735Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 740 745 750Tyr Asp Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 755 760 765Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 770 775
780Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro
Leu785 790 795 800Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ile
Lys Thr Thr Thr 805 810 815Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
Thr Ile Ala Ser Gln Pro 820 825 830Leu Ser Leu Arg Pro Glu Ala Cys
Arg Pro Ala Ala Gly Gly Ala Val 835 840 845His Thr Arg Gly Leu Asp
Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly 850 855 860Cys Ala Ala Phe
Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys865 870 875
880Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly
885 890 895Glu Tyr Met Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser
Arg Leu 900 905 910Thr Asp Val Thr Leu Arg Val Lys Phe Ser Arg Ser
Ala Asp Ala Pro 915 920 925Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
Asn Glu Leu Asn Leu Gly 930 935 940Arg Arg Glu Glu Tyr Asp Val Leu
Asp Lys Arg Arg Gly Arg Asp Pro945 950 955 960Glu Met Gly Gly Lys
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu 965 970 975Tyr Asn Glu
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 980 985 990Gly
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 995
1000 1005Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
His 1010 1015 1020Met Gln Ala Leu Pro Pro Arg1025
1030237237PRTArtificial SequencehuJ591 VH-VK scfv 237Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly
Thr Thr Val Thr 100 105 110Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Ser Gly Gly 115 120 125Gly Ser Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser 130 135 140Val Gly Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asp Val Gly145 150 155 160Thr Ala Val
Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu 165 170 175Leu
Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe 180 185
190Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
195 200 205Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn
Ser Tyr 210 215 220Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Asp Ile
Lys225 230 235238711DNAArtificial SequencehuJ591 VH-VK scfv
238gaggtccagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata cacattcact gaatacacca tccactgggt
gaggcaggcc 120cctggaaagg gccttgagtg gattggaaac attaatccta
acaatggtgg tactacctac 180aaccagaagt tcgaggacag agtcacaatc
actgtagaca agtccaccag cacagcctac 240atggagctca gcagcctgag
atctgaggat actgcagtct attactgtgc agctggttgg 300aactttgact
actggggcca aggcaccacg gtcaccgtct cctcaggagg cggaggatct
360ggcggcggag gaagttctgg cggaggcagc gacattcaga tgacccagtc
tcccagcacc 420ctgtccgcat cagtaggaga cagggtcacc atcacttgca
aggccagtca ggatgtgggt 480actgctgtag actggtatca acagaaacca
gggcaagctc ctaaactact gatttactgg 540gcatccaccc ggcacactgg
agtccctgat cgcttcagcg gcagtggatc tgggacagat 600ttcactctca
ccatcagcag actgcagcct gaagactttg cagtttatta ctgtcagcaa
660tataacagct atcctctcac gttcggccag gggaccaagg tggatatcaa a
711239237PRTArtificial SequencehuJ591 VK-VH scFv 239Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30Val Asp
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro65
70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro
Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Gly Gly Gly
Gly Ser 100 105 110Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Glu Val
Gln Leu Val Gln 115 120 125Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
Ser Val Lys Val Ser Cys 130 135 140Lys Ala Ser Gly Tyr Thr Phe Thr
Glu Tyr Thr Ile His Trp Val Arg145 150 155 160Gln Ala Pro Gly Lys
Gly Leu Glu Trp Ile Gly Asn Ile Asn Pro Asn 165 170 175Asn Gly Gly
Thr Thr Tyr Asn Gln Lys Phe Glu Asp Arg Val Thr Ile 180 185 190Thr
Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu 195 200
205Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe
210 215 220Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser225
230 235240711DNAArtificial SequencehuJ591 VK-VH scfv 240gacattcaga
tgacccagtc tcccagcacc ctgtccgcat cagtaggaga cagggtcacc 60atcacttgca
aggccagtca ggatgtgggt actgctgtag actggtatca acagaaacca
120gggcaagctc ctaaactact gatttactgg gcatccaccc ggcacactgg
agtccctgat 180cgcttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag actgcagcct 240gaagactttg cagtttatta ctgtcagcaa
tataacagct atcctctcac gttcggccag 300gggaccaagg tggatatcaa
aggaggcgga ggatctggcg gcggaggaag ttctggcgga 360ggcagcgagg
tccagctggt gcagtctgga gctgaggtga agaagcctgg ggcctcagtg
420aaggtctcct gcaaggcttc tggatacaca ttcactgaat acaccatcca
ctgggtgagg 480caggcccctg gaaagggcct tgagtggatt ggaaacatta
atcctaacaa tggtggtact 540acctacaacc agaagttcga ggacagagtc
acaatcactg tagacaagtc caccagcaca 600gcctacatgg agctcagcag
cctgagatct gaggatactg cagtctatta ctgtgcagct 660ggttggaact
ttgactactg gggccaaggc accacggtca ccgtctcctc a
711241222PRTArtificial SequencehuJ591 VK-VH scfv sequence without
linker 241Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val
Gly Thr Ala 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro
Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Asp Ile Lys Glu Val Gln Leu Val 100 105 110Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser 115 120 125Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp Val 130 135 140Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Asn Ile Asn Pro145 150
155 160Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu Asp Arg Val
Thr 165 170 175Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu
Leu Ser Ser 180 185 190Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Gly Trp Asn 195 200 205Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 210 215 220242345DNAArtificial SequencehuJ591
VH 242gaggtccagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata cacattcact gaatacacca tccactgggt
gaggcaggcc 120cctggaaagg gccttgagtg gattggaaac attaatccta
acaatggtgg tactacctac 180aaccagaagt tcgaggacag agtcacaatc
actgtagaca agtccaccag cacagcctac 240atggagctca gcagcctgag
atctgaggat actgcagtct attactgtgc agctggttgg 300aactttgact
actggggcca aggcaccacg gtcaccgtct cctca 3452435PRTArtificial
SequencehuJ591 CDR1 243Glu Tyr Thr Ile His1 5244321DNAArtificial
SequencehuJ591 VL 244gacattcaga tgacccagtc tcccagcacc ctgtccgcat
cagtaggaga cagggtcacc 60atcacttgca aggccagtca ggatgtgggt actgctgtag
actggtatca acagaaacca 120gggcaagctc ctaaactact gatttactgg
gcatccaccc ggcacactgg agtccctgat 180cgcttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag actgcagcct 240gaagactttg
cagtttatta ctgtcagcaa tataacagct atcctctcac gttcggccag
300gggaccaagg tggatatcaa a 321245482PRTArtificial
SequencehuJ591VHVK.BBZ CAR 245Met Ala Leu Pro Val Thr Ala Leu Leu
Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gly Glu Val
Gln Leu Val Gln Ser Gly Ala Glu 20 25 30Val Lys Lys Pro Gly Ala Ser
Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45Tyr Thr Phe Thr Glu Tyr
Thr Ile His Trp Val Arg Gln Ala Pro Gly 50 55 60Lys Gly Leu Glu Trp
Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr65 70 75 80Thr Tyr Asn
Gln Lys Phe Glu Asp Arg Val Thr Ile Thr Val Asp Lys 85 90 95Ser Thr
Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 100 105
110Thr Ala Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
115 120 125Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Gln Met
Thr Gln Ser Pro145 150 155 160Ser Thr Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Lys 165 170 175Ala Ser Gln Asp Val Gly Thr
Ala Val Asp Trp Tyr Gln Gln Lys Pro 180 185 190Gly Gln Ala Pro Lys
Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr 195 200 205Gly Val Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 210 215 220Leu
Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys225 230
235 240Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys
Val 245 250 255Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
Pro Ala Pro 260 265 270Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
Glu Ala Cys Arg Pro 275 280 285Ala Ala Gly Gly Ala Val His Thr Arg
Gly Leu Asp Phe Ala Cys Asp 290 295 300Ile Tyr Ile Trp Ala Pro Leu
Ala Gly Thr Cys Gly Val Leu Leu Leu305 310 315 320Ser Leu Val Ile
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu 325 330 335Tyr Ile
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 340 345
350Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
355 360 365Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
Tyr Lys 370 375 380Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
Gly Arg Arg Glu385 390 395 400Glu Tyr Asp Val Leu Asp Lys Arg Arg
Gly Arg Asp Pro Glu Met Gly 405 410 415Gly Lys Pro Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn Glu Leu 420 425 430Gln Lys Asp Lys Met
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 435 440 445Glu Arg Arg
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 450 455 460Thr
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro465 470
475 480Pro Arg2461446DNAArtificial SequencehuJ591VHVK.BBZ CAR
246atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca
cgccgccaga 60cctggagagg tccagctggt gcagtctgga gctgaggtga agaagcctgg
ggcctcagtg 120aaggtctcct gcaaggcttc tggatacaca ttcactgaat
acaccatcca ctgggtgagg 180caggcccctg gaaagggcct tgagtggatt
ggaaacatta atcctaacaa tggtggtact 240acctacaacc agaagttcga
ggacagagtc acaatcactg tagacaagtc caccagcaca 300gcctacatgg
agctcagcag cctgagatct gaggatactg cagtctatta ctgtgcagct
360ggttggaact ttgactactg gggccaaggc accacggtca ccgtctcctc
aggaggcgga 420ggatctggcg gcggaggaag ttctggcgga ggcagcgaca
ttcagatgac ccagtctccc 480agcaccctgt ccgcatcagt aggagacagg
gtcaccatca cttgcaaggc cagtcaggat 540gtgggtactg ctgtagactg
gtatcaacag aaaccagggc aagctcctaa actactgatt 600tactgggcat
ccacccggca cactggagtc cctgatcgct tcagcggcag tggatctggg
660acagatttca ctctcaccat cagcagactg cagcctgaag actttgcagt
ttattactgt 720cagcaatata acagctatcc tctcacgttc ggccagggga
ccaaggtgga tatcaaaacc 780acgacgccag cgccgcgacc accaacaccg
gcgcccacca tcgcgtcgca gcccctgtcc 840ctgcgcccag aggcgtgccg
gccagcggcg gggggcgcag tgcacacgag ggggctggac 900ttcgcctgtg
atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg
960tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta
tatattcaaa 1020caaccattta tgagaccagt acaaactact caagaggaag
acggctgtag ctgccgattt 1080ccagaagaag aagaaggagg atgtgaactg
agagtgaagt tcagcaggag cgcagacgcc 1140cccgcgtaca agcagggcca
gaaccagctc tataacgagc tcaatctagg acgaagagag 1200gagtacgacg
ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga
1260aggaagaacc ctcaggaagg cctgtacaac gaactgcaga aagataagat
ggcggaggcc 1320tacagtgaga ttgggatgaa aggcgagcgc cggaggggca
aggggcacga cggcctttac 1380cagggtctca gtacagccac caaggacacc
tacgacgccc ttcacatgca ggccctgccc 1440cctcgc 1446247482PRTArtificial
SequencehuJ591VKVH.BBZ CAR 247Met Ala Leu Pro Val Thr Ala Leu Leu
Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gly Asp Ile
Gln Met Thr Gln Ser Pro Ser Thr 20 25 30Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45Gln Asp Val Gly Thr Ala
Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60Ala Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val65 70 75 80Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser
Arg Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 100 105
110Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Asp Ile
115 120 125Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly
Gly Ser 130 135 140Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala145 150 155 160Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Glu Tyr 165 170 175Thr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 180 185 190Gly Asn Ile Asn Pro
Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 195 200 205Glu Asp Arg
Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 210 215 220Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys225 230
235 240Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val
Thr 245 250 255Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
Pro Ala Pro 260 265 270Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
Glu Ala Cys Arg Pro 275 280 285Ala Ala Gly Gly Ala Val His Thr Arg
Gly Leu Asp Phe Ala Cys Asp 290 295 300Ile Tyr Ile Trp Ala Pro Leu
Ala Gly Thr Cys Gly Val Leu Leu Leu305 310 315 320Ser Leu Val Ile
Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu 325 330 335Tyr Ile
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 340 345
350Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
355 360 365Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
Tyr Lys 370 375 380Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
Gly Arg Arg Glu385 390 395 400Glu Tyr Asp Val Leu Asp Lys Arg Arg
Gly Arg Asp Pro Glu Met Gly 405 410 415Gly Lys Pro Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn Glu Leu 420 425 430Gln Lys Asp Lys Met
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 435 440 445Glu Arg Arg
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 450 455 460Thr
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro465
470
475 480Pro Arg2481446DNAArtificial SequencehuJ591VKVH.BBZ CAR
248atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca
cgccgccaga 60cctggagaca ttcagatgac ccagtctccc agcaccctgt ccgcatcagt
aggagacagg 120gtcaccatca cttgcaaggc cagtcaggat gtgggtactg
ctgtagactg gtatcaacag 180aaaccagggc aagctcctaa actactgatt
tactgggcat ccacccggca cactggagtc 240cctgatcgct tcagcggcag
tggatctggg acagatttca ctctcaccat cagcagactg 300cagcctgaag
actttgcagt ttattactgt cagcaatata acagctatcc tctcacgttc
360ggccagggga ccaaggtgga tatcaaagga ggcggaggat ctggcggcgg
aggaagttct 420ggcggaggca gcgaggtcca gctggtgcag tctggagctg
aggtgaagaa gcctggggcc 480tcagtgaagg tctcctgcaa ggcttctgga
tacacattca ctgaatacac catccactgg 540gtgaggcagg cccctggaaa
gggccttgag tggattggaa acattaatcc taacaatggt 600ggtactacct
acaaccagaa gttcgaggac agagtcacaa tcactgtaga caagtccacc
660agcacagcct acatggagct cagcagcctg agatctgagg atactgcagt
ctattactgt 720gcagctggtt ggaactttga ctactggggc caaggcacca
cggtcaccgt ctcctcaacc 780acgacgccag cgccgcgacc accaacaccg
gcgcccacca tcgcgtcgca gcccctgtcc 840ctgcgcccag aggcgtgccg
gccagcggcg gggggcgcag tgcacacgag ggggctggac 900ttcgcctgtg
atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg
960tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta
tatattcaaa 1020caaccattta tgagaccagt acaaactact caagaggaag
acggctgtag ctgccgattt 1080ccagaagaag aagaaggagg atgtgaactg
agagtgaagt tcagcaggag cgcagacgcc 1140cccgcgtaca agcagggcca
gaaccagctc tataacgagc tcaatctagg acgaagagag 1200gagtacgacg
ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga
1260aggaagaacc ctcaggaagg cctgtacaac gaactgcaga aagataagat
ggcggaggcc 1320tacagtgaga ttgggatgaa aggcgagcgc cggaggggca
aggggcacga cggcctttac 1380cagggtctca gtacagccac caaggacacc
tacgacgccc ttcacatgca ggccctgccc 1440cctcgc 1446249517PRTArtificial
SequencehJ591VKVH.ICOSBBZ CAR 249Met Ala Leu Pro Val Thr Ala Leu
Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gly Asp
Ile Gln Met Thr Gln Ser Pro Ser Thr 20 25 30Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45Gln Asp Val Gly Thr
Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60Ala Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val65 70 75 80Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile
Ser Arg Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 100 105
110Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Asp Ile
115 120 125Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly
Gly Ser 130 135 140Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala145 150 155 160Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Glu Tyr 165 170 175Thr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 180 185 190Gly Asn Ile Asn Pro
Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 195 200 205Glu Asp Arg
Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 210 215 220Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys225 230
235 240Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val
Thr 245 250 255Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
Pro Ala Pro 260 265 270Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
Glu Ala Cys Arg Pro 275 280 285Ala Ala Gly Gly Ala Val His Thr Arg
Gly Leu Asp Phe Ala Cys Asp 290 295 300Phe Trp Leu Pro Ile Gly Cys
Ala Ala Phe Val Val Val Cys Ile Leu305 310 315 320Gly Cys Ile Leu
Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser 325 330 335Val His
Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr 340 345
350Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu Lys Arg Gly Arg Lys
355 360 365Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
Gln Thr 370 375 380Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
Glu Glu Glu Glu385 390 395 400Gly Gly Cys Glu Leu Arg Val Lys Phe
Ser Arg Ser Ala Asp Ala Pro 405 410 415Ala Tyr Lys Gln Gly Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly 420 425 430Arg Arg Glu Glu Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 435 440 445Glu Met Gly
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 450 455 460Asn
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly465 470
475 480Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
Gln 485 490 495Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
His Met Gln 500 505 510Ala Leu Pro Pro Arg 5152501551DNAArtificial
SequencehJ591VKVH.ICOSBBZ CAR 250atggccctgc ctgtgacagc cctgctgctg
cctctggctc tgctgctgca cgccgccaga 60cctggagaca ttcagatgac ccagtctccc
agcaccctgt ccgcatcagt aggagacagg 120gtcaccatca cttgcaaggc
cagtcaggat gtgggtactg ctgtagactg gtatcaacag 180aaaccagggc
aagctcctaa actactgatt tactgggcat ccacccggca cactggagtc
240cctgatcgct tcagcggcag tggatctggg acagatttca ctctcaccat
cagcagactg 300cagcctgaag actttgcagt ttattactgt cagcaatata
acagctatcc tctcacgttc 360ggccagggga ccaaggtgga tatcaaagga
ggcggaggat ctggcggcgg aggaagttct 420ggcggaggca gcgaggtcca
gctggtgcag tctggagctg aggtgaagaa gcctggggcc 480tcagtgaagg
tctcctgcaa ggcttctgga tacacattca ctgaatacac catccactgg
540gtgaggcagg cccctggaaa gggccttgag tggattggaa acattaatcc
taacaatggt 600ggtactacct acaaccagaa gttcgaggac agagtcacaa
tcactgtaga caagtccacc 660agcacagcct acatggagct cagcagcctg
agatctgagg atactgcagt ctattactgt 720gcagctggtt ggaactttga
ctactggggc caaggcacca cggtcaccgt ctcctcaacc 780acgacgccag
cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc
840ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag
ggggctggac 900ttcgcctgtg atttctggtt acccatagga tgtgcagcct
ttgttgtagt ctgcattttg 960ggatgcatac ttatttgttg gcttacaaaa
aagaagtatt catccagtgt gcacgaccct 1020aacggtgaat acatgttcat
gagagcagtg aacacagcca aaaaatccag actcacagat 1080gtgaccctaa
aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga
1140ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga
agaagaagaa 1200ggaggatgtg aactgagagt gaagttcagc aggagcgcag
acgcccccgc gtacaagcag 1260ggccagaacc agctctataa cgagctcaat
ctaggacgaa gagaggagta cgacgttttg 1320gacaagagac gtggccggga
ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1380gaaggcctgt
acaacgaact gcagaaagat aagatggcgg aggcctacag tgagattggg
1440atgaaaggcg agcgccggag gggcaagggg cacgacggcc tttaccaggg
tctcagtaca 1500gccaccaagg acacctacga cgcccttcac atgcaggccc
tgccccctcg c 1551251517PRTArtificial SequencehJ591VKVH.ICOSBBZYMNM
CAR 251Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu
Leu1 5 10 15His Ala Ala Arg Pro Gly Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser 35 40 45Gln Asp Val Gly Thr Ala Val Asp Trp Tyr Gln Gln
Lys Pro Gly Gln 50 55 60Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg His Thr Gly Val65 70 75 80Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Arg Leu Gln Pro Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln 100 105 110Tyr Asn Ser Tyr Pro Leu
Thr Phe Gly Gln Gly Thr Lys Val Asp Ile 115 120 125Lys Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser 130 135 140Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala145 150 155
160Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
165 170 175Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Ile 180 185 190Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr
Asn Gln Lys Phe 195 200 205Glu Asp Arg Val Thr Ile Thr Val Asp Lys
Ser Thr Ser Thr Ala Tyr 210 215 220Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys225 230 235 240Ala Ala Gly Trp Asn
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 245 250 255Val Ser Ser
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 260 265 270Thr
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 275 280
285Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
290 295 300Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys
Ile Leu305 310 315 320Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys
Lys Tyr Ser Ser Ser 325 330 335Val His Asp Pro Asn Gly Glu Tyr Met
Asn Met Arg Ala Val Asn Thr 340 345 350Ala Lys Lys Ser Arg Leu Thr
Asp Val Thr Leu Lys Arg Gly Arg Lys 355 360 365Lys Leu Leu Tyr Ile
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 370 375 380Thr Gln Glu
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu385 390 395
400Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
405 410 415Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
Leu Gly 420 425 430Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
Gly Arg Asp Pro 435 440 445Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr 450 455 460Asn Glu Leu Gln Lys Asp Lys Met
Ala Glu Ala Tyr Ser Glu Ile Gly465 470 475 480Met Lys Gly Glu Arg
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 485 490 495Gly Leu Ser
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 500 505 510Ala
Leu Pro Pro Arg 5152521551DNAArtificial
SequencehJ591VKVH.ICOSBBZYMNM CAR 252atggccctgc ctgtgacagc
cctgctgctg cctctggctc tgctgctgca cgccgccaga 60cctggagaca ttcagatgac
ccagtctccc agcaccctgt ccgcatcagt aggagacagg 120gtcaccatca
cttgcaaggc cagtcaggat gtgggtactg ctgtagactg gtatcaacag
180aaaccagggc aagctcctaa actactgatt tactgggcat ccacccggca
cactggagtc 240cctgatcgct tcagcggcag tggatctggg acagatttca
ctctcaccat cagcagactg 300cagcctgaag actttgcagt ttattactgt
cagcaatata acagctatcc tctcacgttc 360ggccagggga ccaaggtgga
tatcaaagga ggcggaggat ctggcggcgg aggaagttct 420ggcggaggca
gcgaggtcca gctggtgcag tctggagctg aggtgaagaa gcctggggcc
480tcagtgaagg tctcctgcaa ggcttctgga tacacattca ctgaatacac
catccactgg 540gtgaggcagg cccctggaaa gggccttgag tggattggaa
acattaatcc taacaatggt 600ggtactacct acaaccagaa gttcgaggac
agagtcacaa tcactgtaga caagtccacc 660agcacagcct acatggagct
cagcagcctg agatctgagg atactgcagt ctattactgt 720gcagctggtt
ggaactttga ctactggggc caaggcacca cggtcaccgt ctcctcaacc
780acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca
gcccctgtcc 840ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag
tgcacacgag ggggctggac 900ttcgcctgtg atttctggtt acccatagga
tgtgcagcct ttgttgtagt ctgcattttg 960ggatgcatac ttatttgttg
gcttacaaaa aagaagtatt catccagtgt gcacgaccct 1020aacggtgaat
acatgaacat gagagcagtg aacacagcca aaaaatccag actcacagat
1080gtgaccctaa aacggggcag aaagaaactc ctgtatatat tcaaacaacc
atttatgaga 1140ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc
gatttccaga agaagaagaa 1200ggaggatgtg aactgagagt gaagttcagc
aggagcgcag acgcccccgc gtacaagcag 1260ggccagaacc agctctataa
cgagctcaat ctaggacgaa gagaggagta cgacgttttg 1320gacaagagac
gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag
1380gaaggcctgt acaacgaact gcagaaagat aagatggcgg aggcctacag
tgagattggg 1440atgaaaggcg agcgccggag gggcaagggg cacgacggcc
tttaccaggg tctcagtaca 1500gccaccaagg acacctacga cgcccttcac
atgcaggccc tgccccctcg c 1551253475PRTArtificial
SequencehJ591VKVH.ICOSZ CAR 253Met Ala Leu Pro Val Thr Ala Leu Leu
Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gly Asp Ile
Gln Met Thr Gln Ser Pro Ser Thr 20 25 30Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45Gln Asp Val Gly Thr Ala
Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60Ala Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val65 70 75 80Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser
Arg Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 100 105
110Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Asp Ile
115 120 125Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly
Gly Ser 130 135 140Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala145 150 155 160Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Glu Tyr 165 170 175Thr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 180 185 190Gly Asn Ile Asn Pro
Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 195 200 205Glu Asp Arg
Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 210 215 220Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys225 230
235 240Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val
Thr 245 250 255Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
Pro Ala Pro 260 265 270Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
Glu Ala Cys Arg Pro 275 280 285Ala Ala Gly Gly Ala Val His Thr Arg
Gly Leu Asp Phe Ala Cys Asp 290 295 300Phe Trp Leu Pro Ile Gly Cys
Ala Ala Phe Val Val Val Cys Ile Leu305 310 315 320Gly Cys Ile Leu
Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser 325 330 335Val His
Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr 340 345
350Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu Arg Val Lys Phe Ser
355 360 365Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
Leu Tyr 370 375 380Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
Val Leu Asp Lys385 390 395 400Arg Arg Gly Arg Asp Pro Glu Met Gly
Gly Lys Pro Arg Arg Lys Asn 405 410 415Pro Gln Glu Gly Leu Tyr Asn
Glu Leu Gln Lys Asp Lys Met Ala Glu 420 425 430Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 435 440 445His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 450 455 460Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg465 470
4752541425DNAArtificial SequencehJ591VKVH.ICOSZ CAR 254atggccctgc
ctgtgacagc cctgctgctg cctctggctc tgctgctgca cgccgccaga 60cctggagaca
ttcagatgac ccagtctccc agcaccctgt ccgcatcagt aggagacagg
120gtcaccatca cttgcaaggc cagtcaggat gtgggtactg ctgtagactg
gtatcaacag 180aaaccagggc aagctcctaa actactgatt tactgggcat
ccacccggca cactggagtc 240cctgatcgct tcagcggcag tggatctggg
acagatttca ctctcaccat cagcagactg 300cagcctgaag actttgcagt
ttattactgt cagcaatata acagctatcc tctcacgttc 360ggccagggga
ccaaggtgga tatcaaagga ggcggaggat ctggcggcgg aggaagttct
420ggcggaggca gcgaggtcca gctggtgcag tctggagctg aggtgaagaa
gcctggggcc 480tcagtgaagg tctcctgcaa ggcttctgga tacacattca
ctgaatacac catccactgg 540gtgaggcagg cccctggaaa gggccttgag
tggattggaa acattaatcc taacaatggt 600ggtactacct acaaccagaa
gttcgaggac agagtcacaa tcactgtaga caagtccacc 660agcacagcct
acatggagct cagcagcctg agatctgagg atactgcagt ctattactgt
720gcagctggtt ggaactttga ctactggggc caaggcacca cggtcaccgt
ctcctcaacc 780acgacgccag cgccgcgacc accaacaccg gcgcccacca
tcgcgtcgca gcccctgtcc 840ctgcgcccag aggcgtgccg gccagcggcg
gggggcgcag tgcacacgag ggggctggac 900ttcgcctgtg
atttctggtt acccatagga tgtgcagcct ttgttgtagt ctgcattttg
960ggatgcatac ttatttgttg gcttacaaaa aagaagtatt catccagtgt
gcacgaccct 1020aacggtgaat acatgttcat gagagcagtg aacacagcca
aaaaatccag actcacagat 1080gtgaccctaa gagtgaagtt cagcaggagc
gcagacgccc ccgcgtacaa gcagggccag 1140aaccagctct ataacgagct
caatctagga cgaagagagg agtacgacgt tttggacaag 1200agacgtggcc
gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc
1260ctgtacaacg aactgcagaa agataagatg gcggaggcct acagtgagat
tgggatgaaa 1320ggcgagcgcc ggaggggcaa ggggcacgac ggcctttacc
agggtctcag tacagccacc 1380aaggacacct acgacgccct tcacatgcag
gccctgcccc ctcgc 1425255475PRTArtificial
SequencehJ591VKVH.ICOSZYMNM CAR 255Met Ala Leu Pro Val Thr Ala Leu
Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gly Asp
Ile Gln Met Thr Gln Ser Pro Ser Thr 20 25 30Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45Gln Asp Val Gly Thr
Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60Ala Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val65 70 75 80Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile
Ser Arg Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 100 105
110Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Asp Ile
115 120 125Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly
Gly Ser 130 135 140Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala145 150 155 160Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Glu Tyr 165 170 175Thr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 180 185 190Gly Asn Ile Asn Pro
Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 195 200 205Glu Asp Arg
Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 210 215 220Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys225 230
235 240Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val
Thr 245 250 255Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
Pro Ala Pro 260 265 270Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
Glu Ala Cys Arg Pro 275 280 285Ala Ala Gly Gly Ala Val His Thr Arg
Gly Leu Asp Phe Ala Cys Asp 290 295 300Phe Trp Leu Pro Ile Gly Cys
Ala Ala Phe Val Val Val Cys Ile Leu305 310 315 320Gly Cys Ile Leu
Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser 325 330 335Val His
Asp Pro Asn Gly Glu Tyr Met Asn Met Arg Ala Val Asn Thr 340 345
350Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu Arg Val Lys Phe Ser
355 360 365Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
Leu Tyr 370 375 380Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
Val Leu Asp Lys385 390 395 400Arg Arg Gly Arg Asp Pro Glu Met Gly
Gly Lys Pro Arg Arg Lys Asn 405 410 415Pro Gln Glu Gly Leu Tyr Asn
Glu Leu Gln Lys Asp Lys Met Ala Glu 420 425 430Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 435 440 445His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 450 455 460Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg465 470
4752561425DNAArtificial SequencehJ591VKVH.ICOSZYMNM CAR
256atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca
cgccgccaga 60cctggagaca ttcagatgac ccagtctccc agcaccctgt ccgcatcagt
aggagacagg 120gtcaccatca cttgcaaggc cagtcaggat gtgggtactg
ctgtagactg gtatcaacag 180aaaccagggc aagctcctaa actactgatt
tactgggcat ccacccggca cactggagtc 240cctgatcgct tcagcggcag
tggatctggg acagatttca ctctcaccat cagcagactg 300cagcctgaag
actttgcagt ttattactgt cagcaatata acagctatcc tctcacgttc
360ggccagggga ccaaggtgga tatcaaagga ggcggaggat ctggcggcgg
aggaagttct 420ggcggaggca gcgaggtcca gctggtgcag tctggagctg
aggtgaagaa gcctggggcc 480tcagtgaagg tctcctgcaa ggcttctgga
tacacattca ctgaatacac catccactgg 540gtgaggcagg cccctggaaa
gggccttgag tggattggaa acattaatcc taacaatggt 600ggtactacct
acaaccagaa gttcgaggac agagtcacaa tcactgtaga caagtccacc
660agcacagcct acatggagct cagcagcctg agatctgagg atactgcagt
ctattactgt 720gcagctggtt ggaactttga ctactggggc caaggcacca
cggtcaccgt ctcctcaacc 780acgacgccag cgccgcgacc accaacaccg
gcgcccacca tcgcgtcgca gcccctgtcc 840ctgcgcccag aggcgtgccg
gccagcggcg gggggcgcag tgcacacgag ggggctggac 900ttcgcctgtg
atttctggtt acccatagga tgtgcagcct ttgttgtagt ctgcattttg
960ggatgcatac ttatttgttg gcttacaaaa aagaagtatt catccagtgt
gcacgaccct 1020aacggtgaat acatgaacat gagagcagtg aacacagcca
aaaaatccag actcacagat 1080gtgaccctaa gagtgaagtt cagcaggagc
gcagacgccc ccgcgtacaa gcagggccag 1140aaccagctct ataacgagct
caatctagga cgaagagagg agtacgacgt tttggacaag 1200agacgtggcc
gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc
1260ctgtacaacg aactgcagaa agataagatg gcggaggcct acagtgagat
tgggatgaaa 1320ggcgagcgcc ggaggggcaa ggggcacgac ggcctttacc
agggtctcag tacagccacc 1380aaggacacct acgacgccct tcacatgcag
gccctgcccc ctcgc 1425257706PRTArtificial
SequencednTGF.hJ591VHVK.BBZ CAR 257Met Gly Arg Gly Leu Leu Arg Gly
Leu Trp Pro Leu His Ile Val Leu1 5 10 15Trp Thr Arg Ile Ala Ser Thr
Ile Pro Pro His Val Gln Lys Ser Val 20 25 30Asn Asn Asp Met Ile Val
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45Gln Leu Cys Lys Phe
Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60Lys Ser Cys Met
Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro65 70 75 80Gln Glu
Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95Leu
Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105
110Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys
115 120 125Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu
Cys Asn 130 135 140Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser
Asn Pro Asp Leu145 150 155 160Leu Leu Val Ile Phe Gln Val Thr Gly
Ile Ser Leu Leu Pro Pro Leu 165 170 175Gly Val Ala Ile Ser Val Ile
Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190Arg Gln Gln Lys Leu
Ser Ser Ser Gly Arg Ser Gly Gly Gly Glu Gly 195 200 205Arg Gly Ser
Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 210 215 220Met
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu225 230
235 240His Ala Ala Arg Pro Gly Glu Val Gln Leu Val Gln Ser Gly Ala
Glu 245 250 255Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly 260 265 270Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp Val
Arg Gln Ala Pro Gly 275 280 285Lys Gly Leu Glu Trp Ile Gly Asn Ile
Asn Pro Asn Asn Gly Gly Thr 290 295 300Thr Tyr Asn Gln Lys Phe Glu
Asp Arg Val Thr Ile Thr Val Asp Lys305 310 315 320Ser Thr Ser Thr
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 325 330 335Thr Ala
Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly 340 345
350Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
355 360 365Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
Ser Pro 370 375 380Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Lys385 390 395 400Ala Ser Gln Asp Val Gly Thr Ala Val
Asp Trp Tyr Gln Gln Lys Pro 405 410 415Gly Gln Ala Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg His Thr 420 425 430Gly Val Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 435 440 445Leu Thr Ile
Ser Arg Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys 450 455 460Gln
Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val465 470
475 480Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
Pro 485 490 495Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
Cys Arg Pro 500 505 510Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
Asp Phe Ala Cys Asp 515 520 525Ile Tyr Ile Trp Ala Pro Leu Ala Gly
Thr Cys Gly Val Leu Leu Leu 530 535 540Ser Leu Val Ile Thr Leu Tyr
Cys Lys Arg Gly Arg Lys Lys Leu Leu545 550 555 560Tyr Ile Phe Lys
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 565 570 575Glu Asp
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 580 585
590Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
595 600 605Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
Arg Glu 610 615 620Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
Pro Glu Met Gly625 630 635 640Gly Lys Pro Arg Arg Lys Asn Pro Gln
Glu Gly Leu Tyr Asn Glu Leu 645 650 655Gln Lys Asp Lys Met Ala Glu
Ala Tyr Ser Glu Ile Gly Met Lys Gly 660 665 670Glu Arg Arg Arg Gly
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 675 680 685Thr Ala Thr
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 690 695 700Pro
Arg7052582118DNAArtificial SequencednTGF.hJ591VHVK.BBZ CAR
258atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg
gacgcgtatc 60gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat
agtcactgac 120aacaacggtg cagtcaagtt tccacaactg tgtaaatttt
gtgatgtgag attttccacc 180tgtgacaacc agaaatcctg catgagcaac
tgcagcatca cctccatctg tgagaagcca 240caggaagtct gtgtggctgt
atggagaaag aatgacgaga acataacact agagacagtt 300tgccatgacc
ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag
360tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc
ctgtagctct 420gatgagtgca atgacaacat catcttctca gaagaatata
acaccagcaa tcctgacttg 480ttgctagtca tatttcaagt gacaggcatc
agcctcctgc caccactggg agttgccata 540tctgtcatca tcatcttcta
ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600ggaagatctg
gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag
660aatcccggcc ctatggccct gcctgtgaca gccctgctgc tgcctctggc
tctgctgctg 720cacgccgcca gacctggaga ggtccagctg gtgcagtctg
gagctgaggt gaagaagcct 780ggggcctcag tgaaggtctc ctgcaaggct
tctggataca cattcactga atacaccatc 840cactgggtga ggcaggcccc
tggaaagggc cttgagtgga ttggaaacat taatcctaac 900aatggtggta
ctacctacaa ccagaagttc gaggacagag tcacaatcac tgtagacaag
960tccaccagca cagcctacat ggagctcagc agcctgagat ctgaggatac
tgcagtctat 1020tactgtgcag ctggttggaa ctttgactac tggggccaag
gcaccacggt caccgtctcc 1080tcaggaggcg gaggatctgg cggcggagga
agttctggcg gaggcagcga cattcagatg 1140acccagtctc ccagcaccct
gtccgcatca gtaggagaca gggtcaccat cacttgcaag 1200gccagtcagg
atgtgggtac tgctgtagac tggtatcaac agaaaccagg gcaagctcct
1260aaactactga tttactgggc atccacccgg cacactggag tccctgatcg
cttcagcggc 1320agtggatctg ggacagattt cactctcacc atcagcagac
tgcagcctga agactttgca 1380gtttattact gtcagcaata taacagctat
cctctcacgt tcggccaggg gaccaaggtg 1440gatatcaaaa ccacgacgcc
agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1500cagcccctgt
ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg
1560agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg
gacttgtggg 1620gtccttctcc tgtcactggt tatcaccctt tactgcaaac
ggggcagaaa gaaactcctg 1680tatatattca aacaaccatt tatgagacca
gtacaaacta ctcaagagga agacggctgt 1740agctgccgat ttccagaaga
agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1800agcgcagacg
cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta
1860ggacgaagag aggagtacga cgttttggac aagagacgtg gccgggaccc
tgagatgggg 1920ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca
acgaactgca gaaagataag 1980atggcggagg cctacagtga gattgggatg
aaaggcgagc gccggagggg caaggggcac 2040gacggccttt accagggtct
cagtacagcc accaaggaca cctacgacgc ccttcacatg 2100caggccctgc cccctcgc
2118259706PRTArtificial SequencednTGF.hJ591VKVH.BBZ CAR 259Met Gly
Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu1 5 10 15Trp
Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25
30Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro
35 40 45Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn
Gln 50 55 60Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu
Lys Pro65 70 75 80Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp
Glu Asn Ile Thr 85 90 95Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro
Tyr His Asp Phe Ile 100 105 110Leu Glu Asp Ala Ala Ser Pro Lys Cys
Ile Met Lys Glu Lys Lys Lys 115 120 125Pro Gly Glu Thr Phe Phe Met
Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140Asp Asn Ile Ile Phe
Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu145 150 155 160Leu Leu
Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170
175Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn
180 185 190Arg Gln Gln Lys Leu Ser Ser Ser Gly Arg Ser Gly Gly Gly
Glu Gly 195 200 205Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu
Asn Pro Gly Pro 210 215 220Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro Leu Ala Leu Leu Leu225 230 235 240His Ala Ala Arg Pro Gly Asp
Ile Gln Met Thr Gln Ser Pro Ser Thr 245 250 255Leu Ser Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 260 265 270Gln Asp Val
Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln 275 280 285Ala
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val 290 295
300Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr305 310 315 320Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln 325 330 335Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln
Gly Thr Lys Val Asp Ile 340 345 350Lys Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Ser Gly Gly Gly Ser 355 360 365Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 370 375 380Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr385 390 395 400Thr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 405 410
415Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe
420 425 430Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr
Ala Tyr 435 440 445Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 450 455 460Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Val Thr465 470 475 480Val Ser Ser Thr Thr Thr Pro
Ala Pro Arg Pro Pro Thr Pro Ala Pro 485 490 495Thr Ile Ala Ser Gln
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 500 505 510Ala Ala Gly
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 515 520 525Ile
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 530 535
540Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu
Leu545 550 555 560Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
Thr Thr Gln Glu 565 570
575Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
580 585 590Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
Tyr Lys 595 600 605Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
Gly Arg Arg Glu 610 615 620Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg Asp Pro Glu Met Gly625 630 635 640Gly Lys Pro Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn Glu Leu 645 650 655Gln Lys Asp Lys Met
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 660 665 670Glu Arg Arg
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 675 680 685Thr
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 690 695
700Pro Arg7052602118DNAArtificial SequencednTGF.hJ591VKVH.BBZ CAR
260atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg
gacgcgtatc 60gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat
agtcactgac 120aacaacggtg cagtcaagtt tccacaactg tgtaaatttt
gtgatgtgag attttccacc 180tgtgacaacc agaaatcctg catgagcaac
tgcagcatca cctccatctg tgagaagcca 240caggaagtct gtgtggctgt
atggagaaag aatgacgaga acataacact agagacagtt 300tgccatgacc
ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag
360tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc
ctgtagctct 420gatgagtgca atgacaacat catcttctca gaagaatata
acaccagcaa tcctgacttg 480ttgctagtca tatttcaagt gacaggcatc
agcctcctgc caccactggg agttgccata 540tctgtcatca tcatcttcta
ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600ggaagatctg
gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag
660aatcccggcc ctatggccct gcctgtgaca gccctgctgc tgcctctggc
tctgctgctg 720cacgccgcca gacctggaga cattcagatg acccagtctc
ccagcaccct gtccgcatca 780gtaggagaca gggtcaccat cacttgcaag
gccagtcagg atgtgggtac tgctgtagac 840tggtatcaac agaaaccagg
gcaagctcct aaactactga tttactgggc atccacccgg 900cacactggag
tccctgatcg cttcagcggc agtggatctg ggacagattt cactctcacc
960atcagcagac tgcagcctga agactttgca gtttattact gtcagcaata
taacagctat 1020cctctcacgt tcggccaggg gaccaaggtg gatatcaaag
gaggcggagg atctggcggc 1080ggaggaagtt ctggcggagg cagcgaggtc
cagctggtgc agtctggagc tgaggtgaag 1140aagcctgggg cctcagtgaa
ggtctcctgc aaggcttctg gatacacatt cactgaatac 1200accatccact
gggtgaggca ggcccctgga aagggccttg agtggattgg aaacattaat
1260cctaacaatg gtggtactac ctacaaccag aagttcgagg acagagtcac
aatcactgta 1320gacaagtcca ccagcacagc ctacatggag ctcagcagcc
tgagatctga ggatactgca 1380gtctattact gtgcagctgg ttggaacttt
gactactggg gccaaggcac cacggtcacc 1440gtctcctcaa ccacgacgcc
agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1500cagcccctgt
ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg
1560agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg
gacttgtggg 1620gtccttctcc tgtcactggt tatcaccctt tactgcaaac
ggggcagaaa gaaactcctg 1680tatatattca aacaaccatt tatgagacca
gtacaaacta ctcaagagga agacggctgt 1740agctgccgat ttccagaaga
agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1800agcgcagacg
cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta
1860ggacgaagag aggagtacga cgttttggac aagagacgtg gccgggaccc
tgagatgggg 1920ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca
acgaactgca gaaagataag 1980atggcggagg cctacagtga gattgggatg
aaaggcgagc gccggagggg caaggggcac 2040gacggccttt accagggtct
cagtacagcc accaaggaca cctacgacgc ccttcacatg 2100caggccctgc cccctcgc
2118261699PRTArtificial SequencednTGF.hJ591VKVH.ICOSZYMNM CAR
261Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu1
5 10 15Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser
Val 20 25 30Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys
Phe Pro 35 40 45Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys
Asp Asn Gln 50 55 60Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile
Cys Glu Lys Pro65 70 75 80Gln Glu Val Cys Val Ala Val Trp Arg Lys
Asn Asp Glu Asn Ile Thr 85 90 95Leu Glu Thr Val Cys His Asp Pro Lys
Leu Pro Tyr His Asp Phe Ile 100 105 110Leu Glu Asp Ala Ala Ser Pro
Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125Pro Gly Glu Thr Phe
Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140Asp Asn Ile
Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu145 150 155
160Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu
165 170 175Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg
Val Asn 180 185 190Arg Gln Gln Lys Leu Ser Ser Ser Gly Arg Ser Gly
Gly Gly Glu Gly 195 200 205Arg Gly Ser Leu Leu Thr Cys Gly Asp Val
Glu Glu Asn Pro Gly Pro 210 215 220Met Ala Leu Pro Val Thr Ala Leu
Leu Leu Pro Leu Ala Leu Leu Leu225 230 235 240His Ala Ala Arg Pro
Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Thr 245 250 255Leu Ser Ala
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 260 265 270Gln
Asp Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln 275 280
285Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val
290 295 300Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr305 310 315 320Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln 325 330 335Tyr Asn Ser Tyr Pro Leu Thr Phe Gly
Gln Gly Thr Lys Val Asp Ile 340 345 350Lys Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ser Gly Gly Gly Ser 355 360 365Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 370 375 380Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr385 390 395
400Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
405 410 415Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln
Lys Phe 420 425 430Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr
Ser Thr Ala Tyr 435 440 445Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 450 455 460Ala Ala Gly Trp Asn Phe Asp Tyr
Trp Gly Gln Gly Thr Thr Val Thr465 470 475 480Val Ser Ser Thr Thr
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 485 490 495Thr Ile Ala
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 500 505 510Ala
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 515 520
525Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu
530 535 540Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser
Ser Ser545 550 555 560Val His Asp Pro Asn Gly Glu Tyr Met Asn Met
Arg Ala Val Asn Thr 565 570 575Ala Lys Lys Ser Arg Leu Thr Asp Val
Thr Leu Arg Val Lys Phe Ser 580 585 590Arg Ser Ala Asp Ala Pro Ala
Tyr Lys Gln Gly Gln Asn Gln Leu Tyr 595 600 605Asn Glu Leu Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 610 615 620Arg Arg Gly
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn625 630 635
640Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
645 650 655Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
Lys Gly 660 665 670His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
Lys Asp Thr Tyr 675 680 685Asp Ala Leu His Met Gln Ala Leu Pro Pro
Arg 690 6952622097DNAArtificial SequencednTGF.hJ591VKVH.ICOSZYMNM
CAR 262atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg
gacgcgtatc 60gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat
agtcactgac 120aacaacggtg cagtcaagtt tccacaactg tgtaaatttt
gtgatgtgag attttccacc 180tgtgacaacc agaaatcctg catgagcaac
tgcagcatca cctccatctg tgagaagcca 240caggaagtct gtgtggctgt
atggagaaag aatgacgaga acataacact agagacagtt 300tgccatgacc
ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag
360tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc
ctgtagctct 420gatgagtgca atgacaacat catcttctca gaagaatata
acaccagcaa tcctgacttg 480ttgctagtca tatttcaagt gacaggcatc
agcctcctgc caccactggg agttgccata 540tctgtcatca tcatcttcta
ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600ggaagatctg
gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag
660aatcccggcc ctatggccct gcctgtgaca gccctgctgc tgcctctggc
tctgctgctg 720cacgccgcca gacctggaga cattcagatg acccagtctc
ccagcaccct gtccgcatca 780gtaggagaca gggtcaccat cacttgcaag
gccagtcagg atgtgggtac tgctgtagac 840tggtatcaac agaaaccagg
gcaagctcct aaactactga tttactgggc atccacccgg 900cacactggag
tccctgatcg cttcagcggc agtggatctg ggacagattt cactctcacc
960atcagcagac tgcagcctga agactttgca gtttattact gtcagcaata
taacagctat 1020cctctcacgt tcggccaggg gaccaaggtg gatatcaaag
gaggcggagg atctggcggc 1080ggaggaagtt ctggcggagg cagcgaggtc
cagctggtgc agtctggagc tgaggtgaag 1140aagcctgggg cctcagtgaa
ggtctcctgc aaggcttctg gatacacatt cactgaatac 1200accatccact
gggtgaggca ggcccctgga aagggccttg agtggattgg aaacattaat
1260cctaacaatg gtggtactac ctacaaccag aagttcgagg acagagtcac
aatcactgta 1320gacaagtcca ccagcacagc ctacatggag ctcagcagcc
tgagatctga ggatactgca 1380gtctattact gtgcagctgg ttggaacttt
gactactggg gccaaggcac cacggtcacc 1440gtctcctcaa ccacgacgcc
agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1500cagcccctgt
ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg
1560agggggctgg acttcgcctg tgatttctgg ttacccatag gatgtgcagc
ctttgttgta 1620gtctgcattt tgggatgcat acttatttgt tggcttacaa
aaaagaagta ttcatccagt 1680gtgcacgacc ctaacggtga atacatgaac
atgagagcag tgaacacagc caaaaaatcc 1740agactcacag atgtgaccct
aagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1800aagcagggcc
agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgac
1860gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag
aaggaagaac 1920cctcaggaag gcctgtacaa cgaactgcag aaagataaga
tggcggaggc ctacagtgag 1980attgggatga aaggcgagcg ccggaggggc
aaggggcacg acggccttta ccagggtctc 2040agtacagcca ccaaggacac
ctacgacgcc cttcacatgc aggccctgcc ccctcgc 2097
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