U.S. patent application number 16/395754 was filed with the patent office on 2020-01-16 for cell.
The applicant listed for this patent is UCL BUSINESS PLC. Invention is credited to Shaun Cordoba, Khai Kong, Martin Pule.
Application Number | 20200016204 16/395754 |
Document ID | / |
Family ID | 52003983 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200016204 |
Kind Code |
A1 |
Pule; Martin ; et
al. |
January 16, 2020 |
CELL
Abstract
The present invention provides a cell which co-expresses a first
chimeric antigen receptor (CAR) and second CAR at the cell surface,
each CAR comprising: (i) an antigen-binding domain; (ii) a spacer
(iii) a trans-membrane domain; and (iv) an endodomain wherein the
antigen binding domains of the first and second CARs bind to
different antigens, wherein the spacer of the first CAR is
different to the spacer of the second CAR and wherein one of the
first or second CARs is an activating CAR comprising an activating
endodomain and the other CAR is an inhibitory CAR comprising a
ligation-off inhibitory endodomain.
Inventors: |
Pule; Martin; (London,
GB) ; Kong; Khai; (London, GB) ; Cordoba;
Shaun; (London, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCL BUSINESS PLC |
London |
|
GB |
|
|
Family ID: |
52003983 |
Appl. No.: |
16/395754 |
Filed: |
April 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16192414 |
Nov 15, 2018 |
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16395754 |
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15037414 |
May 18, 2016 |
10172886 |
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PCT/GB2014/053452 |
Nov 21, 2014 |
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16192414 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 2740/10043 20130101; A61K 39/0011 20130101; C07K 2319/03
20130101; C12N 5/0637 20130101; C12N 5/0646 20130101; C07K 2319/74
20130101; C12N 5/0636 20130101; C07K 16/2803 20130101; A61P 37/02
20180101; C07K 2319/02 20130101; C12N 5/0638 20130101; C07K 2317/52
20130101; C12N 2740/15043 20130101; A61K 38/00 20130101; C07K
14/70517 20130101; A61P 37/06 20180101; A61P 37/08 20180101; C07K
14/7051 20130101; A61P 35/02 20180101; C07K 14/70521 20130101; C12Y
301/03048 20130101; C07K 2317/622 20130101; C12N 9/16 20130101;
A61P 31/00 20180101; C12N 2510/00 20130101; A61K 35/17 20130101;
C07K 14/70503 20130101; C07K 14/70589 20130101; C12N 15/86
20130101; C07K 2319/01 20130101; A61K 2039/5158 20130101; A61P
31/12 20180101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; A61K 39/00 20060101 A61K039/00; C07K 14/725 20060101
C07K014/725; C07K 14/705 20060101 C07K014/705; C07K 16/28 20060101
C07K016/28; C12N 5/0783 20060101 C12N005/0783; C12N 15/86 20060101
C12N015/86; C12N 9/16 20060101 C12N009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 21, 2013 |
GB |
1320573.7 |
Jun 19, 2014 |
GB |
1410934.2 |
Claims
1. A T cell which co-expresses a first chimeric antigen receptor
(CAR) and second CAR at the cell surface, each CAR comprising: (i)
an antigen-binding domain; (ii) a spacer (iii) a trans-membrane
domain; and (iv) an endodomain wherein the antigen binding domains
of the first and second CARs bind to different antigens, wherein
the spacer of the first CAR is different to the spacer of the
second CAR and wherein one of the first or second CARs is an
activating CAR comprising an activating endodomain and the other
CAR is an inhibitory CAR comprising a ligation-off inhibitory
endodomain.
2-7. (canceled)
8. A nucleic acid sequence encoding both the first and second
chimeric antigen receptors (CARs) as defined in claim 1.
9. A nucleic acid sequence according to claim 8, which has the
following structure:
AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2 in which AgB1
is a nucleic acid sequence encoding the antigen-binding domain of
the first CAR; spacer 1 is a nucleic acid sequence encoding the
spacer of the first CAR; TM1 is a nucleic acid sequence encoding
the transmembrane domain of the first CAR; endo 1 is a nucleic acid
sequence encoding the endodomain of the first CAR; coexpr is a
nucleic acid sequence enabling co-expression of both CARs AgB2 is a
nucleic acid sequence encoding the antigen-binding domain of the
second CAR; spacer 2 is a nucleic acid sequence encoding the spacer
of the second CAR; TM2 is a nucleic acid sequence encoding the
transmembrane domain of the second CAR; endo 2 is a nucleic acid
sequence encoding the endodomain of the second CAR; which nucleic
acid sequence, when expressed in a T cell, encodes a polypeptide
which is cleaved at the cleavage site such that the first and
second CARs are co-expressed at the T cell surface.
10-14. (canceled)
15. A vector comprising a nucleic acid sequence according to claim
8.
16-18. (canceled)
19. A pharmaceutical composition comprising a plurality of T cells
according to claim 1.
20. A method for treating and/or preventing a disease, which
comprises the step of administering a pharmaceutical composition
according to claim 19 to a subject.
21. (canceled)
22. A method according to claim 20, wherein the disease is a
cancer.
23-24. (canceled)
25. A natural killer (NK) cell which co-expresses a first chimeric
antigen receptor (CAR) and second CAR at the cell surface, each CAR
comprising: (i) an antigen-binding domain; (ii) a spacer (iii) a
trans-membrane domain; and (iv) an endodomain wherein the antigen
binding domains of the first and second CARs bind to different
antigens, wherein the spacer of the first CAR is different to the
spacer of the second CAR and wherein one of the first or second
CARs is an activating CAR comprising an activating endodomain and
the other CAR is an inhibitory CAR comprising a ligation-off
inhibitory endodomain.
26. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a cell which comprises more
than one chimeric antigen receptor (CAR). The cell may be capable
of specifically recognising a target cell, due to a differential
pattern of expression (or non-expression) of two or more antigens
by the target cell.
BACKGROUND TO THE INVENTION
[0002] A number of immunotherapeutic agents have been described for
use in cancer treatment, including therapeutic monoclonal
antibodies (mAbs), immunoconjugated mAbs, radioconjugated mAbs and
bi-specific T-cell engagers.
[0003] Typically these immunotherapeutic agents target a single
antigen: for instance, Rituximab targets CD20; Myelotarg targets
CD33; and Alemtuzumab targets CD52.
[0004] However, it is relatively rare for the presence (or absence)
of a single antigen effectively to describe a cancer, which can
lead to a lack of specificity.
[0005] Most cancers cannot be differentiated from normal tissues on
the basis of a single antigen. Hence, considerable "on-target
off-tumour" toxicity occurs whereby normal tissues are damaged by
the therapy. For instance, whilst targeting CD20 to treat B-cell
lymphomas with Rituximab, the entire normal B-cell compartment is
depleted, whilst targeting CD52 to treat chronic lymphocytic
leukaemia, the entire lymphoid compartment is depleted, whilst
targeting CD33 to treat acute myeloid leukaemia, the entire myeloid
compartment is damaged etc. The predicted problem of "on-target
off-tumour" toxicity has been borne out by clinical trials. For
example, an approach targeting ERBB2 caused death to a patient with
colon cancer metastatic to the lungs and liver. ERBB2 is
over-expressed in colon cancer in some patients, but it is also
expressed on several normal tissues, including heart and normal
vasculature.
[0006] For some cancers, targeting the presence of two cancer
antigens may be more selective and therefore effective than
targeting one. For example, B-chronic lymphocytic leukaemia (B-CLL)
is a common leukaemia which is currently treated by targeting CD19.
This treats the lymphoma but also depletes the entire B-cell
compartment such that the treatment has a considerable toxic
effect. B-CLL has an unusual phenotype in that CD5 and CD19 are
co-expressed. By targeting only cells which express CD5 and CD19,
it would be possible to considerably reduce on-target off-tumour
toxicity.
[0007] There is thus a need for immunotherapeutic agents which are
capable of more targeting to reflect the complex pattern of marker
expression that is associated with many cancers.
Chimeric Antigen Receptors (CARs)
[0008] Chimeric antigen receptors are proteins which graft the
specificity of a monoclonal antibody (mAb) to the effector function
of a T-cell. Their usual form is that of a type I transmembrane
domain protein with an antigen recognizing amino terminus, a
spacer, a transmembrane domain all connected to a compound
endodomain which transmits T-cell survival and activation signals
(see FIG. 1A).
[0009] The most common form of these molecules are fusions of
single-chain variable fragments (scFv) derived from monoclonal
antibodies which recognize a target antigen, fused via a spacer and
a trans-membrane domain to a signaling endodomain. Such molecules
result in activation of the T-cell in response to recognition by
the scFv of its target. When T cells express such a CAR, they
recognize and kill target cells that express the target antigen.
Several CARs have been developed against tumour associated
antigens, and adoptive transfer approaches using such
CAR-expressing T cells are currently in clinical trial for the
treatment of various cancers.
[0010] However, the use of CAR-expressing T cells is also
associated with on-target, off tumour toxicity. For example, a
CAR-based approach targeting carboxy anyhydrase-IX (CAIX) to treat
renal cell carcinoma resulted in liver toxicity which is thought to
be caused by the specific attack on bile duct epithelial cells
(Lamers et al (2013) Mol. Ther. 21:904-912).
Dual Targeting CAR Approaches
[0011] In order to address the problem of "on target, off tumour"
toxicity, CAR T cells have been developed with dual antigen
specificity. In the "dual targeting" approach, two complementary
CARs are co-expressed in the same T-cell population, each directed
to a distant tumour target and engineered to provide complementary
signals.
[0012] Wlikie et al (2012 J Clin Immunol 32:1059-1070) describe a
dual targeting approach in which ErbB2- and MUC1-specific CARs are
co-expressed. The ErbB2-specific CAR provided the CD3.zeta. signal
only and the MUC1-specific CAR provided the CD28 co-stimulatory
signal only. It was found that complementary signalling occurred in
the presence of both antigens, leading to IL-2 production. However,
IL-2 production was modest when compared to control CAR-engineered
T cells in which signaling is delivered by a fused CD28+CD3.zeta.
endodomain.
[0013] A similar approach was described by Kloss et al (2013 Nature
Biotechnol. 31:71-75) in which a CD-19 specific CAR was used which
provides a CD3.zeta.-mediated activation signal in combination with
a chimeric co-stimulatory receptor specific for PSMA. With this
`co-CAR` design, the CAR T-cell receives an activation signal when
it encounters a target cell with one antigen, and a co-stimulatory
signal when it encounters a target cell with the other antigen, and
only receives both activatory and co-stimulatory signals upon
encountering target cells bearing both antigens.
[0014] This represents an early attempt at restricting CAR activity
to only a target cell bearing two antigens. This approach however
is limited: although CAR T-cell activity will be greatest against
targets expressing both antigens, CAR T-cells will still kill
targets expressing only antigen recognized by the activatory CAR;
further, co-stimulation results in prolonged effects on T-cells
which last long after release of target cell. Hence, activity
against single-antigen positive T-cells equal to that against
double-positives might be possible for example in a situation where
single-positive tissues are adjacent to, or in a migratory path
from double positive tumour.
[0015] There is thus a need for improved CAR-based therapeutic
approaches with reduced on-target off-tumour toxicity where T-cell
activation is wholly restricted to target cells which express both
antigens.
DESCRIPTION OF THE FIGURES
[0016] FIG. 1: (a) Generalized architecture of a CAR: A binding
domain recognizes antigen; the spacer elevates the binding domain
from the cell surface; the trans-membrane domain anchors the
protein to the membrane and the endodomain transmits signals. (b)
to (d): Different generations and permutations of CAR endodomains:
(b) initial designs transmitted ITAM signals alone through
Fc.epsilon.R1-.gamma. or CD3.zeta. endodomain, while later designs
transmitted additional (c) one or (d) two co-stimulatory signals in
cis.
[0017] FIG. 2: Schematic diagram illustrating the invention
[0018] The invention relates to engineering T-cells to respond to
logical rules of target cell antigen expression. This is best
illustrated with an imaginary FACS scatter-plot. Target cell
populations express both, either or neither of antigens "A" and
"B". Different target populations (marked in red) are killed by
T-cells transduced with a pair of CARs connected by different
gates. With OR gated receptors, both single-positive and
double-positive cells will be killed. With AND gated receptors,
only double-positive target cells are killed. With AND NOT gating,
double-positive targets are preserved while single-positive
targets
[0019] FIG. 3: Creation of target cell populations
[0020] SupT1 cells were used as target cells. These cells were
transduced to express either CD19, CD33 or both CD19 and CD33.
Target cells were stained with appropriate antibodies and analysed
by flow cytometry.
[0021] FIG. 4: Cassette design for an OR gate
[0022] A single open reading frame provides both CARs with an
in-frame FMD-2A sequence resulting in two proteins. Signal1 is a
signal peptide derived from IgG1 (but can be any effective signal
peptide). scFv1 is the single-chain variable segment which
recognizes CD19 (but can be a scFv or peptide loop or ligand or in
fact any domain which recognizes any desired arbitrary target). STK
is the CD8 stalk but may be any suitable extracellular domain.
CD28tm is the
[0023] CD28 trans-membrane domain but can by any stable type I
protein transmembrane domain and CD3Z is the CD3 Zeta endodomain
but can be any endodomain which contains ITAMs. Signal2 is a signal
peptide derived from CD8 but can be any effective signal peptide
which is different in DNA sequence from signal1. scFv recognizes
CD33 but as for scFv1 is arbitrary. HC2CH3 is the hinge-CH2-CH3 of
human IgG1 but can be any extracellular domain which does not
cross-pair with the spacer used in the first CAR. CD28tm' and CD3Z'
code for the same protein sequence as CD28tm and CD3Z but are
codon-wobbled to prevent homologous recombination.
[0024] FIG. 5: Schematic representation of the chimeric antigen
receptors (CARs) for an OR gate
[0025] Stimulatory CARs were constructed consisting of either an
N-terminal A) anti-CD19 scFv domain followed by the extracellular
hinge region of human CD8 or B) anti-CD33 scFv domain followed by
the extracellular hinge, CH2 and CH3 (containing a pvaa mutation to
reduce FcR binding) region of human IgG1. Both receptors contain a
human CD28 transmembrane domain and a human CD3 Zeta (CD247)
intracellular domain. "S" depicts the presence of disulphide
bonds.
[0026] FIG. 6: Expression data showing co-expression of both CARs
on the surface of one T-cell.
[0027] FIG. 7: Functional analysis of the OR gate
[0028] Effector cells (5.times.10{circumflex over ( )}4 cells)
expressing the OR gate construct were co-incubated with a varying
number of target cells and IL-2 was analysed after 16 hours by
ELISA. The graph displays the average maximum IL-2 secretion from a
chemical stimulation (PMA and lonomycin) of the effector cells
alone and the average background IL-2 from effector cells without
any stimulus from three replicates.
[0029] FIG. 8: Cartoon showing both versions of the cassette used
to express both AND gates
[0030] Activating and inhibiting CARs were co-expressed once again
using a FMD-2A sequence. Signal1 is a signal peptide derived from
IgG1 (but can be any effective signal peptide). scFv1 is the
single-chain variable segment which recognizes CD19 (but can be a
scFv or peptide loop or ligand or in fact any domain which
recognizes any desired arbitrary target). STK is the CD8 stalk but
may be any non-bulky extracellular domain. CD28tm is the CD28
trans-membrane domain but can by any stable type I protein
transmembrane domain and CD3Z is the CD3 Zeta endodomain but can be
any endodomain which contains ITAMs. Signal2 is a signal peptide
derived from CD8 but can be any effective signal peptide which is
different in DNA sequence from signal1. scFv recognizes CD33 but as
for scFv1 is arbitrary. HC2CH3 is the hinge-CH2-CH3 of human IgG1
but can be any bulky extracellular domain. CD45 and CD148 are the
transmembrane and endodomains of CD45 and CD148 respectively but
can be derived from any of this class of protein.
[0031] FIG. 9: Schematic representation of the protein structure of
chimeric antigen receptors (CARs) for the AND gates
[0032] The stimulatory CAR consisting of an N-terminal anti-CD19
scFv domain followed by the extracellular stalk region of human
CD8, human CD28 transmembrane domain and human CD3 Zeta (CD247)
intracellular domain. Two inhibitory CARs were tested. These
consist of an N-terminal anti-CD33 scFv domain followed by the
extracellular hinge, CH2 and CH3 (containing a pvaa mutation to
reduce FcR binding) region of human IgG1 followed by the
transmembrane and intracellular domain of either human CD148 or
CD45. "S" depicts the presence of disulphide bonds.
[0033] FIG. 10: Co-expression of activation and inhibitory CARs
[0034] BW5147 cells were used as effector cells and were transduced
to express both the activation anti-CD19 CAR and one of the
inhibitory anti-CD33 CARs. Effector cells were stained with
CD19-mouse-Fc and CD33-rabbit-Fc and with appropriate secondary
antibodies and analysed by flow cytometry.
[0035] FIG. 11: Functional analysis of the AND gates
[0036] Effector cells (5.times.10{circumflex over ( )}4 cells)
expressing activation anti-CD19 CAR and the inhibitory anti-CD33
CAR with the A) CD148 or B) CXD45 intracellular domain were
co-incubated with a varying number of target cells and IL-2 was
analysed after 16 hours by ELISA. The graph displays the maximum
IL-2 secretion from a chemical stimulation (PMA and lonomycin) of
the effector cells alone and the background IL-2 from effector
cells without any stimulus from three replicates.
[0037] FIG. 12: Cartoon showing three versions of the cassette used
to generate the AND NOT gate
[0038] Activating and inhibiting CARs were co-expressed once again
using a FMD-2A sequence. Signal1 is a signal peptide derived from
IgG1 (but can be any effective signal peptide). scFv1 is the
single-chain variable segment which recognizes CD19 (but can be a
scFv or peptide loop or ligand or in fact any domain which
recognizes any desired arbitrary target). STK is the human CD8
stalk but may be any non-bulky extracellular domain. CD28tm is the
CD28 trans-membrane domain but can by any stable type I protein
transmembrane domain and CD3Z is the CD3 Zeta endodomain but can be
any endodomain which contains ITAMs. Signal2 is a signal peptide
derived from CD8 but can be any effective signal peptide which is
different in DNA sequence from signal1. scFv recognizes CD33 but as
for scFv1 is arbitrary. muSTK is the mouse CD8 stalk but can be any
spacer which co-localises but does not cross-pair with that of the
activating CAR. dPTPN6 is the phosphatase domain of PTPN6. LAIR1 is
the transmembrane and endodomain of LAIR1. 2Aw is a codon-wobbled
version of the FMD-2A sequence. SH2-CD148 is the SH2 domain of
PTPN6 fused with the phosphatase domain of CD148.
[0039] FIG. 13: Schematic representation of the chimeric antigen
receptors (CARs) for the NOT AND gates
[0040] A) A stimulatory CAR consisting of an N-terminal anti-CD19
scFv domain followed by the stalk region of human CD8, human CD28
transmembrane domain and human CD247 intracellular domain. B) An
inhibitory CAR consisting of an N-terminal anti-CD33 scFv domain
followed by the stalk region of mouse CD8, transmembrane region of
mouse CD8 and the phosphatase domain of PTPN6. C) an inhibitory CAR
consisting of an N-terminal anti-CD33 scFv domain followed by the
stalk region of mouse CD8 and the transmembrane and intracellular
segments of LAIR1. D) An inhibitory CAR identical to previous CAR
except it is co-expressed with a fusion protein of the PTPN6 SH2
domain and the CD148 phosphatase domain.
[0041] FIG. 14: Functional analysis of the NOT AND gate
[0042] Effector cells (5.times.10{circumflex over ( )}4 cells)
expressing the A) full length SHP-1 or B) truncated form of SHP-1
were co-incubated with a varying number of target cells and IL-2
was analysed after 16 hours by ELISA. The graph displays the
average maximum IL-2 secretion from a chemical stimulation (PMA and
lonomycin) of the effector cells alone and the average background
IL-2 from effector cells without any stimulus from three
replicates.
[0043] FIG. 15: Amino acid sequence of an OR gate
[0044] FIG. 16: Amino acid sequence of a CD148 and a CD145 based
AND gate
[0045] FIG. 17: Amino acid sequence of two AND NOT gates
[0046] FIG. 18: Dissection of AND gate function
[0047] A. The prototype AND gate is illustrated on the right and
its function in response to CD19, CD33 single and CD19, CD33 double
positive targets is shown on the left. B. The scFvs are swapped so
the activating endodomain is triggered by CD33 and the inhibitory
endodomain is activated by CD19. This AND gate remains functional
despite this scFv swap. C. The CD8 mouse stalk replaced Fc in the
spacer of the inhibitory CAR. With this modification, the gate
fails to respond to either CD19 single positive or CD19, CD33
double positive targets.
[0048] FIG. 19: Expression of target antigens on artificial target
cells A. Shows flow cytometry scatter plots CD19 vs CD33 of the
original set of artificial target cells derived from SupT1 cells.
From left to right: double negative SupT1 cells, SupT1 cells
positive for CD19, positive for CD33 and positive for both CD19 and
CD33. B. Shows flow cytometry scatter plots CD19 vs GD2 of the
artificial target cells generated to test the CD19 AND GD2 gate:
From left to right: negative SupT1 cells, SupT1 cells expressing
CD19, SupT1 cells transduced with GD2 and GM3 synthase vectors
which become GD2 positive and SupT1 cells transduced with CD19 as
well as GD2 and GM3 synthase which are positive for both GD2 and
CD19. C. Shows flow cytometry scatter plots of CD19 vs EGFRvIII of
the artificial targets generated to test the CD19 AND EGFRvIII
gate. From left to right: negative SupT1 cells, SupT1 cells
expressing CD19, SupT1 cells transduced with EGFRvIII and SupT1
cells transduced with both CD19 and EGFRvIII. D. Shows flow
cytometry scatter plots of CD19 vs CD5 of the artificial targets
generated to test the CD19 AND CD5 gate. From left to right:
negative 293T cells, 293T cells transduced with CD19, 293T cells
transduced with CD5, 293T cells transduced with both CD5 and CD19
vectors.
[0049] FIG. 20: Generalizability of the AND gate
[0050] A. Cartoon of AND gate modified so the second CAR's
specificity is changed from the original specificity of CD33, to
generate 3 new CARs: CD19 AND GD2, CD19 AND EGFRvIII, CD19 AND CD5.
B. CD19 AND GD2 AND gate: Left: expression of AND gate is shown
recombinant CD19-Fc staining (x-axis) for the CD19 CAR, versus
anti-human-Fc staining (Y-axis) for the GD2 CAR. Right: function in
response to single positive and double positive targets. C. CD19
AND EGFRvIII AND gate: Left: expression of AND gate is shown
recombinant CD19-Fc staining (x-axis) for the CD19 CAR, versus
anti-human-Fc staining (Y-axis) for the EGFRvIII CAR. Right:
function in response to single positive and double positive
targets. D. CD19 AND CD5 AND gate: Left: expression of AND gate is
shown recombinant CD19-Fc staining (x-axis) for the CD19 CAR,
versus anti-human-Fc staining (Y-axis) for the CD5 CAR. Right:
function in response to single positive and double positive
targets.
[0051] FIG. 21: Function of the AND NOT gates
[0052] Function of the three implementations of an AND NOT gate is
shown. A cartoon of the gates tested is shown to the right, and
function in response to single positive and double positive targets
is shown to the left. A. PTPN6 based AND NOT gate whereby the first
CAR recognizes CD19, has a human CD8 stalk spacer and an ITAM
containing activating endodomain; is co-expressed with a second CAR
that recognizes CD33, has a mouse CD8 stalk spacer and has an
endodomain comprising of a PTPN6 phosphatase domain. B. ITIM based
AND NOT gate is identical to the PTPN6 gate, except the endodomain
is replaced by the endodomain from LAIR1. C. CD148 boosted AND NOT
gate is identical to the ITIM based gate except an additional
fusion between the PTPN6 SH2 and the endodomain of CD148 is
expressed. All three gates work as expected with activation in
response to CD19 but not in response to CD19 and CD33 together.
[0053] FIG. 22: Dissection of PTPN6 based AND NOT gate function
[0054] The original PTPN6 based AND NOT gate is compared with
several controls to demonstrate the model. A cartoon of the gates
tested is shown to the right, and function in response to single
positive and double positive targets is shown to the left. A.
Original AND NOT gate whereby the first CAR recognizes CD19, has a
human CD8 stalk spacer and an ITAM containing activating
endodomain; is co-expressed with a second CAR recognizes CD33, has
a mouse CD8 stalk spacer and has an endodomain comprising of a
PTPN6 phosphatase domain. B. AND NOT gate modified so the mouse CD8
stalk spacer is replaced with an Fc spacer. C AND NOT gate modified
so that the PTPN6 phosphatase domain is replaced with the
endodomain from CD148. Original AND NOT gate (A.) functions as
expected triggering in response to CD19, but not in response to
both CD19 and CD33. The gate in B. triggers both in response to
CD19 along or CD19 and CD33 together. The gate in C. does not
trigger in response to one or both targets.
[0055] FIG. 23: Dissection of LAIR1 based AND NOT gate
[0056] Functional activity against CD19 positive, CD33 positive and
CD19, CD33 double-positive targets is shown. A. Structure and
activity of the original ITIM based AND NOT gate. This gate is
composed of two CARs: the first recognizes CD19, has a human CD8
stalk spacer and an ITAM containing endodomain; the second CAR
recognizes CD33, has a mouse CD8 stalk spacer and an ITIM
containing endodomain. B Structure and activity of the control ITIM
based gate where the mouse CD8 stalk spacer has been replaced by an
Fc domain. This gate is composed of two CARs: the first recognizes
CD19, has a human CD8 stalk spacer and an ITAM containing
endodomain; the second CAR recognizes CD33, has an Fc spacer and an
ITIM containing endodomain. Both gates respond to CD19 single
positive targets, while only the original gate is inactive in
response to CD19 and CD33 double positive targets.
[0057] FIG. 24: Kinetic segregation model of CAR logic gates
[0058] Model of kinetic segregation and behaviour of AND gate, NOT
AND gate and controls. CARs recognize either CD19 or CD33. The
immunological synapse can be imagined between the blue line, which
represents the target cell membrane and the red line, which
represents the T-cell membrane. `45` is the native CD45 protein
present on T-cells. `H8` is a CAR ectodomain with human CD8 stalk
as the spacer. `Fc` is a CAR ectodomain with human HCH2CH3 as the
spacer. `M8` is a CAR ectodomain with murine CD8 stalk as the
spacer. `19` represents CD19 on the target cell surface. `33`
represents CD33 on the target cell surface. The symbol `.sym.`
represents an activating endodomain containing ITAMS. The symbol
`.crclbar.` represents a phosphatase with slow kinetics--a
`ligation on` endodomain such as one comprising of the catalytic
domain of PTPN6 or an ITIM. The symbol `O` represents a phosphatase
with fast kinetics--a `ligation off` endodomain such as the
endodomain of CD45 or CD148. This symbol is enlarged in the figure
to emphasize its potent activity.
(a) Shows the postulated behaviour of the functional AND gate which
comprises of a pair of CARs whereby the first CAR recognizes CD19,
has a human CD8 stalk spacer and an activating endodomain; and the
second CAR recognizes CD33, has an Fc spacer and a CD148
endodomain; (b) Shows the postulated behaviour of the control AND
gate. Here, the first CAR recognizes CD19, has a human CD8 stalk
spacer and an activating endodomain; and the second CAR recognizes
CD33, but has a mouse CD8 stalk spacer and a CD148 endodomain; (c)
Shows the behaviour of a functional AND NOT gate which comprises of
a pair of CARs whereby the first CAR recognizes CD19, has a human
CD8 stalk spacer and an activating endodomain; and the second CAR
recognizes CD33, has a mouse CD8 stalk spacer and a PTPN6
endodomain; (d) Shows the postulated behaviour of the control AND
NOT gate which comprises of a pair of CARs whereby the first CAR
recognizes CD19, has a human CD8 stalk spacer and an activating
endodomain; and the second CAR recognizes CD33, but has an Fc
spacer and a PTPN6 endodomain;
[0059] In the first column, target cells are both CD19 and CD33
negative. In the second column, targets are CD19 negative and CD33
positive. In the third column, target cells are CD19 positive and
CD33 negative. In the fourth column, target cells are positive for
both CD19 and CD33.
[0060] FIG. 25: Design of APRIL-based CARs.
[0061] The CAR design was modified so that the scFv was replaced
with a modified form of A proliferation-inducing ligand (APRIL),
which interacts with interacts with BCMA, TACI and proteoglycans,
to act as an antigen binding domain: APRIL was truncated so that
the proteoglycan binding amino-terminus is absent. A signal peptide
was then attached to truncated APRIL amino-terminus to direct the
protein to the cell surface. Three CARs were generated with this
APRIL based binding domain: A. In the first CAR, the human CD8
stalk domain was used as a spacer domain. B. In the second CAR, the
hinge from IgG1 was used as a spacer domain. C. In the third CAR,
the hinge, CH2 and CH3 domains of human IgG1 modified with the
pva/a mutations described by Hombach et al (2010 Gene Ther.
17:1206-1213) to reduce Fc Receptor binding was used as a spacer
(henceforth referred as Fc-pvaa). In all CARs, these spacers were
connected to the CD28 transmembrane domain and then to a tripartite
endodomain containing a fusion of the CD28, OX40 and the CD3-Zeta
endodomain (Pule et al, Molecular therapy, 2005: Volume 12; Issue
5; Pages 933-41).
[0062] FIG. 26: Annotated Amino acid sequence of the above three
APRIL-CARS
[0063] A: Shows the annotated amino acid sequence of the CD8 stalk
APRIL CAR; B: Shows the annotated amino acid sequence of the APRIL
IgG1 hinge based CAR; C: Shows the annotated amino acid sequence of
the APRIL Fc-pvaa based CAR.
[0064] FIG. 27: Expression and ligand binding of different APRIL
based CARs
[0065] A. The receptors were co-expressed with a marker gene
truncated CD34 in a retroviral gene vector. Expression of the
marker gene on transduced cells allows confirmation of
transduction. B. T-cells were transduced with APRIL based CARs with
either the CD8 stalk spacer, IgG1 hinge or Fc spacer. To test
whether these receptors could be stably expressed on the cell
surface, T-cells were then stained with
anti-APRIL-biotin/Streptavidin APC and anti-CD34. Flow-cytometric
analysis was performed. APRIL was equally detected on the cell
surface in the three CARs suggesting they are equally stably
expressed. C. Next, the capacity of the CARs to recognize TACI and
BCMA was determined. The transduced T-cells were stained with
either recombinant BCMA or TACI fused to mouse IgG2a Fc fusion
along with an anti-mouse secondary and anti-CD34. All three
receptor formats showed binding to both BCMA and TACI. A surprising
finding was that binding to BCMA seemed greater than to TACI. A
further surprising finding was that although all three CARs were
equally expressed, the CD8 stalk and IgG1 hinge CARs appeared
better at recognizing BCMA and TACI than that with the Fc
spacer.
[0066] FIG. 28: Function of the different CAR constructs.
[0067] Functional assays were performed with the three different
APRIL based CARs. Normal donor peripheral blood T-cells either
non-transduced (NT), or transduced to express the different CARs.
Transduction was performed using equal titer supernatant. These
T-cells were then CD56 depleted to remove non-specific NK activity
and used as effectors. SupT1 cells either non-transduced (NT), or
transduced to express BCMA or TACI were used as targets. Data shown
is mean and standard deviation from 5 independent experiments. A.
Specific killing of BCMA and TACI expressing T-cells was determined
using Chromium release. B. Interferon-.mu. release was also
determined. Targets and effectors were co-cultured at a ratio of
1:1. After 24 hours, Interferon-.mu. in the supernatant was assayed
by ELISA. C. Proliferation/survival of CAR T-cells were also
determined by counting number of CAR T-cells in the same co-culture
incubated for a further 6 days. All 3 CARs direct responses against
BCMA and TACI expressing targets. The responses to BCMA were
greater than for TACI.
[0068] FIG. 29: AND gate functionality in primary cells
[0069] PBMCs were isolated from blood and stimulated using PHA and
IL-2. Two days later the cells were transduced on retronectin
coated plates with retro virus containing the CD19:CD33 AND gate
construct. On day 5 the expression level of the two CARs translated
by the AND gate construct was evaluated via flow cytometry and the
cells were depleted of CD56+ cells (predominantly NK cells). On day
6 the PBMCs were placed in a co-culture with target cells at a 1:2
effector to target cell ratio. On day 8 the supernatant was
collected and analysed for IFN-gamma secretion via ELISA.
[0070] FIG. 30: A selection/hierarchy of possible spacer domains of
increasing size is shown. The ectodomain of CD3-Zeta is suggested
as the shortest possible spacer, followed by the (b) the IgG1
hinge. (c) murine or human CD8 stalk and the CD28 ectodomains are
considered intermediate in size and co-segregate. (d) The hinge,
CH2 and CH3 domain of IgG1 is bigger and bulkier, and (e) the
hinge, CH2, CH3 and CH4 domain of IgM is bigger still. Given the
properties of the target molecules, and the epitope of the binding
domains on said target molecules, it is possible to use this
hierarchy of spacers to create a CAR signaling system which either
co-segregates or segregates apart upon synapse formation.
[0071] FIG. 31: Design rules for building logic gated CAR
T-cells.
[0072] OR, AND NOT and AND gated CARs are shown in cartoon format
with the target cell on top, and the T-cell at the bottom with the
synapse in the middle. Target cells express arbitrary target
antigens A, and B.
[0073] T-cells express two CARs which comprise of anti-A and anti-B
recognition domains, spacers and endodomains. An OR gate requires
(1) spacers simply which allow antigen recognition and CAR
activation, and (2) both CARs to have activatory endodomains; An
AND NOT gate requires (1) spacers which result in co-segregation of
both CARs upon recognition of both antigens and (2) one CAR with an
activatory endodomain, and the other whose endodomain comprises or
recruits a weak phosphatase; An AND gate requires (1) spacers which
result in segregation of both CARs into different parts of the
immunological synapse upon recognition of both antigens and (2) one
CAR with an activatory endodomain, and the other whose endodomain
comprises of a potent phosphatase.
SUMMARY OF ASPECTS OF THE INVENTION
[0074] The present inventors have developed a panel of
"logic-gated" chimeric antigen receptor pairs which, when expressed
by a cell, such as a T cell, are capable of detecting a particular
pattern of expression of at least two target antigens. If the at
least two target antigens are arbitrarily denoted as antigen A and
antigen B, the three possible options are as follows:
"OR GATE"--T cell triggers when either antigen A or antigen B is
present on the target cell "AND GATE"--T cell triggers only when
both antigens A and B are present on the target cell "AND NOT
GATE"--T cell triggers if antigen A is present alone on the target
cell, but not if both antigens A and B are present on the target
cell
[0075] Engineered T cells expressing these CAR combinations can be
tailored to be exquisitely specific for cancer cells, based on
their particular expression (or lack of expression) of two or more
markers.
[0076] Thus in a first aspect, the present invention provides a
cell which co-expresses a first chimeric antigen receptor (CAR) and
second CAR at the cell surface, each CAR comprising: [0077] (i) an
antigen-binding domain; [0078] (ii) a spacer [0079] (iii) a
trans-membrane domain; and [0080] (iv) an intracellular T cell
signaling domain (endodomain) wherein the antigen binding domains
of the first and second CARs bind to different antigens, and
wherein the spacer of the first CAR is different to the spacer of
the second CAR, such that the first and second CARs do not form
heterodimers, and wherein one of the first or second CARs is an
activating CAR comprising an activating intracellular T cell
signaling domain and the other CAR is an inhibitory CAR comprising
a "ligation-off" (as defined herein) inhibitory intracellular T
cell signaling domain.
[0081] The cell may be an immune effector cell, such as a T-cell or
natural killer (NK) cell. Features mentioned herein in connection
with a T cell apply equally to other immune effector cells, such as
NK cells.
[0082] The spacer of the first CAR may have a different length
and/or charge and/or shape and/or configuration and/or
glycosylation to the spacer of the second CAR, such that when the
first CAR and the second CAR bind their respective target antigens,
the first CAR and second CAR become spatially separated on the T
cell. Ligation of the first and second CARs to their respective
antigens causes them to be compartmentalized together or separately
in the immunological synapse resulting in control of activation.
This may be understood when one considers the kinetic separation
model of T-cell activation (see below).
[0083] The first spacer or the second spacer may comprise a CD8
stalk and the other spacer may comprise the hinge, CH2 and CH3
domain of an IgG1.
[0084] In the present invention, which relates to the "AND" gate,
one of the first or second CARs is an activating CAR comprising an
activating endodomain, and the other CAR is a "ligation-off"
inhibitory CAR comprising an inhibitory endodomain. The
ligation-off inhibitory CAR inhibits T-cell activation by the
activating CAR in the absence of inhibitory CAR ligation, but does
not significantly inhibit T-cell activation by the activating CAR
when the inhibitory CAR is ligated. Since the spacer of the first
CAR has a different length and/or charge and/or shape and/or
configuration and/or glycosylation from the spacer of the second
CAR, when both CARs are ligated they segregate. This causes the
inhibitory CAR to be spatially separated from the activating CAR,
so that T cell activation can occur. T cell activation therefore
only occurs in response to a target cell bearing both cognate
antigens.
[0085] The inhibitory endodomain may comprise all or part of the
endodomain from a receptor-like tyrosine phosphatase, such as CD148
or CD45.
[0086] The antigen-binding domain of the first CAR may bind CD5 and
the antigen-binding domain of the second CAR may bind CD19. This is
of use in targeting chronic lymphocytic leukaemia (CLL). This
disease can be treated by targeting CD19 alone, but at the cost of
depleting the entire B-cell compartment. CLL cells are unusual in
that they co-express CD5 and CD19. Targeting this pair of antigens
with an AND gate will increase specificity and reduce toxicity.
[0087] In a second aspect, the present invention provides a nucleic
acid sequence encoding both the first and second chimeric antigen
receptors (CARs) as defined in the first aspect of the
invention.
[0088] The nucleic acid sequence according may have the following
structure:AgB1-spacer1-TM1-endo1-coexpr-AgB2-spacer2-TM2-endo2
in which AgB1 is a nucleic acid sequence encoding the
antigen-binding domain of the first CAR; spacer 1 is a nucleic acid
sequence encoding the spacer of the first CAR; TM1 is a nucleic
acid sequence encoding the transmembrane domain of the first CAR;
endo 1 is a nucleic acid sequence encoding the endodomain of the
first CAR; coexpr is a nucleic acid sequence allowing co-expression
of two CARs (e.g. a cleavage site); AgB2 is a nucleic acid sequence
encoding the antigen-binding domain of the second CAR; spacer 2 is
a nucleic acid sequence encoding the spacer of the second CAR; TM2
is a nucleic acid sequence encoding the transmembrane domain of the
second CAR; endo 2 is a nucleic acid sequence encoding the
endodomain of the second CAR; which nucleic acid sequence, when
expressed in a T cell, encodes a polypeptide which is cleaved at
the cleavage site such that the first and second CARs are
co-expressed at the T cell surface.
[0089] The nucleic acid sequence allowing co-expression of two CARs
may encode a self-cleaving peptide or a sequence which allows
alternative means of co-expressing two CARs such as an internal
ribosome entry sequence or a 2.sup.nd promoter or other such means
whereby one skilled in the art can express two proteins from the
same vector.
[0090] Alternative codons may be used in regions of sequence
encoding the same or similar amino acid sequences, in order to
avoid homologous recombination.
[0091] In a third aspect, the present invention provides a kit
which comprises [0092] (i) a first nucleic acid sequence encoding
the first chimeric antigen receptor (CAR) as defined in the first
aspect of the invention, which nucleic acid sequence has the
following structure: AgB1-spacer1-TM1-endo1 in which AgB1 is a
nucleic acid sequence encoding the antigen-binding domain of the
first CAR; spacer 1 is a nucleic acid sequence encoding the spacer
of the first CAR; TM1 is a nucleic acid sequence encoding the
transmembrane domain of the first CAR; endo 1 is a nucleic acid
sequence encoding the endodomain of the first CAR; and [0093] (ii)
a second nucleic acid sequence encoding the second chimeric antigen
receptor (CAR) as defined in the first aspect of the invention,
which nucleic acid sequence has the following structure:
AgB2-spacer2-TM2-endo2 AgB2 is a nucleic acid sequence encoding the
antigen-binding domain of the second CAR; spacer 2 is a nucleic
acid sequence encoding the spacer of the second CAR; TM2 is a
nucleic acid sequence encoding the transmembrane domain of the
second CAR; endo 2 is a nucleic acid sequence encoding the
endodomain of the second CAR.
[0094] In a fourth aspect, the present invention provides a kit
comprising: a first vector which comprises the first nucleic acid
sequence as defined above; and a second vector which comprises the
first nucleic acid sequence as defined above.
[0095] The vectors may be plasmid vectors, retroviral vectors or
transposon vectors. The vectors may be lentiviral vectors.
[0096] In a fifth aspect, the present invention provides a vector
comprising a nucleic acid sequence according to the second aspect
of the invention. The vector may be a lentiviral vector.
[0097] The vector may be a plasmid vector, a retroviral vector or a
transposon vector.
[0098] In a sixth aspect, the present invention involves
co-expressing more than two CARs in such a fashion that a complex
pattern of more than two antigens can be recognized on the target
cell.
[0099] In a seventh aspect, the present invention provides a method
for making a T cell according to the first aspect of the invention,
which comprises the step of introducing one or more nucleic acid
sequence (s) encoding the first and second CARs; or one or more
vector(s) as defined above into a T cell.
[0100] The T cell may be from a sample isolated from a patient, a
related or unrelated haematopoietic transplant donor, a completely
unconnected donor, from cord blood, differentiated from an
embryonic cell line, differentiated from an inducible progenitor
cell line, or derived from a transformed T cell line.
[0101] In an eighth aspect, the present invention provides a
pharmaceutical composition comprising a plurality of T cells
according to the first aspect of the invention.
[0102] In a ninth aspect, the present invention provides a method
for treating and/or preventing a disease, which comprises the step
of administering a pharmaceutical composition according to the
eighth aspect of the invention to a subject.
[0103] The method may comprise the following steps: [0104] (i)
isolation of a T cell as listed above. [0105] (ii) transduction or
transfection of the T cells with one or more nucleic acid
sequence(s) encoding the first and second CAR or one or more
vector(s) comprising such nucleic acid sequence(s); and [0106]
(iii) administering the T cells from (ii) to the subject.
[0107] The disease may be a cancer.
[0108] In a tenth aspect, the present invention provides a
pharmaceutical composition according to the eighth aspect of the
invention for use in treating and/or preventing a disease.
[0109] The disease may be a cancer.
[0110] In an eleventh aspect, the present invention provides use of
a T cell according to the first aspect of the invention in the
manufacture of a medicament for treating and/or preventing a
disease.
[0111] The disease may be a cancer.
[0112] The present invention also provides a nucleic acid sequence
which comprises:
a) a first nucleotide sequence encoding a first chimeric antigen
receptor (CAR); b) a second nucleotide sequence encoding a second
CAR; c) a sequence encoding a self-cleaving peptide positioned
between the first and second nucleotide sequences, such that the
two CARs are expressed as separate entities.
[0113] Alternative codons may be used in one or more portion(s) of
the first and second nucleotide sequences in regions which encode
the same or similar amino acid sequence(s).
[0114] The present invention also provides a vector and a cell
comprising such a nucleic acid.
[0115] The kinetic-segregation based AND gate of the present
invention offers a significant technical advantage to the
previously described "co-CAR", i.e. the dual targeting approach in
which two antigens are recognized by two CARs which supply either
an activating or a co-stimulating signal to the T-cell.
[0116] With the co-CAR approach, although greatest activity might
be expected against target cells bearing both antigens,
considerable activity against tissues bearing only antigen
recognized by the activating CAR can be expected. This activity can
be expected to be at least that of a first-generation CAR. First
generation CARs have resulted in considerable toxicity: for
instance biliary toxicity was observed in clinical testing of a
first generation CAR recognizing Carbonic anhydrase IX which was
unexpectedly expressed on biliary epithelium (Rotterdam ref).
Notably, terminally differentiated effectors do not require or
respond to co-stimulatory signals, so any terminally differentiated
CAR T-cells would act maximally despite the absence of a
co-stimulatory CAR signal.
[0117] Further, co-stimulatory signals lead to long-lasting effects
on the T-cell population. These effects long outlast the
T-cell/target synapse interaction. Consequently, CAR T-cells which
become fully activated within the tumour and migrate could have
maximally potent activity against single-antigen bearing normal
tissues. This "spill-over" effect may be most pronounced in tissues
within, near or which drain from the tumour. In fact, strategies
based on the concept of the activity of a first generation CAR
being enhanced by co-stimulatory signals engaged not CAR activation
but through a distinct receptor, have been proposed and tested
(Rossig, Blood. 2002 Mar. 15; 99(6):2009-16.).
[0118] The co-CAR approach hence can be expected to result at best
to a reduction but not abolition of toxicity towards single antigen
expressing normal tissue. The present invention uses kinetic
segregation at the immunological synapse formed between the
T-cell/target cell to regulate T-cell triggering itself.
Consequently tight absolute control of triggering in the absence of
the second antigen is achieved. Hence the totality of T-cell
activation is restricted to target cells expressing both antigens,
the AND gate should function irrespective of the effector cell type
or differentiation state, and no "spill-over" effect AND gate
T-cell activation is possible.
Further Aspects of the Invention
[0119] The present invention also relates to the aspects listed in
the following numbered paragraphs:
1. A T cell which co-expresses a first chimeric antigen receptor
(CAR) and second CAR at the cell surface, each CAR comprising:
[0120] (i) an antigen-binding domain; [0121] (ii) a spacer [0122]
(iii) a trans-membrane domain; and [0123] (iv) an endodomain
wherein the antigen binding domains of the first and second CARs
bind to different antigens, wherein the spacer of the first CAR is
different to the spacer of the second CAR and wherein one of the
first or second CARs is an activating CAR comprising an activating
endodomain and the other CAR is either an activating CAR comprising
an activating endodomain or an inhibitory CAR comprising a
ligation-on or ligation-off inhibitory endodomain. 2. A T cell
according to paragraph 1, wherein the spacer of the first CAR has a
different length and/or charge and/or size and/or configuration
and/or glycosylation of the spacer of the second CAR, such that
when the first CAR and the second CAR bind their respective target
antigens, the first CAR and second CAR become spatially separated
on the T cell membrane. 3. A T cell according to paragraph 2,
wherein either the first spacer or the second spacer comprises a
CD8 stalk and the other spacer comprises the hinge, CH2 and CH3
domain of IgG1. 4. A T cell according to paragraph 1, wherein both
the first and second CARs are activating CARs. 5. A T cell
according to paragraph 4, wherein one CAR binds CD19 and the other
CAR binds CD20. 6. A T cell according to paragraph 2 or 3, wherein
one of the first or second CARs is an activating CAR comprising an
activating endodomain, and the other CAR is an inhibitory CAR
comprising a ligation-off inhibitory endodomain, which inhibitory
CAR inhibits T-cell activation by the activating CAR in the absence
of inhibitory CAR ligation, but does not significantly inhibit
T-cell activation by the activating CAR when the inhibitory CAR is
ligated. 7. A T cell according to paragraph 6, wherein the
inhibitory endodomain comprises all or part of the endodomain from
CD148 or CD45. 8. A T cell according to paragraph 6 or 7, wherein
the antigen-binding domain of the first CAR binds CD5 and the
antigen-binding domain of the second CAR binds CD19. 9. A T cell
according to paragraph 1 wherein the first and second spacers are
sufficiently different so as to prevent cross-pairing of the first
and second CARs but are sufficiently similar to result in
co-localisation of the first and second CARs following ligation.
10. A T cell according to paragraph 9, wherein one of the first or
second CARs in an activating CAR comprising an activating
endodomain, and the other CAR is an inhibitory CAR comprising a
ligation-on inhibitory endodomain, which inhibitory CAR does not
significantly inhibit T-cell activation by the activating CAR in
the absence of inhibitory CAR ligation, but inhibits T-cell
activation by the activating CAR when the inhibitory CAR is
ligated. 11. A T cell according to paragraph 10, wherein the
ligation-on inhibitory endodomain comprises at least part of a
phosphatase. 12. A T cell according to paragraph 11, wherein the
ligation-on inhibitory endodomain comprises all or part of PTPN6.
13. A T cell according to paragraph 10, wherein the ligation-on
inhibitory endodomain comprises at least one ITIM domain. 14. A T
cell according to paragraph 13, wherein activity of the ligation-on
inhibitory endodomain is enhanced by co-expression of a PTPN6-CD45
or -CD148 fusion protein. 15. A T cell according to any of
paragraphs 10 to 14, wherein the CAR comprising the activating
endodomain comprises an antigen-binding domain which binds CD33 and
the CAR which comprises the ligation-on inhibitory endodomain
comprises an antigen-binding domain which binds CD34. 16. A T cell
which comprises more than two CARs as defined in the preceding
paragraphs such that it is specifically stimulated by a cell, such
as a T cell, bearing a distinct pattern of more than two antigens.
17. A nucleic acid sequence encoding both the first and second
chimeric antigen receptors (CARs) as defined in any of paragraphs 1
to 16. 18. A nucleic acid sequence according to paragraph 17, which
has the following structure:
AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2 in which AgB1
is a nucleic acid sequence encoding the antigen-binding domain of
the first CAR; spacer 1 is a nucleic acid sequence encoding the
spacer of the first CAR; TM1 is a nucleic acid sequence encoding
the transmembrane domain of the first CAR; endo 1 is a nucleic acid
sequence encoding the endodomain of the first CAR; coexpr is a
nucleic acid sequence enabling co-expression of both CARs AgB2 is a
nucleic acid sequence encoding the antigen-binding domain of the
second CAR; spacer 2 is a nucleic acid sequence encoding the spacer
of the second CAR; TM2 is a nucleic acid sequence encoding the
transmembrane domain of the second CAR; endo 2 is a nucleic acid
sequence encoding the endodomain of the second CAR; which nucleic
acid sequence, when expressed in a T cell, encodes a polypeptide
which is cleaved at the cleavage site such that the first and
second CARs are co-expressed at the T cell surface. 19. A nucleic
acid sequence according to paragraph 18, wherein coexpr encodes a
sequence comprising a self-cleaving peptide. 20. A nucleic acid
sequence according to paragraph 18 or 19, wherein alternative
codons are used in regions of sequence encoding the same or similar
amino acid sequences, in order to avoid homologous recombination.
21. A kit which comprises [0124] (i) a first nucleic acid sequence
encoding the first chimeric antigen receptor (CAR) as defined in
any of paragraphs 1 to 16, which nucleic acid sequence has the
following structure: AgB1-spacer1-TM1-endo1 in which AgB1 is a
nucleic acid sequence encoding the antigen-binding domain of the
first CAR; spacer 1 is a nucleic acid sequence encoding the spacer
of the first CAR; TM1 is a nucleic acid sequence encoding the
transmembrane domain of the first CAR; endo 1 is a nucleic acid
sequence encoding the endodomain of the first CAR; and [0125] (ii)
a second nucleic acid sequence encoding the second chimeric antigen
receptor (CAR) as defined in any of paragraphs 1 to 16, which
nucleic acid sequence has the following structure:
AgB2-spacer2-TM2-endo2 AgB2 is a nucleic acid sequence encoding the
antigen-binding domain of the second CAR; spacer 2 is a nucleic
acid sequence encoding the spacer of the second CAR; TM2 is a
nucleic acid sequence encoding the transmembrane domain of the
second CAR; endo 2 is a nucleic acid sequence encoding the
endodomain of the second CAR. 22. A kit comprising: a first vector
which comprises the first nucleic acid sequence as defined in
paragraph 21; and a second vector which comprises the first nucleic
acid sequence as defined in paragraph 21. 23. A kit according to
paragraph 22, wherein the vectors are integrating viral vectors or
transposons. 24. A vector comprising a nucleic acid sequence
according to any of paragraphs 17 to 20. 25. A retroviral vector or
a lentiviral vector or a transposon according to paragraph 24. 26.
A method for making a T cell according to any of paragraphs 1 to
16, which comprises the step of introducing: a nucleic acid
sequence according to any of paragraphs 17 to 20; a first nucleic
acid sequence and a second nucleic acid sequence as defined in
paragraph 21; and/or a first vector and a second vector as defined
in paragraph 22 or a vector according to paragraph 24 or 25, into a
T cell. 27. A method according to paragraph 24, wherein the T cell
is from a sample isolated from a subject. 28. A pharmaceutical
composition comprising a plurality of T cells according to any of
paragraphs 1 to 16. 29. A method for treating and/or preventing a
disease, which comprises the step of administering a pharmaceutical
composition according to paragraph 28 to a subject. 30. A method
according to paragraph 29, which comprises the following steps:
[0126] (i) isolation of a T cell-containing sample from a subject;
[0127] (ii) transduction or transfection of the T cells with: a
nucleic acid sequence according to any of paragraphs 17 to 20; a
first nucleic acid sequence and a second nucleic acid sequence as
defined in paragraph 21; a first vector and a second vector as
defined in paragraph 22 or 23 or a vector according to paragraph 24
or 25; and [0128] (iii) administering the T cells from (ii) to a
the subject. 31. A method according to paragraph 29 or 30, wherein
the disease is a cancer. 32. A pharmaceutical composition according
to paragraph 28 for use in treating and/or preventing a disease.
33. The use of a T cell according to any of paragraphs 1 to 16 in
the manufacture of a medicament for treating and/or preventing a
disease.
DETAILED DESCRIPTION
Chimeric Antigen Receptors (CARs)
[0129] CARs, which are shown schematically in FIG. 1, are chimeric
type I trans-membrane proteins which connect an extracellular
antigen-recognizing domain (binder) to an intracellular signalling
domain (endodomain). The binder is typically a single-chain
variable fragment (scFv) derived from a monoclonal antibody (mAb),
but it can be based on other formats which comprise an
antibody-like antigen binding site. A spacer domain is usually
necessary to isolate the binder from the membrane and to allow it a
suitable orientation. A common spacer domain used is the Fc of
IgG1. More compact spacers can suffice e.g. the stalk from
CD8.alpha. and even just the IgG1 hinge alone, depending on the
antigen. A trans-membrane domain anchors the protein in the cell
membrane and connects the spacer to the endodomain.
[0130] Early CAR designs had endodomains derived from the
intracellular parts of either the .gamma. chain of the
Fc.epsilon.R1 or CD3.zeta.. Consequently, these first generation
receptors transmitted immunological signal 1, which was sufficient
to trigger T-cell killing of cognate target cells but failed to
fully activate the T-cell to proliferate and survive. To overcome
this limitation, compound endodomains have been constructed: fusion
of the intracellular part of a T-cell co-stimulatory molecule to
that of CD3.zeta. results in second generation receptors which can
transmit an activating and co-stimulatory signal simultaneously
after antigen recognition. The co-stimulatory domain most commonly
used is that of CD28. This supplies the most potent co-stimulatory
signal--namely immunological signal 2, which triggers T-cell
proliferation. Some receptors have also been described which
include TNF receptor family endodomains, such as the closely
related OX40 and 41 BB which transmit survival signals. Even more
potent third generation CARs have now been described which have
endodomains capable of transmitting activation, proliferation and
survival signals.
[0131] CAR-encoding nucleic acids may be transferred to T cells
using, for example, retroviral vectors. Lentiviral vectors may be
employed. In this way, a large number of cancer-specific T cells
can be generated for adoptive cell transfer. When the CAR binds the
target-antigen, this results in the transmission of an activating
signal to the T-cell it is expressed on. Thus the CAR directs the
specificity and cytotoxicity of the T cell towards tumour cells
expressing the targeted antigen.
[0132] The first aspect of the invention relates to a T-cell which
co-expresses a first CAR and a second CAR such that a T-cell can
recognize a desired pattern of expression on target cells in the
manner of a logic gate as detailed in the truth tables: table 1, 2
and 3.
[0133] Both the first and second (and optionally subsequent) CARs
comprise:
(i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane
domain; and (iii) an intracellular domain.
TABLE-US-00001 TABLE 1 Truth Table for CAR OR GATE Antigen A
Antigen B Response Absent Absent No activation Absent Present
Activation Present Absent Activation Present Present Activation
TABLE-US-00002 TABLE 2 Truth Table for CAR AND GATE Antigen A
Antigen B Response Absent Absent No activation Absent Present No
Activation Present Absent No Activation Present Present
Activation
TABLE-US-00003 TABLE 3 Truth Table for CAR AND NOT GATE Antigen A
Antigen B Response Absent Absent No activation Absent Present No
Activation Present Absent Activation Present Present No
Activation
[0134] The first and second CAR of the T cell of the present
invention may be produced as a polypeptide comprising both CARs,
together with a cleavage site.
[0135] SEQ ID No. 1 to 5 give examples of such polypeptides, which
each comprise two CARs. The CAR may therefore comprise one or other
part of the following amino acid sequences, which corresponds to a
single CAR.
SEQ ID No 1 is a CAR OR gate which recognizes CD19 OR CD33 SEQ ID
No 2 Is a CAR AND gate which recognizes CD19 AND CD33 using a CD148
phosphatase SEQ ID No 3 Is an alternative implementation of the CAR
AND GATE which recognizes CD19 AND CD33 which uses a CD45
phosphatase SEQ ID No 4 Is a CAR AND NOT GATE which recognizes CD19
AND NOT CD33 based on PTPN6 phosphatase SEQ ID No 5 Is an
alternative implementation of the CAR AND NOT gate which recognizes
CD19 AND NOT CD33 and is based on an ITIM containing endodomain
from LAIR1 SEQ ID No 6. Is a further alternative implementation of
the CAR AND NOT gate which recognizes CD19 AND NOT CD33 and
recruits a PTPN6-CD148 fusion protein to an ITIM containing
endodomain.
TABLE-US-00004 SEQ ID No. 1
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK
LEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG
VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC
AKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQ
VLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLI
YDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRS
GGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMH
WIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYC
AAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPK
DTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA
YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID No. 2
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK
LEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG
VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC
AKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQ
VLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLI
YDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRS
GGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMH
WIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYC
AAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPK
DTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPK
KSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPY
DISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLT
KCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHF
TSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENEN
TVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAP
VTTFGKTNGYIA SEQ ID No. 3
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK
LEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG
VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC
AKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQ
VLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLI
YDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRS
GGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMH
WIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYC
AAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPK
DTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGKKDPKALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVER
DDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYV
DILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKAT
VIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKAT
GREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDA
MLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYL
HNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKH
ELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFWQMI
FQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKR
KDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDG
SQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNG
QVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSV
NGPASPALNQGS SEQ ID No. 4
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK
LEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG
VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC
AKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQ
VLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLI
YDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRS
GGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMH
WIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYC
AAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRG
SVKGTGLDFACDIYWAPLAGICVALLLSLIITLICYHRSRKRVCKSGGGSFWEEFESLQKQEV
KNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENA
KTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPY
SVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQ
RQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTE
AQYKFIYVAIAQFIETTKKKL SEQ ID No. 5
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK
LEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG
VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC
AKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQ
VLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLI
YDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRS
GGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMH
WIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYC
AAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRG
SVKGTGLDFACDILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVD
VLERTADKATVNGLPEKDRETDTSALAAGSSQEVIYAQLDHWALTQRTARAVSPQSTKPM
AESITYAAVARH SEQ ID No. 6
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK
LEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG
VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC
AKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG
AVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQ
VLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLI
YDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRS
GGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMH
WIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYC
AAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRG
SVKGTGLDFACDILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVD
VLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPM
AESITYAAVARHRAEGRGSLLTCGDVEENPGPWYHGHMSGGQAETLLQAKGEPWTFLVR
ESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKT
GIEEASGAFVYLRQPYSGGGGSFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAE
NRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWR
MVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNI
QTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGT
FIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIY
QNTTAMTIYENLAPVTTFGKTNGYIASGS
[0136] The CAR may comprise a variant of the CAR-encoding part of
the sequence shown as SEQ ID No. 1, 2, 3, 4, 5 or 6 having at least
80, 85, 90, 95, 98 or 99% sequence identity, provided that the
variant sequence is a CAR having the required properties.
[0137] Methods of sequence alignment are well known in the art and
are accomplished using suitable alignment programs. The % sequence
identity refers to the percentage of amino acid or nucleotide
residues that are identical in the two sequences when they are
optimally aligned. Nucleotide and protein sequence homology or
identity may be determined using standard algorithms such as a
BLAST program (Basic Local Alignment Search Tool at the National
Center for Biotechnology Information) using default parameters,
which is publicly available at http://blast.ncbi.nlm.nih.gov. Other
algorithms for determining sequence identity or homology include:
LALIGN (http://www.ebi.ac.uk/Tools/psa/lalign/ and
http://www.ebi.ac.uk/Tools/psa/lalign/nucleotide.html), AMAS
(Analysis of Multiply Aligned Sequences, at
http://www.compbio.dundee.ac.uk/Software/Amas/amas.html), FASTA
(http://www.ebi.ac.uk/Tools/sss/fasta/), Clustal Omega
(http://www.ebi.ac.uk/Tools/msa/clustalo/), SIM
(http://web.expasy.org/sim/), and EMBOSS Needle
(http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html).
CAR Logical or Gate
[0138] In this embodiment, the antigen binding domains of the first
and second CARs of the present invention bind to different antigens
and both CARs comprise an activating endodomain. Both CARs have
different spacer domains to prevent cross-pairing of the two
different receptors. A T cell can hence be engineered to activate
upon recognition of either or both antigens. This is useful in the
field of oncology as indicated by the Goldie-Coldman hypothesis:
sole targeting of a single antigen may result in tumour escape by
modulation of said antigen due to the high mutation rate inherent
in most cancers. By simultaneously targeting two antigens, the
probably of such escape is exponentially reduced.
[0139] Various tumour associated antigens are known as shown in the
following Table 4. For a given disease, the first CAR and second
CAR may bind to two different TAAs associated with that disease. In
this way, tumour escape by modulating a single antigen is
prevented, since a second antigen is also targeted. For example,
when targeting a B-cell malignancy, both CD19 and CD20 can be
simultaneously targeted. In this embodiment, it is important that
the two CARs do not heterodimerize.
TABLE-US-00005 TABLE 4 Cancer type TAA Diffuse Large B-cell
Lymphoma CD19, CD20 Breast cancer ErbB2, MUC1 AML CD13, CD33
Neuroblastoma GD2, NCAM B-CLL CD19, CD52 Colorectal cancer Folate
binding protein, CA-125
Kinetic Segregation Model
[0140] Subsequent pairing of CARs to generate the AND gate and the
AND NOT gate are based on the kinetic segregation model (KS) of
T-cell activation. This is a functional model, backed by
experimental data, which explains how antigen recognition by a
T-cell receptor is converted into down-stream activation signals.
Briefly: at the ground state, the signalling components on the
T-cell membrane are in dynamic homeostasis whereby dephosphorylated
ITAMs are favoured over phosphorylated ITAMs. This is due to
greater activity of the transmembrane CD45/CD148 phosphatases over
membrane-tethered kinases such as Ick. When a T-cell engages a
target cell through a T-cell receptor (or CAR) recognition of
cognate antigen, tight immunological synapses form. This close
juxtapositioning of the T-cell and target membranes excludes
CD45/CD148 due to their large ectodomains which cannot fit into the
synapse. Segregation of a high concentration of T-cell receptor
associated ITAMs and kinases in the synapse, in the absence of
phosphatases, leads to a state whereby phosphorylated ITAMs are
favoured. ZAP70 recognizes a threshold of phosphorylated ITAMs and
propagates a T-cell activation signal. This advanced understanding
of T-cell activation is exploited by the present invention. In
particular, the invention is based on this understanding of how
ectodomains of different length and/or bulk and/or charge and/or
configuration and/or glycosylation result in differential
segregation upon synapse formation.
The Car Logical and Gate
[0141] In this embodiment, one CAR comprises an activating
endodomain and one CAR comprises an inhibitory endodomain whereby
the inhibitory CAR constitutively inhibits the first activating
CAR, but upon recognition of its cognate antigen releases its
inhibition of the activating CAR. In this manner, a T-cell can be
engineered to trigger only if a target cell expresses both cognate
antigens. This behaviour is achieved by the activating CAR
comprising an activating endodomain containing ITAM domains for
example the endodomain of CD3 Zeta, and the inhibitory CAR
comprising the endodomain from a phosphatase able to
dephosphorylate an ITAM (e.g. CD45 or CD148). Crucially, the spacer
domains of both CARs are significantly different in size and/or
shape and/or charge etc. When only the activating CAR is ligated,
the inhibitory CAR is in solution on the T-cell surface and can
diffuse in and out of the synapse inhibiting the activating CAR.
When both CARs are ligated, due to differences in spacer
properties, the activating and inhibiting CAR are differentially
segregated allowing the activating CAR to trigger T-cell activation
unhindered by the inhibiting CAR.
[0142] This is of considerable utility in the field of cancer
therapy. Currently, immunotherapies typically target a single
antigen. Most cancers cannot be differentiated from normal tissues
on the basis of a single antigen. Hence, considerable "on-target
off-tumour" toxicity occurs whereby normal tissues are damaged by
the therapy. For instance, whilst targeting CD20 to treat B-cell
lymphomas with Rituximab, the entire normal B-cell compartment is
depleted. For instance, whilst targeting CD52 to treat chronic
lymphocytic leukaemia, the entire lymphoid compartment is depleted.
For instance, whilst targeting CD33 to treat acute myeloid
leukaemia, the entire myeloid compartment is damaged etc. By
restricting activity to a pair of antigens, much more refined
targeting, and hence less toxic therapy can be developed. A
practical example is targeting of CLL which expresses both CD5 and
CD19. Only a small proportion of normal B-cells express both
antigens, so the off-target toxicity of targeting both antigens
with a logical AND gate is substantially less than targeting each
antigen individually.
[0143] The design of the present invention is a considerable
improvement on previous implementation as described by Wilkie et
al. ((2012). J. Clin. Immunol. 32, 1059-1070) and then tested in
vivo (Kloss et al (2013) Nat. Biotechnol. 31, 71-75). In this
implementation, the first CAR comprises of an activating
endodomain, and the second a co-stimulatory domain. This way, a
T-cell only receives an activating and co-stimulatory signal when
both antigens are present. However, the T-cell still will activate
in the sole presence of the first antigen resulting in the
potential for off-target toxicity. Further, the implementation of
the present invention allows for multiple compound linked gates
whereby a cell can interpret a complex pattern of antigens.
TABLE-US-00006 TABLE 5 Cancer Type Antigens Chronic Lymphocytic
Leukaemia CD5, CD19 Neuroblastoma ALK, GD2 Glioma EGFR, Vimentin
Multiple myeloma BCMA, CD138 Renal Cell Carcinoma Carbonic
anhydrase IX, G250 T-ALL CD2, N-Cadherin Prostate Cancer PSMA,
hepsin (or others)
The Car Logical and not Gate
[0144] In this embodiment, one CAR comprises an activating
endodomain and one CAR comprises an inhibitory endodomain such that
this inhibitory CAR is only active when it recognizes its cognate
antigen. Hence a T-cell engineered in this manner is activated in
response to the sole presence of the first antigen but is not
activated when both antigens are present. This invention is
implemented by inhibitory CARs with a spacer that co-localise with
the first CAR but either the phosphatase activity of the inhibitory
CAR should not be so potent that it inhibits in solution, or the
inhibitory endodomain in fact recruits a phosphatase solely when
the inhibitory CAR recognizes its cognate target. Such endodomains
are termed "ligation-on" or semi-inhibitory herein.
[0145] This invention is of use in refining targeting when a tumour
can be distinguished from normal tissue by the presence of tumour
associated antigen and the loss of an antigen expressed on normal
tissue. The AND NOT gate is of considerable utility in the field of
oncology as it allows targeting of an antigen which is expressed by
a normal cell, which normal cell also expresses the antigen
recognised by the CAR comprising the activating endodomain. An
example of such an antigen is CD33 which is expressed by normal
stem cells and acute myeloid leukaemia (AML) cells. CD34 is
expressed on stem cells but not typically expressed on AML cells. A
T-cell recognizing CD33 AND NOT CD34 would result in destruction of
leukaemia cells but sparing of normal stem cells.
[0146] Potential antigen pairs for use with AND NOT gates are shown
in Table 6.
TABLE-US-00007 TABLE 6 Antigen expressed by Normal cell which
normal cell but not Disease TAA expresses TAA cancer cell AML CD33
stem cells CD34 Myeloma BCMA Dendritic cells CD1c B-CLL CD160
Natural Killer cells CD56 Prostate PSMA Neural Tissue NCAM cancer
Bowel A33 Normal bowel HLA class I cancer epithelium
Compound Gates
[0147] The kinetic segregation model with the above components
allows compound gates to be made e.g. a T-cell which triggers in
response to patterns of more than two target antigens. For example,
it is possible to make a T cell which only triggers when three
antigens are present (A AND B AND C). Here, a cell expresses three
CARs, each recognizing antigens A, B and C. One CAR is excitatory
and two are inhibitory, which each CAR having spacer domains which
result in differential segregation. Only when all three are
ligated, will the T-cell activate. A further example: (A OR B) AND
C: here, CARs recognizing antigens A and B are activating and have
spacers which co-localise, while CAR recognizing antigen C is
inhibitory and has a spacer which results in different
co-segregation. A further example (A AND NOT B) AND C: Here CAR
against antigen A has an activating endodomain and co-localises
with CAR against antigen B which has a conditionally inhibiting
endodomain. CAR against antigen C has a spacer who segregates
differently from A or B and is inhibitory. In fact, ever more
complex boolean logic can be programmed with these simple
components of the invention with any number of CARs and
spacers.
Signal Peptide
[0148] The CARs of the T cell of the present invention may comprise
a signal peptide so that when the CAR is expressed inside a cell,
such as a T-cell, the nascent protein is directed to the
endoplasmic reticulum and subsequently to the cell surface, where
it is expressed.
[0149] The core of the signal peptide may contain a long stretch of
hydrophobic amino acids that has a tendency to form a single
alpha-helix. The signal peptide may begin with a short positively
charged stretch of amino acids, which helps to enforce proper
topology of the polypeptide during translocation. At the end of the
signal peptide there is typically a stretch of amino acids that is
recognized and cleaved by signal peptidase. Signal peptidase may
cleave either during or after completion of translocation to
generate a free signal peptide and a mature protein. The free
signal peptides are then digested by specific proteases.
[0150] The signal peptide may be at the amino terminus of the
molecule.
[0151] The signal peptide may comprise the SEQ ID No. 7, 8 or 9 or
a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations
(insertions, substitutions or additions) provided that the signal
peptide still functions to cause cell surface expression of the
CAR.
TABLE-US-00008 SEQ ID No. 7: MGTSLLCWMALCLLGADHADG
[0152] The signal peptide of SEQ ID No. 7 is compact and highly
efficient. It is predicted to give about 95% cleavage after the
terminal glycine, giving efficient removal by signal peptidase.
TABLE-US-00009 SEQ ID No. 8: MSLPVTALLLPLALLLHAARP
[0153] The signal peptide of SEQ ID No. 8 is derived from IgG1.
TABLE-US-00010 SEQ ID No. 9: MAVPTQVLGLLLLWLTDARC
[0154] The signal peptide of SEQ ID No. 9 is derived from CD8.
[0155] The signal peptide for the first CAR may have a different
sequence from the signal peptide of the second CAR (and from the
3.sup.rd CAR and 4.sup.th CAR etc).
Antigen Binding Domain
[0156] The antigen binding domain is the portion of the CAR which
recognizes antigen. Numerous antigen-binding domains are known in
the art, including those based on the antigen binding site of an
antibody, antibody mimetics, and T-cell receptors. For example, the
antigen-binding domain may comprise: a single-chain variable
fragment (scFv) derived from a monoclonal antibody; a natural
ligand of the target antigen; a peptide with sufficient affinity
for the target; a single domain antibody; an artificial single
binder such as a Darpin (designed ankyrin repeat protein); or a
single-chain derived from a T-cell receptor.
[0157] The antigen binding domain may comprise a domain which is
not based on the antigen binding site of an antibody. For example
the antigen binding domain may comprise a domain based on a
protein/peptide which is a soluble ligand for a tumour cell surface
receptor (e.g. a soluble peptide such as a cytokine or a
chemokine); or an extracellular domain of a membrane anchored
ligand or a receptor for which the binding pair counterpart is
expressed on the tumour cell.
[0158] Examples 11 to 13 relate to a CAR which binds BCMA, in which
the antigen binding domain comprises APRIL, a ligand for BCMA.
[0159] The antigen binding domain may be based on a natural ligand
of the antigen.
[0160] The antigen binding domain may comprise an affinity peptide
from a combinatorial library or a de novo designed affinity
protein/peptide.
Spacer Domain
[0161] CARs comprise a spacer sequence to connect the
antigen-binding domain with the transmembrane domain and spatially
separate the antigen-binding domain from the endodomain. A flexible
spacer allows the antigen-binding domain to orient in different
directions to facilitate binding.
[0162] In the T cell of the present invention, the first and second
CARs comprise different spacer molecules. For example, the spacer
sequence may, for example, comprise an IgG1 Fc region, an IgG1
hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer may
alternatively comprise an alternative linker sequence which has
similar length and/or domain spacing properties as an IgG1 Fc
region, an IgG1 hinge or a CD8 stalk. A human IgG1 spacer may be
altered to remove Fc binding motifs.
[0163] Examples of amino acid sequences for these spacers are given
below:
TABLE-US-00011 (hinge-CH2CH3 of human IgG1) SEQ ID No. 10
AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD SEQ ID No. 11 (human CD8 stalk):
ITTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHIRGLDFACDI SEQ ID No. 12 (human
IgG1 hinge): AEPKSPDKTHTCPPCPKDPK (CD2 ectodomain) SEQ ID No. 13
KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKE
KETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDL
KIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITH
KWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD (CD34 ectodomain) SEQ ID no. 14
SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNE
ATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPE
TTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIR
EVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSL
LLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVA SHQSYSQKT
[0164] Since CARs are typically homodimers (see FIG. 1a),
cross-pairing may result in a heterodimeric chimeric antigen
receptor. This is undesirable for various reasons, for example: (1)
the epitope may not be at the same "level" on the target cell so
that a cross-paired CAR may only be able to bind to one antigen;
(2) the VH and VL from the two different scFv could swap over and
either fail to recognize target or worse recognize an unexpected
and unpredicted antigen. For the "OR" gate and the "AND NOT" gate,
the spacer of the first CAR is sufficiently different from the
spacer of the second CAR in order to avoid cross-pairing. The amino
acid sequence of the first spacer may share less that 50%, 40%, 30%
or 20% identity at the amino acid level with the second spacer.
[0165] In other aspects of the invention (for example the AND gate)
it is important that the spacer of the first CAR has a different
length, and/or charge and/or shape and/or configuration and/or
glycosylation, such that when both first and second CARs bind their
target antigen, the difference in spacer charge or dimensions
results in spatial separation of the two types of CAR to different
parts of the membrane to result in activation as predicted by the
kinetic separation model. In these aspects, the different length,
shape and/or configuration of the spacers is carefully chosen
bearing in mind the size and binding epitope on the target antigen
to allow differential segregation upon cognate target recognition.
For example the IgG1 Hinge, CD8 stalk, IgG1 Fc, ectodomain of CD34,
ectodomain of CD45 are expected to differentially segregate.
[0166] Examples of spacer pairs which differentially segregate and
are therefore suitable for use with the AND gate are shown in the
following Table:
TABLE-US-00012 Stimulatory CAR spacer Inhibitory CAR spacer
Human-CD8STK Human-IgG-Hinge-CH2CH3 Human-CD3z ectodomain
Human-IgG-Hinge-CH2CH3 Human-IgG-Hinge Human-IgG-Hinge-CH2CH3
Human-CD28STK Human-IgG-Hinge-CH2CH3 Human-CD8STK
Human-IgM-Hinge-CH2CH3CD4 Human-CD3z ectodomain
Human-IgM-Hinge-CH2CH3CD4 Human-IgG-Hinge Human-IgM-Hinge-CH2CH3CD4
Human-CD28STK Human-IgM-Hinge-CH2CH3CD4
[0167] In other aspects of the invention (for example the AND NOT
gate), it is important that the spacer be sufficiently different as
to prevent cross-pairing, but to be sufficiently similar to
co-localise. Pairs of orthologous spacer sequences may be employed.
Examples are murine and human CD8 stalks, or alternatively spacer
domains which are monomeric--for instance the ectodomain of
CD2.
[0168] Examples of spacer pairs which co-localise and are therefore
suitable for use with the AND NOT gate are shown in the following
Table:
TABLE-US-00013 Stimulatory CAR spacer Inhibitory CAR spacer
Human-CD8aSTK Mouse CD8aSTK Human-CD28STK Mouse CD8aSTK
Human-IgG-Hinge Human-CD3z ectodomain Human-CD8aSTK Mouse CD28STK
Human-CD28STK Mouse CD28STK Human-IgG-Hinge-CH2CH3
Human-IgM-Hinge-CH2CH3CD4
[0169] All the spacer domains mentioned above form homodimers.
However the mechanism is not limited to using homodimeric receptors
and should work with monomeric receptors as long as the spacer is
sufficiently rigid. An example of such a spacer is CD2 or truncated
CD22.
Transmembrane Domain
[0170] The transmembrane domain is the sequence of the CAR that
spans the membrane.
[0171] A transmembrane domain may be any protein structure which is
thermodynamically stable in a membrane. This is typically an alpha
helix comprising of several hydrophobic residues. The transmembrane
domain of any transmembrane protein can be used to supply the
transmembrane portion of the invention. The presence and span of a
transmembrane domain of a protein can be determined by those
skilled in the art using the TMHMM algorithm
(http://www.cbs.dtu.dk/services/TMHMM-2.0/). Further, given that
the transmembrane domain of a protein is a relatively simple
structure, i.e a polypeptide sequence predicted to form a
hydrophobic alpha helix of sufficient length to span the membrane,
an artificially designed TM domain may also be used (U.S. Pat. No.
7,052,906 B1 describes synthetic transmembrane components).
[0172] The transmembrane domain may be derived from CD28, which
gives good receptor stability.
Activating Endodomain
[0173] The endodomain is the signal-transmission portion of the
CAR. After antigen recognition, receptors cluster, native CD45 and
CD148 are excluded from the synapse and a signal is transmitted to
the cell. The most commonly used endodomain component is that of
CD3-zeta which contains 3 ITAMs. This transmits an activation
signal to the T cell after antigen is bound. CD3-zeta may not
provide a fully competent activation signal and additional
co-stimulatory signaling may be needed. For example, chimeric CD28
and OX40 can be used with CD3-Zeta to transmit a
proliferative/survival signal, or all three can be used
together.
[0174] Where the T cell of the present invention comprises a CAR
with an activating endodomain, it may comprise the CD3-Zeta
endodomain alone, the CD3-Zeta endodomain with that of either CD28
or OX40 or the CD28 endodomain and OX40 and CD3-Zeta
endodomain.
[0175] Any endodomain which contains an ITAM motif can act as an
activation endodomain in this invention. Several proteins are known
to contain endodomains with one or more ITAM motifs. Examples of
such proteins include the CD3 epsilon chain, the CD3 gamma chain
and the CD3 delta chain to name a few. The ITAM motif can be easily
recognized as a tyrosine separated from a leucine or isoleucine by
any two other amino acids, giving the signature YxxL/I. Typically,
but not always, two of these motifs are separated by between 6 and
8 amino acids in the tail of the molecule (YxxL/Ix(6-8)YxxL/I).
Hence, one skilled in the art can readily find existing proteins
which contain one or more ITAM to transmit an activation signal.
Further, given the motif is simple and a complex secondary
structure is not required, one skilled in the art can design
polypeptides containing artificial ITAMs to transmit an activation
signal (see WO 2000063372, which relates to synthetic signalling
molecules).
[0176] The transmembrane and intracellular T-cell signalling domain
(endodomain) of a CAR with an activating endodomain may comprise
the sequence shown as SEQ ID No. 15, 16 or 17 or a variant thereof
having at least 80% sequence identity.
TABLE-US-00014 comprising CD28 transmembrane domain and CD3 Z
endodomain SEQ ID No. 15
FWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYN
ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR comprising CD28
transmembrane domain and CD28 and CD3 Zeta endodomains SEQ ID No.
16 FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT
RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY
DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGHDGLYQGLSTATKDTYDALHMQALPPR comprising CD28 transmembrane domain
and CD28, OX40 and CD3 Zeta endodomains. SEQ ID No. 17
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT
RKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHST
LAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG
GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
TKDTYDALHMQALPPR
[0177] A variant sequence may have at least 80%, 85%, 90%, 95%, 98%
or 99% sequence identity to SEQ ID No. 15, 16 or 17, provided that
the sequence provides an effective trans-membrane domain and an
effective intracellular T cell signaling domain.
"Ligation-Off" Inhibitory Endodomain
[0178] In the embodiment referred above as the AND gate, one of the
CARs comprises an inhibitory endodomain such that the inhibitory
CAR inhibits T-cell activation by the activating CAR in the absence
of inhibitory CAR ligation, but does not significantly inhibit
T-cell activation by the activating CAR when the inhibitory CAR is
ligated. This is termed a "ligation-off" inhibitory endodomain.
[0179] In this case, the spacer of the inhibitory CAR is of a
different length, charge, shape and/or configuration and/or
glycosylation from the spacer of the activating CAR, such that when
both receptors are ligated, the difference in spacer dimensions
results in isolation of the activating CARs and the inhibitory CARs
in different membrane compartments of the immunological synapse, so
that the activating endodomain is released from inhibition by the
inhibitory endodomain.
[0180] The inhibitory endodomains for use in a ligation-off
inhibitory CAR may therefore comprise any sequence which inhibits
T-cell signaling by the activating CAR when it is in the same
membrane compartment (i.e. in the absence of the antigen for the
inhibitory CAR) but which does not significantly inhibit T cell
signaling when it is isolated in a separate part of the membrane
from the inhibitory CAR.
[0181] The ligation-off inhibitory endodomain may be or comprise a
tyrosine phosphatase, such as a receptor-like tyrosine phosphatase.
An inhibitory endodomain may be or comprise any tyrosine
phosphatase that is capable of inhibiting the TCR signalling when
only the stimulatory receptor is ligated. An inhibitory endodomain
may be or comprise any tyrosine phosphatase with a sufficiently
fast catalytic rate for phosphorylated ITAMs that is capable of
inhibiting the TCR signalling when only the stimulatory receptor is
ligated.
[0182] For example, the inhibitory endodomain of an AND gate may
comprise the endodomain of CD148 or CD45. CD148 and CD45 have been
shown to act naturally on the phosphorylated tyrosines up-stream of
TCR signalling.
[0183] CD148 is a receptor-like protein tyrosine phosphatase which
negatively regulates TCR signaling by interfering with the
phosphorylation and function of PLC.gamma.1 and LAT.
[0184] CD45 present on all hematopoetic cells, is a protein
tyrosine phosphatase which is capable of regulating signal
transduction and functional responses, again by phosphorylating PLC
.gamma.1.
[0185] An inhibitory endodomain may comprise all of part of a
receptor-like tyrosine phosphatase. The phosphatase may interfere
with the phosphorylation and/or function of elements involved in
T-cell signalling, such as PLC.gamma.1 and/or LAT.
[0186] The transmembrane and endodomain of CD45 and CD148 is shown
as SEQ ID No. 18 and No. 19 respectively.
TABLE-US-00015 -CD45 trans-membrane and endodomain sequence SEQ ID
18 ALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVERDDEKQL
MNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFN
QNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQG
PRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFG
DVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPED
PHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAEN
KVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHP
YLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNV
IPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYW
KPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWG
EGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSV
EQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTG
IFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIAS
TYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQ
AEGSEPTSGTEGPEHSVNGPASPALNQGS -CD148 trans-membrane and endodomain
sequence SEQ ID 19
AVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVEN
FEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVL
PYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWR
MVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLP
EWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDY
MKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLR
MHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAP VTTFGKTNGYIA
[0187] An inhibitory CAR may comprise all or part of SEQ ID No 18
or 19 (for example, it may comprise the phosphatase function of the
endodomain). It may comprise a variant of the sequence or part
thereof having at least 80% sequence identity, as long as the
variant retains the capacity to basally inhibit T cell signalling
by the activating CAR.
[0188] Other spacers and endodomains may be tested for example
using the model system exemplified herein. Target cell populations
can be created by transducing a suitable cell line such as a SupT1
cell line either singly or doubly to establish cells negative for
both antigens (the wild-type), positive for either and positive for
both (e.g. CD19-CD33-, CD19+CD33-, CD19-CD33+ and CD19+CD33+). T
cells such as the mouse T cell line BW5147 which releases IL-2 upon
activation may be transduced with pairs of CARs and their ability
to function in a logic gate measured through measurement of IL-2
release (for example by ELISA). For example, it is shown in Example
4 that both CD148 and CD45 endodomains can function as inhibitory
CARs in combination with an activating CAR containing a CD3 Zeta
endodomain.
[0189] These CARs rely upon a short/non-bulky CD8 stalk spacer on
one CAR and a bulky Fc spacer on the other CAR to achieve AND
gating. When both receptors are ligated, the difference in spacer
dimensions results in isolation of the different receptors in
different membrane compartments, releasing the CD3 Zeta receptor
from inhibition by the CD148 or CD45 endodomains. In this way,
activation only occurs once both receptors are activated. It can be
readily seen that this modular system can be used to test
alternative spacer pairs and inhibitory endodomains. If the spacers
do not achieve isolation following ligation of both receptors, the
inhibition would not be released and so no activation would occur.
If the inhibitory endodomain under test is ineffective, activation
would be expected in the presence of ligation of the activating CAR
irrespective of the ligation status of the inhibitory CAR.
"Ligation-On" Endodomain
[0190] In the embodiment referred above as the AND NOT gate, one of
the CARs comprises a "ligation-on" inhibitory endodomain such that
the inhibitory CAR does not significantly inhibit T-cell activation
by the activating CAR in the absence of inhibitory CAR ligation,
but inhibits T-cell activation by the activating CAR when the
inhibitory CAR is ligated.
[0191] The "ligation-on" inhibitory endodomain may be or comprise a
tyrosine phosphatase that is incapable of inhibiting the TCR
signalling when only the stimulatory receptor is ligated.
[0192] The "ligation-on" inhibitory endodomain may be or comprise a
tyrosine phosphatase with a sufficiently slow catalytic rate for
phosphorylated ITAMs that is incapable of inhibiting the TCR
signalling when only the stimulatory receptor is ligated but it is
capable of inhibiting the TCR signalling response when concentrated
at the synapse. Concentration at the synapse is achieved through
inhibitory receptor ligation.
[0193] If a tyrosine phosphatase has a catalytic rate which is too
fast for a "ligation-on" inhibitory endodomain, then it is possible
to tune-down the catalytic rates of phosphatase through
modification such as point mutations and short linkers (which cause
steric hindrance) to make it suitable for a "ligation-on"
inhibitory endodomain.
[0194] In this first embodiment the endodomain may be or comprise a
phosphatase which is considerably less active than CD45 or CD148,
such that significant dephosphorylation of ITAMS only occurs when
activating and inhibitory endodomains are co-localised. Many
suitable sequences are known in the art. For example, the
inhibitory endodomain of a NOT AND gate may comprise all or part of
a protein-tyrosine phosphatase such as PTPN6.
[0195] Protein tyrosine phosphatases (PTPs) are signaling molecules
that regulate a variety of cellular processes including cell
growth, differentiation, mitotic cycle, and oncogenic
transformation. The N-terminal part of this PTP contains two tandem
Src homolog (SH2) domains, which act as protein phospho-tyrosine
binding domains, and mediate the interaction of this PTP with its
substrates. This PTP is expressed primarily in hematopoietic cells,
and functions as an important regulator of multiple signaling
pathways in hematopoietic cells.
[0196] The inhibitor domain may comprise all of PTPN6 (SEQ ID No.
20) or just the phosphatase domain (SEQ ID No. 21).
TABLE-US-00016 -sequence of PTPN6 SEQ ID 20
MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVT
HIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL
NCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVL
SDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIE
EASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFES
LQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSD
YINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMT
TREVEKGRNKCVPYWPEVGMQRAYGPYSVINCGEHDTTEYKLRTLQVSPL
DNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPII
VHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQ
TEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHA
KASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK -sequence of
phosphatase domain of PTPN6 SEQ ID 21
FWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRD
SNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQEN
SRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVINCGEHDTTEYKLR
TLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESL
PHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRA
QRSGMVQTEAQYKFIYVAIAQF
[0197] A second embodiment of a ligation-on inhibitory endodomain
is an ITIM (Immunoreceptor Tyrosine-based Inhibition motif)
containing endodomain such as that from CD22, LAIR-1, the Killer
inhibitory receptor family (KIR), LILRB1, CTLA4, PD-1, BTLA etc.
When phosphorylated, ITIMs recruits endogenous PTPN6 through its
SH2 domain. If co-localised with an ITAM containing endodomain,
dephosphorylation occurs and the activating CAR is inhibited.
[0198] An ITIM is a conserved sequence of amino acids
(S/I/V/LxYxxl/V/L) that is found in the cytoplasmic tails of many
inhibitory receptors of the immune system. One skilled in the art
can easily find protein domains containing an ITIM. A list of human
candidate ITIM-containing proteins has been generated by
proteome-wide scans (Staub, et al (2004) Cell. Signal. 16,
435-456). Further, since the consensus sequence is well known and
little secondary structure appears to be required, one skilled in
the art could generate an artificial ITIM.
[0199] ITIM endodomains from PDCD1, BTLA4, LILRB1, LAIR1, CTLA4,
KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 and KIR3DL3 are shown in SEQ ID
22 to 31 respectively
TABLE-US-00017 PDCD1 endodomain SEQ ID 22
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPC
VPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL BTLA4 SEQ ID 23
KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMM
EDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGD
YENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH LILRB1 SEQ ID 24
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENL
YAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLS
GEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPS
QEGPSPAVPSIYATLAIH LAIR1 SEQ ID 25
HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRET
DTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVAR H CTLA4 SEQ ID
26 FLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPEC EKQFQPYFIPIN
KIR2DL1 SEQ ID 27
GNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGNRTV
NREDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPN AESRSKVVSCP
KIR2DL4 SEQ ID 28
GIARHLHAVIRYSVAIILFTILPFFLLHRWCSKKKENAAVMNQEPAGHRT
VNREDSDEQDPQEVTYAQLDHCIFTQRKITGPSQRSKRPSTDTSVCIELP
NAEPRALSPAHEHHSQALMGSSRETTALSQTQLASSNVPAAGI KIR2DL5 SEQ ID 29
TGIRRHLHILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRT
VNREDSDDQDPQEVTYAQLDHCVFTQTKITSPSQRPKTPPTDTTMYMELP
NAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI KIR3DL1 SEQ ID 30
KDPRHLHILIGTSVVIILFILLLFFLLHLWCSNKKNAAVMDQEPAGNRTA
NSEDSDEQDPEEVTYAQLDHCVFTQRKITRPSQRPKTPPTDTILYTELPN AKPRSKVVSCP
KIR3DL3 SEQ ID 31
KDPGNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGN
RTVNREDSDEQDPQEVTYAQLNHCVFTQRKITRPSQRPKTPPTDTSV
[0200] A third embodiment of a ligation-on inhibitory endodomain is
an ITIM containing endodomain co-expressed with a fusion protein.
The fusion protein may comprise at least part of a protein-tyrosine
phosphatase and at least part of a receptor-like tyrosine
phosphatase. The fusion may comprise one or more SH2 domains from
the protein-tyrosine phosphatase. For example, the fusion may be
between a PTPN6 SH2 domain and CD45 endodomain or between a PTPN6
SH2 domain and CD148 endodomain. When phosphorylated, the ITIM
domains recruit the fusion protein bring the highly potent CD45 or
CD148 phosphatase to proximity to the activating endodomain
blocking activation.
[0201] SEQUENCES of fusion proteins are listed 32 and 33
TABLE-US-00018 PTPN6-CD45 fusion protein SEQ ID 32
WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPG
SPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYL
RQPYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRK
IADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVE
LSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKA
TVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQ
KLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFF
SGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLM
VQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLE
AEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVLKHELEMSKE
SEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDF
MIQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTY
TLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLP
QKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQ
VVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFD
NEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPAS PALNQGS
PTPN6-CD148 fusion SEQ ID 33
ETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVM
CEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYRKKRKDA
KNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGI
SQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGY
HSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYW
PSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSW
PDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAID
RLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQK
DSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA
[0202] A ligation-on inhibitory CAR may comprise all or part of SEQ
ID No 20 or 21. It may comprise all or part of SEQ ID 22 to 31. It
may comprise all or part of SEQ ID 22 to 31 co-expressed with
either SEQ ID 32 or 33. It may comprise a variant of the sequence
or part thereof having at least 80% sequence identity, as long as
the variant retains the capacity to inhibit T cell signaling by the
activating CAR upon ligation of the inhibitory CAR.
[0203] As above, alternative spacers and endodomains may be tested
for example using the model system exemplified herein. It is shown
in Example 5 that the PTPN6 endodomain can function as a
semi-inhibitory CAR in combination with an activating CAR
containing a CD3 Zeta endodomain. These CARs rely upon a human CD8
stalk spacer on one CAR and a mouse CD8 stalk spacer on the other
CAR. The orthologous sequences prevent cross pairing. However, when
both receptors are ligated, the similarity between the spacers
results in co-segregation of the different receptors in the same
membrane compartments. This results in inhibition of the CD3 Zeta
receptor by the PTPN6 endodomain. If only the activating CAR is
ligated the PTPN6 endodomain is not sufficiently active to prevent
T cell activation. In this way, activation only occurs if the
activating CAR is ligated and the inhibitory CAR is not ligated
(AND NOT gating). It can be readily seen that this modular system
can be used to test alternative spacer pairs and inhibitory
domains. If the spacers do not achieve co-segregation following
ligation of both receptors, the inhibition would not be effective
and so activation would occur. If the semi-inhibitory endodomain
under test is ineffective, activation would be expected in the
presence of ligation of the activating CAR irrespective of the
ligation status of the semi-inhibitory CAR.
Co-Expression Site
[0204] The second aspect of the invention relates to a nucleic acid
which encodes the first and second CARs.
[0205] The nucleic acid may produce a polypeptide which comprises
the two CAR molecules joined by a cleavage site. The cleavage site
may be self-cleaving, such that when the polypeptide is produced,
it is immediately cleaved into the first and second CARs without
the need for any external cleavage activity.
[0206] Various self-cleaving sites are known, including the
Foot-and-Mouth disease virus (FMDV) 2a self-cleaving peptide, which
has the sequence shown as SEQ ID No. 34:
TABLE-US-00019 SEQ ID No. 34 RAEGRGSLLTCGDVEENPGP.
[0207] The co-expressing sequence may be an internal ribosome entry
sequence (IRES). The co-expressing sequence may be an internal
promoter.
Cell
[0208] The first aspect of the invention relates to a cell which
co-expresses a first CAR and a second CAR at the cell surface.
[0209] The cell may be any eukaryotic cell capable of expressing a
CAR at the cell surface, such as an immunological cell.
[0210] In particular the cell may be an immune effector cell such
as a T cell or a natural killer (NK) cell
[0211] T cells or T lymphocytes are a type of lymphocyte that play
a central role in cell-mediated immunity. They can be distinguished
from other lymphocytes, such as B cells and natural killer cells
(NK cells), by the presence of a T-cell receptor (TCR) on the cell
surface. There are various types of T cell, as summarised
below.
[0212] Helper T helper cells (TH cells) assist other white blood
cells in immunologic processes, including maturation of B cells
into plasma cells and memory B cells, and activation of cytotoxic T
cells and macrophages. TH cells express CD4 on their surface. TH
cells become activated when they are presented with peptide
antigens by MHC class II molecules on the surface of antigen
presenting cells (APCs). These cells can differentiate into one of
several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which
secrete different cytokines to facilitate different types of immune
responses.
[0213] Cytotoxic T cells (TC cells, or CTLs) destroy virally
infected cells and tumor cells, and are also implicated in
transplant rejection. CTLs express the CD8 at their surface. These
cells recognize their targets by binding to antigen associated with
MHC class I, which is present on the surface of all nucleated
cells. Through IL-10, adenosine and other molecules secreted by
regulatory T cells, the CD8+ cells can be inactivated to an anergic
state, which prevent autoimmune diseases such as experimental
autoimmune encephalomyelitis.
[0214] Memory T cells are a subset of antigen-specific T cells that
persist long-term after an infection has resolved. They quickly
expand to large numbers of effector T cells upon re-exposure to
their cognate antigen, thus providing the immune system with
"memory" against past infections. Memory T cells comprise three
subtypes: central memory T cells (TCM cells) and two types of
effector memory T cells (TEM cells and TEMRA cells). Memory cells
may be either CD4+ or CD8+. Memory T cells typically express the
cell surface protein CD45RO.
[0215] Regulatory T cells (Treg cells), formerly known as
suppressor T cells, are crucial for the maintenance of
immunological tolerance. Their major role is to shut down T
cell-mediated immunity toward the end of an immune reaction and to
suppress auto-reactive T cells that escaped the process of negative
selection in the thymus.
[0216] Two major classes of CD4+ Treg cells have been
described--naturally occurring Treg cells and adaptive Treg
cells.
[0217] Naturally occurring Treg cells (also known as
CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been
linked to interactions between developing T cells with both myeloid
(CD11c+) and plasmacytoid (CD123+) dendritic cells that have been
activated with TSLP. Naturally occurring Treg cells can be
distinguished from other T cells by the presence of an
intracellular molecule called FoxP3. Mutations of the FOXP3 gene
can prevent regulatory T cell development, causing the fatal
autoimmune disease IPEX.
[0218] Adaptive Treg cells (also known as Tr1 cells or Th3 cells)
may originate during a normal immune response.
[0219] The T cell of the invention may be any of the T cell types
mentioned above, in particular a CTL.
[0220] Natural killer (NK) cells are a type of cytolytic cell which
forms part of the innate immune system. NK cells provide rapid
responses to innate signals from virally infected cells in an MHC
independent manner
[0221] NK cells (belonging to the group of innate lymphoid cells)
are defined as large granular lymphocytes (LGL) and constitute the
third kind of cells differentiated from the common lymphoid
progenitor generating B and T lymphocytes. NK cells are known to
differentiate and mature in the bone marrow, lymph node, spleen,
tonsils and thymus where they then enter into the circulation.
[0222] The CAR cells of the invention may be any of the cell types
mentioned above.
[0223] CAR-expressing cells, such as CAR-expressing T or NK cells,
may either be created ex vivo either from a patient's own
peripheral blood (1.sup.st party), or in the setting of a
haematopoietic stem cell transplant from donor peripheral blood
(2.sup.nd party), or peripheral blood from an unconnected donor
(3.sup.rd party).
[0224] The present invention also provide a cell composition
comprising CAR expressing T cells and/or CAR expressing NK cells
according to the present invention. The cell composition may be
made by tranducing or transfecting a blood-sample ex vivo with a
nucleic acid according to the present invention.
[0225] Alternatively, CAR-expressing cells may be derived from ex
vivo differentiation of inducible progenitor cells or embryonic
progenitor cells to the relevant cell type, such as T cells.
Alternatively, an immortalized cell line such as a T-cell line
which retains its lytic function and could act as a therapeutic may
be used.
[0226] In all these embodiments, CAR cells are generated by
introducing DNA or RNA coding for the CARs by one of many means
including transduction with a viral vector, transfection with DNA
or RNA.
[0227] A CAR T cell of the invention may be an ex vivo T cell from
a subject. The T cell may be from a peripheral blood mononuclear
cell (PBMC) sample. T cells may be activated and/or expanded prior
to being transduced with CAR-encoding nucleic acid, for example by
treatment with an anti-CD3 monoclonal antibody.
[0228] A CAR T cell of the invention may be made by: [0229] (i)
isolation of a T cell-containing sample from a subject or other
sources listed above; and [0230] (ii) transduction or transfection
of the T cells with one or more nucleic acid sequence(s) encoding
the first and second CAR.
[0231] The T cells may then by purified, for example, selected on
the basis of co-expression of the first and second CAR.
Nucleic Acid Sequences
[0232] The second aspect of the invention relates to one or more
nucleic acid sequence(s) which codes for a first CAR and a second
CAR as defined in the first aspect of the invention.
[0233] The nucleic acid sequence may comprise one of the following
sequences, or a variant thereof
TABLE-US-00020 SEQ ID 35 OR gate SEQ ID 36 AND gate using CD45 SEQ
ID 37 AND gate using CD148 SEQ ID 38 AND NOT gate using PTPN6 as
endodomain SEQ ID 39 AND NOT gate using LAIR1 endodomain SEQ ID 40
AND NOT gate using LAIR1 and PTPN6 SH2 fusion with CD148
phosphatase SEQ ID No. 35:
>MP13974.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-
CD28tmZw ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCC
AGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGAC
CGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACC
AGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACA
GCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC
ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCC
TGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAG
GCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGT
GAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGAC
CTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCC
CCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTA
CAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGT
GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAG
CACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTG
ACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC
ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG
GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGT
GGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTT
TCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG
GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT
GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC
CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA
GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG
GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCC
TCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC
CGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGAT
GCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGA
TCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCA
GCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGAT
GGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAA
GTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGC
TCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAG
GAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTG
CAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCC
TGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTC
CAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTAT
CGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCT
ACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA
GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTC
TCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA
CACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA
ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG
CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG
CAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCT
GAGTCTGAGCCCAGGCAAGAAGGACCCCAAGTTCTGGGTCCTGGTGGTGGTGGGAGG
CGTGCTGGCCTGTTACTCTCTCCTGGTGACCGTGGCCTTCATCATCTTTTGGGTGCGCT
CCCGGGTGAAGTTTTCTCGCTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAATCA
GCTGTACAATGAACTGAACCTGGGCAGGCGGGAGGAGTACGACGTGCTGGATAAGCG
GAGAGGCAGAGACCCCGAGATGGGCGGCAAACCACGGCGCAAAAATCCCCAGGAGG
GACTCTATAACGAGCTGCAGAAGGACAAAATGGCCGAGGCCTATTCCGAGATCGGCAT
GAAGGGAGAGAGAAGACGCGGAAAGGGCCACGACGGCCTGTATCAGGGATTGTCCAC
CGCTACAAAAGATACATATGATGCCCTGCACATGCAGGCCCTGCCACCCAGATGA
>MP14802.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-
dCD45 SEQ ID No. 36
ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCC
AGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGAC
CGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACC
AGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACA
GCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC
ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCC
TGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAG
GCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGT
GAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGAC
CTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCC
CCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTA
CAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGT
GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAG
CACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTG
ACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC
ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG
GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGT
GGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTT
TCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG
GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT
GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC
CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA
GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG
GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCC
TCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC
CGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGAT
GCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGA
TCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCA
GCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGAT
GGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAA
GTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGC
TCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAG
GAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTG
CAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCC
TGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTC
CAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTAT
CGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCT
ACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA
GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTC
TCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA
CACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA
ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG
CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG
CAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCT
GAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCACTGATAGCATTTCTGGCATTTCTG
ATTATTGTGACATCAATAGCCCTGCTTGTTGTTCTCTACAAAATCTATGATCTACATAAGA
AAAGATCCTGCAATTTAGATGAACAGCAGGAGCTTGTTGAAAGGGATGATGAAAAACAA
CTGATGAATGTGGAGCCAATCCATGCAGATATTTTGTTGGAAACTTATAAGAGGAAGAT
TGCTGATGAAGGAAGACTTTTTCTGGCTGAATTTCAGAGCATCCCGCGGGTGTTCAGCA
AGTTTCCTATAAAGGAAGCTCGAAAGCCCTTTAACCAGAATAAAAACCGTTATGTTGACA
TTCTTCCTTATGATTATAACCGTGTTGAACTCTCTGAGATAAACGGAGATGCAGGGTCAA
ACTACATAAATGCCAGCTATATTGATGGTTTCAAAGAACCCAGGAAATACATTGCTGCAC
AAGGTCCCAGGGATGAAACTGTTGATGATTTCTGGAGGATGATTTGGGAACAGAAAGC
CACAGTTATTGTCATGGTCACTCGATGTGAAGAAGGAAACAGGAACAAGTGTGCAGAAT
ACTGGCCGTCAATGGAAGAGGGCACTCGGGCTTTTGGAGATGTTGTTGTAAAGATCAA
CCAGCACAAAAGATGTCCAGATTACATCATTCAGAAATTGAACATTGTAAATAAAAAAGA
AAAAGCAACTGGAAGAGAGGTGACTCACATTCAGTTCACCAGCTGGCCAGACCACGGG
GTGCCTGAGGATCCTCACTTGCTCCTCAAACTGAGAAGGAGAGTGAATGCCTTCAGCA
ATTTCTTCAGTGGTCCCATTGTGGTGCACTGCAGTGCTGGTGTTGGGCGCACAGGAAC
CTATATCGGAATTGATGCCATGCTAGAAGGCCTGGAAGCCGAGAACAAAGTGGATGTTT
ATGGTTATGTTGTCAAGCTAAGGCGACAGAGATGCCTGATGGTTCAAGTAGAGGCCCA
GTACATCTTGATCCATCAGGCTTTGGTGGAATACAATCAGTTTGGAGAAACAGAAGTGA
ATTTGTCTGAATTACATCCATATCTACATAACATGAAGAAAAGGGATCCACCCAGTGAGC
CGTCTCCACTAGAGGCTGAATTCCAGAGACTTCCTTCATATAGGAGCTGGAGGACACA
GCACATTGGAAATCAAGAAGAAAATAAAAGTAAAAACAGGAATTCTAATGTCATCCCATA
TGACTATAACAGAGTGCCACTTAAACATGAGCTGGAAATGAGTAAAGAGAGTGAGCATG
ATTCAGATGAATCCTCTGATGATGACAGTGATTCAGAGGAACCAAGCAAATACATCAAT
GCATCTTTTATAATGAGCTACTGGAAACCTGAAGTGATGATTGCTGCTCAGGGACCACT
GAAGGAGACCATTGGTGACTTTTGGCAGATGATCTTCCAAAGAAAAGTCAAAGTTATTG
TTATGCTGACAGAACTGAAACATGGAGACCAGGAAATCTGTGCTCAGTACTGGGGAGA
AGGAAAGCAAACATATGGAGATATTGAAGTTGACCTGAAAGACACAGACAAATCTTCAA
CTTATACCCTTCGTGTCTTTGAACTGAGACATTCCAAGAGGAAAGACTCTCGAACTGTG
TACCAGTACCAATATACAAACTGGAGTGTGGAGCAGCTTCCTGCAGAACCCAAGGAATT
AATCTCTATGATTCAGGTCGTCAAACAAAAACTTCCCCAGAAGAATTCCTCTGAAGGGA
ACAAGCATCACAAGAGTACACCTCTACTCATTCACTGCAGGGATGGATCTCAGCAAACG
GGAATATTTTGTGCTTTGTTAAATCTCTTAGAAAGTGCGGAAACAGAAGAGGTAGTGGA
TATTTTTCAAGTGGTAAAAGCTCTACGCAAAGCTAGGCCAGGCATGGTTTCCACATTCG
AGCAATATCAATTCCTATATGACGTCATTGCCAGCACCTACCCTGCTCAGAATGGACAA
GTAAAGAAAAACAACCATCAAGAAGATAAAATTGAATTTGATAATGAAGTGGACAAAGTA
AAGCAGGATGCTAATTGTGTTAATCCACTTGGTGCCCCAGAAAAGCTCCCTGAAGCAAA
GGAACAGGCTGAAGGTTCTGAACCCACGAGTGGCACTGAGGGGCCAGAACATTCTGTC
AATGGTCCTGCAAGTCCAGCTTTAAATCAAGGTTCATAG SEQ ID No. 37:
>MP14801.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-
dCD148 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCC
AGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGAC
CGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACC
AGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACA
GCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC
ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCC
TGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAG
GCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGT
GAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGAC
CTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCC
CCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTA
CAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGT
GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAG
CACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTG
ACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC
ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG
GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGT
GGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTT
TCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG
GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT
GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC
CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA
GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG
GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCC
TCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC
CGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGAT
GCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGA
TCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCA
GCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGAT
GGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAA
GTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGC
TCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAG
GAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTG
CAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCC
TGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTC
CAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTAT
CGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCT
ACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA
GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTC
TCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA
CACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA
ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG
CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG
CAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCT
GAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCC
CTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAA
GAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGA
GAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAG
GAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGC
TGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAA
ACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCT
ACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATT
TTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTG
AACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGG
AGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATT
TCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACC
TCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACC
TCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGT
GCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAG
AGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCT
TTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTC
AGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAAT
CTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAA
>16076.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-tm-dPTPN6
SEQ ID No. 38
ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCC
AGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGAC
CGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACC
AGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACA
GCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC
ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCC
TGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAG
GCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGT
GAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGAC
CTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCC
CCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTA
CAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGT
GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAG
CACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTG
ACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC
ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG
GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGT
GGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTT
TCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG
GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT
GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC
CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA
GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG
GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCC
TCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC
CGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGAT
GCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGA
TCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCA
GCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGAT
GGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAA
GTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGC
TCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAG
GAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTG
CAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCC
TGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTC
CAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTAT
CGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCT
ACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA
GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTC
TCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGC
ACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGC
GTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATCTACTGGGCACCTCTGGCCGGA
ATATGCGTGGCACTGCTGCTGAGCCTCATCATCACCCTGATCTGTTATCACCGAAGCCG
CAAGCGGGTGTGTAAAAGTGGAGGCGGAAGCTTCTGGGAGGAGTTTGAGAGTTTGCA
GAAGCAGGAGGTGAAGAACTTGCACCAGCGTCTGGAAGGGCAGCGGCCAGAGAACAA
GGGCAAGAACCGCTACAAGAACATTCTCCCCTTTGACCACAGCCGAGTGATCCTGCAG
GGACGGGACAGTAACATCCCCGGGTCCGACTACATCAATGCCAACTACATCAAGAACC
AGCTGCTAGGCCCTGATGAGAACGCTAAGACCTACATCGCCAGCCAGGGCTGTCTGGA
GGCCACGGTCAATGACTTCTGGCAGATGGCGTGGCAGGAGAACAGCCGTGTCATCGT
CATGACCACCCGAGAGGTGGAGAAAGGCCGGAACAAATGCGTCCCATACTGGCCCGA
GGTGGGCATGCAGCGTGCTTATGGGCCCTACTCTGTGACCAACTGCGGGGAGCATGA
CACAACCGAATACAAACTCCGTACCTTACAGGTCTCCCCGCTGGACAATGGAGACCTG
ATTCGGGAGATCTGGCATTACCAGTACCTGAGCTGGCCCGACCACGGGGTCCCCAGT
GAGCCTGGGGGTGTCCTCAGCTTCCTGGACCAGATCAACCAGCGGCAGGAAAGTCTG
CCTCACGCAGGGCCCATCATCGTGCACTGCAGCGCCGGCATCGGCCGCACAGGCACC
ATCATTGTCATCGACATGCTCATGGAGAACATCTCCACCAAGGGCCTGGACTGTGACAT
TGACATCCAGAAGACCATCCAGATGGTGCGGGCGCAGCGCTCGGGCATGGTGCAGAC
GGAGGCGCAGTACAAGTTCATCTACGTGGCCATCGCCCAGTTCATTGAAACCACTAAG
AAGAAGCTGTGA
>MP16091.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1tm-end-
o SEQ ID No. 39
ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCC
AGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGAC
CGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACC
AGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACA
GCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC
ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCC
TGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAG
GCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGT
GAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGAC
CTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCC
CCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTA
CAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGT
GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAG
CACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTG
ACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC
ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG
GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGT
GGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTT
TCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG
GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT
GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC
CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA
GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG
GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCC
TCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC
CGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGAT
GCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGA
TCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCA
GCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGAT
GGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAA
GTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGC
TCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAG
GAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTG
CAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCC
TGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTC
CAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTAT
CGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCT
ACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA
GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTC
TCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGC
ACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGC
GTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATTCTCATCGGGGTCTCAGTGGTCT
TCCTCTTCTGTCTCCTCCTCCTGGTCCTCTTCTGCCTCCATCGCCAGAATCAGATAAAG
CAGGGGCCCCCCAGAAGCAAGGACGAGGAGCAGAAGCCACAGCAGAGGCCTGACCT
GGCTGTTGATGTTCTAGAGAGGACAGCAGACAAGGCCACAGTCAATGGACTTCCTGAG
AAGGACCGGGAGACCGACACCAGCGCCCTGGCTGCAGGGAGTTCCCAGGAGGTGAC
GTATGCTCAGCTGGACCACTGGGCCCTCACACAGAGGACAGCCCGGGCTGTGTCCCC
ACAGTCCACAAAGCCCATGGCCGAGTCCATCACGTATGCAGCCGTTGCCAGACACTGA
>MP16092.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1tm-
endo-2A-PTPN6SH2-dCD148 SEQ ID no. 40
ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCC
AGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGAC
CGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACC
AGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACA
GCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC
ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCC
TGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAG
GCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGT
GAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGAC
CTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCC
CCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTA
CAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGT
GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAG
CACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTG
ACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC
ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG
GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGT
GGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTT
TCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG
GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT
GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC
CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA
GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG
GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCC
TCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC
CGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGAT
GCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGA
TCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCA
GCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGAT
GGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAA
GTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGC
TCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAG
GAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTG
CAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCC
TGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTC
CAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTAT
CGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCT
ACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA
GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTC
TCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGC
ACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGC
GTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATTCTCATCGGGGTCTCAGTGGTCT
TCCTCTTCTGTCTCCTCCTCCTGGTCCTCTTCTGCCTCCATCGCCAGAATCAGATAAAG
CAGGGGCCCCCCAGAAGCAAGGACGAGGAGCAGAAGCCACAGCAGAGGCCTGACCT
GGCTGTTGATGTTCTAGAGAGGACAGCAGACAAGGCCACAGTCAATGGACTTCCTGAG
AAGGACCGGGAGACCGACACCAGCGCCCTGGCTGCAGGGAGTTCCCAGGAGGTGAC
GTATGCTCAGCTGGACCACTGGGCCCTCACACAGAGGACAGCCCGGGCTGTGTCCCC
ACAGTCCACAAAGCCCATGGCCGAGTCCATCACGTATGCAGCCGTTGCCAGACACAGG
GCAGAAGGAAGAGGTAGCCTGCTGACTTGCGGGGACGTGGAAGAGAACCCAGGGCCA
TGGTATCATGGCCACATGTCTGGCGGGCAGGCAGAGACGCTGCTGCAGGCCAAGGGC
GAGCCCTGGACGTTTCTTGTGCGTGAGAGCCTCAGCCAGCCTGGAGACTTCGTGCTTT
CTGTGCTCAGTGACCAGCCCAAGGCTGGCCCAGGCTCCCCGCTCAGGGTCACCCACA
TCAAGGTCATGTGCGAGGGTGGACGCTACACAGTGGGTGGTTTGGAGACCTTCGACAG
CCTCACGGACCTGGTGGAGCATTTCAAGAAGACGGGGATTGAGGAGGCCTCAGGCGC
CTTTGTCTACCTGCGGCAGCCGTACAGCGGTGGCGGTGGCAGCTTTGAGGCCTACTTC
AAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGC
TTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAAT
CGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCAT
TCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTT
TATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGG
AGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAAT
GTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAAT
GACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCC
AGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGG
TGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGA
AGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGA
CGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTG
GATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGA
GGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTC
AAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCC
CGTGACCACATTTGGAAAGACCAATGGTTACATCGCCAGCGGTAGCTAA
[0234] The nucleic acid sequence may encode the same amino acid
sequence as that encoded by SEQ ID No. 35, 36, 37, 38, 39 or 40,
but may have a different nucleic acid sequence, due to the
degeneracy of the genetic code. The nucleic acid sequence may have
at least 80, 85, 90, 95, 98 or 99% identity to the sequence shown
as SEQ ID No. 35, 36, 37, 38, 39 or 40, provided that it encodes a
first CAR and a second CAR as defined in the first aspect of the
invention.
Vector
[0235] The present invention also provides a vector, or kit of
vectors which comprises one or more CAR-encoding nucleic acid
sequence(s). Such a vector may be used to introduce the nucleic
acid sequence(s) into a host cell so that it expresses the first
and second CARs.
[0236] The vector may, for example, be a plasmid or a viral vector,
such as a retroviral vector or a lentiviral vector, or a transposon
based vector or synthetic mRNA.
[0237] The vector may be capable of transfecting or transducing a T
cell.
Pharmaceutical Composition
[0238] The present invention also relates to a pharmaceutical
composition containing a plurality of CAR-expressing cells, such as
T cells or NK cells according to the first aspect of the invention.
The pharmaceutical composition may additionally comprise a
pharmaceutically acceptable carrier, diluent or excipient. The
pharmaceutical composition may optionally comprise one or more
further pharmaceutically active polypeptides and/or compounds. Such
a formulation may, for example, be in a form suitable for
intravenous infusion.
Method of Treatment
[0239] The T cells of the present invention may be capable of
killing target cells, such as cancer cells. The target cell may be
recognisable by a defined pattern of antigen expression, for
example the expression of antigen A AND antigen B; the expression
of antigen A OR antigen B; or the expression of antigen A AND NOT
antigen B or complex iterations of these gates.
[0240] T cells of the present invention may be used for the
treatment of an infection, such as a viral infection.
[0241] T cells of the invention may also be used for the control of
pathogenic immune responses, for example in autoimmune diseases,
allergies and graft-vs-host rejection.
[0242] T cells of the invention may be used for the treatment of a
cancerous disease, such as bladder cancer, breast cancer, colon
cancer, endometrial cancer, kidney cancer (renal cell), leukemia,
lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer,
prostate cancer and thyroid cancer.
[0243] It is particularly suited for treatment of solid tumours
where the availability of good selective single targets is
limited.
[0244] T cells of the invention may be used to treat: cancers of
the oral cavity and pharynx which includes cancer of the tongue,
mouth and pharynx; cancers of the digestive system which includes
oesophageal, gastric and colorectal cancers; cancers of the liver
and biliary tree which includes hepatocellular carcinomas and
cholangiocarcinomas; cancers of the respiratory system which
includes bronchogenic cancers and cancers of the larynx; cancers of
bone and joints which includes osteosarcoma; cancers of the skin
which includes melanoma; breast cancer; cancers of the genital
tract which include uterine, ovarian and cervical cancer in women,
prostate and testicular cancer in men; cancers of the renal tract
which include renal cell carcinoma and transitional cell carcinomas
of the utterers or bladder; brain cancers including gliomas,
glioblastoma multiforme and medullobastomas; cancers of the
endocrine system including thyroid cancer, adrenal carcinoma and
cancers associated with multiple endocrine neoplasm syndromes;
lymphomas including Hodgkin's lymphoma and non-Hodgkin lymphoma;
Multiple Myeloma and plasmacytomas; leukaemias both acute and
chronic, myeloid or lymphoid; and cancers of other and unspecified
sites including neuroblastoma.
[0245] Treatment with the T cells of the invention may help prevent
the escape or release of tumour cells which often occurs with
standard approaches.
[0246] The invention will now be further described by way of
Examples, which are meant to serve to assist one of ordinary skill
in the art in carrying out the invention and are not intended in
any way to limit the scope of the invention.
EXAMPLES
Example 1--Creation of Target Cell Populations
[0247] For the purposes of proving the principle of the invention,
receptors based on anti-CD19 and anti-CD33 were arbitrarily chosen.
Using retroviral vectors, CD19 and CD33 were cloned. These proteins
were truncated so that they do not signal and could be stably
expressed for prolonged periods. Next, these vectors were used to
transduce the SupT1 cell line either singly or doubly to establish
cells negative for both antigen (the wild-type), positive for
either and positive for both. The expression data are shown in FIG.
3.
Example 2--Design and Function of the OR Gate
[0248] To construct the OR gate, a pair of receptors recognizing
CD19 and CD33 were co-expressed. Different spacers were used to
prevent cross-pairing. Both receptors had a trans-membrane domain
derived from CD28 to improve surface stability and an endodomain
derived from that of CD3 Zeta to provide a simple activating
signal. In this way, a pair of independent 1.sup.st generation CARs
were co-expressed. The retroviral vector cassette used to
co-express the sequences utilizes a foot-and-mouth 2A self-cleaving
peptide to allow co-expression 1:1 of both receptors. The cassette
design is shown in FIG. 4, and the protein structures in FIG. 5.
The nucleotide sequence of homologous regions was codon-wobbled to
prevent recombination during retroviral vector reverse
transcription.
Example 3--Testing the OR Gate
[0249] Expression of both CARs was tested on the T-cell surface by
staining with cognate antigen fused to Fc. By using different
species of Fc domains (mouse for CD19 and rabbit for CD33),
co-expression of both CARs was determined on the cell surface by
staining with different secondary antibodies conjugated with
different fluorophores. This is shown in FIG. 6.
[0250] Functional testing was then carried out using the mouse
T-cell line BW5147. This cell line releases IL2 upon activation
allowing a simple quantitative readout. These T-cells were
co-cultured with increasing amounts of the artificial target cells
described above. T-cells responded to target cells expressing
either antigen, as shown by IL2 release measured by ELISA. Both
CARs were shown to be expressed on the cell surfaces and the
T-cells were shown to respond to either or both antigens. These
data are show in FIG. 7.
Example 4--Design and Function of the AND Gate
[0251] The AND gate combines a simple activating receptor with a
receptor which basally inhibits activity, but whose inhibition is
turned off once the receptor is ligated. This was achieved by
combining a standard 1.sup.st generation CAR with a short/non-bulky
CD8 stalk spacer and a CD3 Zeta endodomain with a second receptor
with a bulky Fc spacer whose endodomain contained either CD148 or
CD45 endodomains. When both receptors are ligated, the difference
in spacer dimensions results in isolation of the different
receptors in different membrane compartments, releasing the CD3
Zeta receptor from inhibition by the CD148 or CD45 endodomains. In
this way, activation only occurs once both receptors are activated.
CD148 and CD45 were chosen for this as they function in this manner
natively: for instance, the very bulky CD45 ectodomain excludes the
entire receptor from the immunological synapse. The expression
cassette is depicted in FIG. 8 and the subsequent proteins in FIG.
9.
[0252] Surface staining for the different specificity showed that
both receptor pairs could be effectively expressed on the cell
surface shown in FIG. 10. Function in BW5147 shows that the T-cell
is only activated in the presence of both antigens (FIG. 11).
Example 5: Demonstration of Generalizability of the AND Gate
[0253] To ensure that the observations were not a manifestation of
some specific characteristic of CD19/CD33 and their binders which
had been used, the two targeting scFvs were swapped such that now,
the activation (ITAM) signal was transmitted upon recognition of
CD33, rather than CD19; and the inhibitory (CD148) signal was
transmitted upon recognition of CD19, rather than of CD33. Since
CD45 and CD148 endodomains are considered to be functionally
similar, experimentation was restricted to AND gates with CD148
endodomain. This should still result in a functional AND gate.
T-cells expressing the new logic gate where challenged with targets
bearing either CD19 or CD33 alone, or both. The T-cells responded
to targets expressing both CD19 and CD33, but not to targets
expressing only one or none of these antigens. This shows that the
AND gate is still functional in this format (FIG. 18B).
[0254] On the same lines, it was sought to establish how
generalizable our AND gate is: the AND gate should be generalizable
across different targets. While there may be lesser or greater
fidelity of the gate given relative antigen density, cognate scFv
binding kinetics and precise distance of the scFv binding epitope,
one would expect to see some AND gate manifestations with a wide
set of targets and binders. To test this, three additional AND
gates were generated. Once again, experimentation was restricted to
the CD148 version of the AND gate. The second scFv from the
original CD148 AND gate was replaced with the anti-GD2 scFv huK666
(SEQ ID 41 and SEQ ID 42), or with the anti-CD5 scFv (SEQ ID 43 and
SEQ ID 44), or the anti-EGFRvIII scFv MR1.1 (SEQ ID 45 AND SEQ ID
46) to generate the following CAR AND gates: CD19 AND GD2; CD19 AND
CD5; CD19 AND EGFRvIII. The following artificial antigen expressing
cell lines were also generated: by transducing SupT1, and our
SupT1.CD19 with GM3 and GD2 synthases SupT1.GD2 and SupT1.CD19.GD2
were generated. By transducing SupT1 and SupT1.CD19 with a
retroviral vector coding for EGFRvIII SupT1.EGFRvIII and
SupT1.CD19.EGFRvIII were generated. Since CD5 is expressed on SupT1
cells, a different cell line was used to generate the target cells:
293T cells were generated which express CD19 alone, CD5 alone and
both CD5 and CD19 together. Expression was confirmed by
flow-cytometry (FIG. 19). T-cells expressing the three new CAR AND
gates were challenged with SupT1.CD19 and respective cognate double
positive and single positive target cells. All three AND gates
demonstrated reduced activation by the double positive cell lines
in comparison with the single positive targets (FIG. 20). This
demonstrates generalizability of the AND gate design to arbitrary
targets and cognate binders.
Example 6: Experimental Proof of Kinetic Segregation Model of CAR
AND Gate
[0255] The aim was to prove the model that differential segregation
caused by different spacers is the central mechanism behind the
ability to generate these logic CAR gates. The model is that if
only the activating CAR is ligated, the potent inhibiting `ligation
off` type CAR is in solution in the membrane and can inhibit the
activating CAR. Once both CARs are ligated, if both CAR spacers are
sufficiently different, they will segregate within the synapse and
not co-localize.
[0256] Hence, a key requirement is that the spacers are
sufficiently different. If the model is correct, if both spacers
are sufficiently similar so they co-localize when both receptors
are ligated, the gate will fail to function. To test this, the
"bulky" Fc spacer in the original CAR we replaced with a murine CD8
spacer. It was predicted that this has the similar length, bulk and
charge as human CD8 but so should not cross-pair with it. Hence,
the new gate had a first CAR which recognizes CD19, a human CD8
stalk spacer and an activatory endodomain; while the second CAR
recognizes CD33, has a mouse CD8 stalk spacer and a CD148
endodomain (FIG. 18C). T-cells were transduced to express this new
CAR gate. These T-cells were then challenged with SupT1 cells
expressing CD19 alone, CD33 alone or CD19 and CD33 together.
T-cells did not respond to SupT1 cells expressing either antigen
alone as per the original AND gate. However, CAR T-cells failed to
respond to SupT1 cells expressing both antigens, thereby confirming
the model (FIG. 18C). A functional AND gate requires both CARs to
have spacers sufficiently different so that they do not co-localize
within an immunological synapse (FIGS. 23A and B).
Example 7--Design and Function of an AND NOT Gate
[0257] Phosphatases such as CD45 and CD148 are so potent that even
a small amount entering an immunological synapse can inhibit ITAM
activation. This is the basis of inhibition of the logical AND
gate. Other classes of phosphatases are not as potent e.g. PTPN6
and related phosphatases. It was predicted that a small amount of
PTPN6 entering a synapse by diffusion would not inhibit activation.
In addition, it was predicted that if an inhibitory CAR had a
sufficiently similar spacer to an activating CAR, it could
co-localize within a synapse if both CARs were ligated. In this
case, large amounts of the inhibitory endodomain would be
sufficient to stop the ITAMS from activating when both antigens
were present. In this way, an AND NOT gate could be created.
[0258] For the NOT AND gate, the second signal needs to "veto"
activation. This is done by bringing an inhibitory signal into the
immunological synapse, for example by bringing in the phosphatase
of an enzyme such as PTPN6. We hence generated an initial AND NOT
gate as follows: two CARs co-expressed whereby the first recognizes
CD19, has a human CD8 stalk spacer and an activating endodomain;
co-expressed with an anti-CD33 CAR with a mouse CD8 stalk spacer
and an endodomain comprising of the catalytic domain of PTPN6 (SEQ
ID 38, FIG. 13 A with B). A suitable cassette is shown in FIG. 12
and preliminary functional data are shown in FIG. 14.
[0259] In addition, an alternative strategy was developed for
generating an AND NOT gate. Immune Tyrosinase Inhibitory Motifs
(ITIMs) are activated in a similar manner to ITAMS, in that they
become phosphorylated by Ick upon clustering and exclusion of
phosphatases. Instead of triggering activation by binding ZAP70,
phosphorylated ITIMs recruit phosphatases like PTPN6 through their
cognate SH2 domains. An ITIM can function as an inhibitory
endodomain, as long as the spacers on the activating and inhibiting
CARs can co-localize. To generate this construct, an AND NOT gate
was generated as follows: two CARs co-expressed--the first
recognizes CD19, has a human CD8 stalk spacer and an activating
endodomain; co-expressed with an anti-CD33 CAR with a mouse CD8
stalk spacer and an ITIM containing endodomain derived from that of
LAIR1 (SEQ ID 39, FIG. 13 A with C).
[0260] A further, more complex AND NOT gate was also developed,
whereby an ITIM is enhanced by the presence of an additional
chimeric protein: an intracellular fusion of the SH2 domain of
PTPN6 and the endodomain of CD148. In this design three proteins
are expressed--the first recognizes CD19, has a human CD8 stalk
spacer and an activating endodomain; co-expressed with an anti-CD33
CAR with a mouse CD8 stalk spacer and an ITIM containing endodomain
derived from that of LAIR1. A further 2A peptide, allows
co-expression of the PTPN6-CD148 fusion (SEQ ID 40, FIGS. 13 A and
D). It was predicted that these AND NOT gates would have a
different range of inhibition:
PTPN6-CD148>PTPN6>>ITIM.
[0261] T-cells were transduced with these gates and challenged with
targets expressing either CD19 or CD33 alone, or both CD19 and CD33
together. All three gates responded to targets expressing only
CD19, but not targets expressing both CD19 and CD33 together (FIG.
21), confirming that all three of the AND NOT gates were
functional.
Example 8: Experimental Proof of Kinetic Segregation Model of PTPN6
Based AND NOT Gate
[0262] The model of the AND NOT gate centres around the fact that
the nature of the spacers used in both CARs is pivotal for the
correct function of the gate. In the functional AND NOT gate with
PTPN6, both CAR spacers are sufficiently similar that when both
CARs are ligated, both co-localize within the synapse so the high
concentration even the weak PTPN6 is sufficient to inhibit
activation. If the spacers were different, segregation in the
synapse will isolate the PTPN6 from the ITAM allowing activation
disrupting the AND NOT gate. To test this, a control was generated
replacing the murine CD8 stalk spacer with that of Fc. In this
case, the test gate consisted of two CARs, the first recognizes
CD19, has a human CD8 stalk spacer and an ITAM endodomain; while
the second CAR recognizes CD33, has an Fc spacer and an endodomain
comprising of the phosphatase from PTPN6. This gate activates in
response to CD19, but also activates in response to CD19 and CD33
together (FIG. 22B, where function of this gate is compared with
that of the original AND NOT, and the control AND gate variant
described in Example 6). This experimental data proves the model
that for a functional AND NOT gate with PTPN6, co-localizing
spacers are needed.
Example 9: Experimental Proof of Kinetic Segregation Model of ITIM
Based AND NOT Gate
[0263] Similar to the PTPN6 based AND NOT gate, the ITIM based gate
also requires co-localization in an immunological synapse to
function as an AND NOT gate. To prove this hypothesis, a control
ITIM based gate was generated as follows: two CARs
co-expressed--the first recognizes CD19, has a human CD8 stalk
spacer and an activating endodomain; co-expressed with an anti-CD33
CAR with an Fc spacer and an ITIM containing endodomain derived
from that of LAIR1. The activity of this gate was compared with
that of the original ITIM based AND NOT gate. In this case, the
modified gate activated in response to targets expressing CD19, but
also activated in response to cells expressing both CD19 and CD33.
These data indicate that ITIM based AND NOT gates follow the
kinetic segregation based model and a correct spacer must be
selected to create a functional gate (FIG. 23B).
Example 10: Summary of Model of CAR Logic Gates Generated by
Kinetic Segregation
[0264] Based on current understanding of the kinetic-segregation
model and the experimental data described herein, a summary of the
model for a two-CAR gate is presented in FIG. 24. The Figure shows
a cell expressing two CARs, each recognizing a different antigen.
When either or both CARs recognize a target antigen on a cell, a
synapse forms and native CD45 and CD148 are excluded from the
synapse due to the bulk of their ectodomain. This sets the stage
for T-cell activation. In the case that the target cell bears only
one cognate antigen, the cognate CAR is ligated and the cognate CAR
segregates into the synapse. The unligated CAR remains in solution
on the T-cell membrane and can diffuse in and out of the synapse so
that an area of high local concentration of ligated CAR with low
concentration of unligated CAR forms. In this case, if the ligated
CAR has an ITAM and the non-ligated CAR has `ligation off" type
inhibitory endodomain such as that of CD148, the amount of
non-ligated CAR is sufficient to inhibit activation and the gate is
off. In contrast, in this case, if the ligated CAR has an ITAM and
the non-ligated CAR has a `ligation on` type inhibitory endodomain
such as PTPN6, the amount of non-ligated CAR is insufficient to
inhibit and the gate is on. When challenged by a target cell
bearing both cognate antigens, both cognate CARs are ligated and
form part of an immunological synapse. Importantly, if the CAR
spacers are sufficiently similar, the CARs co-localize in the
synapse but if the CAR spacers are sufficiently different the CARs
segregate within the synapse. In this latter case, areas of
membrane form whereby high concentrations of one CAR are present
but the other CAR is absent. In this case since segregation is
complete, even if the inhibitory endodomain is a `ligation off`
type, the gate is on. In the former case, areas of membrane form
with high concentrations of both CARs mixed together. In this case,
since both endodomains are concentrated, even if the inhibitory
endodomain is `ligation on` type, the gate is off. By selecting the
correct combination of spacer and endodomain logic can be
programmed into a CAR T-cell.
[0265] Based on our work above, we have established a series of
design rules to allow generation of logic-gated CARs (illustrated
in FIG. 31). To generate an "antigen A OR antigen B" gated CAR
T-cell, anti-A and anti-B CARs must be generated such that (1) each
CAR has a spacer which simply allows antigen access and synapse
formation such that the CAR functions, and (2) Each CAR has an
activating endodomain; To generate an "antigen A AND NOT B" gated
CAR T-cell, anti-A and anti-B CARs must be generated such that (1)
both CARs have spacers which do not cross-pair, but which will
allow the CARs to co-segregate upon recognition of both cognate
antigens on the target cell, (2) and one CAR has an activating
endodomain, while the other CAR has an endodomain which comprises
or recruits a weak phosphatase (e.g. PTPN6); (3) To generate an
"antigen A AND antigen B" gated CAR T-cell, anti-A and anti-B CARs
must be generated such that (1) one CAR has a spacer sufficiently
different from the other CAR such that both CARs will not
co-segregate upon recognition of both cognate antigens on the
target cell, (2) one CAR has an activating endodomain, while the
other car has an endodomain which comprises of a potent phosphatase
(e.g. that of CD45 or CD148). The correct spacers to achieve the
desired effect can be selected from a set of spacers with known
size/shape etc as well as taking into consideration size/shape etc
of the target antigen (for instance see FIG. 30) and the location
of the cognate epitope on the target antigen.
TABLE-US-00021 SEQ ID No 41:
SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa-dCD148
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL
IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSG
GGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS
ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDP
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAF
IIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL
QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENP
GPMETDTLLLWVLLLWVPGSTGQVQLQESGPGLVKPSQTLSITCTVSGFSLASYNIHWVRQPPGKGLE
WLGVIWAGGSTNYNSALMSRLTISKDNSKNQVFLKMSSLTAADTAVYYCAKRSDDYSWFAYWGQGTLV
TVSSGGGGSGGGGSGGGGSENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKVWI
YSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPITFGQGTKVEIKRSDPAEPKS
PDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD
ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPK
KSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKL
SVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEY
WPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRY
LVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQY
VFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA SEQ ID No. 42:
SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa-dCD148
ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACAT
CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAG
CCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTG
ATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGA
CTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACA
CCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGC
GGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCC
AGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACT
ACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGC
GAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCA
GGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACT
ACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCC
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG
CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATA
TCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT
ATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA
GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG
GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG
GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGG
CCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCC
GGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCCA
GGTGCAGCTGCAGGAGTCTGGCCCAGGCCTGGTGAAGCCCAGCCAGACCCTGAGCATCACCTGCACCG
TGAGCGGCTTCAGCCTGGCCAGCTACAACATCCACTGGGTGCGGCAGCCCCCAGGCAAGGGCCTGGAG
TGGCTGGGCGTGATCTGGGCTGGCGGCAGCACCAACTACAACAGCGCCCTGATGAGCCGGCTGACCAT
CAGCAAGGACAACAGCAAGAACCAGGTGTTCCTGAAGATGAGCAGCCTGACAGCCGCCGACACCGCCG
TGTACTACTGCGCCAAGCGGAGCGACGACTACAGCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTG
ACCGTGAGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGAACCAGAT
GACCCAGAGCCCCAGCAGCTTGAGCGCCAGCGTGGGCGACCGGGTGACCATGACCTGCAGAGCCAGCA
GCAGCGTGAGCAGCAGCTACCTGCACTGGTACCAGCAGAAGAGCGGCAAGGCCCCAAAGGTGTGGATC
TACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTA
CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCGGCT
ACCCCATCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGTCGGATCCCGCCGAGCCCAAATCT
CCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTT
CCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG
ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAT
GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA
TGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAA
GAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCA
TCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAA
AAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTG
TGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAAC
TGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTT
TCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAA
AGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGA
AAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTAT
TGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCC
GGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGT
TCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTAC
CTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGT
CGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGG
ATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTAT
GTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTA
CCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATG
GTTACATCGCCTAA SEQ ID No. 43:
SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa-dCD148
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL
IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSG
GGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS
ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDP
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAF
IIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL
QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENP
GPMETDTLLLWVLLLWVPGSTGQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKG
LEWLAHIWWDDDVYYNPSLKNQLTISKDASRDQVFLKITNLDTADTATYYCVRRRATGTGFDYWGQGT
TLTVSSGGGGSGGGGSGGGGSNIVMTQSHKFMSTSVGDRVSIACKASQDVGTAVAWYQQKPGQSPKLL
IYWTSTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCHQYNSYNTFGSGTRLELKRSDPAEPKS
PDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD
ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPK
KSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKL
SVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEY
WPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRY
LVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQY
VFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA SEQ ID No. 44:
SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa-dCD148
ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACAT
CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAG
CCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTG
ATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGA
CTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACA
CCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGC
GGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCC
AGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACT
ACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGC
GAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCA
GGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACT
ACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCC
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG
CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATA
TCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT
ATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA
GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG
GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG
GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGG
CCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCC
GGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCCA
GGTGACCCTGAAGGAGAGCGGTCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCT
TCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATTCGGCAGCCCAGCGGCAAGGGC
CTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACGTGTACTACAACCCCAGCCTGAAGAACCAGCT
GACCATCAGCAAGGACGCCAGCCGGGACCAGGTGTTCCTGAAGATCACCAACCTGGACACCGCCGACA
CCGCCACCTACTACTGCGTGCGGCGCCGGGCCACCGGCACCGGCTTCGACTACTGGGGCCAGGGCACC
ACCCTGACCGTGAGCAGCGGTGGCGGTGGCAGCGGCGGCGGCGGAAGCGGAGGTGGTGGCAGCAACAT
CGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGACCGGGTGAGCATCGCCTGCAAGG
CCAGCCAGGACGTGGGCACCGCCGTGGCCTGGTACCAGCAGAAGCCTGGCCAGAGCCCCAAGCTGCTG
ATCTACTGGACCAGCACCCGGCACACCGGCGTGCCCGACCGGTTCACCGGCAGCGGCAGCGGCACCGA
CTTCACCCTGACCATCACCAACGTGCAGAGCGAGGACCTGGCCGACTACTTCTGCCACCAGTACAACA
GCTACAACACCTTCGGCAGCGGCACCCGGCTGGAGCTGAAGCGGTCGGATCCCGCCGAGCCCAAATCT
CCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTT
CCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG
ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAT
GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA
TGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAA
GAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCA
TCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAA
AAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTG
TGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAAC
TGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTT
TCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAA
AGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGA
AAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTAT
TGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCC
GGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGT
TCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTAC
CTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGT
CGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGG
ATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTAT
GTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTA
CCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATG
GTTACATCGCCTAA SEQ ID No. 45:
SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa- dCD148
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL
IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSG
GGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS
ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDP
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAF
IIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL
QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENP
GPMETDTLLLWVLLLWVPGSTGQVKLQQSGGGLVKPGASLKLSCVTSGFTFRKFGMSWVRQTSDKRLE
WVASISTGGYNTYYSDNVKGRFTISRENAKNTLYLQMSSLKSEDTALYYCTRGYSSTSYAMDYWGQGT
TVTVSSGGGGSGGGGSGGGGSDIELTQSPASLSVATGEKVTIRCMTSTDIDDDMNWYQQKPGEPPKFL
ISEGNTLRPGVPSRFSSSGTGTDFVFTIENTLSEDVGDYYCLQSFNVPLTFGDGTKLEIKRSDPAEPK
SPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKP
KKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVK
LSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEE
YWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFR
YLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQ
YVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA SEQ ID No. 46:
SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa- dCD148
ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACAT
CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAG
CCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTG
ATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGA
CTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACA
CCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGC
GGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCC
AGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACT
ACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGC
GAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCA
GGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACT
ACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCC
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG
CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATA
TCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT
ATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA
GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG
GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG
GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGG
CCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCC
GGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCCA
GGTGAAGCTGCAGCAGAGCGGCGGAGGCCTGGTGAAGCCCGGCGCCAGCCTGAAGCTGAGCTGCGTGA
CCAGCGGCTTCACCTTCCGGAAGTTCGGCATGAGCTGGGTGCGGCAGACCAGCGACAAGCGGCTGGAG
TGGGTGGCCAGCATCAGCACCGGCGGCTACAACACCTACTACAGCGACAACGTGAAGGGCCGGTTCAC
CATCAGCCGGGAGAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGAAGAGCGAGGACACCG
CCCTGTACTACTGCACCCGGGGCTACAGCAGCACCAGCTACGCTATGGACTACTGGGGCCAGGGCACC
ACCGTGACAGTGAGCAGCGGCGGAGGAGGCAGTGGTGGGGGTGGATCTGGCGGAGGTGGCAGCGACAT
CGAGCTGACCCAGAGCCCCGCCAGCCTGAGCGTGGCCACCGGCGAGAAGGTGACCATCCGGTGCATGA
CCAGCACCGACATCGACGACGACATGAACTGGTACCAGCAGAAGCCCGGCGAGCCCCCAAAGTTCCTG
ATCAGCGAGGGCAACACCCTGCGGCCCGGCGTGCCCAGCCGGTTCAGCAGCAGCGGCACCGGCACCGA
CTTCGTGTTCACCATCGAGAACACCCTGAGCGAGGACGTGGGCGACTACTACTGCCTGCAGAGCTTCA
ACGTGCCCCTGACCTTCGGCGACGGCACCAAGCTGGAGATCAAGCGGTCGGATCCCGCCGAGCCCAAA
TCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCT
CTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGG
ACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG
AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG
GATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGG
CAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCT
TCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCT
AAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAA
CTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAG
AACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAA
CTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAA
GAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGG
AGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAG
TATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCT
TCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGAC
AGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGG
TACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGG
GGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCG
TGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAG
TATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTAT
CTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCA
ATGGTTACATCGCCTAA
Example 11: Design and Construction of APRIL Based CARs
[0266] APRIL in its natural form is a secreted type II protein. The
use of APRIL as a BCMA binding domain for a CAR requires conversion
of this type II secreted protein to a type I membrane bound protein
and for this protein to be stable and to retain binding to BCMA in
this form. To generate candidate molecules, the extreme
amino-terminus of APRIL was deleted to remove binding to
proteoglycans. Next, a signal peptide was added to direct the
nascent protein to the endoplasmic reticulum and hence the cell
surface. Also, because the nature of spacer used can alter the
function of a CAR, three different spacer domains were tested: an
APRIL based CAR was generated comprising (i) a human IgG1 spacer
altered to remove Fc binding motifs; (ii) a CD8 stalk; and (iii)
the IgG1 hinge alone (cartoon in FIG. 25 and amino acid sequences
in FIG. 26). These CARs were expressed in a bicistronic retroviral
vector (FIG. 27A) so that a marker protein--truncated CD34 could be
co-expressed as a convenient marker gene.
Example 12: Expression and Function of APRIL Based CARs
[0267] The aim of this study was to test whether the APRIL based
CARs which had been constructed were expressed on the cell surface
and whether APRIL had folded to form the native protein. T-cells
were transduced with these different CAR constructs and stained
using a commercially available anti-APRIL mAb, along with staining
for the marker gene and analysed by flow-cytometry. The results of
this experiment are shown in FIG. 27B where APRIL binding is
plotting against marker gene fluorescence. These data show that in
this format, the APRIL based CARs are expressed on the cell surface
and APRIL folds sufficiently to be recognized by an anti-APRIL
mAb.
[0268] Next, it was determined whether APRIL in this format could
recognize BCMA and TACI. Recombinant BCMA and TACI were generated
as fusions with mouse IgG2a-Fc. These recombinant proteins were
incubated with the transduced T-cells. After this, the cells were
washed and stained with an anti-mouse fluorophore conjugated
antibody and an antibody to detect the marker gene conjugated to a
different fluorophore. The cells were analysed by flow cytometry
and the results are presented in FIG. 27C. The different CARs were
able to bind both BCMA and TACI. Surprisingly, the CARs were better
able to bind BCMA than TACI. Also, surprisingly CARs with a CD8
stalk or IgG1 hinge spacer were better able to bind BCMA and TACI
than CAR with an Fc spacer.
Example 13: APRIL Based Chimeric Antigen Receptors are Active
Against BCMA Expressing Cells
[0269] T-cells from normal donors were transduced with the
different APRIL CARs and tested against SupT1 cells either
wild-type, or engineered to express BCMA and TACI. Several
different assays were used to determine function. A classical
chromium release assay was performed. Here, the target cells (the
SupT1 cells) were labelled with 51Cr and mixed with effectors (the
transduced T-cells) at different ratio. Lysis of target cells was
determined by counting 51Cr in the co-culture supernatant (FIG. 28A
shows the cumulative data).
[0270] In addition, supernatant from T-cells cultured 1:1 with
SupT1 cells was assayed by ELISA for Interferon-gamma (FIG. 28B
shows cumulative data). Measurement of T-cell expansion after one
week of co-culture with SupT1 cells was also performed (FIG. 28C).
T-cells were counted by flow-cytometry calibrated with counting
beads. These experimental data show that APRIL based CARs can kill
BCMA expressing targets. Further, these data show that CARs based
on the CD8 stalk or IgG1 hinge performed better than the Fc-pvaa
based CAR.
Example 14: Functional Analysis of the AND Gate in Primary
Cells
[0271] PBMCs were isolated from blood and stimulated using PHA and
IL-2. Two days later the cells were transduced on retronectin
coated plates with retro virus containing the CD19:CD33 AND gate
construct. On day 5 the expression level of the two CARs translated
by the AND gate construct was evaluated via flow cytometry and the
cells were depleted of CD56+ cells (predominantly NK cells). On day
6 the PBMCs were placed in a co-culture with target cells at a 1:2
effector to target cell ratio. On day 8 the supernatant was
collected and analysed for IFN-gamma secretion via ELISA (FIG.
29).
[0272] These data demonstrate that the AND gate functions in
primary cells.
[0273] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in molecular biology, cell
biology or related fields are intended to be within the scope of
the following claims.
Sequence CWU 1
1
5311129PRTArtificial SequenceChimeric antigen receptor (CAR) 1Met
Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10
15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser
Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr
Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly145 150 155 160Leu
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly 165 170
175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg
180 185 190Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr
Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
Lys Asp Asn Ser 210 215 220Lys Ser Gln Val Phe Leu Lys Met Asn Ser
Leu Gln Thr Asp Asp Thr225 230 235 240Ala Ile Tyr Tyr Cys Ala Lys
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255Met Asp Tyr Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265 270Thr Thr Thr
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280 285Ser
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 290 295
300Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Phe
Trp305 310 315 320Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile Ile Phe Trp Val Arg
Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp Ala Pro Ala Tyr Gln
Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu Leu Asn Leu Gly Arg
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380Arg Gly Arg Asp
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro385 390 395 400Gln
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 405 410
415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp 435 440 445Ala Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala
Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr Cys Gly Asp Val Glu Glu
Asn Pro Gly Pro Met Ala465 470 475 480Val Pro Thr Gln Val Leu Gly
Leu Leu Leu Leu Trp Leu Thr Asp Ala 485 490 495Arg Cys Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 500 505 510Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr 515 520 525Phe
Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 530 535
540Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp Gly Val Pro Ser Arg
Phe545 550 555 560Ser Gly Ser Gly Ser Gly Thr Gln Tyr Thr Leu Thr
Ile Ser Ser Leu 565 570 575Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln His Tyr Lys Asn Tyr 580 585 590Pro Leu Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg Ser Gly 595 600 605Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615 620Gly Gly Ser Arg
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu625 630 635 640Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 645 650
655Thr Leu Ser Asn Tyr Gly Met His Trp Ile Arg Gln Ala Pro Gly Lys
660 665 670Gly Leu Glu Trp Val Ser Ser Ile Ser Leu Asn Gly Gly Ser
Thr Tyr 675 680 685Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala 690 695 700Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr705 710 715 720Ala Val Tyr Tyr Cys Ala Ala
Gln Asp Ala Tyr Thr Gly Gly Tyr Phe 725 730 735Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Met Asp Pro 740 745 750Ala Glu Pro
Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 755 760 765Ala
Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 770 775
780Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val
Val785 790 795 800Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val 805 810 815Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln 820 825 830Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln 835 840 845Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 850 855 860Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro865 870 875 880Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 885 890
895Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
900 905 910Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr 915 920 925Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr 930 935 940Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe945 950 955 960Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys 965 970 975Ser Leu Ser Leu Ser
Pro Gly Lys Lys Asp Pro Lys Phe Trp Val Leu 980 985 990Val Val Val
Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 995 1000
1005Ala Phe Ile Ile Phe Trp Val Arg Ser Arg Val Lys Phe Ser Arg
1010 1015 1020Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
Leu Tyr 1025 1030 1035Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
Asp Val Leu Asp 1040 1045 1050Lys Arg Arg Gly Arg Asp Pro Glu Met
Gly Gly Lys Pro Arg Arg 1055 1060 1065Lys Asn Pro Gln Glu Gly Leu
Tyr Asn Glu Leu Gln Lys Asp Lys 1070 1075 1080Met Ala Glu Ala Tyr
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 1085 1090 1095Arg Gly Lys
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 1100 1105 1110Thr
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 1115 1120
1125Arg21350PRTArtificial SequenceChimeric antigen receptor (CAR)
2Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5
10 15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Asp Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His
Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala
Thr Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly
Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly145 150 155
160Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly
165 170 175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro
Pro Arg 180 185 190Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
Glu Thr Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr
Ile Ile Lys Asp Asn Ser 210 215 220Lys Ser Gln Val Phe Leu Lys Met
Asn Ser Leu Gln Thr Asp Asp Thr225 230 235 240Ala Ile Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255Met Asp Tyr
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265 270Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280
285Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
Phe Trp305 310 315 320Val Leu Val Val Val Gly Gly Val Leu Ala Cys
Tyr Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile Ile Phe Trp Val
Arg Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp Ala Pro Ala Tyr
Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro385 390 395
400Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
405 410 415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His 420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp 435 440 445Ala Leu His Met Gln Ala Leu Pro Pro Arg
Arg Ala Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr Cys Gly Asp Val
Glu Glu Asn Pro Gly Pro Met Ala465 470 475 480Val Pro Thr Gln Val
Leu Gly Leu Leu Leu Leu Trp Leu Thr Asp Ala 485 490 495Arg Cys Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 500 505 510Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr 515 520
525Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
530 535 540Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp Gly Val Pro Ser
Arg Phe545 550 555 560Ser Gly Ser Gly Ser Gly Thr Gln Tyr Thr Leu
Thr Ile Ser Ser Leu 565 570 575Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln His Tyr Lys Asn Tyr 580 585 590Pro Leu Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg Ser Gly 595 600 605Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615 620Gly Gly Ser
Arg Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu625 630 635
640Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
645 650 655Thr Leu Ser Asn Tyr Gly Met His Trp Ile Arg Gln Ala Pro
Gly Lys 660 665 670Gly Leu Glu Trp Val Ser Ser Ile Ser Leu Asn Gly
Gly Ser Thr Tyr 675 680 685Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala 690 695 700Lys Ser Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr705 710 715 720Ala Val Tyr Tyr Cys
Ala Ala Gln Asp Ala Tyr Thr Gly Gly Tyr Phe 725 730 735Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Met Asp Pro 740 745 750Ala
Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 755 760
765Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
770 775 780Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys
Val Val785 790 795 800Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val 805 810 815Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln 820 825 830Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 835 840 845Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 850 855 860Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro865 870 875
880Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
885 890 895Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 900 905 910Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr 915 920 925Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr 930 935 940Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe945 950 955 960Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 965 970 975Ser Leu Ser
Leu Ser Pro Gly Lys Lys Asp Pro Lys Ala Val Phe Gly 980 985 990Cys
Ile Phe Gly Ala Leu Val Ile Val Thr Val Gly Gly Phe Ile Phe 995
1000 1005Trp Arg Lys Lys Arg Lys Asp Ala Lys Asn Asn Glu Val Ser
Phe 1010 1015 1020Ser Gln Ile Lys Pro Lys Lys Ser Lys Leu Ile Arg
Val Glu Asn 1025 1030 1035Phe Glu Ala Tyr Phe Lys Lys Gln Gln Ala
Asp Ser Asn Cys Gly 1040 1045 1050Phe Ala Glu Glu Tyr Glu Asp Leu
Lys Leu Val Gly Ile Ser Gln 1055 1060 1065Pro Lys Tyr Ala Ala Glu
Leu Ala Glu Asn Arg Gly Lys Asn Arg 1070 1075 1080Tyr Asn Asn Val
Leu Pro Tyr Asp Ile Ser Arg Val Lys Leu Ser 1085 1090 1095Val Gln
Thr His Ser Thr Asp Asp Tyr Ile Asn Ala Asn Tyr Met 1100 1105
1110Pro Gly Tyr His Ser Lys Lys Asp Phe Ile Ala Thr Gln Gly Pro
1115 1120 1125Leu Pro Asn Thr Leu Lys Asp Phe Trp Arg Met Val Trp
Glu Lys 1130 1135 1140Asn Val Tyr Ala Ile Ile Met Leu Thr Lys Cys
Val Glu Gln Gly 1145 1150 1155Arg Thr Lys Cys Glu Glu Tyr Trp Pro
Ser Lys Gln Ala Gln Asp 1160 1165 1170Tyr Gly Asp Ile Thr Val Ala
Met Thr Ser Glu Ile Val Leu Pro 1175 1180 1185Glu Trp Thr Ile Arg
Asp Phe Thr Val Lys Asn Ile Gln Thr Ser 1190 1195 1200Glu Ser His
Pro Leu Arg Gln Phe His Phe Thr Ser Trp Pro Asp 1205 1210 1215His
Gly Val Pro Asp Thr Thr Asp Leu Leu Ile Asn Phe Arg Tyr 1220 1225
1230Leu Val Arg Asp Tyr Met Lys Gln Ser Pro Pro Glu Ser Pro Ile
1235 1240 1245Leu Val His Cys Ser Ala Gly Val Gly Arg Thr Gly Thr
Phe Ile 1250 1255 1260Ala Ile Asp Arg Leu Ile Tyr Gln Ile Glu Asn
Glu Asn Thr Val 1265 1270 1275Asp Val Tyr Gly Ile Val Tyr Asp Leu
Arg Met His Arg Pro Leu 1280 1285 1290Met Val Gln Thr Glu Asp Gln
Tyr Val Phe Leu Asn Gln Cys Val 1295 1300 1305Leu Asp Ile Val Arg
Ser Gln Lys Asp Ser Lys Val Asp Leu Ile 1310 1315
1320Tyr Gln Asn Thr Thr Ala Met Thr Ile Tyr Glu Asn Leu Ala Pro
1325 1330 1335Val Thr Thr Phe Gly Lys Thr Asn Gly Tyr Ile Ala 1340
1345 135031717PRTArtificial SequenceChimeric antigen receptor (CAR)
3Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5
10 15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Asp Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His
Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala
Thr Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly
Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly145 150 155
160Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly
165 170 175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro
Pro Arg 180 185 190Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
Glu Thr Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr
Ile Ile Lys Asp Asn Ser 210 215 220Lys Ser Gln Val Phe Leu Lys Met
Asn Ser Leu Gln Thr Asp Asp Thr225 230 235 240Ala Ile Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255Met Asp Tyr
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265 270Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280
285Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
Phe Trp305 310 315 320Val Leu Val Val Val Gly Gly Val Leu Ala Cys
Tyr Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile Ile Phe Trp Val
Arg Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp Ala Pro Ala Tyr
Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro385 390 395
400Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
405 410 415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His 420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp 435 440 445Ala Leu His Met Gln Ala Leu Pro Pro Arg
Arg Ala Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr Cys Gly Asp Val
Glu Glu Asn Pro Gly Pro Met Ala465 470 475 480Val Pro Thr Gln Val
Leu Gly Leu Leu Leu Leu Trp Leu Thr Asp Ala 485 490 495Arg Cys Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 500 505 510Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr 515 520
525Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
530 535 540Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp Gly Val Pro Ser
Arg Phe545 550 555 560Ser Gly Ser Gly Ser Gly Thr Gln Tyr Thr Leu
Thr Ile Ser Ser Leu 565 570 575Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln His Tyr Lys Asn Tyr 580 585 590Pro Leu Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg Ser Gly 595 600 605Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615 620Gly Gly Ser
Arg Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu625 630 635
640Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
645 650 655Thr Leu Ser Asn Tyr Gly Met His Trp Ile Arg Gln Ala Pro
Gly Lys 660 665 670Gly Leu Glu Trp Val Ser Ser Ile Ser Leu Asn Gly
Gly Ser Thr Tyr 675 680 685Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala 690 695 700Lys Ser Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr705 710 715 720Ala Val Tyr Tyr Cys
Ala Ala Gln Asp Ala Tyr Thr Gly Gly Tyr Phe 725 730 735Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Met Asp Pro 740 745 750Ala
Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 755 760
765Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
770 775 780Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys
Val Val785 790 795 800Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val 805 810 815Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln 820 825 830Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 835 840 845Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 850 855 860Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro865 870 875
880Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
885 890 895Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 900 905 910Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr 915 920 925Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr 930 935 940Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe945 950 955 960Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 965 970 975Ser Leu Ser
Leu Ser Pro Gly Lys Lys Asp Pro Lys Ala Leu Ile Ala 980 985 990Phe
Leu Ala Phe Leu Ile Ile Val Thr Ser Ile Ala Leu Leu Val Val 995
1000 1005Leu Tyr Lys Ile Tyr Asp Leu His Lys Lys Arg Ser Cys Asn
Leu 1010 1015 1020Asp Glu Gln Gln Glu Leu Val Glu Arg Asp Asp Glu
Lys Gln Leu 1025 1030 1035Met Asn Val Glu Pro Ile His Ala Asp Ile
Leu Leu Glu Thr Tyr 1040 1045 1050Lys Arg Lys Ile Ala Asp Glu Gly
Arg Leu Phe Leu Ala Glu Phe 1055 1060 1065Gln Ser Ile Pro Arg Val
Phe Ser Lys Phe Pro Ile Lys Glu Ala 1070 1075 1080Arg Lys Pro Phe
Asn Gln Asn Lys Asn Arg Tyr Val Asp Ile Leu 1085 1090 1095Pro Tyr
Asp Tyr Asn Arg Val Glu Leu Ser Glu Ile Asn Gly Asp 1100 1105
1110Ala Gly Ser Asn Tyr Ile Asn Ala Ser Tyr Ile Asp Gly Phe Lys
1115 1120 1125Glu Pro Arg Lys Tyr Ile Ala Ala Gln Gly Pro Arg Asp
Glu Thr 1130 1135 1140Val Asp Asp Phe Trp Arg Met Ile Trp Glu Gln
Lys Ala Thr Val 1145 1150 1155Ile Val Met Val Thr Arg Cys Glu Glu
Gly Asn Arg Asn Lys Cys 1160 1165 1170Ala Glu Tyr Trp Pro Ser Met
Glu Glu Gly Thr Arg Ala Phe Gly 1175 1180 1185Asp Val Val Val Lys
Ile Asn Gln His Lys Arg Cys Pro Asp Tyr 1190 1195 1200Ile Ile Gln
Lys Leu Asn Ile Val Asn Lys Lys Glu Lys Ala Thr 1205 1210 1215Gly
Arg Glu Val Thr His Ile Gln Phe Thr Ser Trp Pro Asp His 1220 1225
1230Gly Val Pro Glu Asp Pro His Leu Leu Leu Lys Leu Arg Arg Arg
1235 1240 1245Val Asn Ala Phe Ser Asn Phe Phe Ser Gly Pro Ile Val
Val His 1250 1255 1260Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Tyr
Ile Gly Ile Asp 1265 1270 1275Ala Met Leu Glu Gly Leu Glu Ala Glu
Asn Lys Val Asp Val Tyr 1280 1285 1290Gly Tyr Val Val Lys Leu Arg
Arg Gln Arg Cys Leu Met Val Gln 1295 1300 1305Val Glu Ala Gln Tyr
Ile Leu Ile His Gln Ala Leu Val Glu Tyr 1310 1315 1320Asn Gln Phe
Gly Glu Thr Glu Val Asn Leu Ser Glu Leu His Pro 1325 1330 1335Tyr
Leu His Asn Met Lys Lys Arg Asp Pro Pro Ser Glu Pro Ser 1340 1345
1350Pro Leu Glu Ala Glu Phe Gln Arg Leu Pro Ser Tyr Arg Ser Trp
1355 1360 1365Arg Thr Gln His Ile Gly Asn Gln Glu Glu Asn Lys Ser
Lys Asn 1370 1375 1380Arg Asn Ser Asn Val Ile Pro Tyr Asp Tyr Asn
Arg Val Pro Leu 1385 1390 1395Lys His Glu Leu Glu Met Ser Lys Glu
Ser Glu His Asp Ser Asp 1400 1405 1410Glu Ser Ser Asp Asp Asp Ser
Asp Ser Glu Glu Pro Ser Lys Tyr 1415 1420 1425Ile Asn Ala Ser Phe
Ile Met Ser Tyr Trp Lys Pro Glu Val Met 1430 1435 1440Ile Ala Ala
Gln Gly Pro Leu Lys Glu Thr Ile Gly Asp Phe Trp 1445 1450 1455Gln
Met Ile Phe Gln Arg Lys Val Lys Val Ile Val Met Leu Thr 1460 1465
1470Glu Leu Lys His Gly Asp Gln Glu Ile Cys Ala Gln Tyr Trp Gly
1475 1480 1485Glu Gly Lys Gln Thr Tyr Gly Asp Ile Glu Val Asp Leu
Lys Asp 1490 1495 1500Thr Asp Lys Ser Ser Thr Tyr Thr Leu Arg Val
Phe Glu Leu Arg 1505 1510 1515His Ser Lys Arg Lys Asp Ser Arg Thr
Val Tyr Gln Tyr Gln Tyr 1520 1525 1530Thr Asn Trp Ser Val Glu Gln
Leu Pro Ala Glu Pro Lys Glu Leu 1535 1540 1545Ile Ser Met Ile Gln
Val Val Lys Gln Lys Leu Pro Gln Lys Asn 1550 1555 1560Ser Ser Glu
Gly Asn Lys His His Lys Ser Thr Pro Leu Leu Ile 1565 1570 1575His
Cys Arg Asp Gly Ser Gln Gln Thr Gly Ile Phe Cys Ala Leu 1580 1585
1590Leu Asn Leu Leu Glu Ser Ala Glu Thr Glu Glu Val Val Asp Ile
1595 1600 1605Phe Gln Val Val Lys Ala Leu Arg Lys Ala Arg Pro Gly
Met Val 1610 1615 1620Ser Thr Phe Glu Gln Tyr Gln Phe Leu Tyr Asp
Val Ile Ala Ser 1625 1630 1635Thr Tyr Pro Ala Gln Asn Gly Gln Val
Lys Lys Asn Asn His Gln 1640 1645 1650Glu Asp Lys Ile Glu Phe Asp
Asn Glu Val Asp Lys Val Lys Gln 1655 1660 1665Asp Ala Asn Cys Val
Asn Pro Leu Gly Ala Pro Glu Lys Leu Pro 1670 1675 1680Glu Ala Lys
Glu Gln Ala Glu Gly Ser Glu Pro Thr Ser Gly Thr 1685 1690 1695Glu
Gly Pro Glu His Ser Val Asn Gly Pro Ala Ser Pro Ala Leu 1700 1705
1710Asn Gln Gly Ser 171541114PRTArtificial SequenceChimeric antigen
receptor (CAR) 4Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr
Thr Ser Ser Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Asp Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser
Arg Leu His Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135
140Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro
Gly145 150 155 160Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
Thr Val Ser Gly 165 170 175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp
Ile Arg Gln Pro Pro Arg 180 185 190Lys Gly Leu Glu Trp Leu Gly Val
Ile Trp Gly Ser Glu Thr Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser 210 215 220Lys Ser Gln Val
Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr225 230 235 240Ala
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250
255Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro
260 265 270Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
Ile Ala 275 280 285Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
Pro Ala Ala Gly 290 295 300Gly Ala Val His Thr Arg Gly Leu Asp Phe
Ala Cys Asp Ile Phe Trp305 310 315 320Val Leu Val Val Val Gly Gly
Val Leu Ala Cys Tyr Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile
Ile Phe Trp Val Arg Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375
380Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
Pro385 390 395 400Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
Met Ala Glu Ala 405 410 415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
Arg Arg Gly Lys Gly His 420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser
Thr Ala Thr Lys Asp Thr Tyr Asp 435 440 445Ala Leu His Met Gln Ala
Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr
Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala465 470 475 480Val
Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr Asp Ala 485 490
495Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
500 505 510Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp
Ile Tyr 515 520 525Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu 530 535 540Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp
Gly Val Pro Ser Arg Phe545 550 555 560Ser Gly Ser Gly Ser Gly Thr
Gln Tyr Thr Leu Thr Ile Ser Ser Leu 565 570 575Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln His Tyr Lys Asn Tyr 580 585 590Pro Leu Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ser Gly 595 600 605Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615
620Gly Gly Ser Arg Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu625 630 635 640Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe 645 650 655Thr Leu Ser Asn Tyr Gly Met His Trp Ile
Arg Gln Ala Pro Gly Lys 660 665 670Gly Leu Glu Trp Val Ser Ser Ile
Ser Leu Asn Gly Gly Ser Thr Tyr 675 680 685Tyr Arg Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 690 695
700Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr705 710 715 720Ala Val Tyr Tyr Cys Ala Ala Gln Asp Ala Tyr Thr
Gly Gly Tyr Phe 725 730 735Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Met Asp Pro 740 745 750Ala Thr Thr Thr Lys Pro Val Leu
Arg Thr Pro Ser Pro Val His Pro 755 760 765Thr Gly Thr Ser Gln Pro
Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly 770 775 780Ser Val Lys Gly
Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Trp Ala785 790 795 800Pro
Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr 805 810
815Leu Ile Cys Tyr His Arg Ser Arg Lys Arg Val Cys Lys Ser Gly Gly
820 825 830Gly Ser Phe Trp Glu Glu Phe Glu Ser Leu Gln Lys Gln Glu
Val Lys 835 840 845Asn Leu His Gln Arg Leu Glu Gly Gln Arg Pro Glu
Asn Lys Gly Lys 850 855 860Asn Arg Tyr Lys Asn Ile Leu Pro Phe Asp
His Ser Arg Val Ile Leu865 870 875 880Gln Gly Arg Asp Ser Asn Ile
Pro Gly Ser Asp Tyr Ile Asn Ala Asn 885 890 895Tyr Ile Lys Asn Gln
Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr Tyr 900 905 910Ile Ala Ser
Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp Gln 915 920 925Met
Ala Trp Gln Glu Asn Ser Arg Val Ile Val Met Thr Thr Arg Glu 930 935
940Val Glu Lys Gly Arg Asn Lys Cys Val Pro Tyr Trp Pro Glu Val
Gly945 950 955 960Met Gln Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn
Cys Gly Glu His 965 970 975Asp Thr Thr Glu Tyr Lys Leu Arg Thr Leu
Gln Val Ser Pro Leu Asp 980 985 990Asn Gly Asp Leu Ile Arg Glu Ile
Trp His Tyr Gln Tyr Leu Ser Trp 995 1000 1005Pro Asp His Gly Val
Pro Ser Glu Pro Gly Gly Val Leu Ser Phe 1010 1015 1020Leu Asp Gln
Ile Asn Gln Arg Gln Glu Ser Leu Pro His Ala Gly 1025 1030 1035Pro
Ile Ile Val His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr 1040 1045
1050Ile Ile Val Ile Asp Met Leu Met Glu Asn Ile Ser Thr Lys Gly
1055 1060 1065Leu Asp Cys Asp Ile Asp Ile Gln Lys Thr Ile Gln Met
Val Arg 1070 1075 1080Ala Gln Arg Ser Gly Met Val Gln Thr Glu Ala
Gln Tyr Lys Phe 1085 1090 1095Ile Tyr Val Ala Ile Ala Gln Phe Ile
Glu Thr Thr Lys Lys Lys 1100 1105 1110Leu5918PRTArtificial
SequenceChimeric antigen receptor (CAR) 5Met Ser Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30Ser Ala Ser Leu
Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser
Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60Val Lys
Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro65 70 75
80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Thr 115 120 125Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Glu Val Lys
Leu Gln Glu Ser Gly Pro Gly145 150 155 160Leu Val Ala Pro Ser Gln
Ser Leu Ser Val Thr Cys Thr Val Ser Gly 165 170 175Val Ser Leu Pro
Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg 180 185 190Lys Gly
Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr 195 200
205Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser
210 215 220Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp
Asp Thr225 230 235 240Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr
Gly Gly Ser Tyr Ala 245 250 255Met Asp Tyr Trp Gly Gln Gly Thr Ser
Val Thr Val Ser Ser Asp Pro 260 265 270Thr Thr Thr Pro Ala Pro Arg
Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280 285Ser Gln Pro Leu Ser
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 290 295 300Gly Ala Val
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Phe Trp305 310 315
320Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
325 330 335Thr Val Ala Phe Ile Ile Phe Trp Val Arg Arg Val Lys Phe
Ser Arg 340 345 350Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
Gln Leu Tyr Asn 355 360 365Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
Asp Val Leu Asp Lys Arg 370 375 380Arg Gly Arg Asp Pro Glu Met Gly
Gly Lys Pro Arg Arg Lys Asn Pro385 390 395 400Gln Glu Gly Leu Tyr
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 405 410 415Tyr Ser Glu
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 420 425 430Asp
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 435 440
445Ala Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly
450 455 460Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro
Met Ala465 470 475 480Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu
Trp Leu Thr Asp Ala 485 490 495Arg Cys Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser 500 505 510Val Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Glu Asp Ile Tyr 515 520 525Phe Asn Leu Val Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 530 535 540Leu Ile Tyr
Asp Thr Asn Arg Leu Ala Asp Gly Val Pro Ser Arg Phe545 550 555
560Ser Gly Ser Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Ser Ser Leu
565 570 575Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Lys
Asn Tyr 580 585 590Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Arg Ser Gly 595 600 605Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 610 615 620Gly Gly Ser Arg Ser Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu625 630 635 640Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 645 650 655Thr Leu Ser
Asn Tyr Gly Met His Trp Ile Arg Gln Ala Pro Gly Lys 660 665 670Gly
Leu Glu Trp Val Ser Ser Ile Ser Leu Asn Gly Gly Ser Thr Tyr 675 680
685Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
690 695 700Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr705 710 715 720Ala Val Tyr Tyr Cys Ala Ala Gln Asp Ala Tyr
Thr Gly Gly Tyr Phe 725 730 735Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Met Asp Pro 740 745 750Ala Thr Thr Thr Lys Pro Val
Leu Arg Thr Pro Ser Pro Val His Pro 755 760 765Thr Gly Thr Ser Gln
Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly 770 775 780Ser Val Lys
Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Leu Ile Gly785 790 795
800Val Ser Val Val Phe Leu Phe Cys Leu Leu Leu Leu Val Leu Phe Cys
805 810 815Leu His Arg Gln Asn Gln Ile Lys Gln Gly Pro Pro Arg Ser
Lys Asp 820 825 830Glu Glu Gln Lys Pro Gln Gln Arg Pro Asp Leu Ala
Val Asp Val Leu 835 840 845Glu Arg Thr Ala Asp Lys Ala Thr Val Asn
Gly Leu Pro Glu Lys Asp 850 855 860Arg Glu Thr Asp Thr Ser Ala Leu
Ala Ala Gly Ser Ser Gln Glu Val865 870 875 880Thr Tyr Ala Gln Leu
Asp His Trp Ala Leu Thr Gln Arg Thr Ala Arg 885 890 895Ala Val Ser
Pro Gln Ser Thr Lys Pro Met Ala Glu Ser Ile Thr Tyr 900 905 910Ala
Ala Val Ala Arg His 91561363PRTArtificial SequenceChimeric antigen
receptor (CAR) 6Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr
Thr Ser Ser Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Asp Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser
Arg Leu His Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135
140Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro
Gly145 150 155 160Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
Thr Val Ser Gly 165 170 175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp
Ile Arg Gln Pro Pro Arg 180 185 190Lys Gly Leu Glu Trp Leu Gly Val
Ile Trp Gly Ser Glu Thr Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser 210 215 220Lys Ser Gln Val
Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr225 230 235 240Ala
Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250
255Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro
260 265 270Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
Ile Ala 275 280 285Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
Pro Ala Ala Gly 290 295 300Gly Ala Val His Thr Arg Gly Leu Asp Phe
Ala Cys Asp Ile Phe Trp305 310 315 320Val Leu Val Val Val Gly Gly
Val Leu Ala Cys Tyr Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile
Ile Phe Trp Val Arg Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375
380Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
Pro385 390 395 400Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
Met Ala Glu Ala 405 410 415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
Arg Arg Gly Lys Gly His 420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser
Thr Ala Thr Lys Asp Thr Tyr Asp 435 440 445Ala Leu His Met Gln Ala
Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr
Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala465 470 475 480Val
Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr Asp Ala 485 490
495Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
500 505 510Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp
Ile Tyr 515 520 525Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu 530 535 540Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp
Gly Val Pro Ser Arg Phe545 550 555 560Ser Gly Ser Gly Ser Gly Thr
Gln Tyr Thr Leu Thr Ile Ser Ser Leu 565 570 575Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln His Tyr Lys Asn Tyr 580 585 590Pro Leu Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ser Gly 595 600 605Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615
620Gly Gly Ser Arg Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu625 630 635 640Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe 645 650 655Thr Leu Ser Asn Tyr Gly Met His Trp Ile
Arg Gln Ala Pro Gly Lys 660 665 670Gly Leu Glu Trp Val Ser Ser Ile
Ser Leu Asn Gly Gly Ser Thr Tyr 675 680 685Tyr Arg Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 690 695 700Lys Ser Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr705 710 715 720Ala
Val Tyr Tyr Cys Ala Ala Gln Asp Ala Tyr Thr Gly Gly Tyr Phe 725 730
735Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Met Asp Pro
740 745 750Ala Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val
His Pro 755 760 765Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys
Arg Pro Arg Gly 770 775 780Ser Val Lys Gly Thr Gly Leu Asp Phe Ala
Cys Asp Ile Leu Ile Gly785 790 795 800Val Ser Val Val Phe Leu Phe
Cys Leu Leu Leu Leu Val Leu Phe Cys 805 810 815Leu His Arg Gln Asn
Gln Ile Lys Gln Gly Pro Pro Arg Ser Lys Asp 820 825 830Glu Glu Gln
Lys Pro Gln Gln Arg Pro Asp Leu Ala Val Asp Val Leu 835 840 845Glu
Arg Thr Ala Asp Lys Ala Thr Val Asn Gly Leu Pro Glu Lys Asp 850 855
860Arg Glu Thr Asp Thr Ser Ala Leu Ala Ala Gly Ser Ser Gln Glu
Val865 870 875 880Thr Tyr Ala Gln Leu Asp His Trp Ala Leu Thr Gln
Arg Thr Ala Arg 885 890 895Ala Val Ser Pro Gln Ser Thr Lys Pro Met
Ala Glu Ser Ile Thr Tyr 900 905 910Ala Ala Val Ala Arg His Arg Ala
Glu Gly Arg Gly Ser Leu Leu Thr 915 920 925Cys Gly Asp Val Glu Glu
Asn Pro Gly Pro Trp Tyr His Gly His Met 930 935 940Ser Gly Gly Gln
Ala Glu Thr Leu Leu Gln Ala Lys Gly Glu Pro Trp945 950 955 960Thr
Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp Phe Val Leu 965 970
975Ser Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser Pro Leu Arg
980 985 990Val Thr His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr Thr
Val Gly 995 1000 1005Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu
Val Glu His Phe 1010 1015 1020Lys Lys Thr Gly Ile Glu Glu Ala Ser
Gly Ala Phe Val Tyr Leu 1025 1030 1035Arg Gln Pro Tyr Ser Gly Gly
Gly Gly Ser Phe Glu Ala Tyr Phe 1040 1045 1050Lys Lys Gln Gln Ala
Asp Ser Asn Cys Gly Phe Ala Glu Glu Tyr 1055 1060 1065Glu Asp Leu
Lys Leu Val Gly Ile Ser Gln Pro Lys Tyr Ala Ala 1070 1075 1080Glu
Leu Ala Glu Asn Arg Gly Lys Asn Arg Tyr Asn Asn Val Leu 1085 1090
1095Pro Tyr Asp Ile Ser Arg Val Lys Leu Ser Val Gln Thr His Ser
1100 1105 1110Thr Asp Asp Tyr Ile Asn Ala Asn Tyr Met Pro Gly Tyr
His Ser 1115
1120 1125Lys Lys Asp Phe Ile Ala Thr Gln Gly Pro Leu Pro Asn Thr
Leu 1130 1135 1140Lys Asp Phe Trp Arg Met Val Trp Glu Lys Asn Val
Tyr Ala Ile 1145 1150 1155Ile Met Leu Thr Lys Cys Val Glu Gln Gly
Arg Thr Lys Cys Glu 1160 1165 1170Glu Tyr Trp Pro Ser Lys Gln Ala
Gln Asp Tyr Gly Asp Ile Thr 1175 1180 1185Val Ala Met Thr Ser Glu
Ile Val Leu Pro Glu Trp Thr Ile Arg 1190 1195 1200Asp Phe Thr Val
Lys Asn Ile Gln Thr Ser Glu Ser His Pro Leu 1205 1210 1215Arg Gln
Phe His Phe Thr Ser Trp Pro Asp His Gly Val Pro Asp 1220 1225
1230Thr Thr Asp Leu Leu Ile Asn Phe Arg Tyr Leu Val Arg Asp Tyr
1235 1240 1245Met Lys Gln Ser Pro Pro Glu Ser Pro Ile Leu Val His
Cys Ser 1250 1255 1260Ala Gly Val Gly Arg Thr Gly Thr Phe Ile Ala
Ile Asp Arg Leu 1265 1270 1275Ile Tyr Gln Ile Glu Asn Glu Asn Thr
Val Asp Val Tyr Gly Ile 1280 1285 1290Val Tyr Asp Leu Arg Met His
Arg Pro Leu Met Val Gln Thr Glu 1295 1300 1305Asp Gln Tyr Val Phe
Leu Asn Gln Cys Val Leu Asp Ile Val Arg 1310 1315 1320Ser Gln Lys
Asp Ser Lys Val Asp Leu Ile Tyr Gln Asn Thr Thr 1325 1330 1335Ala
Met Thr Ile Tyr Glu Asn Leu Ala Pro Val Thr Thr Phe Gly 1340 1345
1350Lys Thr Asn Gly Tyr Ile Ala Ser Gly Ser 1355
1360721PRTArtificial SequenceSignal peptide 7Met Gly Thr Ser Leu
Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala1 5 10 15Asp His Ala Asp
Gly 20821PRTArtificial SequenceSignal peptide 8Met Ser Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro 20920PRTArtificial SequenceSignal peptide 9Met Ala Val Pro Thr
Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr1 5 10 15Asp Ala Arg Cys
2010234PRTArtificial SequenceSpacer (hinge-CH2CH3 of human IgG1)
10Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro1
5 10 15Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 20 25 30Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys
Val Val 35 40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val 50 55 60Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln 85 90 95Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala 100 105 110Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150 155
160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr 180 185 190Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe 195 200 205Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys 210 215 220Ser Leu Ser Leu Ser Pro Gly Lys
Lys Asp225 2301146PRTArtificial SequenceSpacer (human CD8 stalk)
11Thr 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
35 40 451220PRTArtificial SequenceSpacer (human IgG1 hinge) 12Ala
Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10
15Lys Asp Pro Lys 2013185PRTArtificial SequenceSpacer (CD2
ectodomain) 13Lys Glu Ile Thr Asn Ala Leu Glu Thr Trp Gly Ala Leu
Gly Gln Asp1 5 10 15Ile Asn Leu Asp Ile Pro Ser Phe Gln Met Ser Asp
Asp Ile Asp Asp 20 25 30Ile Lys Trp Glu Lys Thr Ser Asp Lys Lys Lys
Ile Ala Gln Phe Arg 35 40 45Lys Glu Lys Glu Thr Phe Lys Glu Lys Asp
Thr Tyr Lys Leu Phe Lys 50 55 60Asn Gly Thr Leu Lys Ile Lys His Leu
Lys Thr Asp Asp Gln Asp Ile65 70 75 80Tyr Lys Val Ser Ile Tyr Asp
Thr Lys Gly Lys Asn Val Leu Glu Lys 85 90 95Ile Phe Asp Leu Lys Ile
Gln Glu Arg Val Ser Lys Pro Lys Ile Ser 100 105 110Trp Thr Cys Ile
Asn Thr Thr Leu Thr Cys Glu Val Met Asn Gly Thr 115 120 125Asp Pro
Glu Leu Asn Leu Tyr Gln Asp Gly Lys His Leu Lys Leu Ser 130 135
140Gln Arg Val Ile Thr His Lys Trp Thr Thr Ser Leu Ser Ala Lys
Phe145 150 155 160Lys Cys Thr Ala Gly Asn Lys Val Ser Lys Glu Ser
Ser Val Glu Pro 165 170 175Val Ser Cys Pro Glu Lys Gly Leu Asp 180
18514259PRTArtificial SequenceSpacer (CD34 ectodomain) 14Ser Leu
Asp Asn Asn Gly Thr Ala Thr Pro Glu Leu Pro Thr Gln Gly1 5 10 15Thr
Phe Ser Asn Val Ser Thr Asn Val Ser Tyr Gln Glu Thr Thr Thr 20 25
30Pro Ser Thr Leu Gly Ser Thr Ser Leu His Pro Val Ser Gln His Gly
35 40 45Asn Glu Ala Thr Thr Asn Ile Thr Glu Thr Thr Val Lys Phe Thr
Ser 50 55 60Thr Ser Val Ile Thr Ser Val Tyr Gly Asn Thr Asn Ser Ser
Val Gln65 70 75 80Ser Gln Thr Ser Val Ile Ser Thr Val Phe Thr Thr
Pro Ala Asn Val 85 90 95Ser Thr Pro Glu Thr Thr Leu Lys Pro Ser Leu
Ser Pro Gly Asn Val 100 105 110Ser Asp Leu Ser Thr Thr Ser Thr Ser
Leu Ala Thr Ser Pro Thr Lys 115 120 125Pro Tyr Thr Ser Ser Ser Pro
Ile Leu Ser Asp Ile Lys Ala Glu Ile 130 135 140Lys Cys Ser Gly Ile
Arg Glu Val Lys Leu Thr Gln Gly Ile Cys Leu145 150 155 160Glu Gln
Asn Lys Thr Ser Ser Cys Ala Glu Phe Lys Lys Asp Arg Gly 165 170
175Glu Gly Leu Ala Arg Val Leu Cys Gly Glu Glu Gln Ala Asp Ala Asp
180 185 190Ala Gly Ala Gln Val Cys Ser Leu Leu Leu Ala Gln Ser Glu
Val Arg 195 200 205Pro Gln Cys Leu Leu Leu Val Leu Ala Asn Arg Thr
Glu Ile Ser Ser 210 215 220Lys Leu Gln Leu Met Lys Lys His Gln Ser
Asp Leu Lys Lys Leu Gly225 230 235 240Ile Leu Asp Phe Thr Glu Gln
Asp Val Ala Ser His Gln Ser Tyr Ser 245 250 255Gln Lys
Thr15140PRTArtificial SequenceCD28 transmembrane domain and CD3 Z
endodomains 15Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
Tyr Ser Leu1 5 10 15Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg
Arg Val Lys Phe 20 25 30Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
Gly Gln Asn Gln Leu 35 40 45Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
Glu Tyr Asp Val Leu Asp 50 55 60Lys Arg Arg Gly Arg Asp Pro Glu Met
Gly Gly Lys Pro Arg Arg Lys65 70 75 80Asn Pro Gln Glu Gly Leu Tyr
Asn Glu Leu Gln Lys Asp Lys Met Ala 85 90 95Glu Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 100 105 110Gly His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 115 120 125Tyr Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg 130 135
14016180PRTArtificial SequenceCD28 transmembrane domain and CD28
and CD3 Zeta endodomains 16Phe Trp Val Leu Val Val Val Gly Gly Val
Leu Ala Cys Tyr Ser Leu1 5 10 15Leu Val Thr Val Ala Phe Ile Ile Phe
Trp Val Arg Ser Lys Arg Ser 20 25 30Arg Leu Leu His Ser Asp Tyr Met
Asn Met Thr Pro Arg Arg Pro Gly 35 40 45Pro Thr Arg Lys His Tyr Gln
Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60Ala Tyr Arg Ser Arg Val
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala65 70 75 80Tyr Gln Gln Gly
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 85 90 95Arg Glu Glu
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 100 105 110Met
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 115 120
125Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
130 135 140Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
Gln Gly145 150 155 160Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
Leu His Met Gln Ala 165 170 175Leu Pro Pro Arg
18017216PRTArtificial SequenceCD28 transmembrane domain and CD28,
OX40 and CD3 Zeta endodomains 17Phe Trp Val Leu Val Val Val Gly Gly
Val Leu Ala Cys Tyr Ser Leu1 5 10 15Leu Val Thr Val Ala Phe Ile Ile
Phe Trp Val Arg Ser Lys Arg Ser 20 25 30Arg Leu Leu His Ser Asp Tyr
Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45Pro Thr Arg Lys His Tyr
Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60Ala Tyr Arg Ser Arg
Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro65 70 75 80Pro Gly Gly
Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp 85 90 95Ala His
Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala 100 105
110Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
115 120 125Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
Arg Gly 130 135 140Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
Asn Pro Gln Glu145 150 155 160Gly Leu Tyr Asn Glu Leu Gln Lys Asp
Lys Met Ala Glu Ala Tyr Ser 165 170 175Glu Ile Gly Met Lys Gly Glu
Arg Arg Arg Gly Lys Gly His Asp Gly 180 185 190Leu Tyr Gln Gly Leu
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 195 200 205His Met Gln
Ala Leu Pro Pro Arg 210 21518729PRTArtificial SequenceCD45
transmembrane and endodomain 18Ala Leu Ile Ala Phe Leu Ala Phe Leu
Ile Ile Val Thr Ser Ile Ala1 5 10 15Leu Leu Val Val Leu Tyr Lys Ile
Tyr Asp Leu His Lys Lys Arg Ser 20 25 30Cys Asn Leu Asp Glu Gln Gln
Glu Leu Val Glu Arg Asp Asp Glu Lys 35 40 45Gln Leu Met Asn Val Glu
Pro Ile His Ala Asp Ile Leu Leu Glu Thr 50 55 60Tyr Lys Arg Lys Ile
Ala Asp Glu Gly Arg Leu Phe Leu Ala Glu Phe65 70 75 80Gln Ser Ile
Pro Arg Val Phe Ser Lys Phe Pro Ile Lys Glu Ala Arg 85 90 95Lys Pro
Phe Asn Gln Asn Lys Asn Arg Tyr Val Asp Ile Leu Pro Tyr 100 105
110Asp Tyr Asn Arg Val Glu Leu Ser Glu Ile Asn Gly Asp Ala Gly Ser
115 120 125Asn Tyr Ile Asn Ala Ser Tyr Ile Asp Gly Phe Lys Glu Pro
Arg Lys 130 135 140Tyr Ile Ala Ala Gln Gly Pro Arg Asp Glu Thr Val
Asp Asp Phe Trp145 150 155 160Arg Met Ile Trp Glu Gln Lys Ala Thr
Val Ile Val Met Val Thr Arg 165 170 175Cys Glu Glu Gly Asn Arg Asn
Lys Cys Ala Glu Tyr Trp Pro Ser Met 180 185 190Glu Glu Gly Thr Arg
Ala Phe Gly Asp Val Val Val Lys Ile Asn Gln 195 200 205His Lys Arg
Cys Pro Asp Tyr Ile Ile Gln Lys Leu Asn Ile Val Asn 210 215 220Lys
Lys Glu Lys Ala Thr Gly Arg Glu Val Thr His Ile Gln Phe Thr225 230
235 240Ser Trp Pro Asp His Gly Val Pro Glu Asp Pro His Leu Leu Leu
Lys 245 250 255Leu Arg Arg Arg Val Asn Ala Phe Ser Asn Phe Phe Ser
Gly Pro Ile 260 265 270Val Val His Cys Ser Ala Gly Val Gly Arg Thr
Gly Thr Tyr Ile Gly 275 280 285Ile Asp Ala Met Leu Glu Gly Leu Glu
Ala Glu Asn Lys Val Asp Val 290 295 300Tyr Gly Tyr Val Val Lys Leu
Arg Arg Gln Arg Cys Leu Met Val Gln305 310 315 320Val Glu Ala Gln
Tyr Ile Leu Ile His Gln Ala Leu Val Glu Tyr Asn 325 330 335Gln Phe
Gly Glu Thr Glu Val Asn Leu Ser Glu Leu His Pro Tyr Leu 340 345
350His Asn Met Lys Lys Arg Asp Pro Pro Ser Glu Pro Ser Pro Leu Glu
355 360 365Ala Glu Phe Gln Arg Leu Pro Ser Tyr Arg Ser Trp Arg Thr
Gln His 370 375 380Ile Gly Asn Gln Glu Glu Asn Lys Ser Lys Asn Arg
Asn Ser Asn Val385 390 395 400Ile Pro Tyr Asp Tyr Asn Arg Val Pro
Leu Lys His Glu Leu Glu Met 405 410 415Ser Lys Glu Ser Glu His Asp
Ser Asp Glu Ser Ser Asp Asp Asp Ser 420 425 430Asp Ser Glu Glu Pro
Ser Lys Tyr Ile Asn Ala Ser Phe Ile Met Ser 435 440 445Tyr Trp Lys
Pro Glu Val Met Ile Ala Ala Gln Gly Pro Leu Lys Glu 450 455 460Thr
Ile Gly Asp Phe Trp Gln Met Ile Phe Gln Arg Lys Val Lys Val465 470
475 480Ile Val Met Leu Thr Glu Leu Lys His Gly Asp Gln Glu Ile Cys
Ala 485 490 495Gln Tyr Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp Ile
Glu Val Asp 500 505 510Leu Lys Asp Thr Asp Lys Ser Ser Thr Tyr Thr
Leu Arg Val Phe Glu 515 520 525Leu Arg His Ser Lys Arg Lys Asp Ser
Arg Thr Val Tyr Gln Tyr Gln 530 535 540Tyr Thr Asn Trp Ser Val Glu
Gln Leu Pro Ala Glu Pro Lys Glu Leu545 550 555 560Ile Ser Met Ile
Gln Val Val Lys Gln Lys Leu Pro Gln Lys Asn Ser 565 570 575Ser Glu
Gly Asn Lys His His Lys Ser Thr Pro Leu Leu Ile His Cys 580 585
590Arg Asp Gly Ser Gln Gln Thr Gly Ile Phe Cys Ala Leu Leu Asn Leu
595 600 605Leu Glu Ser Ala Glu Thr Glu Glu Val Val Asp Ile Phe Gln
Val Val 610 615 620Lys Ala Leu Arg Lys Ala Arg Pro Gly Met Val Ser
Thr Phe Glu Gln625 630 635 640Tyr Gln Phe Leu Tyr Asp Val Ile Ala
Ser Thr Tyr Pro Ala Gln Asn 645 650 655Gly Gln Val Lys Lys Asn Asn
His Gln Glu Asp Lys Ile Glu Phe Asp 660 665 670Asn Glu Val Asp Lys
Val Lys Gln Asp Ala Asn Cys Val Asn Pro Leu 675 680 685Gly Ala Pro
Glu Lys Leu Pro Glu Ala Lys Glu Gln Ala Glu Gly Ser 690 695 700Glu
Pro Thr Ser Gly Thr Glu Gly Pro Glu His Ser Val Asn Gly Pro705 710
715 720Ala Ser Pro Ala Leu Asn Gln Gly Ser 72519362PRTArtificial
SequenceCD148 transmembrane and endodomain 19Ala Val Phe Gly Cys
Ile Phe Gly Ala Leu Val Ile Val Thr Val Gly1 5 10 15Gly Phe Ile Phe
Trp Arg Lys Lys Arg Lys Asp Ala Lys Asn Asn Glu 20 25 30Val Ser Phe
Ser Gln Ile Lys Pro Lys Lys Ser Lys Leu Ile Arg Val 35 40
45Glu Asn Phe Glu Ala Tyr Phe Lys Lys Gln Gln Ala Asp Ser Asn Cys
50 55 60Gly Phe Ala Glu Glu Tyr Glu Asp Leu Lys Leu Val Gly Ile Ser
Gln65 70 75 80Pro Lys Tyr Ala Ala Glu Leu Ala Glu Asn Arg Gly Lys
Asn Arg Tyr 85 90 95Asn Asn Val Leu Pro Tyr Asp Ile Ser Arg Val Lys
Leu Ser Val Gln 100 105 110Thr His Ser Thr Asp Asp Tyr Ile Asn Ala
Asn Tyr Met Pro Gly Tyr 115 120 125His Ser Lys Lys Asp Phe Ile Ala
Thr Gln Gly Pro Leu Pro Asn Thr 130 135 140Leu Lys Asp Phe Trp Arg
Met Val Trp Glu Lys Asn Val Tyr Ala Ile145 150 155 160Ile Met Leu
Thr Lys Cys Val Glu Gln Gly Arg Thr Lys Cys Glu Glu 165 170 175Tyr
Trp Pro Ser Lys Gln Ala Gln Asp Tyr Gly Asp Ile Thr Val Ala 180 185
190Met Thr Ser Glu Ile Val Leu Pro Glu Trp Thr Ile Arg Asp Phe Thr
195 200 205Val Lys Asn Ile Gln Thr Ser Glu Ser His Pro Leu Arg Gln
Phe His 210 215 220Phe Thr Ser Trp Pro Asp His Gly Val Pro Asp Thr
Thr Asp Leu Leu225 230 235 240Ile Asn Phe Arg Tyr Leu Val Arg Asp
Tyr Met Lys Gln Ser Pro Pro 245 250 255Glu Ser Pro Ile Leu Val His
Cys Ser Ala Gly Val Gly Arg Thr Gly 260 265 270Thr Phe Ile Ala Ile
Asp Arg Leu Ile Tyr Gln Ile Glu Asn Glu Asn 275 280 285Thr Val Asp
Val Tyr Gly Ile Val Tyr Asp Leu Arg Met His Arg Pro 290 295 300Leu
Met Val Gln Thr Glu Asp Gln Tyr Val Phe Leu Asn Gln Cys Val305 310
315 320Leu Asp Ile Val Arg Ser Gln Lys Asp Ser Lys Val Asp Leu Ile
Tyr 325 330 335Gln Asn Thr Thr Ala Met Thr Ile Tyr Glu Asn Leu Ala
Pro Val Thr 340 345 350Thr Phe Gly Lys Thr Asn Gly Tyr Ile Ala 355
36020595PRTArtificial Sequencesequence of PTPN6 20Met Val Arg Trp
Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr1 5 10 15Leu Leu Lys
Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 20 25 30Arg Lys
Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 35 40 45Val
Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 50 55
60Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr65
70 75 80Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His
Leu 85 90 95Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser Glu Arg Trp
Tyr His 100 105 110Gly His Met Ser Gly Gly Gln Ala Glu Thr Leu Leu
Gln Ala Lys Gly 115 120 125Glu Pro Trp Thr Phe Leu Val Arg Glu Ser
Leu Ser Gln Pro Gly Asp 130 135 140Phe Val Leu Ser Val Leu Ser Asp
Gln Pro Lys Ala Gly Pro Gly Ser145 150 155 160Pro Leu Arg Val Thr
His Ile Lys Val Met Cys Glu Gly Gly Arg Tyr 165 170 175Thr Val Gly
Gly Leu Glu Thr Phe Asp Ser Leu Thr Asp Leu Val Glu 180 185 190His
Phe Lys Lys Thr Gly Ile Glu Glu Ala Ser Gly Ala Phe Val Tyr 195 200
205Leu Arg Gln Pro Tyr Tyr Ala Thr Arg Val Asn Ala Ala Asp Ile Glu
210 215 220Asn Arg Val Leu Glu Leu Asn Lys Lys Gln Glu Ser Glu Asp
Thr Ala225 230 235 240Lys Ala Gly Phe Trp Glu Glu Phe Glu Ser Leu
Gln Lys Gln Glu Val 245 250 255Lys Asn Leu His Gln Arg Leu Glu Gly
Gln Arg Pro Glu Asn Lys Gly 260 265 270Lys Asn Arg Tyr Lys Asn Ile
Leu Pro Phe Asp His Ser Arg Val Ile 275 280 285Leu Gln Gly Arg Asp
Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala 290 295 300Asn Tyr Ile
Lys Asn Gln Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr305 310 315
320Tyr Ile Ala Ser Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp
325 330 335Gln Met Ala Trp Gln Glu Asn Ser Arg Val Ile Val Met Thr
Thr Arg 340 345 350Glu Val Glu Lys Gly Arg Asn Lys Cys Val Pro Tyr
Trp Pro Glu Val 355 360 365Gly Met Gln Arg Ala Tyr Gly Pro Tyr Ser
Val Thr Asn Cys Gly Glu 370 375 380His Asp Thr Thr Glu Tyr Lys Leu
Arg Thr Leu Gln Val Ser Pro Leu385 390 395 400Asp Asn Gly Asp Leu
Ile Arg Glu Ile Trp His Tyr Gln Tyr Leu Ser 405 410 415Trp Pro Asp
His Gly Val Pro Ser Glu Pro Gly Gly Val Leu Ser Phe 420 425 430Leu
Asp Gln Ile Asn Gln Arg Gln Glu Ser Leu Pro His Ala Gly Pro 435 440
445Ile Ile Val His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile
450 455 460Val Ile Asp Met Leu Met Glu Asn Ile Ser Thr Lys Gly Leu
Asp Cys465 470 475 480Asp Ile Asp Ile Gln Lys Thr Ile Gln Met Val
Arg Ala Gln Arg Ser 485 490 495Gly Met Val Gln Thr Glu Ala Gln Tyr
Lys Phe Ile Tyr Val Ala Ile 500 505 510Ala Gln Phe Ile Glu Thr Thr
Lys Lys Lys Leu Glu Val Leu Gln Ser 515 520 525Gln Lys Gly Gln Glu
Ser Glu Tyr Gly Asn Ile Thr Tyr Pro Pro Ala 530 535 540Met Lys Asn
Ala His Ala Lys Ala Ser Arg Thr Ser Ser Lys His Lys545 550 555
560Glu Asp Val Tyr Glu Asn Leu His Thr Lys Asn Lys Arg Glu Glu Lys
565 570 575Val Lys Lys Gln Arg Ser Ala Asp Lys Glu Lys Ser Lys Gly
Ser Leu 580 585 590Lys Arg Lys 59521272PRTArtificial
Sequencesequence of phosphatase domain of PTPN6 21Phe Trp Glu Glu
Phe Glu Ser Leu Gln Lys Gln Glu Val Lys Asn Leu1 5 10 15His Gln Arg
Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly Lys Asn Arg 20 25 30Tyr Lys
Asn Ile Leu Pro Phe Asp His Ser Arg Val Ile Leu Gln Gly 35 40 45Arg
Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala Asn Tyr Ile 50 55
60Lys Asn Gln Leu Leu Gly Pro Asp Glu Asn Ala Lys Thr Tyr Ile Ala65
70 75 80Ser Gln Gly Cys Leu Glu Ala Thr Val Asn Asp Phe Trp Gln Met
Ala 85 90 95Trp Gln Glu Asn Ser Arg Val Ile Val Met Thr Thr Arg Glu
Val Glu 100 105 110Lys Gly Arg Asn Lys Cys Val Pro Tyr Trp Pro Glu
Val Gly Met Gln 115 120 125Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn
Cys Gly Glu His Asp Thr 130 135 140Thr Glu Tyr Lys Leu Arg Thr Leu
Gln Val Ser Pro Leu Asp Asn Gly145 150 155 160Asp Leu Ile Arg Glu
Ile Trp His Tyr Gln Tyr Leu Ser Trp Pro Asp 165 170 175His Gly Val
Pro Ser Glu Pro Gly Gly Val Leu Ser Phe Leu Asp Gln 180 185 190Ile
Asn Gln Arg Gln Glu Ser Leu Pro His Ala Gly Pro Ile Ile Val 195 200
205His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Ile Ile Val Ile Asp
210 215 220Met Leu Met Glu Asn Ile Ser Thr Lys Gly Leu Asp Cys Asp
Ile Asp225 230 235 240Ile Gln Lys Thr Ile Gln Met Val Arg Ala Gln
Arg Ser Gly Met Val 245 250 255Gln Thr Glu Ala Gln Tyr Lys Phe Ile
Tyr Val Ala Ile Ala Gln Phe 260 265 2702297PRTArtificial
SequencePDCD1 endodomain 22Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly
Ala Arg Arg Thr Gly Gln1 5 10 15Pro Leu Lys Glu Asp Pro Ser Ala Val
Pro Val Phe Ser Val Asp Tyr 20 25 30Gly Glu Leu Asp Phe Gln Trp Arg
Glu Lys Thr Pro Glu Pro Pro Val 35 40 45Pro Cys Val Pro Glu Gln Thr
Glu Tyr Ala Thr Ile Val Phe Pro Ser 50 55 60Gly Met Gly Thr Ser Ser
Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro65 70 75 80Arg Ser Ala Gln
Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro 85 90
95Leu23141PRTArtificial SequenceBTLA4 endodomain 23Lys Leu Gln Arg
Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly Leu Gln1 5 10 15Glu Asn Ser
Ser Gly Gln Ser Phe Phe Val Arg Asn Lys Lys Val Arg 20 25 30Arg Ala
Pro Leu Ser Glu Gly Pro His Ser Leu Gly Cys Tyr Asn Pro 35 40 45Met
Met Glu Asp Gly Ile Ser Tyr Thr Thr Leu Arg Phe Pro Glu Met 50 55
60Asn Ile Pro Arg Thr Gly Asp Ala Glu Ser Ser Glu Met Gln Arg Pro65
70 75 80Pro Pro Asp Cys Asp Asp Thr Val Thr Tyr Ser Ala Leu His Lys
Arg 85 90 95Gln Val Gly Asp Tyr Glu Asn Val Ile Pro Asp Phe Pro Glu
Asp Glu 100 105 110Gly Ile His Tyr Ser Glu Leu Ile Gln Phe Gly Val
Gly Glu Arg Pro 115 120 125Gln Ala Gln Glu Asn Val Asp Tyr Val Ile
Leu Lys His 130 135 14024168PRTArtificial SequenceLILRB1 endodomain
24Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln Arg Lys1
5 10 15Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro Thr
Asp 20 25 30Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln
Glu Glu 35 40 45Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp
Gly Val Glu 50 55 60Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln
Ala Val Thr Tyr65 70 75 80Ala Glu Val Lys His Ser Arg Pro Arg Arg
Glu Met Ala Ser Pro Pro 85 90 95Ser Pro Leu Ser Gly Glu Phe Leu Asp
Thr Lys Asp Arg Gln Ala Glu 100 105 110Glu Asp Arg Gln Met Asp Thr
Glu Ala Ala Ala Ser Glu Ala Pro Gln 115 120 125Asp Val Thr Tyr Ala
Gln Leu His Ser Leu Thr Leu Arg Arg Glu Ala 130 135 140Thr Glu Pro
Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val Pro Ser145 150 155
160Ile Tyr Ala Thr Leu Ala Ile His 16525101PRTArtificial
SequenceLAIR1 endodomain 25His Arg Gln Asn Gln Ile Lys Gln Gly Pro
Pro Arg Ser Lys Asp Glu1 5 10 15Glu Gln Lys Pro Gln Gln Arg Pro Asp
Leu Ala Val Asp Val Leu Glu 20 25 30Arg Thr Ala Asp Lys Ala Thr Val
Asn Gly Leu Pro Glu Lys Asp Arg 35 40 45Glu Thr Asp Thr Ser Ala Leu
Ala Ala Gly Ser Ser Gln Glu Val Thr 50 55 60Tyr Ala Gln Leu Asp His
Trp Ala Leu Thr Gln Arg Thr Ala Arg Ala65 70 75 80Val Ser Pro Gln
Ser Thr Lys Pro Met Ala Glu Ser Ile Thr Tyr Ala 85 90 95Ala Val Ala
Arg His 1002662PRTArtificial SequenceCTLA4 endodomain 26Phe Leu Leu
Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe Tyr1 5 10 15Ser Phe
Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys Arg 20 25 30Ser
Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro 35 40
45Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn 50 55
6027111PRTArtificial SequenceKIR2DL1 endodomain 27Gly Asn Ser Arg
His Leu His Val Leu Ile Gly Thr Ser Val Val Ile1 5 10 15Ile Pro Phe
Ala Ile Leu Leu Phe Phe Leu Leu His Arg Trp Cys Ala 20 25 30Asn Lys
Lys Asn Ala Val Val Met Asp Gln Glu Pro Ala Gly Asn Arg 35 40 45Thr
Val Asn Arg Glu Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr 50 55
60Tyr Thr Gln Leu Asn His Cys Val Phe Thr Gln Arg Lys Ile Thr Arg65
70 75 80Pro Ser Gln Arg Pro Lys Thr Pro Pro Thr Asp Ile Ile Val Tyr
Thr 85 90 95Glu Leu Pro Asn Ala Glu Ser Arg Ser Lys Val Val Ser Cys
Pro 100 105 11028143PRTArtificial SequenceKIR2DL4 endodomain 28Gly
Ile Ala Arg His Leu His Ala Val Ile Arg Tyr Ser Val Ala Ile1 5 10
15Ile Leu Phe Thr Ile Leu Pro Phe Phe Leu Leu His Arg Trp Cys Ser
20 25 30Lys Lys Lys Glu Asn Ala Ala Val Met Asn Gln Glu Pro Ala Gly
His 35 40 45Arg Thr Val Asn Arg Glu Asp Ser Asp Glu Gln Asp Pro Gln
Glu Val 50 55 60Thr Tyr Ala Gln Leu Asp His Cys Ile Phe Thr Gln Arg
Lys Ile Thr65 70 75 80Gly Pro Ser Gln Arg Ser Lys Arg Pro Ser Thr
Asp Thr Ser Val Cys 85 90 95Ile Glu Leu Pro Asn Ala Glu Pro Arg Ala
Leu Ser Pro Ala His Glu 100 105 110His His Ser Gln Ala Leu Met Gly
Ser Ser Arg Glu Thr Thr Ala Leu 115 120 125Ser Gln Thr Gln Leu Ala
Ser Ser Asn Val Pro Ala Ala Gly Ile 130 135 14029143PRTArtificial
SequenceKIR2DL5 endodomain 29Thr Gly Ile Arg Arg His Leu His Ile
Leu Ile Gly Thr Ser Val Ala1 5 10 15Ile Ile Leu Phe Ile Ile Leu Phe
Phe Phe Leu Leu His Cys Cys Cys 20 25 30Ser Asn Lys Lys Asn Ala Ala
Val Met Asp Gln Glu Pro Ala Gly Asp 35 40 45Arg Thr Val Asn Arg Glu
Asp Ser Asp Asp Gln Asp Pro Gln Glu Val 50 55 60Thr Tyr Ala Gln Leu
Asp His Cys Val Phe Thr Gln Thr Lys Ile Thr65 70 75 80Ser Pro Ser
Gln Arg Pro Lys Thr Pro Pro Thr Asp Thr Thr Met Tyr 85 90 95Met Glu
Leu Pro Asn Ala Lys Pro Arg Ser Leu Ser Pro Ala His Lys 100 105
110His His Ser Gln Ala Leu Arg Gly Ser Ser Arg Glu Thr Thr Ala Leu
115 120 125Ser Gln Asn Arg Val Ala Ser Ser His Val Pro Ala Ala Gly
Ile 130 135 14030111PRTArtificial SequenceKIR3DL1 endodomain 30Lys
Asp Pro Arg His Leu His Ile Leu Ile Gly Thr Ser Val Val Ile1 5 10
15Ile Leu Phe Ile Leu Leu Leu Phe Phe Leu Leu His Leu Trp Cys Ser
20 25 30Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala Gly Asn
Arg 35 40 45Thr Ala Asn Ser Glu Asp Ser Asp Glu Gln Asp Pro Glu Glu
Val Thr 50 55 60Tyr Ala Gln Leu Asp His Cys Val Phe Thr Gln Arg Lys
Ile Thr Arg65 70 75 80Pro Ser Gln Arg Pro Lys Thr Pro Pro Thr Asp
Thr Ile Leu Tyr Thr 85 90 95Glu Leu Pro Asn Ala Lys Pro Arg Ser Lys
Val Val Ser Cys Pro 100 105 1103197PRTArtificial SequenceKIR3DL3
endodomain 31Lys Asp Pro Gly Asn Ser Arg His Leu His Val Leu Ile
Gly Thr Ser1 5 10 15Val Val Ile Ile Pro Phe Ala Ile Leu Leu Phe Phe
Leu Leu His Arg 20 25 30Trp Cys Ala Asn Lys Lys Asn Ala Val Val Met
Asp Gln Glu Pro Ala 35 40 45Gly Asn Arg Thr Val Asn Arg Glu Asp Ser
Asp Glu Gln Asp Pro Gln 50 55 60Glu Val Thr Tyr Ala Gln Leu Asn His
Cys Val Phe Thr Gln Arg Lys65 70 75 80Ile Thr Arg Pro Ser Gln Arg
Pro Lys Thr Pro Pro Thr Asp Thr Ser 85 90 95Val32807PRTArtificial
SequencePTPN6-CD45 fusion protein 32Trp Tyr His Gly His Met Ser Gly
Gly Gln Ala Glu Thr Leu Leu Gln1 5 10 15Ala Lys Gly Glu Pro Trp Thr
Phe Leu Val Arg Glu Ser Leu Ser Gln 20 25
30Pro Gly Asp Phe Val Leu Ser Val Leu Ser Asp Gln Pro Lys Ala Gly
35 40 45Pro Gly Ser Pro Leu Arg Val Thr His Ile Lys Val Met Cys Glu
Gly 50 55 60Gly Arg Tyr Thr Val Gly Gly Leu Glu Thr Phe Asp Ser Leu
Thr Asp65 70 75 80Leu Val Glu His Phe Lys Lys Thr Gly Ile Glu Glu
Ala Ser Gly Ala 85 90 95Phe Val Tyr Leu Arg Gln Pro Tyr Lys Ile Tyr
Asp Leu His Lys Lys 100 105 110Arg Ser Cys Asn Leu Asp Glu Gln Gln
Glu Leu Val Glu Arg Asp Asp 115 120 125Glu Lys Gln Leu Met Asn Val
Glu Pro Ile His Ala Asp Ile Leu Leu 130 135 140Glu Thr Tyr Lys Arg
Lys Ile Ala Asp Glu Gly Arg Leu Phe Leu Ala145 150 155 160Glu Phe
Gln Ser Ile Pro Arg Val Phe Ser Lys Phe Pro Ile Lys Glu 165 170
175Ala Arg Lys Pro Phe Asn Gln Asn Lys Asn Arg Tyr Val Asp Ile Leu
180 185 190Pro Tyr Asp Tyr Asn Arg Val Glu Leu Ser Glu Ile Asn Gly
Asp Ala 195 200 205Gly Ser Asn Tyr Ile Asn Ala Ser Tyr Ile Asp Gly
Phe Lys Glu Pro 210 215 220Arg Lys Tyr Ile Ala Ala Gln Gly Pro Arg
Asp Glu Thr Val Asp Asp225 230 235 240Phe Trp Arg Met Ile Trp Glu
Gln Lys Ala Thr Val Ile Val Met Val 245 250 255Thr Arg Cys Glu Glu
Gly Asn Arg Asn Lys Cys Ala Glu Tyr Trp Pro 260 265 270Ser Met Glu
Glu Gly Thr Arg Ala Phe Gly Asp Val Val Val Lys Ile 275 280 285Asn
Gln His Lys Arg Cys Pro Asp Tyr Ile Ile Gln Lys Leu Asn Ile 290 295
300Val Asn Lys Lys Glu Lys Ala Thr Gly Arg Glu Val Thr His Ile
Gln305 310 315 320Phe Thr Ser Trp Pro Asp His Gly Val Pro Glu Asp
Pro His Leu Leu 325 330 335Leu Lys Leu Arg Arg Arg Val Asn Ala Phe
Ser Asn Phe Phe Ser Gly 340 345 350Pro Ile Val Val His Cys Ser Ala
Gly Val Gly Arg Thr Gly Thr Tyr 355 360 365Ile Gly Ile Asp Ala Met
Leu Glu Gly Leu Glu Ala Glu Asn Lys Val 370 375 380Asp Val Tyr Gly
Tyr Val Val Lys Leu Arg Arg Gln Arg Cys Leu Met385 390 395 400Val
Gln Val Glu Ala Gln Tyr Ile Leu Ile His Gln Ala Leu Val Glu 405 410
415Tyr Asn Gln Phe Gly Glu Thr Glu Val Asn Leu Ser Glu Leu His Pro
420 425 430Tyr Leu His Asn Met Lys Lys Arg Asp Pro Pro Ser Glu Pro
Ser Pro 435 440 445Leu Glu Ala Glu Phe Gln Arg Leu Pro Ser Tyr Arg
Ser Trp Arg Thr 450 455 460Gln His Ile Gly Asn Gln Glu Glu Asn Lys
Ser Lys Asn Arg Asn Ser465 470 475 480Asn Val Ile Pro Tyr Asp Tyr
Asn Arg Val Leu Lys His Glu Leu Glu 485 490 495Met Ser Lys Glu Ser
Glu His Asp Ser Asp Glu Ser Ser Asp Asp Asp 500 505 510Ser Asp Ser
Glu Glu Pro Ser Lys Tyr Ile Asn Ala Ser Phe Ile Met 515 520 525Ser
Tyr Trp Lys Pro Glu Val Met Ile Ala Ala Gln Gly Pro Leu Lys 530 535
540Glu Thr Ile Gly Asp Phe Met Ile Gln Arg Lys Val Lys Val Ile
Val545 550 555 560Met Leu Thr Glu Leu Lys His Gly Asp Gln Glu Ile
Cys Ala Gln Tyr 565 570 575Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp
Ile Glu Val Asp Leu Lys 580 585 590Asp Thr Asp Lys Ser Ser Thr Tyr
Thr Leu Arg Val Phe Glu Leu Arg 595 600 605His Ser Lys Arg Lys Asp
Ser Arg Thr Val Tyr Gln Tyr Gln Tyr Thr 610 615 620Asn Trp Ser Val
Glu Gln Leu Pro Ala Glu Pro Lys Glu Leu Ile Ser625 630 635 640Met
Ile Gln Val Val Lys Gln Lys Leu Pro Gln Lys Asn Ser Ser Glu 645 650
655Gly Asn Lys His His Lys Ser Thr Pro Leu Leu Ile His Cys Arg Asp
660 665 670Gly Ser Gln Gln Thr Gly Ile Phe Cys Ala Leu Leu Asn Leu
Leu Glu 675 680 685Ser Ala Glu Thr Glu Glu Val Val Asp Ile Phe Gln
Val Val Lys Ala 690 695 700Leu Arg Lys Ala Arg Pro Gly Met Val Ser
Thr Phe Glu Gln Tyr Gln705 710 715 720Phe Leu Tyr Asp Val Ile Ala
Ser Thr Tyr Pro Ala Gln Asn Gly Gln 725 730 735Val Lys Lys Asn Asn
His Gln Glu Asp Lys Ile Glu Phe Asp Asn Glu 740 745 750Val Asp Lys
Val Lys Gln Asp Ala Asn Cys Val Asn Pro Leu Gly Ala 755 760 765Pro
Glu Lys Leu Pro Glu Ala Lys Glu Gln Ala Glu Gly Ser Glu Pro 770 775
780Thr Ser Gly Thr Glu Gly Pro Glu His Ser Val Asn Gly Pro Ala
Ser785 790 795 800Pro Ala Leu Asn Gln Gly Ser 80533434PRTArtificial
SequencePTPN6-CD148 fusion protein 33Glu Thr Leu Leu Gln Ala Lys
Gly Glu Pro Trp Thr Phe Leu Val Arg1 5 10 15Glu Ser Leu Ser Gln Pro
Gly Asp Phe Val Leu Ser Val Leu Ser Asp 20 25 30Gln Pro Lys Ala Gly
Pro Gly Ser Pro Leu Arg Val Thr His Ile Lys 35 40 45Val Met Cys Glu
Gly Gly Arg Tyr Thr Val Gly Gly Leu Glu Thr Phe 50 55 60Asp Ser Leu
Thr Asp Leu Val Glu His Phe Lys Lys Thr Gly Ile Glu65 70 75 80Glu
Ala Ser Gly Ala Phe Val Tyr Leu Arg Gln Pro Tyr Arg Lys Lys 85 90
95Arg Lys Asp Ala Lys Asn Asn Glu Val Ser Phe Ser Gln Ile Lys Pro
100 105 110Lys Lys Ser Lys Leu Ile Arg Val Glu Asn Phe Glu Ala Tyr
Phe Lys 115 120 125Lys Gln Gln Ala Asp Ser Asn Cys Gly Phe Ala Glu
Glu Tyr Glu Asp 130 135 140Leu Lys Leu Val Gly Ile Ser Gln Pro Lys
Tyr Ala Ala Glu Leu Ala145 150 155 160Glu Asn Arg Gly Lys Asn Arg
Tyr Asn Asn Val Leu Pro Tyr Asp Ile 165 170 175Ser Arg Val Lys Leu
Ser Val Gln Thr His Ser Thr Asp Asp Tyr Ile 180 185 190Asn Ala Asn
Tyr Met Pro Gly Tyr His Ser Lys Lys Asp Phe Ile Ala 195 200 205Thr
Gln Gly Pro Leu Pro Asn Thr Leu Lys Asp Phe Trp Arg Met Val 210 215
220Trp Glu Lys Asn Val Tyr Ala Ile Ile Met Leu Thr Lys Cys Val
Glu225 230 235 240Gln Gly Arg Thr Lys Cys Glu Glu Tyr Trp Pro Ser
Lys Gln Ala Gln 245 250 255Asp Tyr Gly Asp Ile Thr Val Ala Met Thr
Ser Glu Ile Val Leu Pro 260 265 270Glu Trp Thr Ile Arg Asp Phe Thr
Val Lys Asn Ile Gln Thr Ser Glu 275 280 285Ser His Pro Leu Arg Gln
Phe His Phe Thr Ser Trp Pro Asp His Gly 290 295 300Val Pro Asp Thr
Thr Asp Leu Leu Ile Asn Phe Arg Tyr Leu Val Arg305 310 315 320Asp
Tyr Met Lys Gln Ser Pro Pro Glu Ser Pro Ile Leu Val His Cys 325 330
335Ser Ala Gly Val Gly Arg Thr Gly Thr Phe Ile Ala Ile Asp Arg Leu
340 345 350Ile Tyr Gln Ile Glu Asn Glu Asn Thr Val Asp Val Tyr Gly
Ile Val 355 360 365Tyr Asp Leu Arg Met His Arg Pro Leu Met Val Gln
Thr Glu Asp Gln 370 375 380Tyr Val Phe Leu Asn Gln Cys Val Leu Asp
Ile Val Arg Ser Gln Lys385 390 395 400Asp Ser Lys Val Asp Leu Ile
Tyr Gln Asn Thr Thr Ala Met Thr Ile 405 410 415Tyr Glu Asn Leu Ala
Pro Val Thr Thr Phe Gly Lys Thr Asn Gly Tyr 420 425 430Ile
Ala3420PRTFoot-and-mouth disease virus 34Arg Ala Glu Gly Arg Gly
Ser Leu Leu Thr Cys Gly Asp Val Glu Glu1 5 10 15Asn Pro Gly Pro
20353390DNAArtificial SequenceNucleic acid sequences coding for
CARs
(MP13974.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-CD
28tmZw) 35atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca
cgccgccaga 60ccagacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg
cgaccgggtg 120accatcagct gcagagccag ccaggacatc agcaagtacc
tgaactggta ccagcagaag 180cccgacggca ccgtgaagct gctgatctac
cacaccagcc ggctgcacag cggcgtgccc 240agccggttca gcggcagcgg
cagcggcacc gactacagcc tgaccatcag caacctggag 300caggaggaca
tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcgga
360ggcggcacca agctggagat caccaaggcc ggaggcggag gctctggcgg
aggcggctct 420ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc
tgcaggagtc tggcccaggc 480ctggtggccc caagccagag cctgagcgtg
acctgcaccg tgagcggcgt gagcctgccc 540gactacggcg tgagctggat
caggcagccc ccacggaagg gcctggagtg gctgggcgtg 600atctggggca
gcgagaccac ctactacaac agcgccctga agagccggct gaccatcatc
660aaggacaaca gcaagagcca ggtgttcctg aagatgaaca gcctgcagac
cgacgacacc 720gccatctact actgcgccaa gcactactac tatggcggca
gctacgctat ggactactgg 780ggccagggca ccagcgtgac cgtgagctca
gatcccacca cgacgccagc gccgcgacca 840ccaacaccgg cgcccaccat
cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900ccagcggcgg
ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatcttttgg
960gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac
agtggccttt 1020attattttct gggtgaggag agtgaagttc agcaggagcg
cagacgcccc cgcgtaccag 1080cagggccaga accagctcta taacgagctc
aatctaggac gaagagagga gtacgatgtt 1140ttggacaaga gacgtggccg
ggaccctgag atggggggaa agccgagaag gaagaaccct 1200caggaaggcc
tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt
1260gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca
gggtctcagt 1320acagccacca aggacaccta cgacgccctt cacatgcagg
ccctgcctcc tcgcagagcc 1380gagggcaggg gaagtcttct aacatgcggg
gacgtggagg aaaatcccgg gcccatggcc 1440gtgcccactc aggtcctggg
gttgttgcta ctgtggctta cagatgccag atgtgacatc 1500cagatgacac
agtctccatc ttccctgtct gcatctgtcg gagatcgcgt caccatcacc
1560tgtcgagcaa gtgaggacat ttattttaat ttagtgtggt atcagcagaa
accaggaaag 1620gcccctaagc tcctgatcta tgatacaaat cgcttggcag
atggggtccc atcacggttc 1680agtggctctg gatctggcac acagtatact
ctaaccataa gtagcctgca acccgaagat 1740ttcgcaacct attattgtca
acactataag aattatccgc tcacgttcgg tcaggggacc 1800aagctggaaa
tcaaaagatc tggtggcgga gggtcaggag gcggaggcag cggaggcggt
1860ggctcgggag gcggaggctc gagatctgag gtgcagttgg tggagtctgg
gggcggcttg 1920gtgcagcctg gagggtccct gaggctctcc tgtgcagcct
caggattcac tctcagtaat 1980tatggcatgc actggatcag gcaggctcca
gggaagggtc tggagtgggt ctcgtctatt 2040agtcttaatg gtggtagcac
ttactatcga gactccgtga agggccgatt cactatctcc 2100agggacaatg
caaaaagcac cctctacctt caaatgaata gtctgagggc cgaggacacg
2160gccgtctatt actgtgcagc acaggacgct tatacgggag gttactttga
ttactggggc 2220caaggaacgc tggtcacagt ctcgtctatg gatcccgccg
agcccaaatc tcctgacaaa 2280actcacacat gcccaccgtg cccagcacct
cccgtggccg gcccgtcagt cttcctcttc 2340cccccaaaac ccaaggacac
cctcatgatc gcccggaccc ctgaggtcac atgcgtggtg 2400gtggacgtga
gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag
2460gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta
ccgtgtggtc 2520agcgtcctca ccgtcctgca ccaggactgg ctgaatggca
aggagtacaa gtgcaaggtc 2580tccaacaaag ccctcccagc ccccatcgag
aaaaccatct ccaaagccaa agggcagccc 2640cgagaaccac aggtgtacac
cctgccccca tcccgggatg agctgaccaa gaaccaggtc 2700agcctgacct
gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc
2760aatgggcaac cggagaacaa ctacaagacc acgcctcccg tgctggactc
cgacggctcc 2820ttcttcctct acagcaagct caccgtggac aagagcaggt
ggcagcaggg gaacgtcttc 2880tcatgctccg tgatgcatga ggccctgcac
aatcactata cccagaaatc tctgagtctg 2940agcccaggca agaaggaccc
caagttctgg gtcctggtgg tggtgggagg cgtgctggcc 3000tgttactctc
tcctggtgac cgtggccttc atcatctttt gggtgcgctc ccgggtgaag
3060ttttctcgct ctgccgatgc cccagcctat cagcagggcc agaatcagct
gtacaatgaa 3120ctgaacctgg gcaggcggga ggagtacgac gtgctggata
agcggagagg cagagacccc 3180gagatgggcg gcaaaccacg gcgcaaaaat
ccccaggagg gactctataa cgagctgcag 3240aaggacaaaa tggccgaggc
ctattccgag atcggcatga agggagagag aagacgcgga 3300aagggccacg
acggcctgta tcagggattg tccaccgcta caaaagatac atatgatgcc
3360ctgcacatgc aggccctgcc acccagatga 3390365154DNAArtificial
SequenceNucleic acid sequences coding for CARs
(MP14802.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3p
vaa-dCD45) 36atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca
cgccgccaga 60ccagacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg
cgaccgggtg 120accatcagct gcagagccag ccaggacatc agcaagtacc
tgaactggta ccagcagaag 180cccgacggca ccgtgaagct gctgatctac
cacaccagcc ggctgcacag cggcgtgccc 240agccggttca gcggcagcgg
cagcggcacc gactacagcc tgaccatcag caacctggag 300caggaggaca
tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcgga
360ggcggcacca agctggagat caccaaggcc ggaggcggag gctctggcgg
aggcggctct 420ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc
tgcaggagtc tggcccaggc 480ctggtggccc caagccagag cctgagcgtg
acctgcaccg tgagcggcgt gagcctgccc 540gactacggcg tgagctggat
caggcagccc ccacggaagg gcctggagtg gctgggcgtg 600atctggggca
gcgagaccac ctactacaac agcgccctga agagccggct gaccatcatc
660aaggacaaca gcaagagcca ggtgttcctg aagatgaaca gcctgcagac
cgacgacacc 720gccatctact actgcgccaa gcactactac tatggcggca
gctacgctat ggactactgg 780ggccagggca ccagcgtgac cgtgagctca
gatcccacca cgacgccagc gccgcgacca 840ccaacaccgg cgcccaccat
cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900ccagcggcgg
ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatcttttgg
960gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac
agtggccttt 1020attattttct gggtgaggag agtgaagttc agcaggagcg
cagacgcccc cgcgtaccag 1080cagggccaga accagctcta taacgagctc
aatctaggac gaagagagga gtacgatgtt 1140ttggacaaga gacgtggccg
ggaccctgag atggggggaa agccgagaag gaagaaccct 1200caggaaggcc
tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt
1260gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca
gggtctcagt 1320acagccacca aggacaccta cgacgccctt cacatgcagg
ccctgcctcc tcgcagagcc 1380gagggcaggg gaagtcttct aacatgcggg
gacgtggagg aaaatcccgg gcccatggcc 1440gtgcccactc aggtcctggg
gttgttgcta ctgtggctta cagatgccag atgtgacatc 1500cagatgacac
agtctccatc ttccctgtct gcatctgtcg gagatcgcgt caccatcacc
1560tgtcgagcaa gtgaggacat ttattttaat ttagtgtggt atcagcagaa
accaggaaag 1620gcccctaagc tcctgatcta tgatacaaat cgcttggcag
atggggtccc atcacggttc 1680agtggctctg gatctggcac acagtatact
ctaaccataa gtagcctgca acccgaagat 1740ttcgcaacct attattgtca
acactataag aattatccgc tcacgttcgg tcaggggacc 1800aagctggaaa
tcaaaagatc tggtggcgga gggtcaggag gcggaggcag cggaggcggt
1860ggctcgggag gcggaggctc gagatctgag gtgcagttgg tggagtctgg
gggcggcttg 1920gtgcagcctg gagggtccct gaggctctcc tgtgcagcct
caggattcac tctcagtaat 1980tatggcatgc actggatcag gcaggctcca
gggaagggtc tggagtgggt ctcgtctatt 2040agtcttaatg gtggtagcac
ttactatcga gactccgtga agggccgatt cactatctcc 2100agggacaatg
caaaaagcac cctctacctt caaatgaata gtctgagggc cgaggacacg
2160gccgtctatt actgtgcagc acaggacgct tatacgggag gttactttga
ttactggggc 2220caaggaacgc tggtcacagt ctcgtctatg gatcccgccg
agcccaaatc tcctgacaaa 2280actcacacat gcccaccgtg cccagcacct
cccgtggccg gcccgtcagt cttcctcttc 2340cccccaaaac ccaaggacac
cctcatgatc gcccggaccc ctgaggtcac atgcgtggtg 2400gtggacgtga
gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag
2460gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta
ccgtgtggtc 2520agcgtcctca ccgtcctgca ccaggactgg ctgaatggca
aggagtacaa gtgcaaggtc 2580tccaacaaag ccctcccagc ccccatcgag
aaaaccatct ccaaagccaa agggcagccc 2640cgagaaccac aggtgtacac
cctgccccca tcccgggatg agctgaccaa gaaccaggtc 2700agcctgacct
gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc
2760aatgggcaac cggagaacaa ctacaagacc acgcctcccg tgctggactc
cgacggctcc 2820ttcttcctct acagcaagct caccgtggac aagagcaggt
ggcagcaggg gaacgtcttc 2880tcatgctccg tgatgcatga ggccctgcac
aatcactata cccagaaatc tctgagtctg 2940agcccaggca agaaggaccc
caaggcactg atagcatttc tggcatttct gattattgtg 3000acatcaatag
ccctgcttgt tgttctctac aaaatctatg atctacataa gaaaagatcc
3060tgcaatttag atgaacagca ggagcttgtt gaaagggatg atgaaaaaca
actgatgaat 3120gtggagccaa tccatgcaga tattttgttg gaaacttata
agaggaagat tgctgatgaa 3180ggaagacttt ttctggctga atttcagagc
atcccgcggg tgttcagcaa gtttcctata 3240aaggaagctc gaaagccctt
taaccagaat aaaaaccgtt atgttgacat tcttccttat 3300gattataacc
gtgttgaact ctctgagata aacggagatg cagggtcaaa ctacataaat
3360gccagctata ttgatggttt caaagaaccc aggaaataca ttgctgcaca
aggtcccagg 3420gatgaaactg ttgatgattt ctggaggatg atttgggaac
agaaagccac agttattgtc 3480atggtcactc gatgtgaaga aggaaacagg
aacaagtgtg cagaatactg gccgtcaatg 3540gaagagggca ctcgggcttt
tggagatgtt gttgtaaaga tcaaccagca caaaagatgt 3600ccagattaca
tcattcagaa attgaacatt gtaaataaaa aagaaaaagc aactggaaga
3660gaggtgactc acattcagtt caccagctgg ccagaccacg gggtgcctga
ggatcctcac 3720ttgctcctca aactgagaag gagagtgaat gccttcagca
atttcttcag
tggtcccatt 3780gtggtgcact gcagtgctgg tgttgggcgc acaggaacct
atatcggaat tgatgccatg 3840ctagaaggcc tggaagccga gaacaaagtg
gatgtttatg gttatgttgt caagctaagg 3900cgacagagat gcctgatggt
tcaagtagag gcccagtaca tcttgatcca tcaggctttg 3960gtggaataca
atcagtttgg agaaacagaa gtgaatttgt ctgaattaca tccatatcta
4020cataacatga agaaaaggga tccacccagt gagccgtctc cactagaggc
tgaattccag 4080agacttcctt catataggag ctggaggaca cagcacattg
gaaatcaaga agaaaataaa 4140agtaaaaaca ggaattctaa tgtcatccca
tatgactata acagagtgcc acttaaacat 4200gagctggaaa tgagtaaaga
gagtgagcat gattcagatg aatcctctga tgatgacagt 4260gattcagagg
aaccaagcaa atacatcaat gcatctttta taatgagcta ctggaaacct
4320gaagtgatga ttgctgctca gggaccactg aaggagacca ttggtgactt
ttggcagatg 4380atcttccaaa gaaaagtcaa agttattgtt atgctgacag
aactgaaaca tggagaccag 4440gaaatctgtg ctcagtactg gggagaagga
aagcaaacat atggagatat tgaagttgac 4500ctgaaagaca cagacaaatc
ttcaacttat acccttcgtg tctttgaact gagacattcc 4560aagaggaaag
actctcgaac tgtgtaccag taccaatata caaactggag tgtggagcag
4620cttcctgcag aacccaagga attaatctct atgattcagg tcgtcaaaca
aaaacttccc 4680cagaagaatt cctctgaagg gaacaagcat cacaagagta
cacctctact cattcactgc 4740agggatggat ctcagcaaac gggaatattt
tgtgctttgt taaatctctt agaaagtgcg 4800gaaacagaag aggtagtgga
tatttttcaa gtggtaaaag ctctacgcaa agctaggcca 4860ggcatggttt
ccacattcga gcaatatcaa ttcctatatg acgtcattgc cagcacctac
4920cctgctcaga atggacaagt aaagaaaaac aaccatcaag aagataaaat
tgaatttgat 4980aatgaagtgg acaaagtaaa gcaggatgct aattgtgtta
atccacttgg tgccccagaa 5040aagctccctg aagcaaagga acaggctgaa
ggttctgaac ccacgagtgg cactgagggg 5100ccagaacatt ctgtcaatgg
tcctgcaagt ccagctttaa atcaaggttc atag 5154374053DNAArtificial
SequenceNucleic acid sequences coding for CARs
(MP14801.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3p
vaa-dCD148) 37atgagcctgc ccgtgaccgc cctgctgctg cccctggccc
tgctgctgca cgccgccaga 60ccagacatcc agatgaccca gaccaccagc agcctgagcg
ccagcctggg cgaccgggtg 120accatcagct gcagagccag ccaggacatc
agcaagtacc tgaactggta ccagcagaag 180cccgacggca ccgtgaagct
gctgatctac cacaccagcc ggctgcacag cggcgtgccc 240agccggttca
gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag
300caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta
caccttcgga 360ggcggcacca agctggagat caccaaggcc ggaggcggag
gctctggcgg aggcggctct 420ggcggaggcg gctctggcgg aggcggcagc
gaggtgaagc tgcaggagtc tggcccaggc 480ctggtggccc caagccagag
cctgagcgtg acctgcaccg tgagcggcgt gagcctgccc 540gactacggcg
tgagctggat caggcagccc ccacggaagg gcctggagtg gctgggcgtg
600atctggggca gcgagaccac ctactacaac agcgccctga agagccggct
gaccatcatc 660aaggacaaca gcaagagcca ggtgttcctg aagatgaaca
gcctgcagac cgacgacacc 720gccatctact actgcgccaa gcactactac
tatggcggca gctacgctat ggactactgg 780ggccagggca ccagcgtgac
cgtgagctca gatcccacca cgacgccagc gccgcgacca 840ccaacaccgg
cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg
900ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga
tatcttttgg 960gtgctggtgg tggttggtgg agtcctggct tgctatagct
tgctagtaac agtggccttt 1020attattttct gggtgaggag agtgaagttc
agcaggagcg cagacgcccc cgcgtaccag 1080cagggccaga accagctcta
taacgagctc aatctaggac gaagagagga gtacgatgtt 1140ttggacaaga
gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct
1200caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta
cagtgagatt 1260gggatgaaag gcgagcgccg gaggggcaag gggcacgatg
gcctttacca gggtctcagt 1320acagccacca aggacaccta cgacgccctt
cacatgcagg ccctgcctcc tcgcagagcc 1380gagggcaggg gaagtcttct
aacatgcggg gacgtggagg aaaatcccgg gcccatggcc 1440gtgcccactc
aggtcctggg gttgttgcta ctgtggctta cagatgccag atgtgacatc
1500cagatgacac agtctccatc ttccctgtct gcatctgtcg gagatcgcgt
caccatcacc 1560tgtcgagcaa gtgaggacat ttattttaat ttagtgtggt
atcagcagaa accaggaaag 1620gcccctaagc tcctgatcta tgatacaaat
cgcttggcag atggggtccc atcacggttc 1680agtggctctg gatctggcac
acagtatact ctaaccataa gtagcctgca acccgaagat 1740ttcgcaacct
attattgtca acactataag aattatccgc tcacgttcgg tcaggggacc
1800aagctggaaa tcaaaagatc tggtggcgga gggtcaggag gcggaggcag
cggaggcggt 1860ggctcgggag gcggaggctc gagatctgag gtgcagttgg
tggagtctgg gggcggcttg 1920gtgcagcctg gagggtccct gaggctctcc
tgtgcagcct caggattcac tctcagtaat 1980tatggcatgc actggatcag
gcaggctcca gggaagggtc tggagtgggt ctcgtctatt 2040agtcttaatg
gtggtagcac ttactatcga gactccgtga agggccgatt cactatctcc
2100agggacaatg caaaaagcac cctctacctt caaatgaata gtctgagggc
cgaggacacg 2160gccgtctatt actgtgcagc acaggacgct tatacgggag
gttactttga ttactggggc 2220caaggaacgc tggtcacagt ctcgtctatg
gatcccgccg agcccaaatc tcctgacaaa 2280actcacacat gcccaccgtg
cccagcacct cccgtggccg gcccgtcagt cttcctcttc 2340cccccaaaac
ccaaggacac cctcatgatc gcccggaccc ctgaggtcac atgcgtggtg
2400gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga
cggcgtggag 2460gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
acagcacgta ccgtgtggtc 2520agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca aggagtacaa gtgcaaggtc 2580tccaacaaag ccctcccagc
ccccatcgag aaaaccatct ccaaagccaa agggcagccc 2640cgagaaccac
aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc
2700agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga
gtgggagagc 2760aatgggcaac cggagaacaa ctacaagacc acgcctcccg
tgctggactc cgacggctcc 2820ttcttcctct acagcaagct caccgtggac
aagagcaggt ggcagcaggg gaacgtcttc 2880tcatgctccg tgatgcatga
ggccctgcac aatcactata cccagaaatc tctgagtctg 2940agcccaggca
agaaggaccc caaggcggtt tttggctgta tctttggtgc cctggttatt
3000gtgactgtgg gaggcttcat cttctggaga aagaagagga aagatgcaaa
gaataatgaa 3060gtgtcctttt ctcaaattaa acctaaaaaa tctaagttaa
tcagagtgga gaattttgag 3120gcctacttca agaagcagca agctgactcc
aactgtgggt tcgcagagga atacgaagat 3180ctgaagcttg ttggaattag
tcaacctaaa tatgcagcag aactggctga gaatagagga 3240aagaatcgct
ataataatgt tctgccctat gatatttccc gtgtcaaact ttcggtccag
3300acccattcaa cggatgacta catcaatgcc aactacatgc ctggctacca
ctccaagaaa 3360gattttattg ccacacaagg acctttaccg aacactttga
aagatttttg gcgtatggtt 3420tgggagaaaa atgtatatgc catcattatg
ttgactaaat gtgttgaaca gggaagaacc 3480aaatgtgagg agtattggcc
ctccaagcag gctcaggact atggagacat aactgtggca 3540atgacatcag
aaattgttct tccggaatgg accatcagag atttcacagt gaaaaatatc
3600cagacaagtg agagtcaccc tctgagacag ttccatttca cctcctggcc
agaccacggt 3660gttcccgaca ccactgacct gctcatcaac ttccggtacc
tcgttcgtga ctacatgaag 3720cagagtcctc ccgaatcgcc gattctggtg
cattgcagtg ctggggtcgg aaggacgggc 3780actttcattg ccattgatcg
tctcatctac cagatagaga atgagaacac cgtggatgtg 3840tatgggattg
tgtatgacct tcgaatgcat aggcctttaa tggtgcagac agaggaccag
3900tatgttttcc tcaatcagtg tgttttggat attgtcagat cccagaaaga
ctcaaaagta 3960gatcttatct accagaacac aactgcaatg acaatctatg
aaaaccttgc gcccgtgacc 4020acatttggaa agaccaatgg ttacatcgcc taa
4053383345DNAArtificial SequenceNucleic acid sequences coding for
CARs
(16076.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-tm-dPTP
N6) 38atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca
cgccgccaga 60ccagacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg
cgaccgggtg 120accatcagct gcagagccag ccaggacatc agcaagtacc
tgaactggta ccagcagaag 180cccgacggca ccgtgaagct gctgatctac
cacaccagcc ggctgcacag cggcgtgccc 240agccggttca gcggcagcgg
cagcggcacc gactacagcc tgaccatcag caacctggag 300caggaggaca
tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcgga
360ggcggcacca agctggagat caccaaggcc ggaggcggag gctctggcgg
aggcggctct 420ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc
tgcaggagtc tggcccaggc 480ctggtggccc caagccagag cctgagcgtg
acctgcaccg tgagcggcgt gagcctgccc 540gactacggcg tgagctggat
caggcagccc ccacggaagg gcctggagtg gctgggcgtg 600atctggggca
gcgagaccac ctactacaac agcgccctga agagccggct gaccatcatc
660aaggacaaca gcaagagcca ggtgttcctg aagatgaaca gcctgcagac
cgacgacacc 720gccatctact actgcgccaa gcactactac tatggcggca
gctacgctat ggactactgg 780ggccagggca ccagcgtgac cgtgagctca
gatcccacca cgacgccagc gccgcgacca 840ccaacaccgg cgcccaccat
cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900ccagcggcgg
ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatcttttgg
960gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac
agtggccttt 1020attattttct gggtgaggag agtgaagttc agcaggagcg
cagacgcccc cgcgtaccag 1080cagggccaga accagctcta taacgagctc
aatctaggac gaagagagga gtacgatgtt 1140ttggacaaga gacgtggccg
ggaccctgag atggggggaa agccgagaag gaagaaccct 1200caggaaggcc
tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt
1260gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca
gggtctcagt 1320acagccacca aggacaccta cgacgccctt cacatgcagg
ccctgcctcc tcgcagagcc 1380gagggcaggg gaagtcttct aacatgcggg
gacgtggagg aaaatcccgg gcccatggcc 1440gtgcccactc aggtcctggg
gttgttgcta ctgtggctta cagatgccag atgtgacatc 1500cagatgacac
agtctccatc ttccctgtct gcatctgtcg gagatcgcgt caccatcacc
1560tgtcgagcaa gtgaggacat ttattttaat ttagtgtggt atcagcagaa
accaggaaag 1620gcccctaagc tcctgatcta tgatacaaat cgcttggcag
atggggtccc atcacggttc 1680agtggctctg gatctggcac acagtatact
ctaaccataa gtagcctgca acccgaagat 1740ttcgcaacct attattgtca
acactataag aattatccgc tcacgttcgg tcaggggacc 1800aagctggaaa
tcaaaagatc tggtggcgga gggtcaggag gcggaggcag cggaggcggt
1860ggctcgggag gcggaggctc gagatctgag gtgcagttgg tggagtctgg
gggcggcttg 1920gtgcagcctg gagggtccct gaggctctcc tgtgcagcct
caggattcac tctcagtaat 1980tatggcatgc actggatcag gcaggctcca
gggaagggtc tggagtgggt ctcgtctatt 2040agtcttaatg gtggtagcac
ttactatcga gactccgtga agggccgatt cactatctcc 2100agggacaatg
caaaaagcac cctctacctt caaatgaata gtctgagggc cgaggacacg
2160gccgtctatt actgtgcagc acaggacgct tatacgggag gttactttga
ttactggggc 2220caaggaacgc tggtcacagt ctcgtctatg gatcccgcca
ccacaaccaa gcccgtgctg 2280cggaccccaa gccctgtgca ccctaccggc
accagccagc ctcagagacc cgaggactgc 2340cggcctcggg gcagcgtgaa
gggcaccggc ctggacttcg cctgcgacat ctactgggca 2400cctctggccg
gaatatgcgt ggcactgctg ctgagcctca tcatcaccct gatctgttat
2460caccgaagcc gcaagcgggt gtgtaaaagt ggaggcggaa gcttctggga
ggagtttgag 2520agtttgcaga agcaggaggt gaagaacttg caccagcgtc
tggaagggca gcggccagag 2580aacaagggca agaaccgcta caagaacatt
ctcccctttg accacagccg agtgatcctg 2640cagggacggg acagtaacat
ccccgggtcc gactacatca atgccaacta catcaagaac 2700cagctgctag
gccctgatga gaacgctaag acctacatcg ccagccaggg ctgtctggag
2760gccacggtca atgacttctg gcagatggcg tggcaggaga acagccgtgt
catcgtcatg 2820accacccgag aggtggagaa aggccggaac aaatgcgtcc
catactggcc cgaggtgggc 2880atgcagcgtg cttatgggcc ctactctgtg
accaactgcg gggagcatga cacaaccgaa 2940tacaaactcc gtaccttaca
ggtctccccg ctggacaatg gagacctgat tcgggagatc 3000tggcattacc
agtacctgag ctggcccgac cacggggtcc ccagtgagcc tgggggtgtc
3060ctcagcttcc tggaccagat caaccagcgg caggaaagtc tgcctcacgc
agggcccatc 3120atcgtgcact gcagcgccgg catcggccgc acaggcacca
tcattgtcat cgacatgctc 3180atggagaaca tctccaccaa gggcctggac
tgtgacattg acatccagaa gaccatccag 3240atggtgcggg cgcagcgctc
gggcatggtg cagacggagg cgcagtacaa gttcatctac 3300gtggccatcg
cccagttcat tgaaaccact aagaagaagc tgtga 3345392757DNAArtificial
SequenceNucleic acid sequences coding for CARs
(MP16091.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1
tm-endo) 39atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca
cgccgccaga 60ccagacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg
cgaccgggtg 120accatcagct gcagagccag ccaggacatc agcaagtacc
tgaactggta ccagcagaag 180cccgacggca ccgtgaagct gctgatctac
cacaccagcc ggctgcacag cggcgtgccc 240agccggttca gcggcagcgg
cagcggcacc gactacagcc tgaccatcag caacctggag 300caggaggaca
tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcgga
360ggcggcacca agctggagat caccaaggcc ggaggcggag gctctggcgg
aggcggctct 420ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc
tgcaggagtc tggcccaggc 480ctggtggccc caagccagag cctgagcgtg
acctgcaccg tgagcggcgt gagcctgccc 540gactacggcg tgagctggat
caggcagccc ccacggaagg gcctggagtg gctgggcgtg 600atctggggca
gcgagaccac ctactacaac agcgccctga agagccggct gaccatcatc
660aaggacaaca gcaagagcca ggtgttcctg aagatgaaca gcctgcagac
cgacgacacc 720gccatctact actgcgccaa gcactactac tatggcggca
gctacgctat ggactactgg 780ggccagggca ccagcgtgac cgtgagctca
gatcccacca cgacgccagc gccgcgacca 840ccaacaccgg cgcccaccat
cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900ccagcggcgg
ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatcttttgg
960gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac
agtggccttt 1020attattttct gggtgaggag agtgaagttc agcaggagcg
cagacgcccc cgcgtaccag 1080cagggccaga accagctcta taacgagctc
aatctaggac gaagagagga gtacgatgtt 1140ttggacaaga gacgtggccg
ggaccctgag atggggggaa agccgagaag gaagaaccct 1200caggaaggcc
tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt
1260gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca
gggtctcagt 1320acagccacca aggacaccta cgacgccctt cacatgcagg
ccctgcctcc tcgcagagcc 1380gagggcaggg gaagtcttct aacatgcggg
gacgtggagg aaaatcccgg gcccatggcc 1440gtgcccactc aggtcctggg
gttgttgcta ctgtggctta cagatgccag atgtgacatc 1500cagatgacac
agtctccatc ttccctgtct gcatctgtcg gagatcgcgt caccatcacc
1560tgtcgagcaa gtgaggacat ttattttaat ttagtgtggt atcagcagaa
accaggaaag 1620gcccctaagc tcctgatcta tgatacaaat cgcttggcag
atggggtccc atcacggttc 1680agtggctctg gatctggcac acagtatact
ctaaccataa gtagcctgca acccgaagat 1740ttcgcaacct attattgtca
acactataag aattatccgc tcacgttcgg tcaggggacc 1800aagctggaaa
tcaaaagatc tggtggcgga gggtcaggag gcggaggcag cggaggcggt
1860ggctcgggag gcggaggctc gagatctgag gtgcagttgg tggagtctgg
gggcggcttg 1920gtgcagcctg gagggtccct gaggctctcc tgtgcagcct
caggattcac tctcagtaat 1980tatggcatgc actggatcag gcaggctcca
gggaagggtc tggagtgggt ctcgtctatt 2040agtcttaatg gtggtagcac
ttactatcga gactccgtga agggccgatt cactatctcc 2100agggacaatg
caaaaagcac cctctacctt caaatgaata gtctgagggc cgaggacacg
2160gccgtctatt actgtgcagc acaggacgct tatacgggag gttactttga
ttactggggc 2220caaggaacgc tggtcacagt ctcgtctatg gatcccgcca
ccacaaccaa gcccgtgctg 2280cggaccccaa gccctgtgca ccctaccggc
accagccagc ctcagagacc cgaggactgc 2340cggcctcggg gcagcgtgaa
gggcaccggc ctggacttcg cctgcgacat tctcatcggg 2400gtctcagtgg
tcttcctctt ctgtctcctc ctcctggtcc tcttctgcct ccatcgccag
2460aatcagataa agcaggggcc ccccagaagc aaggacgagg agcagaagcc
acagcagagg 2520cctgacctgg ctgttgatgt tctagagagg acagcagaca
aggccacagt caatggactt 2580cctgagaagg accgggagac cgacaccagc
gccctggctg cagggagttc ccaggaggtg 2640acgtatgctc agctggacca
ctgggccctc acacagagga cagcccgggc tgtgtcccca 2700cagtccacaa
agcccatggc cgagtccatc acgtatgcag ccgttgccag acactga
2757404092DNAArtificial SequenceNucleic acid sequences coding for
CARs
(MP16092.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1
tm-endo-2A-PTPN6_SH2-dCD148) 40atgagcctgc ccgtgaccgc cctgctgctg
cccctggccc tgctgctgca cgccgccaga 60ccagacatcc agatgaccca gaccaccagc
agcctgagcg ccagcctggg cgaccgggtg 120accatcagct gcagagccag
ccaggacatc agcaagtacc tgaactggta ccagcagaag 180cccgacggca
ccgtgaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc
240agccggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag
caacctggag 300caggaggaca tcgccaccta cttctgccag cagggcaaca
ccctgcccta caccttcgga 360ggcggcacca agctggagat caccaaggcc
ggaggcggag gctctggcgg aggcggctct 420ggcggaggcg gctctggcgg
aggcggcagc gaggtgaagc tgcaggagtc tggcccaggc 480ctggtggccc
caagccagag cctgagcgtg acctgcaccg tgagcggcgt gagcctgccc
540gactacggcg tgagctggat caggcagccc ccacggaagg gcctggagtg
gctgggcgtg 600atctggggca gcgagaccac ctactacaac agcgccctga
agagccggct gaccatcatc 660aaggacaaca gcaagagcca ggtgttcctg
aagatgaaca gcctgcagac cgacgacacc 720gccatctact actgcgccaa
gcactactac tatggcggca gctacgctat ggactactgg 780ggccagggca
ccagcgtgac cgtgagctca gatcccacca cgacgccagc gccgcgacca
840ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga
ggcgtgccgg 900ccagcggcgg ggggcgcagt gcacacgagg gggctggact
tcgcctgtga tatcttttgg 960gtgctggtgg tggttggtgg agtcctggct
tgctatagct tgctagtaac agtggccttt 1020attattttct gggtgaggag
agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1080cagggccaga
accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt
1140ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag
gaagaaccct 1200caggaaggcc tgtacaatga actgcagaaa gataagatgg
cggaggccta cagtgagatt 1260gggatgaaag gcgagcgccg gaggggcaag
gggcacgatg gcctttacca gggtctcagt 1320acagccacca aggacaccta
cgacgccctt cacatgcagg ccctgcctcc tcgcagagcc 1380gagggcaggg
gaagtcttct aacatgcggg gacgtggagg aaaatcccgg gcccatggcc
1440gtgcccactc aggtcctggg gttgttgcta ctgtggctta cagatgccag
atgtgacatc 1500cagatgacac agtctccatc ttccctgtct gcatctgtcg
gagatcgcgt caccatcacc 1560tgtcgagcaa gtgaggacat ttattttaat
ttagtgtggt atcagcagaa accaggaaag 1620gcccctaagc tcctgatcta
tgatacaaat cgcttggcag atggggtccc atcacggttc 1680agtggctctg
gatctggcac acagtatact ctaaccataa gtagcctgca acccgaagat
1740ttcgcaacct attattgtca acactataag aattatccgc tcacgttcgg
tcaggggacc 1800aagctggaaa tcaaaagatc tggtggcgga gggtcaggag
gcggaggcag cggaggcggt 1860ggctcgggag gcggaggctc gagatctgag
gtgcagttgg tggagtctgg gggcggcttg 1920gtgcagcctg gagggtccct
gaggctctcc tgtgcagcct caggattcac tctcagtaat 1980tatggcatgc
actggatcag gcaggctcca gggaagggtc tggagtgggt ctcgtctatt
2040agtcttaatg gtggtagcac ttactatcga gactccgtga agggccgatt
cactatctcc 2100agggacaatg caaaaagcac cctctacctt caaatgaata
gtctgagggc cgaggacacg 2160gccgtctatt actgtgcagc acaggacgct
tatacgggag gttactttga ttactggggc 2220caaggaacgc tggtcacagt
ctcgtctatg gatcccgcca ccacaaccaa gcccgtgctg 2280cggaccccaa
gccctgtgca ccctaccggc accagccagc ctcagagacc cgaggactgc
2340cggcctcggg gcagcgtgaa gggcaccggc ctggacttcg cctgcgacat
tctcatcggg 2400gtctcagtgg tcttcctctt ctgtctcctc ctcctggtcc
tcttctgcct ccatcgccag 2460aatcagataa agcaggggcc ccccagaagc
aaggacgagg agcagaagcc acagcagagg 2520cctgacctgg ctgttgatgt
tctagagagg acagcagaca aggccacagt caatggactt 2580cctgagaagg
accgggagac cgacaccagc gccctggctg cagggagttc ccaggaggtg
2640acgtatgctc agctggacca ctgggccctc acacagagga cagcccgggc
tgtgtcccca 2700cagtccacaa agcccatggc cgagtccatc acgtatgcag
ccgttgccag acacagggca 2760gaaggaagag gtagcctgct gacttgcggg
gacgtggaag agaacccagg gccatggtat 2820catggccaca tgtctggcgg
gcaggcagag acgctgctgc aggccaaggg cgagccctgg 2880acgtttcttg
tgcgtgagag cctcagccag cctggagact tcgtgctttc tgtgctcagt
2940gaccagccca aggctggccc aggctccccg ctcagggtca cccacatcaa
ggtcatgtgc 3000gagggtggac gctacacagt gggtggtttg gagaccttcg
acagcctcac ggacctggtg 3060gagcatttca agaagacggg gattgaggag
gcctcaggcg cctttgtcta cctgcggcag 3120ccgtacagcg gtggcggtgg
cagctttgag gcctacttca agaagcagca agctgactcc 3180aactgtgggt
tcgcagagga atacgaagat ctgaagcttg ttggaattag tcaacctaaa
3240tatgcagcag aactggctga gaatagagga aagaatcgct ataataatgt
tctgccctat 3300gatatttccc gtgtcaaact ttcggtccag acccattcaa
cggatgacta catcaatgcc 3360aactacatgc ctggctacca ctccaagaaa
gattttattg ccacacaagg acctttaccg 3420aacactttga aagatttttg
gcgtatggtt tgggagaaaa atgtatatgc catcattatg 3480ttgactaaat
gtgttgaaca gggaagaacc aaatgtgagg agtattggcc ctccaagcag
3540gctcaggact atggagacat aactgtggca atgacatcag aaattgttct
tccggaatgg 3600accatcagag atttcacagt gaaaaatatc cagacaagtg
agagtcaccc tctgagacag 3660ttccatttca cctcctggcc agaccacggt
gttcccgaca ccactgacct gctcatcaac 3720ttccggtacc tcgttcgtga
ctacatgaag cagagtcctc ccgaatcgcc gattctggtg 3780cattgcagtg
ctggggtcgg aaggacgggc actttcattg ccattgatcg tctcatctac
3840cagatagaga atgagaacac cgtggatgtg tatgggattg tgtatgacct
tcgaatgcat 3900aggcctttaa tggtgcagac agaggaccag tatgttttcc
tcaatcagtg tgttttggat 3960attgtcagat cccagaaaga ctcaaaagta
gatcttatct accagaacac aactgcaatg 4020acaatctatg aaaaccttgc
gcccgtgacc acatttggaa agaccaatgg ttacatcgcc 4080agcggtagct aa
4092411341PRTArtificial SequenceSingle-chain variable fragment
(scFv) SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa-dCD148 41Met
Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10
15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser
Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr
Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly145 150 155 160Leu
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly 165 170
175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg
180 185 190Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr
Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
Lys Asp Asn Ser 210 215 220Lys Ser Gln Val Phe Leu Lys Met Asn Ser
Leu Gln Thr Asp Asp Thr225 230 235 240Ala Ile Tyr Tyr Cys Ala Lys
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255Met Asp Tyr Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265 270Thr Thr Thr
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280 285Ser
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 290 295
300Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Phe
Trp305 310 315 320Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr
Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile Ile Phe Trp Val Arg
Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp Ala Pro Ala Tyr Gln
Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu Leu Asn Leu Gly Arg
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380Arg Gly Arg Asp
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro385 390 395 400Gln
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 405 410
415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp 435 440 445Ala Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala
Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr Cys Gly Asp Val Glu Glu
Asn Pro Gly Pro Met Glu465 470 475 480Thr Asp Thr Leu Leu Leu Trp
Val Leu Leu Leu Trp Val Pro Gly Ser 485 490 495Thr Gly Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 500 505 510Ser Gln Thr
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ala 515 520 525Ser
Tyr Asn Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu 530 535
540Trp Leu Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser
Ala545 550 555 560Leu Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser
Lys Asn Gln Val 565 570 575Phe Leu Lys Met Ser Ser Leu Thr Ala Ala
Asp Thr Ala Val Tyr Tyr 580 585 590Cys Ala Lys Arg Ser Asp Asp Tyr
Ser Trp Phe Ala Tyr Trp Gly Gln 595 600 605Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 610 615 620Gly Ser Gly Gly
Gly Gly Ser Glu Asn Gln Met Thr Gln Ser Pro Ser625 630 635 640Ser
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala 645 650
655Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Ser
660 665 670Gly Lys Ala Pro Lys Val Trp Ile Tyr Ser Thr Ser Asn Leu
Ala Ser 675 680 685Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Tyr Thr 690 695 700Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys705 710 715 720Gln Gln Tyr Ser Gly Tyr Pro
Ile Thr Phe Gly Gln Gly Thr Lys Val 725 730 735Glu Ile Lys Arg Ser
Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr 740 745 750His Thr Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 755 760 765Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr 770 775
780Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu785 790 795 800Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 805 810 815Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser 820 825 830Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 835 840 845Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 850 855 860Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro865 870 875 880Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 885 890
895Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
900 905 910Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser 915 920 925Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 930 935 940Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu945 950 955 960His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys Lys 965 970 975Asp Pro Lys Ala Val
Phe Gly Cys Ile Phe Gly Ala Leu Val Ile Val 980 985 990Thr Val Gly
Gly Phe Ile Phe Trp Arg Lys Lys Arg Lys Asp Ala Lys 995 1000
1005Asn Asn Glu Val Ser Phe Ser Gln Ile Lys Pro Lys Lys Ser Lys
1010 1015 1020Leu Ile Arg Val Glu Asn Phe Glu Ala Tyr Phe Lys Lys
Gln Gln 1025 1030 1035Ala Asp Ser Asn Cys Gly Phe Ala Glu Glu Tyr
Glu Asp Leu Lys 1040 1045 1050Leu Val Gly Ile Ser Gln Pro Lys Tyr
Ala Ala Glu Leu Ala Glu 1055 1060 1065Asn Arg Gly Lys Asn Arg Tyr
Asn Asn Val Leu Pro Tyr Asp Ile 1070 1075 1080Ser Arg Val Lys Leu
Ser Val Gln Thr His Ser Thr Asp Asp Tyr 1085 1090 1095Ile Asn Ala
Asn Tyr Met Pro Gly Tyr His Ser Lys Lys Asp Phe 1100 1105 1110Ile
Ala Thr Gln Gly Pro Leu Pro Asn Thr Leu Lys Asp Phe Trp 1115 1120
1125Arg Met Val Trp Glu Lys Asn Val Tyr Ala Ile Ile Met Leu Thr
1130 1135 1140Lys Cys Val Glu Gln Gly Arg Thr Lys Cys Glu Glu Tyr
Trp Pro 1145 1150 1155Ser Lys Gln Ala Gln Asp Tyr Gly Asp Ile Thr
Val Ala Met Thr 1160 1165 1170Ser Glu Ile Val Leu Pro Glu Trp Thr
Ile Arg Asp Phe Thr Val 1175 1180 1185Lys Asn Ile Gln Thr Ser Glu
Ser His Pro Leu Arg Gln Phe His 1190 1195 1200Phe Thr Ser Trp Pro
Asp His Gly Val Pro Asp Thr Thr Asp Leu 1205 1210 1215Leu Ile Asn
Phe Arg Tyr Leu Val Arg Asp Tyr Met Lys Gln Ser 1220 1225 1230Pro
Pro Glu Ser Pro Ile Leu Val His Cys Ser Ala Gly Val Gly 1235 1240
1245Arg Thr Gly Thr Phe Ile Ala Ile Asp Arg Leu Ile Tyr Gln Ile
1250 1255 1260Glu Asn Glu Asn Thr Val Asp Val Tyr Gly Ile Val Tyr
Asp Leu 1265 1270 1275Arg Met His Arg Pro Leu Met Val Gln Thr Glu
Asp Gln Tyr Val 1280 1285 1290Phe Leu Asn Gln Cys Val Leu Asp Ile
Val Arg Ser Gln Lys Asp 1295 1300 1305Ser Lys Val Asp Leu Ile Tyr
Gln Asn Thr Thr Ala Met Thr Ile 1310 1315 1320Tyr Glu Asn Leu Ala
Pro Val Thr Thr Phe Gly Lys Thr Asn Gly 1325 1330 1335Tyr Ile Ala
1340424026DNAArtificial SequenceSingle-chain variable fragment
(scFv) SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa-dCD148
42atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccaga
60ccagacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgaccgggtg
120accatcagct gcagagccag ccaggacatc agcaagtacc tgaactggta
ccagcagaag 180cccgacggca ccgtgaagct gctgatctac cacaccagcc
ggctgcacag cggcgtgccc 240agccggttca gcggcagcgg cagcggcacc
gactacagcc tgaccatcag caacctggag 300caggaggaca tcgccaccta
cttctgccag cagggcaaca ccctgcccta caccttcgga 360ggcggcacca
agctggagat caccaaggcc ggaggcggag gctctggcgg aggcggctct
420ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc tgcaggagtc
tggcccaggc 480ctggtggccc caagccagag cctgagcgtg acctgcaccg
tgagcggcgt gagcctgccc 540gactacggcg tgagctggat caggcagccc
ccacggaagg gcctggagtg gctgggcgtg 600atctggggca gcgagaccac
ctactacaac agcgccctga agagccggct gaccatcatc 660aaggacaaca
gcaagagcca ggtgttcctg aagatgaaca gcctgcagac cgacgacacc
720gccatctact actgcgccaa gcactactac tatggcggca gctacgctat
ggactactgg 780ggccagggca ccagcgtgac cgtgagctca gatcccacca
cgacgccagc gccgcgacca 840ccaacaccgg cgcccaccat cgcgtcgcag
cccctgtccc tgcgcccaga ggcgtgccgg 900ccagcggcgg ggggcgcagt
gcacacgagg gggctggact tcgcctgtga tatcttttgg 960gtgctggtgg
tggttggtgg agtcctggct tgctatagct tgctagtaac agtggccttt
1020attattttct gggtgaggag agtgaagttc agcaggagcg cagacgcccc
cgcgtaccag 1080cagggccaga accagctcta taacgagctc aatctaggac
gaagagagga gtacgatgtt 1140ttggacaaga gacgtggccg ggaccctgag
atggggggaa agccgagaag gaagaaccct 1200caggaaggcc tgtacaatga
actgcagaaa gataagatgg cggaggccta cagtgagatt 1260gggatgaaag
gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt
1320acagccacca aggacaccta cgacgccctt cacatgcagg ccctgcctcc
tcgcagagcc 1380gagggcaggg gaagtcttct aacatgcggg gacgtggagg
aaaatcccgg gcccatggag 1440accgacaccc tgctgctgtg ggtgctgctg
ctgtgggtgc caggcagcac cggccaggtg 1500cagctgcagg agtctggccc
aggcctggtg aagcccagcc agaccctgag catcacctgc 1560accgtgagcg
gcttcagcct ggccagctac aacatccact gggtgcggca gcccccaggc
1620aagggcctgg agtggctggg cgtgatctgg gctggcggca gcaccaacta
caacagcgcc 1680ctgatgagcc ggctgaccat cagcaaggac aacagcaaga
accaggtgtt cctgaagatg 1740agcagcctga cagccgccga caccgccgtg
tactactgcg ccaagcggag cgacgactac 1800agctggttcg cctactgggg
ccagggcacc ctggtgaccg tgagctctgg cggaggcggc 1860tctggcggag
gcggctctgg cggaggcggc agcgagaacc agatgaccca gagccccagc
1920agcttgagcg ccagcgtggg cgaccgggtg accatgacct gcagagccag
cagcagcgtg 1980agcagcagct acctgcactg gtaccagcag aagagcggca
aggccccaaa ggtgtggatc 2040tacagcacca gcaacctggc cagcggcgtg
cccagccggt tcagcggcag cggcagcggc 2100accgactaca ccctgaccat
cagcagcctg cagcccgagg acttcgccac ctactactgc 2160cagcagtaca
gcggctaccc catcaccttc ggccagggca ccaaggtgga gatcaagcgg
2220tcggatcccg ccgagcccaa atctcctgac aaaactcaca catgcccacc
gtgcccagca 2280cctcccgtgg ccggcccgtc agtcttcctc ttccccccaa
aacccaagga caccctcatg 2340atcgcccgga cccctgaggt cacatgcgtg
gtggtggacg tgagccacga agaccctgag 2400gtcaagttca actggtacgt
ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 2460gaggagcagt
acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac
2520tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc
agcccccatc 2580gagaaaacca tctccaaagc caaagggcag ccccgagaac
cacaggtgta caccctgccc 2640ccatcccggg atgagctgac caagaaccag
gtcagcctga cctgcctggt caaaggcttc 2700tatcccagcg acatcgccgt
ggagtgggag agcaatgggc aaccggagaa caactacaag 2760accacgcctc
ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg
2820gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca
tgaggccctg 2880cacaatcact atacccagaa atctctgagt ctgagcccag
gcaagaagga ccccaaggcg 2940gtttttggct gtatctttgg tgccctggtt
attgtgactg tgggaggctt catcttctgg 3000agaaagaaga ggaaagatgc
aaagaataat gaagtgtcct tttctcaaat taaacctaaa 3060aaatctaagt
taatcagagt ggagaatttt gaggcctact tcaagaagca gcaagctgac
3120tccaactgtg ggttcgcaga ggaatacgaa gatctgaagc ttgttggaat
tagtcaacct 3180aaatatgcag cagaactggc tgagaataga ggaaagaatc
gctataataa tgttctgccc 3240tatgatattt cccgtgtcaa actttcggtc
cagacccatt caacggatga ctacatcaat 3300gccaactaca tgcctggcta
ccactccaag aaagatttta ttgccacaca aggaccttta 3360ccgaacactt
tgaaagattt ttggcgtatg gtttgggaga aaaatgtata tgccatcatt
3420atgttgacta aatgtgttga acagggaaga accaaatgtg aggagtattg
gccctccaag 3480caggctcagg actatggaga cataactgtg gcaatgacat
cagaaattgt tcttccggaa 3540tggaccatca gagatttcac agtgaaaaat
atccagacaa gtgagagtca ccctctgaga 3600cagttccatt tcacctcctg
gccagaccac ggtgttcccg acaccactga cctgctcatc 3660aacttccggt
acctcgttcg tgactacatg aagcagagtc ctcccgaatc gccgattctg
3720gtgcattgca gtgctggggt cggaaggacg ggcactttca ttgccattga
tcgtctcatc 3780taccagatag agaatgagaa caccgtggat gtgtatggga
ttgtgtatga ccttcgaatg 3840cataggcctt taatggtgca gacagaggac
cagtatgttt tcctcaatca gtgtgttttg 3900gatattgtca gatcccagaa
agactcaaaa gtagatctta tctaccagaa cacaactgca 3960atgacaatct
atgaaaacct tgcgcccgtg accacatttg gaaagaccaa tggttacatc 4020gcctaa
4026431341PRTArtificial SequenceSingle-chain variable fragment
(scFv) SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa-dCD148 43Met
Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10
15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser
Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr
Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly145 150 155 160Leu
Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly 165 170
175Val Ser Leu Pro Asp Tyr Gly
Val Ser Trp Ile Arg Gln Pro Pro Arg 180 185 190Lys Gly Leu Glu Trp
Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr 195 200 205Tyr Asn Ser
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser 210 215 220Lys
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr225 230
235 240Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr
Ala 245 250 255Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser Asp Pro 260 265 270Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
Ala Pro Thr Ile Ala 275 280 285Ser Gln Pro Leu Ser Leu Arg Pro Glu
Ala Cys Arg Pro Ala Ala Gly 290 295 300Gly Ala Val His Thr Arg Gly
Leu Asp Phe Ala Cys Asp Ile Phe Trp305 310 315 320Val Leu Val Val
Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 325 330 335Thr Val
Ala Phe Ile Ile Phe Trp Val Arg Arg Val Lys Phe Ser Arg 340 345
350Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
355 360 365Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
Lys Arg 370 375 380Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
Arg Lys Asn Pro385 390 395 400Gln Glu Gly Leu Tyr Asn Glu Leu Gln
Lys Asp Lys Met Ala Glu Ala 405 410 415Tyr Ser Glu Ile Gly Met Lys
Gly Glu Arg Arg Arg Gly Lys Gly His 420 425 430Asp Gly Leu Tyr Gln
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 435 440 445Ala Leu His
Met Gln Ala Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly 450 455 460Ser
Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu465 470
475 480Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly
Ser 485 490 495Thr Gly Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile
Leu Lys Pro 500 505 510Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser
Gly Phe Ser Leu Ser 515 520 525Thr Ser Gly Met Gly Val Gly Trp Ile
Arg Gln Pro Ser Gly Lys Gly 530 535 540Leu Glu Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Val Tyr Tyr Asn545 550 555 560Pro Ser Leu Lys
Asn Gln Leu Thr Ile Ser Lys Asp Ala Ser Arg Asp 565 570 575Gln Val
Phe Leu Lys Ile Thr Asn Leu Asp Thr Ala Asp Thr Ala Thr 580 585
590Tyr Tyr Cys Val Arg Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr Trp
595 600 605Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly 610 615 620Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ile Val
Met Thr Gln Ser625 630 635 640His Lys Phe Met Ser Thr Ser Val Gly
Asp Arg Val Ser Ile Ala Cys 645 650 655Lys Ala Ser Gln Asp Val Gly
Thr Ala Val Ala Trp Tyr Gln Gln Lys 660 665 670Pro Gly Gln Ser Pro
Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg His 675 680 685Thr Gly Val
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe 690 695 700Thr
Leu Thr Ile Thr Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe705 710
715 720Cys His Gln Tyr Asn Ser Tyr Asn Thr Phe Gly Ser Gly Thr Arg
Leu 725 730 735Glu Leu Lys Arg Ser Asp Pro Ala Glu Pro Lys Ser Pro
Asp Lys Thr 740 745 750His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser Val 755 760 765Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ala Arg Thr 770 775 780Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu785 790 795 800Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 805 810 815Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 820 825
830Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
835 840 845Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 850 855 860Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro865 870 875 880Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu 885 890 895Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn 900 905 910Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 915 920 925Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 930 935 940Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu945 950
955 960His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
Lys 965 970 975Asp Pro Lys Ala Val Phe Gly Cys Ile Phe Gly Ala Leu
Val Ile Val 980 985 990Thr Val Gly Gly Phe Ile Phe Trp Arg Lys Lys
Arg Lys Asp Ala Lys 995 1000 1005Asn Asn Glu Val Ser Phe Ser Gln
Ile Lys Pro Lys Lys Ser Lys 1010 1015 1020Leu Ile Arg Val Glu Asn
Phe Glu Ala Tyr Phe Lys Lys Gln Gln 1025 1030 1035Ala Asp Ser Asn
Cys Gly Phe Ala Glu Glu Tyr Glu Asp Leu Lys 1040 1045 1050Leu Val
Gly Ile Ser Gln Pro Lys Tyr Ala Ala Glu Leu Ala Glu 1055 1060
1065Asn Arg Gly Lys Asn Arg Tyr Asn Asn Val Leu Pro Tyr Asp Ile
1070 1075 1080Ser Arg Val Lys Leu Ser Val Gln Thr His Ser Thr Asp
Asp Tyr 1085 1090 1095Ile Asn Ala Asn Tyr Met Pro Gly Tyr His Ser
Lys Lys Asp Phe 1100 1105 1110Ile Ala Thr Gln Gly Pro Leu Pro Asn
Thr Leu Lys Asp Phe Trp 1115 1120 1125Arg Met Val Trp Glu Lys Asn
Val Tyr Ala Ile Ile Met Leu Thr 1130 1135 1140Lys Cys Val Glu Gln
Gly Arg Thr Lys Cys Glu Glu Tyr Trp Pro 1145 1150 1155Ser Lys Gln
Ala Gln Asp Tyr Gly Asp Ile Thr Val Ala Met Thr 1160 1165 1170Ser
Glu Ile Val Leu Pro Glu Trp Thr Ile Arg Asp Phe Thr Val 1175 1180
1185Lys Asn Ile Gln Thr Ser Glu Ser His Pro Leu Arg Gln Phe His
1190 1195 1200Phe Thr Ser Trp Pro Asp His Gly Val Pro Asp Thr Thr
Asp Leu 1205 1210 1215Leu Ile Asn Phe Arg Tyr Leu Val Arg Asp Tyr
Met Lys Gln Ser 1220 1225 1230Pro Pro Glu Ser Pro Ile Leu Val His
Cys Ser Ala Gly Val Gly 1235 1240 1245Arg Thr Gly Thr Phe Ile Ala
Ile Asp Arg Leu Ile Tyr Gln Ile 1250 1255 1260Glu Asn Glu Asn Thr
Val Asp Val Tyr Gly Ile Val Tyr Asp Leu 1265 1270 1275Arg Met His
Arg Pro Leu Met Val Gln Thr Glu Asp Gln Tyr Val 1280 1285 1290Phe
Leu Asn Gln Cys Val Leu Asp Ile Val Arg Ser Gln Lys Asp 1295 1300
1305Ser Lys Val Asp Leu Ile Tyr Gln Asn Thr Thr Ala Met Thr Ile
1310 1315 1320Tyr Glu Asn Leu Ala Pro Val Thr Thr Phe Gly Lys Thr
Asn Gly 1325 1330 1335Tyr Ile Ala 1340444026DNAArtificial
SequenceSingle-chain variable fragment (scFv)
SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa-dCD148 44atgagcctgc
ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccaga 60ccagacatcc
agatgaccca gaccaccagc agcctgagcg ccagcctggg cgaccgggtg
120accatcagct gcagagccag ccaggacatc agcaagtacc tgaactggta
ccagcagaag 180cccgacggca ccgtgaagct gctgatctac cacaccagcc
ggctgcacag cggcgtgccc 240agccggttca gcggcagcgg cagcggcacc
gactacagcc tgaccatcag caacctggag 300caggaggaca tcgccaccta
cttctgccag cagggcaaca ccctgcccta caccttcgga 360ggcggcacca
agctggagat caccaaggcc ggaggcggag gctctggcgg aggcggctct
420ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc tgcaggagtc
tggcccaggc 480ctggtggccc caagccagag cctgagcgtg acctgcaccg
tgagcggcgt gagcctgccc 540gactacggcg tgagctggat caggcagccc
ccacggaagg gcctggagtg gctgggcgtg 600atctggggca gcgagaccac
ctactacaac agcgccctga agagccggct gaccatcatc 660aaggacaaca
gcaagagcca ggtgttcctg aagatgaaca gcctgcagac cgacgacacc
720gccatctact actgcgccaa gcactactac tatggcggca gctacgctat
ggactactgg 780ggccagggca ccagcgtgac cgtgagctca gatcccacca
cgacgccagc gccgcgacca 840ccaacaccgg cgcccaccat cgcgtcgcag
cccctgtccc tgcgcccaga ggcgtgccgg 900ccagcggcgg ggggcgcagt
gcacacgagg gggctggact tcgcctgtga tatcttttgg 960gtgctggtgg
tggttggtgg agtcctggct tgctatagct tgctagtaac agtggccttt
1020attattttct gggtgaggag agtgaagttc agcaggagcg cagacgcccc
cgcgtaccag 1080cagggccaga accagctcta taacgagctc aatctaggac
gaagagagga gtacgatgtt 1140ttggacaaga gacgtggccg ggaccctgag
atggggggaa agccgagaag gaagaaccct 1200caggaaggcc tgtacaatga
actgcagaaa gataagatgg cggaggccta cagtgagatt 1260gggatgaaag
gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt
1320acagccacca aggacaccta cgacgccctt cacatgcagg ccctgcctcc
tcgcagagcc 1380gagggcaggg gaagtcttct aacatgcggg gacgtggagg
aaaatcccgg gcccatggag 1440accgacaccc tgctgctgtg ggtgctgctg
ctgtgggtgc ccggcagcac cggccaggtg 1500accctgaagg agagcggtcc
cggcatcctg aagcccagcc agaccctgag cctgacctgc 1560agcttcagcg
gcttcagcct gagcaccagc ggcatgggcg tgggctggat tcggcagccc
1620agcggcaagg gcctggagtg gctggcccac atctggtggg acgacgacgt
gtactacaac 1680cccagcctga agaaccagct gaccatcagc aaggacgcca
gccgggacca ggtgttcctg 1740aagatcacca acctggacac cgccgacacc
gccacctact actgcgtgcg gcgccgggcc 1800accggcaccg gcttcgacta
ctggggccag ggcaccaccc tgaccgtgag cagcggtggc 1860ggtggcagcg
gcggcggcgg aagcggaggt ggtggcagca acatcgtgat gacccagagc
1920cacaagttca tgagcaccag cgtgggcgac cgggtgagca tcgcctgcaa
ggccagccag 1980gacgtgggca ccgccgtggc ctggtaccag cagaagcctg
gccagagccc caagctgctg 2040atctactgga ccagcacccg gcacaccggc
gtgcccgacc ggttcaccgg cagcggcagc 2100ggcaccgact tcaccctgac
catcaccaac gtgcagagcg aggacctggc cgactacttc 2160tgccaccagt
acaacagcta caacaccttc ggcagcggca cccggctgga gctgaagcgg
2220tcggatcccg ccgagcccaa atctcctgac aaaactcaca catgcccacc
gtgcccagca 2280cctcccgtgg ccggcccgtc agtcttcctc ttccccccaa
aacccaagga caccctcatg 2340atcgcccgga cccctgaggt cacatgcgtg
gtggtggacg tgagccacga agaccctgag 2400gtcaagttca actggtacgt
ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 2460gaggagcagt
acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac
2520tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc
agcccccatc 2580gagaaaacca tctccaaagc caaagggcag ccccgagaac
cacaggtgta caccctgccc 2640ccatcccggg atgagctgac caagaaccag
gtcagcctga cctgcctggt caaaggcttc 2700tatcccagcg acatcgccgt
ggagtgggag agcaatgggc aaccggagaa caactacaag 2760accacgcctc
ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg
2820gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca
tgaggccctg 2880cacaatcact atacccagaa atctctgagt ctgagcccag
gcaagaagga ccccaaggcg 2940gtttttggct gtatctttgg tgccctggtt
attgtgactg tgggaggctt catcttctgg 3000agaaagaaga ggaaagatgc
aaagaataat gaagtgtcct tttctcaaat taaacctaaa 3060aaatctaagt
taatcagagt ggagaatttt gaggcctact tcaagaagca gcaagctgac
3120tccaactgtg ggttcgcaga ggaatacgaa gatctgaagc ttgttggaat
tagtcaacct 3180aaatatgcag cagaactggc tgagaataga ggaaagaatc
gctataataa tgttctgccc 3240tatgatattt cccgtgtcaa actttcggtc
cagacccatt caacggatga ctacatcaat 3300gccaactaca tgcctggcta
ccactccaag aaagatttta ttgccacaca aggaccttta 3360ccgaacactt
tgaaagattt ttggcgtatg gtttgggaga aaaatgtata tgccatcatt
3420atgttgacta aatgtgttga acagggaaga accaaatgtg aggagtattg
gccctccaag 3480caggctcagg actatggaga cataactgtg gcaatgacat
cagaaattgt tcttccggaa 3540tggaccatca gagatttcac agtgaaaaat
atccagacaa gtgagagtca ccctctgaga 3600cagttccatt tcacctcctg
gccagaccac ggtgttcccg acaccactga cctgctcatc 3660aacttccggt
acctcgttcg tgactacatg aagcagagtc ctcccgaatc gccgattctg
3720gtgcattgca gtgctggggt cggaaggacg ggcactttca ttgccattga
tcgtctcatc 3780taccagatag agaatgagaa caccgtggat gtgtatggga
ttgtgtatga ccttcgaatg 3840cataggcctt taatggtgca gacagaggac
cagtatgttt tcctcaatca gtgtgttttg 3900gatattgtca gatcccagaa
agactcaaaa gtagatctta tctaccagaa cacaactgca 3960atgacaatct
atgaaaacct tgcgcccgtg accacatttg gaaagaccaa tggttacatc 4020gcctaa
4026451342PRTArtificial SequenceSingle-chain variable fragment
(scFv) SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa-dCD148
45Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1
5 10 15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala
Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
Asp Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His
Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp Ile Ala
Thr Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly
Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly145 150 155
160Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly
165 170 175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro
Pro Arg 180 185 190Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
Glu Thr Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr
Ile Ile Lys Asp Asn Ser 210 215 220Lys Ser Gln Val Phe Leu Lys Met
Asn Ser Leu Gln Thr Asp Asp Thr225 230 235 240Ala Ile Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255Met Asp Tyr
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265 270Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280
285Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
Phe Trp305 310 315 320Val Leu Val Val Val Gly Gly Val Leu Ala Cys
Tyr Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile Ile Phe Trp Val
Arg Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp Ala Pro Ala Tyr
Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro385 390 395
400Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
405 410 415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His 420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp 435 440 445Ala Leu His Met Gln Ala Leu Pro Pro Arg
Arg Ala Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr Cys Gly Asp Val
Glu Glu Asn Pro Gly Pro Met Glu465 470 475 480Thr Asp Thr Leu Leu
Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser 485 490 495Thr Gly Gln
Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro 500 505 510Gly
Ala Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Arg 515 520
525Lys Phe Gly Met Ser Trp Val Arg Gln Thr Ser Asp Lys Arg Leu Glu
530 535 540Trp Val Ala Ser Ile Ser Thr Gly Gly Tyr Asn Thr Tyr Tyr
Ser Asp545 550 555 560Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Glu
Asn Ala Lys Asn Thr 565 570 575Leu Tyr Leu Gln Met Ser Ser Leu Lys
Ser Glu Asp Thr Ala Leu Tyr 580 585 590Tyr Cys Thr Arg Gly Tyr Ser
Ser Thr Ser Tyr Ala Met Asp Tyr Trp 595
600 605Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly 610 615 620Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu
Thr Gln Ser625 630 635 640Pro Ala Ser Leu Ser Val Ala Thr Gly Glu
Lys Val Thr Ile Arg Cys 645 650 655Met Thr Ser Thr Asp Ile Asp Asp
Asp Met Asn Trp Tyr Gln Gln Lys 660 665 670Pro Gly Glu Pro Pro Lys
Phe Leu Ile Ser Glu Gly Asn Thr Leu Arg 675 680 685Pro Gly Val Pro
Ser Arg Phe Ser Ser Ser Gly Thr Gly Thr Asp Phe 690 695 700Val Phe
Thr Ile Glu Asn Thr Leu Ser Glu Asp Val Gly Asp Tyr Tyr705 710 715
720Cys Leu Gln Ser Phe Asn Val Pro Leu Thr Phe Gly Asp Gly Thr Lys
725 730 735Leu Glu Ile Lys Arg Ser Asp Pro Ala Glu Pro Lys Ser Pro
Asp Lys 740 745 750Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser 755 760 765Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ala Arg 770 775 780Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro785 790 795 800Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 805 810 815Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 820 825 830Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 835 840
845Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
850 855 860Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu865 870 875 880Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 885 890 895Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 900 905 910Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 915 920 925Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 930 935 940Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala945 950 955
960Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970 975Lys Asp Pro Lys Ala Val Phe Gly Cys Ile Phe Gly Ala Leu
Val Ile 980 985 990Val Thr Val Gly Gly Phe Ile Phe Trp Arg Lys Lys
Arg Lys Asp Ala 995 1000 1005Lys Asn Asn Glu Val Ser Phe Ser Gln
Ile Lys Pro Lys Lys Ser 1010 1015 1020Lys Leu Ile Arg Val Glu Asn
Phe Glu Ala Tyr Phe Lys Lys Gln 1025 1030 1035Gln Ala Asp Ser Asn
Cys Gly Phe Ala Glu Glu Tyr Glu Asp Leu 1040 1045 1050Lys Leu Val
Gly Ile Ser Gln Pro Lys Tyr Ala Ala Glu Leu Ala 1055 1060 1065Glu
Asn Arg Gly Lys Asn Arg Tyr Asn Asn Val Leu Pro Tyr Asp 1070 1075
1080Ile Ser Arg Val Lys Leu Ser Val Gln Thr His Ser Thr Asp Asp
1085 1090 1095Tyr Ile Asn Ala Asn Tyr Met Pro Gly Tyr His Ser Lys
Lys Asp 1100 1105 1110Phe Ile Ala Thr Gln Gly Pro Leu Pro Asn Thr
Leu Lys Asp Phe 1115 1120 1125Trp Arg Met Val Trp Glu Lys Asn Val
Tyr Ala Ile Ile Met Leu 1130 1135 1140Thr Lys Cys Val Glu Gln Gly
Arg Thr Lys Cys Glu Glu Tyr Trp 1145 1150 1155Pro Ser Lys Gln Ala
Gln Asp Tyr Gly Asp Ile Thr Val Ala Met 1160 1165 1170Thr Ser Glu
Ile Val Leu Pro Glu Trp Thr Ile Arg Asp Phe Thr 1175 1180 1185Val
Lys Asn Ile Gln Thr Ser Glu Ser His Pro Leu Arg Gln Phe 1190 1195
1200His Phe Thr Ser Trp Pro Asp His Gly Val Pro Asp Thr Thr Asp
1205 1210 1215Leu Leu Ile Asn Phe Arg Tyr Leu Val Arg Asp Tyr Met
Lys Gln 1220 1225 1230Ser Pro Pro Glu Ser Pro Ile Leu Val His Cys
Ser Ala Gly Val 1235 1240 1245Gly Arg Thr Gly Thr Phe Ile Ala Ile
Asp Arg Leu Ile Tyr Gln 1250 1255 1260Ile Glu Asn Glu Asn Thr Val
Asp Val Tyr Gly Ile Val Tyr Asp 1265 1270 1275Leu Arg Met His Arg
Pro Leu Met Val Gln Thr Glu Asp Gln Tyr 1280 1285 1290Val Phe Leu
Asn Gln Cys Val Leu Asp Ile Val Arg Ser Gln Lys 1295 1300 1305Asp
Ser Lys Val Asp Leu Ile Tyr Gln Asn Thr Thr Ala Met Thr 1310 1315
1320Ile Tyr Glu Asn Leu Ala Pro Val Thr Thr Phe Gly Lys Thr Asn
1325 1330 1335Gly Tyr Ile Ala 1340464029DNAArtificial
SequenceSingle-chain variable fragment (scFv)
SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa-dCD148
46atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccaga
60ccagacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgaccgggtg
120accatcagct gcagagccag ccaggacatc agcaagtacc tgaactggta
ccagcagaag 180cccgacggca ccgtgaagct gctgatctac cacaccagcc
ggctgcacag cggcgtgccc 240agccggttca gcggcagcgg cagcggcacc
gactacagcc tgaccatcag caacctggag 300caggaggaca tcgccaccta
cttctgccag cagggcaaca ccctgcccta caccttcgga 360ggcggcacca
agctggagat caccaaggcc ggaggcggag gctctggcgg aggcggctct
420ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc tgcaggagtc
tggcccaggc 480ctggtggccc caagccagag cctgagcgtg acctgcaccg
tgagcggcgt gagcctgccc 540gactacggcg tgagctggat caggcagccc
ccacggaagg gcctggagtg gctgggcgtg 600atctggggca gcgagaccac
ctactacaac agcgccctga agagccggct gaccatcatc 660aaggacaaca
gcaagagcca ggtgttcctg aagatgaaca gcctgcagac cgacgacacc
720gccatctact actgcgccaa gcactactac tatggcggca gctacgctat
ggactactgg 780ggccagggca ccagcgtgac cgtgagctca gatcccacca
cgacgccagc gccgcgacca 840ccaacaccgg cgcccaccat cgcgtcgcag
cccctgtccc tgcgcccaga ggcgtgccgg 900ccagcggcgg ggggcgcagt
gcacacgagg gggctggact tcgcctgtga tatcttttgg 960gtgctggtgg
tggttggtgg agtcctggct tgctatagct tgctagtaac agtggccttt
1020attattttct gggtgaggag agtgaagttc agcaggagcg cagacgcccc
cgcgtaccag 1080cagggccaga accagctcta taacgagctc aatctaggac
gaagagagga gtacgatgtt 1140ttggacaaga gacgtggccg ggaccctgag
atggggggaa agccgagaag gaagaaccct 1200caggaaggcc tgtacaatga
actgcagaaa gataagatgg cggaggccta cagtgagatt 1260gggatgaaag
gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt
1320acagccacca aggacaccta cgacgccctt cacatgcagg ccctgcctcc
tcgcagagcc 1380gagggcaggg gaagtcttct aacatgcggg gacgtggagg
aaaatcccgg gcccatggag 1440accgacaccc tgctgctgtg ggtgctgctg
ctgtgggtgc ccggcagcac cggccaggtg 1500aagctgcagc agagcggcgg
aggcctggtg aagcccggcg ccagcctgaa gctgagctgc 1560gtgaccagcg
gcttcacctt ccggaagttc ggcatgagct gggtgcggca gaccagcgac
1620aagcggctgg agtgggtggc cagcatcagc accggcggct acaacaccta
ctacagcgac 1680aacgtgaagg gccggttcac catcagccgg gagaacgcca
agaacaccct gtacctgcag 1740atgagcagcc tgaagagcga ggacaccgcc
ctgtactact gcacccgggg ctacagcagc 1800accagctacg ctatggacta
ctggggccag ggcaccaccg tgacagtgag cagcggcgga 1860ggaggcagtg
gtgggggtgg atctggcgga ggtggcagcg acatcgagct gacccagagc
1920cccgccagcc tgagcgtggc caccggcgag aaggtgacca tccggtgcat
gaccagcacc 1980gacatcgacg acgacatgaa ctggtaccag cagaagcccg
gcgagccccc aaagttcctg 2040atcagcgagg gcaacaccct gcggcccggc
gtgcccagcc ggttcagcag cagcggcacc 2100ggcaccgact tcgtgttcac
catcgagaac accctgagcg aggacgtggg cgactactac 2160tgcctgcaga
gcttcaacgt gcccctgacc ttcggcgacg gcaccaagct ggagatcaag
2220cggtcggatc ccgccgagcc caaatctcct gacaaaactc acacatgccc
accgtgccca 2280gcacctcccg tggccggccc gtcagtcttc ctcttccccc
caaaacccaa ggacaccctc 2340atgatcgccc ggacccctga ggtcacatgc
gtggtggtgg acgtgagcca cgaagaccct 2400gaggtcaagt tcaactggta
cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 2460cgggaggagc
agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag
2520gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct
cccagccccc 2580atcgagaaaa ccatctccaa agccaaaggg cagccccgag
aaccacaggt gtacaccctg 2640cccccatccc gggatgagct gaccaagaac
caggtcagcc tgacctgcct ggtcaaaggc 2700ttctatccca gcgacatcgc
cgtggagtgg gagagcaatg ggcaaccgga gaacaactac 2760aagaccacgc
ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc
2820gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat
gcatgaggcc 2880ctgcacaatc actataccca gaaatctctg agtctgagcc
caggcaagaa ggaccccaag 2940gcggtttttg gctgtatctt tggtgccctg
gttattgtga ctgtgggagg cttcatcttc 3000tggagaaaga agaggaaaga
tgcaaagaat aatgaagtgt ccttttctca aattaaacct 3060aaaaaatcta
agttaatcag agtggagaat tttgaggcct acttcaagaa gcagcaagct
3120gactccaact gtgggttcgc agaggaatac gaagatctga agcttgttgg
aattagtcaa 3180cctaaatatg cagcagaact ggctgagaat agaggaaaga
atcgctataa taatgttctg 3240ccctatgata tttcccgtgt caaactttcg
gtccagaccc attcaacgga tgactacatc 3300aatgccaact acatgcctgg
ctaccactcc aagaaagatt ttattgccac acaaggacct 3360ttaccgaaca
ctttgaaaga tttttggcgt atggtttggg agaaaaatgt atatgccatc
3420attatgttga ctaaatgtgt tgaacaggga agaaccaaat gtgaggagta
ttggccctcc 3480aagcaggctc aggactatgg agacataact gtggcaatga
catcagaaat tgttcttccg 3540gaatggacca tcagagattt cacagtgaaa
aatatccaga caagtgagag tcaccctctg 3600agacagttcc atttcacctc
ctggccagac cacggtgttc ccgacaccac tgacctgctc 3660atcaacttcc
ggtacctcgt tcgtgactac atgaagcaga gtcctcccga atcgccgatt
3720ctggtgcatt gcagtgctgg ggtcggaagg acgggcactt tcattgccat
tgatcgtctc 3780atctaccaga tagagaatga gaacaccgtg gatgtgtatg
ggattgtgta tgaccttcga 3840atgcataggc ctttaatggt gcagacagag
gaccagtatg ttttcctcaa tcagtgtgtt 3900ttggatattg tcagatccca
gaaagactca aaagtagatc ttatctacca gaacacaact 3960gcaatgacaa
tctatgaaaa ccttgcgccc gtgaccacat ttggaaagac caatggttac
4020atcgcctaa 4029476PRTArtificial SequenceImmunoreceptor
tyrosine-based inhibition motif (ITIM)MISC_FEATURE(1)..(1)Xaa may
be Ser, Ile, Val or Leumisc_feature(2)..(2)Xaa can be any naturally
occurring amino acidmisc_feature(4)..(5)Xaa can be any naturally
occurring amino acidMISC_FEATURE(6)..(6)Xaa may be Ile, Val or Leu
47Xaa Xaa Tyr Xaa Xaa Xaa1 5481114PRTArtificial SequenceAmino acid
sequence of a AND NOT gate
(16076.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-tm-dPTP
N6) 48Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu
Leu1 5 10 15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser
Ser Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Asp Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu
His Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp Ile
Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly
Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly145 150 155
160Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly
165 170 175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro
Pro Arg 180 185 190Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
Glu Thr Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr
Ile Ile Lys Asp Asn Ser 210 215 220Lys Ser Gln Val Phe Leu Lys Met
Asn Ser Leu Gln Thr Asp Asp Thr225 230 235 240Ala Ile Tyr Tyr Cys
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255Met Asp Tyr
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265 270Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 275 280
285Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
290 295 300Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
Phe Trp305 310 315 320Val Leu Val Val Val Gly Gly Val Leu Ala Cys
Tyr Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile Ile Phe Trp Val
Arg Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp Ala Pro Ala Tyr
Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro385 390 395
400Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
405 410 415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His 420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp 435 440 445Ala Leu His Met Gln Ala Leu Pro Pro Arg
Arg Ala Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr Cys Gly Asp Val
Glu Glu Asn Pro Gly Pro Met Ala465 470 475 480Val Pro Thr Gln Val
Leu Gly Leu Leu Leu Leu Trp Leu Thr Asp Ala 485 490 495Arg Cys Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 500 505 510Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr 515 520
525Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
530 535 540Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp Gly Val Pro Ser
Arg Phe545 550 555 560Ser Gly Ser Gly Ser Gly Thr Gln Tyr Thr Leu
Thr Ile Ser Ser Leu 565 570 575Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln His Tyr Lys Asn Tyr 580 585 590Pro Leu Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg Ser Gly 595 600 605Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615 620Gly Gly Ser
Arg Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu625 630 635
640Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
645 650 655Thr Leu Ser Asn Tyr Gly Met His Trp Ile Arg Gln Ala Pro
Gly Lys 660 665 670Gly Leu Glu Trp Val Ser Ser Ile Ser Leu Asn Gly
Gly Ser Thr Tyr 675 680 685Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala 690 695 700Lys Ser Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr705 710 715 720Ala Val Tyr Tyr Cys
Ala Ala Gln Asp Ala Tyr Thr Gly Gly Tyr Phe 725 730 735Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Met Asp Pro 740 745 750Ala
Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro 755 760
765Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly
770 775 780Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr
Trp Ala785 790 795 800Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu
Ser Leu Ile Ile Thr 805 810 815Leu Ile Cys Tyr His Arg Ser Arg Lys
Arg Val Cys Lys Ser Gly Gly 820 825 830Gly Ser Phe Trp Glu Glu Phe
Glu Ser Leu Gln Lys Gln Glu Val Lys 835 840 845Asn Leu His Gln Arg
Leu Glu Gly Gln Arg Pro Glu Asn Lys Gly Lys 850 855 860Asn Arg Tyr
Lys Asn Ile Leu Pro Phe Asp His Ser Arg Val Ile Leu865 870 875
880Gln Gly Arg Asp Ser Asn Ile Pro Gly Ser Asp Tyr Ile Asn Ala Asn
885 890 895Tyr Ile Lys Asn Gln Leu Leu Gly Pro Asp Glu Asn Ala Lys
Thr Tyr 900 905 910Ile Ala Ser Gln Gly Cys Leu Glu Ala Thr Val Asn
Asp Phe Trp Gln 915 920 925Met Ala Trp Gln Glu Asn Ser Arg Val Ile
Val Met Thr Thr Arg Glu 930 935 940Val Glu Lys Gly Arg Asn Lys Cys
Val Pro Tyr Trp Pro Glu Val Gly945 950
955 960Met Gln Arg Ala Tyr Gly Pro Tyr Ser Val Thr Asn Cys Gly Glu
His 965 970 975Asp Thr Thr Glu Tyr Lys Leu Arg Thr Leu Gln Val Ser
Pro Leu Asp 980 985 990Asn Gly Asp Leu Ile Arg Glu Ile Trp His Tyr
Gln Tyr Leu Ser Trp 995 1000 1005Pro Asp His Gly Val Pro Ser Glu
Pro Gly Gly Val Leu Ser Phe 1010 1015 1020Leu Asp Gln Ile Asn Gln
Arg Gln Glu Ser Leu Pro His Ala Gly 1025 1030 1035Pro Ile Ile Val
His Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr 1040 1045 1050Ile Ile
Val Ile Asp Met Leu Met Glu Asn Ile Ser Thr Lys Gly 1055 1060
1065Leu Asp Cys Asp Ile Asp Ile Gln Lys Thr Ile Gln Met Val Arg
1070 1075 1080Ala Gln Arg Ser Gly Met Val Gln Thr Glu Ala Gln Tyr
Lys Phe 1085 1090 1095Ile Tyr Val Ala Ile Ala Gln Phe Ile Glu Thr
Thr Lys Lys Lys 1100 1105 1110Leu49918PRTArtificial SequenceAmino
acid sequence of a AND NOT gate
(MP16091.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1
tm-endo) 49Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
Leu Leu1 5 10 15His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr
Ser Ser Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys
Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln
Lys Pro Asp Gly Thr 50 55 60Val Lys Leu Leu Ile Tyr His Thr Ser Arg
Leu His Ser Gly Val Pro65 70 75 80Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn Leu Glu Gln Glu Asp
Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110Asn Thr Leu Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125Lys Ala Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly145 150
155 160Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser
Gly 165 170 175Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln
Pro Pro Arg 180 185 190Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly
Ser Glu Thr Thr Tyr 195 200 205Tyr Asn Ser Ala Leu Lys Ser Arg Leu
Thr Ile Ile Lys Asp Asn Ser 210 215 220Lys Ser Gln Val Phe Leu Lys
Met Asn Ser Leu Gln Thr Asp Asp Thr225 230 235 240Ala Ile Tyr Tyr
Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala 245 250 255Met Asp
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro 260 265
270Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
275 280 285Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
Ala Gly 290 295 300Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
Asp Ile Phe Trp305 310 315 320Val Leu Val Val Val Gly Gly Val Leu
Ala Cys Tyr Ser Leu Leu Val 325 330 335Thr Val Ala Phe Ile Ile Phe
Trp Val Arg Arg Val Lys Phe Ser Arg 340 345 350Ser Ala Asp Ala Pro
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365Glu Leu Asn
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380Arg
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro385 390
395 400Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
Ala 405 410 415Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
Lys Gly His 420 425 430Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
Lys Asp Thr Tyr Asp 435 440 445Ala Leu His Met Gln Ala Leu Pro Pro
Arg Arg Ala Glu Gly Arg Gly 450 455 460Ser Leu Leu Thr Cys Gly Asp
Val Glu Glu Asn Pro Gly Pro Met Ala465 470 475 480Val Pro Thr Gln
Val Leu Gly Leu Leu Leu Leu Trp Leu Thr Asp Ala 485 490 495Arg Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 500 505
510Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr
515 520 525Phe Asn Leu Val Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu 530 535 540Leu Ile Tyr Asp Thr Asn Arg Leu Ala Asp Gly Val
Pro Ser Arg Phe545 550 555 560Ser Gly Ser Gly Ser Gly Thr Gln Tyr
Thr Leu Thr Ile Ser Ser Leu 565 570 575Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln His Tyr Lys Asn Tyr 580 585 590Pro Leu Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg Ser Gly 595 600 605Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 610 615 620Gly
Gly Ser Arg Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu625 630
635 640Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe 645 650 655Thr Leu Ser Asn Tyr Gly Met His Trp Ile Arg Gln Ala
Pro Gly Lys 660 665 670Gly Leu Glu Trp Val Ser Ser Ile Ser Leu Asn
Gly Gly Ser Thr Tyr 675 680 685Tyr Arg Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala 690 695 700Lys Ser Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr705 710 715 720Ala Val Tyr Tyr
Cys Ala Ala Gln Asp Ala Tyr Thr Gly Gly Tyr Phe 725 730 735Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Met Asp Pro 740 745
750Ala Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro
755 760 765Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro
Arg Gly 770 775 780Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp
Ile Leu Ile Gly785 790 795 800Val Ser Val Val Phe Leu Phe Cys Leu
Leu Leu Leu Val Leu Phe Cys 805 810 815Leu His Arg Gln Asn Gln Ile
Lys Gln Gly Pro Pro Arg Ser Lys Asp 820 825 830Glu Glu Gln Lys Pro
Gln Gln Arg Pro Asp Leu Ala Val Asp Val Leu 835 840 845Glu Arg Thr
Ala Asp Lys Ala Thr Val Asn Gly Leu Pro Glu Lys Asp 850 855 860Arg
Glu Thr Asp Thr Ser Ala Leu Ala Ala Gly Ser Ser Gln Glu Val865 870
875 880Thr Tyr Ala Gln Leu Asp His Trp Ala Leu Thr Gln Arg Thr Ala
Arg 885 890 895Ala Val Ser Pro Gln Ser Thr Lys Pro Met Ala Glu Ser
Ile Thr Tyr 900 905 910Ala Ala Val Ala Arg His
915501362PRTArtificial SequenceAmino acid sequence of a AND NOT
gate
(MP16092.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1
tm-endo-2A-PTPN6_SH2-dCD148) 50Met Ser Leu Pro Val Thr Ala Leu Leu
Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Asp Ile Gln
Met Thr Gln Thr Thr Ser Ser Leu 20 25 30Ser Ala Ser Leu Gly Asp Arg
Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45Asp Ile Ser Lys Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60Val Lys Leu Leu Ile
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro65 70 75 80Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95Ser Asn
Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105
110Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
Ser Gly Pro Gly145 150 155 160Leu Val Ala Pro Ser Gln Ser Leu Ser
Val Thr Cys Thr Val Ser Gly 165 170 175Val Ser Leu Pro Asp Tyr Gly
Val Ser Trp Ile Arg Gln Pro Pro Arg 180 185 190Lys Gly Leu Glu Trp
Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr 195 200 205Tyr Asn Ser
Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser 210 215 220Lys
Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr225 230
235 240Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr
Ala 245 250 255Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser Asp Pro 260 265 270Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
Ala Pro Thr Ile Ala 275 280 285Ser Gln Pro Leu Ser Leu Arg Pro Glu
Ala Cys Arg Pro Ala Ala Gly 290 295 300Gly Ala Val His Thr Arg Gly
Leu Asp Phe Ala Cys Asp Ile Phe Trp305 310 315 320Val Leu Val Val
Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 325 330 335Thr Val
Ala Phe Ile Ile Phe Trp Val Arg Arg Val Lys Phe Ser Arg 340 345
350Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
355 360 365Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
Lys Arg 370 375 380Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
Arg Lys Asn Pro385 390 395 400Gln Glu Gly Leu Tyr Asn Glu Leu Gln
Lys Asp Lys Met Ala Glu Ala 405 410 415Tyr Ser Glu Ile Gly Met Lys
Gly Glu Arg Arg Arg Gly Lys Gly His 420 425 430Asp Gly Leu Tyr Gln
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 435 440 445Ala Leu His
Met Gln Ala Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly 450 455 460Ser
Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala465 470
475 480Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr Asp
Ala 485 490 495Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser 500 505 510Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Glu Asp Ile Tyr 515 520 525Phe Asn Leu Val Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu 530 535 540Leu Ile Tyr Asp Thr Asn Arg
Leu Ala Asp Gly Val Pro Ser Arg Phe545 550 555 560Ser Gly Ser Gly
Ser Gly Thr Gln Tyr Thr Leu Thr Ile Ser Ser Leu 565 570 575Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Lys Asn Tyr 580 585
590Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ser Gly
595 600 605Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 610 615 620Gly Gly Ser Arg Ser Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu625 630 635 640Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe 645 650 655Thr Leu Ser Asn Tyr Gly Met
His Trp Ile Arg Gln Ala Pro Gly Lys 660 665 670Gly Leu Glu Trp Val
Ser Ser Ile Ser Leu Asn Gly Gly Ser Thr Tyr 675 680 685Tyr Arg Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 690 695 700Lys
Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr705 710
715 720Ala Val Tyr Tyr Cys Ala Ala Gln Asp Ala Tyr Thr Gly Gly Tyr
Phe 725 730 735Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Met Asp Pro 740 745 750Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser
Pro Val His Pro Thr 755 760 765Gly Thr Ser Gln Pro Gln Arg Pro Glu
Asp Cys Arg Pro Arg Gly Ser 770 775 780Val Lys Gly Thr Gly Leu Asp
Phe Ala Cys Asp Ile Leu Ile Gly Val785 790 795 800Ser Val Val Phe
Leu Phe Cys Leu Leu Leu Leu Val Leu Phe Cys Leu 805 810 815His Arg
Gln Asn Gln Ile Lys Gln Gly Pro Pro Arg Ser Lys Asp Glu 820 825
830Glu Gln Lys Pro Gln Gln Arg Pro Asp Leu Ala Val Asp Val Leu Glu
835 840 845Arg Thr Ala Asp Lys Ala Thr Val Asn Gly Leu Pro Glu Lys
Asp Arg 850 855 860Glu Thr Asp Thr Ser Ala Leu Ala Ala Gly Ser Ser
Gln Glu Val Thr865 870 875 880Tyr Ala Gln Leu Asp His Trp Ala Leu
Thr Gln Arg Thr Ala Arg Ala 885 890 895Val Ser Pro Gln Ser Thr Lys
Pro Met Ala Glu Ser Ile Thr Tyr Ala 900 905 910Ala Val Ala Arg His
Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys 915 920 925Gly Asp Val
Glu Glu Asn Pro Gly Pro Trp Tyr His Gly His Met Ser 930 935 940Gly
Gly Gln Ala Glu Thr Leu Leu Gln Ala Lys Gly Glu Pro Trp Thr945 950
955 960Phe Leu Val Arg Glu Ser Leu Ser Gln Pro Gly Asp Phe Val Leu
Ser 965 970 975Val Leu Ser Asp Gln Pro Lys Ala Gly Pro Gly Ser Pro
Leu Arg Val 980 985 990Thr His Ile Lys Val Met Cys Glu Gly Gly Arg
Tyr Thr Val Gly Gly 995 1000 1005Leu Glu Thr Phe Asp Ser Leu Thr
Asp Leu Val Glu His Phe Lys 1010 1015 1020Lys Thr Gly Ile Glu Glu
Ala Ser Gly Ala Phe Val Tyr Leu Arg 1025 1030 1035Gln Pro Tyr Ser
Gly Gly Gly Gly Ser Phe Glu Ala Tyr Phe Lys 1040 1045 1050Lys Gln
Gln Ala Asp Ser Asn Cys Gly Phe Ala Glu Glu Tyr Glu 1055 1060
1065Asp Leu Lys Leu Val Gly Ile Ser Gln Pro Lys Tyr Ala Ala Glu
1070 1075 1080Leu Ala Glu Asn Arg Gly Lys Asn Arg Tyr Asn Asn Val
Leu Pro 1085 1090 1095Tyr Asp Ile Ser Arg Val Lys Leu Ser Val Gln
Thr His Ser Thr 1100 1105 1110Asp Asp Tyr Ile Asn Ala Asn Tyr Met
Pro Gly Tyr His Ser Lys 1115 1120 1125Lys Asp Phe Ile Ala Thr Gln
Gly Pro Leu Pro Asn Thr Leu Lys 1130 1135 1140Asp Phe Trp Arg Met
Val Trp Glu Lys Asn Val Tyr Ala Ile Ile 1145 1150 1155Met Leu Thr
Lys Cys Val Glu Gln Gly Arg Thr Lys Cys Glu Glu 1160 1165 1170Tyr
Trp Pro Ser Lys Gln Ala Gln Asp Tyr Gly Asp Ile Thr Val 1175 1180
1185Ala Met Thr Ser Glu Ile Val Leu Pro Glu Trp Thr Ile Arg Asp
1190 1195 1200Phe Thr Val Lys Asn Ile Gln Thr Ser Glu Ser His Pro
Leu Arg 1205 1210 1215Gln Phe His Phe Thr Ser Trp Pro Asp His Gly
Val Pro Asp Thr 1220 1225 1230Thr Asp Leu Leu Ile Asn Phe Arg Tyr
Leu Val Arg Asp Tyr Met 1235 1240 1245Lys Gln Ser Pro Pro Glu Ser
Pro Ile Leu Val His Cys Ser Ala 1250 1255 1260Gly Val Gly Arg Thr
Gly Thr Phe Ile Ala Ile Asp Arg Leu Ile 1265 1270 1275Tyr Gln Ile
Glu Asn Glu Asn Thr Val Asp Val Tyr Gly Ile Val 1280 1285 1290Tyr
Asp Leu Arg Met His Arg Pro Leu Met Val Gln Thr Glu Asp 1295 1300
1305Gln Tyr Val Phe Leu Asn Gln Cys Val Leu Asp Ile Val Arg Ser
1310 1315 1320Gln Lys Asp Ser Lys Val Asp Leu Ile Tyr Gln Asn Thr
Thr Ala 1325 1330 1335Met Thr Ile Tyr
Glu Asn Leu Ala Pro Val Thr Thr Phe Gly Lys 1340 1345 1350Thr Asn
Gly Tyr Ile Ala Ser Gly Ser 1355 136051424PRTArtificial
SequenceAPRIL-based (A proliferation-inducing ligand- based) CAR,
CD8 stalk APRIL CAR 51Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Ser Val Leu His Leu Val
Pro Ile Asn Ala Thr Ser 20 25 30Lys Asp Asp Ser Asp Val Thr Glu Val
Met Trp Gln Pro Ala Leu Arg 35 40 45Arg Gly Arg Gly Leu Gln Ala Gln
Gly Tyr Gly Val Arg Ile Gln Asp 50 55 60Ala Gly Val Tyr Leu Leu Tyr
Ser Gln Val Leu Phe Gln Asp Val Thr65 70 75 80Phe Thr Met Gly Gln
Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu 85 90 95Thr Leu Phe Arg
Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala 100 105 110Tyr Asn
Ser Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp 115 120
125Ile Leu Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser
130 135 140Pro His Gly Thr Phe Leu Gly Phe Val Lys Leu Ser Gly Gly
Gly Ser145 150 155 160Asp Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro
Thr Pro Ala Pro Thr 165 170 175Ile Ala Ser Gln Pro Leu Ser Leu Arg
Pro Glu Ala Cys Arg Pro Ala 180 185 190Ala Gly Gly Ala Val His Thr
Arg Gly Leu Asp Phe Ala Cys Asp Ile 195 200 205Phe Trp Val Leu Val
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 210 215 220Leu Val Thr
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser225 230 235
240Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
245 250 255Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
Phe Ala 260 265 270Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp
Ala His Lys Pro 275 280 285Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile
Gln Glu Glu Gln Ala Asp 290 295 300Ala His Ser Thr Leu Ala Lys Ile
Arg Val Lys Phe Ser Arg Ser Ala305 310 315 320Asp Ala Pro Ala Tyr
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 325 330 335Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 340 345 350Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 355 360
365Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
370 375 380Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
Asp Gly385 390 395 400Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
Thr Tyr Asp Ala Leu 405 410 415His Met Gln Ala Leu Pro Pro Arg
42052398PRTArtificial SequenceAPRIL-based (A proliferation-inducing
ligand- based) CAR, APRIL IgG1 hinge based CAR 52Met Glu Thr Asp
Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr
Gly Ser Val Leu His Leu Val Pro Ile Asn Ala Thr Ser 20 25 30Lys Asp
Asp Ser Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu Arg 35 40 45Arg
Gly Arg Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp 50 55
60Ala Gly Val Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr65
70 75 80Phe Thr Met Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln
Glu 85 90 95Thr Leu Phe Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp
Arg Ala 100 105 110Tyr Asn Ser Cys Tyr Ser Ala Gly Val Phe His Leu
His Gln Gly Asp 115 120 125Ile Leu Ser Val Ile Ile Pro Arg Ala Arg
Ala Lys Leu Asn Leu Ser 130 135 140Pro His Gly Thr Phe Leu Gly Phe
Val Lys Leu Ser Gly Gly Gly Ser145 150 155 160Asp Pro Ala Glu Pro
Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro 165 170 175Cys Pro Lys
Asp Pro Lys Phe Trp Val Leu Val Val Val Gly Gly Val 180 185 190Leu
Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp 195 200
205Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
210 215 220Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
Tyr Ala225 230 235 240Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg
Asp Gln Arg Leu Pro 245 250 255Pro Asp Ala His Lys Pro Pro Gly Gly
Gly Ser Phe Arg Thr Pro Ile 260 265 270Gln Glu Glu Gln Ala Asp Ala
His Ser Thr Leu Ala Lys Ile Arg Val 275 280 285Lys Phe Ser Arg Ser
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 290 295 300Gln Leu Tyr
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val305 310 315
320Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
325 330 335Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
Asp Lys 340 345 350Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
Glu Arg Arg Arg 355 360 365Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
Leu Ser Thr Ala Thr Lys 370 375 380Asp Thr Tyr Asp Ala Leu His Met
Gln Ala Leu Pro Pro Arg385 390 39553614PRTArtificial
SequenceAPRIL-based (A proliferation-inducing ligand- based) CAR,
APRIL Fc-pvaa based CAR 53Met Glu Thr Asp Thr Leu Leu Leu Trp Val
Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Ser Val Leu His Leu
Val Pro Ile Asn Ala Thr Ser 20 25 30Lys Asp Asp Ser Asp Val Thr Glu
Val Met Trp Gln Pro Ala Leu Arg 35 40 45Arg Gly Arg Gly Leu Gln Ala
Gln Gly Tyr Gly Val Arg Ile Gln Asp 50 55 60Ala Gly Val Tyr Leu Leu
Tyr Ser Gln Val Leu Phe Gln Asp Val Thr65 70 75 80Phe Thr Met Gly
Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu 85 90 95Thr Leu Phe
Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala 100 105 110Tyr
Asn Ser Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp 115 120
125Ile Leu Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser
130 135 140Pro His Gly Thr Phe Leu Gly Phe Val Lys Leu Ser Gly Gly
Gly Ser145 150 155 160Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr
His Thr Cys Pro Pro 165 170 175Cys Pro Ala Pro Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro 180 185 190Lys Pro Lys Asp Thr Leu Met
Ile Ala Arg Thr Pro Glu Val Thr Cys 195 200 205Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 210 215 220Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu225 230 235
240Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
245 250 255His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 260 265 270Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 275 280 285Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu 290 295 300Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr305 310 315 320Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 325 330 335Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 340 345 350Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 355 360
365Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
370 375 380Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Pro Lys
Phe Trp385 390 395 400Val Leu Val Val Val Gly Gly Val Leu Ala Cys
Tyr Ser Leu Leu Val 405 410 415Thr Val Ala Phe Ile Ile Phe Trp Val
Arg Ser Lys Arg Ser Arg Leu 420 425 430Leu His Ser Asp Tyr Met Asn
Met Thr Pro Arg Arg Pro Gly Pro Thr 435 440 445Arg Lys His Tyr Gln
Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr 450 455 460Arg Ser Arg
Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly465 470 475
480Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His
485 490 495Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala
Asp Ala 500 505 510Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
Glu Leu Asn Leu 515 520 525Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
Lys Arg Arg Gly Arg Asp 530 535 540Pro Glu Met Gly Gly Lys Pro Arg
Arg Lys Asn Pro Gln Glu Gly Leu545 550 555 560Tyr Asn Glu Leu Gln
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 565 570 575Gly Met Lys
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 580 585 590Gln
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 595 600
605Gln Ala Leu Pro Pro Arg 610
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References