U.S. patent application number 16/879636 was filed with the patent office on 2020-11-26 for engineered expression of cell surface and secreted sialidase by car t cells for increased efficacy in solid tumors.
The applicant listed for this patent is The Trustees of the University of Pennsylvania. Invention is credited to Tiffany King, Avery D. Posey.
Application Number | 20200370013 16/879636 |
Document ID | / |
Family ID | 1000004860016 |
Filed Date | 2020-11-26 |
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United States Patent
Application |
20200370013 |
Kind Code |
A1 |
Posey; Avery D. ; et
al. |
November 26, 2020 |
Engineered Expression of Cell Surface and Secreted Sialidase by CAR
T Cells for Increased Efficacy in Solid Tumors
Abstract
The present disclosure provides modified immune cells or
precursors thereof (e.g. modified T cells) comprising a chimeric
cell surface sialidase or a variant sialidase precursor protein.
Compositions and methods of treatment are also provided.
Inventors: |
Posey; Avery D.;
(Philadelphia, PA) ; King; Tiffany; (Philadelphia,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania |
Philadelphia |
PA |
US |
|
|
Family ID: |
1000004860016 |
Appl. No.: |
16/879636 |
Filed: |
May 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62850014 |
May 20, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/73 20130101;
C07K 2319/33 20130101; C07K 2317/622 20130101; C07K 2319/03
20130101; C07K 16/2803 20130101; C07K 2317/53 20130101; C07K
16/3069 20130101; C07K 2317/24 20130101; C12N 9/2402 20130101; C07K
2317/55 20130101; C07K 14/7051 20130101; C07K 14/7151 20130101;
C07K 14/70507 20130101; A61P 35/00 20180101; C12N 7/00 20130101;
C07K 2317/524 20130101; C12Y 302/01018 20130101; A61K 35/17
20130101; C12N 2740/15043 20130101; C07K 14/70517 20130101; C12N
5/0646 20130101; C12N 5/0636 20130101; C07K 2319/02 20130101; C12N
15/86 20130101; C07K 2317/526 20130101 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783; C12N 9/24 20060101 C12N009/24; C07K 16/28 20060101
C07K016/28; C07K 14/705 20060101 C07K014/705; C07K 14/715 20060101
C07K014/715; C07K 14/725 20060101 C07K014/725; C07K 16/30 20060101
C07K016/30; C12N 15/86 20060101 C12N015/86; C12N 7/00 20060101
C12N007/00; A61K 35/17 20060101 A61K035/17; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
CA214278 awarded by the National Institute of Health and
IK2BX004183 awarded by the Department of Veterans Affairs. The
government has certain rights in the invention.
Claims
1. A modified immune cell or precursor cell thereof, comprising:
(a) a chimeric cell surface sialidase (neuraminidase) enzyme
comprising an extracellular portion comprising a sialidase
(neuraminidase) or an enzymatically functional portion thereof, and
(b) a heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface.
2. A modified immune cell or precursor cell thereof, comprising:
(a) a chimeric cell surface sialidase consisting of the amino acid
sequence set forth in any one of SEQ ID NOs: 10, 37, or 38; and/or
(b) a variant sialidase precursor protein comprising a heterologous
secretory sequence operably linked to a sialidase (neuraminidase)
or an enzymatically functional portion thereof, wherein the variant
sialidase precursor protein lacks a transmembrane domain, and
wherein the sialidase or enzymatically functional portion thereof
is capable of being secreted from an immune or precursor cell
thereof when the variant sialidase precursor protein is expressed
in the cell; and/or (c) a variant sialidase precursor protein
comprising a heterologous secretory sequence operably linked to a
sialidase (neuraminidase) or an enzymatically functional portion
thereof, wherein the sialidase comprises the amino acid sequence
set forth in SEQ ID NO: 34; and/or (d) a chimeric cell surface
sialidase (neuraminidase) enzyme comprising an extracellular
portion comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface;
and a chimeric antigen receptor (CAR) and/or a T cell receptor
(TCR); and/or (e) a variant sialidase precursor protein comprising
a heterologous secretory sequence operably linked to a sialidase
(neuraminidase) or an enzymatically functional portion thereof,
wherein the variant sialidase precursor protein lacks a
transmembrane domain, and wherein the sialidase or enzymatically
functional portion thereof is capable of being secreted from an
immune or precursor cell thereof when the variant sialidase
precursor protein is expressed in the cell; and a chimeric antigen
receptor (CAR) and/or a T cell receptor (TCR); and/or (f) a
chimeric cell surface sialidase (neuraminidase) enzyme comprising
an extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface;
and a chimeric antigen receptor (CAR); and/or (g) a chimeric cell
surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface;
and a chimeric antigen receptor (CAR) having specificity for
TnMUC1, CD19, or PSMA; and/or (h) a chimeric cell surface sialidase
(neuraminidase) enzyme comprising an extracellular portion
comprising Neu2 or an enzymatically functional portion thereof, a
heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface, a hinge domain, and an
intracellular region comprising a costimulatory signaling domain
and an intracellular signaling domain; and a chimeric antigen
receptor (CAR); and/or (i) a chimeric cell surface sialidase
(neuraminidase) enzyme comprising an extracellular portion
comprising Neu2 or an enzymatically functional portion thereof, a
heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface, a hinge domain, and an
intracellular region comprising a costimulatory signaling domain
and an intracellular signaling domain; and a chimeric antigen
receptor (CAR) having specificity for TnMUC1, CD19, or PSMA; and/or
(j) a variant sialidase precursor protein comprising a heterologous
secretory sequence operably linked to a sialidase (neuraminidase)
or an enzymatically functional portion thereof, wherein the variant
sialidase precursor protein lacks a transmembrane domain, and
wherein the sialidase or enzymatically functional portion thereof
is capable of being secreted from an immune or precursor cell
thereof when the variant sialidase precursor protein is expressed
in the cell; and a chimeric antigen receptor (CAR) having
specificity for TnMUC1, CD19, or PSMA.
3. The modified cell of claim 1, wherein: (a) the sialidase is a
human or humanized sialidase; and/or (b) the sialidase is a human
or humanized sialidase and wherein the human sialidase is selected
from the group consisting of Neu1, Neu2, Neu3, and Neu4; and/or (c)
the sialidase is a human or humanized sialidase and wherein the
human sialidase is Neu2; and/or (d) the sialidase comprises the
amino acid sequence set forth in SEQ ID NO: 13; and/or (e) the
sialidase comprises the amino acid sequence set forth in SEQ ID NO:
10; and/or (f) the sialidase consists of the amino acid sequence
set forth in SEQ ID NO: 10; and/or (g) the sialidase comprises the
amino acid sequence set forth in any one of SEQ ID NOs: 10, 37, or
38; and/or (h) the sialidase consists of the amino acid sequence
set forth in any one of SEQ ID NOs: 10, 37, or 38; and/or (i) the
sialidase comprises the amino acid sequence set forth in SEQ ID NO:
34; and/or (j) the sialidase consists of the amino acid sequence
set forth in SEQ ID NO: 34; and/or (k) the transmembrane domain is
selected from the group consisting of an artificial hydrophobic
sequence, and a transmembrane domain of a type I transmembrane
protein, an alpha, beta, or zeta chain of a T cell receptor, CD28,
CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37,
CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154; and/or
(l) the transmembrane domain comprises a transmembrane domain of
CD8; and/or (m) the transmembrane domain comprises a transmembrane
domain of CD8 alpha; and/or (n) the transmembrane domain comprises
the amino acid sequence set forth in SEQ ID NO: 16.
4. The modified cell of claim 2, wherein: (a) the sialidase is a
human or humanized sialidase; and/or (b) the sialidase is a human
or humanized sialidase and wherein the human sialidase is selected
from the group consisting of Neu1, Neu2, Neu3, and Neu4; and/or (c)
the sialidase is a human or humanized sialidase and wherein the
human sialidase is Neu2; and/or (d) the sialidase comprises the
amino acid sequence set forth in SEQ ID NO: 13; and/or (e) the
sialidase comprises the amino acid sequence set forth in SEQ ID NO:
10; and/or (f) the sialidase consists of the amino acid sequence
set forth in SEQ ID NO: 10; and/or (g) the sialidase comprises the
amino acid sequence set forth in any one of SEQ ID NOs: 10, 37, or
38; and/or (h) the sialidase consists of the amino acid sequence
set forth in any one of SEQ ID NOs: 10, 37, or 38; and/or (i) the
sialidase comprises the amino acid sequence set forth in SEQ ID NO:
34; and/or (j) the sialidase consists of the amino acid sequence
set forth in SEQ ID NO: 34; and/or (k) the variant sialidase
precursor protein comprises the amino acid sequence set forth in
SEQ ID NO: 13; and/or (l) the variant sialidase precursor protein
consists of the amino acid sequence set forth in SEQ ID NO: 13 (m)
the transmembrane domain is selected from the group consisting of
an artificial hydrophobic sequence, and a transmembrane domain of a
type I transmembrane protein, an alpha, beta, or zeta chain of a T
cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,
CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137),
and CD154; and/or (n) the transmembrane domain comprises a
transmembrane domain of CD8; and/or (o) the transmembrane domain
comprises a transmembrane domain of CD8 alpha; and/or (p) the
transmembrane domain comprises the amino acid sequence set forth in
SEQ ID NO: 16.
5. The modified cell of claim 1, wherein the chimeric cell surface
sialidase further comprises: (a) a hinge domain; and/or (b) an
intracellular region comprising a costimulatory signaling domain
and an intracellular signaling domain.
6. The modified cell of claim 5, wherein: (a) the hinge domain is
selected from the group consisting of an Fc fragment of an
antibody, a hinge region of an antibody, a CH2 region of an
antibody, a CH3 region of an antibody, an artificial hinge domain,
a hinge comprising an amino acid sequence of CD8, or any
combination thereof; and/or (b) the hinge domain is a hinge
comprising an amino acid sequence of CD8; and/or (c) the hinge
domain is a hinge comprising an amino acid sequence of CD8 alpha;
and/or (d) the hinge domain comprises the amino acid sequence set
forth in SEQ ID NO: 15; and/or (e) the costimulatory signaling
domain comprises a costimulatory domain of a protein selected from
the group consisting of proteins in the TNFR superfamily, CD28,
4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12,
CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFI-II, Fas, CD30,
CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof; and/or
(f) the costimulatory signaling domain comprises a costimulatory
domain of 4-1BB; and/or (g) the costimulatory signaling domain
comprises the amino acid sequence set forth in SEQ ID NO: 17;
and/or (h) the costimulatory signaling domain comprises a
costimulatory domain of CD2; and/or (i) the intracellular signaling
domain comprises an intracellular domain selected from the group
consisting of cytoplasmic signaling domains of a human CD3 zeta
chain (CD3z), Fc.gamma.RIII, FcsRI, a cytoplasmic tail of an Fc
receptor, an immunoreceptor tyrosine-based activation motif (ITAM)
bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3
delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a
variant thereof; and/or (j) the intracellular signaling domain
comprises an intracellular domain of CD3z; and/or (k) the
intracellular signaling domain comprises the amino acid sequence
set forth in SEQ ID NO: 18.
7. The modified immune cell of claim 1, further comprising a
chimeric antigen receptor (CAR) and/or a T cell receptor (TCR).
8. The modified cell of claim 2, wherein: (a) the CAR comprises an
antigen binding domain, a transmembrane domain, and an
intracellular region; and/or (b) the CAR comprises an antigen
binding domain selected from the group consisting of an antibody,
an scFv, and a Fab; and/or (c) the CAR comprises an antigen binding
domain comprising specificity for a tumor associated antigen (TAA);
and/or (d) the CAR comprises an antigen binding domain comprising
specificity for TnMUC1; and/or (e) the CAR comprises an antigen
binding domain comprising specificity for CD19; and/or (f) the CAR
comprises an antigen binding domain comprising specificity for
PSMA; and/or (g) the CAR further comprises a hinge domain.
9. The modified cell of claim 7, wherein the TCR: (a) is specific
for a tumor associated antigen (TAA); and/or (b) is selected from
the group consisting of a wild-type TCR, a high affinity TCR, and a
chimeric TCR; and/or (c) comprises a TCR alpha chain and a TCR beta
chain.
10. The modified cell of claim 1, wherein the modified cell is: (a)
a modified immune cell; and/or (b) a modified T cell; and/or (c) an
autologous cell; and/or (d) an autologous cell obtained from a
human subject.
11. A pharmaceutical composition comprising a therapeutically
effective amount of the modified cell of claim 1.
12. The composition of claim 11, further comprising a
therapeutically effective population of innate immune cells.
13. The composition of claim 12, wherein: (a) the innate immune
cells are NK cells; and/or (b) the innate immune cells are NK cells
and wherein the NK cells are autologous NK cells; and/or (c) the
innate immune cells are NK cells and wherein the NK cells are
autologous NK cells obtained from a human subject.
14. The pharmaceutical composition of claim 11, comprising: (a) a
therapeutically effective amount of a modified immune cell or
precursor cell thereof, wherein the modified cell comprises a
chimeric cell surface sialidase (neuraminidase) enzyme comprising
an extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface,
and a chimeric antigen receptor (CAR); and a therapeutically
effective amount of an NK cell; and/or (b) a therapeutically
effective amount of a modified immune cell or precursor cell
thereof, wherein the modified cell comprises a chimeric cell
surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface, a
hinge domain, and an intracellular region comprising a
costimulatory signaling domain and an intracellular signaling
domain, and a chimeric antigen receptor (CAR); and a
therapeutically effective amount of an NK cell; and/or (c) a
therapeutically effective amount of a modified immune cell or
precursor cell thereof, wherein the modified cell comprises a
variant sialidase precursor protein comprising a heterologous
secretory sequence operably linked to a sialidase (neuraminidase)
or an enzymatically functional portion thereof, wherein the variant
sialidase precursor protein lacks a transmembrane domain, and
wherein the sialidase or enzymatically functional portion thereof
is capable of being secreted from an immune or precursor cell
thereof when the variant sialidase precursor protein is expressed
in the cell, and a chimeric antigen receptor (CAR); and a
therapeutically effective amount of an NK cell.
15. A chimeric cell surface sialidase (neuraminidase) enzyme
comprising an extracellular portion comprising a sialidase
(neuraminidase) or an enzymatically functional portion thereof, and
a heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface.
16. The cell surface sialidase of claim 15, wherein: (a) the
sialidase is a human or humanized sialidase; and/or (b) the
sialidase is a human or humanized sialidase and wherein the human
sialidase is selected from the group consisting of Neu1, Neu2,
Neu3, and Neu4; and/or (c) the sialidase is a human or humanized
sialidase and wherein the human sialidase is Neu2; and/or (d) the
sialidase comprises the amino acid sequence set forth in SEQ ID NO:
13; and/or (e) the cell surface sialidase comprises the amino acid
sequence set forth in SEQ ID NO: 10; and/or (f) the cell surface
sialidase comprises the amino acid sequence set forth any one of
SEQ ID NOs: 10, 37, or 38; and/or (g) the transmembrane domain is
selected from the group consisting of an artificial hydrophobic
sequence, and a transmembrane domain of a type I transmembrane
protein, an alpha, beta, or zeta chain of a T cell receptor, CD28,
CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37,
CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154; and/or
(h) the transmembrane domain comprises a transmembrane domain of
CD8; and/or (i) the transmembrane domain comprises a transmembrane
domain of CD8 alpha; and/or (j) the transmembrane domain comprises
the amino acid sequence set forth in SEQ ID NO: 16.
17. The cell surface sialidase of claim 15, further comprising: (a)
a hinge domain; and/or (b) an intracellular region comprising a
costimulatory signaling domain and an intracellular signaling
domain.
18. The cell surface sialidase of claim 17, wherein: (a) the hinge
domain is selected from the group consisting of an Fc fragment of
an antibody, a hinge region of an antibody, a CH2 region of an
antibody, a CH3 region of an antibody, an artificial hinge domain,
a hinge comprising an amino acid sequence of CD8, or any
combination thereof; and/or (b) the hinge domain is a hinge
comprising an amino acid sequence of CD8; and/or (c) the hinge
domain is a hinge comprising an amino acid sequence of CD8 alpha;
and/or (d) the hinge domain comprises the amino acid sequence set
forth in SEQ ID NO: 15; and/or (e) the costimulatory signaling
domain comprises a costimulatory domain of a protein selected from
the group consisting of proteins in the TNFR superfamily, CD28,
4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12,
CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFI-II, Fas, CD30,
CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof; and/or
(f) the costimulatory signaling domain comprises a costimulatory
domain of 4-1BB; and/or (g) the costimulatory signaling domain
comprises the amino acid sequence set forth in SEQ ID NO: 17;
and/or (h) the intracellular signaling domain comprises an
intracellular domain selected from the group consisting of
cytoplasmic signaling domains of a human CD3 zeta chain (CD3z),
Fc.gamma.RIII, FcsRI, a cytoplasmic tail of an Fc receptor, an
immunoreceptor tyrosine-based activation motif (ITAM) bearing
cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta,
CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant
thereof; and/or (i) the intracellular signaling domain comprises an
intracellular domain of CD3z; and/or (j) the intracellular
signaling domain comprises the amino acid sequence set forth in SEQ
ID NO: 18.
19. A variant sialidase precursor protein, comprising: (a) a
heterologous secretory sequence operably linked to a sialidase
(neuraminidase) or an enzymatically functional portion thereof,
wherein the variant sialidase precursor protein lacks a
transmembrane domain, and wherein the sialidase or enzymatically
functional portion thereof is capable of being secreted from an
immune or precursor cell thereof when the variant sialidase
precursor protein is expressed in the cell; and/or (b) a
heterologous secretory sequence operably linked to a sialidase
(neuraminidase) or an enzymatically functional portion thereof,
wherein the sialidase comprises the amino acid sequence set forth
in SEQ ID NO: 34.
20. The variant sialidase of claim 19, wherein: (a) the sialidase
is a human sialidase; and/or (b) the sialidase is a human sialidase
and wherein the human sialidase is selected from the group
consisting of Neu1, Neu2, Neu3, and Neu4; and/or (c) the variant
sialidase is a variant Neu2; and/or (d) the variant sialidase is a
variant Neu2 and wherein the variant Neu2 comprises the amino acid
sequence set forth in SEQ ID NO: 13.
21. A nucleic acid comprising: (a) a first nucleic acid encoding a
chimeric cell surface sialidase (neuraminidase) enzyme comprising
an extracellular portion comprising a sialidase (neuraminidase) or
an enzymatically functional portion thereof, and a heterologous
transmembrane domain capable of tethering the extracellular portion
to a cell surface; and/or (b) a first nucleic acid sequence
encoding a variant sialidase precursor protein comprising SEQ ID
NO: 30; and/or (c) first nucleic acid sequence encoding a variant
sialidase precursor protein comprising SEQ ID NO: 30; and/or (d) a
first nucleic acid sequence encoding a chimeric cell surface
sialidase comprising SEQ ID NO: 1 or 40; and/or (e) a first nucleic
acid sequence encoding a variant sialidase precursor protein
comprising a heterologous secretory sequence operably linked to a
sialidase (neuraminidase) or an enzymatically functional portion
thereof, wherein the variant sialidase precursor protein lacks a
transmembrane domain, and wherein the sialidase or enzymatically
functional portion thereof is capable of being secreted from an
immune or precursor cell thereof when the variant sialidase
precursor protein is expressed in the cell; and/or (f) a first
nucleic acid sequence encoding a chimeric cell surface sialidase
(neuraminidase) enzyme comprising an extracellular portion
comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface,
and a second nucleic acid sequence encoding a chimeric antigen
receptor (CAR) and/or a T cell receptor (TCR); and/or (g) a first
nucleic acid sequence encoding a variant sialidase precursor
protein comprising a heterologous secretory sequence operably
linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof, wherein the variant sialidase precursor
protein lacks a transmembrane domain, and wherein the sialidase or
enzymatically functional portion thereof is capable of being
secreted from an immune or precursor cell thereof when the variant
sialidase precursor protein is expressed in the cell, and a second
nucleic acid sequence encoding a chimeric antigen receptor (CAR)
and/or a T cell receptor (TCR).
22. The nucleic acid of claim 21, wherein: (a) the sialidase is a
human or humanized sialidase; and/or (b) the sialidase is a human
or humanized sialidase and wherein the human sialidase is selected
from the group consisting of Neu1, Neu2, Neu3, and Neu4; and/or (c)
the sialidase is a human or humanized sialidase and wherein the
sialidase is Neu2; and/or (d) the sialidase is encoded by a nucleic
acid sequence comprising SEQ ID NO: 4; and/or (e) the transmembrane
domain is selected from the group consisting of an artificial
hydrophobic sequence, and a transmembrane domain of a type I
transmembrane protein, an alpha, beta, or zeta chain of a T cell
receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22,
CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and
CD154; and/or (f) the transmembrane domain comprises a
transmembrane domain of CD8; and/or (g) the transmembrane domain
comprises a transmembrane domain of CD8 alpha; and/or (h) the
transmembrane domain is encoded by a nucleic acid sequence
comprising SEQ ID NO: 7; and/or (i) the nucleic acid further
comprises a leader sequence; and/or (j) the nucleic acid further
comprises a leader sequence and wherein the leader sequence is a
CD8 alpha leader sequence; and/or (k) the nucleic acid further
comprises a leader sequence and wherein the leader sequence is
encoded by a nucleic acid sequence comprising SEQ ID NO: 2; and/or
(l) the nucleic acid further comprises a hinge domain; and/or (m)
the nucleic acid further comprises a hinge domain and wherein the
hinge domain is selected from the group consisting of an Fc
fragment of an antibody, a hinge region of an antibody, a CH2
region of an antibody, a CH3 region of an antibody, an artificial
hinge domain, a hinge comprising an amino acid sequence of CD8, or
any combination thereof; and/or (n) the nucleic acid further
comprises a hinge domain and wherein the hinge domain is a hinge
comprising an amino acid sequence of CD8; and/or (o) the nucleic
acid further comprises a hinge domain and wherein the hinge domain
is a hinge comprising an amino acid sequence of CD8 alpha; and/or
(p) the nucleic acid further comprises a hinge domain and wherein
the hinge domain is encoded by a nucleic acid sequence comprising
SEQ ID NO: 6; and/or (q) the nucleic acid further comprises an
intracellular region comprising a costimulatory signaling domain
and an intracellular signaling domain, and optionally wherein: (i)
the costimulatory signaling domain comprises a costimulatory domain
of a protein selected from the group consisting of proteins in the
TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7,
LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck,
TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276),
or a variant thereof; and/or (ii) the costimulatory signaling
domain comprises a costimulatory domain of 4-1BB; and/or (iii) the
costimulatory signaling domain is encoded by a nucleic acid
sequence comprising SEQ ID NO: 8; and/or (iv) the intracellular
signaling domain comprises an intracellular domain selected from
the group consisting of cytoplasmic signaling domains of a human
CD3 zeta chain (CD3z), Fc.gamma.RIII, FcsRI, a cytoplasmic tail of
an Fc receptor, an immunoreceptor tyrosine-based activation motif
(ITAM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3
gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d,
or a variant thereof; and/or (v) the intracellular signaling domain
comprises an intracellular domain of CD3z; and/or (vi) the
intracellular signaling domain is encoded by a nucleic acid
sequence comprising SEQ ID NO: 9.
23. The nucleic acid of claim 21, further comprising a second
nucleic acid sequence encoding an exogenous T cell receptor (TCR)
and/or chimeric antigen receptor (CAR).
24. The nucleic acid of claim 23, wherein: (a) the CAR comprises an
antigen binding domain, a transmembrane domain, and an
intracellular region, and optionally wherein: (i) the transmembrane
domain is selected from the group consisting of an artificial
hydrophobic sequence and transmembrane domain of a type I
transmembrane protein, an alpha, beta, or zeta chain of a T cell
receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22,
CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; and/or (ii)
the intracellular region comprises a costimulatory signaling domain
and an intracellular signaling domain; and/or (iii) the
costimulatory signaling domain comprises a costimulatory domain of
a protein selected from the group consisting of proteins in the
TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7,
LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck,
TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276),
or a variant thereof; and/or (iv) the costimulatory signaling
domain comprises a costimulatory domain of CD2; and/or (v) the
costimulatory signaling domain comprises a costimulatory domain of
4-1BB; and/or (vi) the intracellular signaling domain comprises an
intracellular domain selected from the group consisting of
cytoplasmic signaling domains of a human CD3 zeta chain (CD3z),
Fc.gamma.RIII, FcsRI, a cytoplasmic tail of an Fc receptor, an
immunoreceptor tyrosine-based activation motif (ITAM) bearing
cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta,
CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant
thereof; and/or (vii) the intracellular signaling domain comprises
an intracellular domain of CD3z; and/or (b) the CAR comprises an
antigen binding domain selected from the group consisting of an
antibody, an scFv, and a Fab; and/or (c) the CAR comprises an
antigen binding domain comprising specificity for a tumor
associated antigen (TAA); and/or (d) the CAR comprises an antigen
binding domain comprising specificity for TnMUC1; and/or (e) the
CAR comprises an antigen binding domain comprising specificity for
CD19; and/or (f) the CAR comprises an antigen binding domain
comprising specificity for PSMA; and/or (g) the CAR further
comprises a hinge domain, and optionally wherein the hinge domain
is a hinge domain selected from the group consisting of an Fc
fragment of an antibody, a hinge region of an antibody, a CH2
region of an antibody, a CH3 region of an antibody, an artificial
hinge domain, a hinge comprising an amino acid sequence of CD8, or
any combination thereof; and/or (h) the TCR is specific for a tumor
associated antigen (TAA); and/or (i) the TCR is selected from the
group consisting of a wild-type TCR, a high affinity TCR, and a
chimeric TCR; and/or (j) the TCR comprises a TCR alpha chain coding
sequence and a TCR beta chain coding sequence; and/or (k) the TCR
alpha chain coding sequence and the TCR beta chain coding sequence
are separated by a first linker, and optionally wherein: (i) the
first linker comprises a nucleic acid sequence encoding an internal
ribosome entry site (IRES), a furin cleavage site, a self-cleaving
peptide, or any combination thereof; and/or (ii) the first linker
comprises a furin cleavage site and a self-cleaving peptide, and
optionally wherein the self-cleaving peptide is a 2A peptide
selected from the group consisting of porcine teschovirus-1 2A
(P2A), Thoseaasigna virus 2A (T2A), equine rhinitis A virus 2A
(E2A), and foot-and-mouth disease virus 2A (F2A); and/or (l) the
first nucleic acid sequence and the second nucleic acid sequence
are separated by a second linker, and optionally wherein: (i) the
second linker comprises a nucleic acid sequence encoding an
internal ribosome entry site (IRES); and/or (ii) the second linker
comprises a cleavage site and/or a self-cleaving peptide, and
optionally wherein the cleavage site is a furin cleavage site
and/or wherein the self-cleaving peptide is a 2A peptide, and
optionally wherein the 2A peptide (A) is selected from the group
consisting of porcine teschovirus-1 2A (P2A), Thoseaasigna virus 2A
(T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease
virus 2A (F2A); (B) is P2A; and/or (C) is T2A; and/or (m) the
nucleic acid comprises from 5' to 3': the first nucleic acid
sequence, the second linker, and the second nucleic acid sequence;
and/or (n) the nucleic acid comprises from 5' to 3': the second
nucleic acid sequence, the second linker, and the first nucleic
acid sequence.
25. An expression vector comprising the nucleic acid of claim
21.
26. The expression vector of claim 25, wherein: (a) the expression
vector is a viral vector selected from the group consisting of a
retroviral vector, a lentiviral vector, an adenoviral vector, and
an adeno-associated viral vector; and/or (b) the expression vector
is a lentiviral vector, and optionally wherein the lentiviral
vector is a self-inactivating lentiviral vector.
27. A method of treating cancer in a subject in need thereof, the
method comprising: (a) administering the modified cell of claim 1
or the pharmaceutical composition of claim 11 to the subject;
and/or (b) administering a modified T cell comprising a chimeric
cell surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising a sialidase (neuraminidase) or an
enzymatically functional portion thereof, and a heterologous
transmembrane domain capable of tethering the extracellular portion
to a cell surface, and a chimeric antigen receptor (CAR); and/or
(c) administering a modified T cell comprising a variant sialidase
precursor protein comprising a heterologous secretory sequence
operably linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof, wherein the variant sialidase precursor
protein lacks a transmembrane domain, and wherein the sialidase or
enzymatically functional portion thereof is capable of being
secreted from an immune or precursor cell thereof when the variant
sialidase precursor protein is expressed in the cell, and a
chimeric antigen receptor (CAR); and/or (d) administering to the
subject a therapeutically effective amount of a modified T cell
comprising a chimeric cell surface sialidase (neuraminidase) enzyme
comprising an extracellular portion comprising a sialidase
(neuraminidase) or an enzymatically functional portion thereof, and
a heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface, and a chimeric antigen
receptor (CAR); and administering to the subject a therapeutically
effective amount of a NK cell; and/or (e) administering to the
subject a therapeutically effective amount of a modified T cell
comprising a variant sialidase precursor protein comprising a
heterologous secretory sequence operably linked to a sialidase
(neuraminidase) or an enzymatically functional portion thereof,
wherein the variant sialidase precursor protein lacks a
transmembrane domain, and wherein the sialidase or enzymatically
functional portion thereof is capable of being secreted from an
immune or precursor cell thereof when the variant sialidase
precursor protein is expressed in the cell, and a chimeric antigen
receptor (CAR); and administering to the subject a therapeutically
effective amount of a NK cell.
28. The method of claim 27, wherein: (a) the CAR comprises
specificity for TnMUC1; and/or (b) the CAR comprises specificity
for CD19; and/or (c) the CAR comprises specificity for PSMA; and/or
(d) the method further comprises administering to the subject a
population of innate immune cells, and optionally wherein: (i) the
innate immune cells are NK cells; and/or (ii) the innate immune
cells are NK cells and wherein the NK cells are autologous NK cells
obtained from a human subject; and/or (e) the modified T cell and
the NK cell are administered simultaneously; and/or (f) the
modified T cell and the NK cell are administered separately; and/or
(g) the NK cell is autologous; and/or (h) the modified T cell is
autologous; and/or (i) the subject is human.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is entitled to priority under 35
U.S.C. .sctn. 119(e) to U.S. Provisional Patent Application No.
62/850,014, filed May 20, 2019, which is hereby incorporated by
reference in its entirety herein.
BACKGROUND OF THE INVENTION
[0003] Chimeric antigen receptors (CARs) are antibody-based,
artificial T cell receptors that endow synthetic retargeting of
patient T cells towards cancer-specific epitopes. CAR T cells
targeting CD19 are now FDA approved for the treatment of pediatric
acute lymphoblastic leukemia (ALL) and adult diffuse large B cell
lymphoma. While these immunotherapy advancements are revolutionary
for the treatment of blood cancers, leukemia and lymphomas
represent just 8% of all cancer indications and 7% of
cancer-related deaths. The greatest unmet burdens for cancer
treatments are solid tumors, particularly prostate, breast,
colorectal and lung cancers, which account for 45% of all
cancer-related deaths. Translating CAR T cell therapy into solid
tumors requires overcoming significant tumor-associated challenges,
including specific target identification, tumor heterogeneity, and
dense immunosuppressive stroma, which are associated with
increasing tumor aggressiveness.
[0004] There is a need in the art for improving CAR T cell
therapies, specifically CAR T anti-tumor efficacy in solid tumors.
The present invention addresses this need.
SUMMARY OF THE INVENTION
[0005] The present invention is based on the discovery that CAR T
therapies may be improved by endowing glycoediting activity to the
T cells.
[0006] In one aspect, the invention includes a modified immune cell
or precursor cell thereof, comprising: (a) a chimeric cell surface
sialidase (neuraminidase) enzyme comprising an extracellular
portion comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and (b) a heterologous transmembrane
domain capable of tethering the extracellular portion to a cell
surface.
[0007] In another aspect, the invention includes a modified immune
cell or precursor cell thereof, comprising: (a) a chimeric cell
surface sialidase consisting of the amino acid sequence set forth
in any one of SEQ ID NOs: 10, 37, or 38; and/or (b) a variant
sialidase precursor protein comprising a heterologous secretory
sequence operably linked to a sialidase (neuraminidase) or an
enzymatically functional portion thereof, wherein the variant
sialidase precursor protein lacks a transmembrane domain, and
wherein the sialidase or enzymatically functional portion thereof
is capable of being secreted from an immune or precursor cell
thereof when the variant sialidase precursor protein is expressed
in the cell; and/or (c) a variant sialidase precursor protein
comprising a heterologous secretory sequence operably linked to a
sialidase (neuraminidase) or an enzymatically functional portion
thereof, wherein the sialidase comprises the amino acid sequence
set forth in SEQ ID NO: 34; and/or (d) a chimeric cell surface
sialidase (neuraminidase) enzyme comprising an extracellular
portion comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface;
and a chimeric antigen receptor (CAR) and/or a T cell receptor
(TCR); and/or (e) a variant sialidase precursor protein comprising
a heterologous secretory sequence operably linked to a sialidase
(neuraminidase) or an enzymatically functional portion thereof,
wherein the variant sialidase precursor protein lacks a
transmembrane domain, and wherein the sialidase or enzymatically
functional portion thereof is capable of being secreted from an
immune or precursor cell thereof when the variant sialidase
precursor protein is expressed in the cell; and a chimeric antigen
receptor (CAR) and/or a T cell receptor (TCR); and/or (f) a
chimeric cell surface sialidase (neuraminidase) enzyme comprising
an extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface;
and a chimeric antigen receptor (CAR); and/or (g) a chimeric cell
surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface;
and a chimeric antigen receptor (CAR) having specificity for
TnMUC1, CD19, or PSMA; and/or (h) a chimeric cell surface sialidase
(neuraminidase) enzyme comprising an extracellular portion
comprising Neu2 or an enzymatically functional portion thereof, a
heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface, a hinge domain, and an
intracellular region comprising a costimulatory signaling domain
and an intracellular signaling domain; and a chimeric antigen
receptor (CAR); and/or (i) a chimeric cell surface sialidase
(neuraminidase) enzyme comprising an extracellular portion
comprising Neu2 or an enzymatically functional portion thereof, a
heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface, a hinge domain, and an
intracellular region comprising a costimulatory signaling domain
and an intracellular signaling domain; and a chimeric antigen
receptor (CAR) having specificity for TnMUC1, CD19, or PSMA; and/or
(j) a variant sialidase precursor protein comprising a heterologous
secretory sequence operably linked to a sialidase (neuraminidase)
or an enzymatically functional portion thereof, wherein the variant
sialidase precursor protein lacks a transmembrane domain, and
wherein the sialidase or enzymatically functional portion thereof
is capable of being secreted from an immune or precursor cell
thereof when the variant sialidase precursor protein is expressed
in the cell; and a chimeric antigen receptor (CAR) having
specificity for TnMUC1, CD19, or PSMA.
[0008] In various embodiments of the above aspects or any other
aspect of the invention delineated herein, the sialidase is a human
or humanized sialidase. In certain embodiments, the sialidase is a
human or humanized sialidase wherein the human sialidase is
selected from the group consisting of Neu1, Neu2, Neu3, and Neu4.
In certain embodiments, the sialidase is a human or humanized
sialidase wherein the human sialidase is Neu2.
[0009] In certain embodiments, the sialidase comprises the amino
acid sequence set forth in SEQ ID NO: 13. In certain embodiments,
the sialidase comprises the amino acid sequence set forth in SEQ ID
NO: 10. In certain embodiments the sialidase consists of the amino
acid sequence set forth in SEQ ID NO: 10. In certain embodiments,
the sialidase comprises the amino acid sequence set forth in any
one of SEQ ID NOs: 10, 37, or 38. In certain embodiments, the
sialidase consists of the amino acid sequence set forth in any one
of SEQ ID NOs: 10, 37, or 38. In certain embodiments, the sialidase
comprises the amino acid sequence set forth in SEQ ID NO: 34. In
certain embodiments, the sialidase consists of the amino acid
sequence set forth in SEQ ID NO: 34.
[0010] In certain embodiments, the variant sialidase precursor
protein comprises the amino acid sequence set forth in SEQ ID NO:
13. In certain embodiments, the variant sialidase precursor protein
consists of the amino acid sequence set forth in SEQ ID NO: 13.
[0011] In certain embodiments, the transmembrane domain is selected
from the group consisting of an artificial hydrophobic sequence,
and a transmembrane domain of a type I transmembrane protein, an
alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon,
CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,
OX40 (CD134), 4-1BB (CD137), and CD154. In certain embodiments, the
transmembrane domain comprises a transmembrane domain of CD8. In
certain embodiments, the transmembrane domain comprises a
transmembrane domain of CD8 alpha. In certain embodiments, the
transmembrane domain comprises the amino acid sequence set forth in
SEQ ID NO: 16.
[0012] In certain embodiments, the chimeric cell surface sialidase
further comprises a hinge domain. In certain embodiments, the hinge
domain is selected from the group consisting of an Fc fragment of
an antibody, a hinge region of an antibody, a CH2 region of an
antibody, a CH3 region of an antibody, an artificial hinge domain,
a hinge comprising an amino acid sequence of CD8, or any
combination thereof. In certain embodiments, the hinge domain is a
hinge comprising an amino acid sequence of CD8. In certain
embodiments, the hinge domain is a hinge comprising an amino acid
sequence of CD8 alpha. In certain embodiments, the hinge domain
comprises the amino acid sequence set forth in SEQ ID NO: 15.
[0013] In certain embodiments, the chimeric cell surface sialidase
further comprises intracellular region comprising a costimulatory
signaling domain and an intracellular signaling domain. In certain
embodiments, the costimulatory signaling domain comprises a
costimulatory domain of a protein selected from the group
consisting of proteins in the TNFR superfamily, CD28, 4-1BB
(CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27,
CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFI-II, Fas, CD30, CD40,
ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof. In certain
embodiments, the costimulatory signaling domain comprises a
costimulatory domain of 4-1BB. In certain embodiments, the
costimulatory signaling domain comprises the amino acid sequence
set forth in SEQ ID NO: 17. In certain embodiments, the
costimulatory signaling domain comprises a costimulatory domain of
CD2.
[0014] In certain embodiments, the intracellular signaling domain
comprises an intracellular domain selected from the group
consisting of cytoplasmic signaling domains of a human CD3 zeta
chain (CD3z), Fc.gamma.RIII, FcsRI, a cytoplasmic tail of an Fc
receptor, an immunoreceptor tyrosine-based activation motif (ITAM)
bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3
delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a
variant thereof. In certain embodiments, the intracellular
signaling domain comprises an intracellular domain of CD3zeta. In
certain embodiments, the intracellular signaling domain comprises
the amino acid sequence set forth in SEQ ID NO: 18.
[0015] In certain embodiments, the modified immune cell further
comprises a chimeric antigen receptor (CAR) and/or a T cell
receptor (TCR).
[0016] In certain embodiments, the CAR comprises an antigen binding
domain, a transmembrane domain, and an intracellular region. In
certain embodiments, the CAR comprises an antigen binding domain
selected from the group consisting of an antibody, an scFv, and a
Fab.
[0017] In certain embodiments, the CAR comprises an antigen binding
domain comprising specificity for a tumor associated antigen (TAA).
In certain embodiments, the CAR comprises an antigen binding domain
comprising specificity for TnMUC1. In certain embodiments, the CAR
comprises an antigen binding domain comprising specificity for
CD19. In certain embodiments, the CAR comprises an antigen binding
domain comprising specificity for PSMA. In certain embodiments, the
CAR further comprises a hinge domain.
[0018] In certain embodiments, the TCR is specific for a tumor
associated antigen (TAA). In certain embodiments, the TCR is
selected from the group consisting of a wild-type TCR, a high
affinity TCR, and a chimeric TCR. In certain embodiments, the TCR
comprises a TCR alpha chain and a TCR beta chain.
[0019] In certain embodiments, the modified cell is a modified
immune cell. In certain embodiments, the modified cell is a
modified T cell. In certain embodiments, the modified cell is an
autologous cell. In certain embodiments, the modified cell is an
autologous cell obtained from a human subject.
[0020] In another aspect, the invention includes a pharmaceutical
composition comprising a therapeutically effective amount of any of
the modified cells disclosed herein. In certain embodiments, the
composition further comprises a therapeutically effective
population of innate immune cells. In certain embodiments, the
innate immune cells are NK cells. In certain embodiments, the
innate immune cells are NK cells and the NK cells are autologous NK
cells. In certain embodiments, the innate immune cells are NK cells
and the NK cells are autologous NK cells obtained from a human
subject.
[0021] In certain embodiments, the pharmaceutical composition
comprises a therapeutically effective amount of a modified immune
cell or precursor cell thereof, wherein the modified cell comprises
a chimeric cell surface sialidase (neuraminidase) enzyme comprising
an extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface,
and a chimeric antigen receptor (CAR); and a therapeutically
effective amount of an NK cell.
[0022] In certain embodiments, the pharmaceutical composition
comprises a therapeutically effective amount of a modified immune
cell or precursor cell thereof, wherein the modified cell comprises
a chimeric cell surface sialidase (neuraminidase) enzyme comprising
an extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface, a
hinge domain, and an intracellular region comprising a
costimulatory signaling domain and an intracellular signaling
domain, and a chimeric antigen receptor (CAR); and a
therapeutically effective amount of an NK cell.
[0023] In certain embodiments, the pharmaceutical composition
comprises a therapeutically effective amount of a modified immune
cell or precursor cell thereof, wherein the modified cell comprises
a variant sialidase precursor protein comprising a heterologous
secretory sequence operably linked to a sialidase (neuraminidase)
or an enzymatically functional portion thereof, wherein the variant
sialidase precursor protein lacks a transmembrane domain, and
wherein the sialidase or enzymatically functional portion thereof
is capable of being secreted from an immune or precursor cell
thereof when the variant sialidase precursor protein is expressed
in the cell, and a chimeric antigen receptor (CAR); and a
therapeutically effective amount of an NK cell.
[0024] In another aspect, the invention includes a chimeric cell
surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising a sialidase (neuraminidase) or an
enzymatically functional portion thereof, and a heterologous
transmembrane domain capable of tethering the extracellular portion
to a cell surface.
[0025] In certain embodiments, the sialidase is a human or
humanized sialidase. In certain embodiments, the sialidase is a
human or humanized sialidase and the human sialidase is selected
from the group consisting of Neu1, Neu2, Neu3, and Neu4. In certain
embodiments, the sialidase is a human or humanized sialidase and
the human sialidase is Neu2. In certain embodiments, the sialidase
comprises the amino acid sequence set forth in SEQ ID NO: 13. In
certain embodiments, the cell surface sialidase comprises the amino
acid sequence set forth in SEQ ID NO: 10. In certain embodiments,
the cell surface sialidase comprises the amino acid sequence set
forth any one of SEQ ID NOs: 10, 37, or 38.
[0026] In certain embodiments, the transmembrane domain is selected
from the group consisting of an artificial hydrophobic sequence,
and a transmembrane domain of a type I transmembrane protein, an
alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon,
CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,
OX40 (CD134), 4-1BB (CD137), and CD154. In certain embodiments, the
transmembrane domain comprises a transmembrane domain of CD8. In
certain embodiments, the transmembrane domain comprises a
transmembrane domain of CD8 alpha. In certain embodiments, the
transmembrane domain comprises the amino acid sequence set forth in
SEQ ID NO: 16.
[0027] In certain embodiments, the cell surface sialidase further
comprises a hinge domain; and/or an intracellular region comprising
a costimulatory signaling domain and an intracellular signaling
domain.
[0028] In certain embodiments, the hinge domain is selected from
the group consisting of an Fc fragment of an antibody, a hinge
region of an antibody, a CH2 region of an antibody, a CH3 region of
an antibody, an artificial hinge domain, a hinge comprising an
amino acid sequence of CD8, or any combination thereof. In certain
embodiments, the hinge domain is a hinge comprising an amino acid
sequence of CD8. In certain embodiments, the hinge domain is a
hinge comprising an amino acid sequence of CD8 alpha. In certain
embodiments, the hinge domain comprises the amino acid sequence set
forth in SEQ ID NO: 15.
[0029] In certain embodiments, the costimulatory signaling domain
comprises a costimulatory domain of a protein selected from the
group consisting of proteins in the TNFR superfamily, CD28, 4-1BB
(CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27,
CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFI-II, Fas, CD30, CD40,
ICOS, NKG2C, and B7-H3 (CD276). In certain embodiments, the
costimulatory signaling domain comprises a costimulatory domain of
4-1BB. In certain embodiments, the costimulatory signaling domain
comprises the amino acid sequence set forth in SEQ ID NO: 17.
[0030] In certain embodiments, the intracellular signaling domain
comprises an intracellular domain selected from the group
consisting of cytoplasmic signaling domains of a human CD3 zeta
chain (CD3z), Fc.gamma.RIII, FcsRI, a cytoplasmic tail of an Fc
receptor, an immunoreceptor tyrosine-based activation motif (ITAM)
bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3
delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a
variant thereof. In certain embodiments, the intracellular
signaling domain comprises an intracellular domain of CD3z. In
certain embodiments, the intracellular signaling domain comprises
the amino acid sequence set forth in SEQ ID NO: 18.
[0031] In another aspect, the invention includes a variant
sialidase precursor protein, comprising a heterologous secretory
sequence operably linked to a sialidase (neuraminidase) or an
enzymatically functional portion thereof, wherein the variant
sialidase precursor protein lacks a transmembrane domain, and
wherein the sialidase or enzymatically functional portion thereof
is capable of being secreted from an immune or precursor cell
thereof when the variant sialidase precursor protein is expressed
in the cell; and/or a heterologous secretory sequence operably
linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof, wherein the sialidase comprises the
amino acid sequence set forth in SEQ ID NO: 34.
[0032] In certain embodiments, the sialidase is a human sialidase.
In certain embodiments, the sialidase is a human sialidase and
wherein the human sialidase is selected from the group consisting
of Neu1, Neu2, Neu3, and Neu4. In certain embodiments, the variant
sialidase is a variant Neu2. In certain embodiments, the variant
sialidase is a variant Neu2 and the variant Neu2 comprises the
amino acid sequence set forth in SEQ ID NO: 13.
[0033] In another aspect, the invention includes a nucleic acid
comprising (a) a first nucleic acid encoding a chimeric cell
surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising a sialidase (neuraminidase) or an
enzymatically functional portion thereof, and a heterologous
transmembrane domain capable of tethering the extracellular portion
to a cell surface; and/or (b) a first nucleic acid sequence
encoding any of the cell surface sialidases or the variant
sialidases disclosed herein; and/or (c) a first nucleic acid
sequence encoding a variant sialidase precursor protein comprising
SEQ ID NO: 30; and/or (d) a first nucleic acid sequence encoding a
variant sialidase precursor protein comprising SEQ ID NO: 30;
and/or (e) a first nucleic acid sequence encoding a chimeric cell
surface sialidase comprising SEQ ID NO: 1 or 40; and/or (f) a first
nucleic acid sequence encoding a variant sialidase precursor
protein comprising a heterologous secretory sequence operably
linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof, wherein the variant sialidase precursor
protein lacks a transmembrane domain, and wherein the sialidase or
enzymatically functional portion thereof is capable of being
secreted from an immune or precursor cell thereof when the variant
sialidase precursor protein is expressed in the cell; and/or (g) a
first nucleic acid sequence encoding a chimeric cell surface
sialidase (neuraminidase) enzyme comprising an extracellular
portion comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface,
and a second nucleic acid sequence encoding a chimeric antigen
receptor (CAR) and/or a T cell receptor (TCR); and/or (h) a first
nucleic acid sequence encoding a variant sialidase precursor
protein comprising a heterologous secretory sequence operably
linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof, wherein the variant sialidase precursor
protein lacks a transmembrane domain, and wherein the sialidase or
enzymatically functional portion thereof is capable of being
secreted from an immune or precursor cell thereof when the variant
sialidase precursor protein is expressed in the cell, and a second
nucleic acid sequence encoding a chimeric antigen receptor (CAR)
and/or a T cell receptor (TCR).
[0034] In certain embodiments, (a) the sialidase is a human or
humanized sialidase; and/or (b) the sialidase is a human or
humanized sialidase and wherein the human sialidase is selected
from the group consisting of Neu1, Neu2, Neu3, and Neu4; and/or (c)
the sialidase is a human or humanized sialidase and wherein the
sialidase is Neu2; and/or (d) the sialidase is encoded by a nucleic
acid sequence comprising SEQ ID NO: 4; and/or (e) the transmembrane
domain is selected from the group consisting of an artificial
hydrophobic sequence, and a transmembrane domain of a type I
transmembrane protein, an alpha, beta, or zeta chain of a T cell
receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22,
CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and
CD154; and/or (f) the transmembrane domain comprises a
transmembrane domain of CD8; and/or (g) the transmembrane domain
comprises a transmembrane domain of CD8 alpha; and/or (h) the
transmembrane domain is encoded by a nucleic acid sequence
comprising SEQ ID NO: 7; and/or (i) the nucleic acid further
comprises a leader sequence; and/or (j) the nucleic acid further
comprises a leader sequence and wherein the leader sequence is a
CD8 alpha leader sequence; and/or (k) the nucleic acid further
comprises a leader sequence and wherein the leader sequence is
encoded by a nucleic acid sequence comprising SEQ ID NO: 2; and/or
(1) the nucleic acid further comprises a hinge domain; and/or (m)
the nucleic acid further comprises a hinge domain and wherein the
hinge domain is selected from the group consisting of an Fc
fragment of an antibody, a hinge region of an antibody, a CH2
region of an antibody, a CH3 region of an antibody, an artificial
hinge domain, a hinge comprising an amino acid sequence of CD8, or
any combination thereof; and/or (n) the nucleic acid further
comprises a hinge domain and wherein the hinge domain is a hinge
comprising an amino acid sequence of CD8; and/or (o) the nucleic
acid further comprises a hinge domain and wherein the hinge domain
is a hinge comprising an amino acid sequence of CD8 alpha; and/or
(p) the nucleic acid further comprises a hinge domain and wherein
the hinge domain is encoded by a nucleic acid sequence comprising
SEQ ID NO: 6; and/or (q) the nucleic acid further comprises an
intracellular region comprising a costimulatory signaling domain
and an intracellular signaling domain.
[0035] In certain embodiments, (i) the costimulatory signaling
domain comprises a costimulatory domain of a protein selected from
the group consisting of proteins in the TNFR superfamily, CD28,
4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12,
CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFI-II, Fas, CD30,
CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof; and/or
(ii) the costimulatory signaling domain comprises a costimulatory
domain of 4-1BB; and/or (iii) the costimulatory signaling domain is
encoded by a nucleic acid sequence comprising SEQ ID NO: 8; and/or
(iv) the intracellular signaling domain comprises an intracellular
domain selected from the group consisting of cytoplasmic signaling
domains of a human CD3 zeta chain (CD3z), Fc.gamma.RIII, FcsRI, a
cytoplasmic tail of an Fc receptor, an immunoreceptor
tyrosine-based activation motif (ITAM) bearing cytoplasmic
receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon,
CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof; and/or
(v) the intracellular signaling domain comprises an intracellular
domain of CD3z; and/or (vi) the intracellular signaling domain is
encoded by a nucleic acid sequence comprising SEQ ID NO: 9.
[0036] In certain embodiments, the nucleic acid further comprises a
second nucleic acid sequence encoding an exogenous T cell receptor
(TCR) and/or chimeric antigen receptor (CAR).
[0037] In certain embodiments, (a) the CAR comprises an antigen
binding domain, a transmembrane domain, and an intracellular
region; and/or (b) the CAR comprises an antigen binding domain
selected from the group consisting of an antibody, an scFv, and a
Fab; and/or (c) the CAR comprises an antigen binding domain
comprising specificity for a tumor associated antigen (TAA); and/or
(d) the CAR comprises an antigen binding domain comprising
specificity for TnMUC1; and/or (e) the CAR comprises an antigen
binding domain comprising specificity for CD19; and/or (f) the CAR
comprises an antigen binding domain comprising specificity for
PSMA; and/or (g) the CAR further comprises a hinge domain, and
optionally the hinge domain is a hinge domain selected from the
group consisting of an Fc fragment of an antibody, a hinge region
of an antibody, a CH2 region of an antibody, a CH3 region of an
antibody, an artificial hinge domain, a hinge comprising an amino
acid sequence of CD8, or any combination thereof.
[0038] In certain embodiments, (i) the transmembrane domain is
selected from the group consisting of an artificial hydrophobic
sequence and transmembrane domain of a type I transmembrane
protein, an alpha, beta, or zeta chain of a T cell receptor, CD28,
CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37,
CD64, CD80, CD86, CD134, CD137, and CD154; and/or (ii) the
intracellular region comprises a costimulatory signaling domain and
an intracellular signaling domain; and/or (iii) the costimulatory
signaling domain comprises a costimulatory domain of a protein
selected from the group consisting of proteins in the TNFR
superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT,
CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I,
TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a
variant thereof; and/or (iv) the costimulatory signaling domain
comprises a costimulatory domain of CD2; and/or (v) the
costimulatory signaling domain comprises a costimulatory domain of
4-1BB; and/or (vi) the intracellular signaling domain comprises an
intracellular domain selected from the group consisting of
cytoplasmic signaling domains of a human CD3 zeta chain (CD3z),
Fc.gamma.RIII, FcsRI, a cytoplasmic tail of an Fc receptor, an
immunoreceptor tyrosine-based activation motif (ITAM) bearing
cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta,
CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant
thereof and/or (vii) the intracellular signaling domain comprises
an intracellular domain of CD3z.
[0039] In certain embodiments, the TCR is specific for a tumor
associated antigen (TAA); and/or the TCR is selected from the group
consisting of a wild-type TCR, a high affinity TCR, and a chimeric
TCR; and/or the TCR comprises a TCR alpha chain coding sequence and
a TCR beta chain coding sequence; and/or the TCR alpha chain coding
sequence and the TCR beta chain coding sequence are separated by a
first linker.
[0040] In certain embodiments, (i) the first linker comprises a
nucleic acid sequence encoding an internal ribosome entry site
(IRES), a furin cleavage site, a self-cleaving peptide, or any
combination thereof; and/or (ii) the first linker comprises a furin
cleavage site and a self-cleaving peptide, and optionally wherein
the self-cleaving peptide is a 2A peptide selected from the group
consisting of porcine teschovirus-1 2A (P2A), Thoseaasigna virus 2A
(T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease
virus 2A (F2A); and/or
[0041] In certain embodiments, the first nucleic acid sequence and
the second nucleic acid sequence are separated by a second linker,
optionally wherein: (i) the second linker comprises a nucleic acid
sequence encoding an internal ribosome entry site (IRES); and/or
(ii) the second linker comprises a cleavage site and/or a
self-cleaving peptide, and optionally wherein the cleavage site is
a furin cleavage site and/or wherein the self-cleaving peptide is a
2A peptide, and optionally wherein the 2A peptide (A) is selected
from the group consisting of porcine teschovirus-1 2A (P2A),
Thoseaasigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and
foot-and-mouth disease virus 2A (F2A); (B) is P2A; and/or (C) is
T2A; and/or
[0042] In certain embodiments, the nucleic acid comprises from 5'
to 3': the first nucleic acid sequence, the second linker, and the
second nucleic acid sequence. In certain embodiments, the nucleic
acid comprises from 5' to 3': the second nucleic acid sequence, the
second linker, and the first nucleic acid sequence.
[0043] In another aspect, the invention includes an expression
vector comprising any of the nucleic acids disclosed herein. In
certain embodiments, (a) the expression vector is a viral vector
selected from the group consisting of a retroviral vector, a
lentiviral vector, an adenoviral vector, and an adeno-associated
viral vector; and/or (b) the expression vector is a lentiviral
vector, and optionally wherein the lentiviral vector is a
self-inactivating lentiviral vector.
[0044] In another aspect, the invention includes a method of
treating cancer in a subject in need thereof. The method comprises:
(a) administering any of the modified cells or any of the
pharmaceutical composition disclosed herein to the subject; and/or
(b) administering a modified T cell comprising a chimeric cell
surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising a sialidase (neuraminidase) or an
enzymatically functional portion thereof, and a heterologous
transmembrane domain capable of tethering the extracellular portion
to a cell surface, and a chimeric antigen receptor (CAR); and/or
(c) administering a modified T cell comprising a variant sialidase
precursor protein comprising a heterologous secretory sequence
operably linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof, wherein the variant sialidase precursor
protein lacks a transmembrane domain, and wherein the sialidase or
enzymatically functional portion thereof is capable of being
secreted from an immune or precursor cell thereof when the variant
sialidase precursor protein is expressed in the cell, and a
chimeric antigen receptor (CAR); and/or (d) administering to the
subject a therapeutically effective amount of a modified T cell
comprising a chimeric cell surface sialidase (neuraminidase) enzyme
comprising an extracellular portion comprising a sialidase
(neuraminidase) or an enzymatically functional portion thereof, and
a heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface, and a chimeric antigen
receptor (CAR); and administering to the subject a therapeutically
effective amount of a NK cell; and/or (e) administering to the
subject a therapeutically effective amount of a modified T cell
comprising a variant sialidase precursor protein comprising a
heterologous secretory sequence operably linked to a sialidase
(neuraminidase) or an enzymatically functional portion thereof,
wherein the variant sialidase precursor protein lacks a
transmembrane domain, and wherein the sialidase or enzymatically
functional portion thereof is capable of being secreted from an
immune or precursor cell thereof when the variant sialidase
precursor protein is expressed in the cell, and a chimeric antigen
receptor (CAR); and administering to the subject a therapeutically
effective amount of a NK cell.
[0045] In certain embodiments, the CAR comprises specificity for
TnMUC1; and/or the CAR comprises specificity for CD19; and/or the
CAR comprises specificity for PSMA.
[0046] In certain embodiments, the method further comprises
administering to the subject a population of innate immune cells,
optionally wherein the innate immune cells are NK cells; and/or the
innate immune cells are NK cells and the NK cells are autologous NK
cells obtained from a human subject; and/or the modified T cell and
the NK cell are administered simultaneously; and/or the modified T
cell and the NK cell are administered separately; and/or the NK
cell is autologous; and/or the modified T cell is autologous;
and/or the subject is human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The foregoing and other features and advantages of the
present invention will be more fully understood from the following
detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings.
[0048] FIGS. 1A-1C depict schematics of CAR-T cells with sialidase
enzymatic activity. T cells bearing sialidase/neuraminidase
activity can cleave inhibitory sialic acids on tumor cells, thereby
enhancing the anti-tumor efficacy of Siglec-expressing innate
immune cells, such as NK and monocytes. FIG. 1A illustrates an
iteration of a chimeric sialidase cell-surface receptor that
includes T cell signaling domains from 4-1BB and CD3zeta. FIG. 1B
illustrates a chimeric sialidase cell-surface receptor without
intracellular T cell signaling domains. FIG. 1C illustrates
secreted sialidase activity.
[0049] FIG. 2 depicts chimeric siladase/neuraminidase constructs.
Constructs include pTRPE-Neu2-BBz, pTRPE-Neu2-Dz,
pTRPE-Myc-Neu2-BBz, pTRPE-Myc-Neu2-Dz, Neu2, Neu1, Neu3, Neu4, and
5E5-P2A-Neu2.
[0050] FIG. 3 depicts surface detection of 293T cells transfected
with the Myc-Tagged Neu2-BBz construct.
[0051] FIG. 4 depicts surface expression of sialic acid on PC3
cells after purified sialidase treatment and recovery periods. This
figure demonstrates cleavage of sialic acid from the surface of PC3
tumor cells using sialidase enzyme derived from Clostridium
perfringens. The bottom-most flow cytometry histogram represents
SNA lectin staining on PC3 cells that were not treated with
sialidase, a staining control for sialic acid on PC3 cells. The
histogram third from the top represents PC3 cells that were treated
with sialidase for lhr at 37 degrees C. and given no recovery time
to restore surface sialic acid expression. There is a significant
decrease in MFI for SNA staining on these cells. SNA staining after
one hour and 2-hour recovery periods demonstrate sialic acid
increase with recovery time (the top two histograms respectively).
The changes in MFI are plotted in the bar graph.
[0052] FIGS. 5A-5F depict the finding that engineered Neu2-BBz T
cells demonstrate sialidase activity after 24 hr co-culture. Human
sialidase/neuraminidase-expressing T cells exhibit the ability to
reduce surface sialic acid expression after co-culture with PC3 and
DU145 tumor cells, as evidenced by SNA staining. There is a
decrease in SNA staining after co-culture with Neu2-BBz T cells
compared with NTD T cells (FIG. 5B compared to FIG. 5A; FIG. 5E
compared to FIG. 5D). FIGS. 5B and 5E represent at 10:1
effector:target ratio of Neu2-BBz T cells:tumor, and show greater
activity compared with FIGS. 5C and 5F, which represent a 5:1
effector:target ratio of Neu2-BBz T cells to tumor cells.
[0053] FIG. 6 depicts 24 hr co-culture analysis repeated comparing
MFI.
[0054] FIG. 7 depicts the finding that addition of sialidase and NK
cells enhances IFN-g production of CART cells targeting prostate
cancer PC3 cells. 5E5-CD2z CART cells demonstrate reactivity to PC3
prostate cancer cells in co-culture alone. Interestingly, IFN-g
secretion is elevated when both sialidase and NK cells are also
added at the time of co-culture with 5E5-CD2z CART cells. This
combination approach shows greater reactivity than with each
effector condition alone.
[0055] FIG. 8A-8C depicts the finding that sialidase activity
promotes synergistic cytotoxicity effects of CART and NK cells.
Results from cytotoxicity assays of PC3 prostate cancer cells
cultured with human T cells are shown. FIG. 8A illustrates at 1:1
effector:target ratio, 5E5-CD2z CAR T cells and NTD T cells exhibit
no cytotoxic effects against PC3 tumor cells. FIG. 8B illustrates
that there is no increased cytotoxicity through the addition of
sialidase or NK cells to the 5E5-CART cells. However, when
sialidase AND NK cells are added to the 5E5-CART cells, there is
virtually complete lysis of PC3 cells, approximating that of the
positive lysis control, Triton. Of note, this effect is not
observed with 5E5-CART alone, NK cells alone, 5E5-CART+ sialidase,
or 5E5-CART+NK cells. FIG. 8C shows the synergy of T cells, NK
cells, and sialidase activity is not observed with NTD T cells,
demonstrating that CAR activity is required for increased
cytotoxicity from this combination. This data suggests that
5E5-CART cell cytotoxicity can cooperate with unmodified innate
immune cells, such as NK cells, through the addition of sialidase
activity.
[0056] FIG. 9 depicts the finding that engineered Neu2-BBz T cells
or sialidase activity can also enhance the anti-tumor activity of
CD19-BBz treatment against leukemic cells. CD19-BBz CART cell
effector function can be enhanced with the addition of
neuraminidase-expressing T cells and NK cells, much like in
cultures with bacterial sialidase and NK cells (no significant
difference between treatment with the addition of Neu2-BBz+NK and
Sialidase+NK). This data demonstrates the potential for the
invention to enhance CAR-T cell immunotherapies beyond the
5E5-CAR.
[0057] FIG. 10 depicts the finding that engineered Neu2-BBz T cells
can enhance the anti-tumor activity of prostate cancer CART
treatment. The reactivity of PSMA-BBz CART cells and 5E5-CART cells
against aggressive prostate cancer cell line PC3 is significantly
increased by human sialidase/neuraminidase T cells and NK
cells.
[0058] FIG. 11 depicts results from a neuraminidase activity assay
that compares the enzymatic function of engineered receptors. A
fluorometric neuraminidase functional assay using the artificial
substrate 4-MU-NANA. Neuraminidase catalyzes the release of
fluorescent 4-MU from 4-MU-NANA, and the fluorescence emissions is
quantified. The panel to the left shows the normalized sialidase
function of various forms of the neuraminidase receptors present on
transfected 293T cells compared to non-transfected. The
Neu1-Dz(delta zeta) receptor showed the greatest enzymatic activity
followed by Neu2-BBz. The panel to the right shows various units of
bacterial sialidase and corresponding function as determined by
fluorescence emission. This information was used as a standard when
calculating fluorescent readout for activity produced by the
receptors.
[0059] FIGS. 12A-12E depict the finding that dual expressing human
sialidase-5E5-CAR-T cells exhibit rapid synergistic cytotoxicity
against PC3 tumor cells when co-cultured with NK cells. Normal
donor human T cells were lentivirually transduced with the
construct indicated. Non-transduced (NTD) cells were used as a
control. FIG. 12A depicts NTD T cells gated on protein L and
anti-Myc tag antibody staining. FIG. 12B depicts T cells transduced
with the single lentiviral vector 5E5-BBz, gated on protein L and
anti-Myc tag stainings. FIG. 12C depicts T cells single transduced
with lentiviral vector Myc-Neu1-Dz, gated on protein L and anti-Myc
tag staining. FIG. 12D depicts T cells double-transduced with both
the 5E5-BBz and Myc-Neu1-Dz lentiviral vectors (referred to as Dual
Expressing Sialidase-5E5 T cells), and gated on protein L and
anti-Myc tag antibodies. FIG. 12E depicts cytotoxic ability
assessed using xCELLigence RTCA. NK cells and effectors listed were
co-cultured at a 1:1 ratio with PC3 tumor cells. The
5E5+NK+Sialidase-T cell group is a 3-product co-culture; whereas
the Dual Expressing Sialidase-5E5 T cell+NK group is a 2-cell
product consisting of NK cells co-cultured with T cells expressing
both the Neuraminidase receptor and the 5E5-CAR.
[0060] FIGS. 13A-13H depict nucleotide and amino acid sequences of
constructs disclosed herein.
[0061] FIGS. 14A-14C depict additional sialidase nucleotide and
amino acid sequences.
DETAILED DESCRIPTION
[0062] The present invention is based on the discovery that CAR T
therapies may be improved by endowing glycoediting activity to the
T cell, which promotes synergistic cytotoxic effects of the
modified and endogenous immune system, mitigating some of the
boundaries to T cell infiltration and anti-tumor efficacy seen in
solid tumors.
[0063] In certain aspects, the present invention provides a
chimeric cell surface sialidase (neuraminidase) enzyme comprising
an extracellular portion comprising a sialidase (neuraminidase) or
an enzymatically functional portion thereof, and a heterologous
transmembrane domain capable of tethering the extracellular portion
to a cell surface. In another aspect, the invention provides a
variant sialidase precursor protein comprising a heterologous
secretory sequence operably linked to a sialidase (neuraminidase)
or an enzymatically functional portion thereof, and lacks a
transmembrane domain. The sialidase or enzymatically functional
portion thereof is capable of being secreted from an immune or
precursor cell thereof when the variant sialidase precursor protein
is expressed in the cell.
[0064] Also provided are compositions and methods for modified
immune cells or precursors thereof (e.g., modified T cells)
comprising a variant sialidase precursor protein or a chimeric cell
surface sialidase (neuraminidase) enzyme. In certain embodiments,
the modified immune cells or precursors thereof further comprise a
chimeric antigen receptor (CAR) and/or a T cell receptor (TCR).
[0065] Pharmaceutical compositions comprising a chimeric cell
surface sialidase or a variant sialidase precursor protein, nucleic
acids encoding a chimeric cell surface sialidase or a variant
sialidase precursor protein, and expression vectors comprising
nucleic acids comprising a chimeric cell surface sialidase or a
variant sialidase precursor protein, are also provided. The
invention also provides methods of treating cancer in a subject in
need thereof, comprising administering to the subject a modified T
cell comprising a CAR and a variant sialidase precursor protein or
a variant sialidase precursor protein. In certain embodiments, the
method further comprises administering a population of innate
immune cells (e.g. NK cells) to the subject.
[0066] It is to be understood that the methods described in this
disclosure are not limited to particular methods and experimental
conditions disclosed herein as such methods and conditions may
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0067] Furthermore, the experiments described herein, unless
otherwise indicated, use conventional molecular and cellular
biological and immunological techniques within the skill of the
art. Such techniques are well known to the skilled worker, and are
explained fully in the literature. See, e.g., Ausubel, et al., ed.,
Current Protocols in Molecular Biology, John Wiley & Sons,
Inc., NY, N.Y. (1987-2008), including all supplements, Molecular
Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J.
Sambrook and Harlow et al., Antibodies: A Laboratory Manual,
Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor
(2013, 2nd edition).
A. Definitions
[0068] Unless otherwise defined, scientific and technical terms
used herein have the meanings that are commonly understood by those
of ordinary skill in the art. In the event of any latent ambiguity,
definitions provided herein take precedent over any dictionary or
extrinsic definition. Unless otherwise required by context,
singular terms shall include pluralities and plural terms shall
include the singular. The use of "or" means "and/or" unless stated
otherwise. The use of the term "including," as well as other forms,
such as "includes" and "included," is not limiting.
[0069] Generally, nomenclature used in connection with cell and
tissue culture, molecular biology, immunology, microbiology,
genetics and protein and nucleic acid chemistry and hybridization
described herein is well-known and commonly used in the art. The
methods and techniques provided herein are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification unless
otherwise indicated. Enzymatic reactions and purification
techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well-known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0070] That the disclosure may be more readily understood, select
terms are defined below.
[0071] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0072] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0073] "Activation," as used herein, refers to the state of a T
cell that has been sufficiently stimulated to induce detectable
cellular proliferation. Activation can also be associated with
induced cytokine production, and detectable effector functions. The
term "activated T cells" refers to, among other things, T cells
that are undergoing cell division.
[0074] As used herein, to "alleviate" a disease means reducing the
severity of one or more symptoms of the disease.
[0075] The term "antigen" as used herein is defined as a molecule
that provokes an immune response. This immune response may involve
either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen.
[0076] Furthermore, antigens can be derived from recombinant or
genomic DNA. A skilled artisan will understand that any DNA, which
comprises a nucleotide sequences or a partial nucleotide sequence
encoding a protein that elicits an immune response therefore
encodes an "antigen" as that term is used herein. Furthermore, one
skilled in the art will understand that an antigen need not be
encoded solely by a full-length nucleotide sequence of a gene. It
is readily apparent that the present invention includes, but is not
limited to, the use of partial nucleotide sequences of more than
one gene and that these nucleotide sequences are arranged in
various combinations to elicit the desired immune response.
Moreover, a skilled artisan will understand that an antigen need
not be encoded by a "gene" at all. It is readily apparent that an
antigen can be generated synthesized or can be derived from a
biological sample. Such a biological sample can include, but is not
limited to a tissue sample, a tumor sample, a cell or a biological
fluid.
[0077] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to which it is later
to be re-introduced into the individual.
[0078] A "co-stimulatory molecule" refers to the cognate binding
partner on a T cell that specifically binds with a co-stimulatory
ligand, thereby mediating a co-stimulatory response by the T cell,
such as, but not limited to, proliferation. Co-stimulatory
molecules include, but are not limited to an WIC class I molecule,
BTLA and a Toll ligand receptor.
[0079] A "co-stimulatory signal", as used herein, refers to a
signal, which in combination with a primary signal, such as TCR/CD3
ligation, leads to T cell proliferation and/or upregulation or
downregulation of key molecules.
[0080] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in
which the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0081] The term "downregulation" as used herein refers to the
decrease or elimination of gene expression of one or more
genes.
[0082] "Effective amount" or "therapeutically effective amount" are
used interchangeably herein, and refer to an amount of a compound,
formulation, material, or composition, as described herein
effective to achieve a particular biological result or provides a
therapeutic or prophylactic benefit. Such results may include, but
are not limited to an amount that when administered to a mammal,
causes a detectable level of immune suppression or tolerance
compared to the immune response detected in the absence of the
composition of the invention. The immune response can be readily
assessed by a plethora of art-recognized methods. The skilled
artisan would understand that the amount of the composition
administered herein varies and can be readily determined based on a
number of factors such as the disease or condition being treated,
the age and health and physical condition of the mammal being
treated, the severity of the disease, the particular compound being
administered, and the like.
[0083] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0084] As used herein "endogenous" refers to any material from or
produced inside an organism, cell, tissue or system.
[0085] The term "epitope" as used herein is defined as a small
chemical molecule on an antigen that can elicit an immune response,
inducing B and/or T cell responses. An antigen can have one or more
epitopes. Most antigens have many epitopes; i.e., they are
multivalent. In general, an epitope is roughly about 10 amino acids
and/or sugars in size. Preferably, the epitope is about 4-18 amino
acids, more preferably about 5-16 amino acids, and even more most
preferably 6-14 amino acids, more preferably about 7-12, and most
preferably about 8-10 amino acids. One skilled in the art
understands that generally the overall three-dimensional structure,
rather than the specific linear sequence of the molecule, is the
main criterion of antigenic specificity and therefore distinguishes
one epitope from another. Based on the present disclosure, a
peptide used in the present invention can be an epitope.
[0086] As used herein, the term "exogenous" refers to any material
introduced from or produced outside an organism, cell, tissue or
system.
[0087] The term "expand" as used herein refers to increasing in
number, as in an increase in the number of T cells. In one
embodiment, the T cells that are expanded ex vivo increase in
number relative to the number originally present in the culture. In
another embodiment, the T cells that are expanded ex vivo increase
in number relative to other cell types in the culture. The term "ex
vivo," as used herein, refers to cells that have been removed from
a living organism, (e.g., a human) and propagated outside the
organism (e.g., in a culture dish, test tube, or bioreactor).
[0088] The term "expression" as used herein is defined as the
transcription and/or translation of a particular nucleotide
sequence driven by its promoter.
[0089] "Expression vector" refers to a vector comprising a
recombinant polynucleotide comprising expression control sequences
operatively linked to a nucleotide sequence to be expressed. An
expression vector comprises sufficient cis-acting elements for
expression; other elements for expression can be supplied by the
host cell or in an in vitro expression system. Expression vectors
include all those known in the art, such as cosmids, plasmids
(e.g., naked or contained in liposomes) and viruses (e.g., Sendai
viruses, lentiviruses, retroviruses, adenoviruses, and
adeno-associated viruses) that incorporate the recombinant
polynucleotide.
[0090] "Identity" as used herein refers to the subunit sequence
identity between two polymeric molecules particularly between two
amino acid molecules, such as, between two polypeptide molecules.
When two amino acid sequences have the same residues at the same
positions; e.g., if a position in each of two polypeptide molecules
is occupied by an arginine, then they are identical at that
position. The identity or extent to which two amino acid sequences
have the same residues at the same positions in an alignment is
often expressed as a percentage. The identity between two amino
acid sequences is a direct function of the number of matching or
identical positions; e.g., if half (e.g., five positions in a
polymer ten amino acids in length) of the positions in two
sequences are identical, the two sequences are 50% identical; if
90% of the positions (e.g., 9 of 10), are matched or identical, the
two amino acids sequences are 90% identical.
[0091] The term "immune response" as used herein is defined as a
cellular response to an antigen that occurs when lymphocytes
identify antigenic molecules as foreign and induce the formation of
antibodies and/or activate lymphocytes to remove the antigen.
[0092] The term "immunosuppressive" is used herein to refer to
reducing overall immune response.
[0093] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a peptide naturally present in a
living animal is not "isolated," but the same nucleic acid or
peptide partially or completely separated from the coexisting
materials of its natural state is "isolated." An isolated nucleic
acid or protein can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a host
cell.
[0094] A "lentivirus" as used herein refers to a genus of the
Retroviridae family. Lentiviruses are unique among the retroviruses
in being able to infect non-dividing cells; they can deliver a
significant amount of genetic information into the DNA of the host
cell, so they are one of the most efficient methods of a gene
delivery vector. HIV, SIV, and FIV are all examples of
lentiviruses. Vectors derived from lentiviruses offer the means to
achieve significant levels of gene transfer in vivo.
[0095] By the term "modified" as used herein, is meant a changed
state or structure of a molecule or cell of the invention.
Molecules may be modified in many ways, including chemically,
structurally, and functionally. Cells may be modified through the
introduction of nucleic acids.
[0096] By the term "modulating," as used herein, is meant mediating
a detectable increase or decrease in the level of a response in a
subject compared with the level of a response in the subject in the
absence of a treatment or compound, and/or compared with the level
of a response in an otherwise identical but untreated subject. The
term encompasses perturbing and/or affecting a native signal or
response thereby mediating a beneficial therapeutic response in a
subject, preferably, a human.
[0097] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0098] The term "oligonucleotide" typically refers to short
polynucleotides. It will be understood that when a nucleotide
sequence is represented by a DNA sequence (i.e., A, T, C, G), this
also includes an RNA sequence (i.e., A, U, C, G) in which "U"
replaces "T."
[0099] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or an RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0100] "Parenteral" administration of an immunogenic composition
includes, e.g., subcutaneous (s.c.), intravenous (i.v.),
intramuscular (i.m.), or intrasternal injection, or infusion
techniques.
[0101] The term "polynucleotide" as used herein is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, nucleic acids and polynucleotides as used herein
are interchangeable. One skilled in the art has the general
knowledge that nucleic acids are polynucleotides, which can be
hydrolyzed into the monomeric "nucleotides." The monomeric
nucleotides can be hydrolyzed into nucleosides. As used herein
polynucleotides include, but are not limited to, all nucleic acid
sequences which are obtained by any means available in the art,
including, without limitation, recombinant means, i.e., the cloning
of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR, and the like,
and by synthetic means.
[0102] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptides, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0103] By the term "specifically binds," as used herein with
respect to an antibody, is meant an antibody which recognizes a
specific antigen, but does not substantially recognize or bind
other molecules in a sample. For example, an antibody that
specifically binds to an antigen from one species may also bind to
that antigen from one or more species. But, such cross-species
reactivity does not itself alter the classification of an antibody
as specific. In another example, an antibody that specifically
binds to an antigen may also bind to different allelic forms of the
antigen. However, such cross reactivity does not itself alter the
classification of an antibody as specific. In some instances, the
terms "specific binding" or "specifically binding," can be used in
reference to the interaction of an antibody, a protein, or a
peptide with a second chemical species, to mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0104] By the term "stimulation," is meant a primary response
induced by binding of a stimulatory molecule (e.g., a TCR/CD3
complex) with its cognate ligand thereby mediating a signal
transduction event, such as, but not limited to, signal
transduction via the TCR/CD3 complex. Stimulation can mediate
altered expression of certain molecules, such as downregulation of
TGF-beta, and/or reorganization of cytoskeletal structures, and the
like.
[0105] A "stimulatory molecule," as the term is used herein, means
a molecule on a T cell that specifically binds with a cognate
stimulatory ligand present on an antigen presenting cell.
[0106] A "stimulatory ligand," as used herein, means a ligand that
when present on an antigen presenting cell (e.g., an aAPC, a
dendritic cell, a B-cell, and the like) can specifically bind with
a cognate binding partner (referred to herein as a "stimulatory
molecule") on a T cell, thereby mediating a primary response by the
T cell, including, but not limited to, activation, initiation of an
immune response, proliferation, and the like. Stimulatory ligands
are well-known in the art and encompass, inter alia, an MHC Class I
molecule loaded with a peptide, an anti-CD3 antibody, a
superagonist anti-CD28 antibody, and a superagonist anti-CD2
antibody.
[0107] The term "subject" is intended to include living organisms
in which an immune response can be elicited (e.g., mammals). A
"subject" or "patient," as used therein, may be a human or
non-human mammal. Non-human mammals include, for example, livestock
and pets, such as ovine, bovine, porcine, canine, feline and murine
mammals. Preferably, the subject is human.
[0108] A "target site" or "target sequence" refers to a nucleic
acid sequence that defines a portion of a nucleic acid to which a
binding molecule may specifically bind under conditions sufficient
for binding to occur. In some embodiments, a target sequence refers
to a genomic nueleic acid sequence that defines a portion of a
nucleic acid to which a binding molecule may specifically bind
under conditions sufficient for binding to occur.
[0109] As used herein, the term "T cell receptor" or "TCR" refers
to a complex of membrane proteins that participate in the
activation of T cells in response to the presentation of antigen.
The TCR is responsible for recognizing antigens bound to major
histocompatibility complex molecules. TCR is composed of a
heterodimer of an alpha (.alpha.) and beta (.beta.) chain, although
in some cells the TCR consists of gamma and delta (.gamma./.delta.)
chains. TCRs may exist in alpha/beta and gamma/delta forms, which
are structurally similar but have distinct anatomical locations and
functions. Each chain is composed of two extracellular domains, a
variable and constant domain. In some embodiments, the TCR may be
modified on any cell comprising a TCR, including, for example, a
helper T cell, a cytotoxic T cell, a memory T cell, regulatory T
cell, natural killer T cell, and gamma delta T cell.
[0110] The term "therapeutic" as used herein means a treatment
and/or prophylaxis. A therapeutic effect is obtained by
suppression, remission, or eradication of a disease state.
[0111] The term "transfected" or "transformed" or "transduced" as
used herein refers to a process by which exogenous nucleic acid is
transferred or introduced into the host cell. A "transfected" or
"transformed" or "transduced" cell is one which has been
transfected, transformed or transduced with exogenous nucleic acid.
The cell includes the primary subject cell and its progeny.
[0112] To "treat" a disease as the term is used herein, means to
reduce the frequency or severity of at least one sign or symptom of
a disease or disorder experienced by a subject.
[0113] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. The term should also
be construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for
example, polylysine compounds, liposomes, and the like. Examples of
viral vectors include, but are not limited to, Sendai viral
vectors, adenoviral vectors, adeno-associated virus vectors,
retroviral vectors, lentiviral vectors, and the like.
[0114] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
B. Chimeric Cell Surface Sialidases
[0115] The present invention provides a chimeric cell surface
sialidase (neuraminidase) enzyme comprising an extracellular
portion comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell
surface.
[0116] The sialidase may be derived from any species. In certain
embodiments, the sialidase is from a human. Examples of human
sialidases include, but are not limited to, sialidase 1 (Neul),
sialidase 2 (Neu2), sialidase 3 (Neu3), and sialidase 4 (Neu4). In
certain embodiments, the sialidase is Neu2. In certain embodiments,
the sialidase is humanized.
[0117] In certain embodiments, the sialidase comprises Neu2
comprising the amino acid sequence set forth in SEQ ID NO: 13 and
may be encoded by the nucleotide sequence set forth in SEQ ID NO:
4. In certain embodiments, the sialidase comprises Neu2 (without a
signal peptide) comprising the amino acid sequence set forth in SEQ
ID NO: 34 and may be encoded by the nucleotide sequence set forth
in SEQ ID NO: 30. In certain embodiments, the sialidase comprises
Neul comprising the amino acid sequence set forth in SEQ ID NO: 33
and may be encoded by the nucleotide sequence set forth in SEQ ID
NO: 29. In certain embodiments, the sialidase comprises Neu3
comprising the amino acid sequence set forth in SEQ ID NO: 35 and
may be encoded by the nucleotide sequence set forth in SEQ ID NO:
31. In certain embodiments, the sialidase comprises Neu4 comprising
the amino acid sequence set forth in SEQ ID NO: 36 and may be
encoded by the nucleotide sequence set forth in SEQ ID NO: 32.
[0118] Tolerable variations of the sialidase or an enzymatically
functional portion thereof will be known to those of skill in the
art. For example, in some embodiments the sialidase or an
enzymatically functional portion thereof comprises an amino acid
sequence that has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% sequence identity to any of the amino
acid sequences set forth in SEQ ID NO: 13, 33, 34, 35, or 36. In
some embodiments the sialidase or an enzymatically functional
portion thereof is encoded by a nucleic acid sequence that has at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least or
99% sequence identity to the nucleic acid sequence set forth in SEQ
ID NO: 4, 29, 30, 31 or 32.
[0119] In some embodiments, the sialidase or an enzymatically
functional portion thereof consists of an amino acid sequence set
forth in SEQ ID NO: 13, 33, 34, 35, or 36. In some embodiments, the
sialidase or an enzymatically functional portion thereof is encoded
by a nucleic acid sequence set forth in SEQ ID NO: 4, 29, 30, 31 or
32.
[0120] The chimeric cell surface sialidase comprises a heterologous
transmembrane domain capable of tethering the extracellular portion
to a cell surface. The transmemebrane domain of the chimeric cell
surface sialidase may comprise any of the transmembrane domains
described elsewhere herein (e.g. any CAR transmembrane domain). In
certain embodiments, the transmembrane domain is selected from the
group consisting of an artificial hydrophobic sequence, and a
transmembrane domain of a type I transmembrane protein, an alpha,
beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45,
CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40
(CD134), 4-1BB (CD137), and CD154. In certain embodiments, the
transmembrane domain comprises a transmembrane domain of CD8. In
certain embodiments, the transmembrane domain comprises a
transmembrane domain of CD8 alpha. In certain embodiments, the
transmembrane domain comprises the amino acid sequence set forth in
SEQ ID NO: 16. In certain embodiments, the transmembrane domain
consists of the amino acid sequence set forth in SEQ ID NO: 16. In
certain embodiments, the transmembrane domain is encoded by the
nucleotide sequence set forth in SEQ ID NO: 7.
[0121] The chimeric cell surface sialidase may optionally comprise
a hinge domain. The hinge domain may comprise any of the hinge
domains described in detail elsewhere herein. Hinge domains that
may be included in the chimeric cell surface sialidase include, but
are not limited, to an Fc fragment of an antibody, a hinge region
of an antibody, a CH2 region of an antibody, a CH3 region of an
antibody, an artificial hinge domain, a hinge comprising an amino
acid sequence of CD8, or any combination thereof. In certain
embodiments, the hinge domain is a hinge comprising an amino acid
sequence of CD8 or encoded by a nucleotide sequence of CD8. In
certain exemplary embodiments, the hinge domain is a hinge
comprising an amino acid sequence of CD8 alpha or encoded by a
nucleotide sequence of CD8 alpha. In certain exemplary embodiments,
the hinge domain comprises the amino acid sequence set forth in SEQ
ID NO: 15. In some embodiments, the hinge domain consists of the
amino acid sequence set forth in SEQ ID NO: 15. In certain
exemplary embodiments, the hinge domain is encoded by the
nucleotide sequence set forth in SEQ ID NO: 6.
[0122] The chimeric cell surface sialidase may optionally comprise
an intracellular region comprising a costimulatory signaling domain
and/or an intracellular signaling domain. The intracellular region
may comprise any of the intracellular domains or any of the
costimulatory signaling domains or any of the intracellular
signaling domains described herein. In certain embodiments, the
costimulatory signaling domain comprises a costimulatory domain of
a protein selected from the group consisting of proteins in the
TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7,
LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck,
TNFR-I, TNFI-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276),
or a variant thereof. In certain embodiments, the costimulatory
signaling domain comprises a costimulatory domain of 4-1BB. In
certain embodiments, the costimulatory signaling domain comprises
the amino acid sequence set forth in SEQ ID NO: 17. In some
embodiments, the costimulatory signaling domain consists of the
amino acid sequence set forth in SEQ ID NO: 17. In certain
embodiments, the costimulatory signaling domain is encoded by the
nucleotide sequence set forth in SEQ ID NO: 8.
[0123] In certain embodiments, the intracellular signaling domain
of the chimeric cell surface sialidase comprises an intracellular
domain selected from the group consisting of cytoplasmic signaling
domains of a human CD3 zeta chain (CD3z), Fc.gamma.RIII, FcsRI, a
cytoplasmic tail of an Fc receptor, an immunoreceptor
tyrosine-based activation motif (ITAM) bearing cytoplasmic
receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon,
CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. In
certain embodiments, the intracellular signaling domain comprises
an intracellular domain of CD3z. In certain embodiments, the
intracellular signaling domain comprises the amino acid sequence
set forth in SEQ ID NO: 18. In some embodiments, the intracellular
signaling domain consists of the amino acid sequence set forth in
SEQ ID NO: 18. In certain embodiments, the intracellular signaling
domain is encoded by the nucleotide sequence set forth in SEQ ID
NO: 9.
[0124] Tolerable variations of the hinge or transmembrane domain or
costimulatory signaling domain or intracellular signaling domain
sequences will be known to those of skill in the art. For example,
in some embodiments the hinge or transmembrane domain or
costimulatory signaling domain or intracellular signaling domain
comprises an amino acid sequence that has at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to any of the amino acid sequences set forth in SEQ ID NO:
15, 16, 17, or 18. In some embodiments hinge or transmembrane
domain or costimulatory signaling domain or intracellular signaling
domain is encoded by a nucleic acid sequence that has at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least or 99%
sequence identity to the nucleic acid sequence set forth in SEQ ID
NO: 6, 7, 8, or 9.
[0125] In some embodiments, the hinge or transmembrane domain or
costimulatory signaling domain or intracellular signaling domain
consists of the amino acid sequence set forth in SEQ ID NO: 15, 16,
17, or 18. In some embodiments, the hinge or transmembrane domain
or costimulatory signaling domain or intracellular signaling domain
is encoded by the nucleic acid sequence set forth in SEQ ID NO: 6,
7, 8, or 9.
[0126] In some embodiments, the chimeric cell surface sialidase
comprises an amino acid sequence that has at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to any one of SEQ ID NOs: 10, 21, 25, 28, 37, or 38. In
some embodiments, the chimeric cell surface sialidase comprises is
encoded by a nucleic acid sequence that has at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to any one of SEQ ID NOs: 1, 19, 23, 27, 39 or 40. In
certain embodiments, the chimeric cell surface sialidase comprises
the amino acid sequence set forth in any one of SEQ ID NOs: 10, 21,
25, 28, 37, or 38. In certain embodiments, the chimeric cell
surface sialidase consists of the amino acid sequence set forth in
any one of SEQ ID NOs: 10, 21, 25, 28, 37, or 38. In certain
embodiments, the cell surface sialidase is encoded by the nucleic
acid set forth in any one of SEQ ID NOs: 1, 19, 23, 27, 39 or
40.
TABLE-US-00001 Neu2-BBz AA sequence (SEQ ID NO: 10)
MALPVTALLLPLALLLHAARPGSMASLPVLQKESVFQSGAHAYRIPALLY
LPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQA
RLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQV
TSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVP
AYAYRKLHPIQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETG
EQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVI
SFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLL
AKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEY
LPQSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED
GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDV
LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK
GHDGLYQGLSTATKDTYDALHMQALPPR Neu2-BBz (no leader) AA sequence (SEQ
ID NO: 37) MASLPVLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAE
LIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFL
FFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPA
YREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPIQRPIPSAFCFLSH
DHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTN
DGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTH
SWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLF
GCLYEANDYEEIVFLMFTLKQAFPAEYLPQSGTTTPAPRPPTPAPTIASQ
PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY
CKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
ADAPAYKQGQNOLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG
LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR
Neu2-hinge-Tm AA sequence (SEQ ID NO: 38)
MALPVTALLLPLALLLHAARPGSMASLPVLQKESVFQSGAHAYRIPALLY
LPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTFIQVQWQAQEVVAQ
ARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQ
VTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVV
PAYAYRKLHPIQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVET
GEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSV
ISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVL
LAKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAE
YLPQSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC
DIYIWAPLAGTCGVLLLSLVITLYC Neu2-BBz nucleotide sequence (SEQ ID NO:
1) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCA
CGCCGCCAGGCCGGGATCCATGGCTTCCTTGCCGGTGCTGCAAAAAGAGA
GCGTATTCCAGTCAGGCGCCCATGCGTATAGAATCCCGGCACTTCTCTAT
TTGCCGGGCCAACAAAGTCTCTTGGCGTTCGCGGAACAGCGGGCGTCCAA
AAAAGACGAACACGCCGAGTTGATTGTGCTCCGCCGCGGGGATTATGATG
CCCCAACGCATCAGGTTCAGTGGCAGGCACAAGAGGTAGTCGCTCAGGCG
CGACTGGATGGACATCGGTCAATGAACCCATGTCCACTGTACGATGCTCA
GACAGGTACGTTGTTTCTGTTCTTCATCGCTATCCCTGGGCAAGTAACAG
AACAACAACAACTGCAAACCAGAGCCAATGTAACAAGACTCTGCCAGGTA
ACTAGCACTGACCACGGACGAACGTGGTCTTCCCCTAGAGATCTTACTGA
CGCCGCAATCGGGCCTGCATATCGCGAATGGAGCACTTTCGCAGTAGGCC
CTGGTCATTGCCTGCAACTCCATGATCGCGCCCGATCACTTGTGGTGCCA
GCGTACGCATACCGGAAGCTCCATCCAATACAACGCCCCATCCCGTCCGC
TTTTTGTTTCCTCTCCCATGACCACGGGCGGACTTGGGCGCGGGGTCATT
TCGTCGCACAGGATACGTTGGAGTGTCAGGTAGCGGAAGTAGAAACCGGG
GAGCAGAGAGTGGTCACTCTCAACGCGCGCAGTCATCTTCGCGCCCGCGT
ACAGGCGCAGAGCACTAATGACGGGCTTGATTTTCAAGAAAGTCAACTCG
TCAAAAAGTTGGTTGAACCGCCCCCGCAGGGCTGTCAAGGTTCAGTTATA
AGTTTTCCAAGTCCACGCTCCGGTCCAGGATCACCAGCACAGTGGCTTCT
CTACACCCATCCCACCCACAGCTGGCAGCGGGCAGATCTTGGTGCTTACT
TGAATCCCAGGCCACCGGCCCCCGAAGCCTGGAGCGAGCCTGTACTGCTT
GCAAAGGGGAGCTGTGCGTACTCTGATCTCCAGTCAATGGGTACTGGACC
AGATGGGAGTCCATTGTTTGGTTGTCTCTACGAGGCGAACGATTATGAGG
AAATCGTTTTTCTTATGTTTACTTTGAAACAGGCGTTCCCAGCCGAATAT
TTGCCTCAGTCCGGAACCACGACGCCAGCGCCGCGACCACCAACACCGGC
GCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC
CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTC
ACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATA
TATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAG
AGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGA
ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT
TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG
GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGG
CGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAG
GGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTA
CGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC Neu2-BBz (no leader) nucleotide
sequence (SEQ ID NO: 39)
ATGGCTTCCTTGCCGGTGCTGCAAAAAGAGAGCGTATTCCAGTCAGGCGC
CCATGCGTATAGAATCCCGGCACTTCTCTATTTGCCGGGCCAACAAAGTC
TCTTGGCGTTCGCGGAACAGCGGGCGTCCAAAAAAGACGAACACGCCGAG
TTGATTGTGCTCCGCCGCGGGGATTATGATGCCCCAACGCATCAGGTTCA
GTGGCAGGCACAAGAGGTAGTCGCTCAGGCGCGACTGGATGGACATCGGT
CAATGAACCCATGTCCACTGTACGATGCTCAGACAGGTACGTTGTTTCTG
TTCTTCATCGCTATCCCTGGGCAAGTAACAGAACAACAACAACTGCAAAC
CAGAGCCAATGTAACAAGACTCTGCCAGGTAACTAGCACTGACCACGGAC
GAACGTGGTCTTCCCCTAGAGATCTTACTGACGCCGCAATCGGGCCTGCA
TATCGCGAATGGAGCACTTTCGCAGTAGGCCCTGGTCATTGCCTGCAACT
CCATGATCGCGCCCGATCACTTGTGGTGCCAGCGTACGCATACCGGAAGC
TCCATCCAATACAACGCCCCATCCCGTCCGCTTTTTGTTTCCTCTCCCAT
GACCACGGGCGGACTTGGGCGCGGGGTCATTTCGTCGCACAGGATACGTT
GGAGTGTCAGGTAGCGGAAGTAGAAACCGGGGAGCAGAGAGTGGTCACTC
TCAACGCGCGCAGTCATCTTCGCGCCCGCGTACAGGCGCAGAGCACTAAT
GACGGGCTTGATTTTCAAGAAAGTCAACTCGTCAAAAAGTTGGTTGAACC
GCCCCCGCAGGGCTGTCAAGGTTCAGTTATAAGTTTTCCAAGTCCACGCT
CCGGTCCAGGATCACCAGCACAGTGGCTTCTCTACACCCATCCCACCCAC
AGCTGGCAGCGGGCAGATCTTGGTGCTTACTTGAATCCCAGGCCACCGGC
CCCCGAAGCCTGGAGCGAGCCTGTACTGCTTGCAAAGGGGAGCTGTGCGT
ACTCTGATCTCCAGTCAATGGGTACTGGACCAGATGGGAGTCCATTGTTT
GGTTGTCTCTACGAGGCGAACGATTATGAGGAAATCGTTTTTCTTATGTT
TACTTTGAAACAGGCGTTCCCAGCCGAATATTTGCCTCAGTCCGGAACCA
CGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG
CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGT
GCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCT
TGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTAC
TGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTAT
GAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC
CAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGC
GCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCT
CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC
GGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC
CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGAT
TGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACC
AGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGC
Neu2-hinge-Tm nucleotide sequence (SEQ ID NO: 40)
ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCA
CGCCGCCAGGCCGGGATCCATGGCTTCCTTGCCGGTGCTGCAAAAAGAGA
GCGTATTCCAGTCAGGCGCCCATGCGTATAGAATCCCGGCACTTCTCTAT
TTGCCGGGCCAACAAAGTCTCTTGGCGTTCGCGGAACAGCGGGCGTCCAA
AAAAGACGAACACGCCGAGTTGATTGTGCTCCGCCGCGGGGATTATGATG
CCCCAACGCATCAGGTTCAGTGGCAGGCACAAGAGGTAGTCGCTCAGGCG
CGACTGGATGGACATCGGTCAATGAACCCATGTCCACTGTACGATGCTCA
GACAGGTACGTTGTTTCTGTTCTTCATCGCTATCCCTGGGCAAGTAACAG
AACAACAACAACTGCAAACCAGAGCCAATGTAACAAGACTCTGCCAGGTA
ACTAGCACTGACCACGGACGAACGTGGTCTTCCCCTAGAGATCTTACTGA
CGCCGCAATCGGGCCTGCATATCGCGAATGGAGCACTTTCGCAGTAGGCC
CTGGTCATTGCCTGCAACTCCATGATCGCGCCCGATCACTTGTGGTGCCA
GCGTACGCATACCGGAAGCTCCATCCAATACAACGCCCCATCCCGTCCGC
TTTTTGTTTCCTCTCCCATGACCACGGGCGGACTTGGGCGCGGGGTCATT
TCGTCGCACAGGATACGTTGGAGTGTCAGGTAGCGGAAGTAGAAACCGGG
GAGCAGAGAGTGGTCACTCTCAACGCGCGCAGTCATCTTCGCGCCCGCGT
ACAGGCGCAGAGCACTAATGACGGGCTTGATTTTCAAGAAAGTCAACTCG
TCAAAAAGTTGGTTGAACCGCCCCCGCAGGGCTGTCAAGGTTCAGTTATA
AGTTTTCCAAGTCCACGCTCCGGTCCAGGATCACCAGCACAGTGGCTTCT
CTACACCCATCCCACCCACAGCTGGCAGCGGGCAGATCTTGGTGCTTACT
TGAATCCCAGGCCACCGGCCCCCGAAGCCTGGAGCGAGCCTGTACTGCTT
GCAAAGGGGAGCTGTGCGTACTCTGATCTCCAGTCAATGGGTACTGGACC
AGATGGGAGTCCATTGTTTGGTTGTCTCTACGAGGCGAACGATTATGAGG
AAATCGTTTTTCTTATGTTTACTTTGAAACAGGCGTTCCCAGCCGAATAT
TTGCCTCAGTCCGGAACCACGACGCCAGCGCCGCGACCACCAACACCGGC
GCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGC
CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTC
ACTGGTTATCACCCTTTACTGC
C. Variant Sialidase Precursor Protein
[0127] The present invention provides a variant sialidase precursor
protein, comprising a heterologous secretory sequence operably
linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof. In some embodiments, the variant
sialidase precursor protein comprises a sialidase or an
enzymatically functional portion thereof, wherein the sialidase
lacks a transmembrane domain, and is capable of being secreted from
an immune or precursor cell thereof when the variant sialidase
precursor protein is expressed in the cell.
[0128] As used herein, a "heterologous secretory sequence" refers
to a peptide sequence that functions to prompt a cell to
translocate a protein (e.g., sialidase) that the heterologous
secretory sequence is operably linked to. A heterologous secretory
sequence may also be referred to as a signal peptide, a signal
sequence, a targeting sequence, a targeting signal, a localization
signal, a localization sequence, a transit peptide, a leader
sequence, a leader peptide, or any other term known in the art. The
heterologous secretory sequence may be a short peptide about of
16-30 amino acids in length, e.g., about 12, about 13, about 14,
about 15, about 16, about 17, about 18, about 19, about 20, about
21, about 22, about 23, about 24, about 25, about 26, about 27,
about 28, about 29, about 30, about 31, about 32, about 33, about
34, about 35, about 36 amino acids in length.
[0129] In some embodiments, the heterologous secretory sequence is
derived from a naturally occuring protein, for example, a naturally
secreted protein. The naturally secreted protein from which a
heterologous secretory sequence may be derived may be of, without
limitation, human, murine, bovine, camelid, bacterial, or yeast
origin. The skilled artisan would be able to choose the appropriate
naturally occuring protein and origin from which to derive a
heterologous secretory sequence. In certain embodiments, the
heterologous secretory sequence is derived from a naturally
secreted human protein. Examples of human secreted proteins may be
found in, e.g., U.S. Pat. No. 7,368,531, the contents of which are
hereby incorporated by reference in its entirety. Examples of
signal peptide-containing proteins may be found in, e.g., U.S. Pat.
No. 8,716,445, the contents of which are hereby incorporated by
reference in its entirety. In some embodiments, the heterologous
secretory sequence is a synthetic sequence, see, e.g., Clerico et
al., Biopolymers (2010) 90(3):307-319, the contents of which are
hereby incorporated by reference in its entirety.
[0130] In some embodiments, a variant sialidase precursor protein
comprises a heterologous secretory sequence operably linked
N-terminal to a sialidase or enzymatically functional protein
thereof. In some embodiments, a variant sialidase precursor protein
comprises a heterologous secretory sequence operably linked
C-terminal to a sialidase or enzymatically functional protein
thereof. The skilled artisan would readily be able to determine the
appropriate location of the heterologous secretory sequence in
order for it to perform its intended function, e.g., prompt the
cell to translocate the sialidase outside of the cell.
[0131] In some embodiments, a variant sialidase precursor protein
comprises a heterologous secretory sequence operably linked to a
sialidase or enzymatically functional portion thereof. The
sialidase or enzymatically functional portion thereof may be
derived from any species. Sialidases are found in various species,
including mammalian, murine, bacterial, and viral species. In
certain embodiments, the sialidase or enzymatically functional
portion is from a human. Examples of human sialidases include, but
are not limited to, sialidase 1 (Neul), sialidase 2 (Neu2),
sialidase 3 (Neu3), and sialidase 4 (Neu4). In certain embodiments,
the sialidase is Neu2 or an enzymatically functional portion of
Neu2. Accordingly, in some embodiments, a variant sialidase
precursor protein comprises a heterologous secretory sequence
operably linked to a sialidase or enzymatically functional portion
thereof selected from Neu1, Neu2, Neu3, or Neu4. In certain
embodiments, the sialidase is humanized.
[0132] In certain embodiments, a variant sialidase precursor
protein comprises a sialidase or enzymatically functional portion
thereof, wherein the sialidase comprises Neu2 comprising the amino
acid sequence set forth in SEQ ID NO: 34 or 13, and may be encoded
by the nucleotide sequence set forth in SEQ ID NO: 30 or 4. In
certain embodiments, a variant sialidase precursor protein
comprises a sialidase or enzymatically functional portion thereof,
wherein the sialidase comprises Neul comprising the amino acid
sequence set forth in SEQ ID NO: 33, and may be encoded by the
nucleotide sequence set forth in SEQ ID NO: 29. In certain
embodiments, a variant sialidase precursor protein comprises a
sialidase or enzymatically functional portion thereof, wherein the
sialidase comprises Neu3 comprising the amino acid sequence set
forth in SEQ ID NO: 35, and may be encoded by the nucleotide
sequence set forth in SEQ ID NO: 31. In certain embodiments, a
variant sialidase precursor protein comprises a sialidase or
enzymatically functional portion thereof, wherein the sialidase
comprises Neu4 comprising the amino acid sequence set forth in SEQ
ID NO: 36, and may be encoded by the nucleotide sequence set forth
in SEQ ID NO: 32.
[0133] In some embodiments, a variant sialidase precursor protein
comprises a heterologous secretory sequence operably linked to an
amino acid sequence that has at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% sequence identity to SEQ
ID NO: 33, 34, 35, or 36. In some embodiments, a variant sialidase
precursor protein comprises a heterologous secretory sequence
operably linked to an amino acid sequence comprising SEQ ID NO: 33,
34, 35, or 36. In some embodiments, a variant sialidase precursor
protein comprises a heterologous secretory sequence operably linked
to an amino acid sequence consisting of SEQ ID NO: 13, 33, 34, 35,
or 36.
[0134] In some embodiments, a variant sialidase precursor protein
comprises a heterologous secretory sequence operably linked to a
sequence encoded by a nucleic acid sequence that has at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%
sequence identity to SEQ ID NO: 29, 30, 31, or 32. In some
embodiments, a variant sialidase precursor protein comprises a
heterologous secretory sequence operably linked to a sequence
encoded by a nucleic acid sequence comprising SEQ ID NO: 29, 30,
31, or 32. In some embodiments, a variant sialidase precursor
protein comprises a heterologous secretory sequence operably linked
to a sequence encoded by a nucleic acid sequence consisting of SEQ
ID NO: 4, 29, 30, 31, or 32.
D. Modified Immune Cells
[0135] In one aspect, the present invention provides a modified
immune cell or precursor thereof (e.g., a T cell) comprising a
chimeric cell surface sialidase (neuraminidase) enzyme. The
chimeric cell surface silidase comprises an extracellular portion
comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell
surface.
[0136] Also provided is a modified immune cell or precursor cell
thereof, comprising a chimeric cell surface sialidase
(neuraminidase) enzyme and a chimeric antigen receptor (CAR) and/or
a T cell receptor (TCR). The chimeric cell surface silidase
comprises an extracellular portion comprising a sialidase
(neuraminidase) or an enzymatically functional portion thereof, and
a heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface.
[0137] In another aspect, the invention provides a modified immune
cell or precursor cell thereof, comprising a variant sialidase
precursor protein comprising a heterologous secretory sequence
operably linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof. The variant sialidase precursor protein
lacks a transmembrane domain, and the sialidase or enzymatically
functional portion thereof is capable of being secreted from an
immune or precursor cell when the variant sialidase precursor
protein is expressed in the cell.
[0138] Also provided is a modified immune cell or precursor cell
thereof, comprising a variant sialidase precursor protein and a
chimeric antigen receptor (CAR) and/or a T cell receptor (TCR). The
variant sialidase precursor protein comprises a heterologous
secretory sequence operably linked to a sialidase (neuraminidase)
or an enzymatically functional portion thereof and lacks a
transmembrane domain. The sialidase or enzymatically functional
portion thereof is capable of being secreted from an immune or
precursor cell thereof when the variant sialidase precursor protein
is expressed in the cell.
[0139] The modified immune cells or precursor cells thereof may
comprise any of the chimeric cell surface sialidase (neuraminidase)
enzymes disclosed and described in detail elsewhere herein.
[0140] The modified immune cells or precursor cells thereof may
comprise any of the CARS and/or any of the TCRs disclosed and
described in detail elsewhere herein.
[0141] In one aspect, the invention includes a modified immune cell
or precursor cell thereof, comprising a chimeric cell surface
sialidase (neuraminidase) enzyme and a chimeric antigen receptor
(CAR). The chimeric cell surface sialidase comprises an
extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell
surface.
[0142] In another aspect, the invention includes a modified immune
cell or precursor cell thereof, comprising a chimeric cell surface
sialidase (neuraminidase) enzyme and a chimeric antigen receptor
(CAR) having specificity for TnMUC1, CD19, or PSMA. The chimeric
cell surface sialidase comprises an extracellular portion
comprising Neu2 or an enzymatically functional portion thereof, and
a heterologous transmembrane domain capable of tethering the
extracellular portion to a cell surface.
[0143] In yet another aspect, the invention includes a modified
immune cell or precursor cell thereof, comprising a chimeric cell
surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface, a
hinge domain, and an intracellular region comprising a
costimulatory signaling domain and an intracellular signaling
domain; and a chimeric antigen receptor (CAR).
[0144] In still another aspect, the invention includes a modified
immune cell or precursor cell thereof, comprising: a chimeric cell
surface sialidase (neuraminidase) enzyme comprising an
extracellular portion comprising Neu2 or an enzymatically
functional portion thereof, a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface, a
hinge domain, and an intracellular region comprising a
costimulatory signaling domain and an intracellular signaling
domain; and a chimeric antigen receptor (CAR) having specificity
for TnMUC1, CD19, or PSMA.
[0145] Another aspect of the invention includes a modified immune
cell or precursor cell thereof, comprising a variant sialidase
precursor protein and a chimeric antigen receptor (CAR) having
specificity for TnMUC1, CD19, or PSMA. The variant sialidase
precursor protein comprises a heterologous secretory sequence
operably linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof and lacks a transmembrane domain, and
the sialidase or enzymatically functional portion thereof is
capable of being secreted from an immune or precursor cell thereof
when the variant sialidase precursor protein is expressed in the
cell.
[0146] In certain embodiments, the modified cell is a modified
immune cell. In certain embodiments, the modified cell is a
modified T cell. In certain embodiments, the modified cell is an
autologous cell. In certain embodiments, the modified cell is an
autologous cell obtained from a human subject.
E. Chimeric Antigen Receptors
[0147] The present invention provides compositions and methods for
modified immune cells or precursors thereof (e.g., modified T cells
comprising a chimeric cell surface sialidase or a variant sialidase
precursor protein), comprising a chimeric antigen receptor (CAR).
Thus, in some embodiments, the immune cell has been genetically
modified to express the CAR. CARs of the present invention comprise
an antigen binding domain, a transmembrane domain, and an
intracellular domain.
[0148] The antigen binding domain may be operably linked to another
domain of the CAR, such as the transmembrane domain or the
intracellular domain, both described elsewhere herein, for
expression in the cell. In one embodiment, a first nucleic acid
sequence encoding the antigen binding domain is operably linked to
a second nucleic acid encoding a transmembrane domain, and further
operably linked to a third a nucleic acid sequence encoding an
intracellular domain.
[0149] The antigen binding domains described herein can be combined
with any of the transmembrane domains described herein, any of the
intracellular domains or cytoplasmic domains described herein, or
any of the other domains described herein that may be included in a
CAR of the present invention. A subject CAR of the present
invention may also include a hinge domain as described herein. A
subject CAR of the present invention may also include a spacer
domain as described herein. In some embodiments, each of the
antigen binding domain, transmembrane domain, and intracellular
domain is separated by a linker.
Antigen Binding Domain
[0150] The antigen binding domain of a CAR is an extracellular
region of the CAR for binding to a specific target antigen
including proteins, carbohydrates, and glycolipids. In some
embodiments, the CAR comprises affinity to a target antigen on a
target cell. The target antigen may include any type of protein, or
epitope thereof, associated with the target cell. For example, the
CAR may comprise affinity to a target antigen on a target cell that
indicates a particular disease state of the target cell.
[0151] In one embodiment, the target cell antigen is a tumor
associated antigen (TAA). Examples of tumor associated antigens
(TAAs), include but are not limited to, differentiation antigens
such as MART-1/MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1,
TRP-2 and tumor-specific multilineage antigens such as MAGE-1,
MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens
such as CEA; overexpressed oncogenes and mutated tumor-suppressor
genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting
from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET,
IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr
virus antigens EBVA and the human papillomavirus (HPV) antigens E6
and E7. Other large, protein-based antigens include TSP-180,
MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,
nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,
beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,
alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA
27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5,
G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,
NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin
C-associated protein, TAAL6, TAG72, TLP, and TPS. In a preferred
embodiment, the antigen binding domain of the CAR targets an
antigen that includes but is not limited to CD19, CD20, CD22, ROR1,
Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77,
EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.
[0152] Depending on the desired antigen to be targeted, the CAR of
the invention can be engineered to include the appropriate antigen
binding domain that is specific to the desired antigen target. For
example, if CD19 is the desired antigen that is to be targeted, an
antibody for CD19 can be used as the antigen bind moiety for
incorporation into the CAR of the invention.
[0153] In one embodiment, the target cell antigen is TnMUC1. As
such, in one embodiment, a CAR of the present disclosure has
affinity for TnMUC1 on a target cell. In one embodiment, the target
cell antigen is CD19. As such, in one embodiment, a CAR of the
present disclosure has affinity for CD19 on a target cell. This
should not be construed as limiting in any way, as a CAR having
affinity for any target antigen is suitable for use in a
composition or method of the present invention. In one embodiment,
the target cell antigen is PSMA. As such, in one embodiment, a CAR
of the present disclosure has affinity for PSMA on a target
cell.
[0154] As described herein, a CAR of the present disclosure having
affinity for a specific target antigen on a target cell may
comprise a target-specific binding domain. In some embodiments, the
target-specific binding domain is a murine target-specific binding
domain, e.g., the target-specific binding domain is of murine
origin. In some embodiments, the target-specific binding domain is
a human target-specific binding domain, e.g., the target-specific
binding domain is of human origin. In one embodiment, a CAR of the
present disclosure having affinity for CD19 on a target cell may
comprise a CD19 binding domain.
[0155] In some embodiments, a CAR of the present disclosure may
have affinity for one or more target antigens on one or more target
cells. In some embodiments, a CAR may have affinity for one or more
target antigens on a target cell. In such embodiments, the CAR is a
bispecific CAR, or a multispecific CAR. In some embodiments, the
CAR comprises one or more target-specific binding domains that
confer affinity for one or more target antigens. In some
embodiments, the CAR comprises one or more target-specific binding
domains that confer affinity for the same target antigen. For
example, a CAR comprising one or more target-specific binding
domains having affinity for the same target antigen could bind
distinct epitopes of the target antigen. When a plurality of
target-specific binding domains is present in a CAR, the binding
domains may be arranged in tandem and may be separated by linker
peptides. For example, in a CAR comprising two target-specific
binding domains, the binding domains are connected to each other
covalently on a single polypeptide chain, through an oligo- or
polypeptide linker, an Fc hinge region, or a membrane hinge
region.
[0156] In some embodiments, the antigen binding domain is selected
from the group consisting of an antibody, an antigen binding
fragment (Fab), and a single-chain variable fragment (scFv). In
some embodiments, a TnMUC1 binding domain of the present invention
is selected from the group consisting of a TnMUC1-specific
antibody, a TnMUC1-specific Fab, and a TnMUC1-specific scFv. In one
embodiment, a PSCA binding domain is a PSMA-specific antibody. In
one embodiment, a PSMA binding domain is a PSMA-specific Fab. In
one embodiment, a PSMA binding domain is a PSMA-specific scFv. In
some embodiments, a CD19 binding domain of the present invention is
selected from the group consisting of a CD19-specific antibody, a
CD19-specific Fab, and a CD19-specific scFv. In one embodiment, a
CD19 binding domain is a CD19-specific antibody. In one embodiment,
a CD19 binding domain is a CD19-specific Fab. In one embodiment, a
CD19 binding domain is a CD19-specific scFv.
[0157] The antigen binding domain can include any domain that binds
to the antigen and may include, but is not limited to, a monoclonal
antibody, a polyclonal antibody, a synthetic antibody, a human
antibody, a humanized antibody, a non-human antibody, and any
fragment thereof. In some embodiments, the antigen binding domain
portion comprises a mammalian antibody or a fragment thereof. The
choice of antigen binding domain may depend upon the type and
number of antigens that are present on the surface of a target
cell.
[0158] As used herein, the term "single-chain variable fragment" or
"scFv" is a fusion protein of the variable regions of the heavy
(VH) and light chains (VL) of an immunoglobulin (e.g., mouse or
human) covalently linked to form a VH::VL heterodimer. The heavy
(VH) and light chains (VL) are either joined directly or joined by
a peptide-encoding linker, which connects the N-terminus of the VH
with the C-terminus of the VL, or the C-terminus of the VH with the
N-terminus of the VL. In some embodiments, the antigen binding
domain (e.g., PSCA binding domain) comprises an scFv having the
configuration from N-terminus to C-terminus, VH-linker-VL. In some
embodiments, the antigen binding domain comprises an scFv having
the configuration from N-terminus to C-terminus, VL-linker-VH.
Those of skill in the art would be able to select the appropriate
configuration for use in the present invention.
[0159] The linker is usually rich in glycine for flexibility, as
well as serine or threonine for solubility. The linker can link the
heavy chain variable region and the light chain variable region of
the extracellular antigen-binding domain. Non-limiting examples of
linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917
(2008) and WO 2014/087010, the contents of which are hereby
incorporated by reference in their entireties. Various linker
sequences are known in the art, including, without limitation,
glycine serine (GS) linkers such as (GS).sub.n, (GSGGS).sub.n (SEQ
ID NO:12), (GGGS).sub.n (SEQ ID NO:14), and (GGGGS).sub.n (SEQ ID
NO:48), where n represents an integer of at least 1. Exemplary
linker sequences can comprise amino acid sequences including,
without limitation, GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:5), GSGSG
(SEQ ID NO:41), GSGGG (SEQ ID NO:42), GGGSG (SEQ ID NO:43), GSSSG
(SEQ ID NO:44), GGGGS (SEQ ID NO:45), GGGGSGGGGSGGGGS (SEQ ID
NO:46) and the like. Those of skill in the art would be able to
select the appropriate linker sequence for use in the present
invention. In one embodiment, an antigen binding domain of the
present invention comprises a heavy chain variable region (VH) and
a light chain variable region (VL), wherein the VH and VL is
separated by the linker sequence having the amino acid sequence
GGGGSGGGGSGGGGS (SEQ ID NO:46), which may be encoded by the nucleic
acid sequence GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQ ID
NO:47).
[0160] Despite removal of the constant regions and the introduction
of a linker, scFv proteins retain the specificity of the original
immunoglobulin. Single chain Fv polypeptide antibodies can be
expressed from a nucleic acid comprising VH- and VL-encoding
sequences as described by Huston, et al. (Proc. Nat. Acad. Sci.
USA, 85:5879-5883, 1988). See, also, U.S. Pat. Nos. 5,091,513,
5,132,405 and 4,956,778; and U.S. Patent Publication Nos.
20050196754 and 20050196754. Antagonistic scFvs having inhibitory
activity have been described (see, e.g., Zhao et al., Hyrbidoma
(Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia
Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85;
Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J
Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology
1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40).
Agonistic scFvs having stimulatory activity have been described
(see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie
et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev
Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003
1638(3):257-66).
[0161] As used herein, "Fab" refers to a fragment of an antibody
structure that binds to an antigen but is monovalent and does not
have a Fc portion, for example, an antibody digested by the enzyme
papain yields two Fab fragments and an Fc fragment (e.g., a heavy
(H) chain constant region; Fc region that does not bind to an
antigen).
[0162] As used herein, "F(ab')2" refers to an antibody fragment
generated by pepsin digestion of whole IgG antibodies, wherein this
fragment has two antigen binding (ab') (bivalent) regions, wherein
each (ab') region comprises two separate amino acid chains, a part
of a H chain and a light (L) chain linked by an S--S bond for
binding an antigen and where the remaining H chain portions are
linked together. A "F(ab')2" fragment can be split into two
individual Fab' fragments.
[0163] In some embodiments, the antigen binding domain may be
derived from the same species in which the CAR will ultimately be
used. For example, for use in humans, the antigen binding domain of
the CAR may comprise a human antibody or a fragment thereof. In
some embodiments, the antigen binding domain may be derived from a
different species in which the CAR will ultimately be used. For
example, for use in humans, the antigen binding domain of the CAR
may comprise a murine antibody or a fragment thereof.
Transmembrane Domain
[0164] CARs of the present invention may comprise a transmembrane
domain that connects the antigen binding domain of the CAR to the
intracellular domain of the CAR. The transmembrane domain of a
subject CAR is a region that is capable of spanning the plasma
membrane of a cell (e.g., an immune cell or precursor thereof). The
transmembrane domain is for insertion into a cell membrane, e.g., a
eukaryotic cell membrane. In some embodiments, the transmembrane
domain is interposed between the antigen binding domain and the
intracellular domain of a CAR.
[0165] In some embodiments, the transmembrane domain is naturally
associated with one or more of the domains in the CAR. In some
embodiments, the transmembrane domain can be selected or modified
by one or more amino acid substitutions to avoid binding of such
domains to the transmembrane domains of the same or different
surface membrane proteins, to minimize interactions with other
members of the receptor complex.
[0166] The transmembrane domain may be derived either from a
natural or a synthetic source. Where the source is natural, the
domain may be derived from any membrane-bound or transmembrane
protein, e.g., a Type I transmembrane protein. Where the source is
synthetic, the transmembrane domain may be any artificial sequence
that facilitates insertion of the CAR into a cell membrane, e.g.,
an artificial hydrophobic sequence. Examples of the transmembrane
domain of particular use in this invention include, without
limitation, transmembrane domains derived from (i.e. comprise at
least the transmembrane region(s) of) the alpha, beta or zeta chain
of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7,
CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40),
CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2,
TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments,
the transmembrane domain may be synthetic, in which case it will
comprise predominantly hydrophobic residues such as leucine and
valine. Preferably a triplet of phenylalanine, tryptophan and
valine will be found at each end of a synthetic transmembrane
domain.
[0167] The transmembrane domains described herein can be combined
with any of the antigen binding domains described herein, any of
the intracellular domains described herein, or any of the other
domains described herein that may be included in a subject CAR.
[0168] In some embodiments, the transmembrane domain further
comprises a hinge region. A subject CAR of the present invention
may also include a hinge region. The hinge region of the CAR is a
hydrophilic region which is located between the antigen binding
domain and the transmembrane domain. In some embodiments, this
domain facilitates proper protein folding for the CAR. The hinge
region is an optional component for the CAR. The hinge region may
include a domain selected from Fc fragments of antibodies, hinge
regions of antibodies, CH2 regions of antibodies, CH3 regions of
antibodies, artificial hinge sequences or combinations thereof.
Examples of hinge regions include, without limitation, a CD8a
hinge, artificial hinges made of polypeptides which may be as small
as, three glycines (Gly), as well as CH1 and CH3 domains of IgGs
(such as human IgG4).
[0169] In some embodiments, a subject CAR of the present disclosure
includes a hinge region that connects the antigen binding domain
with the transmembrane domain, which, in turn, connects to the
intracellular domain. The hinge region is preferably capable of
supporting the antigen binding domain to recognize and bind to the
target antigen on the target cells (see, e.g., Hudecek et al.,
Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments,
the hinge region is a flexible domain, thus allowing the antigen
binding domain to have a structure to optimally recognize the
specific structure and density of the target antigens on a cell
such as tumor cell (Hudecek et al., supra). The flexibility of the
hinge region permits the hinge region to adopt many different
conformations.
[0170] In some embodiments, the hinge region is an immunoglobulin
heavy chain hinge region. In some embodiments, the hinge region is
a hinge region polypeptide derived from a receptor (e.g., a
CD8-derived hinge region).
[0171] The hinge region can have a length of from about 4 amino
acids to about 50 amino acids, e.g., from about 4 aa to about 10
aa, from about 10 aa to about 15 aa, from about 15 aa to about 20
aa, from about 20 aa to about 25 aa, from about 25 aa to about 30
aa, from about 30 aa to about 40 aa, or from about 40 aa to about
50 aa. In some embodiments, the hinge region can have a length of
greater than 5 aa, greater than 10 aa, greater than 15 aa, greater
than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35
aa, greater than 40 aa, greater than 45 aa, greater than 50 aa,
greater than 55 aa, or more.
[0172] Suitable hinge regions can be readily selected and can be of
any of a number of suitable lengths, such as from 1 amino acid
(e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino
acids, from 3 amino acids to 12 amino acids, including 4 amino
acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino
acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can
be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can
have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60
or more amino acids).
[0173] For example, hinge regions include glycine polymers
(G).sub.n, glycine-serine polymers (including, for example,
(GS).sub.n, (GSGGS).sub.n (SEQ ID NO:12) and (GGGS).sub.n (SEQ ID
NO:14), where n is an integer of at least one), glycine-alanine
polymers, alanine-serine polymers, and other flexible linkers known
in the art. Glycine and glycine-serine polymers can be used; both
Gly and Ser are relatively unstructured, and therefore can serve as
a neutral tether between components. Glycine polymers can be used;
glycine accesses significantly more phi-psi space than even
alanine, and is much less restricted than residues with longer side
chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2:
73-142). Exemplary hinge regions can comprise amino acid sequences
including, but not limited to, GGSG (SEQ ID NO:3), GGSGG (SEQ ID
NO:5), GSGSG (SEQ ID NO:41), GSGGG (SEQ ID NO:42), GGGSG (SEQ ID
NO:43), GSSSG (SEQ ID NO:44), and the like.
[0174] In some embodiments, the hinge region is an immunoglobulin
heavy chain hinge region. Immunoglobulin hinge region amino acid
sequences are known in the art; see, e.g., Tan et al., Proc. Natl.
Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids
Res. (1986) 14(4): 1779-1789. As non-limiting examples, an
immunoglobulin hinge region can include one of the following amino
acid sequences: DKTHT (SEQ ID NO:49); CPPC (SEQ ID NO:50);
CPEPKSCDTPPPCPR (SEQ ID NO:51) (see, e.g., Glaser et al., J. Biol.
Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO:52);
KSCDKTHTCP (SEQ ID NO:53); KCCVDCP (SEQ ID NO:54); KYGPPCP (SEQ ID
NO:55); EPKSCDKTHTCPPCP (SEQ ID NO:56) (human IgG1 hinge);
ERKCCVECPPCP (SEQ ID NO:57) (human IgG2 hinge); ELKTPLGDTTHTCPRCP
(SEQ ID NO:58) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:59)
(human IgG4 hinge); and the like.
[0175] The hinge region can comprise an amino acid sequence of a
human IgG1, IgG2, IgG3, or IgG4, hinge region. In one embodiment,
the hinge region can include one or more amino acid substitutions
and/or insertions and/or deletions compared to a wild-type
(naturally-occurring) hinge region. For example, His229 of human
IgG1 hinge can be substituted with Tyr, so that the hinge region
comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO:116); see, e.g.,
Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one
embodiment, the hinge region can comprise an amino acid sequence
derived from human CD8, or a variant thereof
Intracellular Region
[0176] A subject CAR of the present invention also includes an
intracellular region. The intracellular region comprises a
costimulatory signaling domain and an intracellular signaling
domain". The intracellular regoin of the CAR is responsible for
activation of at least one of the effector functions of the cell in
which the CAR is expressed (e.g., immune cell). The intracellular
region transduces the effector function signal and directs the cell
(e.g., immune cell) to perform its specialized function, e.g.,
harming and/or destroying a target cell.
[0177] Examples of an intracellular region for use in the invention
include, but are not limited to, the cytoplasmic portion of a
surface receptor, co-stimulatory molecule, and any molecule that
acts in concert to initiate signal transduction in the T cell, as
well as any derivative or variant of these elements and any
synthetic sequence that has the same functional capability.
[0178] Examples of the intracellular signaling domain include,
without limitation, the .zeta. chain of the T cell receptor complex
or any of its homologs, e.g., .eta. chain, FcsI.gamma. and .beta.
chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta
chain, CD3 polypeptides (.DELTA., .delta. and .epsilon.), syk
family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine
kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T
cell transduction, such as CD2, CD5 and CD28. In one embodiment,
the intracellular signaling domain may be human CD3 zeta chain,
Fc.gamma.RIII, FcsRI, cytoplasmic tails of Fc receptors, an
immunoreceptor tyrosine-based activation motif (ITAM) bearing
cytoplasmic receptors, and combinations thereof.
[0179] In one embodiment, the costimulatory signaling domain of the
CAR includes any portion of one or more co-stimulatory molecules,
such as at least one signaling domain from CD2, CD3, CD8, CD27,
CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any
synthetic sequence thereof that has the same functional capability,
and any combination thereof.
[0180] Other examples of the costimulatory signaling domain include
a fragment or domain from one or more molecules or receptors
including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta,
CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon RIb),
CD79a, CD79b, Fcgamma RIIa, DAP10, DAP12, T cell receptor (TCR),
CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS,
a KIR family protein, lymphocyte function-associated antigen-1
(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically
binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7,
NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R
beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4,
CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,
CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,
LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244,
2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160
(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM
(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT,
GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like
receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,
other co-stimulatory molecules described herein, any derivative,
variant, or fragment thereof, any synthetic sequence of a
co-stimulatory molecule that has the same functional capability,
and any combination thereof.
[0181] Additional examples of intracellular domains include,
without limitation, intracellular signaling domains of several
types of various other immune signaling receptors, including, but
not limited to, first, second, and third generation T cell
signaling proteins including CD3, B7 family costimulatory, and
Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see,
e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653).
Additionally, intracellular signaling domains may include signaling
domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman,
Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30
(B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5):
2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol.
(2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and
CD3z.
[0182] Intracellular signaling domains suitable for use in a
subject CAR of the present invention include any desired signaling
domain that provides a distinct and detectable signal (e.g.,
increased production of one or more cytokines by the cell; change
in transcription of a target gene; change in activity of a protein;
change in cell behavior, e.g., cell death; cellular proliferation;
cellular differentiation; cell survival; modulation of cellular
signaling responses; etc.) in response to activation of the CAR
(i.e., activated by antigen and dimerizing agent). In some
embodiments, the intracellular signaling domain includes at least
one (e.g., one, two, three, four, five, six, etc.) ITAM motifs as
described below. In some embodiments, the intracellular signaling
domain includes DAP10/CD28 type signaling chains. In some
embodiments, the intracellular signaling domain is not covalently
attached to the membrane bound CAR, but is instead diffused in the
cytoplasm.
[0183] Intracellular signaling domains suitable for use in a
subject CAR of the present invention include immunoreceptor
tyrosine-based activation motif (ITAM)-containing intracellular
signaling polypeptides. In some embodiments, an ITAM motif is
repeated twice in an intracellular signaling domain, where the
first and second instances of the ITAM motif are separated from one
another by 6 to 8 amino acids. In one embodiment, the intracellular
signaling domain of a subject CAR comprises 3 ITAM motifs.
[0184] In some embodiments, intracellular signaling domains
includes the signaling domains of human immunoglobulin receptors
that contain immunoreceptor tyrosine based activation motifs
(ITAMs) such as, but not limited to, FcgammaRl, FcgammaRIIA,
FcgammaRllC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis et al., Front.
Immunol. (2014) 5:254).
[0185] A suitable intracellular signaling domain can be an ITAM
motif-containing portion that is derived from a polypeptide that
contains an ITAM motif. For example, a suitable intracellular
signaling domain can be an ITAM motif-containing domain from any
ITAM motif-containing protein. Thus, a suitable intracellular
signaling domain need not contain the entire sequence of the entire
protein from which it is derived. Examples of suitable ITAM
motif-containing polypeptides include, but are not limited to:
DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3
delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and
CD79A (antigen receptor complex-associated protein alpha
chain).
[0186] In one embodiment, the intracellular signaling domain is
derived from DAP12 (also known as TYROBP; TYRO protein tyrosine
kinase binding protein; KARAP; PLOSL; DNAX-activation protein 12;
KAR-associated protein; TYRO protein tyrosine kinase-binding
protein; killer activating receptor associated protein;
killer-activating receptor-associated protein; etc.). In one
embodiment, the intracellular signaling domain is derived from
FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc
receptor gamma-chain; fc-epsilon RI-gamma; fcRgamma; fceRl gamma;
high affinity immunoglobulin epsilon receptor subunit gamma;
immunoglobulin E receptor, high affinity, gamma chain; etc.). In
one embodiment, the intracellular signaling domain is derived from
T-cell surface glycoprotein CD3 delta chain (also known as CD3D;
CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d
antigen, delta polypeptide (TiT3 complex); OKT3, delta chain;
T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3
delta chain; etc.). In one embodiment, the intracellular signaling
domain is derived from T-cell surface glycoprotein CD3 epsilon
chain (also known as CD3e, T-cell surface antigen T3/Leu-4 epsilon
chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783,
CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular
signaling domain is derived from T-cell surface glycoprotein CD3
gamma chain (also known as CD3G, T-cell receptor T3 gamma chain,
CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one
embodiment, the intracellular signaling domain is derived from
T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z,
T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z,
TCRZ, etc.). In one embodiment, the intracellular signaling domain
is derived from CD79A (also known as B-cell antigen receptor
complex-associated protein alpha chain; CD79a antigen
(immunoglobulin-associated alpha); MB-1 membrane glycoprotein;
ig-alpha; membrane-bound immunoglobulin-associated protein; surface
IgM-associated protein; etc.). In one embodiment, an intracellular
signaling domain suitable for use in an FN3 CAR of the present
disclosure includes a DAP10/CD28 type signaling chain. In one
embodiment, an intracellular signaling domain suitable for use in
an FN3 CAR of the present disclosure includes a ZAP70 polypeptide.
In some embodiments, the intracellular signaling domain includes a
cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3
gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d.
In one embodiment, the intracellular signaling domain in the CAR
includes a cytoplasmic signaling domain of human CD3 zeta.
[0187] While usually the entire intracellular signaling domain can
be employed, in many cases it is not necessary to use the entire
chain. To the extent that a truncated portion of the intracellular
signaling domain is used, such truncated portion may be used in
place of the intact chain as long as it transduces the effector
function signal. The intracellular signaling domain includes any
truncated portion of the intracellular signaling domain sufficient
to transduce the effector function signal.
[0188] The intracellular regions described herein can be combined
with any of the antigen binding domains described herein, any of
the transmembrane domains described herein, or any of the other
domains described herein that may be included in the CAR.
F. T Cell Receptors
[0189] The present invention provides compositions and methods for
modified immune cells or precursors thereof (e.g., modified T cells
comprising a chimeric cell surface sialidase or a variant sialidase
precursor protein) comprising a T cell receptor (TCR). In some
embodiments, the cell has been altered to contain specific T cell
receptor (TCR) genes (e.g., a nucleic acid encoding an alpha/beta
TCR). TCRs or antigen-binding portions thereof include those that
recognize a peptide epitope or T cell epitope of a target
polypeptide, such as an antigen of a tumor, viral or autoimmune
protein. In certain embodiments, the TCR has binding specificity
for a tumor associated antigen, e.g., human NY-ESO-1.
[0190] A TCR is a disulfide-linked heterodimeric protein comprised
of six different membrane bound chains that participate in the
activation of T cells in response to an antigen. There exists
alpha/beta TCRs and gamma/delta TCRs. An alpha/beta TCR comprises a
TCR alpha chain and a TCR beta chain. T cells expressing a TCR
comprising a TCR alpha chain and a TCR beta chain are commonly
referred to as alpha/beta T cells. Gamma/delta TCRs comprise a TCR
gamma chain and a TCR delta chain. T cells expressing a TCR
comprising a TCR gamma chain and a TCR delta chain are commonly
referred to as gamma/delta T cells. A TCR of the present disclosure
is a TCR comprising a TCR alpha chain and a TCR beta chain.
[0191] The TCR alpha chain and the TCR beta chain are each
comprised of two extracellular domains, a variable region and a
constant region. The TCR alpha chain variable region and the TCR
beta chain variable region are required for the affinity of a TCR
to a target antigen. Each variable region comprises three
hypervariable or complementarity-determining regions (CDRs) which
provide for binding to a target antigen. The constant region of the
TCR alpha chain and the constant region of the TCR beta chain are
proximal to the cell membrane. A TCR further comprises a
transmembrane region and a short cytoplasmic tail. CD3 molecules
are assembled together with the TCR heterodimer. CD3 molecules
comprise a characteristic sequence motif for tyrosine
phosphorylation, known as immunoreceptor tyrosine-based activation
motifs (ITAMs). Proximal signaling events are mediated through the
CD3 molecules, and accordingly, TCR-CD3 complex interaction plays
an important role in mediating cell recognition events.
[0192] Stimulation of TCR is triggered by major histocompatibility
complex molecules (MHCs) on antigen presenting cells that present
antigen peptides to T cells and interact with TCRs to induce a
series of intracellular signaling cascades. Engagement of the TCR
initiates both positive and negative signaling cascades that result
in cellular proliferation, cytokine production, and/or
activation-induced cell death.
[0193] A TCR of the present invention can be a wild-type TCR, a
high affinity TCR, and/or a chimeric TCR. A high affinity TCR may
be the result of modifications to a wild-type TCR that confers a
higher affinity for a target antigen compared to the wild-type TCR.
A high affinity TCR may be an affinity-matured TCR. Methods for
modifying TCRs and/or the affinity-maturation of TCRs are known to
those of skill in the art. Techniques for engineering and
expressing TCRs include, but are not limited to, the production of
TCR heterodimers which include the native disulphide bridge which
connects the respective subunits (Garboczi, et al., (1996), Nature
384(6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12):
5403-10; Chang et al., (1994), PNAS USA 91: 11408-11412; Davodeau
et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al.,
(1997), J. Imm. Meth. 206: 163-169; U.S. Pat. No. 6,080,840).
[0194] In some embodiments, the exogenous TCR is a full TCR or an
antigen-binding portion or antigen-binding fragment thereof. In
some embodiments, the TCR is an intact or full-length TCR,
including TCRs in the .alpha..beta. form or .gamma..delta. form. In
some embodiments, the TCR is an antigen-binding portion that is
less than a full-length TCR but that binds to a specific peptide
bound in an MHC molecule, such as binds to an MHC-peptide complex.
In some cases, an antigen-binding portion or fragment of a TCR can
contain only a portion of the structural domains of a full-length
or intact TCR, but yet is able to bind the peptide epitope, such as
MHC-peptide complex, to which the full TCR binds. In some cases, an
antigen-binding portion contains the variable domains of a TCR,
such as variable .alpha. chain and variable .beta. chain of a TCR,
sufficient to form a binding site for binding to a specific
MHC-peptide complex. Generally, the variable chains of a TCR
contain complementarity determining regions (CDRs) involved in
recognition of the peptide, MHC and/or MHC-peptide complex.
[0195] In some embodiments, the variable domains of the TCR contain
hypervariable loops, or CDRs, which generally are the primary
contributors to antigen recognition and binding capabilities and
specificity. In some embodiments, a CDR of a TCR or combination
thereof forms all or substantially all of the antigen-binding site
of a given TCR molecule. The various CDRs within a variable region
of a TCR chain generally are separated by framework regions (FRs),
which generally display less variability among TCR molecules as
compared to the CDRs (see, e.g., Jores et al, Proc. Nat'l Acad.
Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988;
see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some
embodiments, CDR3 is the main CDR responsible for antigen binding
or specificity, or is the most important among the three CDRs on a
given TCR variable region for antigen recognition, and/or for
interaction with the processed peptide portion of the peptide-MHC
complex. In some contexts, the CDR1 of the alpha chain can interact
with the N-terminal part of certain antigenic peptides. In some
contexts, CDR1 of the beta chain can interact with the C-terminal
part of the peptide. In some contexts, CDR2 contributes most
strongly to or is the primary CDR responsible for the interaction
with or recognition of the MHC portion of the MHC-peptide complex.
In some embodiments, the variable region of the (3-chain can
contain a further hypervariable region (CDR4 or HVR4), which
generally is involved in superantigen binding and not antigen
recognition (Kotb (1995) Clinical Microbiology Reviews,
8:411-426).
[0196] In some embodiments, a TCR contains a variable alpha domain
(V.sub..alpha.) and/or a variable beta domain (V.sub..beta.) or
antigen-binding fragments thereof. In some embodiments, the
.alpha.-chain and/or .beta.-chain of a TCR also can contain a
constant domain, a transmembrane domain and/or a short cytoplasmic
tail (see, e.g., Janeway et al., Immunobiology: The Immune System
in Health and Disease, 3 Ed., Current Biology Publications, p.
4:33, 1997). In some embodiments, the .alpha. chain constant domain
is encoded by the TRAC gene (IMGT nomenclature) or is a variant
thereof. In some embodiments, the (3 chain constant region is
encoded by TRBC1 or TRBC2 genes (IMGT nomenclature) or is a variant
thereof. In some embodiments, the constant domain is adjacent to
the cell membrane. For example, in some cases, the extracellular
portion of the TCR formed by the two chains contains two
membrane-proximal constant domains, and two membrane-distal
variable domains, which variable domains each contain CDRs.
[0197] It is within the level of a skilled artisan to determine or
identify the various domains or regions of a TCR. In some aspects,
residues of a TCR are known or can be identified according to the
International Immunogenetics Information System (IMGT) numbering
system (see e.g. www.imgt.org; see also, Lefranc et al. (2003)
Developmental and Comparative Immunology, 2& 55-77; and The T
Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press
2001). Using this system, the CDR1 sequences within a TCR Va chain
and/or V.beta. chain correspond to the amino acids present between
residue numbers 27-38, inclusive, the CDR2 sequences within a TCR
Va chain and/or V.beta. chain correspond to the amino acids present
between residue numbers 56-65, inclusive, and the CDR3 sequences
within a TCR Va chain and/or V.beta. chain correspond to the amino
acids present between residue numbers 105-117, inclusive. The IMGT
numbering system should not be construed as limiting in any way, as
there are other numbering systems known to those of skill in the
art, and it is within the level of the skilled artisan to use any
of the numbering systems available to identify the various domains
or regions of a TCR.
[0198] In some embodiments, the TCR may be a heterodimer of two
chains .alpha. and .beta. (or optionally .gamma. and .delta.) that
are linked, such as by a disulfide bond or disulfide bonds. In some
embodiments, the constant domain of the TCR may contain short
connecting sequences in which a cysteine residue forms a disulfide
bond, thereby linking the two chains of the TCR. In some
embodiments, a TCR may have an additional cysteine residue in each
of the .alpha. and .beta. chains, such that the TCR contains two
disulfide bonds in the constant domains. In some embodiments, each
of the constant and variable domains contain disulfide bonds formed
by cysteine residues.
[0199] In some embodiments, the TCR for engineering cells as
described is one generated from a known TCR sequence(s), such as
sequences of V.alpha.,.beta. chains, for which a substantially
full-length coding sequence is readily available. Methods for
obtaining full-length TCR sequences, including V chain sequences,
from cell sources are well known. In some embodiments, nucleic
acids encoding the TCR can be obtained from a variety of sources,
such as by polymerase chain reaction (PCR) amplification of
TCR-encoding nucleic acids within or isolated from a given cell or
cells, or synthesis of publicly available TCR DNA sequences. In
some embodiments, the TCR is obtained from a biological source,
such as from cells such as from a T cell (e.g. cytotoxic T cell), T
cell hybridomas or other publicly available source. In some
embodiments, the T cells can be obtained from in vivo isolated
cells. In some embodiments, the T-cells can be a cultured T cell
hybridoma or clone. In some embodiments, the TCR or antigen-binding
portion thereof can be synthetically generated from knowledge of
the sequence of the TCR. In some embodiments, a high-affinity T
cell clone for a target antigen (e.g., a cancer antigen) is
identified, isolated from a patient, and introduced into the cells.
In some embodiments, the TCR clone for a target antigen has been
generated in transgenic mice engineered with human immune system
genes (e.g., the human leukocyte antigen system, or HLA). See,
e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin
Cancer Res. 15: 169-180 and Cohen et al. (2005) J Immunol.
175:5799-5808. In some embodiments, phage display is used to
isolate TCRs against a target antigen (see, e.g., Varela-Rohena et
al. (2008) Nat Med. 14: 1390-1395 and Li (2005) Nat Biotechnol.
23:349-354.
[0200] In some embodiments, the TCR or antigen-binding portion
thereof is one that has been modified or engineered. In some
embodiments, directed evolution methods are used to generate TCRs
with altered properties, such as with higher affinity for a
specific MHC-peptide complex. In some embodiments, directed
evolution is achieved by display methods including, but not limited
to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62;
Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage
display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell
display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In
some embodiments, display approaches involve engineering, or
modifying, a known, parent or reference TCR. For example, in some
cases, a wild-type TCR can be used as a template for producing
mutagenized TCRs in which in one or more residues of the CDRs are
mutated, and mutants with an desired altered property, such as
higher affinity for a desired target antigen, are selected.
[0201] In some embodiments as described, the TCR can contain an
introduced disulfide bond or bonds. In some embodiments, the native
disulfide bonds are not present. In some embodiments, the one or
more of the native cysteines (e.g. in the constant domain of the
.alpha. chain and .beta. chain) that form a native interchain
disulfide bond are substituted with another residue, such as with a
serine or alanine. In some embodiments, an introduced disulfide
bond can be formed by mutating non-cysteine residues on the alpha
and beta chains, such as in the constant domain of the .alpha.
chain and .beta. chain, to cysteine. Exemplary non-native disulfide
bonds of a TCR are described in published International PCT No.
WO2006/000830 and WO2006/037960. In some embodiments, cysteines can
be introduced at residue Thr48 of the .alpha. chain and Ser57 of
the .beta. chain, at residue Thr45 of the .alpha. chain and Ser77
of the .beta. chain, at residue Tyr10 of the .alpha. chain and
Ser17 of the .beta. chain, at residue Thr45 of the .alpha. chain
and Asp59 of the .beta. chain and/or at residue Ser15 of the
.alpha. chain and Glu15 of the .beta. chain. In some embodiments,
the presence of non-native cysteine residues (e.g. resulting in one
or more non-native disulfide bonds) in a recombinant TCR can favor
production of the desired recombinant TCR in a cell in which it is
introduced over expression of a mismatched TCR pair containing a
native TCR chain.
[0202] In some embodiments, the TCR chains contain a transmembrane
domain. In some embodiments, the transmembrane domain is positively
charged. In some cases, the TCR chain contains a cytoplasmic tail.
In some aspects, each chain (e.g. alpha or beta) of the TCR can
possess one N-terminal immunoglobulin variable domain, one
immunoglobulin constant domain, a transmembrane region, and a short
cytoplasmic tail at the C-terminal end. In some embodiments, a TCR,
for example via the cytoplasmic tail, is associated with invariant
proteins of the CD3 complex involved in mediating signal
transduction. In some cases, the structure allows the TCR to
associate with other molecules like CD3 and subunits thereof. For
example, a TCR containing constant domains with a transmembrane
region may anchor the protein in the cell membrane and associate
with invariant subunits of the CD3 signaling apparatus or complex.
The intracellular tails of CD3 signaling subunits (e.g. CD3y, CD35,
CD3s and CD3.zeta. chains) contain one or more immunoreceptor
tyrosine-based activation motif or IT AM that are involved in the
signaling capacity of the TCR complex.
[0203] In some embodiments, the TCR is a full-length TCR. In some
embodiments, the TCR is an antigen-binding portion. In some
embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments,
the TCR is a single-chain TCR (sc-TCR). A TCR may be cell-bound or
in soluble form. In some embodiments, for purposes of the provided
methods, the TCR is in cell-bound form expressed on the surface of
a cell. In some embodiments a dTCR contains a first polypeptide
wherein a sequence corresponding to a TCR .alpha. chain variable
region sequence is fused to the N terminus of a sequence
corresponding to a TCR .alpha. chain constant region extracellular
sequence, and a second polypeptide wherein a sequence corresponding
to a TCR .beta. chain variable region sequence is fused to the N
terminus a sequence corresponding to a TCR .beta. chain constant
region extracellular sequence, the first and second polypeptides
being linked by a disulfide bond. In some embodiments, the bond can
correspond to the native interchain disulfide bond present in
native dimeric .alpha..beta. TCRs. In some embodiments, the
interchain disulfide bonds are not present in a native TCR. For
example, in some embodiments, one or more cysteines can be
incorporated into the constant region extracellular sequences of
dTCR polypeptide pair. In some cases, both a native and a
non-native disulfide bond may be desirable. In some embodiments,
the TCR contains a transmembrane sequence to anchor to the
membrane. In some embodiments, a dTCR contains a TCR .alpha. chain
containing a variable .alpha. domain, a constant .alpha. domain and
a first dimerization motif attached to the C-terminus of the
constant .alpha. domain, and a TCR .beta. chain comprising a
variable .beta. domain, a constant .beta. domain and a first
dimerization motif attached to the C-terminus of the constant
.beta. domain, wherein the first and second dimerization motifs
easily interact to form a covalent bond between an amino acid in
the first dimerization motif and an amino acid in the second
dimerization motif linking the TCR .alpha. chain and TCR chain
together.
[0204] In some embodiments, the TCR is a scTCR, which is a single
amino acid strand containing an .alpha. chain and a .beta. chain
that is able to bind to MHC-peptide complexes. Typically, a scTCR
can be generated using methods known to those of skill in the art,
See e.g., International published PCT Nos. WO 96/13593, WO
96/18105, WO99/18129, WO04/033685, WO2006/037960, WO2011/044186;
U.S. Pat. No. 7,569,664; and Schlueter, C. J. et al. J. Mol. Biol.
256, 859 (1996). In some embodiments, a scTCR contains a first
segment constituted by an amino acid sequence corresponding to a
TCR .alpha. chain variable region, a second segment constituted by
an amino acid sequence corresponding to a TCR .beta. chain variable
region sequence fused to the N terminus of an amino acid sequence
corresponding to a TCR .beta. chain constant domain extracellular
sequence, and a linker sequence linking the C terminus of the first
segment to the N terminus of the second segment. In some
embodiments, a scTCR contains a first segment constituted by an
amino acid sequence corresponding to a TCR .beta. chain variable
region, a second segment constituted by an amino acid sequence
corresponding to a TCR .alpha. chain variable region sequence fused
to the N terminus of an amino acid sequence corresponding to a TCR
.alpha. chain constant domain extracellular sequence, and a linker
sequence linking the C terminus of the first segment to the N
terminus of the second segment. In some embodiments, a scTCR
contains a first segment constituted by an .alpha. chain variable
region sequence fused to the N terminus of an .alpha. chain
extracellular constant domain sequence, and a second segment
constituted by a .beta. chain variable region sequence fused to the
N terminus of a sequence .beta. chain extracellular constant and
transmembrane sequence, and, optionally, a linker sequence linking
the C terminus of the first segment to the N terminus of the second
segment. In some embodiments, a scTCR contains a first segment
constituted by a TCR .beta. chain variable region sequence fused to
the N terminus of a .beta. chain extracellular constant domain
sequence, and a second segment constituted by an .alpha. chain
variable region sequence fused to the N terminus of a sequence
comprising an .alpha. chain extracellular constant domain sequence
and transmembrane sequence, and, optionally, a linker sequence
linking the C terminus of the first segment to the N terminus of
the second segment. In some embodiments, for the scTCR to bind an
MHC-peptide complex, the .alpha. and .beta. chains must be paired
so that the variable region sequences thereof are orientated for
such binding. Various methods of promoting pairing of an .alpha.
and .beta. in a scTCR are well known in the art. In some
embodiments, a linker sequence is included that links the .alpha.
and .beta. chains to form the single polypeptide strand. In some
embodiments, the linker should have sufficient length to span the
distance between the C terminus of the .alpha. chain and the N
terminus of the .beta. chain, or vice versa, while also ensuring
that the linker length is not so long so that it blocks or reduces
bonding of the scTCR to the target peptide-MHC complex. In some
embodiments, the linker of a scTCRs that links the first and second
TCR segments can be any linker capable of forming a single
polypeptide strand, while retaining TCR binding specificity. In
some embodiments, the linker sequence may, for example, have the
formula -P-AA-P-, wherein P is proline and AA represents an amino
acid sequence wherein the amino acids are glycine and serine. In
some embodiments, the first and second segments are paired so that
the variable region sequences thereof are orientated for such
binding. Hence, in some cases, the linker has a sufficient length
to span the distance between the C terminus of the first segment
and the N terminus of the second segment, or vice versa, but is not
too long to block or reduces bonding of the scTCR to the target
ligand. In some embodiments, the linker can contain from or from
about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to
41 amino acids residues, for example 29, 30, 31 or 32 amino acids.
In some embodiments, a scTCR contains a disulfide bond between
residues of the single amino acid strand, which, in some cases, can
promote stability of the pairing between the .alpha. and .beta.
regions of the single chain molecule (see e.g. U.S. Pat. No.
7,569,664). In some embodiments, the scTCR contains a covalent
disulfide bond linking a residue of the immunoglobulin region of
the constant domain of the .alpha. chain to a residue of the
immunoglobulin region of the constant domain of the .beta. chain of
the single chain molecule. In some embodiments, the disulfide bond
corresponds to the native disulfide bond present in a native dTCR.
In some embodiments, the disulfide bond in a native TCR is not
present. In some embodiments, the disulfide bond is an introduced
non-native disulfide bond, for example, by incorporating one or
more cysteines into the constant region extracellular sequences of
the first and second chain regions of the scTCR polypeptide.
Exemplary cysteine mutations include any as described above. In
some cases, both a native and a non-native disulfide bond may be
present.
[0205] In some embodiments, any of the TCRs, including a dTCR or
scTCR, can be linked to signaling domains that yield an active TCR
on the surface of a T cell. In some embodiments, the TCR is
expressed on the surface of cells. In some embodiments, the TCR
does contain a sequence corresponding to a transmembrane sequence.
In some embodiments, the transmembrane domain can be a Ca or CP
transmembrane domain. In some embodiments, the transmembrane domain
can be from a non-TCR origin, for example, a transmembrane region
from CD3z, CD28 or B7.1. In some embodiments, the TCR does contain
a sequence corresponding to cytoplasmic sequences. In some
embodiments, the TCR contains a CD3z signaling domain. In some
embodiments, the TCR is capable of forming a TCR complex with CD3.
In some embodiments, the TCR or antigen binding portion thereof may
be a recombinantly produced natural protein or mutated form thereof
in which one or more property, such as binding characteristic, has
been altered. In some embodiments, a TCR may be derived from one of
various animal species, such as human, mouse, rat, or other
mammal.
[0206] In some embodiments, the TCR comprises affinity to a target
antigen on a target cell. The target antigen may include any type
of protein, or epitope thereof, associated with the target cell.
For example, the TCR may comprise affinity to a target antigen on a
target cell that indicates a particular disease state of the target
cell. In some embodiments, the target antigen is processed and
presented by MHCs.
[0207] In one embodiment, the target cell antigen is a New York
esophageal-1 (NY-ESO-1) peptide. NY-ESO-1 belongs to the
cancer-testis (CT) antigen group of proteins. NY-ESO-1 is a highly
immunogenic antigen in vitro and is presented to T cells via the
MHC. CTLs recognizing the A2 presented epitope NY-ESO.sub.157-165,
SLLMWITQC (SEQ ID NO:60), have been grown from the blood and lymph
nodes of myeloma patients. T cell clones specific for this epitope
have been shown to kill tumor cells. A high affinity TCR
recognizing the NY-E50.sub.157165 epitope may recognize
HLA-A2-positive, NY-ESO-1 positive cell lines (but not to cells
that lack either HLA-A2 or NY-ESO). Accordingly, a TCR of the
present disclosure may be a HLA-A2-restricted NY-ESO-1 (SLLMWITQC;
SEQ ID NO:60)-specific TCR. In one embodiment, an NY-ESO-1 TCR of
the present disclosure is a wild-type NY-ESO-1 TCR. A wild-type
NY-ESO-1 TCR may include, without limitation, the 8F NY-ESO-1 TCR
(also referred to herein as "8F" or "8F TCR"), and the 1G4 NY-ESO-1
TCR (also referred to herein as "1G4" or "1G4 TCR"). In one
embodiment, an NY-ESO-1 TCR of the present disclosure is an
affinity enhanced 1G4 TCR, also called Ly95. 1G4 TCR and affinity
enhanced 1G4 TCR is described in U.S. Pat. No. 8,143,376. This
should not be construed as limiting in any way, as a TCR having
affinity for any target antigen is suitable for use in a
composition or method of the present invention.
[0208] The TCR alpha chain coding sequence and the TCR beta chain
coding sequence may be separated by a linker. In certain
embodiments, the linker comprises a nucleic acid sequence encoding
an internal ribosome entry site (IRES), a furin cleavage site, a
self-cleaving peptide, or any combination thereof. In certain
embodiments, the first linker comprises a furin cleavage site and a
self-cleaving peptide. In certain embodiments, the self-cleaving
peptide is a 2A peptide selected from the group consisting of
porcine teschovirus-1 2A (P2A), Thoseaasigna virus 2A (T2A), equine
rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A
(F2A).
G. Nucleic Acids and Expression Vectors
[0209] The present disclosure provides a nucleic acid comprising a
nucleic acid sequence encoding a chimeric cell surface sialidase
(neuraminidase) enzyme. Also provided is a nucleic acid comprising
a nucleic acid sequence encoding a variant sialidase precursor
protein.
[0210] In one embodiment, a nucleic acid of the present disclosure
further comprises a nucleic acid sequence encoding an exogenous TCR
(e.g., an NY-ESO-1 TCR). In one embodiment, a nucleic acid of the
present disclosure further comprises a nucleic acid sequence
encoding an exogenous CAR (e.g., a PSMA CAR).
[0211] In certain embodiments, a nucleic acid of the present
disclosure comprises a first nucleic acid encoding a chimeric cell
surface sialidase and a second nucleic acid sequence encoding an
exogenous TCR. In certain embodiments, a nucleic acid of the
present disclosure comprises a first nucleic acid encoding a
chimeric cell surface sialidase and a second nucleic acid sequence
encoding an exogenous CAR. In certain embodiments, a nucleic acid
of the present disclosure comprises a first nucleic acid encoding a
chimeric cell surface sialidase and a second nucleic acid sequence
encoding an exogenous TCR and an exogenous CAR.
[0212] In certain embodiments, a nucleic acid of the present
disclosure comprises a first nucleic acid sequence encoding a
variant sialidase precursor protein and a second nucleic acid
sequence encoding an exogenous TCR. In certain embodiments, a
nucleic acid of the present disclosure comprises a first nucleic
acid sequence encoding a variant sialidase precursor protein and a
second nucleic acid sequence encoding an exogenous CAR. In certain
embodiments, a nucleic acid of the present disclosure comprises a
first nucleic acid sequence encoding a variant sialidase precursor
protein and a second nucleic acid sequence encoding an exogenous
TCR and an exogeneous CAR.
[0213] In certain embodiments, the first nucleic acid sequence and
the second nucleic acid sequence are separated by a linker.
[0214] In some embodiments, the linker comprises a nucleic acid
sequence that encodes for an internal ribosome entry site (IRES).
As used herein, "an internal ribosome entry site" or "IRES" refers
to an element that promotes direct internal ribosome entry to the
initiation codon, such as ATG, of a protein coding region, thereby
leading to cap-independent translation of the gene. Various
internal ribosome entry sites are known to those of skill in the
art, including, without limitation, IRES obtainable from viral or
cellular mRNA sources, e.g., immunogloublin heavy-chain binding
protein (BiP); vascular endothelial growth factor (VEGF);
fibroblast growth factor 2; insulin-like growth factor;
translational initiation factor eIF4G; yeast transcription factors
TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus,
rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV),
and Moloney murine leukemia virus (MoMLV). Those of skill in the
art would be able to select the appropriate IRES for use in the
present invention.
[0215] In some embodiments, the linker comprises a nucleic acid
sequence that encodes for a self-cleaving peptide. As used herein,
a "self-cleaving peptide" or "2A peptide" refers to an oligopeptide
that allow multiple proteins to be encoded as polyproteins, which
dissociate into component proteins upon translation. Use of the
term "self-cleaving" is not intended to imply a proteolytic
cleavage reaction. Various self-cleaving or 2A peptides are known
to those of skill in the art, including, without limitation, those
found in members of the Picornaviridae virus family, e.g.,
foot-and-mouth disease virus (FMDV), equine rhinitis A virus
(ERAVO, Thosea asigna virus (TaV), and porcine tescho virus-1
(PTV-1); and carioviruses such as Theilovirus and
encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV,
PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and
"T2A," respectively. Those of skill in the art would be able to
select the appropriate self-cleaving peptide for use in the present
invention.
[0216] In some embodiments, a linker further comprises a nucleic
acid sequence that encodes a furin cleavage site. Furin is a
ubiquitously expressed protease that resides in the trans-golgi and
processes protein precursors before their secretion. Furin cleaves
at the COOH-- terminus of its consensus recognition sequence.
Various furin consensus recognition sequences (or "furin cleavage
sites") are known to those of skill in the art, including, without
limitation, Arg-X1-Lys-Arg (SEQ ID NO:117) or Arg-X1-Arg-Arg (SEQ
ID NO:118), X2-Arg-X1-X3-Arg (SEQ ID NO:119) and Arg-X1-X1-Arg (SEQ
ID NO:120), such as an Arg-Gln-Lys-Arg (SEQ ID NO:121), where X1 is
any naturally occurring amino acid, X2 is Lys or Arg, and X3 is Lys
or Arg. Those of skill in the art would be able to select the
appropriate Furin cleavage site for use in the present
invention.
[0217] In some embodiments, the linker comprises a nucleic acid
sequence encoding a combination of a Furin cleavage site and a 2A
peptide. Examples include, without limitation, a linker comprising
a nucleic acid sequence encoding Furin and F2A, a linker comprising
a nucleic acid sequence encoding Furin and E2A, a linker comprising
a nucleic acid sequence encoding Furin and P2A, a linker comprising
a nucleic acid sequence encoding Furin and T2A. Those of skill in
the art would be able to select the appropriate combination for use
in the present invention. In such embodiments, the linker may
further comprise a spacer sequence between the Furin and 2A
peptide. Various spacer sequences are known in the art, including,
without limitation, glycine serine (GS) spacers such as (GS)n,
(GSGGS)n (SEQ ID NO:12) and (GGGS)n (SEQ ID NO:14), where n
represents an integer of at least 1. Exemplary spacer sequences can
comprise amino acid sequences including, without limitation, GGSG
(SEQ ID NO:3), GGSGG (SEQ ID NO:5), GSGSG (SEQ ID NO:41), GSGGG
(SEQ ID NO:42), GGGSG (SEQ ID NO:43), GSSSG (SEQ ID NO:44), and the
like. Those of skill in the art would be able to select the
appropriate spacer sequence for use in the present invention.
[0218] In some embodiments, a nucleic acid of the present
disclosure is provided for the production of a chimeric cell
surface sialidase or a variant sialidase precursor protein or a TCR
or a CAR as described herein, e.g., in a mammalian cell. In some
embodiments, a nucleic acid of the present disclosure provides for
amplification of the sialidase- or TCR- or CAR-encoding nucleic
acid.
[0219] As described herein, a TCR of the present disclosure
comprises a TCR alpha chain and a TCR beta chain. Accordingly, the
present disclosure provides a nucleic acid encoding a TCR alpha
chain, and a nucleic acid encoding a TCR beta chain. In some
embodiments, the nucleic acid encoding a TCR alpha chain is
separate from the nucleic acid encoding a TCR beta chain. In an
exemplary embodiment, the nucleic acid encoding a TCR alpha chain,
and the nucleic acid encoding a TCR beta chain, resides within the
same nucleic acid.
[0220] In some embodiments, a nucleic acid of the present
disclosure comprises a nucleic acid comprising a TCR alpha chain
coding sequence and a TCR beta chain coding sequence. In some
embodiments, a nucleic acid of the present disclosure comprises a
nucleic acid comprising a TCR alpha chain coding sequence and a TCR
beta chain coding sequence that is separated by a linker. A linker
for use in the present disclosure allows for multiple proteins to
be encoded by the same nucleic acid sequence (e.g., a
multicistronic or bicistronic sequence), which are translated as a
polyprotein that is dissociated into separate protein components.
For example, a linker for use in a nucleic acid of the present
disclosure comprising a TCR alpha chain coding sequence and a TCR
beta chain coding sequence, allows for the TCR alpha chain and TCR
beta chain to be translated as a polyprotein that is dissociated
into separate TCR alpha chain and TCR beta chain components.
[0221] In some embodiments, a nucleic acid of the present
disclosure may comprise a leader sequence. Suitable leader
sequences are known to those of skill in the art. In one
embodiment, the leader sequence is a CD8 alpha leader sequence. In
one embodiment, the leader sequence is encoded by a nucleic acid
sequence comprising SEQ ID NO: 2.
[0222] In some embodiments, a nucleic acid of the present
disclosure may be operably linked to a transcriptional control
element, e.g., a promoter, and enhancer, etc. Suitable promoter and
enhancer elements are known to those of skill in the art.
[0223] In certain embodiments, the nucleic acid is in operable
linkage with a promoter. In certain embodiments, the promoter is a
phosphoglycerate kinase-1 (PGK) promoter.
[0224] For expression in a bacterial cell, suitable promoters
include, but are not limited to, lad, lacZ, T3, T7, gpt, lambda P
and trc. For expression in a eukaryotic cell, suitable promoters
include, but are not limited to, light and/or heavy chain
immunoglobulin gene promoter and enhancer elements; cytomegalovirus
immediate early promoter; herpes simplex virus thymidine kinase
promoter; early and late SV40 promoters; promoter present in long
terminal repeats from a retrovirus; mouse metallothionein-I
promoter; and various art-known tissue specific promoters. Suitable
reversible promoters, including reversible inducible promoters are
known in the art. Such reversible promoters may be isolated and
derived from many organisms, e.g., eukaryotes and prokaryotes.
Modification of reversible promoters derived from a first organism
for use in a second organism, e.g., a first prokaryote and a second
a eukaryote, a first eukaryote and a second a prokaryote, etc., is
well known in the art. Such reversible promoters, and systems based
on such reversible promoters but also comprising additional control
proteins, include, but are not limited to, alcohol regulated
promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter,
promoters responsive to alcohol transactivator proteins (A1cR),
etc.), tetracycline regulated promoters, (e.g., promoter systems
including TetActivators, TetON, TetOFF, etc.), steroid regulated
promoters (e.g., rat glucocorticoid receptor promoter systems,
human estrogen receptor promoter systems, retinoid promoter
systems, thyroid promoter systems, ecdysone promoter systems,
mifepristone promoter systems, etc.), metal regulated promoters
(e.g., metallothionein promoter systems, etc.),
pathogenesis-related regulated promoters (e.g., salicylic acid
regulated promoters, ethylene regulated promoters, benzothiadiazole
regulated promoters, etc.), temperature regulated promoters (e.g.,
heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat
shock promoter, etc.), light regulated promoters, synthetic
inducible promoters, and the like.
[0225] In some embodiments, the promoter is a CD8 cell-specific
promoter, a CD4 cell-specific promoter, a neutrophil-specific
promoter, or an NK-specific promoter. For example, a CD4 gene
promoter can be used; see, e.g., Salmon et al. Proc. Natl. Acad.
Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.
As another example, a CD8 gene promoter can be used. NK
cell-specific expression can be achieved by use of an NcrI (p46)
promoter; see, e.g., Eckelhart et al. Blood (2011) 117:1565.
[0226] For expression in a yeast cell, a suitable promoter is a
constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an
ENO promoter, a PYK1 promoter and the like; or a regulatable
promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2
promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a
MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter,
an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1
promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO
promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia).
Selection of the appropriate vector and promoter is well within the
level of ordinary skill in the art. Suitable promoters for use in
prokaryotic host cells include, but are not limited to, a
bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac
operon promoter; a hybrid promoter, e.g., a lac/tac hybrid
promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac
promoter; a trc promoter; a tac promoter, and the like; an araBAD
promoter; in vivo regulated promoters, such as an ssaG promoter or
a related promoter (see, e.g., U.S. Patent Publication No.
20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol.
(1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci.
USA (1992) 89(21): 10079-83), a nirB promoter (Harborne et al. Mol.
Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan et
al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine
(2004) 22:3243-3255; and Chatfield et al., Biotechnol. (1992)
10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter
(see, e.g., GenBank Accession Nos. AX798980, AX798961, and
AX798183); a stationary phase promoter, e.g., a dps promoter, an
spy promoter, and the like; a promoter derived from the
pathogenicity island SPI-2 (see, e.g., WO96/17951); an actA
promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002)
70:1087-1096); an rpsM promoter (see, e.g., Valdivia and Falkow
Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g., Hillen,
W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds),
Topics in Molecular and Structural Biology, Protein--Nucleic Acid
Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6
promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984)
12:7035); and the like. Suitable strong promoters for use in
prokaryotes such as Escherichia coli include, but are not limited
to Trc, Tac, T5, T7, and PLambda. Non-limiting examples of
operators for use in bacterial host cells include a lactose
promoter operator (Lad repressor protein changes conformation when
contacted with lactose, thereby preventing the Lad repressor
protein from binding to the operator), a tryptophan promoter
operator (when complexed with tryptophan, TrpR repressor protein
has a conformation that binds the operator; in the absence of
tryptophan, the TrpR repressor protein has a conformation that does
not bind to the operator), and a tac promoter operator (see, e.g.,
deBoer et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25).
[0227] Other examples of suitable promoters include the immediate
early cytomegalovirus (CMV) promoter sequence. This promoter
sequence is a strong constitutive promoter sequence capable of
driving high levels of expression of any polynucleotide sequence
operatively linked thereto. Other constitutive promoter sequences
may also be used, including, but not limited to a simian virus 40
(SV40) early promoter, a mouse mammary tumor virus (MMTV) or human
immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a
MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr
virus immediate early promoter, a Rous sarcoma virus promoter, the
EF-1 alpha promoter, as well as human gene promoters such as, but
not limited to, an actin promoter, a myosin promoter, a hemoglobin
promoter, and a creatine kinase promoter. Further, the invention
should not be limited to the use of constitutive promoters.
Inducible promoters are also contemplated as part of the invention.
The use of an inducible promoter provides a molecular switch
capable of turning on expression of the polynucleotide sequence
which it is operatively linked when such expression is desired, or
turning off the expression when expression is not desired. Examples
of inducible promoters include, but are not limited to a
metallothionine promoter, a glucocorticoid promoter, a progesterone
promoter, and a tetracycline promoter.
[0228] In some embodiments, the locus or construct or transgene
containing the suitable promoter is irreversibly switched through
the induction of an inducible system. Suitable systems for
induction of an irreversible switch are well known in the art,
e.g., induction of an irreversible switch may make use of a
Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et
al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of
which is incorporated herein by reference). Any suitable
combination of recombinase, endonuclease, ligase, recombination
sites, etc. known to the art may be used in generating an
irreversibly switchable promoter. Methods, mechanisms, and
requirements for performing site-specific recombination, described
elsewhere herein, find use in generating irreversibly switched
promoters and are well known in the art, see, e.g., Grindley et al.
Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular
Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.),
the disclosures of which are incorporated herein by reference.
[0229] In some embodiments, a nucleic acid of the present
disclosure further comprises a nucleic acid sequence encoding a TCR
and/or CAR inducible expression cassette. In one embodiment, the
TCR and/or CAR inducible expression cassette is for the production
of a transgenic polypeptide product that is released upon TCR
and/or CAR signaling. See, e.g., Chmielewski and Abken, Expert
Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy
(2015) 7(5): 535-544. In some embodiments, a nucleic acid of the
present disclosure further comprises a nucleic acid sequence
encoding a cytokine operably linked to a T-cell activation
responsive promoter. In some embodiments, the cytokine operably
linked to a T-cell activation responsive promoter is present on a
separate nucleic acid sequence. In one embodiment, the cytokine is
IL-12.
[0230] A nucleic acid of the present disclosure may be present
within an expression vector and/or a cloning vector. An expression
vector can include a selectable marker, an origin of replication,
and other features that provide for replication and/or maintenance
of the vector. Suitable expression vectors include, e.g., plasmids,
viral vectors, and the like. Large numbers of suitable vectors and
promoters are known to those of skill in the art; many are
commercially available for generating a subject recombinant
construct. The following vectors are provided by way of example,
and should not be construed in anyway as limiting: Bacterial: pBs,
phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a,
pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A,
pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3,
pBPV, pMSG and pSVL (Pharmacia).
[0231] Expression vectors generally have convenient restriction
sites located near the promoter sequence to provide for the
insertion of nucleic acid sequences encoding heterologous proteins.
A selectable marker operative in the expression host may be
present. Suitable expression vectors include, but are not limited
to, viral vectors (e.g. viral vectors based on vaccinia virus;
poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol.
Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6:
515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92:
7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO
94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO
95/00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene
Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA
(1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci.
(1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690,
Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al.,
Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239,
Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al.,
Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad.
Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human
immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl.
Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol.
(1999) 73: 7812-7816); a retroviral vector (e.g., Murine Leukemia
Virus, spleen necrosis virus, and vectors derived from retroviruses
such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis
virus, human immunodeficiency virus, myeloproliferative sarcoma
virus, and mammary tumor virus); and the like.
[0232] Additional expression vectors suitable for use are, e.g.,
without limitation, a lentivirus vector, a gamma retrovirus vector,
a foamy virus vector, an adeno-associated virus vector, an
adenovirus vector, a pox virus vector, a herpes virus vector, an
engineered hybrid virus vector, a transposon mediated vector, and
the like. Viral vector technology is well known in the art and is
described, for example, in Sambrook et al., 2012, Molecular
Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor
Press, NY), and in other virology and molecular biology manuals.
Viruses, which are useful as vectors include, but are not limited
to, retroviruses, adenoviruses, adeno-associated viruses, herpes
viruses, and lentiviruses.
[0233] In general, a suitable vector contains an origin of
replication functional in at least one organism, a promoter
sequence, convenient restriction endonuclease sites, and one or
more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S.
Pat. No. 6,326,193).
[0234] In some embodiments, an expression vector (e.g., a
lentiviral vector) may be used to introduce the chimeric cell
surface sialidase or variant sialidase precursor protein into an
immune cell or precursor thereof (e.g., a T cell). Accordingly, an
expression vector (e.g., a lentiviral vector) of the present
invention may comprise a nucleic acid encoding for a chimeric cell
surface sialidase or variant sialidase precursor protein (and
optionally a TCR and/or CAR). In some embodiments, the expression
vector (e.g., lentiviral vector) will comprise additional elements
that will aid in the functional expression of the chimeric cell
surface sialidase or variant sialidase precursor protein encoded
therein. In some embodiments, an expression vector comprising a
nucleic acid encoding for a chimeric cell surface sialidase or
variant sialidase precursor protein further comprises a mammalian
promoter. In one embodiment, the vector further comprises an
elongation-factor-1-alpha promoter (EF-1.alpha. promoter). Use of
an EF-1.alpha. promoter may increase the efficiency in expression
of downstream transgenes (e.g., a chimeric cell surface sialidase
or variant sialidase precursor protein or TCR or CAR encoding
nucleic acid sequence). Physiologic promoters (e.g., an EF-1.alpha.
promoter) may be less likely to induce integration mediated
genotoxicity, and may abrogate the ability of the retroviral vector
to transform stem cells. Other physiological promoters suitable for
use in a vector (e.g., lentiviral vector) are known to those of
skill in the art and may be incorporated into a vector of the
present invention. In some embodiments, the vector (e.g.,
lentiviral vector) further comprises a non-requisite cis acting
sequence that may improve titers and gene expression. One
non-limiting example of a non-requisite cis acting sequence is the
central polypurine tract and central termination sequence
(cPPT/CTS) which is important for efficient reverse transcription
and nuclear import. Other non-requisite cis acting sequences are
known to those of skill in the art and may be incorporated into a
vector (e.g., lentiviral vector) of the present invention.
[0235] In some embodiments, the vector further comprises a
posttranscriptional regulatory element. Posttranscriptional
regulatory elements may improve RNA translation, improve transgene
expression and stabilize RNA transcripts. One example of a
posttranscriptional regulatory element is the woodchuck hepatitis
virus posttranscriptional regulatory element (WPRE). Accordingly,
in some embodiments a vector for the present invention further
comprises a WPRE sequence. Various posttranscriptional regulator
elements are known to those of skill in the art and may be
incorporated into a vector (e.g., lentiviral vector) of the present
invention. A vector of the present invention may further comprise
additional elements such as a rev response element (RRE) for RNA
transport, packaging sequences, and 5' and 3' long terminal repeats
(LTRs). The term "long terminal repeat" or "LTR" refers to domains
of base pairs located at the ends of retroviral DNAs which comprise
U3, R and U5 regions. LTRs generally provide functions required for
the expression of retroviral genes (e.g., promotion, initiation and
polyadenylation of gene transcripts) and to viral replication. In
one embodiment, a vector (e.g., lentiviral vector) of the present
invention includes a 3' U3 deleted LTR. Accordingly, a vector
(e.g., lentiviral vector) of the present invention may comprise any
combination of the elements described herein to enhance the
efficiency of functional expression of transgenes. For example, a
vector (e.g., lentiviral vector) of the present invention may
comprise a WPRE sequence, cPPT sequence, RRE sequence, 5'LTR, 3' U3
deleted LTR' in addition to a nucleic acid encoding for a chimeric
cell surface sialidase or variant sialidase precursor protein.
[0236] Vectors of the present invention may be self-inactivating
vectors. As used herein, the term "self-inactivating vector" refers
to vectors in which the 3' LTR enhancer promoter region (U3 region)
has been modified (e.g., by deletion or substitution). A
self-inactivating vector may prevent viral transcription beyond the
first round of viral replication. Consequently, a self-inactivating
vector may be capable of infecting and then integrating into a host
genome (e.g., a mammalian genome) only once, and cannot be passed
further. Accordingly, self-inactivating vectors may greatly reduce
the risk of creating a replication-competent virus.
[0237] In some embodiments, a nucleic acid of the present invention
may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro
synthesis of RNA are known to those of skill in the art; any known
method can be used to synthesize RNA comprising a sequence encoding
a chimeric cell surface sialidase or variant sialidase precursor
protein and/or TCR and/or CAR of the present disclosure. Methods
for introducing RNA into a host cell are known in the art. See,
e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA
comprising a nucleotide sequence encoding a chimeric cell surface
sialidase or variant sialidase precursor protein and/or TCR and/or
CAR of the present disclosure into a host cell can be carried out
in vitro, ex vivo or in vivo. For example, a host cell (e.g., an NK
cell, a cytotoxic T lymphocyte, etc.) can be electroporated in
vitro or ex vivo with RNA comprising a nucleotide sequence encoding
a chimeric cell surface sialidase or variant sialidase precursor
protein and/or TCR and/or CAR of the present disclosure.
[0238] In order to assess the expression of a polypeptide or
portions thereof, the expression vector to be introduced into a
cell may also contain either a selectable marker gene or a reporter
gene, or both, to facilitate identification and selection of
expressing cells from the population of cells sought to be
transfected or infected through viral vectors. In some embodiments,
the selectable marker may be carried on a separate piece of DNA and
used in a co-transfection procedure. Both selectable markers and
reporter genes may be flanked with appropriate regulatory sequences
to enable expression in the host cells. Useful selectable markers
include, without limitation, antibiotic-resistance genes.
[0239] Reporter genes are used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. In general, a reporter gene is a gene that is
not present in or expressed by the recipient organism or tissue and
that encodes a polypeptide whose expression is manifested by some
easily detectable property, e.g., enzymatic activity. Expression of
the reporter gene is assessed at a suitable time after the DNA has
been introduced into the recipient cells. Suitable reporter genes
may include, without limitation, genes encoding luciferase,
beta-galactosidase, chloramphenicol acetyl transferase, secreted
alkaline phosphatase, or the green fluorescent protein gene (e.g.,
Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
H. Methods of Treatment
[0240] The modified cells (e.g., T cells comprising a chimeric cell
surface sialidase or a variant sialidase precursor protein)
described herein may be included in a composition for
immunotherapy. The composition may include a pharmaceutical
composition and further include a pharmaceutically acceptable
carrier. A therapeutically effective amount of the pharmaceutical
composition comprising the modified T cells may be
administered.
[0241] In one aspect, the invention includes a method for adoptive
cell transfer therapy comprising administering to a subject in need
thereof a modified T cell of the present invention. In another
aspect, the invention includes a method of treating a disease or
condition (e.g. cancer) in a subject comprising administering to a
subject in need thereof a population of modified T cells.
[0242] Methods for administration of immune cells for adoptive cell
therapy are known and may be used in connection with the provided
methods and compositions. For example, adoptive T cell therapy
methods are described, e.g., in US Patent Application Publication
No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to
Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See,
e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933;
Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9;
Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments,
the cell therapy, e.g., adoptive T cell therapy is carried out by
autologous transfer, in which the cells are isolated and/or
otherwise prepared from the subject who is to receive the cell
therapy, or from a sample derived from such a subject. Thus, in
some aspects, the cells are derived from a subject, e.g., patient,
in need of a treatment and the cells, following isolation and
processing are administered to the same subject.
[0243] In some embodiments, the cell therapy, e.g., adoptive T cell
therapy, is carried out by allogeneic transfer, in which the cells
are isolated and/or otherwise prepared from a subject other than a
subject who is to receive or who ultimately receives the cell
therapy, e.g., a first subject. In such embodiments, the cells then
are administered to a different subject, e.g., a second subject, of
the same species. In some embodiments, the first and second
subjects are genetically identical. In some embodiments, the first
and second subjects are genetically similar. In some embodiments,
the second subject expresses the same HLA class or supertype as the
first subject.
[0244] In some embodiments, the subject has been treated with a
therapeutic agent targeting the disease or condition, e.g. the
tumor, prior to administration of the cells or composition
containing the cells. In some aspects, the subject is refractory or
non-responsive to the other therapeutic agent. In some embodiments,
the subject has persistent or relapsed disease, e.g., following
treatment with another therapeutic intervention, including
chemotherapy, radiation, and/or hematopoietic stem cell
transplantation (HSCT), e.g., allogenic HSCT. In some embodiments,
the administration effectively treats the subject despite the
subject having become resistant to another therapy.
[0245] In some embodiments, the subject is responsive to the other
therapeutic agent, and treatment with the therapeutic agent reduces
disease burden. In some aspects, the subject is initially
responsive to the therapeutic agent, but exhibits a relapse of the
disease or condition over time. In some embodiments, the subject
has not relapsed. In some such embodiments, the subject is
determined to be at risk for relapse, such as at a high risk of
relapse, and thus the cells are administered prophylactically,
e.g., to reduce the likelihood of or prevent relapse. In some
aspects, the subject has not received prior treatment with another
therapeutic agent.
[0246] In some embodiments, the subject has persistent or relapsed
disease, e.g., following treatment with another therapeutic
intervention, including chemotherapy, radiation, and/or
hematopoietic stem cell transplantation (HSCT), e.g., allogenic
HSCT. In some embodiments, the administration effectively treats
the subject despite the subject having become resistant to another
therapy.
[0247] The modified immune cells of the present invention can be
administered to an animal, preferably a mammal, even more
preferably a human, to treat a cancer. In addition, the cells of
the present invention can be used for the treatment of any
condition related to a cancer, especially a cell-mediated immune
response against a tumor cell(s), where it is desirable to treat or
alleviate the disease. The types of cancers to be treated with the
modified cells or pharmaceutical compositions of the invention
include, carcinoma, blastoma, and sarcoma, and certain leukemia or
lymphoid malignancies, benign and malignant tumors, and
malignancies e.g., sarcomas, carcinomas, and melanomas. Other
exemplary cancers include but are not limited breast cancer,
prostate cancer, ovarian cancer, cervical cancer, skin cancer,
pancreatic cancer, colorectal cancer, renal cancer, liver cancer,
brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and
the like. The cancers may be non-solid tumors (such as
hematological tumors) or solid tumors. Adult tumors/cancers and
pediatric tumors/cancers are also included. In one embodiment, the
cancer is a solid tumor or a hematological tumor. In one
embodiment, the cancer is a carcinoma. In one embodiment, the
cancer is a sarcoma. In one embodiment, the cancer is a leukemia.
In one embodiment the cancer is a solid tumor.
[0248] Solid tumors are abnormal masses of tissue that usually do
not contain cysts or liquid areas. Solid tumors can be benign or
malignant. Different types of solid tumors are named for the type
of cells that form them (such as sarcomas, carcinomas, and
lymphomas). Examples of solid tumors, such as sarcomas and
carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteosarcoma, and other sarcomas, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, lymphoid malignancy, pancreatic cancer, breast
cancer, lung cancers, ovarian cancer, prostate cancer,
hepatocellular carcinoma, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid
carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,
cervical cancer, testicular tumor, seminoma, bladder carcinoma,
melanoma, and CNS tumors (such as a glioma (such as brainstem
glioma and mixed gliomas), glioblastoma (also known as glioblastoma
multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma,
Schwannoma craniopharyogioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,
neuroblastoma, retinoblastoma and brain metastases).
[0249] Carcinomas that can be amenable to therapy by a method
disclosed herein include, but are not limited to, esophageal
carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form
of skin cancer), squamous cell carcinoma (various tissues), bladder
carcinoma, including transitional cell carcinoma (a malignant
neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma,
colorectal carcinoma, gastric carcinoma, lung carcinoma, including
small cell carcinoma and non-small cell carcinoma of the lung,
adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma,
breast carcinoma, ovarian carcinoma, prostate carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma,
medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ
or bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma,
testicular carcinoma, osteogenic carcinoma, epithelial carcinoma,
and nasopharyngeal carcinoma.
[0250] Sarcomas that can be amenable to therapy by a method
disclosed herein include, but are not limited to, fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic
sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma,
and other soft tissue sarcomas.
[0251] In certain exemplary embodiments, the modified immune cells
of the invention are used to treat a myeloma, or a condition
related to myeloma. Examples of myeloma or conditions related
thereto include, without limitation, light chain myeloma,
non-secretory myeloma, monoclonal gamopathy of undertermined
significance (MGUS), plasmacytoma (e.g., solitary, multiple
solitary, extramedullary plasmacytoma), amyloidosis, and multiple
myeloma. In one embodiment, a method of the present disclosure is
used to treat multiple myeloma. In one embodiment, a method of the
present disclosure is used to treat refractory myeloma. In one
embodiment, a method of the present disclosure is used to treat
relapsed myeloma.
[0252] In certain exemplary embodiments, the modified immune cells
of the invention are used to treat a melanoma, or a condition
related to melanoma. Examples of melanoma or conditions related
thereto include, without limitation, superficial spreading
melanoma, nodular melanoma, lentigo maligna melanoma, acral
lentiginous melanoma, amelanotic melanoma, or melanoma of the skin
(e.g., cutaneous, eye, vulva, vagina, rectum melanoma). In one
embodiment, a method of the present disclosure is used to treat
cutaneous melanoma. In one embodiment, a method of the present
disclosure is used to treat refractory melanoma. In one embodiment,
a method of the present disclosure is used to treat relapsed
melanoma.
[0253] In yet other exemplary embodiments, the modified immune
cells of the invention are used to treat a sarcoma, or a condition
related to sarcoma. Examples of sarcoma or conditions related
thereto include, without limitation, angiosarcoma, chondrosarcoma,
Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor,
leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath
tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and
synovial sarcoma. In one embodiment, a method of the present
disclosure is used to treat synovial sarcoma. In one embodiment, a
method of the present disclosure is used to treat liposarcoma such
as myxoid/round cell liposarcoma, differentiated/dedifferentiated
liposarcoma, and pleomorphic liposarcoma. In one embodiment, a
method of the present disclosure is used to treat myxoid/round cell
liposarcoma. In one embodiment, a method of the present disclosure
is used to treat a refractory sarcoma. In one embodiment, a method
of the present disclosure is used to treat a relapsed sarcoma.
[0254] The cells of the invention to be administered may be
autologous, with respect to the subject undergoing therapy.
[0255] The administration of the cells of the invention may be
carried out in any convenient manner known to those of skill in the
art. The cells of the present invention may be administered to a
subject by aerosol inhalation, injection, ingestion, transfusion,
implantation or transplantation. The compositions described herein
may be administered to a patient transarterially, subcutaneously,
intradermally, intratumorally, intranodally, intramedullary,
intramuscularly, by intravenous (i.v.) injection, or
intraperitoneally. In other instances, the cells of the invention
are injected directly into a site of inflammation in the subject, a
local disease site in the subject, alymph node, an organ, a tumor,
and the like.
[0256] In some embodiments, the cells are administered at a desired
dosage, which in some aspects includes a desired dose or number of
cells or cell type(s) and/or a desired ratio of cell types. Thus,
the dosage of cells in some embodiments is based on a total number
of cells (or number per kg body weight) and a desired ratio of the
individual populations or sub-types, such as the CD4+ to CD8+
ratio. In some embodiments, the dosage of cells is based on a
desired total number (or number per kg of body weight) of cells in
the individual populations or of individual cell types. In some
embodiments, the dosage is based on a combination of such features,
such as a desired number of total cells, desired ratio, and desired
total number of cells in the individual populations.
[0257] In some embodiments, the populations or sub-types of cells,
such as CD8.sup.+ and CD4.sup.+ T cells, are administered at or
within a tolerated difference of a desired dose of total cells,
such as a desired dose of T cells. In some aspects, the desired
dose is a desired number of cells or a desired number of cells per
unit of body weight of the subject to whom the cells are
administered, e.g., cells/kg. In some aspects, the desired dose is
at or above a minimum number of cells or minimum number of cells
per unit of body weight. In some aspects, among the total cells,
administered at the desired dose, the individual populations or
sub-types are present at or near a desired output ratio (such as
CD4.sup.+ to CD8.sup.+ ratio), e.g., within a certain tolerated
difference or error of such a ratio.
[0258] In some embodiments, the cells are administered at or within
a tolerated difference of a desired dose of one or more of the
individual populations or sub-types of cells, such as a desired
dose of CD4+ cells and/or a desired dose of CD8+ cells. In some
aspects, the desired dose is a desired number of cells of the
sub-type or population, or a desired number of such cells per unit
of body weight of the subject to whom the cells are administered,
e.g., cells/kg. In some aspects, the desired dose is at or above a
minimum number of cells of the population or subtype, or minimum
number of cells of the population or sub-type per unit of body
weight. Thus, in some embodiments, the dosage is based on a desired
fixed dose of total cells and a desired ratio, and/or based on a
desired fixed dose of one or more, e.g., each, of the individual
sub-types or sub-populations. Thus, in some embodiments, the dosage
is based on a desired fixed or minimum dose of T cells and a
desired ratio of CD4.sup.+ to CD8.sup.+ cells, and/or is based on a
desired fixed or minimum dose of CD4.sup.+ and/or CD8.sup.+
cells.
[0259] In certain embodiments, the cells, or individual populations
of sub-types of cells, are administered to the subject at a range
of about one million to about 100 billion cells, such as, e.g., 1
million to about 50 billion cells (e.g., about 5 million cells,
about 25 million cells, about 500 million cells, about 1 billion
cells, about 5 billion cells, about 20 billion cells, about 30
billion cells, about 40 billion cells, or a range defined by any
two of the foregoing values), such as about 10 million to about 100
billion cells (e.g., about 20 million cells, about 30 million
cells, about 40 million cells, about 60 million cells, about 70
million cells, about 80 million cells, about 90 million cells,
about 10 billion cells, about 25 billion cells, about 50 billion
cells, about 75 billion cells, about 90 billion cells, or a range
defined by any two of the foregoing values), and in some cases
about 100 million cells to about 50 billion cells (e.g., about 120
million cells, about 250 million cells, about 350 million cells,
about 450 million cells, about 650 million cells, about 800 million
cells, about 900 million cells, about 3 billion cells, about 30
billion cells, about 45 billion cells) or any value in between
these ranges.
[0260] In some embodiments, the dose of total cells and/or dose of
individual sub-populations of cells is within a range of between at
or about 1.times.10.sup.5 cells/kg to about 1.times.10.sup.11
cells/kg 10.sup.4 and at or about 10.sup.11 cells/kilograms (kg)
body weight, such as between 10.sup.5 and 10.sup.6 cells/kg body
weight, for example, at or about 1.times.10.sup.5 cells/kg,
1.5.times.10.sup.5 cells/kg, 2.times.10.sup.5 cells/kg, or
1.times.10.sup.6 cells/kg body weight. For example, in some
embodiments, the cells are administered at, or within a certain
range of error of, between at or about 10.sup.4 and at or about
10.sup.9 T cells/kilograms (kg) body weight, such as between
10.sup.5 and 10.sup.6 T cells/kg body weight, for example, at or
about 1.times.10.sup.5 T cells/kg, 1.5.times.10.sup.5 T cells/kg,
2.times.10.sup.5 T cells/kg, or 1.times.10.sup.6 T cells/kg body
weight. In other exemplary embodiments, a suitable dosage range of
modified cells for use in a method of the present disclosure
includes, without limitation, from about 1.times.10.sup.5 cells/kg
to about 1.times.10.sup.6 cells/kg, from about 1.times.10.sup.6
cells/kg to about 1.times.10.sup.7 cells/kg, from about
1.times.10.sup.7 cells/kg about 1.times.10.sup.8 cells/kg, from
about 1.times.10.sup.8 cells/kg about 1.times.10.sup.9 cells/kg,
from about 1.times.10.sup.9 cells/kg about 1.times.10.sup.10
cells/kg, from about 1.times.10.sup.10 cells/kg about
1.times.10.sup.11 cells/kg. In an exemplary embodiment, a suitable
dosage for use in a method of the present disclosure is about
1.times.10.sup.8 cells/kg. In an exemplary embodiment, a suitable
dosage for use in a method of the present disclosure is about
1.times.10.sup.7 cells/kg. In other embodiments, a suitable dosage
is from about 1.times.10.sup.7 total cells to about
5.times.10.sup.7 total cells. In some embodiments, a suitable
dosage is from about 1.times.10.sup.8 total cells to about
5.times.10.sup.8 total cells. In some embodiments, a suitable
dosage is from about 1.4.times.10.sup.7 total cells to about
1.1.times.10.sup.9 total cells. In an exemplary embodiment, a
suitable dosage for use in a method of the present disclosure is
about 7.times.10.sup.9 total cells.
[0261] In some embodiments, the cells are administered at or within
a certain range of error of between at or about 10.sup.4 and at or
about 10.sup.9 CD4.sup.+ and/or CD8.sup.+ cells/kilograms (kg) body
weight, such as between 10.sup.5 and 10.sup.6 CD4.sup.+ and/or
CD8.sup.+ cells/kg body weight, for example, at or about
1.times.10.sup.5 CD4.sup.+ and/or CD8.sup.+ cells/kg,
1.5.times.10.sup.5 CD4.sup.+ and/or CD8.sup.+ cells/kg,
2.times.10.sup.5 CD4.sup.+ and/or CD8.sup.+ cells/kg, or
1.times.10.sup.6 CD4.sup.+ and/or CD8.sup.+ cells/kg body weight.
In some embodiments, the cells are administered at or within a
certain range of error of, greater than, and/or at least about
1.times.10.sup.6, about 2.5.times.10.sup.6, about 5.times.10.sup.6,
about 7.5.times.10.sup.6, or about 9.times.10.sup.6 CD4.sup.+
cells, and/or at least about 1.times.10.sup.6, about
2.5.times.10.sup.6, about 5.times.10.sup.6, about
7.5.times.10.sup.6, or about 9.times.10.sup.6 CD8+ cells, and/or at
least about 1.times.10.sup.6, about 2.5.times.10.sup.6, about
5.times.10.sup.6, about 7.5.times.10.sup.6, or about
9.times.10.sup.6 T cells. In some embodiments, the cells are
administered at or within a certain range of error of between about
10.sup.8 and 10.sup.12 or between about 10.sup.10 and 10.sup.11 T
cells, between about 10.sup.8 and 10.sup.12 or between about
10.sup.10 and 10.sup.11 CD4.sup.+ cells, and/or between about
10.sup.8 and 10.sup.12 or between about 10.sup.10 and 10.sup.11
CD8.sup.+ cells.
[0262] In some embodiments, the cells are administered at or within
a tolerated range of a desired output ratio of multiple cell
populations or sub-types, such as CD4+ and CD8+ cells or sub-types.
In some aspects, the desired ratio can be a specific ratio or can
be a range of ratios, for example, in some embodiments, the desired
ratio (e.g., ratio of CD4.sup.+ to CD8.sup.+ cells) is between at
or about 5:1 and at or about 5:1 (or greater than about 1:5 and
less than about 5:1), or between at or about 1:3 and at or about
3:1 (or greater than about 1:3 and less than about 3:1), such as
between at or about 2:1 and at or about 1:5 (or greater than about
1:5 and less than about 2:1, such as at or about 5:1, 4.5:1, 4:1,
3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,
1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,
1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In
some aspects, the tolerated difference is within about 1%, about
2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of
the desired ratio, including any value in between these ranges.
[0263] In some embodiments, a dose of modified cells is
administered to a subject in need thereof, in a single dose or
multiple doses. In some embodiments, a dose of modified cells is
administered in multiple doses, e.g., once a week or every 7 days,
once every 2 weeks or every 14 days, once every 3 weeks or every 21
days, once every 4 weeks or every 28 days. In an exemplary
embodiment, a single dose of modified cells is administered to a
subject in need thereof. In an exemplary embodiment, a single dose
of modified cells is administered to a subject in need thereof by
rapid intravenous infusion.
[0264] For the prevention or treatment of disease, the appropriate
dosage may depend on the type of disease to be treated, the type of
cells or recombinant receptors, the severity and course of the
disease, whether the cells are administered for preventive or
therapeutic purposes, previous therapy, the subject's clinical
history and response to the cells, and the discretion of the
attending physician. The compositions and cells are in some
embodiments suitably administered to the subject at one time or
over a series of treatments.
[0265] In some embodiments, the cells are administered as part of a
combination treatment, such as simultaneously with or sequentially
with, in any order, another therapeutic intervention, such as an
antibody or engineered cell or receptor or agent, such as a
cytotoxic or therapeutic agent. The cells in some embodiments are
co-administered with one or more additional therapeutic agents or
in connection with another therapeutic intervention, either
simultaneously or sequentially in any order. In some contexts, the
cells are co-administered with another therapy sufficiently close
in time such that the cell populations enhance the effect of one or
more additional therapeutic agents, or vice versa. In some
embodiments, the cells are administered prior to the one or more
additional therapeutic agents. In some embodiments, the cells are
administered after the one or more additional therapeutic agents.
In some embodiments, the one or more additional agents includes a
cytokine, such as IL-2, for example, to enhance persistence. In
some embodiments, the methods comprise administration of a
chemotherapeutic agent.
[0266] In certain embodiments, the modified cells of the invention
may be administered to a subject in combination with an immune
checkpoint antibody (e.g., an anti-PD1, anti-CTLA-4, or anti-PDL1
antibody). For example, the modified cell may be administered in
combination with an antibody or antibody fragment targeting, for
example, PD-1 (programmed death 1 protein). Examples of anti-PD-1
antibodies include, but are not limited to, pembrolizumab
(KEYTRUDA.RTM., formerly lambrolizumab, also known as MK-3475), and
nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA.RTM.) or an
antigen-binding fragment thereof. In certain embodiments, the
modified cell may be administered in combination with an anti-PD-L1
antibody or antigen-binding fragment thereof. Examples of
anti-PD-L1 antibodies include, but are not limited to, BMS-936559,
MPDL3280A (TECENTRIQ.RTM., Atezolizumab), and MEDI4736 (Durvalumab,
Imfinzi). In certain embodiments, the modified cell may be
administered in combination with an anti-CTLA-4 antibody or
antigen-binding fragment thereof. An example of an anti-CTLA-4
antibody includes, but is not limited to, Ipilimumab (trade name
Yervoy). Other types of immune checkpoint modulators may also be
used including, but not limited to, small molecules, siRNA, miRNA,
and CRISPR systems. Immune checkpoint modulators may be
administered before, after, or concurrently with the modified cell
comprising the CAR. In certain embodiments, combination treatment
comprising an immune checkpoint modulator may increase the
therapeutic efficacy of a therapy comprising a modified cell of the
present invention.
[0267] Following administration of the cells, the biological
activity of the engineered cell populations in some embodiments is
measured, e.g., by any of a number of known methods. Parameters to
assess include specific binding of an engineered or natural T cell
or other immune cell to antigen, in vivo, e.g., by imaging, or ex
vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the
ability of the engineered cells to destroy target cells can be
measured using any suitable method known in the art, such as
cytotoxicity assays described in, for example, Kochenderfer et al.,
J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J.
Immunological Methods, 285(1): 25-40 (2004). In certain
embodiments, the biological activity of the cells is measured by
assaying expression and/or secretion of one or more cytokines, such
as CD107a, IFN.gamma., IL-2, and TNF. In some aspects the
biological activity is measured by assessing clinical outcome, such
as reduction in tumor burden or load.
[0268] In certain embodiments, the subject is provided a secondary
treatment. Secondary treatments include but are not limited to
chemotherapy, radiation, surgery, and medications.
[0269] In some embodiments, the subject can be administered a
conditioning therapy prior to CAR T cell therapy. In some
embodiments, the conditioning therapy comprises administering an
effective amount of cyclophosphamide to the subject. In some
embodiments, the conditioning therapy comprises administering an
effective amount of fludarabine to the subject. In preferred
embodiments, the conditioning therapy comprises administering an
effective amount of a combination of cyclophosphamide and
fludarabine to the subject. Administration of a conditioning
therapy prior to CAR T cell therapy may increase the efficacy of
the CAR T cell therapy. Methods of conditioning patients for T cell
therapy are described in U.S. Pat. No. 9,855,298, which is
incorporated herein by reference in its entirety.
[0270] In some embodiments, a specific dosage regimen of the
present disclosure includes a lymphodepletion step prior to the
administration of the modified T cells. In an exemplary embodiment,
the lymphodepletion step includes administration of
cyclophosphamide and/or fludarabine.
[0271] In some embodiments, the lymphodepletion step includes
administration of cyclophosphamide at a dose of between about 200
mg/m.sup.2/day and about 2000 mg/m.sup.2/day (e.g., 200
mg/m.sup.2/day, 300 mg/m.sup.2/day, or 500 mg/m.sup.2/day). In an
exemplary embodiment, the dose of cyclophosphamide is about 300
mg/m.sup.2/day. In some embodiments, the lymphodepletion step
includes administration of fludarabine at a dose of between about
20 mg/m.sup.2/day and about 900 mg/m.sup.2/day (e.g., 20
mg/m.sup.2/day, 25 mg/m.sup.2/day, 30 mg/m.sup.2/day, or 60
mg/m.sup.2/day). In an exemplary embodiment, the dose of
fludarabine is about 30 mg/m.sup.2/day.
[0272] In some embodiment, the lymphodepletion step includes
administration of cyclophosphamide at a dose of between about 200
mg/m.sup.2/day and about 2000 mg/m.sup.2/day (e.g., 200
mg/m.sup.2/day, 300 mg/m.sup.2/day, or 500 mg/m.sup.2/day), and
fludarabine at a dose of between about 20 mg/m.sup.2/day and about
900 mg/m.sup.2/day (e.g., 20 mg/m.sup.2/day, 25 mg/m.sup.2/day, 30
mg/m.sup.2/day, or 60 mg/m.sup.2/day). In an exemplary embodiment,
the lymphodepletion step includes administration of
cyclophosphamide at a dose of about 300 mg/m.sup.2/day, and
fludarabine at a dose of about 30 mg/m.sup.2/day.
[0273] In an exemplary embodiment, the dosing of cyclophosphamide
is 300 mg/m.sup.2/day over three days, and the dosing of
fludarabine is 30 mg/m.sup.2/day over three days.
[0274] Dosing of lymphodepletion chemotherapy may be scheduled on
Days -6 to -4 (with a -1 day window, i.e., dosing on Days -7 to -5)
relative to T cell (e.g., CAR-T, TCR-T, a modified T cell, etc.)
infusion on Day 0.
[0275] In an exemplary embodiment, for a subject having cancer, the
subject receives lymphodepleting chemotherapy including 300
mg/m.sup.2 of cyclophosphamide by intravenous infusion 3 days prior
to administration of the modified T cells. In an exemplary
embodiment, for a subject having cancer, the subject receives
lymphodepleting chemotherapy including 300 mg/m.sup.2 of
cyclophosphamide by intravenous infusion for 3 days prior to
administration of the modified T cells.
[0276] In an exemplary embodiment, for a subject having cancer, the
subject receives lymphodepleting chemotherapy including fludarabine
at a dose of between about 20 mg/m.sup.2/day and about 900
mg/m.sup.2/day (e.g., 20 mg/m.sup.2/day, 25 mg/m.sup.2/day, 30
mg/m.sup.2/day, or 60 mg/m.sup.2/day). In an exemplary embodiment,
for a subject having cancer, the subject receives lymphodepleting
chemotherapy including fludarabine at a dose of 30 mg/m.sup.2 for 3
days.
[0277] In an exemplary embodiment, for a subject having cancer, the
subject receives lymphodepleting chemotherapy including
cyclophosphamide at a dose of between about 200 mg/m.sup.2/day and
about 2000 mg/m.sup.2/day (e.g., 200 mg/m.sup.2/day, 300
mg/m.sup.2/day, or 500 mg/m.sup.2/day), and fludarabine at a dose
of between about 20 mg/m.sup.2/day and about 900 mg/m.sup.2/day
(e.g., 20 mg/m.sup.2/day, 25 mg/m.sup.2/day, 30 mg/m.sup.2/day, or
60 mg/m.sup.2/day). In an exemplary embodiment, for a subject
having cancer, the subject receives lymphodepleting chemotherapy
including cyclophosphamide at a dose of about 300 mg/m.sup.2/day,
and fludarabine at a dose of 30 mg/m.sup.2 for 3 days.
[0278] Cells of the invention can be administered in dosages and
routes and at times to be determined in appropriate pre-clinical
and clinical experimentation and trials. Cell compositions may be
administered multiple times at dosages within these ranges.
Administration of the cells of the invention may be combined with
other methods useful to treat the desired disease or condition as
determined by those of skill in the art.
[0279] It is known in the art that one of the adverse effects
following infusion of CAR T cells is the onset of immune
activation, known as cytokine release syndrome (CRS). CRS is immune
activation resulting in elevated inflammatory cytokines. CRS is a
known on-target toxicity, development of which likely correlates
with efficacy. Clinical and laboratory measures range from mild CRS
(constitutional symptoms and/or grade-2 organ toxicity) to severe
CRS (sCRS; grade>3 organ toxicity, aggressive clinical
intervention, and/or potentially life threatening). Clinical
features include: high fever, malaise, fatigue, myalgia, nausea,
anorexia, tachycardia/hypotension, capillary leak, cardiac
dysfunction, renal impairment, hepatic failure, and disseminated
intravascular coagulation. Dramatic elevations of cytokines
including interferon-gamma, granulocyte macrophage
colony-stimulating factor, IL-10, and IL-6 have been shown
following CAR T-cell infusion. One CRS signature is elevation of
cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha
(moderate), and IL-2 (mild). Elevations in clinically available
markers of inflammation including ferritin and C-reactive protein
(CRP) have also been observed to correlate with the CRS syndrome.
The presence of CRS generally correlates with expansion and
progressive immune activation of adoptively transferred cells. It
has been demonstrated that the degree of CRS severity is dictated
by disease burden at the time of infusion as patients with high
tumor burden experience a more sCRS.
[0280] Accordingly, the invention provides for, following the
diagnosis of CRS, appropriate CRS management strategies to mitigate
the physiological symptoms of uncontrolled inflammation without
dampening the antitumor efficacy of the engineered cells (e.g., CAR
T cells). CRS management strategies are known in the art. For
example, systemic corticosteroids may be administered to rapidly
reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising
initial antitumor response.
[0281] In some embodiments, an anti-IL-6R antibody may be
administered. An example of an anti-IL-6R antibody is the Food and
Drug Administration-approved monoclonal antibody tocilizumab, also
known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab
is a humanized monoclonal antibody against the interleukin-6
receptor (IL-6R). Administration of tocilizumab has demonstrated
near-immediate reversal of CRS.
[0282] CRS is generally managed based on the severity of the
observed syndrome and interventions are tailored as such. CRS
management decisions may be based upon clinical signs and symptoms
and response to interventions, not solely on laboratory values
alone.
[0283] Mild to moderate cases generally are treated with symptom
management with fluid therapy, non-steroidal anti-inflammatory drug
(NSAID) and antihistamines as needed for adequate symptom relief.
More severe cases include patients with any degree of hemodynamic
instability; with any hemodynamic instability, the administration
of tocilizumab is recommended. The first-line management of CRS may
be tocilizumab, in some embodiments, at the labeled dose of 8 mg/kg
IV over 60 minutes (not to exceed 800 mg/dose); tocilizumab can be
repeated Q8 hours. If suboptimal response to the first dose of
tocilizumab, additional doses of tocilizumab may be considered.
Tocilizumab can be administered alone or in combination with
corticosteroid therapy. Patients with continued or progressive CRS
symptoms, inadequate clinical improvement in 12-18 hours or poor
response to tocilizumab, may be treated with high-dose
corticosteroid therapy, generally hydrocortisone 100 mg IV or
methylprednisolone 1-2 mg/kg. In patients with more severe
hemodynamic instability or more severe respiratory symptoms,
patients may be administered high-dose corticosteroid therapy early
in the course of the CRS. CRS management guidance may be based on
published standards (Lee et al. (2019) Biol Blood Marrow
Transplant, doi.org/10.1016/j.bbmt.2018.12.758; Neelapu et al.
(2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer
Discov, 6(6):664-679).
[0284] Features consistent with Macrophage Activation Syndrome
(MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been
observed in patients treated with CAR-T therapy (Henter, 2007),
coincident with clinical manifestations of the CRS. MAS appears to
be a reaction to immune activation that occurs from the CRS, and
should therefore be considered a manifestation of CRS. MAS is
similar to HLH (also a reaction to immune stimulation). The
clinical syndrome of MAS is characterized by high grade
non-remitting fever, cytopenias affecting at least two of three
lineages, and hepatosplenomegaly. It is associated with high serum
ferritin, soluble interleukin-2 receptor, and triglycerides, and a
decrease of circulating natural killer (NK) activity.
[0285] In one aspect, the invention includes a method of treating
cancer in a subject in need thereof, comprising administering to
the subject any one of the modified immune or precursor cells
disclosed herein.
[0286] In another aspect, the invention includes a method of
treating cancer in a subject in need thereof, the method comprising
administering a modified T cell comprising a chimeric cell surface
sialidase (neuraminidase) enzyme comprising an extracellular
portion comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface,
and a chimeric antigen receptor (CAR).
[0287] In another aspect, the invention includes a method of
treating cancer in a subject in need thereof, the method comprising
administering a modified T cell comprising a variant sialidase
precursor protein comprising a heterologous secretory sequence
operably linked to a sialidase (neuraminidase) or an enzymatically
functional portion thereof, wherein the variant sialidase precursor
protein lacks a transmembrane domain, and wherein the sialidase or
enzymatically functional portion thereof is capable of being
secreted from an immune or precursor cell thereof when the variant
sialidase precursor protein is expressed in the cell, and a
chimeric antigen receptor (CAR).
[0288] In certain embodiments, the CAR comprises specificity for
TnMUC1. In certain embodiments, the CAR comprises specificity for
CD19. In certain embodiments, the CAR comprises specificity for
PSMA.
[0289] In certain embodiments, the method further comprises
administering to the subject a population of innate immune cells.
In certain embodiments, the innate immune cells are NK cells. In
certain embodiments, the NK cells are autologous NK cells obtained
from a human subject.
[0290] In another aspect, the invention includes a method of
treating cancer in a subject in need thereof comprising:
administering to the subject a therapeutically effective amount of
a modified T cell comprising a chimeric cell surface sialidase
(neuraminidase) enzyme comprising an extracellular portion
comprising a sialidase (neuraminidase) or an enzymatically
functional portion thereof, and a heterologous transmembrane domain
capable of tethering the extracellular portion to a cell surface,
and a chimeric antigen receptor (CAR); and administering to the
subject a therapeutically effective amount of a NK cell.
[0291] In another aspect, the invention includes a method of
treating cancer in a subject in need thereof comprising:
administering to the subject a therapeutically effective amount of
a modified T cell comprising a variant sialidase precursor protein
comprising a heterologous secretory sequence operably linked to a
sialidase (neuraminidase) or an enzymatically functional portion
thereof, wherein the variant sialidase precursor protein lacks a
transmembrane domain, and wherein the sialidase or enzymatically
functional portion thereof is capable of being secreted from an
immune or precursor cell thereof when the variant sialidase
precursor protein is expressed in the cell, and a chimeric antigen
receptor (CAR); and administering to the subject a therapeutically
effective amount of a NK cell.
[0292] In certain embodiments, the modified T cell and the NK cell
are administered simultaneously. In certain embodiments, the
modified T cell and the NK cell are administered separately. In
certain embodiments, the NK cell is autologous.
I. Sources of Immune Cells
[0293] Prior to expansion, a source of immune cells may be obtained
from a subject for ex vivo manipulation. Sources of target cells
for ex vivo manipulation may also include, e.g., autologous or
heterologous donor blood, cord blood, or bone marrow. For example,
the source of immune cells may be from the subject to be treated
with the modified immune cells of the invention, e.g., the
subject's blood, the subject's cord blood, or the subject's bone
marrow. Non-limiting examples of subjects include humans, dogs,
cats, mice, rats, and transgenic species thereof. Preferably, the
subject is a human.
[0294] Immune cells can be obtained from a number of sources,
including blood, peripheral blood mononuclear cells, bone marrow,
lymph node tissue, spleen tissue, umbilical cord, lymph, or
lymphoid organs. Immune cells are cells of the immune system, such
as cells of the innate or adaptive immunity, e.g., myeloid or
lymphoid cells, including lymphocytes, typically T cells and/or NK
cells. Other exemplary cells include stem cells, such as
multipotent and pluripotent stem cells, including induced
pluripotent stem cells (iPSCs). In some aspects, the cells are
human cells. With reference to the subject to be treated, the cells
may be allogeneic and/or autologous. The cells typically are
primary cells, such as those isolated directly from a subject
and/or isolated from a subject and frozen.
[0295] In certain embodiments, the immune cell is a T cell, e.g., a
CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or
effector memory T cell), a CD4+ T cell, a natural killer T cell
(NKT cells), a regulatory T cell (Treg), a stem cell memory T cell,
a lymphoid progenitor cell a hematopoietic stem cell, a natural
killer cell (NK cell) or a dendritic cell. In some embodiments, the
cells are monocytes or granulocytes, e.g., myeloid cells,
macrophages, neutrophils, dendritic cells, mast cells, eosinophils,
and/or basophils. In an embodiment, the target cell is an induced
pluripotent stem (iPS) cell or a cell derived from an iPS cell,
e.g., an iPS cell generated from a subject, manipulated to alter
(e.g., induce a mutation in) or manipulate the expression of one or
more target genes, and differentiated into, e.g., a T cell, e.g., a
CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or
effector memory T cell), a CD4+ T cell, a stem cell memory T cell,
a lymphoid progenitor cell or a hematopoietic stem cell.
[0296] In some embodiments, the cells include one or more subsets
of T cells or other cell types, such as whole T cell populations,
CD4+ cells, CD8+ cells, and subpopulations thereof, such as those
defined by function, activation state, maturity, potential for
differentiation, expansion, recirculation, localization, and/or
persistence capacities, antigen-specificity, type of antigen
receptor, presence in a particular organ or compartment, marker or
cytokine secretion profile, and/or degree of differentiation. Among
the sub-types and subpopulations of T cells and/or of CD4+ and/or
of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF),
memory T cells and sub-types thereof, such as stem cell memory T
(TSCM), central memory T (TCM), effector memory T (TEM), or
terminally differentiated effector memory T cells,
tumor-infiltrating lymphocytes (TIL), immature T cells, mature T
cells, helper T cells, cytotoxic T cells, mucosa-associated
invariant T (MAIT) cells, naturally occurring and adaptive
regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2
cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular
helper T cells, alpha/beta T cells, and delta/gamma T cells. In
certain embodiments, any number of T cell lines available in the
art, may be used.
[0297] In some embodiments, the methods include isolating immune
cells from the subject, preparing, processing, culturing, and/or
engineering them. In some embodiments, preparation of the
engineered cells includes one or more culture and/or preparation
steps. The cells for engineering as described may be isolated from
a sample, such as a biological sample, e.g., one obtained from or
derived from a subject. In some embodiments, the subject from which
the cell is isolated is one having the disease or condition or in
need of a cell therapy or to which cell therapy will be
administered. The subject in some embodiments is a human in need of
a particular therapeutic intervention, such as the adoptive cell
therapy for which cells are being isolated, processed, and/or
engineered. Accordingly, the cells in some embodiments are primary
cells, e.g., primary human cells. The samples include tissue,
fluid, and other samples taken directly from the subject, as well
as samples resulting from one or more processing steps, such as
separation, centrifugation, genetic engineering (e.g. transduction
with viral vector), washing, and/or incubation. The biological
sample can be a sample obtained directly from a biological source
or a sample that is processed. Biological samples include, but are
not limited to, body fluids, such as blood, plasma, serum,
cerebrospinal fluid, synovial fluid, urine and sweat, tissue and
organ samples, including processed samples derived therefrom.
[0298] In some aspects, the sample from which the cells are derived
or isolated is blood or a blood-derived sample, or is or is derived
from an apheresis or leukapheresis product. Exemplary samples
include whole blood, peripheral blood mononuclear cells (PBMCs),
leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia,
lymphoma, lymph node, gut associated lymphoid tissue, mucosa
associated lymphoid tissue, spleen, other lymphoid tissues, liver,
lung, stomach, intestine, colon, kidney, pancreas, breast, bone,
prostate, cervix, testes, ovaries, tonsil, or other organ, and/or
cells derived therefrom. Samples include, in the context of cell
therapy, e.g., adoptive cell therapy, samples from autologous and
allogeneic sources.
[0299] In some embodiments, the cells are derived from cell lines,
e.g., T cell lines. The cells in some embodiments are obtained from
a xenogeneic source, for example, from mouse, rat, non-human
primate, and pig. In some embodiments, isolation of the cells
includes one or more preparation and/or non-affinity based cell
separation steps. In some examples, cells are washed, centrifuged,
and/or incubated in the presence of one or more reagents, for
example, to remove unwanted components, enrich for desired
components, lyse or remove cells sensitive to particular reagents.
In some examples, cells are separated based on one or more
property, such as density, adherent properties, size, sensitivity
and/or resistance to particular components.
[0300] In some examples, cells from the circulating blood of a
subject are obtained, e.g., by apheresis or leukapheresis. The
samples, in some aspects, contain lymphocytes, including T cells,
monocytes, granulocytes, B cells, other nucleated white blood
cells, red blood cells, and/or platelets, and in some aspects
contains cells other than red blood cells and platelets. In some
embodiments, the blood cells collected from the subject are washed,
e.g., to remove the plasma fraction and to place the cells in an
appropriate buffer or media for subsequent processing steps. In
some embodiments, the cells are washed with phosphate buffered
saline (PBS). In some aspects, a washing step is accomplished by
tangential flow filtration (TFF) according to the manufacturer's
instructions. In some embodiments, the cells are resuspended in a
variety of biocompatible buffers after washing. In certain
embodiments, components of a blood cell sample are removed and the
cells directly resuspended in culture media. In some embodiments,
the methods include density-based cell separation methods, such as
the preparation of white blood cells from peripheral blood by
lysing the red blood cells and centrifugation through a Percoll or
Ficoll gradient.
[0301] In one embodiment, immune are obtained cells from the
circulating blood of an individual are obtained by apheresis or
leukapheresis. The apheresis product typically contains
lymphocytes, including T cells, monocytes, granulocytes, B cells,
other nucleated white blood cells, red blood cells, and platelets.
The cells collected by apheresis may be washed to remove the plasma
fraction and to place the cells in an appropriate buffer or media,
such as phosphate buffered saline (PBS) or wash solution lacks
calcium and may lack magnesium or may lack many if not all divalent
cations, for subsequent processing steps. After washing, the cells
may be resuspended in a variety of biocompatible buffers, such as,
for example, Ca-free, Mg-free PBS. Alternatively, the undesirable
components of the apheresis sample may be removed and the cells
directly resuspended in culture media.
[0302] In some embodiments, the isolation methods include the
separation of different cell types based on the expression or
presence in the cell of one or more specific molecules, such as
surface markers, e.g., surface proteins, intracellular markers, or
nucleic acid. In some embodiments, any known method for separation
based on such markers may be used. In some embodiments, the
separation is affinity- or immunoaffinity-based separation. For
example, the isolation in some aspects includes separation of cells
and cell populations based on the cells' expression or expression
level of one or more markers, typically cell surface markers, for
example, by incubation with an antibody or binding partner that
specifically binds to such markers, followed generally by washing
steps and separation of cells having bound the antibody or binding
partner, from those cells having not bound to the antibody or
binding partner.
[0303] Such separation steps can be based on positive selection, in
which the cells having bound the reagents are retained for further
use, and/or negative selection, in which the cells having not bound
to the antibody or binding partner are retained. In some examples,
both fractions are retained for further use. In some aspects,
negative selection can be particularly useful where no antibody is
available that specifically identifies a cell type in a
heterogeneous population, such that separation is best carried out
based on markers expressed by cells other than the desired
population. The separation need not result in 100% enrichment or
removal of a particular cell population or cells expressing a
particular marker. For example, positive selection of or enrichment
for cells of a particular type, such as those expressing a marker,
refers to increasing the number or percentage of such cells, but
need not result in a complete absence of cells not expressing the
marker. Likewise, negative selection, removal, or depletion of
cells of a particular type, such as those expressing a marker,
refers to decreasing the number or percentage of such cells, but
need not result in a complete removal of all such cells.
[0304] In some examples, multiple rounds of separation steps are
carried out, where the positively or negatively selected fraction
from one step is subjected to another separation step, such as a
subsequent positive or negative selection. In some examples, a
single separation step can deplete cells expressing multiple
markers simultaneously, such as by incubating cells with a
plurality of antibodies or binding partners, each specific for a
marker targeted for negative selection. Likewise, multiple cell
types can simultaneously be positively selected by incubating cells
with a plurality of antibodies or binding partners expressed on the
various cell types.
[0305] In some embodiments, one or more of the T cell populations
is enriched for or depleted of cells that are positive for
(marker+) or express high levels (marker.sup.high) of one or more
particular markers, such as surface markers, or that are negative
for (marker -) or express relatively low levels (marker.sup.low) of
one or more markers. For example, in some aspects, specific
subpopulations of T cells, such as cells positive or expressing
high levels of one or more surface markers, e.g., CD28+, CD62L+,
CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells,
are isolated by positive or negative selection techniques. In some
cases, such markers are those that are absent or expressed at
relatively low levels on certain populations of T cells (such as
non-memory cells) but are present or expressed at relatively higher
levels on certain other populations of T cells (such as memory
cells). In one embodiment, the cells (such as the CD8+ cells or the
T cells, e.g., CD3+ cells) are enriched for (i.e., positively
selected for) cells that are positive or expressing high surface
levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L
and/or depleted of (e.g., negatively selected for) cells that are
positive for or express high surface levels of CD45RA. In some
embodiments, cells are enriched for or depleted of cells positive
or expressing high surface levels of CD 122, CD95, CD25, CD27,
and/or IL7-Ra (CD 127). In some examples, CD8+ T cells are enriched
for cells positive for CD45RO (or negative for CD45RA) and for
CD62L. For example, CD3+, CD28+ T cells can be positively selected
using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS.RTM.
M-450 CD3/CD28 T Cell Expander).
[0306] In some embodiments, T cells are separated from a PBMC
sample by negative selection of markers expressed on non-T cells,
such as B cells, monocytes, or other white blood cells, such as CD
14. In some aspects, a CD4+ or CD8+ selection step is used to
separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+
populations can be further sorted into sub-populations by positive
or negative selection for markers expressed or expressed to a
relatively higher degree on one or more naive, memory, and/or
effector T cell subpopulations. In some embodiments, CD8+ cells are
further enriched for or depleted of naive, central memory, effector
memory, and/or central memory stem cells, such as by positive or
negative selection based on surface antigens associated with the
respective subpopulation. In some embodiments, enrichment for
central memory T (TCM) cells is carried out to increase efficacy,
such as to improve long-term survival, expansion, and/or
engraftment following administration, which in some aspects is
particularly robust in such sub-populations. In some embodiments,
combining TCM-enriched CD8+ T cells and CD4+ T cells further
enhances efficacy.
[0307] In some embodiments, memory T cells are present in both
CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes.
PBMC can be enriched for or depleted of CD62L-CD8+ and/or
CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L
antibodies. In some embodiments, a CD4+ T cell population and a
CD8+ T cell sub-population, e.g., a sub-population enriched for
central memory (TCM) cells. In some embodiments, the enrichment for
central memory T (TCM) cells is based on positive or high surface
expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in
some aspects, it is based on negative selection for cells
expressing or highly expressing CD45RA and/or granzyme B. In some
aspects, isolation of a CD8+ population enriched for TCM cells is
carried out by depletion of cells expressing CD4, CD 14, CD45RA,
and positive selection or enrichment for cells expressing CD62L. In
one aspect, enrichment for central memory T (TCM) cells is carried
out starting with a negative fraction of cells selected based on
CD4 expression, which is subjected to a negative selection based on
expression of CD 14 and CD45RA, and a positive selection based on
CD62L. Such selections in some aspects are carried out
simultaneously and in other aspects are carried out sequentially,
in either order. In some aspects, the same CD4 expression-based
selection step used in preparing the CD8+ cell population or
subpopulation, also is used to generate the CD4+ cell population or
sub-population, such that both the positive and negative fractions
from the CD4-based separation are retained and used in subsequent
steps of the methods, optionally following one or more further
positive or negative selection steps.
[0308] CD4+T helper cells are sorted into naive, central memory,
and effector cells by identifying cell populations that have cell
surface antigens. CD4+ lymphocytes can be obtained by standard
methods. In some embodiments, naive CD4+T lymphocytes are CD45RO-,
CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory
CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector
CD4+ cells are CD62L- and CD45RO. In one example, to enrich for
CD4+ cells by negative selection, a monoclonal antibody cocktail
typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR,
and CD8. In some embodiments, the antibody or binding partner is
bound to a solid support or matrix, such as a magnetic bead or
paramagnetic bead, to allow for separation of cells for positive
and/or negative selection.
[0309] In some embodiments, the cells are incubated and/or cultured
prior to or in connection with genetic engineering. The incubation
steps can include culture, cultivation, stimulation, activation,
and/or propagation. In some embodiments, the compositions or cells
are incubated in the presence of stimulating conditions or a
stimulatory agent. Such conditions include those designed to induce
proliferation, expansion, activation, and/or survival of cells in
the population, to mimic antigen exposure, and/or to prime the
cells for genetic engineering, such as for the introduction of a
recombinant antigen receptor. The conditions can include one or
more of particular media, temperature, oxygen content, carbon
dioxide content, time, agents, e.g., nutrients, amino acids,
antibiotics, ions, and/or stimulatory factors, such as cytokines,
chemokines, antigens, binding partners, fusion proteins,
recombinant soluble receptors, and any other agents designed to
activate the cells. In some embodiments, the stimulating conditions
or agents include one or more agent, e.g., ligand, which is capable
of activating an intracellular signaling domain of a TCR complex.
In some aspects, the agent turns on or initiates TCR/CD3
intracellular signaling cascade in a T cell. Such agents can
include antibodies, such as those specific for a TCR component
and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for
example, bound to solid support such as a bead, and/or one or more
cytokines. Optionally, the expansion method may further comprise
the step of adding anti-CD3 and/or anti CD28 antibody to the
culture medium (e.g., at a concentration of at least about 0.5
ng/ml). In some embodiments, the stimulating agents include IL-2
and/or IL-15, for example, an IL-2 concentration of at least about
10 units/mL.
[0310] In another embodiment, T cells are isolated from peripheral
blood by lysing the red blood cells and depleting the monocytes,
for example, by centrifugation through a PERCOLL.TM. gradient.
Alternatively, T cells can be isolated from an umbilical cord. In
any event, a specific subpopulation of T cells can be further
isolated by positive or negative selection techniques.
[0311] The cord blood mononuclear cells so isolated can be depleted
of cells expressing certain antigens, including, but not limited
to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can
be accomplished using an isolated antibody, a biological sample
comprising an antibody, such as ascites, an antibody bound to a
physical support, and a cell bound antibody.
[0312] Enrichment of a T cell population by negative selection can
be accomplished using a combination of antibodies directed to
surface markers unique to the negatively selected cells. A
preferred method is cell sorting and/or selection via negative
magnetic immunoadherence or flow cytometry that uses a cocktail of
monoclonal antibodies directed to cell surface markers present on
the cells negatively selected. For example, to enrich for CD4.sup.+
cells by negative selection, a monoclonal antibody cocktail
typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR,
and CD8.
[0313] For isolation of a desired population of cells by positive
or negative selection, the concentration of cells and surface
(e.g., particles such as beads) can be varied. In certain
embodiments, it may be desirable to significantly decrease the
volume in which beads and cells are mixed together (i.e., increase
the concentration of cells), to ensure maximum contact of cells and
beads. For example, in one embodiment, a concentration of 2 billion
cells/ml is used. In one embodiment, a concentration of 1 billion
cells/ml is used. In a further embodiment, greater than 100 million
cells/ml is used. In a further embodiment, a concentration of cells
of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
In yet another embodiment, a concentration of cells from 75, 80,
85, 90, 95, or 100 million cells/ml is used. In further
embodiments, concentrations of 125 or 150 million cells/ml can be
used. Using high concentrations can result in increased cell yield,
cell activation, and cell expansion.
[0314] T cells can also be frozen after the washing step, which
does not require the monocyte-removal step. While not wishing to be
bound by theory, the freeze and subsequent thaw step provides a
more uniform product by removing granulocytes and to some extent
monocytes in the cell population. After the washing step that
removes plasma and platelets, the cells may be suspended in a
freezing solution. While many freezing solutions and parameters are
known in the art and will be useful in this context, in a
non-limiting example, one method involves using PBS containing 20%
DMSO and 8% human serum albumin, or other suitable cell freezing
media. The cells are then frozen to -80.degree. C. at a rate of
1.degree. C. per minute and stored in the vapor phase of a liquid
nitrogen storage tank. Other methods of controlled freezing may be
used as well as uncontrolled freezing immediately at -20.degree. C.
or in liquid nitrogen.
[0315] In one embodiment, the population of T cells is comprised
within cells such as peripheral blood mononuclear cells, cord blood
cells, a purified population of T cells, and a T cell line. In
another embodiment, peripheral blood mononuclear cells comprise the
population of T cells. In yet another embodiment, purified T cells
comprise the population of T cells.
[0316] In certain embodiments, T regulatory cells (Tregs) can be
isolated from a sample. The sample can include, but is not limited
to, umbilical cord blood or peripheral blood. In certain
embodiments, the Tregs are isolated by flow-cytometry sorting. The
sample can be enriched for Tregs prior to isolation by any means
known in the art. The isolated Tregs can be cryopreserved, and/or
expanded prior to use. Methods for isolating Tregs are described in
U.S. Pat. Nos. 7,754,482, 8,722,400, and 9,555,105, and U.S. patent
application Ser. No. 13/639,927, contents of which are incorporated
herein in their entirety.
J. Expansion of Immune Cells
[0317] Whether prior to or after modification of cells to express a
a chimeric cell surface sialidase or a variant sialidase precursor
protein and/or a TCR and/or CAR, the cells can be activated and
expanded in number using methods as described, for example, in U.S.
Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358;
6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566;
7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S.
Publication No. 20060121005. For example, the T cells of the
invention may be expanded by contact with a surface having attached
thereto an agent that stimulates a CD3/TCR complex associated
signal and a ligand that stimulates a co-stimulatory molecule on
the surface of the T cells. In particular, T cell populations may
be stimulated by contact with an anti-CD3 antibody, or
antigen-binding fragment thereof, or an anti-CD2 antibody
immobilized on a surface, or by contact with a protein kinase C
activator (e.g., bryostatin) in conjunction with a calcium
ionophore. For co-stimulation of an accessory molecule on the
surface of the T cells, a ligand that binds the accessory molecule
is used. For example, T cells can be contacted with an anti-CD3
antibody and an anti-CD28 antibody, under conditions appropriate
for stimulating proliferation of the T cells. Examples of an
anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon,
France) and these can be used in the invention, as can other
methods and reagents known in the art (see, e.g., ten Berge et al.,
Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp.
Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol.
Methods (1999) 227(1-2): 53-63).
[0318] Expanding T cells by the methods disclosed herein can be
multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60
fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400
fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold,
2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold,
8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold,
10,000,000 fold, or greater, and any and all whole or partial
integers therebetween. In one embodiment, the T cells expand in the
range of about 20 fold to about 50 fold.
[0319] Following culturing, the T cells can be incubated in cell
medium in a culture apparatus for a period of time or until the
cells reach confluency or high cell density for optimal passage
before passing the cells to another culture apparatus. The
culturing apparatus can be of any culture apparatus commonly used
for culturing cells in vitro. Preferably, the level of confluence
is 70% or greater before passing the cells to another culture
apparatus. More preferably, the level of confluence is 90% or
greater. A period of time can be any time suitable for the culture
of cells in vitro. The T cell medium may be replaced during the
culture of the T cells at any time. Preferably, the T cell medium
is replaced about every 2 to 3 days. The T cells are then harvested
from the culture apparatus whereupon the T cells can be used
immediately or cryopreserved to be stored for use at a later time.
In one embodiment, the invention includes cryopreserving the
expanded T cells. The cryopreserved T cells are thawed prior to
introducing nucleic acids into the T cell.
[0320] In another embodiment, the method comprises isolating T
cells and expanding the T cells. In another embodiment, the
invention further comprises cryopreserving the T cells prior to
expansion. In yet another embodiment, the cryopreserved T cells are
thawed for electroporation with the RNA encoding the chimeric
membrane protein.
[0321] Another procedure for ex vivo expansion cells is described
in U.S. Pat. No. 5,199,942 (incorporated herein by reference).
Expansion, such as described in U.S. Pat. No. 5,199,942 can be an
alternative or in addition to other methods of expansion described
herein. Briefly, ex vivo culture and expansion of T cells comprises
the addition to the cellular growth factors, such as those
described in U.S. Pat. No. 5,199,942, or other factors, such as
flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding
the T cells comprises culturing the T cells with a factor selected
from the group consisting of flt3-L, IL-1, IL-3 and c-kit
ligand.
[0322] The culturing step as described herein (contact with agents
as described herein or after electroporation) can be very short,
for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
The culturing step as described further herein (contact with agents
as described herein) can be longer, for example 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, or more days.
[0323] Various terms are used to describe cells in culture. Cell
culture refers generally to cells taken from a living organism and
grown under controlled condition. A primary cell culture is a
culture of cells, tissues or organs taken directly from an organism
and before the first subculture. Cells are expanded in culture when
they are placed in a growth medium under conditions that facilitate
cell growth and/or division, resulting in a larger population of
the cells. When cells are expanded in culture, the rate of cell
proliferation is typically measured by the amount of time required
for the cells to double in number, otherwise known as the doubling
time.
[0324] Each round of subculturing is referred to as a passage. When
cells are subcultured, they are referred to as having been
passaged. A specific population of cells, or a cell line, is
sometimes referred to or characterized by the number of times it
has been passaged. For example, a cultured cell population that has
been passaged ten times may be referred to as a P10 culture. The
primary culture, i.e., the first culture following the isolation of
cells from tissue, is designated P0. Following the first
subculture, the cells are described as a secondary culture (P1 or
passage 1). After the second subculture, the cells become a
tertiary culture (P2 or passage 2), and so on. It will be
understood by those of skill in the art that there may be many
population doublings during the period of passaging; therefore the
number of population doublings of a culture is greater than the
passage number. The expansion of cells (i.e., the number of
population doublings) during the period between passaging depends
on many factors, including but is not limited to the seeding
density, substrate, medium, and time between passaging.
[0325] In one embodiment, the cells may be cultured for several
hours (about 3 hours) to about 14 days or any hourly integer value
in between. Conditions appropriate for T cell culture include an
appropriate media (e.g., Minimal Essential Media or RPMI Media 1640
or, X-vivo 15, (Lonza)) that may contain factors necessary for
proliferation and viability, including serum (e.g., fetal bovine or
human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7,
GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-.alpha. or any other
additives for the growth of cells known to the skilled artisan.
Other additives for the growth of cells include, but are not
limited to, surfactant, plasmanate, and reducing agents such as
N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI
1640, AIM-V, DMEM, MEM, .alpha.-MEM, F-12, X-Vivo 15, and X-Vivo
20, Optimizer, with added amino acids, sodium pyruvate, and
vitamins, either serum-free or supplemented with an appropriate
amount of serum (or plasma) or a defined set of hormones, and/or an
amount of cytokine(s) sufficient for the growth and expansion of T
cells. Antibiotics, e.g., penicillin and streptomycin, are included
only in experimental cultures, not in cultures of cells that are to
be infused into a subject. The target cells are maintained under
conditions necessary to support growth, for example, an appropriate
temperature (e.g., 37.degree. C.) and atmosphere (e.g., air plus 5%
CO.sub.2).
[0326] The medium used to culture the T cells may include an agent
that can co-stimulate the T cells. For example, an agent that can
stimulate CD3 is an antibody to CD3, and an agent that can
stimulate CD28 is an antibody to CD28. A cell isolated by the
methods disclosed herein can be expanded approximately 10 fold, 20
fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90
fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold,
700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000
fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000
fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In
one embodiment, the T cells expand in the range of about 20 fold to
about 50 fold, or more. In one embodiment, human T regulatory cells
are expanded via anti-CD3 antibody coated KT64.86 artificial
antigen presenting cells (aAPCs). Methods for expanding and
activating T cells can be found in U.S. Pat. Nos. 7,754,482,
8,722,400, and 9,555,105, contents of which are incorporated herein
in their entirety.
[0327] In one embodiment, the method of expanding the T cells can
further comprise isolating the expanded T cells for further
applications. In another embodiment, the method of expanding can
further comprise a subsequent electroporation of the expanded T
cells followed by culturing. The subsequent electroporation may
include introducing a nucleic acid encoding an agent, such as a
transducing the expanded T cells, transfecting the expanded T
cells, or electroporating the expanded T cells with a nucleic acid,
into the expanded population of T cells, wherein the agent further
stimulates the T cell. The agent may stimulate the T cells, such as
by stimulating further expansion, effector function, or another T
cell function.
K. Pharmaceutical Compositions and Formulations
[0328] Also provided are populations of modified immune cells of
the invention, compositions containing such cells and/or enriched
for such cells, such as in which cells expressing the chimeric cell
surface sialidase or variant sialidase precursor protein make up at
least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or more of the total cells in the composition or cells of
a certain type such as T cells or CD8+ or CD4+ cells. Among the
compositions are pharmaceutical compositions and formulations for
administration, such as for adoptive cell therapy. Also provided
are therapeutic methods for administering the cells and
compositions to subjects, e.g., patients.
[0329] Also provided are compositions including the cells for
administration, including pharmaceutical compositions and
formulations, such as unit dose form compositions including the
number of cells for administration in a given dose or fraction
thereof. The pharmaceutical compositions and formulations generally
include one or more optional pharmaceutically acceptable carrier or
excipient. In some embodiments, the composition includes at least
one additional therapeutic agent.
[0330] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be administered.
A "pharmaceutically acceptable carrier" refers to an ingredient in
a pharmaceutical formulation, other than an active ingredient,
which is nontoxic to a subject. A pharmaceutically acceptable
carrier includes, but is not limited to, a buffer, excipient,
stabilizer, or preservative. In some aspects, the choice of carrier
is determined in part by the particular cell and/or by the method
of administration. Accordingly, there are a variety of suitable
formulations. For example, the pharmaceutical composition can
contain preservatives. Suitable preservatives may include, for
example, methylparaben, propylparaben, sodium benzoate, and
benzalkonium chloride. In some aspects, a mixture of two or more
preservatives is used. The preservative or mixtures thereof are
typically present in an amount of about 0.0001% to about 2% by
weight of the total composition. Carriers are described, e.g., by
Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980). Pharmaceutically acceptable carriers are generally nontoxic
to recipients at the dosages and concentrations employed, and
include, but are not limited to: buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid and methionine; preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride;
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as polyethylene glycol (PEG).
[0331] Buffering agents in some aspects are included in the
compositions. Suitable buffering agents include, for example,
citric acid, sodium citrate, phosphoric acid, potassium phosphate,
and various other acids and salts. In some aspects, a mixture of
two or more buffering agents is used. The buffering agent or
mixtures thereof are typically present in an amount of about 0.001%
to about 4% by weight of the total composition. Methods for
preparing administrable pharmaceutical compositions are known.
Exemplary methods are described in more detail in, for example,
Remington: The Science and Practice of Pharmacy, Lippincott
Williams & Wilkins; 21st ed. (May 1, 2005).
[0332] The formulations can include aqueous solutions. The
formulation or composition may also contain more than one active
ingredient useful for the particular indication, disease, or
condition being treated with the cells, preferably those with
activities complementary to the cells, where the respective
activities do not adversely affect one another. Such active
ingredients are suitably present in combination in amounts that are
effective for the purpose intended. Thus, in some embodiments, the
pharmaceutical composition further includes other pharmaceutically
active agents or drugs, such as chemotherapeutic agents, e.g.,
asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,
doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,
paclitaxel, rituximab, vinblastine, and/or vincristine. The
pharmaceutical composition in some embodiments contains the cells
in amounts effective to treat or prevent the disease or condition,
such as a therapeutically effective or prophylactically effective
amount. Therapeutic or prophylactic efficacy in some embodiments is
monitored by periodic assessment of treated subjects. The desired
dosage can be delivered by a single bolus administration of the
cells, by multiple bolus administrations of the cells, or by
continuous infusion administration of the cells.
[0333] Formulations include those for oral, intravenous,
intraperitoneal, subcutaneous, pulmonary, transdermal,
intramuscular, intranasal, buccal, sublingual, or suppository
administration. In some embodiments, the cell populations are
administered parenterally. The term "parenteral," as used herein,
includes intravenous, intramuscular, subcutaneous, rectal, vaginal,
and intraperitoneal administration. In some embodiments, the cells
are administered to the subject using peripheral systemic delivery
by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid
preparations, e.g., isotonic aqueous solutions, suspensions,
emulsions, dispersions, or viscous compositions, which may in some
aspects be buffered to a selected pH. Liquid preparations are
normally easier to prepare than gels, other viscous compositions,
and solid compositions. Additionally, liquid compositions are
somewhat more convenient to administer, especially by injection.
Viscous compositions, on the other hand, can be formulated within
the appropriate viscosity range to provide longer contact periods
with specific tissues. Liquid or viscous compositions can comprise
carriers, which can be a solvent or dispersing medium containing,
for example, water, saline, phosphate buffered saline, polyoi (for
example, glycerol, propylene glycol, liquid polyethylene glycol)
and suitable mixtures thereof.
[0334] Sterile injectable solutions can be prepared by
incorporating the cells in a solvent, such as in admixture with a
suitable carrier, diluent, or excipient such as sterile water,
physiological saline, glucose, dextrose, or the like. The
compositions can contain auxiliary substances such as wetting,
dispersing, or emulsifying agents (e.g., methylcellulose), pH
buffering agents, gelling or viscosity enhancing additives,
preservatives, flavoring agents, and/or colors, depending upon the
route of administration and the preparation desired. Standard texts
may in some aspects be consulted to prepare suitable
preparations.
[0335] Various additives which enhance the stability and sterility
of the compositions, including antimicrobial preservatives,
antioxidants, chelating agents, and buffers, can be added.
Prevention of the action of microorganisms can be ensured by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the
injectable pharmaceutical form can be brought about by the use of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0336] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0337] The contents of the articles, patents, and patent
applications, and all other documents and electronically available
information mentioned or cited herein, are hereby incorporated by
reference in their entirety to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference. Applicants reserve the right to
physically incorporate into this application any and all materials
and information from any such articles, patents, patent
applications, or other physical and electronic documents.
L. Methods of Producing Genetically Modified Immune Cells
[0338] The present disclosure provides methods for producing or
generating a modified immune cell or precursor thereof (e.g., a T
cell comprising a chimeric cell surface sialidase or a variant
sialidase precursor protein) of the invention for tumor
immunotherapy, e.g., adoptive immunotherapy.
[0339] In some embodiments, the chimeric cell surface sialidase or
variant sialidase precursor protein is introduced into a cell by an
expression vector. Expression vectors comprising a nucleic acid
sequence encoding a chimeric cell surface sialidase or a variant
sialidase precursor protein of the present invention are provided
herein. Suitable expression vectors include lentivirus vectors,
gamma retrovirus vectors, foamy virus vectors, adeno associated
virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses,
naked DNA, including but not limited to transposon mediated
vectors, such as Sleeping Beauty, Piggybak, and Integrases such as
Phi31. Some other suitable expression vectors include Herpes
simplex virus (HSV) and retrovirus expression vectors.
[0340] Adenovirus expression vectors are based on adenoviruses,
which have a low capacity for integration into genomic DNA but a
high efficiency for transfecting host cells. Adenovirus expression
vectors contain adenovirus sequences sufficient to: (a) support
packaging of the expression vector and (b) to ultimately express
the chimeric cell surface sialidase or a variant sialidase
precursor protein in the host cell. In some embodiments, the
adenovirus genome is a 36 kb, linear, double stranded DNA, where a
foreign DNA sequence (e.g., a nucleic acid encoding a chimeric cell
surface sialidase or a variant sialidase precursor protein) may be
inserted to substitute large pieces of adenoviral DNA in order to
make the expression vector of the present invention (see, e.g.,
Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).
Another expression vector is based on an adeno associated virus
(AAV), which takes advantage of the adenovirus coupled systems.
This AAV expression vector has a high frequency of integration into
the host genome. It can infect nondividing cells, thus making it
useful for delivery of genes into mammalian cells, for example, in
tissue cultures or in vivo. The AAV vector has a broad host range
for infectivity. Details concerning the generation and use of AAV
vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368. In
some embodiments, the nucleic acid encoding a chimeric cell surface
sialidase or a variant sialidase precursor protein is introduced
into the cell via viral transduction. In certain embodiments, the
viral transduction comprises contacting the immune or precursor
cell with a viral vector comprising the nucleic acid encoding a
chimeric cell surface sialidase or a variant sialidase precursor
protein. In certain embodiments, the viral vector is an
adeno-associated viral (AAV) vector. In certain embodiments, the
AAV vector comprises a Woodchuck Hepatitis Virus
post-transcriptional regulatory element (WPRE). In certain
embodiments, the AAV vector comprises a polyadenylation (polyA)
sequence. In certain embodiments, the polyA sequence is a bovine
growth hormone (BGH) polyA sequence.
[0341] Retrovirus expression vectors are capable of integrating
into the host genome, delivering a large amount of foreign genetic
material, infecting a broad spectrum of species and cell types and
being packaged in special cell lines. The retroviral vector is
constructed by inserting a nucleic acid (e.g., a nucleic acid
encoding a chimeric cell surface sialidase or a variant sialidase
precursor protein) into the viral genome at certain locations to
produce a virus that is replication defective. Though the
retroviral vectors are able to infect a broad variety of cell
types, integration and stable expression of the chimeric cell
surface sialidase or variant sialidase precursor protein requires
the division of host cells.
[0342] Lentiviral vectors are derived from lentiviruses, which are
complex retroviruses that, in addition to the common retroviral
genes gag, pol, and env, contain other genes with regulatory or
structural function (see, e.g., U.S. Pat. Nos. 6,013,516 and
5,994,136). Some examples of lentiviruses include the Human
Immunodeficiency Viruses (HIV-1, HIV-2) and the Simian
Immunodeficiency Virus (SIV). Lentiviral vectors have been
generated by multiply attenuating the HIV virulence genes, for
example, the genes env, vif, vpr, vpu and nef are deleted making
the vector biologically safe. Lentiviral vectors are capable of
infecting non-dividing cells and can be used for both in vivo and
ex vivo gene transfer and expression, e.g., of a nucleic acid
encoding a chimeric cell surface sialidase or a variant sialidase
precursor protein (see, e.g., U.S. Pat. No. 5,994,136).
[0343] Expression vectors including a nucleic acid of the present
disclosure can be introduced into a host cell by any means known to
persons skilled in the art. The expression vectors may include
viral sequences for transfection, if desired. Alternatively, the
expression vectors may be introduced by fusion, electroporation,
biolistics, transfection, lipofection, or the like. The host cell
may be grown and expanded in culture before introduction of the
expression vectors, followed by the appropriate treatment for
introduction and integration of the vectors. The host cells are
then expanded and may be screened by virtue of a marker present in
the vectors. Various markers that may be used are known in the art,
and may include hprt, neomycin resistance, thymidine kinase,
hygromycin resistance, etc. As used herein, the terms "cell," "cell
line," and "cell culture" may be used interchangeably. In some
embodiments, the host cell an immune cell or precursor thereof,
e.g., a T cell, an NK cell, or an NKT cell.
[0344] The present invention also provides modified cells which
include and stably express a chimeric cell surface sialidase or a
variant sialidase precursor protein of the present disclosure. In
some embodiments, the modified cells are genetically engineered
T-lymphocytes (T cells), naive T cells (TN), memory T cells (for
example, central memory T cells (TCM), effector memory cells
(TEM)), natural killer cells (NK cells), and macrophages capable of
giving rise to therapeutically relevant progeny. In certain
embodiments, the genetically engineered cells are autologous
cells.
[0345] Modified cells (e.g., comprising a chimeric cell surface
sialidase or a variant sialidase precursor protein) may be produced
by stably transfecting host cells with an expression vector
including a nucleic acid of the present disclosure. Additional
methods for generating a modified cell of the present disclosure
include, without limitation, chemical transformation methods (e.g.,
using calcium phosphate, dendrimers, liposomes and/or cationic
polymers), non-chemical transformation methods (e.g.,
electroporation, optical transformation, gene electrotransfer
and/or hydrodynamic delivery) and/or particle-based methods (e.g.,
impalefection, using a gene gun and/or magnetofection). Transfected
cells expressing a chimeric cell surface sialidase or a variant
sialidase precursor protein of the present disclosure may be
expanded ex vivo.
[0346] Physical methods for introducing an expression vector into
host cells include calcium phosphate precipitation, lipofection,
particle bombardment, microinjection, electroporation, and the
like. Methods for producing cells including vectors and/or
exogenous nucleic acids are well-known in the art. See, e.g.,
Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, New York. Chemical methods for
introducing an expression vector into a host cell include colloidal
dispersion systems, such as macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes.
[0347] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP")
can be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Choi") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform may be used as the only
solvent since it is more readily evaporated than methanol.
"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed
lipid bilayers or aggregates. Liposomes can be characterized as
having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology
5: 505-10). Compositions that have different structures in solution
than the normal vesicular structure are also encompassed. For
example, the lipids may assume a micellar structure or merely exist
as nonuniform aggregates of lipid molecules. Also contemplated are
lipofectamine-nucleic acid complexes.
[0348] Regardless of the method used to introduce exogenous nucleic
acids into a host cell or otherwise expose a cell to the inhibitor
of the present invention, in order to confirm the presence of the
nucleic acids in the host cell, a variety of assays may be
performed. Such assays include, for example, molecular biology
assays well known to those of skill in the art, such as Southern
and Northern blotting, RT-PCR and PCR; biochemistry assays, such as
detecting the presence or absence of a particular peptide, e.g., by
immunological means (ELISAs and Western blots) or by assays
described herein to identify agents falling within the scope of the
invention.
[0349] In one embodiment, the nucleic acids introduced into the
host cell are RNA. In another embodiment, the RNA is mRNA that
comprises in vitro transcribed RNA or synthetic RNA. The RNA may be
produced by in vitro transcription using a polymerase chain
reaction (PCR)-generated template. DNA of interest from any source
can be directly converted by PCR into a template for in vitro mRNA
synthesis using appropriate primers and RNA polymerase. The source
of the DNA may be, for example, genomic DNA, plasmid DNA, phage
DNA, cDNA, synthetic DNA sequence or any other appropriate source
of DNA.
[0350] PCR may be used to generate a template for in vitro
transcription of mRNA which is then introduced into cells. Methods
for performing PCR are well known in the art. Primers for use in
PCR are designed to have regions that are substantially
complementary to regions of the DNA to be used as a template for
the PCR. "Substantially complementary," as used herein, refers to
sequences of nucleotides where a majority or all of the bases in
the primer sequence are complementary. Substantially complementary
sequences are able to anneal or hybridize with the intended DNA
target under annealing conditions used for PCR. The primers can be
designed to be substantially complementary to any portion of the
DNA template. For example, the primers can be designed to amplify
the portion of a gene that is normally transcribed in cells (the
open reading frame), including 5' and 3' UTRs. The primers may also
be designed to amplify a portion of a gene that encodes a
particular domain of interest. In one embodiment, the primers are
designed to amplify the coding region of a human cDNA, including
all or portions of the 5' and 3' UTRs. Primers useful for PCR are
generated by synthetic methods that are well known in the art.
"Forward primers" are primers that contain a region of nucleotides
that are substantially complementary to nucleotides on the DNA
template that are upstream of the DNA sequence that is to be
amplified. "Upstream" is used herein to refer to a location 5, to
the DNA sequence to be amplified relative to the coding strand.
"Reverse primers" are primers that contain a region of nucleotides
that are substantially complementary to a double-stranded DNA
template that are downstream of the DNA sequence that is to be
amplified. "Downstream" is used herein to refer to a location 3' to
the DNA sequence to be amplified relative to the coding strand.
[0351] Chemical structures that have the ability to promote
stability and/or translation efficiency of the RNA may also be
used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the
5' UTR is between zero and 3000 nucleotides in length. The length
of 5' and 3' UTR sequences to be added to the coding region can be
altered by different methods, including, but not limited to,
designing primers for PCR that anneal to different regions of the
UTRs. Using this approach, one of ordinary skill in the art can
modify the 5' and 3' UTR lengths required to achieve optimal
translation efficiency following transfection of the transcribed
RNA.
[0352] The 5' and 3' UTRs can be the naturally occurring,
endogenous 5' and 3' UTRs for the gene of interest. Alternatively,
UTR sequences that are not endogenous to the gene of interest can
be added by incorporating the UTR sequences into the forward and
reverse primers or by any other modifications of the template. The
use of UTR sequences that are not endogenous to the gene of
interest can be useful for modifying the stability and/or
translation efficiency of the RNA. For example, it is known that
AU-rich elements in 3' UTR sequences can decrease the stability of
mRNA. Therefore, 3' UTRs can be selected or designed to increase
the stability of the transcribed RNA based on properties of UTRs
that are well known in the art.
[0353] In one embodiment, the 5' UTR can contain the Kozak sequence
of the endogenous gene. Alternatively, when a 5' UTR that is not
endogenous to the gene of interest is being added by PCR as
described above, a consensus Kozak sequence can be redesigned by
adding the 5' UTR sequence. Kozak sequences can increase the
efficiency of translation of some RNA transcripts, but does not
appear to be required for all RNAs to enable efficient translation.
The requirement for Kozak sequences for many mRNAs is known in the
art. In other embodiments the 5' UTR can be derived from an RNA
virus whose RNA genome is stable in cells. In other embodiments
various nucleotide analogues can be used in the 3' or 5' UTR to
impede exonuclease degradation of the mRNA.
[0354] To enable synthesis of RNA from a DNA template without the
need for gene cloning, a promoter of transcription should be
attached to the DNA template upstream of the sequence to be
transcribed. When a sequence that functions as a promoter for an
RNA polymerase is added to the 5' end of the forward primer, the
RNA polymerase promoter becomes incorporated into the PCR product
upstream of the open reading frame that is to be transcribed. In
one embodiment, the promoter is a T7 polymerase promoter, as
described elsewhere herein. Other useful promoters include, but are
not limited to, T3 and SP6 RNA polymerase promoters. Consensus
nucleotide sequences for T7, T3 and SP6 promoters are known in the
art.
[0355] In one embodiment, the mRNA has both a cap on the 5' end and
a 3' poly(A) tail which determine ribosome binding, initiation of
translation and stability mRNA in the cell. On a circular DNA
template, for instance, plasmid DNA, RNA polymerase produces a long
concatameric product which is not suitable for expression in
eukaryotic cells. The transcription of plasmid DNA linearized at
the end of the 3' UTR results in normal sized mRNA which is not
effective in eukaryotic transfection even if it is polyadenylated
after transcription.
[0356] On a linear DNA template, phage T7 RNA polymerase can extend
the 3' end of the transcript beyond the last base of the template
(Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985);
Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65
(2003).
[0357] The polyA/T segment of the transcriptional DNA template can
be produced during PCR by using a reverse primer containing a polyT
tail, such as 100T tail (size can be 50-5000 T), or after PCR by
any other method, including, but not limited to, DNA ligation or in
vitro recombination. Poly(A) tails also provide stability to RNAs
and reduce their degradation. Generally, the length of a poly(A)
tail positively correlates with the stability of the transcribed
RNA. In one embodiment, the poly(A) tail is between 100 and 5000
adenosines.
[0358] Poly(A) tails of RNAs can be further extended following in
vitro transcription with the use of a poly(A) polymerase, such as
E. coli polyA polymerase (E-PAP). In one embodiment, increasing the
length of a poly(A) tail from 100 nucleotides to between 300 and
400 nucleotides results in about a two-fold increase in the
translation efficiency of the RNA. Additionally, the attachment of
different chemical groups to the 3' end can increase mRNA
stability. Such attachment can contain modified/artificial
nucleotides, aptamers and other compounds. For example, ATP analogs
can be incorporated into the poly(A) tail using poly(A) polymerase.
ATP analogs can further increase the stability of the RNA.
[0359] 5' caps also provide stability to RNA molecules. In a
preferred embodiment, RNAs produced by the methods disclosed herein
include a 5' cap. The 5' cap is provided using techniques known in
the art and described herein (Cougot, et al., Trends in Biochem.
Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001);
Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966
(2005)).
[0360] In some embodiments, the RNA is electroporated into the
cells, such as in vitro transcribed RNA. Any solutes suitable for
cell electroporation, which can contain factors facilitating
cellular permeability and viability such as sugars, peptides,
lipids, proteins, antioxidants, and surfactants can be
included.
[0361] In some embodiments, a nucleic acid encoding a chimeric cell
surface sialidase or a variant sialidase precursor protein of the
present disclosure will be RNA, e.g., in vitro synthesized RNA.
Methods for in vitro synthesis of RNA are known in the art; any
known method can be used to synthesize RNA comprising a sequence
encoding a chimeric cell surface sialidase or a variant sialidase
precursor protein. Methods for introducing RNA into a host cell are
known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15:
9053. Introducing RNA comprising a nucleotide sequence encoding a
chimeric cell surface sialidase or a variant sialidase precursor
protein into a host cell can be carried out in vitro, ex vivo or in
vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T
lymphocyte, etc.) can be electroporated in vitro or ex vivo with
RNA comprising a nucleotide sequence encoding a chimeric cell
surface sialidase or a variant sialidase precursor protein.
[0362] The disclosed methods can be applied to the modulation of T
cell activity in basic research and therapy, in the fields of
cancer, stem cells, acute and chronic infections, and autoimmune
diseases, including the assessment of the ability of the
genetically modified T cell to kill a target cancer cell.
[0363] The methods also provide the ability to control the level of
expression over a wide range by changing, for example, the promoter
or the amount of input RNA, making it possible to individually
regulate the expression level. Furthermore, the PCR-based technique
of mRNA production greatly facilitates the design of the mRNAs with
different structures and combination of their domains.
[0364] One advantage of RNA transfection methods of the invention
is that RNA transfection is essentially transient and a
vector-free. An RNA transgene can be delivered to a lymphocyte and
expressed therein following a brief in vitro cell activation, as a
minimal expressing cassette without the need for any additional
viral sequences. Under these conditions, integration of the
transgene into the host cell genome is unlikely. Cloning of cells
is not necessary because of the efficiency of transfection of the
RNA and its ability to uniformly modify the entire lymphocyte
population.
[0365] Genetic modification of T cells with in vitro-transcribed
RNA (IVT-RNA) makes use of two different strategies both of which
have been successively tested in various animal models. Cells are
transfected with in vitro-transcribed RNA by means of lipofection
or electroporation. It is desirable to stabilize IVT-RNA using
various modifications in order to achieve prolonged expression of
transferred IVT-RNA.
[0366] Some IVT vectors are known in the literature which are
utilized in a standardized manner as template for in vitro
transcription and which have been genetically modified in such a
way that stabilized RNA transcripts are produced. Currently
protocols used in the art are based on a plasmid vector with the
following structure: a 5' RNA polymerase promoter enabling RNA
transcription, followed by a gene of interest which is flanked
either 3' and/or 5' by untranslated regions (UTR), and a 3'
polyadenyl cassette containing 50-70 A nucleotides. Prior to in
vitro transcription, the circular plasmid is linearized downstream
of the polyadenyl cassette by type II restriction enzymes
(recognition sequence corresponds to cleavage site). The polyadenyl
cassette thus corresponds to the later poly(A) sequence in the
transcript. As a result of this procedure, some nucleotides remain
as part of the enzyme cleavage site after linearization and extend
or mask the poly(A) sequence at the 3' end. It is not clear,
whether this nonphysiological overhang affects the amount of
protein produced intracellularly from such a construct.
[0367] In another aspect, the RNA construct is delivered into the
cells by electroporation. See, e.g., the formulations and
methodology of electroporation of nucleic acid constructs into
mammalian cells as taught in US 2004/0014645, US 2005/0052630A1, US
2005/0070841A1, US 2004/0059285A1, US 2004/0092907A1. The various
parameters including electric field strength required for
electroporation of any known cell type are generally known in the
relevant research literature as well as numerous patents and
applications in the field. See e.g., U.S. Pat. Nos. 6,678,556,
7,171,264, and 7,173,116. Apparatus for therapeutic application of
electroporation are available commercially, e.g., the MedPulser.TM.
DNA Electroporation Therapy System (Inovio/Genetronics, San Diego,
Calif.), and are described in patents such as U.S. Pat. Nos.
6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and
6,233,482; electroporation may also be used for transfection of
cells in vitro as described e.g. in US20070128708A1.
Electroporation may also be utilized to deliver nucleic acids into
cells in vitro. Accordingly, electroporation-mediated
administration into cells of nucleic acids including expression
constructs utilizing any of the many available devices and
electroporation systems known to those of skill in the art presents
an exciting new means for delivering an RNA of interest to a target
cell.
[0368] In some embodiments, the immune cells (e.g. T cells) can be
incubated or cultivated prior to, during and/or subsequent to
introducing the nucleic acid molecule encoding the chimeric cell
surface sialidase or a variant sialidase precursor protein (and/or
the TCR and/or CAR). In some embodiments, the cells (e.g. T cells)
can be incubated or cultivated prior to, during or subsequent to
the introduction of the nucleic acid molecule encoding the chimeric
cell surface sialidase or a variant sialidase precursor protein,
such as prior to, during or subsequent to the transduction of the
cells with a viral vector (e.g. lentiviral vector) encoding the
exogenous receptor.
[0369] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. It will be readily apparent
to those skilled in the art that other suitable modifications and
adaptations of the methods described herein may be made using
suitable equivalents without departing from the scope of the
embodiments disclosed herein. In addition, many modifications may
be made to adapt a particular situation, material, composition of
matter, process, process step or steps, to the objective, spirit
and scope of the present invention. All such modifications are
intended to be within the scope of the claims appended hereto.
Having now described certain embodiments in detail, the same will
be more clearly understood by reference to the following examples,
which are included for purposes of illustration only and are not
intended to be limiting.
EXPERIMENTAL EXAMPLES
[0370] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only, and the invention is not limited to these
Examples, but rather encompasses all variations that are evident as
a result of the teachings provided herein.
Materials and Methods
[0371] Molecular Cloning:
[0372] Codon optimized nucleic acid sequences of human Neu1, Neu2,
Neu3, and Neu4 were synthesized as "gBlock Gene Fragments" through
Integrated DNA Technologies (IDT). Each neuraminidase was
sub-cloned into the pCR-BluntII-TOPO vector, then cloned into
destination pTRPE lentiviral vectors, which are third-generation
lentiviral production vectors utilizing the EF1.alpha. promoter,
and containing the CD8.alpha. leader sequence, hinge, transmembrane
domain, and 4-1BB and CD3z endodomains. Myc-Tag sequences were also
cloned into each construct, and other receptor versions were
designed to include a truncated intracellular domain (.DELTA.z
endodomain).
[0373] Transduction and Expansion of Normal Donor T Cells:
[0374] HEK 293T cells were transfected with pTRPE-Neu2-BBz,
pTRPE-Myc-Neu2-BBz, pTRPE-5E5-CD2z, pELPS-CD19-BBz, and
pTRPE-PSMA-BBz in addition to gag/pol, env, and Vsvg packaging mix.
Virus was collected and concentrated at 24 and 48 hrs. Normal donor
T cells were obtained from the Human Immunology Core (HIC) at the
University of Pennsylvania where they were negatively selected from
apheresis. The normal donor T cells were activated in-vitro with
CD3/CD28 magnetic Dynabeads (Thermo Fischer Scientific), transduced
with lentivirus 16 hrs after bead activation, and cultured in RPMI
1640 medium (Gibco) supplemented with 10% FBS, 1%
penicillin-streptomycin (Gibco), 1% HEPES (Gibco), and 1% GlutaMax
(Gibco) (R10 complete growth medium), with the addition of 30 U/mL
of IL-2 for 10-17 days.
[0375] Cell Culture:
[0376] The adherent PC3 and DU145 prostate cancer cells lines were
obtained from ATCC and maintained with R10 complete growth medium.
The human embryonic kidney 293T cell line was also obtained from
ATCC and maintained on R10. All T cells used in assays were
obtained from the Human Immunology Core at the University of
Pennsylvania and activated and transduced in R10, expanded in R10
supplemented with IL-2, and maintained in R10 during use in
assays.
[0377] Flow Cytometry:
[0378] Before each staining, cells were washed in either
phosphate-buffered saline (PBS), or PBS containing 2% FBS, and
stains were performed on ice. SNA expression was assessed in
neuraminidase activity assays by staining PC3 and DU145 cells with
Biotinylated-SNA Lectin (Vector Laboratories) followed by
PE-conjugated streptavidin. Surface Myc-Tag expression was assessed
by staining 293T cells with Myc-Tag (71D10) rabbit mAb-Alexa AF700
conjugated (Cell Signaling Technology). Extracellular Neu2
expression was assessed by staining 293T cells with Neu2 rabbit pAb
(OriGene) and goat anti-rabbit AF488 secondary antibody.
Intracellular CD3z was detected by fixing and permeabilizing the
293T cells (BD Cytofix/Cytoperm), then staining for CD3z/CD247
rabbit pAb (Proteintech), and goat anti rabbit AF488 secondary
antibody. Viability of cells used in assays was assessed using
Live/Dead Violet Stain (Thermo Fischer Scientific). CAR T cells
were detected by staining with biotinylated goat anti mouse
F(ab).sub.2 antibody (Jackson ImmunoResearch) and PE-conjugated
streptavidin. Flow analysis was performed by gating singlets on
FSC-H versus FSC-A and SSC-H versus SSC-A, then on forward versus
side scatter characteristics. All flow cytometry was performed on
LSRFortessa or LSRII multi-laser Becton Dickinson cytometers.
[0379] Cytokine Secretion:
[0380] 5E5-CD2z CAR T cells were were incubated with PC3 target
cells at a 1:1 ratio either alone, or with the addition of
Sialidase and NK cells individually, or the combination of the CAR
T cells, Sialidase, and NK cells (as indicated in FIG. 7) for 16
hrs in R10 medium at 37.degree. C. NTD T cells were used as a
control in the same conditions. CD19-BBz, PSMA-BBz, and 5E5-CD2z
CAR T cells were incubated with NALM6 target cells at a 1:1 ratio
either alone or with Neu2-BBz T cells +NK cells, or Sialidase+NK
cells (as indicated in FIG. 9) for 16 hrs in R10 medium. NTD T
cells were used as a control under the same conditions. CD19-BBz,
PSMA-BBz, and 5E5-CD2z CAR T cells were incubated with PC3 target
cells at a 1:1 ratio either alone or with Neu2-BBz T cells, or with
Neu2-BBz T cells+NK cells (as indicated in FIG. 10). NTD T cells
were used as a control under the same conditions. After 16 hrs,
supernatant was collected and analyzed for IFN-.gamma. production
using the Human DuoSet ELISA kit (R&D Systems).
[0381] Cytotoxicity Assays:
[0382] Cytotoxicity assays were performed using the xCELLigence
real-time cell analysis (RTCA) system, which measures rate of
de-adherence as target cells are lysed/undergo cytolytic responses
after the addition of cytolytic cells. Adherent PC3 target cells in
culture were suspended using trypsin (0.05%) and counted on a
Beckman Coulter multisizer Coulter Counter, then 1E4 PC3 target
cells were plated on an xCELLigence assay E-plate and allowed to
adhere overnight at 37.degree. C. After overnight incubation, T
cells, NK cells and sialidase were added at the indicated
effector:target ratios (FIG. 8), and co-cultures were returned to
37.degree. C., and de-adherence data was monitored and recorded
automatically and continuously for 7 days.
[0383] Neuraminidase (Sialidase) Activity Assays:
[0384] Flow-Based Surface Sialic Acid Detection Using SNA-Lectin
Staining:
[0385] PC3 tumor cells were treated with Clostridium
perfringens-derived sialidase (500 U of .alpha.2-3, 6, 8
Neuraminidase from New England BioLabs) for one hour ate 37.degree.
C. Cells were allowed to recover for either a 2-hour, or 1-hour
period, or no recovery time to restore surface sialic acid
expression. Non-treated PC3 cells were used as a control. Surface
expression of sialic acid was investigated by flow cytometry of
SNA-lectin staining after treatment and recovery periods, and
staining intensity of the non-treated group was compared to
treatment groups. Neuraminidase activity of engineered T cells was
assessed against PC3 and DU145 tumor cells. Cells were co-cultured
at indicated effector:target ratios (FIG. 6) for 24 hrs at
37.degree. C. with Neu2-BBz T cells and NTD T cell controls. Sialic
acid expression was assessed using SNA-lectin staining and flow
cytometric analysis comparing PC3 and DU145 cells co-cultured with
either NTD or Neu2-BBz T cells.
[0386] Fluorometric Assay for Sialidase Activity (Using the
Artificial Substrate 4-MU-NANA):
[0387] 293T cells were transfected with 2.5 ug of the each of the
following lentiviral sialidase expressing constructs and a
lipofectamine/Opti-MEM mixture--pTRPE-Myc-Neu1-Dz,
pTREP-Myc-Neu2-Dz, pTRPE-Myc-Neu3-Dz, pTRPE-Myc-Neu4Dz, and
pTREP-Myc-Neu2-BBz, and one sample was left non-transfected as a
control. Cells were incubated for 24 hours, and then
2.times.10.sup.6 293 T cells from each of the samples were
harvested and washed 3.times. with PBS and pelleted with gentle
centrifugation. Cells were resuspended in assay buffer as described
in Leang and Hurt, 2017, and 100 uL of each sample was plated in a
black flat-bottom 96 well plate in duplicate. In order to generate
a standard curve of relative fluorescence units (RFU) of 4-MU and
an equation for which to derive the unknown sialidase activity of
each receptor, serial dilutions of Clostridium perfringens-derived
sialidase (from 0.5 U to 0 U of enzyme) were added to the plate in
duplicate, and suspended in the same assay buffer. The artificial
substrate 4-MU-NANA was added to each of the wells as described in
Leang and Hurt, 2017. The plate was taped to mix, covered, and
incubated for 1 hour at 37.degree. C. After incubation, the
reaction was terminated with a stop solution (Leang and Hurt, J Vis
Exp. 2017; (122):55570. Published 2017 Apr. 15. doi:10.3791/55570)
and sialidase activity was determined by measuring the fluorescence
intensity of released 4-MU with a fluorescence plate reader
(fluorescence spectrophotometer; excitation at 355 nm; emission at
460 nm).
Example 1: Engineered Expression of Cell Surface and Secreted
Sialidase by CART Cells for Increased Efficacy in Solid Tumors
[0388] CAR T cells have lacked efficacy in the treatment of solid
tumors due to a number of challenges, including overcoming the
dense immunosuppressive tumor stroma and post-translational
modifications on the tumor that favor tumor survival. The work
presented herein improves CAR T cell therapy against solid tumors
by enhancing cytotoxic effects of endogenous and unmodified immune
cells by engineering sialidase function on CAR T cells. T cells
bearing sialidase/neuraminidase activity can cleave inhibitory
sialic acids on tumor cells, thereby enhancing the anti-tumor
efficacy of Siglec-expressing innate immune cells, such as NK and
monocytes (FIGS. 1A-1C). In one iteration, the CAR T cell comprises
a cell-surface receptor comprising a sialidase/neuraminidase domain
along with T cell signaling domains from 4-1BB and CD3zeta (FIG.
1A). In another iteration, the CAR T cell comprises a cell-surface
receptor without intracellular T cell signaling domains (FIG. 1B).
In a third iteration, the CAR T cell comprises secreted sialidase
activity (FIG. 1C).
[0389] Various neuraminidase (Neu) receptor (chimeric cell surface
sialidase) constructs were generated (FIG. 2) including
pTRPE-Neu2-BBz (SEQ ID NOs: 1 & 10), pTRPE-Neu2-Dz (SEQ ID NOs:
19 & 21), pTRPE-Myc-Neu2-BBz (SEQ ID NOs: 23 & 25), and
pTRPE-Myc-Neu2-Dz (SEQ ID NO: 27 & 28). Additional constructs
were generated which replaced the Neu2 domain (SEQ ID NO: 4 or 13)
with a Neul (SEQ ID NO: 29 or 33), Neu3 (SEQ ID NO: 31 or 33), or
Neu4 (SEQ ID NO: 32 or 36) domain (FIG. 2). The construct
5E5-P2A-Neu2 was also generated (FIG. 2). Constructs were
transfected into HEK 293T cells and surface expression was detected
(FIG. 3).
[0390] PC3 tumor cells were treated with the sialidase enzyme
derived from Clostridium perfringens for lhr at 37 degrees C. (FIG.
4). Cells were given either a 2-hour recovery time, a 1-hour
recovery time, or no recovery time to restore surface sialic acid
expression. PC3 cells that were not treated with sialidase were
used as a control. Surface expression of sialic acid was measured
on PC3 cells after the sialidase treatment and recovery periods
using flow cytometry. The bottom-most flow cytometry histogram in
FIG. 4 represents SNA lectin staining on PC3 cells that were not
treated with sialidase, a staining control for sialic acid on PC3
cells. There was a significant decrease in MFI for SNA staining on
PC3 cells that were treated with sialidase for 1 hr at 37 degrees
C. and given no recovery time to restore surface sialic acid
expression (FIG. 4). SNA staining after one hour and 2-hour
recovery periods demonstrated that sialic acid increased with
recovery time (FIG. 4).
[0391] Human sialidase/neuraminidase-expressing T cells exhibited
the ability to reduce surface sialic acid expression after 24 hr
co-culture with PC3 and DU145 tumor cells, as evidenced by SNA
staining (FIGS. 5A-5F). There is a decrease in SNA staining after
co-culture with Neu2-BBz T cells compared with NTD T cells (FIG. 5B
compared to FIG. 5A; FIG. 5E compared to FIG. 5D). FIGS. 5B and 5E
represent a 10:1 effector:target ratio of Neu2-BBz T cells:tumor,
and show greater activity compared with FIGS. 5C and 5F, which
represent a 5:1 effector:target ratio of Neu2-BBz T cells to tumor
cells. The 24 hr co-culture analysis was repeated and MFI compared
(FIG. 6).
[0392] The addition of sialidase and NK cells enhanced IFN-g
production of CART cells targeting prostate cancer PC3 cells (FIG.
7). 5E5-CD2z CART cells demonstrated reactivity to PC3 prostate
cancer cells in co-culture alone. Interestingly, IFN-g secretion
was elevated when both sialidase and NK cells were added at the
time of co-culture with 5E5-CD2z CART cells. This combination
approach showed greater reactivity than with each effector
condition alone (FIG. 7).
[0393] Sialidase activity promoted synergistic cytotoxicity effects
of CART and NK cells (FIGS. 8A-8C). Cytotoxicity assays of PC3
prostate cancer cells cultured with human T cells were performed.
At 1:1 effector:target ratio, 5E5-CD2z CAR T cells and NTD T cells
exhibited no cytotoxic effects against PC3 tumor cells (FIG. 8A).
There was no increased cytotoxicity through the addition of
sialidase or NK cells to the 5E5-CART cells (FIG. 8B). However,
when sialidase AND NK cells were added to the 5E5-CART cells, there
was virtually complete lysis of PC3 cells, approximating that of
the positive lysis control, Triton. Of note, this effect was not
observed with 5E5-CART alone, NK cells alone, 5E5-CART+ sialidase,
or 5E5-CART+NK cells. The synergy of T cells, NK cells, and
sialidase activity was not observed with NTD T cells, demonstrating
that CAR activity was required for increased cytotoxicity from this
combination (FIG. 8C) These data suggests that 5E5-CART cell
cytotoxicity can cooperate with unmodified innate immune cells,
such as NK cells, through the addition of sialidase activity.
[0394] Engineered Neu2-BBz T cells or sialidase activity enhanced
the anti-tumor activity of CD19-BBz treatment against leukemic
cells (FIG. 9). CD19-BBz CART cell effector function was enhanced
with the addition of neuraminidase-expressing T cells and NK cells,
much like in cultures with bacterial sialidase and NK cells (no
significant difference between treatment with the addition of
Neu2-BBz+NK and Sialidase+NK). These data demonstrate the potential
for the invention to enhance CAR-T cell immunotherapies beyond the
5E5-CAR.
[0395] Engineered Neu2-BBz T cells enhanced the anti-tumor activity
of prostate cancer CART treatment (FIG. 10). The reactivity of
PSMA-BBz CART cells and 5E5-CART cells against aggressive prostate
cancer cell line PC3 is significantly increased by human
sialidase/neuraminidase T cells and NK cells (FIG. 10).
[0396] A novel cell-surface human sialidase was designed and
expressed on CART cells targeting Tn-MUC1. Truncated,
cancer-specific glycoforms, such as Tn-MUC1, are thought to play a
role in decreasing cell-cell interactions and increasing the
metastatic potential of cancer cells. Tn-MUC1-CART (or 5E5-CART)
cells induced minimal cytotoxic effects against the aggressive PC3
prostate cancer cell line, when co-cultured alone at equal
effector:target ratios. When PC3 cells were treated with a
Clostridium perfringens-derived sialidase, NK cells and 5E5-CART
cells showed enhanced IFN-.gamma. production and improved cytotoxic
effects are observed compared with NK cells and 5E5-CART cells
alone. Similarly, these effector enhancements were observed in the
presence of T cells presenting a human cell-surface sialidase.
Taken together, these data demonstrated that glycoediting of tumor
sialic acid presents a rational and unique opportunity to overcome
barriers for adoptive immunotherapies in solid tumors, and
engagement of the endogenous immune system by adoptively
transferred cells is an effective strategy for improved anti-tumor
activity.
[0397] The sialidase-expressing CAR presented herein is the first
of its kind, and cleaves inhibitory glycans on the tumor cell
surface, allowing tumors to be recognized and killed by cells of
the endogenous innate immune system, such as NK cells and
monocytes, in combination and/or synergy with CAR T cell
activity.
Example 2: Enzymatic Function of Engineered Receptors is
Demonstrated Using a Fluorometric Neuraminidase (Sialidase)
Activity Assays
[0398] Neuraminidase (Sialidase) activity assays were performed to
compare the enzymatic function of the engineered receptors designed
herein (FIG. 11). A fluorometric neuraminidase functional assay
using the artificial substrate 4-MU-NANA was performed. The
enzymatic activity of neuraminidase catalyzes the release of 4-MU
from 4-MU-NANA, and the florescence emission is quantified. 293T
cells were transfected with various forms of the neuraminidase
receptors and sialidase activity was measured. Normalized sialidase
function of the various receptors compared to non-transfected cells
is shown in FIG. 11, left panel. The Neu1-Dz(delta zeta) receptor
showed the greatest enzymatic activity followed by Neu2-BBz. FIG.
11, right panel shows various units of bacterial sialidase and
corresponding function as determined by fluorescence emission. This
information was used as a standard when calculating fluorescent
readout for activity produced by the receptors. Based on the
activity level demonstrated, Neu1-Dz, Neu2-BBz, Neu1-BBz, and
Neu1-secreated (when generated) receptor versions were chosen for
additional functional assays (both in-vitro and in-vivo).
[0399] Based on results from this assay the Neu1-delta zeta vector
was used to assess if normal donor human T cells could be
successfully transduced, and to evaluate the synergistic effects of
CAR-T cells expressing this receptor co-cultured with NK cells
against prostate tumor cells.
Example 3: Dual Expressing Human Sialidase-5E5-CAR-T Cells Exhibit
Rapid Synergistic Cytotoxicity Against PC3 Tumor Cells when
Co-Cultured with NK Cells
[0400] Normal donor human T cells were transduced with either the
single lentiviral vector 5E5-BBz, the single lentiviral vector
Myc-NeulDz, or double-transduced with both the 5E5-BBz and
Myc-Neu1-Dz lentiviral vectors (referred to as Dual Expressing
Sialidase-5E5 T cells) and analyzed by flow cytometry (FIGS.
12A-12D). Non-transduced (NTD) cells were used as a control. Cells
were gated on protein L and stained with an anti-Myc tagged
antibody (FIG. 12A-12D).
[0401] Cytotoxic ability was also assessed using xCELLigence RTCA
(FIG. 12E). NK cells and effectors listed were co-cultured at a 1:1
ratio with PC3 tumor cells. The 5E5+NK+Sialidase-T cell group is a
3-product co-culture; whereas the Dual Expressing Sialidase-5E5 T
cell+NK group is a 2-cell product consisting of NK cells
co-cultured with T cells expressing both the Neuraminidase receptor
and the 5E5-CAR.
[0402] These data demonstrated that normal donor human T cells can
be successfully transduced with an engineered human neuraminidase
receptor and confer neuraminidase receptor expression on 5E5-CAR-T
cells (FIGS. 12C-12 D). Additionally, these dual expressing
Sialidase-5E5 T cells are functional, and in co-culture with NK
cells show more rapid synergistic cytotoxicity against PC3 tumors
than what is observed with the bacterial sialidase control in
co-culture with NK and 5E5-CAR-T cells (FIG. 12E).
Sequence CWU 1
1
6011884DNAArtificial SequenceNeu2-BBz 1atggccttac cagtgaccgc
cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccgggatcca tggcttcctt
gccggtgctg caaaaagaga gcgtattcca gtcaggcgcc 120catgcgtata
gaatcccggc acttctctat ttgccgggcc aacaaagtct cttggcgttc
180gcggaacagc gggcgtccaa aaaagacgaa cacgccgagt tgattgtgct
ccgccgcggg 240gattatgatg ccccaacgca tcaggttcag tggcaggcac
aagaggtagt cgctcaggcg 300cgactggatg gacatcggtc aatgaaccca
tgtccactgt acgatgctca gacaggtacg 360ttgtttctgt tcttcatcgc
tatccctggg caagtaacag aacaacaaca actgcaaacc 420agagccaatg
taacaagact ctgccaggta actagcactg accacggacg aacgtggtct
480tcccctagag atcttactga cgccgcaatc gggcctgcat atcgcgaatg
gagcactttc 540gcagtaggcc ctggtcattg cctgcaactc catgatcgcg
cccgatcact tgtggtgcca 600gcgtacgcat accggaagct ccatccaata
caacgcccca tcccgtccgc tttttgtttc 660ctctcccatg accacgggcg
gacttgggcg cggggtcatt tcgtcgcaca ggatacgttg 720gagtgtcagg
tagcggaagt agaaaccggg gagcagagag tggtcactct caacgcgcgc
780agtcatcttc gcgcccgcgt acaggcgcag agcactaatg acgggcttga
ttttcaagaa 840agtcaactcg tcaaaaagtt ggttgaaccg cccccgcagg
gctgtcaagg ttcagttata 900agttttccaa gtccacgctc cggtccagga
tcaccagcac agtggcttct ctacacccat 960cccacccaca gctggcagcg
ggcagatctt ggtgcttact tgaatcccag gccaccggcc 1020cccgaagcct
ggagcgagcc tgtactgctt gcaaagggga gctgtgcgta ctctgatctc
1080cagtcaatgg gtactggacc agatgggagt ccattgtttg gttgtctcta
cgaggcgaac 1140gattatgagg aaatcgtttt tcttatgttt actttgaaac
aggcgttccc agccgaatat 1200ttgcctcagt ccggaaccac gacgccagcg
ccgcgaccac caacaccggc gcccaccatc 1260gcgtcgcagc ccctgtccct
gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 1320cacacgaggg
ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact
1380tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg
cagaaagaaa 1440ctcctgtata tattcaaaca accatttatg agaccagtac
aaactactca agaggaagat 1500ggctgtagct gccgatttcc agaagaagaa
gaaggaggat gtgaactgag agtgaagttc 1560agcaggagcg cagacgcccc
cgcgtacaag cagggccaga accagctcta taacgagctc 1620aatctaggac
gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag
1680atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga
actgcagaaa 1740gataagatgg cggaggccta cagtgagatt gggatgaaag
gcgagcgccg gaggggcaag 1800gggcacgatg gcctttacca gggtctcagt
acagccacca aggacaccta cgacgccctt 1860cacatgcagg ccctgccccc tcgc
1884263DNAArtificial SequenceCD8alpha leader 2atggccttac cagtgaccgc
cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccg 6334PRTArtificial
Sequencespacer 3Gly Gly Ser Gly141140DNAArtificial SequenceNeu2
4atggcttcct tgccggtgct gcaaaaagag agcgtattcc agtcaggcgc ccatgcgtat
60agaatcccgg cacttctcta tttgccgggc caacaaagtc tcttggcgtt cgcggaacag
120cgggcgtcca aaaaagacga acacgccgag ttgattgtgc tccgccgcgg
ggattatgat 180gccccaacgc atcaggttca gtggcaggca caagaggtag
tcgctcaggc gcgactggat 240ggacatcggt caatgaaccc atgtccactg
tacgatgctc agacaggtac gttgtttctg 300ttcttcatcg ctatccctgg
gcaagtaaca gaacaacaac aactgcaaac cagagccaat 360gtaacaagac
tctgccaggt aactagcact gaccacggac gaacgtggtc ttcccctaga
420gatcttactg acgccgcaat cgggcctgca tatcgcgaat ggagcacttt
cgcagtaggc 480cctggtcatt gcctgcaact ccatgatcgc gcccgatcac
ttgtggtgcc agcgtacgca 540taccggaagc tccatccaat acaacgcccc
atcccgtccg ctttttgttt cctctcccat 600gaccacgggc ggacttgggc
gcggggtcat ttcgtcgcac aggatacgtt ggagtgtcag 660gtagcggaag
tagaaaccgg ggagcagaga gtggtcactc tcaacgcgcg cagtcatctt
720cgcgcccgcg tacaggcgca gagcactaat gacgggcttg attttcaaga
aagtcaactc 780gtcaaaaagt tggttgaacc gcccccgcag ggctgtcaag
gttcagttat aagttttcca 840agtccacgct ccggtccagg atcaccagca
cagtggcttc tctacaccca tcccacccac 900agctggcagc gggcagatct
tggtgcttac ttgaatccca ggccaccggc ccccgaagcc 960tggagcgagc
ctgtactgct tgcaaagggg agctgtgcgt actctgatct ccagtcaatg
1020ggtactggac cagatgggag tccattgttt ggttgtctct acgaggcgaa
cgattatgag 1080gaaatcgttt ttcttatgtt tactttgaaa caggcgttcc
cagccgaata tttgcctcag 114055PRTArtificial Sequencespacer 5Gly Gly
Ser Gly Gly1 56132DNAArtificial SequenceCD8alpha hinge 6accacgacgc
cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60tccctgcgcc
cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg
120gacttcgcct gt 132775DNAArtificial Sequencetransmembrane domain
7gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt
60atcacccttt actgc 758126DNAArtificial Sequence4-1BB ICD
8aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa
60actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt
120gaactg 1269336DNAArtificial SequenceCD3 zeta 9agagtgaagt
tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60tataacgagc
tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc
120cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg
cctgtacaat 180gaactgcaga aagataagat ggcggaggcc tacagtgaga
ttgggatgaa aggcgagcgc 240cggaggggca aggggcacga tggcctttac
cagggtctca gtacagccac caaggacacc 300tacgacgccc ttcacatgca
ggccctgccc cctcgc 33610628PRTArtificial SequenceNeu2-BBz 10Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His
Ala Ala Arg Pro Gly Ser Met Ala Ser Leu Pro Val Leu Gln Lys 20 25
30Glu Ser Val Phe Gln Ser Gly Ala His Ala Tyr Arg Ile Pro Ala Leu
35 40 45Leu Tyr Leu Pro Gly Gln Gln Ser Leu Leu Ala Phe Ala Glu Gln
Arg 50 55 60Ala Ser Lys Lys Asp Glu His Ala Glu Leu Ile Val Leu Arg
Arg Gly65 70 75 80Asp Tyr Asp Ala Pro Thr His Gln Val Gln Trp Gln
Ala Gln Glu Val 85 90 95Val Ala Gln Ala Arg Leu Asp Gly His Arg Ser
Met Asn Pro Cys Pro 100 105 110Leu Tyr Asp Ala Gln Thr Gly Thr Leu
Phe Leu Phe Phe Ile Ala Ile 115 120 125Pro Gly Gln Val Thr Glu Gln
Gln Gln Leu Gln Thr Arg Ala Asn Val 130 135 140Thr Arg Leu Cys Gln
Val Thr Ser Thr Asp His Gly Arg Thr Trp Ser145 150 155 160Ser Pro
Arg Asp Leu Thr Asp Ala Ala Ile Gly Pro Ala Tyr Arg Glu 165 170
175Trp Ser Thr Phe Ala Val Gly Pro Gly His Cys Leu Gln Leu His Asp
180 185 190Arg Ala Arg Ser Leu Val Val Pro Ala Tyr Ala Tyr Arg Lys
Leu His 195 200 205Pro Ile Gln Arg Pro Ile Pro Ser Ala Phe Cys Phe
Leu Ser His Asp 210 215 220His Gly Arg Thr Trp Ala Arg Gly His Phe
Val Ala Gln Asp Thr Leu225 230 235 240Glu Cys Gln Val Ala Glu Val
Glu Thr Gly Glu Gln Arg Val Val Thr 245 250 255Leu Asn Ala Arg Ser
His Leu Arg Ala Arg Val Gln Ala Gln Ser Thr 260 265 270Asn Asp Gly
Leu Asp Phe Gln Glu Ser Gln Leu Val Lys Lys Leu Val 275 280 285Glu
Pro Pro Pro Gln Gly Cys Gln Gly Ser Val Ile Ser Phe Pro Ser 290 295
300Pro Arg Ser Gly Pro Gly Ser Pro Ala Gln Trp Leu Leu Tyr Thr
His305 310 315 320Pro Thr His Ser Trp Gln Arg Ala Asp Leu Gly Ala
Tyr Leu Asn Pro 325 330 335Arg Pro Pro Ala Pro Glu Ala Trp Ser Glu
Pro Val Leu Leu Ala Lys 340 345 350Gly Ser Cys Ala Tyr Ser Asp Leu
Gln Ser Met Gly Thr Gly Pro Asp 355 360 365Gly Ser Pro Leu Phe Gly
Cys Leu Tyr Glu Ala Asn Asp Tyr Glu Glu 370 375 380Ile Val Phe Leu
Met Phe Thr Leu Lys Gln Ala Phe Pro Ala Glu Tyr385 390 395 400Leu
Pro Gln Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 405 410
415Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
420 425 430Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
Phe Ala 435 440 445Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
Cys Gly Val Leu 450 455 460Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
Lys Arg Gly Arg Lys Lys465 470 475 480Leu Leu Tyr Ile Phe Lys Gln
Pro Phe Met Arg Pro Val Gln Thr Thr 485 490 495Gln Glu Glu Asp Gly
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 500 505 510Gly Cys Glu
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 515 520 525Tyr
Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 530 535
540Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
Glu545 550 555 560Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
Gly Leu Tyr Asn 565 570 575Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
Tyr Ser Glu Ile Gly Met 580 585 590Lys Gly Glu Arg Arg Arg Gly Lys
Gly His Asp Gly Leu Tyr Gln Gly 595 600 605Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp Ala Leu His Met Gln Ala 610 615 620Leu Pro Pro
Arg6251121PRTArtificial SequenceCD8alpha leader 11Met Ala Leu Pro
Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala
Arg Pro 20125PRTArtificial SequencelinkerREPEAT(1)..(5)repeat n
times, where n represents an integer of at least 1 12Gly Ser Gly
Gly Ser1 513380PRTArtificial SequenceNeu2 13Met Ala Ser Leu Pro Val
Leu Gln Lys Glu Ser Val Phe Gln Ser Gly1 5 10 15Ala His Ala Tyr Arg
Ile Pro Ala Leu Leu Tyr Leu Pro Gly Gln Gln 20 25 30Ser Leu Leu Ala
Phe Ala Glu Gln Arg Ala Ser Lys Lys Asp Glu His 35 40 45Ala Glu Leu
Ile Val Leu Arg Arg Gly Asp Tyr Asp Ala Pro Thr His 50 55 60Gln Val
Gln Trp Gln Ala Gln Glu Val Val Ala Gln Ala Arg Leu Asp65 70 75
80Gly His Arg Ser Met Asn Pro Cys Pro Leu Tyr Asp Ala Gln Thr Gly
85 90 95Thr Leu Phe Leu Phe Phe Ile Ala Ile Pro Gly Gln Val Thr Glu
Gln 100 105 110Gln Gln Leu Gln Thr Arg Ala Asn Val Thr Arg Leu Cys
Gln Val Thr 115 120 125Ser Thr Asp His Gly Arg Thr Trp Ser Ser Pro
Arg Asp Leu Thr Asp 130 135 140Ala Ala Ile Gly Pro Ala Tyr Arg Glu
Trp Ser Thr Phe Ala Val Gly145 150 155 160Pro Gly His Cys Leu Gln
Leu His Asp Arg Ala Arg Ser Leu Val Val 165 170 175Pro Ala Tyr Ala
Tyr Arg Lys Leu His Pro Ile Gln Arg Pro Ile Pro 180 185 190Ser Ala
Phe Cys Phe Leu Ser His Asp His Gly Arg Thr Trp Ala Arg 195 200
205Gly His Phe Val Ala Gln Asp Thr Leu Glu Cys Gln Val Ala Glu Val
210 215 220Glu Thr Gly Glu Gln Arg Val Val Thr Leu Asn Ala Arg Ser
His Leu225 230 235 240Arg Ala Arg Val Gln Ala Gln Ser Thr Asn Asp
Gly Leu Asp Phe Gln 245 250 255Glu Ser Gln Leu Val Lys Lys Leu Val
Glu Pro Pro Pro Gln Gly Cys 260 265 270Gln Gly Ser Val Ile Ser Phe
Pro Ser Pro Arg Ser Gly Pro Gly Ser 275 280 285Pro Ala Gln Trp Leu
Leu Tyr Thr His Pro Thr His Ser Trp Gln Arg 290 295 300Ala Asp Leu
Gly Ala Tyr Leu Asn Pro Arg Pro Pro Ala Pro Glu Ala305 310 315
320Trp Ser Glu Pro Val Leu Leu Ala Lys Gly Ser Cys Ala Tyr Ser Asp
325 330 335Leu Gln Ser Met Gly Thr Gly Pro Asp Gly Ser Pro Leu Phe
Gly Cys 340 345 350Leu Tyr Glu Ala Asn Asp Tyr Glu Glu Ile Val Phe
Leu Met Phe Thr 355 360 365Leu Lys Gln Ala Phe Pro Ala Glu Tyr Leu
Pro Gln 370 375 380144PRTArtificial
SequencelinkerREPEAT(1)..(4)repeat n times. where n represents an
integer of at least 1 14Gly Gly Gly Ser11541PRTArtificial
SequenceCD8alpha hinge 15Thr 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 35 401628PRTArtificial Sequencetransmembrane domain 16Phe Ala
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly1 5 10 15Val
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 20 251742PRTArtificial
Sequence4-1BB ICD 17Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
Gln Pro Phe Met1 5 10 15Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
Cys Ser Cys Arg Phe 20 25 30Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 4018112PRTArtificial SequenceCD3 zeta 18Arg Val Lys Phe Ser Arg
Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly1 5 10 15Gln Asn Gln Leu Tyr
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu Asp
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45Pro Arg Arg
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60Asp Lys
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg65 70 75
80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
Arg 100 105 110191461DNAArtificial SequencepTRPE-Neu2-Dz
19atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
60ccgggatcca tggcttcctt gccggtgctg caaaaagaga gcgtattcca gtcaggcgcc
120catgcgtata gaatcccggc acttctctat ttgccgggcc aacaaagtct
cttggcgttc 180gcggaacagc gggcgtccaa aaaagacgaa cacgccgagt
tgattgtgct ccgccgcggg 240gattatgatg ccccaacgca tcaggttcag
tggcaggcac aagaggtagt cgctcaggcg 300cgactggatg gacatcggtc
aatgaaccca tgtccactgt acgatgctca gacaggtacg 360ttgtttctgt
tcttcatcgc tatccctggg caagtaacag aacaacaaca actgcaaacc
420agagccaatg taacaagact ctgccaggta actagcactg accacggacg
aacgtggtct 480tcccctagag atcttactga cgccgcaatc gggcctgcat
atcgcgaatg gagcactttc 540gcagtaggcc ctggtcattg cctgcaactc
catgatcgcg cccgatcact tgtggtgcca 600gcgtacgcat accggaagct
ccatccaata caacgcccca tcccgtccgc tttttgtttc 660ctctcccatg
accacgggcg gacttgggcg cggggtcatt tcgtcgcaca ggatacgttg
720gagtgtcagg tagcggaagt agaaaccggg gagcagagag tggtcactct
caacgcgcgc 780agtcatcttc gcgcccgcgt acaggcgcag agcactaatg
acgggcttga ttttcaagaa 840agtcaactcg tcaaaaagtt ggttgaaccg
cccccgcagg gctgtcaagg ttcagttata 900agttttccaa gtccacgctc
cggtccagga tcaccagcac agtggcttct ctacacccat 960cccacccaca
gctggcagcg ggcagatctt ggtgcttact tgaatcccag gccaccggcc
1020cccgaagcct ggagcgagcc tgtactgctt gcaaagggga gctgtgcgta
ctctgatctc 1080cagtcaatgg gtactggacc agatgggagt ccattgtttg
gttgtctcta cgaggcgaac 1140gattatgagg aaatcgtttt tcttatgttt
actttgaaac aggcgttccc agccgaatat 1200ttgcctcagt ccggaaccac
gacgccagcg ccgcgaccac caacaccggc gcccaccatc 1260gcgtcgcagc
ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg
1320cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt
ggccgggact 1380tgtggggtcc ttctcctgtc actggttatc accctttact
gcagagtgaa gttcagcagg 1440agcgcagacg cccccgcgta a
14612039DNAArtificial Sequencedelta zeta domain 20agagtgaagt
tcagcaggag cgcagacgcc cccgcgtaa 3921486PRTArtificial
SequencepTRPE-Neu2-Dz 21Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro
Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gly Ser Met Ala Ser
Leu Pro Val Leu Gln Lys 20 25 30Glu Ser Val Phe Gln Ser Gly Ala His
Ala Tyr Arg Ile Pro Ala Leu 35 40 45Leu Tyr Leu Pro Gly Gln Gln Ser
Leu Leu Ala Phe Ala Glu Gln Arg 50 55 60Ala Ser Lys Lys Asp Glu His
Ala Glu Leu Ile Val Leu Arg Arg Gly65 70 75 80Asp Tyr Asp Ala Pro
Thr His Gln Val Gln Trp Gln Ala Gln Glu Val 85 90 95Val Ala Gln Ala
Arg Leu Asp Gly His Arg Ser Met Asn Pro Cys Pro 100 105 110Leu Tyr
Asp Ala Gln Thr Gly Thr Leu Phe Leu Phe Phe Ile Ala Ile 115 120
125Pro Gly Gln Val Thr Glu Gln Gln Gln Leu Gln Thr Arg Ala Asn Val
130 135 140Thr Arg Leu Cys Gln Val Thr Ser Thr Asp His
Gly Arg Thr Trp Ser145 150 155 160Ser Pro Arg Asp Leu Thr Asp Ala
Ala Ile Gly Pro Ala Tyr Arg Glu 165 170 175Trp Ser Thr Phe Ala Val
Gly Pro Gly His Cys Leu Gln Leu His Asp 180 185 190Arg Ala Arg Ser
Leu Val Val Pro Ala Tyr Ala Tyr Arg Lys Leu His 195 200 205Pro Ile
Gln Arg Pro Ile Pro Ser Ala Phe Cys Phe Leu Ser His Asp 210 215
220His Gly Arg Thr Trp Ala Arg Gly His Phe Val Ala Gln Asp Thr
Leu225 230 235 240Glu Cys Gln Val Ala Glu Val Glu Thr Gly Glu Gln
Arg Val Val Thr 245 250 255Leu Asn Ala Arg Ser His Leu Arg Ala Arg
Val Gln Ala Gln Ser Thr 260 265 270Asn Asp Gly Leu Asp Phe Gln Glu
Ser Gln Leu Val Lys Lys Leu Val 275 280 285Glu Pro Pro Pro Gln Gly
Cys Gln Gly Ser Val Ile Ser Phe Pro Ser 290 295 300Pro Arg Ser Gly
Pro Gly Ser Pro Ala Gln Trp Leu Leu Tyr Thr His305 310 315 320Pro
Thr His Ser Trp Gln Arg Ala Asp Leu Gly Ala Tyr Leu Asn Pro 325 330
335Arg Pro Pro Ala Pro Glu Ala Trp Ser Glu Pro Val Leu Leu Ala Lys
340 345 350Gly Ser Cys Ala Tyr Ser Asp Leu Gln Ser Met Gly Thr Gly
Pro Asp 355 360 365Gly Ser Pro Leu Phe Gly Cys Leu Tyr Glu Ala Asn
Asp Tyr Glu Glu 370 375 380Ile Val Phe Leu Met Phe Thr Leu Lys Gln
Ala Phe Pro Ala Glu Tyr385 390 395 400Leu Pro Gln Ser Gly Thr Thr
Thr Pro Ala Pro Arg Pro Pro Thr Pro 405 410 415Ala Pro Thr Ile Ala
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 420 425 430Arg Pro Ala
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 435 440 445Cys
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 450 455
460Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser
Arg465 470 475 480Ser Ala Asp Ala Pro Ala 4852212PRTArtificial
Sequencedelta zeta domain 22Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
Pro Ala1 5 10231914DNAArtificial SequencepTRPE-Myc-Neu2-BBz
23atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
60ccggaacaga aacttatctc cgaggaagac ctgggatcca tggcttcctt gccggtgctg
120caaaaagaga gcgtattcca gtcaggcgcc catgcgtata gaatcccggc
acttctctat 180ttgccgggcc aacaaagtct cttggcgttc gcggaacagc
gggcgtccaa aaaagacgaa 240cacgccgagt tgattgtgct ccgccgcggg
gattatgatg ccccaacgca tcaggttcag 300tggcaggcac aagaggtagt
cgctcaggcg cgactggatg gacatcggtc aatgaaccca 360tgtccactgt
acgatgctca gacaggtacg ttgtttctgt tcttcatcgc tatccctggg
420caagtaacag aacaacaaca actgcaaacc agagccaatg taacaagact
ctgccaggta 480actagcactg accacggacg aacgtggtct tcccctagag
atcttactga cgccgcaatc 540gggcctgcat atcgcgaatg gagcactttc
gcagtaggcc ctggtcattg cctgcaactc 600catgatcgcg cccgatcact
tgtggtgcca gcgtacgcat accggaagct ccatccaata 660caacgcccca
tcccgtccgc tttttgtttc ctctcccatg accacgggcg gacttgggcg
720cggggtcatt tcgtcgcaca ggatacgttg gagtgtcagg tagcggaagt
agaaaccggg 780gagcagagag tggtcactct caacgcgcgc agtcatcttc
gcgcccgcgt acaggcgcag 840agcactaatg acgggcttga ttttcaagaa
agtcaactcg tcaaaaagtt ggttgaaccg 900cccccgcagg gctgtcaagg
ttcagttata agttttccaa gtccacgctc cggtccagga 960tcaccagcac
agtggcttct ctacacccat cccacccaca gctggcagcg ggcagatctt
1020ggtgcttact tgaatcccag gccaccggcc cccgaagcct ggagcgagcc
tgtactgctt 1080gcaaagggga gctgtgcgta ctctgatctc cagtcaatgg
gtactggacc agatgggagt 1140ccattgtttg gttgtctcta cgaggcgaac
gattatgagg aaatcgtttt tcttatgttt 1200actttgaaac aggcgttccc
agccgaatat ttgcctcagt ccggaaccac gacgccagcg 1260ccgcgaccac
caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag
1320gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt
cgcctgtgat 1380atctacatct gggcgccctt ggccgggact tgtggggtcc
ttctcctgtc actggttatc 1440accctttact gcaaacgggg cagaaagaaa
ctcctgtata tattcaaaca accatttatg 1500agaccagtac aaactactca
agaggaagat ggctgtagct gccgatttcc agaagaagaa 1560gaaggaggat
gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag
1620cagggccaga accagctcta taacgagctc aatctaggac gaagagagga
gtacgatgtt 1680ttggacaaga gacgtggccg ggaccctgag atggggggaa
agccgagaag gaagaaccct 1740caggaaggcc tgtacaatga actgcagaaa
gataagatgg cggaggccta cagtgagatt 1800gggatgaaag gcgagcgccg
gaggggcaag gggcacgatg gcctttacca gggtctcagt 1860acagccacca
aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc
19142430DNAArtificial SequenceMyc-Tag 24gaacagaaac ttatctccga
ggaagacctg 3025638PRTArtificial SequencepTRPE-Myc-Neu2-BBz 25Met
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10
15His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly
20 25 30Ser Met Ala Ser Leu Pro Val Leu Gln Lys Glu Ser Val Phe Gln
Ser 35 40 45Gly Ala His Ala Tyr Arg Ile Pro Ala Leu Leu Tyr Leu Pro
Gly Gln 50 55 60Gln Ser Leu Leu Ala Phe Ala Glu Gln Arg Ala Ser Lys
Lys Asp Glu65 70 75 80His Ala Glu Leu Ile Val Leu Arg Arg Gly Asp
Tyr Asp Ala Pro Thr 85 90 95His Gln Val Gln Trp Gln Ala Gln Glu Val
Val Ala Gln Ala Arg Leu 100 105 110Asp Gly His Arg Ser Met Asn Pro
Cys Pro Leu Tyr Asp Ala Gln Thr 115 120 125Gly Thr Leu Phe Leu Phe
Phe Ile Ala Ile Pro Gly Gln Val Thr Glu 130 135 140Gln Gln Gln Leu
Gln Thr Arg Ala Asn Val Thr Arg Leu Cys Gln Val145 150 155 160Thr
Ser Thr Asp His Gly Arg Thr Trp Ser Ser Pro Arg Asp Leu Thr 165 170
175Asp Ala Ala Ile Gly Pro Ala Tyr Arg Glu Trp Ser Thr Phe Ala Val
180 185 190Gly Pro Gly His Cys Leu Gln Leu His Asp Arg Ala Arg Ser
Leu Val 195 200 205Val Pro Ala Tyr Ala Tyr Arg Lys Leu His Pro Ile
Gln Arg Pro Ile 210 215 220Pro Ser Ala Phe Cys Phe Leu Ser His Asp
His Gly Arg Thr Trp Ala225 230 235 240Arg Gly His Phe Val Ala Gln
Asp Thr Leu Glu Cys Gln Val Ala Glu 245 250 255Val Glu Thr Gly Glu
Gln Arg Val Val Thr Leu Asn Ala Arg Ser His 260 265 270Leu Arg Ala
Arg Val Gln Ala Gln Ser Thr Asn Asp Gly Leu Asp Phe 275 280 285Gln
Glu Ser Gln Leu Val Lys Lys Leu Val Glu Pro Pro Pro Gln Gly 290 295
300Cys Gln Gly Ser Val Ile Ser Phe Pro Ser Pro Arg Ser Gly Pro
Gly305 310 315 320Ser Pro Ala Gln Trp Leu Leu Tyr Thr His Pro Thr
His Ser Trp Gln 325 330 335Arg Ala Asp Leu Gly Ala Tyr Leu Asn Pro
Arg Pro Pro Ala Pro Glu 340 345 350Ala Trp Ser Glu Pro Val Leu Leu
Ala Lys Gly Ser Cys Ala Tyr Ser 355 360 365Asp Leu Gln Ser Met Gly
Thr Gly Pro Asp Gly Ser Pro Leu Phe Gly 370 375 380Cys Leu Tyr Glu
Ala Asn Asp Tyr Glu Glu Ile Val Phe Leu Met Phe385 390 395 400Thr
Leu Lys Gln Ala Phe Pro Ala Glu Tyr Leu Pro Gln Ser Gly Thr 405 410
415Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
420 425 430Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
Gly Gly 435 440 445Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
Ile Tyr Ile Trp 450 455 460Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
Leu Leu Ser Leu Val Ile465 470 475 480Thr Leu Tyr Cys Lys Arg Gly
Arg Lys Lys Leu Leu Tyr Ile Phe Lys 485 490 495Gln Pro Phe Met Arg
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 500 505 510Ser Cys Arg
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 515 520 525Lys
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn 530 535
540Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
Val545 550 555 560Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
Gly Lys Pro Arg 565 570 575Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
Glu Leu Gln Lys Asp Lys 580 585 590Met Ala Glu Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg Arg Arg 595 600 605Gly Lys Gly His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 610 615 620Asp Thr Tyr Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg625 630 6352610PRTArtificial
SequenceMyc-Tag 26Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu1 5
10271491DNAArtificial SequencepTRPE-Myc-Neu2-Dz 27atggccttac
cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccggaacaga
aacttatctc cgaggaagac ctgggatcca tggcttcctt gccggtgctg
120caaaaagaga gcgtattcca gtcaggcgcc catgcgtata gaatcccggc
acttctctat 180ttgccgggcc aacaaagtct cttggcgttc gcggaacagc
gggcgtccaa aaaagacgaa 240cacgccgagt tgattgtgct ccgccgcggg
gattatgatg ccccaacgca tcaggttcag 300tggcaggcac aagaggtagt
cgctcaggcg cgactggatg gacatcggtc aatgaaccca 360tgtccactgt
acgatgctca gacaggtacg ttgtttctgt tcttcatcgc tatccctggg
420caagtaacag aacaacaaca actgcaaacc agagccaatg taacaagact
ctgccaggta 480actagcactg accacggacg aacgtggtct tcccctagag
atcttactga cgccgcaatc 540gggcctgcat atcgcgaatg gagcactttc
gcagtaggcc ctggtcattg cctgcaactc 600catgatcgcg cccgatcact
tgtggtgcca gcgtacgcat accggaagct ccatccaata 660caacgcccca
tcccgtccgc tttttgtttc ctctcccatg accacgggcg gacttgggcg
720cggggtcatt tcgtcgcaca ggatacgttg gagtgtcagg tagcggaagt
agaaaccggg 780gagcagagag tggtcactct caacgcgcgc agtcatcttc
gcgcccgcgt acaggcgcag 840agcactaatg acgggcttga ttttcaagaa
agtcaactcg tcaaaaagtt ggttgaaccg 900cccccgcagg gctgtcaagg
ttcagttata agttttccaa gtccacgctc cggtccagga 960tcaccagcac
agtggcttct ctacacccat cccacccaca gctggcagcg ggcagatctt
1020ggtgcttact tgaatcccag gccaccggcc cccgaagcct ggagcgagcc
tgtactgctt 1080gcaaagggga gctgtgcgta ctctgatctc cagtcaatgg
gtactggacc agatgggagt 1140ccattgtttg gttgtctcta cgaggcgaac
gattatgagg aaatcgtttt tcttatgttt 1200actttgaaac aggcgttccc
agccgaatat ttgcctcagt ccggaaccac gacgccagcg 1260ccgcgaccac
caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag
1320gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt
cgcctgtgat 1380atctacatct gggcgccctt ggccgggact tgtggggtcc
ttctcctgtc actggttatc 1440accctttact gcagagtgaa gttcagcagg
agcgcagacg cccccgcgta a 149128496PRTArtificial
SequencepTRPE-Myc-Neu2-Dz 28Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Gln Lys Leu
Ile Ser Glu Glu Asp Leu Gly 20 25 30Ser Met Ala Ser Leu Pro Val Leu
Gln Lys Glu Ser Val Phe Gln Ser 35 40 45Gly Ala His Ala Tyr Arg Ile
Pro Ala Leu Leu Tyr Leu Pro Gly Gln 50 55 60Gln Ser Leu Leu Ala Phe
Ala Glu Gln Arg Ala Ser Lys Lys Asp Glu65 70 75 80His Ala Glu Leu
Ile Val Leu Arg Arg Gly Asp Tyr Asp Ala Pro Thr 85 90 95His Gln Val
Gln Trp Gln Ala Gln Glu Val Val Ala Gln Ala Arg Leu 100 105 110Asp
Gly His Arg Ser Met Asn Pro Cys Pro Leu Tyr Asp Ala Gln Thr 115 120
125Gly Thr Leu Phe Leu Phe Phe Ile Ala Ile Pro Gly Gln Val Thr Glu
130 135 140Gln Gln Gln Leu Gln Thr Arg Ala Asn Val Thr Arg Leu Cys
Gln Val145 150 155 160Thr Ser Thr Asp His Gly Arg Thr Trp Ser Ser
Pro Arg Asp Leu Thr 165 170 175Asp Ala Ala Ile Gly Pro Ala Tyr Arg
Glu Trp Ser Thr Phe Ala Val 180 185 190Gly Pro Gly His Cys Leu Gln
Leu His Asp Arg Ala Arg Ser Leu Val 195 200 205Val Pro Ala Tyr Ala
Tyr Arg Lys Leu His Pro Ile Gln Arg Pro Ile 210 215 220Pro Ser Ala
Phe Cys Phe Leu Ser His Asp His Gly Arg Thr Trp Ala225 230 235
240Arg Gly His Phe Val Ala Gln Asp Thr Leu Glu Cys Gln Val Ala Glu
245 250 255Val Glu Thr Gly Glu Gln Arg Val Val Thr Leu Asn Ala Arg
Ser His 260 265 270Leu Arg Ala Arg Val Gln Ala Gln Ser Thr Asn Asp
Gly Leu Asp Phe 275 280 285Gln Glu Ser Gln Leu Val Lys Lys Leu Val
Glu Pro Pro Pro Gln Gly 290 295 300Cys Gln Gly Ser Val Ile Ser Phe
Pro Ser Pro Arg Ser Gly Pro Gly305 310 315 320Ser Pro Ala Gln Trp
Leu Leu Tyr Thr His Pro Thr His Ser Trp Gln 325 330 335Arg Ala Asp
Leu Gly Ala Tyr Leu Asn Pro Arg Pro Pro Ala Pro Glu 340 345 350Ala
Trp Ser Glu Pro Val Leu Leu Ala Lys Gly Ser Cys Ala Tyr Ser 355 360
365Asp Leu Gln Ser Met Gly Thr Gly Pro Asp Gly Ser Pro Leu Phe Gly
370 375 380Cys Leu Tyr Glu Ala Asn Asp Tyr Glu Glu Ile Val Phe Leu
Met Phe385 390 395 400Thr Leu Lys Gln Ala Phe Pro Ala Glu Tyr Leu
Pro Gln Ser Gly Thr 405 410 415Thr Thr Pro Ala Pro Arg Pro Pro Thr
Pro Ala Pro Thr Ile Ala Ser 420 425 430Gln Pro Leu Ser Leu Arg Pro
Glu Ala Cys Arg Pro Ala Ala Gly Gly 435 440 445Ala Val His Thr Arg
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 450 455 460Ala Pro Leu
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile465 470 475
480Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
485 490 495291131DNAArtificial SequenceNeu1 29ggatcctcct ggagtaaagc
cgaaaatgac tttggattgg tgcagccttt ggtcacaatg 60gaacaacttc tgtgggtcag
cgggcggcaa atcggatcag ttgatacttt cagaatcccc 120cttattacag
ccacgccgcg cggcacactt ttggcctttg ccgaggctag gaagatgtct
180agttctgacg aaggcgctaa atttattgca ctcaggagat ccatggacca
gggttccact 240tggagcccta cagcttttat agttaatgac ggcgatgttc
cagacggcct gaatctggga 300gccgttgtct cagatgtaga aacaggagta
gtgtttcttt tttattcact ctgtgcacac 360aaggcgggat gccaggtcgc
gtctacgatg ctggtatggt caaaggacga tggggtatcc 420tggtcaacac
caagaaatct ttcacttgat ataggcacgg aagtgttcgc tccgggaccc
480ggctctggaa tacaaaagca aagagaaccg cgaaaaggca ggctgatagt
ttgtggacat 540gggaccttgg agcgggatgg tgtattttgc ttgcttagcg
atgaccacgg agcatcatgg 600cgctatggca gtggggtgag cgggattccc
tatgggcagc caaaacagga aaatgacttt 660aaccccgatg agtgccagcc
atacgaactt cctgatggtt cagtggtaat aaatgcgagg 720aatcagaaca
actaccattg ccattgtagg attgtcctcc gatcctatga cgcgtgtgac
780actttgcgcc cacgcgatgt aacgttcgat cccgagctgg tagatcctgt
agtggcggct 840ggtgccgtcg ttacaagctc aggtatcgtt ttttttagta
acccggctca ccctgaattt 900agagtcaacc tgaccctgag gtggagcttc
tcaaacggta catcttggag aaaggagact 960gtgcaattgt ggcctggccc
ctcaggctac agttccctcg ctaccctcga aggatcaatg 1020gatggagaag
agcaagctcc ccaactttac gtgctctacg agaagggccg aaaccattat
1080accgaaagca ttagcgtcgc gaagatcagc gtctatggaa ccctttccgg a
1131301038DNAArtificial SequenceNeu2 (with signal peptide removed)
30ggatccgaac aaagagcttc aaaaaaagat gagcacgccg agcttatagt gctccggcgg
60ggggattacg atgcgcccac gcatcaggtg cagtggcagg cacaggaggt cgtagcgcag
120gctcggcttg acggccatag aagtatgaat ccatgcccgc tttacgacgc
acaaactggc 180actctgtttt tgttttttat agcaatccca gggcaggtca
cagagcagca acaactccag 240acgcgagcta acgttacccg cctctgccaa
gtaacttcca cggatcacgg taggacttgg 300tcatctccac gcgacctcac
tgatgcggcc attgggcctg cctaccgcga atggagcaca 360ttcgctgtcg
gaccgggtca ctgtttgcaa ctccacgaca gagcaaggtc cttggtggtg
420ccagcctatg cctacaggaa gctgcatccc attcaaagac ccatcccttc
cgctttttgc 480ttccttagtc acgatcacgg caggacttgg gctagaggcc
attttgtagc gcaggacact 540ttggaatgcc aagtggcaga ggtggaaacc
ggcgaacaac gagtagtaac cctgaacgcc 600agaagtcacc ttcgcgctcg
cgtccaggcc caaagtacaa acgatggact tgacttccag 660gaaagtcagc
tcgttaagaa gctggtggaa ccgccacccc aagggtgtca gggcagtgtt
720ataagttttc catctccccg cagtggccct ggtagcccgg cgcagtggtt
gctgtacaca 780cacccaaccc actcttggca acgggctgac cttggtgctt
atctcaatcc tcgaccgcct 840gcacccgagg catggtcaga acccgttttg
ctggctaaag ggtcctgcgc gtacagcgac 900ctgcagtcta tggggaccgg
tcctgacgga agtccactgt ttgggtgcct ctatgaggca 960aacgattatg
aagaaattgt gtttctcatg ttcaccctta aacaggcatt
tcccgccgaa 1020tatctcccgc agtccgga 1038311302DNAArtificial
SequenceNeu3 31ggatccgagg tgatggaaga agtaactact tgctcattta
attcccctct cttcagacag 60gaagacgatc gaggcattac ttaccggatt cccgcccttt
tgtatatacc acccacacat 120acgtttcttg catttgcaga aaagcgctca
acgcgcaggg atgaggatgc gcttcacctg 180gtgctccggc gagggctccg
aatcggtcaa cttgtccaat ggggccctct gaaacccctc 240atggaggcta
ctttgccagg tcacaggacc atgaatccgt gcccagtctg ggaacagaag
300tcaggatgcg tgtttttgtt tttcatctgc gttcgaggtc atgtgacgga
gcgccagcag 360attgtttcag gtcggaacgc tgcacgcttg tgcttcatat
attcacaaga tgccggttgt 420tcatggtctg aggtcagaga tctgaccgaa
gaggtcattg gcagtgagtt gaagcattgg 480gccacattcg ccgtaggacc
cggacacgga atacagcttc agtctggtag gctcgtcatc 540cctgcttata
cctactatat tccatcatgg ttcttttgct tccaactgcc atgcaagacc
600agacctcact ctttgatgat ttactcagac gatctgggtg tcacatggca
tcacggtcga 660ttgattcggc ctatggtcac ggtagagtgc gaggtagcgg
aggtgacggg acgagctggc 720caccctgtgt tgtattgctc agcgcgcacc
cctaaccggt gcagggcgga agcactgtcc 780acagatcatg gagaaggttt
ccaaaggctc gctctttcac gccaactctg cgaaccacct 840catggttgcc
agggtagtgt cgtcagcttc cgaccattgg agataccgca tcggtgccaa
900gatagctcat ccaaagacgc accgacgatc cagcagagta gtccaggatc
ttccttgcgc 960ttggaagaag aagctggtac cccatcagaa tcatggcttc
tttactctca cccaacgtcc 1020aggaagcaaa gagtagattt ggggatctac
ctgaaccaaa cccccctcga agcggcgtgt 1080tggagtaggc cctggattct
ccattgtggc ccctgtgggt actctgacct tgcagcgttg 1140gaggaagagg
gattgttcgg ttgtcttttt gaatgcggga cgaagcagga gtgtgaacag
1200attgcgttca gactcttcac tcatcgcgaa attctgagtc accttcaagg
ggattgcacg 1260tcaccgggcc gaaacccatc ccaattcaag agcaattccg ga
1302321341DNAArtificial SequenceNeu4 32ggatcccagc gattgtcacc
ggatgactcc catgcgcatc ggttggtttt gcgcagaggc 60accctcgcgg gcgggagtgt
gcgctggggc gctttgcatg tgttgggtac agcagccctc 120gccgagcata
gatcaatgaa cccttgtccc gtgcatgacg cggggacagg aactgtgttt
180ttgttcttta ttgcggtact gggtcatacg cccgaagctg ttcaaattgc
aacgggacga 240aacgcggcca gactgtgttg tgttgcttcc agggatgcag
gacttagctg gggctccgcg 300cgggatctca cggaagaggc cattggcggc
gcagttcagg actgggcaac ctttgctgtg 360ggaccaggac atggagtgca
gcttccgtca ggtcggttgt tggtcccggc ttacacatat 420cgggtcgatc
ggcgcgaatg tttcggaaag atatgccgaa cctctccgca cagttttgca
480ttttactctg acgaccacgg ccggacgtgg aggtgcggag ggttggtgcc
caatcttcgc 540tccggtgaat gtcaattggc agctgtggac gggggacagg
caggctcctt tctgtactgt 600aatgctcgca gtccacttgg ttcccgcgtc
caagctctga gtacagatga agggacttct 660tttttgcctg ctgaacgagt
tgcgtccctc cctgaaacgg cctggggttg ccaaggcagt 720atagtgggtt
ttccagctcc cgcacccaac cgccccaggg atgatagttg gtccgtgggt
780cctggcagcc ctctgcagcc tccccttttg ggaccagggg tacatgagcc
gccagaagag 840gcggccgttg atcctagagg aggccaggtg ccaggtgggc
cttttagcag actccagcct 900agaggcgatg gaccgagaca gccggggcct
agaccagggg taagtggcga tgtgggtagt 960tggacacttg cgctgccaat
gcctttcgct gctcctcccc agtccccaac ttggctgctc 1020tattctcacc
cagtgggtag gcgcgctagg ctgcacatgg ggatacgcct ctcacagtcc
1080ccgcttgatc ctagatcttg gacagaacct tgggtcatat atgaaggccc
gagcggttac 1140agcgacctgg cgagcatcgg tccagcaccg gagggtgggt
tggtcttcgc gtgcttgtac 1200gaaagcggcg cccgcactag ctacgatgag
ataagctttt gcaccttctc attgagggaa 1260gttcttgaaa atgtaccggc
atcccccaaa cccccaaatc tcggggacaa gcccagagga 1320tgctgttggc
cctcctccgg a 134133358PRTArtificial SequenceNeu1 33Met Glu Gln Leu
Leu Trp Val Ser Gly Arg Gln Ile Gly Ser Val Asp1 5 10 15Thr Phe Arg
Ile Pro Leu Ile Thr Ala Thr Pro Arg Gly Thr Leu Leu 20 25 30Ala Phe
Ala Glu Ala Arg Lys Met Ser Ser Ser Asp Glu Gly Ala Lys 35 40 45Phe
Ile Ala Leu Arg Arg Ser Met Asp Gln Gly Ser Thr Trp Ser Pro 50 55
60Thr Ala Phe Ile Val Asn Asp Gly Asp Val Pro Asp Gly Leu Asn Leu65
70 75 80Gly Ala Val Val Ser Asp Val Glu Thr Gly Val Val Phe Leu Phe
Tyr 85 90 95Ser Leu Cys Ala His Lys Ala Gly Cys Gln Val Ala Ser Thr
Met Leu 100 105 110Val Trp Ser Lys Asp Asp Gly Val Ser Trp Ser Thr
Pro Arg Asn Leu 115 120 125Ser Leu Asp Ile Gly Thr Glu Val Phe Ala
Pro Gly Pro Gly Ser Gly 130 135 140Ile Gln Lys Gln Arg Glu Pro Arg
Lys Gly Arg Leu Ile Val Cys Gly145 150 155 160His Gly Thr Leu Glu
Arg Asp Gly Val Phe Cys Leu Leu Ser Asp Asp 165 170 175His Gly Ala
Ser Trp Arg Tyr Gly Ser Gly Val Ser Gly Ile Pro Tyr 180 185 190Gly
Gln Pro Lys Gln Glu Asn Asp Phe Asn Pro Asp Glu Cys Gln Pro 195 200
205Tyr Glu Leu Pro Asp Gly Ser Val Val Ile Asn Ala Arg Asn Gln Asn
210 215 220Asn Tyr His Cys His Cys Arg Ile Val Leu Arg Ser Tyr Asp
Ala Cys225 230 235 240Asp Thr Leu Arg Pro Arg Asp Val Thr Phe Asp
Pro Glu Leu Val Asp 245 250 255Pro Val Val Ala Ala Gly Ala Val Val
Thr Ser Ser Gly Ile Val Phe 260 265 270Phe Ser Asn Pro Ala His Pro
Glu Phe Arg Val Asn Leu Thr Leu Arg 275 280 285Trp Ser Phe Ser Asn
Gly Thr Ser Trp Arg Lys Glu Thr Val Gln Leu 290 295 300Trp Pro Gly
Pro Ser Gly Tyr Ser Ser Leu Ala Thr Leu Glu Gly Ser305 310 315
320Met Asp Gly Glu Glu Gln Ala Pro Gln Leu Tyr Val Leu Tyr Glu Lys
325 330 335Gly Arg Asn His Tyr Thr Glu Ser Ile Ser Val Ala Lys Ile
Ser Val 340 345 350Tyr Gly Thr Leu Ser Gly 35534298PRTArtificial
SequenceNeu2 (with signal peptide removed) 34Met Asn Pro Cys Pro
Leu Tyr Asp Ala Gln Thr Gly Thr Leu Phe Leu1 5 10 15Phe Phe Ile Ala
Ile Pro Gly Gln Val Thr Glu Gln Gln Gln Leu Gln 20 25 30Thr Arg Ala
Asn Val Thr Arg Leu Cys Gln Val Thr Ser Thr Asp His 35 40 45Gly Arg
Thr Trp Ser Ser Pro Arg Asp Leu Thr Asp Ala Ala Ile Gly 50 55 60Pro
Ala Tyr Arg Glu Trp Ser Thr Phe Ala Val Gly Pro Gly His Cys65 70 75
80Leu Gln Leu His Asp Arg Ala Arg Ser Leu Val Val Pro Ala Tyr Ala
85 90 95Tyr Arg Lys Leu His Pro Ile Gln Arg Pro Ile Pro Ser Ala Phe
Cys 100 105 110Phe Leu Ser His Asp His Gly Arg Thr Trp Ala Arg Gly
His Phe Val 115 120 125Ala Gln Asp Thr Leu Glu Cys Gln Val Ala Glu
Val Glu Thr Gly Glu 130 135 140Gln Arg Val Val Thr Leu Asn Ala Arg
Ser His Leu Arg Ala Arg Val145 150 155 160Gln Ala Gln Ser Thr Asn
Asp Gly Leu Asp Phe Gln Glu Ser Gln Leu 165 170 175Val Lys Lys Leu
Val Glu Pro Pro Pro Gln Gly Cys Gln Gly Ser Val 180 185 190Ile Ser
Phe Pro Ser Pro Arg Ser Gly Pro Gly Ser Pro Ala Gln Trp 195 200
205Leu Leu Tyr Thr His Pro Thr His Ser Trp Gln Arg Ala Asp Leu Gly
210 215 220Ala Tyr Leu Asn Pro Arg Pro Pro Ala Pro Glu Ala Trp Ser
Glu Pro225 230 235 240Val Leu Leu Ala Lys Gly Ser Cys Ala Tyr Ser
Asp Leu Gln Ser Met 245 250 255Gly Thr Gly Pro Asp Gly Ser Pro Leu
Phe Gly Cys Leu Tyr Glu Ala 260 265 270Asn Asp Tyr Glu Glu Ile Val
Phe Leu Met Phe Thr Leu Lys Gln Ala 275 280 285Phe Pro Ala Glu Tyr
Leu Pro Gln Ser Gly 290 29535430PRTArtificial SequenceNeu3 35Met
Glu Glu Val Thr Thr Cys Ser Phe Asn Ser Pro Leu Phe Arg Gln1 5 10
15Glu Asp Asp Arg Gly Ile Thr Tyr Arg Ile Pro Ala Leu Leu Tyr Ile
20 25 30Pro Pro Thr His Thr Phe Leu Ala Phe Ala Glu Lys Arg Ser Thr
Arg 35 40 45Arg Asp Glu Asp Ala Leu His Leu Val Leu Arg Arg Gly Leu
Arg Ile 50 55 60Gly Gln Leu Val Gln Trp Gly Pro Leu Lys Pro Leu Met
Glu Ala Thr65 70 75 80Leu Pro Gly His Arg Thr Met Asn Pro Cys Pro
Val Trp Glu Gln Lys 85 90 95Ser Gly Cys Val Phe Leu Phe Phe Ile Cys
Val Arg Gly His Val Thr 100 105 110Glu Arg Gln Gln Ile Val Ser Gly
Arg Asn Ala Ala Arg Leu Cys Phe 115 120 125Ile Tyr Ser Gln Asp Ala
Gly Cys Ser Trp Ser Glu Val Arg Asp Leu 130 135 140Thr Glu Glu Val
Ile Gly Ser Glu Leu Lys His Trp Ala Thr Phe Ala145 150 155 160Val
Gly Pro Gly His Gly Ile Gln Leu Gln Ser Gly Arg Leu Val Ile 165 170
175Pro Ala Tyr Thr Tyr Tyr Ile Pro Ser Trp Phe Phe Cys Phe Gln Leu
180 185 190Pro Cys Lys Thr Arg Pro His Ser Leu Met Ile Tyr Ser Asp
Asp Leu 195 200 205Gly Val Thr Trp His His Gly Arg Leu Ile Arg Pro
Met Val Thr Val 210 215 220Glu Cys Glu Val Ala Glu Val Thr Gly Arg
Ala Gly His Pro Val Leu225 230 235 240Tyr Cys Ser Ala Arg Thr Pro
Asn Arg Cys Arg Ala Glu Ala Leu Ser 245 250 255Thr Asp His Gly Glu
Gly Phe Gln Arg Leu Ala Leu Ser Arg Gln Leu 260 265 270Cys Glu Pro
Pro His Gly Cys Gln Gly Ser Val Val Ser Phe Arg Pro 275 280 285Leu
Glu Ile Pro His Arg Cys Gln Asp Ser Ser Ser Lys Asp Ala Pro 290 295
300Thr Ile Gln Gln Ser Ser Pro Gly Ser Ser Leu Arg Leu Glu Glu
Glu305 310 315 320Ala Gly Thr Pro Ser Glu Ser Trp Leu Leu Tyr Ser
His Pro Thr Ser 325 330 335Arg Lys Gln Arg Val Asp Leu Gly Ile Tyr
Leu Asn Gln Thr Pro Leu 340 345 350Glu Ala Ala Cys Trp Ser Arg Pro
Trp Ile Leu His Cys Gly Pro Cys 355 360 365Gly Tyr Ser Asp Leu Ala
Ala Leu Glu Glu Glu Gly Leu Phe Gly Cys 370 375 380Leu Phe Glu Cys
Gly Thr Lys Gln Glu Cys Glu Gln Ile Ala Phe Arg385 390 395 400Leu
Phe Thr His Arg Glu Ile Leu Ser His Leu Gln Gly Asp Cys Thr 405 410
415Ser Pro Gly Arg Asn Pro Ser Gln Phe Lys Ser Asn Ser Gly 420 425
43036402PRTArtificial SequenceNeu4 36Met Asn Pro Cys Pro Val His
Asp Ala Gly Thr Gly Thr Val Phe Leu1 5 10 15Phe Phe Ile Ala Val Leu
Gly His Thr Pro Glu Ala Val Gln Ile Ala 20 25 30Thr Gly Arg Asn Ala
Ala Arg Leu Cys Cys Val Ala Ser Arg Asp Ala 35 40 45Gly Leu Ser Trp
Gly Ser Ala Arg Asp Leu Thr Glu Glu Ala Ile Gly 50 55 60Gly Ala Val
Gln Asp Trp Ala Thr Phe Ala Val Gly Pro Gly His Gly65 70 75 80Val
Gln Leu Pro Ser Gly Arg Leu Leu Val Pro Ala Tyr Thr Tyr Arg 85 90
95Val Asp Arg Arg Glu Cys Phe Gly Lys Ile Cys Arg Thr Ser Pro His
100 105 110Ser Phe Ala Phe Tyr Ser Asp Asp His Gly Arg Thr Trp Arg
Cys Gly 115 120 125Gly Leu Val Pro Asn Leu Arg Ser Gly Glu Cys Gln
Leu Ala Ala Val 130 135 140Asp Gly Gly Gln Ala Gly Ser Phe Leu Tyr
Cys Asn Ala Arg Ser Pro145 150 155 160Leu Gly Ser Arg Val Gln Ala
Leu Ser Thr Asp Glu Gly Thr Ser Phe 165 170 175Leu Pro Ala Glu Arg
Val Ala Ser Leu Pro Glu Thr Ala Trp Gly Cys 180 185 190Gln Gly Ser
Ile Val Gly Phe Pro Ala Pro Ala Pro Asn Arg Pro Arg 195 200 205Asp
Asp Ser Trp Ser Val Gly Pro Gly Ser Pro Leu Gln Pro Pro Leu 210 215
220Leu Gly Pro Gly Val His Glu Pro Pro Glu Glu Ala Ala Val Asp
Pro225 230 235 240Arg Gly Gly Gln Val Pro Gly Gly Pro Phe Ser Arg
Leu Gln Pro Arg 245 250 255Gly Asp Gly Pro Arg Gln Pro Gly Pro Arg
Pro Gly Val Ser Gly Asp 260 265 270Val Gly Ser Trp Thr Leu Ala Leu
Pro Met Pro Phe Ala Ala Pro Pro 275 280 285Gln Ser Pro Thr Trp Leu
Leu Tyr Ser His Pro Val Gly Arg Arg Ala 290 295 300Arg Leu His Met
Gly Ile Arg Leu Ser Gln Ser Pro Leu Asp Pro Arg305 310 315 320Ser
Trp Thr Glu Pro Trp Val Ile Tyr Glu Gly Pro Ser Gly Tyr Ser 325 330
335Asp Leu Ala Ser Ile Gly Pro Ala Pro Glu Gly Gly Leu Val Phe Ala
340 345 350Cys Leu Tyr Glu Ser Gly Ala Arg Thr Ser Tyr Asp Glu Ile
Ser Phe 355 360 365Cys Thr Phe Ser Leu Arg Glu Val Leu Glu Asn Val
Pro Ala Ser Pro 370 375 380Lys Pro Pro Asn Leu Gly Asp Lys Pro Arg
Gly Cys Cys Trp Pro Ser385 390 395 400Ser Gly37605PRTArtificial
SequenceNeu2- BBz (no leader) 37Met Ala Ser Leu Pro Val Leu Gln Lys
Glu Ser Val Phe Gln Ser Gly1 5 10 15Ala His Ala Tyr Arg Ile Pro Ala
Leu Leu Tyr Leu Pro Gly Gln Gln 20 25 30Ser Leu Leu Ala Phe Ala Glu
Gln Arg Ala Ser Lys Lys Asp Glu His 35 40 45Ala Glu Leu Ile Val Leu
Arg Arg Gly Asp Tyr Asp Ala Pro Thr His 50 55 60Gln Val Gln Trp Gln
Ala Gln Glu Val Val Ala Gln Ala Arg Leu Asp65 70 75 80Gly His Arg
Ser Met Asn Pro Cys Pro Leu Tyr Asp Ala Gln Thr Gly 85 90 95Thr Leu
Phe Leu Phe Phe Ile Ala Ile Pro Gly Gln Val Thr Glu Gln 100 105
110Gln Gln Leu Gln Thr Arg Ala Asn Val Thr Arg Leu Cys Gln Val Thr
115 120 125Ser Thr Asp His Gly Arg Thr Trp Ser Ser Pro Arg Asp Leu
Thr Asp 130 135 140Ala Ala Ile Gly Pro Ala Tyr Arg Glu Trp Ser Thr
Phe Ala Val Gly145 150 155 160Pro Gly His Cys Leu Gln Leu His Asp
Arg Ala Arg Ser Leu Val Val 165 170 175Pro Ala Tyr Ala Tyr Arg Lys
Leu His Pro Ile Gln Arg Pro Ile Pro 180 185 190Ser Ala Phe Cys Phe
Leu Ser His Asp His Gly Arg Thr Trp Ala Arg 195 200 205Gly His Phe
Val Ala Gln Asp Thr Leu Glu Cys Gln Val Ala Glu Val 210 215 220Glu
Thr Gly Glu Gln Arg Val Val Thr Leu Asn Ala Arg Ser His Leu225 230
235 240Arg Ala Arg Val Gln Ala Gln Ser Thr Asn Asp Gly Leu Asp Phe
Gln 245 250 255Glu Ser Gln Leu Val Lys Lys Leu Val Glu Pro Pro Pro
Gln Gly Cys 260 265 270Gln Gly Ser Val Ile Ser Phe Pro Ser Pro Arg
Ser Gly Pro Gly Ser 275 280 285Pro Ala Gln Trp Leu Leu Tyr Thr His
Pro Thr His Ser Trp Gln Arg 290 295 300Ala Asp Leu Gly Ala Tyr Leu
Asn Pro Arg Pro Pro Ala Pro Glu Ala305 310 315 320Trp Ser Glu Pro
Val Leu Leu Ala Lys Gly Ser Cys Ala Tyr Ser Asp 325 330 335Leu Gln
Ser Met Gly Thr Gly Pro Asp Gly Ser Pro Leu Phe Gly Cys 340 345
350Leu Tyr Glu Ala Asn Asp Tyr Glu Glu Ile Val Phe Leu Met Phe Thr
355 360 365Leu Lys Gln Ala Phe Pro Ala Glu Tyr Leu Pro Gln Ser Gly
Thr Thr 370 375 380Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
Ile Ala Ser Gln385 390 395 400Pro Leu Ser Leu Arg Pro Glu Ala Cys
Arg Pro Ala Ala Gly Gly Ala 405 410 415Val His Thr Arg Gly Leu Asp
Phe Ala Cys Asp Ile Tyr Ile Trp Ala 420 425 430Pro Leu Ala Gly Thr
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 435 440 445Leu Tyr Cys
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 450 455 460Pro
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser465 470
475 480Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
Lys 485
490 495Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn
Gln 500 505 510Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
Asp Val Leu 515 520 525Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
Gly Lys Pro Arg Arg 530 535 540Lys Asn Pro Gln Glu Gly Leu Tyr Asn
Glu Leu Gln Lys Asp Lys Met545 550 555 560Ala Glu Ala Tyr Ser Glu
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 565 570 575Lys Gly His Asp
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 580 585 590Thr Tyr
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 595 600
60538474PRTArtificial SequenceNeu2-hinge-Tm 38Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Pro Gly Ser Met Ala Ser Leu Pro Val Leu Gln Lys 20 25 30Glu Ser Val
Phe Gln Ser Gly Ala His Ala Tyr Arg Ile Pro Ala Leu 35 40 45Leu Tyr
Leu Pro Gly Gln Gln Ser Leu Leu Ala Phe Ala Glu Gln Arg 50 55 60Ala
Ser Lys Lys Asp Glu His Ala Glu Leu Ile Val Leu Arg Arg Gly65 70 75
80Asp Tyr Asp Ala Pro Thr His Gln Val Gln Trp Gln Ala Gln Glu Val
85 90 95Val Ala Gln Ala Arg Leu Asp Gly His Arg Ser Met Asn Pro Cys
Pro 100 105 110Leu Tyr Asp Ala Gln Thr Gly Thr Leu Phe Leu Phe Phe
Ile Ala Ile 115 120 125Pro Gly Gln Val Thr Glu Gln Gln Gln Leu Gln
Thr Arg Ala Asn Val 130 135 140Thr Arg Leu Cys Gln Val Thr Ser Thr
Asp His Gly Arg Thr Trp Ser145 150 155 160Ser Pro Arg Asp Leu Thr
Asp Ala Ala Ile Gly Pro Ala Tyr Arg Glu 165 170 175Trp Ser Thr Phe
Ala Val Gly Pro Gly His Cys Leu Gln Leu His Asp 180 185 190Arg Ala
Arg Ser Leu Val Val Pro Ala Tyr Ala Tyr Arg Lys Leu His 195 200
205Pro Ile Gln Arg Pro Ile Pro Ser Ala Phe Cys Phe Leu Ser His Asp
210 215 220His Gly Arg Thr Trp Ala Arg Gly His Phe Val Ala Gln Asp
Thr Leu225 230 235 240Glu Cys Gln Val Ala Glu Val Glu Thr Gly Glu
Gln Arg Val Val Thr 245 250 255Leu Asn Ala Arg Ser His Leu Arg Ala
Arg Val Gln Ala Gln Ser Thr 260 265 270Asn Asp Gly Leu Asp Phe Gln
Glu Ser Gln Leu Val Lys Lys Leu Val 275 280 285Glu Pro Pro Pro Gln
Gly Cys Gln Gly Ser Val Ile Ser Phe Pro Ser 290 295 300Pro Arg Ser
Gly Pro Gly Ser Pro Ala Gln Trp Leu Leu Tyr Thr His305 310 315
320Pro Thr His Ser Trp Gln Arg Ala Asp Leu Gly Ala Tyr Leu Asn Pro
325 330 335Arg Pro Pro Ala Pro Glu Ala Trp Ser Glu Pro Val Leu Leu
Ala Lys 340 345 350Gly Ser Cys Ala Tyr Ser Asp Leu Gln Ser Met Gly
Thr Gly Pro Asp 355 360 365Gly Ser Pro Leu Phe Gly Cys Leu Tyr Glu
Ala Asn Asp Tyr Glu Glu 370 375 380Ile Val Phe Leu Met Phe Thr Leu
Lys Gln Ala Phe Pro Ala Glu Tyr385 390 395 400Leu Pro Gln Ser Gly
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 405 410 415Ala Pro Thr
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 420 425 430Arg
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 435 440
445Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
450 455 460Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys465
470391815DNAArtificial SequenceNeu2- BBz (no leader) 39atggcttcct
tgccggtgct gcaaaaagag agcgtattcc agtcaggcgc ccatgcgtat 60agaatcccgg
cacttctcta tttgccgggc caacaaagtc tcttggcgtt cgcggaacag
120cgggcgtcca aaaaagacga acacgccgag ttgattgtgc tccgccgcgg
ggattatgat 180gccccaacgc atcaggttca gtggcaggca caagaggtag
tcgctcaggc gcgactggat 240ggacatcggt caatgaaccc atgtccactg
tacgatgctc agacaggtac gttgtttctg 300ttcttcatcg ctatccctgg
gcaagtaaca gaacaacaac aactgcaaac cagagccaat 360gtaacaagac
tctgccaggt aactagcact gaccacggac gaacgtggtc ttcccctaga
420gatcttactg acgccgcaat cgggcctgca tatcgcgaat ggagcacttt
cgcagtaggc 480cctggtcatt gcctgcaact ccatgatcgc gcccgatcac
ttgtggtgcc agcgtacgca 540taccggaagc tccatccaat acaacgcccc
atcccgtccg ctttttgttt cctctcccat 600gaccacgggc ggacttgggc
gcggggtcat ttcgtcgcac aggatacgtt ggagtgtcag 660gtagcggaag
tagaaaccgg ggagcagaga gtggtcactc tcaacgcgcg cagtcatctt
720cgcgcccgcg tacaggcgca gagcactaat gacgggcttg attttcaaga
aagtcaactc 780gtcaaaaagt tggttgaacc gcccccgcag ggctgtcaag
gttcagttat aagttttcca 840agtccacgct ccggtccagg atcaccagca
cagtggcttc tctacaccca tcccacccac 900agctggcagc gggcagatct
tggtgcttac ttgaatccca ggccaccggc ccccgaagcc 960tggagcgagc
ctgtactgct tgcaaagggg agctgtgcgt actctgatct ccagtcaatg
1020ggtactggac cagatgggag tccattgttt ggttgtctct acgaggcgaa
cgattatgag 1080gaaatcgttt ttcttatgtt tactttgaaa caggcgttcc
cagccgaata tttgcctcag 1140tccggaacca cgacgccagc gccgcgacca
ccaacaccgg cgcccaccat cgcgtcgcag 1200cccctgtccc tgcgcccaga
ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 1260gggctggact
tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc
1320cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa
actcctgtat 1380atattcaaac aaccatttat gagaccagta caaactactc
aagaggaaga tggctgtagc 1440tgccgatttc cagaagaaga agaaggagga
tgtgaactga gagtgaagtt cagcaggagc 1500gcagacgccc ccgcgtacaa
gcagggccag aaccagctct ataacgagct caatctagga 1560cgaagagagg
agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga
1620aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa
agataagatg 1680gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc
ggaggggcaa ggggcacgat 1740ggcctttacc agggtctcag tacagccacc
aaggacacct acgacgccct tcacatgcag 1800gccctgcccc ctcgc
1815401422DNAArtificial SequenceNeu2-hinge-Tm 40atggccttac
cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60ccgggatcca
tggcttcctt gccggtgctg caaaaagaga gcgtattcca gtcaggcgcc
120catgcgtata gaatcccggc acttctctat ttgccgggcc aacaaagtct
cttggcgttc 180gcggaacagc gggcgtccaa aaaagacgaa cacgccgagt
tgattgtgct ccgccgcggg 240gattatgatg ccccaacgca tcaggttcag
tggcaggcac aagaggtagt cgctcaggcg 300cgactggatg gacatcggtc
aatgaaccca tgtccactgt acgatgctca gacaggtacg 360ttgtttctgt
tcttcatcgc tatccctggg caagtaacag aacaacaaca actgcaaacc
420agagccaatg taacaagact ctgccaggta actagcactg accacggacg
aacgtggtct 480tcccctagag atcttactga cgccgcaatc gggcctgcat
atcgcgaatg gagcactttc 540gcagtaggcc ctggtcattg cctgcaactc
catgatcgcg cccgatcact tgtggtgcca 600gcgtacgcat accggaagct
ccatccaata caacgcccca tcccgtccgc tttttgtttc 660ctctcccatg
accacgggcg gacttgggcg cggggtcatt tcgtcgcaca ggatacgttg
720gagtgtcagg tagcggaagt agaaaccggg gagcagagag tggtcactct
caacgcgcgc 780agtcatcttc gcgcccgcgt acaggcgcag agcactaatg
acgggcttga ttttcaagaa 840agtcaactcg tcaaaaagtt ggttgaaccg
cccccgcagg gctgtcaagg ttcagttata 900agttttccaa gtccacgctc
cggtccagga tcaccagcac agtggcttct ctacacccat 960cccacccaca
gctggcagcg ggcagatctt ggtgcttact tgaatcccag gccaccggcc
1020cccgaagcct ggagcgagcc tgtactgctt gcaaagggga gctgtgcgta
ctctgatctc 1080cagtcaatgg gtactggacc agatgggagt ccattgtttg
gttgtctcta cgaggcgaac 1140gattatgagg aaatcgtttt tcttatgttt
actttgaaac aggcgttccc agccgaatat 1200ttgcctcagt ccggaaccac
gacgccagcg ccgcgaccac caacaccggc gcccaccatc 1260gcgtcgcagc
ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg
1320cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt
ggccgggact 1380tgtggggtcc ttctcctgtc actggttatc accctttact gc
1422415PRTArtificial Sequencelinker 41Gly Ser Gly Ser Gly1
5425PRTArtificial Sequencelinker 42Gly Ser Gly Gly Gly1
5435PRTArtificial Sequencelinker 43Gly Gly Gly Ser Gly1
5445PRTArtificial Sequencelinker 44Gly Ser Ser Ser Gly1
5455PRTArtificial Sequencelinker 45Gly Gly Gly Gly Ser1
54615PRTArtificial Sequencelinker 46Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser1 5 10 154745PRTArtificial
Sequencelinker 47Gly Gly Thr Gly Gly Cys Gly Gly Thr Gly Gly Cys
Thr Cys Gly Gly1 5 10 15Gly Cys Gly Gly Thr Gly Gly Thr Gly Gly Gly
Thr Cys Gly Gly Gly 20 25 30Thr Gly Gly Cys Gly Gly Cys Gly Gly Ala
Thr Cys Thr 35 40 45485PRTArtificial
SequencelinkerREPEAT(1)..(5)repeat n times. where n represents an
integer of at least 1 48Gly Gly Gly Gly Ser1 5495PRTArtificial
Sequencehinge 49Asp Lys Thr His Thr1 5504PRTArtificial
Sequencehinge 50Cys Pro Pro Cys15115PRTArtificial Sequencehinge
51Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg1 5 10
155212PRTArtificial Sequencehinge 52Glu Leu Lys Thr Pro Leu Gly Asp
Thr Thr His Thr1 5 105310PRTArtificial Sequencehinge 53Lys Ser Cys
Asp Lys Thr His Thr Cys Pro1 5 10547PRTArtificial Sequencehinge
54Lys Cys Cys Val Asp Cys Pro1 5557PRTArtificial Sequencehinge
55Lys Tyr Gly Pro Pro Cys Pro1 55615PRTArtificial Sequencehinge
56Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10
155712PRTArtificial Sequencehinge 57Glu Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro1 5 105817PRTArtificial Sequencehinge 58Glu Leu Lys
Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys1 5 10
15Pro5912PRTArtificial Sequencehinge 59Ser Pro Asn Met Val Pro His
Ala His His Ala Gln1 5 10609PRTArtificial SequenceNY-ESO157-165
60Ser Leu Leu Met Trp Ile Thr Gln Cys1 5
* * * * *
References