U.S. patent application number 16/540673 was filed with the patent office on 2020-03-05 for multipartite signaling proteins and uses thereof.
This patent application is currently assigned to bluebird bio, Inc.. The applicant listed for this patent is bluebird bio, Inc.. Invention is credited to Alexander Astrakhan, Michael Certo, Jordan Jarjour.
Application Number | 20200071399 16/540673 |
Document ID | / |
Family ID | 52432340 |
Filed Date | 2020-03-05 |
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United States Patent
Application |
20200071399 |
Kind Code |
A1 |
Jarjour; Jordan ; et
al. |
March 5, 2020 |
MULTIPARTITE SIGNALING PROTEINS AND USES THEREOF
Abstract
The present disclosure relates to compositions and methods for
using cells having chemically-induced fusion protein complexes to
spatially and temporally control immune cell signal initiation and
downstream responses for treating disease.
Inventors: |
Jarjour; Jordan; (Seattle,
WA) ; Astrakhan; Alexander; (Seattle, WA) ;
Certo; Michael; (Medford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
bluebird bio, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
bluebird bio, Inc.
Cambridge
MA
|
Family ID: |
52432340 |
Appl. No.: |
16/540673 |
Filed: |
August 14, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14608098 |
Jan 28, 2015 |
10428142 |
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16540673 |
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PCT/US2014/047852 |
Jul 23, 2014 |
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14608098 |
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61934092 |
Jan 31, 2014 |
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61859697 |
Jul 29, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 207/11001 20130101;
C12Y 502/01008 20130101; C07K 14/7051 20130101; C07K 14/70521
20130101; C07K 16/2896 20130101; C12N 2510/00 20130101; C12N 9/16
20130101; C12N 15/85 20130101; C07K 16/40 20130101; A61P 35/00
20180101; A61K 35/17 20130101; C12N 9/90 20130101; A61P 29/00
20180101; C07K 2319/03 20130101; C07K 14/7056 20130101; C07K
14/70514 20130101; C07K 16/2803 20130101; C07K 14/70535 20130101;
C07K 14/70517 20130101; A61P 37/06 20180101; C07K 14/70596
20130101; C07K 2319/70 20130101; C12N 9/12 20130101; C12N 9/003
20130101; C07K 14/70503 20130101; A61K 45/06 20130101; C07K
2317/622 20130101; C07K 14/70578 20130101; A61P 43/00 20180101;
C07H 21/04 20130101; A61K 39/0005 20130101; A61P 37/02
20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 14/705 20060101 C07K014/705; C07K 14/725 20060101
C07K014/725; C07K 14/73 20060101 C07K014/73; C07K 14/735 20060101
C07K014/735; C12N 9/06 20060101 C12N009/06; C12N 9/16 20060101
C12N009/16; A61K 35/17 20060101 A61K035/17; A61K 39/00 20060101
A61K039/00; A61K 45/06 20060101 A61K045/06; C07K 16/40 20060101
C07K016/40; C12N 9/12 20060101 C12N009/12; C12N 9/90 20060101
C12N009/90; C12N 15/85 20060101 C12N015/85 |
Claims
1. A non-natural cell, comprising: (a) a first nucleic acid
molecule encoding a first fusion protein comprising a first
multimerization domain, a hydrophobic domain, and an actuator
domain, wherein the first multimerization domain localizes
extracellularly when the first fusion protein is expressed; and (b)
a second nucleic acid molecule encoding a second fusion protein
comprising a binding domain and a second multimerization domain,
wherein the second fusion protein localizes extracellularly when
expressed; wherein a first bridging factor promotes the formation
of a polypeptide complex on the non-natural cell surface with the
bridging factor associated with and disposed between the
multimerization domains of the first and second fusion
proteins.
2. The non-natural cell according to claim 1, wherein the first and
second multimerization domains are the same or different.
3. The non-natural cell according to claim 1 or claim 2, wherein
the multimerization domains of the first and second fusion proteins
associate with a bridging factor selected from rapamycin or a
rapalog thereof, coumermycin or a derivative thereof, gibberellin
or a derivative thereof, abscisic acid (ABA) or a derivative
thereof, methotrexate or a derivative thereof, cyclosporin A or a
derivative thereof, FKCsA or a derivative thereof, trimethoprim
(Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or
any combination thereof.
4. The non-natural cell according to any one of the preceding
claims, wherein the first and second multimerization domains are a
pair selected from FKBP and FRB, FKBP and calcineurin, FKBP and
cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin,
PYL1 and ABI1, or GIB1 and GAI, or variants thereof.
5. The non-natural cell according to any one of the preceding
claims, wherein the first multimerization domain comprises a first
FKBP polypeptide or variant thereof, and the second multimerization
domain comprises a first FRB polypeptide or variant thereof
6. The non-natural cell according to any one of the preceding
claims, wherein the first multimerization domain comprises a first
FRB polypeptide or variant thereof, and the second multimerization
domain comprises a first FKBP polypeptide or variant thereof
7. The non-natural cell according to claim 5 or 6, wherein the
bridging factor is sirolimus, everolimus, novolimus, pimecrolimus,
ridaforolimus, tacrolimus, temsirolimus, umirolimus, or
zotarolimus.
8. The non-natural cell according to any one of the preceding
claims, wherein the first nucleic acid molecule encodes a first
fusion protein further comprising a third multimerization
domain.
9. The non-natural cell according to claim 8, wherein the third
multimerization domain of the first fusion protein is a binding
domain for a bridging factor selected from rapamycin or a rapalog
thereof, coumermycin or a derivative thereof, gibberellin or a
derivative thereof, ABA or a derivative thereof, methotrexate or a
derivative thereof, cyclosporin A or a derivative thereof, FKCsA or
a derivative thereof, Tmp-SLF or a derivative thereof, or any
combination thereof.
10. The non-natural cell according to any one of the preceding
claims, wherein a second bridging factor promotes the association
of at least two first fusion proteins with the bridging factor
associated with and disposed between the third multimerization
domains of the first fusion proteins.
11. The non-natural cell according to any one of the preceding
claims, wherein the protein complex is a homocomplex comprising at
least two first fusion proteins.
12. The non-natural cell according to any one of the preceding
claims, wherein the first fusion protein has at least one
multimerization domain of FKBP, DHFR or GyrB.
13. The non-natural cell according to any one of claims 1-12,
wherein the binding domain of the polypeptide complex specifically
binds to a target located on a target cell surface.
14. The non-natural cell according to claim 13, wherein the protein
complex is a heterocomplex comprising one or more first fusion
proteins and one or more second fusion proteins.
15. The non-natural cell according to claim 14, wherein the binding
domain of the protein heterocomplex specifically binds to a target
located on a target cell surface.
16. The non-natural cell according to any one of the preceding
claims, wherein the hydrophobic domain is a transmembrane
domain.
17. The non-natural cell according to any one of the preceding
claims, wherein the transmembrane domain is a CD4, CD8 or CD28
transmembrane domain.
18. The non-natural cell according to any one of the preceding
claims, wherein the actuator domain comprises a lymphocyte receptor
signaling domain.
19. The non-natural cell according to any one of the preceding
claims, wherein the actuator domain comprises one or a plurality of
immunoreceptor tyrosine-based activation motifs (ITAMs).
20. The non-natural cell according to any one of the preceding
claims, wherein the actuator domain comprises CD3.epsilon.,
CD3.delta., CD3.zeta., pT.alpha., TCR.alpha., TCR.beta.,
FcR.alpha., FcR.beta., FcR.gamma., NKG2D, CD22, CD79A, or CD79B, or
any combination thereof.
21. The non-natural cell according to any one of the preceding
claims, wherein the first nucleic acid molecule encodes the first
fusion protein further comprising a different actuator domain, a
costimulatory domain, an adhesion factor, or any combination
thereof.
22. The non-natural cell according to claim 18, wherein the
costimulatory domain is selected from CD27, CD28, CD30, CD40, LAT,
Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn,
Slp76, TRIM, OX40, or any combination thereof.
23. The non-natural cell according to any one of the preceding
claims, wherein the actuator domain comprises a cytoplasmic portion
that associates with a cytoplasmic signaling protein.
24. The non-natural cell according to claim 23, wherein the
cytoplasmic signaling protein is a lymphocyte receptor or signaling
domain thereof, a protein comprising a plurality of immunoreceptor
tyrosine-based activation motifs (ITAMs), a costimulatory domain,
an adhesion factor, or any combination thereof.
25. The non-natural cell according to claim 24, wherein the
lymphocyte receptor or signaling domain thereof is CD3.epsilon.,
CD3.delta., CD3.zeta., pT.alpha., TCR.alpha., TCR.beta.,
FcR.alpha., FcR.beta., FcR.gamma., NKG2D, CD22, CD79A, or CD79B, or
any combination thereof.
26. The non-natural cell according to claim 24, wherein the
costimulatory domain is selected from CD27, CD28, CD30, CD40, LAT,
Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn,
Slp76, TRIM, OX40, or any combination thereof.
27. The non-natural cell according to any one of the preceding
claims, further overexpressing a costimulatory factor, an
immunomodulatoy factor, an agonist for a costimulatory factor, an
agonist for an immunomodulatoy factor, or any combination
thereof.
28. The non-natural cell according to any one of the preceding
claims, wherein the second nucleic acid molecule further encodes a
secretion signal such that the second fusion protein is secreted
from the non-natural cell when expressed, and optionally further
encodes an anchor domain.
29. The non-natural cell according to any one of the preceding
claims, wherein the binding domain of the second fusion protein is
a single chain antibody variable region, a receptor ectodomain, or
a ligand.
30. The non-natural cell according to claim 29, wherein the single
chain antibody variable region is a domain antibody, sFv, scFv,
F(ab').sub.2, or Fab.
31. The non-natural cell according to any one of the preceding
claims, wherein the binding domain of the second fusion protein is
amino terminal to the multimerization domain.
32. The non-natural cell according to any one of the preceding
claims, wherein the binding domain of the second fusion protein is
carboxy terminal to the multimerization domain.
33. The non-natural cell according to any one of the preceding
claims, wherein the second nucleic acid molecule encoding the
second fusion protein further comprises a sequence encoding a
linker disposed between the binding domain and the second
multimerization domain.
34. The non-natural cell according to any one of the preceding
claims, wherein the cell further comprises a third nucleic acid
molecule encoding a third fusion protein comprising a binding
domain and a second multimerization domain, wherein the third
fusion protein localizes extracellularly when expressed.
35. The non-natural cell according to any one of the preceding
claims, wherein the fusion proteins comprising a binding domain
have one, two, three, or four binding domains.
36. The non-natural cell according to any one of the preceding
claims, wherein the one, two, three, or four binding domains are
specific for one target or up to four different targets.
37. The non-natural cell according to any one of the preceding
claims, wherein the binding domain is specific for a target that is
an antigen associated with a cancer, an inflammatory disease, an
autoimmune disease, or a graft versus host disease.
38. The non-natural cell according to claim 37, wherein the cancer
is a solid malignancy or a hematologic malignancy.
39. The non-natural cell according to claim 38, wherein the
hematologic malignancy associated antigen target is CD19, CD20,
CD22, CD33, or CD37.
40. The non-natural cell according to any one of the preceding
claims, wherein the binding domain specifically binds to a target
selected from a-folate receptor, .alpha..sub.v.beta..sub.6
integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33,
CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, DLL4,
EGP-2, EGP-40, CSPG4, EGFR, EGFR family including ErbB2 (HER2),
EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine
receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11R.alpha.,
IL13R-.alpha.2, KDR, .kappa. light chain, .lamda. light chain, LeY,
L1CAM, MAGE-A1, mesothelin, MHC presented peptides, MUC1, MUC16,
NCAM, NKG2D ligands, Notch1, Notch2/3, NY-ESO-1, PRAME, PSCA, PSMA,
Survivin, TAG-72, TEMs, TERT, VEGFR2, and ROR1.
41. The non-natural cell according to any one of the preceding
claims, wherein the first bridging factor is rapamycin or a rapalog
thereof, coumermycin or a derivative thereof, gibberellin or a
derivative thereof, ABA or a derivative thereof, methotrexate or a
derivative thereof, cyclosporin A or a derivative thereof, FKCsA or
a derivative thereof, or Tmp-SLF or a derivative thereof.
42. The non-natural cell according to claim 10, wherein the second
bridging factor is rapamycin or a rapalog thereof, coumermycin or a
derivative thereof, gibberellin or a derivative thereof, ABA or a
derivative thereof, methotrexate or a derivative thereof,
cyclosporin A or a derivative thereof, FKCsA or a derivative
thereof, or Tmp-SLF or a derivative thereof.
43. The non-natural cell according to any one of the preceding
claims, wherein the encoded first fusion protein comprises a first
multimerization domain of FRB T2098L, a transmembrane domain, a
costimulatory domain of 4-1BB, and actuator domain of CD3.zeta.;
wherein the second encoded fusion protein comprises a binding
domain of an scFv specific for CD19 and a second multimerization
domain of FKBP12; and wherein the first bridging factor that
promotes the formation of a polypeptide complex on the non-natural
cell surface is rapalog AP21967.
44. The non-natural cell according to claim 43, wherein the first
fusion protein has an amino acid sequence as set forth in SEQ ID
NO.:15 and the second fusion protein has an amino acid sequence as
set forth in SEQ ID NO.:1.
45. A method for treating a hyperproliferative, inflammatory,
autoimmune, or graft-versus-host disease, comprising: (a)
administering a recombinant cell comprising a first and a second
nucleic acid molecule, wherein the first nucleic acid molecule
encodes a first fusion protein comprising a first multimerization
domain, a hydrophobic domain, and an actuator domain, wherein the
first multimerization domain localizes extracellularly when the
first fusion protein is expressed, and the second nucleic acid
molecule encodes a second fusion protein comprising a binding
domain and a second multimerization domain, wherein the second
fusion protein localizes extracellularly when expressed; and (b)
administering a bridging factor, wherein the bridging factor
promotes the formation of a polypeptide complex on the recombinant
cell surface with the bridging factor associated with and disposed
between the multimerization domains of the first and second fusion
proteins; wherein the binding domain of the polypeptide complex
specifically binds a cell surface target on a hyperproliferative,
inflammatory, autoimmune, or graft-versus-host disease cell to
promote an immunomodulatory response and thereby treats the
hyperproliferative, inflammatory, autoimmune, or graft-versus-host
disease.
46. A method for treating a hyperproliferative, inflammatory,
autoimmune, or graft-versus-host disease, comprising: (a)
administering a non-natural cell comprising a first nucleic acid
molecule encoding a first fusion protein comprising a first
multimerization domain, a hydrophobic domain, and an actuator
domain, wherein the first multimerization domain localizes
extracellularly when the first fusion protein is expressed; (b)
administering a second fusion protein comprising a binding domain
and a second multimerization domain; and (c) administering a
bridging factor, wherein the bridging factor promotes the formation
of a polypeptide complex on the recombinant cell surface with the
bridging factor associated with and disposed between the
multimerization domains of the first and second fusion proteins;
wherein the binding domain of the polypeptide complex specifically
binds a cell surface target on a hyperproliferative, inflammatory,
autoimmune, or graft-versus-host disease cell to promote an
immunomodulatory response and thereby treats the
hyperproliferative, inflammatory, autoimmune, or graft-versus-host
disease.
47. The method according to claim 45 or 46, wherein the first and
second multimerization domains are the same or different.
48. The method according to any one of claims 45-47, wherein the
multimerization domains of the first and second fusion proteins
associate with a bridging factor selected from rapamycin or a
rapalog thereof, coumermycin or a derivative thereof, gibberellin
or a derivative thereof, abscisic acid (ABA) or a derivative
thereof, methotrexate or a derivative thereof, cyclosporin A or a
derivative thereof, FKCsA or a derivative thereof, trimethoprim
(Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or
any combination thereof.
49. The method according to any one of claims 45-48, wherein the
first and second multimerization domains are a pair selected from
FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and
bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1
and GAI, or variants thereof.
50. The method according to any one of claims 45-49, wherein the
first multimerization domain comprises a first FKBP polypeptide or
variant thereof, and the second multimerization domain comprises a
first FRB polypeptide or variant thereof.
51. The method according to any one of claims 45-50, wherein the
first multimerization domain comprises a first FRB polypeptide or
variant thereof, and the second multimerization domain comprises a
first FKBP polypeptide or variant thereof.
52. The method according to claim 50 or 51, wherein the bridging
factor is sirolimus, everolimus, novolimus, pimecrolimus,
ridaforolimus, tacrolimus, temsirolimus, umirolimus, or
zotarolimus.
53. The method according to any one of claims 45-52, wherein the
first nucleic acid molecule encodes a first fusion protein further
comprising a third multimerization domain.
54. The method according to claim 53, wherein the third
multimerization domain of the first fusion protein is a binding
domain for a bridging factor selected from rapamycin or a rapalog
thereof, coumermycin or a derivative thereof, gibberellin or a
derivative thereof, ABA or a derivative thereof, methotrexate or a
derivative thereof, cyclosporin A or a derivative thereof, FKCsA or
a derivative thereof, Tmp-SLF or a derivative thereof, or any
combination thereof.
55. The method according to any one of claims 45-54, wherein a
second bridging factor promotes the association of at least two
first fusion proteins with the bridging factor associated with and
disposed between the third multimerization domains of the first
fusion proteins.
56. The method according to any one of claims 45-55, wherein the
protein complex is a homocomplex comprising at least two first
fusion proteins.
57. The method according to any one of claims 45-56, wherein the
first fusion protein has at least one multimerization domain of
FKBP, DHFR or GyrB.
58. The method according to any one of claims 45-57, wherein the
binding domain of the polypeptide complex specifically binds to a
target located on a target hyperproliferative disease cell
surface.
59. The method according to claim 58, wherein the protein complex
is a heterocomplex comprising one or more first fusion proteins and
one or more second fusion proteins.
60. The method according to claim 59, wherein the binding domain of
the protein heterocomplex specifically binds to a target located on
a target hyperproliferative disease cell surface.
61. The method according to any one of claims 45-60, wherein the
hydrophobic domain is a transmembrane domain.
62. The method according to any one of claims 45-61, wherein the
transmembrane domain is a CD4, CD8 or CD28 transmembrane
domain.
63. The method according to any one of claims 45-62, wherein the
actuator domain comprises a lymphocyte receptor signaling
domain.
64. The method according to any one of claims 45-63, wherein the
actuator domain comprises a plurality of immunoreceptor
tyrosine-based activation motifs (ITAMs).
65. The method according to any one of claims 45-64, wherein the
actuator domain comprises CD3.epsilon., CD3.delta., CD3.zeta.,
pT.alpha., TCR.alpha., TCR.beta., FcR.alpha., FcR.beta.,
FcR.gamma., NKG2D, CD22, CD79A, or CD79B, or any combination
thereof.
66. The method according to any one of claims 45-65, wherein the
first nucleic acid molecule encodes the first fusion protein
further comprising a different actuator domain, a costimulatory
domain, an adhesion factor, or any combination thereof.
67. The method according to claim 66, wherein the costimulatory
domain is selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS,
DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM,
OX40, or any combination thereof.
68. The method according to any one of claims 45-67, wherein the
actuator domain comprises a cytoplasmic portion that associates
with a cytoplasmic signaling protein.
69. The method according to claim 68, wherein the cytoplasmic
signaling protein is a lymphocyte receptor or signaling domain
thereof, a protein comprising one or a plurality of immunoreceptor
tyrosine-based activation motifs (ITAMs), a costimulatory domain,
an adhesion factor, or any combination thereof.
70. The method according to claim 69, wherein the lymphocyte
receptor or signaling domain thereof is CD3.epsilon., CD3,.delta.,
CD3.zeta., pT.alpha., TCR.alpha., TCR.beta., FcR.alpha., FcR.beta.,
FcR.gamma.NKG2D, CD22, CD79A, or CD79B, or any combination
thereof.
71. The method according to claim 69, wherein the costimulatory
domain is selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS,
DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM,
OX40, or any combination thereof.
72. The method according to claim 69, wherein the cytoplasmic
signaling protein is combination of CD3.zeta. and 4-1BB or a
combination of CD3t and OX40.
73. The method according to any one of claims 45-72, wherein the
non-natural cell is further overexpressing a costimulatory factor,
an immunomodulatoy factor, an agonist for a costimulatory factor,
an agonist for an immunomodulatoy factor, or any combination
thereof.
74. The method according to any one of claims 45-73, wherein the
binding domain of the second fusion protein is a single chain
antibody variable region, a receptor ectodomain, or a ligand.
75. The method according to claim 74, wherein the single chain
antibody variable region is a domain antibody, sFv, scFv, F(ab')2,
or Fab.
76. The method according to any one of claims 45-75, wherein the
binding domain of the second fusion protein is amino terminal to
the multimerization domain.
77. The method according to any one of claims 45-76, wherein the
binding domain of the second fusion protein is carboxy terminal to
the multimerization domain.
78. The method according to any one of claims 45-77, wherein the
second fusion protein further comprises a linker disposed between
the binding domain and the second multimerization domain.
79. The method according to any one of claims 45-78, wherein the
cell further comprises a third nucleic acid molecule encoding a
third fusion protein comprising a binding domain and a second
multimerization domain, wherein the third fusion protein localizes
extracellularly when expressed.
80. The method according to any one of claims 45-76, wherein the
fusion proteins comprising a binding domain have one, two, three,
or four binding domains.
81. The method according to any one of claims 45-80, wherein the
one, two, three, or four binding domains are specific for one
target or up to four different targets.
82. The method according to any one of claims 45-81, wherein the
binding domain is specific for a target that is an antigen
associated with a cancer.
83. The method according to claim 82, wherein the cancer is a solid
malignancy or a hematologic malignancy.
84. The method according to claim 83, wherein the hematologic
malignancy associated antigen target is CD19, CD20, CD22, CD33, or
CD37.
85. The method according to any one of claims 45-84, wherein the
binding domain specifically binds to a target selected from
.alpha.-folate receptor, .alpha..sub.v.beta..sub.6 integrin, BCMA,
B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44,
CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2,
EGP-40, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII,
EPCAM, EphA2, EpCAM, FAP, FBP, fetal acetylcholine receptor, Fzd7,
GD2, GD3, Glypican-3 (GPC3), h5T4, IL-11R.alpha., IL13R-.alpha.2,
KDR, .kappa. light chain, .lamda. light chain, LeY, L1CAM, MAGE-A1,
mesothelin, MHC presented peptides, MUC1, MUC16, NCAM, NKG2D
ligands, Notchl, Notch2/3, NY-ESO-1, PRAME, PSCA, PSMA, Survivin,
TAG-72, TEMs, TERT, VEGFR2, and ROR1.
86. The method according to any one of claims 45-85, wherein the
first bridging factor is rapamycin or a rapalog thereof,
coumermycin or a derivative thereof, gibberellin or a derivative
thereof, ABA or a derivative thereof, methotrexate or a derivative
thereof, cyclosporin A or a derivative thereof, FKCsA or a
derivative thereof, or Tmp-SLF or a derivative thereof.
87. The method according to claim 55, wherein the second bridging
factor is rapamycin or a rapalog thereof, coumermycin or a
derivative thereof, gibberellin or a derivative thereof, ABA or a
derivative thereof, methotrexate or a derivative thereof,
cyclosporin A or a derivative thereof, FKCsA or a derivative
thereof, or Tmp-SLF or a derivative thereof.
88. The method according to any one of claims 45-87, wherein the
first fusion protein comprises a first multimerization domain of
FRB T2098L, a transmembrane domain, a costimulatory domain of
4-1BB, and actuator domain of CD3.zeta.;wherein the second fusion
protein comprises a binding domain of an scFv specific for CD19 and
a second multimerization domain of FKBP12; and wherein the first
bridging factor that promotes the formation of a polypeptide
complex on the non-natural cell surface is rapalog AP21967.
89. The method according to claim 88, wherein the first fusion
protein has an amino acid sequence as set forth in SEQ ID NO.:15
and the second fusion protein has an amino acid sequence as set
forth in SEQ ID NO.:1.
90. The method according to any one of claims 45-89, wherein the
method further comprises administering an agent that antagonizes or
blocks an inhibitor of T-cell activation.
91. The method according to claim 90, wherein the agent antagonizes
or blocks a T-cell ligand.
92. The method according to claim 90, wherein the agent antagonizes
or blocks a T-cell receptor.
93. The method according to any one of claims 90-92, wherein the
agent that antagonizes or blocks an inhibitor of T-cell activation
is an anti-PD1 antibody or antigen binding fragment thereof,
anti-PD-L1 antibody or antigen binding fragment thereof, or an
anti-CTLA4 antibody or antigen binding fragment thereof or an
engineered homing endonuclease that targets PD-1.
94. The method according to any one of claims 45-93, wherein the
method further comprises administering a cytokine agonist.
95. A fusion polypeptide heterocomplex, comprising: (a) a first
fusion protein comprising a first multimerization domain, a
hydrophobic domain, and an actuator domain; (b) a second fusion
protein comprising an extracellular binding domain and second
multimerization domain; and (c) a bridging factor; wherein the
first fusion protein, second fusion protein, and bridging factor
associate to form a polypeptide heterocomplex with the bridging
factor associated with and disposed between the multimerization
domains of the first and second fusion proteins.
96. The polypeptide heterocomplex according to claim 95, wherein
the binding domain is a single chain antibody variable region, a
receptor ectodomain, or a ligand.
97. The polypeptide heterocomplex according to claim 96, wherein
the single chain antibody variable region is a domain antibody,
sFv, scFv, F(ab').sub.2, or Fab.
98. The polypeptide heterocomplex according to any one of claims
95-97, wherein the binding domain is amino terminal to the
multimerization domain.
99. The polypeptide heterocomplex according to any one of claims
95-97, wherein the binding domain is carboxy terminal to the
multimerization domain.
100. The polypeptide heterocomplex according to any one of claims
95-99, wherein the first multimerization domain comprises a first
FKBP polypeptide or variant thereof, and the second multimerization
domain comprises a first FRB polypeptide or variant thereof.
101. The polypeptide heterocomplex according to any one of claims
95-99, wherein the first multimerization domain comprises a first
FRB polypeptide or variant thereof, and the second multimerization
domain comprises a first FKBP polypeptide or variant thereof.
102. The polypeptide heterocomplex according to any one of claims
95-101, wherein the hydrophobic domain is a transmembrane
domain.
103. The polypeptide heterocomplex according to any one of claims
95-102, wherein the actuator domain comprises a lymphocyte receptor
chain.
104. The polypeptide heterocomplex according to any one of claims
95-103, wherein the bridging factor is rapamycin or a rapalog
thereof, coumermycin or a derivative thereof, gibberellin or a
derivative thereof, ABA or a derivative thereof, methotrexate or a
derivative thereof, cyclosporin A or a derivative thereof, FKCsA or
a derivative thereof, or Tmp-SLF or a derivative thereof.
105. The polypeptide heterocomplex according to any one of claims
95-104, wherein the second fusion protein further comprises an
anchor domain.
106. The polypeptide heterocomplex according to claim 105, wherein
the anchor domain is a transmembrane domain.
107. The polypeptide heterocomplex according to claim 105 or 106,
wherein the second fusion protein further comprises a sub-threshold
signaling domain.
108. The polypeptide heterocomplex according to claim 105, wherein
the anchor domain is a GPI signal sequence.
109. The polypeptide heterocomplex according to claim 105, wherein
the GPI signal sequence has been altered and the second fusion
protein further comprises a GPI molecule.
110. The polypeptide heterocomplex according to any one of claims
95-109, wherein the binding domain is specific for a target that is
an antigen associated with a cancer, an inflammatory disease, an
autoimmune disease, or a graft versus host disease.
111. The polypeptide heterocomplex according to claim 110, wherein
the cancer is a hematologic malignancy having an antigen target of
CD19, CD20, CD22, CD33, or CD37.
112. A nucleic acid molecule encoding any one or more of the fusion
proteins according to any one of claim 1-44 or 95-109.
113. The nucleic acid molecule of claim 112, wherein the nucleic
acid molecule is disposed between 5' and 3' polynucleotide
sequences homologous to a genomic locus.
114. An expression vector containing a nucleic acid according to
claim 112 or claim 113.
115. The expression vector according to claim 114, wherein the
first and second fusion proteins are encoded as a polycistronic
message or as a single protein separated by a 2A peptide.
116. The expression vector according to claim 115, wherein the
polycistronic message comprises an internal ribosome entry site
(IRES) between the nucleotide sequences that encode the fusion
proteins.
117. A non-natural cell, comprising: (a) a first nucleic acid
molecule encoding a first fusion protein comprising a binding
domain that binds a receptor on a T cell and a first
multimerization domain, wherein the first fusion protein is
secreted from the cell; and (b) a second nucleic acid molecule
encoding a second fusion protein comprising a binding domain that
binds a target located on a target cell surface and a second
multimerization domain, wherein the second fusion protein is
secreted from the cell; wherein a bridging factor promotes the
formation of a polypeptide complex with the bridging factor
associated with and disposed between the multimerization domains of
the first and second fusion proteins.
118. The non-natural cell according to claim 117, wherein the first
and second multimerization domains are the same or different.
119. The non-natural cell according to claim 117 or claim 118,
wherein the multimerization domains of the first and second fusion
proteins associate with a bridging factor selected from rapamycin
or a rapalog thereof, coumermycin or a derivative thereof,
gibberellin or a derivative thereof, abscisic acid (ABA) or a
derivative thereof, methotrexate or a derivative thereof,
cyclosporin A or a derivative thereof, FKCsA or a derivative
thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a
derivative thereof, or any combination thereof.
120. The non-natural cell according to any one of claims 117-119,
wherein the first and second multimerization domains are a pair
selected from FKBP and FRB, FKBP and calcineurin, FKBP and
cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin,
PYL1 and ABI1, or GIB1 and GAI, or variants thereof.
121. The non-natural cell according to any one of claims 117-120,
wherein the first multimerization domain comprises a first FKBP
polypeptide or variant thereof, and the second multimerization
domain comprises a first FRB polypeptide or variant thereof.
122. The non-natural cell according to any one of claims 117-120,
wherein the first multimerization domain comprises a first FRB
polypeptide or variant thereof, and the second multimerization
domain comprises a first FKBP polypeptide or variant thereof.
123. The non-natural cell according to claim 121 or claim 122,
wherein the bridging factor is sirolimus, everolimus, novolimus,
pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus,
or zotarolimus.
124. The non-natural cell according to any one of claims 117-123,
further overexpressing a costimulatory factor, an immunomodulatoy
factor, an agonist for a costimulatory factor, an agonist for an
immunomodulatoy factor, or any combination thereof.
125. The non-natural cell according to any one of claims 117-124,
wherein the binding domain of the first fusion protein and the
binding domain of the second fusion protein are each independently
selected from the group consisting of: a single chain antibody
variable region, a receptor ectodomain, or a ligand.
126. The non-natural cell according to claim 125, wherein the
single chain antibody variable region is a domain antibody, sFv,
scFv, F(ab').sub.2, or Fab.
127. The non-natural cell according to any one of claims 117-126,
wherein the binding domain of the first fusion protein is amino
terminal to the first multimerization domain.
128. The non-natural cell according to any one of claims 117-126,
wherein the binding domain of the first fusion protein is carboxy
terminal to the first multimerization domain.
129. The non-natural cell according to any one of claims 117-126,
wherein the binding domain of the second fusion protein is amino
terminal to the second multimerization domain.
130. The non-natural cell according to any one of claims 117-126,
wherein the binding domain of the second fusion protein is carboxy
terminal to the second multimerization domain.
131. The non-natural cell according to any one of claims 117-130,
wherein the first nucleic acid molecule encoding the first fusion
protein further comprises a sequence encoding a linker disposed
between the binding domain and the first multimerization
domain.
132. The non-natural cell according to any one of claims 117-130,
wherein the second nucleic acid molecule encoding the second fusion
protein further comprises a sequence encoding a linker disposed
between the binding domain and the second multimerization
domain.
133. The non-natural cell according to any one of claims 117-132,
wherein the binding domain of the second nucleic acid molecule is
specific for a target that is an antigen associated with a cancer,
an inflammatory disease, an autoimmune disease, or a graft versus
host disease.
134. The non-natural cell according to claim 133, wherein the
cancer is a solid malignancy or a hematologic malignancy.
135. The non-natural cell according to claim 134, wherein the
hematologic malignancy associated antigen target is CD19, CD20,
CD22, CD33, or CD37.
136. The non-natural cell according to any one of claims 117-132,
wherein the binding domain of the second nucleic acid molecule
specifically binds to a target selected from a-folate receptor,
.alpha..sub.v.beta..sub.6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19,
CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123,
CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family
including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP,
fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3),
h5T4, IL-11R.alpha., IL13R-.alpha.2, KDR, .kappa. light chain,
.lamda. light chain, LeY, L1CAM, MAGE-A1, mesothelin, MHC presented
peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notchl, Notch2/3,
NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2,
and ROR1.
137. The non-natural cell according to any one of claims 117-136,
wherein the bridging factor is rapamycin or a rapalog thereof,
coumermycin or a derivative thereof, gibberellin or a derivative
thereof, ABA or a derivative thereof, methotrexate or a derivative
thereof, cyclosporin A or a derivative thereof, FKCsA or a
derivative thereof, or Tmp-SLF or a derivative thereof.
138. The non-natural cell according to any one of claims 117-137,
wherein the first nucleic acid encodes a first fusion protein
comprising a binding domain of an scFv specific for CD3 and a first
multimerization domain of FRB T2098L; wherein the second nucleic
acid encodes a second fusion protein comprising a binding domain of
an scFv specific for CD19 and a second multimerization domain of
FKBP12; and wherein the bridging factor that promotes the formation
of a polypeptide complex is rapalog AP21967.
139. The non-natural cell according to any one of claims 117-137,
wherein the first nucleic acid encodes a first fusion protein
comprising a binding domain of an scFv specific for CD3 and a first
multimerization domain of FRB T2098L; wherein the second nucleic
acid encodes a second fusion protein comprising a binding domain of
an scFv specific for BCMA and a second multimerization domain of
FKBP12; and wherein the bridging factor that promotes the formation
of a polypeptide complex is rapalog AP21967.
140. A method for treating a hyperproliferative, inflammatory,
autoimmune, or graft-versus-host disease, comprising administering
a non-natural cell according to any one of claims 117-139 and
administering a bridging factor, wherein the bridging factor
promotes the formation of a polypeptide complex with the bridging
factor associated with and disposed between the multimerization
domains of the first and second fusion proteins; wherein the
binding domain of the second fusion polypeptide specifically binds
a cell surface target on a hyperproliferative disease cell to
promote an immunomodulatory response and thereby treats the
hyperproliferative disease.
141. A method for treating a hyperproliferative, inflammatory,
autoimmune, or graft-versus-host disease, comprising: (a)
administering a first fusion protein comprising a binding domain
that binds a receptor on a T cell and a first multimerization
domain; and a second fusion protein comprising a binding domain
that binds a cell surface target on a hyperproliferative,
inflammatory, autoimmune, or graft-versus-host disease cell and a
second multimerization domain; and (b) administering a bridging
factor that promotes the formation of a polypeptide complex with
the bridging factor associated with and disposed between the
multimerization domains of the first and second fusion proteins;
thereby treating the hyperproliferative, inflammatory, autoimmune,
or graft-versus-host disease.
142. A fusion polypeptide heterocomplex, comprising: (a) a first
fusion protein comprising a binding domain that binds a receptor on
a T cell and a first multimerization domain; (b) a second fusion
protein comprising a binding domain that binds a cell surface
target on a target cell; and (c) a bridging factor; wherein the
first fusion protein, second fusion protein, and bridging factor
associate to form a polypeptide heterocomplex with the bridging
factor associated with and disposed between the multimerization
domains of the first and second fusion proteins.
143. The fusion polypeptide heterocomplex according to claim 142,
wherein the first and second multimerization domains are the same
or different.
144. The fusion polypeptide heterocomplex according to claim 142 or
claim 143, wherein the multimerization domains of the first and
second fusion proteins associate with a bridging factor selected
from rapamycin or a rapalog thereof, coumermycin or a derivative
thereof, gibberellin or a derivative thereof, abscisic acid (ABA)
or a derivative thereof, methotrexate or a derivative thereof,
cyclosporin A or a derivative thereof, FKCsA or a derivative
thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a
derivative thereof, or any combination thereof.
145. The fusion polypeptide heterocomplex according to any one of
claims 142-144, wherein the first and second multimerization
domains are a pair selected from FKBP and FRB, FKBP and
calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR,
calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or
variants thereof.
146. The fusion polypeptide heterocomplex according to any one of
claims 142-145, wherein the first multimerization domain comprises
a first FKBP polypeptide or variant thereof, and the second
multimerization domain comprises a first FRB polypeptide or variant
thereof.
147. The fusion polypeptide heterocomplex according to any one of
claims 142-145, wherein the first multimerization domain comprises
a first FRB polypeptide or variant thereof, and the second
multimerization domain comprises a first FKBP polypeptide or
variant thereof.
148. The fusion polypeptide heterocomplex according to any one of
claims 146 or claim 147, wherein the bridging factor is sirolimus,
everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,
temsirolimus, umirolimus, or zotarolimus.
149. The fusion polypeptide heterocomplex according to any one of
claims 142-148, wherein the binding domain of the first fusion
protein and the binding domain of the second fusion protein are
each independently selected from the group consisting of: a single
chain antibody variable region, a receptor ectodomain, or a
ligand.
150. The fusion polypeptide heterocomplex according to claim 149,
wherein the single chain antibody variable region is a domain
antibody, sFv, scFv, F(ab').sub.2, or Fab.
151. The fusion polypeptide heterocomplex according to any one of
claims 142-150, wherein the binding domain of the second fusion
polypeptide specifically binds to a target selected from a-folate
receptor, 46 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22,
CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138,
CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family including
ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP, fetal
acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3), h5T4,
IL-11R.alpha., IL13R-.alpha.2, KDR, .kappa. light chain, .lamda.
light chain, LeY, L1CAM, MAGE-A1, mesothelin, MHC presented
peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notchl, Notch2/3,
NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2,
and ROR1.
152. The fusion polypeptide heterocomplex according to any one of
claims 141-150, wherein the first fusion protein comprises a
binding domain of an scFv specific for CD3 and a first
multimerization domain of FRB T2098L; wherein the second fusion
protein comprises a binding domain of an scFv specific for CD19 and
a second multimerization domain of FKBP12; and wherein the bridging
factor is rapalog AP21967.
153. The fusion polypeptide heterocomplex according to any one of
claims 141-150, wherein the first fusion protein comprises a
binding domain of an scFv specific for CD3 and a first
multimerization domain of FRB T2098L; wherein the second fusion
protein comprises a binding domain of an scFv specific for BCMA and
a second multimerization domain of FKBP12; and wherein the bridging
factor is rapalog AP21967.
154. A nucleic acid molecule encoding any one or more of the fusion
proteins according to any one of claims 117-153.
155. The nucleic acid molecule of claim 154, wherein the nucleic
acid molecule is disposed between 5' and 3' polynucleotide
sequences homologous to a genomic locus.
156. An expression vector containing a nucleic acid according to
claim 154 or claim 155.
157. The expression vector according to claim 156, wherein the
first and second fusion proteins are encoded as a polycistronic
message or as a single protein separated by a 2A peptide.
158. The expression vector according to claim 157, wherein the
polycistronic message comprises an internal ribosome entry site
(IRES) between the nucleotide sequences that encode the fusion
proteins.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/608,098, filed Jan. 28, 2015, which is a
continuation-in-part of PCT/US2014/047852, filed Jul. 23, 2014,
which in turn claims the benefit of U.S. Provisional Application
No. 61/934,092, filed Jan. 31, 2014, and U.S. Provisional
Application No. 61/859,697, filed Jul. 29, 2013, each of which is
incorporated by reference in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is BLBD 036 05US
ST25.txt. The text file is about 634 KB, was created on Aug. 13,
2019, and is being submitted electronically via EFS-Web.
BACKGROUND
Technical Field
[0003] The present disclosure relates to compositions and methods
for using multi-component proteins in immunotherapy and, more
particularly, using chemically induced multimerization to generate
chimeric antigen receptor proteins for modulating spatial and
temporal control of cellular signal initiation and downstream
responses during adoptive immunotherapy.
Description of the Related Art
[0004] Cellular therapy is emerging as a powerful paradigm for
delivering complex signals for biological action. In contrast to
small molecule and biologic drug compositions, cells have the
potential to execute unique therapeutic tasks owing to their myriad
sensory and response programs and increasingly defined mechanisms
of genetic control. To achieve such therapeutic value, cells need
to be outfitted with machinery for sensing and integrating chemical
and/or biological information associated with local physiological
environments.
[0005] The most clinically advanced example of engineered
sensory-response machinery is chimeric antigen receptors (CARs) in
genetically engineered T cells for use in adoptive cellular
immunotherapy (see June et al., Nat. Biotechnol. 30:611, 2012;
Restifo et al., Nat. Rev. Immunol. 12:269, 2012). Antigen binding
stimulates the signaling domains on the intracellular segment of
the CAR, thereby transducing signals that unleash inflammatory and
cytotoxicity mechanisms. CAR-based adoptive cellular immunotherapy
has been used to treat cancer patients with tumors refractory to
conventional standard-of-care treatments (see Grupp et al., N.
Engl. J. Med. 368:1509, 2013; Kalos et al., Sci. Transl. Med.
3:95ra73, 2011).
[0006] In addition to targeting and initiating T cell activation,
an effective adoptive cellular immunotherapy would preferably also
modulate T cell expansion and persistence, as well as the strength
and quality of T cell signaling. But, current CAR-mediated T cell
responses do not realize the full potential of T cell activation
and proliferation. Improvement of CAR function has been achieved by
including costimulatory signaling domains into the CAR structure
(see, e.g., Kowolik et al., Cancer Res. 66:10995, 2006; Milone et
al., Mol. Ther. 17:1453, 2009; Pule et al., Mol. Ther. 12:933,
2005; Carpenito et al., Proc. Nat'l Acad. Sci. U.S.A. 106:3360,
2009), but the clinical results have been mixed (see, e.g.,
Brentjens et al., Blood 118:4817, 2011; Till et al., Blood
119:3940, 2012; Kochenderfer and Rosenberg, Nat. Rev. Clin. Oncol.
10:267, 2013). Others have included, in addition to a CAR,
co-expression of costimulatory ligands (see, e.g., Stephan et al.,
Nat. Med. 13:1440, 2007), costimulatory receptors (see, e.g., Duong
et al., Immunother. 3:33, 2011; Wilkie et al., J. Clin. Immunol.
32:1059, 2012), and cytokines (see, e.g., Hsu et al., J. Immunol.
175:7226, 2005; Quintarelli et al., Blood 110:2793, 2007).
[0007] A concern with the use of CARs is toxicity, which arises in
two forms: one is the targeted destruction of normal tissue and the
second is cytokine-release associated adverse events (e.g.,
cytokine storm). For example, collateral damage observed with
CD19-targeted CARs is B-cell aplasia (Kalos et al., 2011;
Kochenderfer et al., Blood 119:2709, 2012). Such off-target effects
could be very dangerous, particularly if the target antigen is
found on other tissues, such as the heart or lung. The cytokine
storms associated with large numbers of activated T cells can be
life threatening (Kalos et al., 2011; Kochenderfer et al., 2012).
Unlike conventional drug treatments where reducing drug dosage can
control toxicity, the proliferation of T cells cannot be controlled
with current CAR technologies and, therefore, immunopathology will
result once a threshold level of T cells is reached.
[0008] In view of the limitations associated with CAR-mediated T
cell responses, there is a need in the art for alternative
compositions and methods useful for immunotherapy in which
modulation of immune cell signal initiation and expansion is
controllable. The present disclosure meets such needs, and further
provides other related advantages.
SUMMARY OF THE INVENTION
[0009] The present disclosure describes improved chimeric antigen
receptor signaling complexes and non-natural cell compositions
having signal transduction systems that are controlled--both in
their activation and deactivation--by pharmacological agents.
Numerous pharmacologically controlled, multipartite signal
transduction systems are contemplated herein.
[0010] In various embodiments, the present invention contemplates,
in part, a non-natural cell, comprising: a first nucleic acid
molecule encoding a first fusion protein comprising a first
multimerization domain, a first hydrophobic domain, and an actuator
domain, wherein the first multimerization domain localizes
extracellularly when the first fusion protein is expressed; and a
second nucleic acid molecule encoding a second fusion protein
comprising a binding domain and a second multimerization domain,
and a second hydrophobic domain; wherein a first bridging factor
promotes the formation of a polypeptide complex on the non-natural
cell surface with the bridging factor associated with and disposed
between the multimerization domains of the first and second fusion
proteins.
[0011] In particular embodiments, the first and second
multimerization domains are the same or different.
[0012] In additional embodiments, the multimerization domains of
the first and second fusion proteins associate with a bridging
factor selected from rapamycin or a rapalog thereof, coumermycin or
a derivative thereof, gibberellin or a derivative thereof, abscisic
acid (ABA) or a derivative thereof, methotrexate or a derivative
thereof, cyclosporin A or a derivative thereof, FKCsA or a
derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP
(SLF) or a derivative thereof, or any combination thereof.
[0013] In certain embodiments, the first and second multimerization
domains are a pair selected from FKBP and FRB, FKBP and
calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR,
calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or
variants thereof.
[0014] In certain embodiments, the first multimerization domain
comprises a first FKBP polypeptide or variant thereof, and the
second multimerization domain comprises a first FRB polypeptide or
variant thereof.
[0015] In further embodiments, the first multimerization domain
comprises a first FRB polypeptide or variant thereof, and the
second multimerization domain comprises a first FKBP polypeptide or
variant thereof.
[0016] In some embodiments, the bridging factor is sirolimus,
everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,
temsirolimus, umirolimus, or zotarolimus.
[0017] In additional embodiments, the first fusion protein has at
least one multimerization domain of FKBP, DHFR or GyrB.
[0018] In particular embodiments, the binding domain of the
polypeptide complex specifically binds to a target located on a
target cell surface.
[0019] In particular embodiments, the first hydrophobic domain is a
transmembrane domain selected from the group of a CD4, CD8, AMN, or
CD28 transmembrane domain.
[0020] In some embodiments, the second hydrophobic domain comprises
a CD154 transmembrane domain.
[0021] In certain embodiments, the second hydrophobic domain
comprises a CD71 transmembrane domain.
[0022] In particular embodiments, a particular transmembrane domain
may be included in the first or second fusion proteins as a type I
or type II transmembrane domain.
[0023] In further embodiments, the first hydrophobic domain and the
second hydrophobic domain do not increase cytotoxic activity of the
non-natural cell in the absence of the bridging factor.
[0024] In additional embodiments, the first hydrophobic domain and
the second hydrophobic domain increase cytotoxic activity of the
non-natural cell in the absence of the bridging factor, wherein the
increase in cytotoxic activity is less than the cytotoxic activity
in the presence of the bridging factor.
[0025] In certain embodiments, the actuator domain comprises a
lymphocyte receptor signaling domain.
[0026] In additional embodiments, the actuator domain comprises one
or a plurality of immunoreceptor tyrosine-based activation motifs
(ITAMs).
[0027] In some embodiments, the actuator domain comprises
CD3.epsilon., CD3.delta., CD3.zeta., pT.alpha., TCR.alpha.,
TCR.beta., FcR.alpha., FcR.beta., FcR.gamma., NKG2D, CD22, CD79A,
or CD79B, or any combination thereof.
[0028] In particular embodiments, the first nucleic acid molecule
encodes the first fusion protein further comprising a different
actuator domain, a costimulatory domain, an adhesion factor, or any
combination thereof.
[0029] In further embodiments, the costimulatory domain is selected
from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB,
CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any
combination thereof.
[0030] In additional embodiments, the actuator domain comprises a
cytoplasmic portion that associates with a cytoplasmic signaling
protein.
[0031] In some embodiments, the cytoplasmic signaling protein is a
lymphocyte receptor or signaling domain thereof, a protein
comprising a plurality of immunoreceptor tyrosine-based activation
motifs (ITAMs), a costimulatory domain, an adhesion factor, or any
combination thereof.
[0032] In particular embodiments, the lymphocyte receptor or
signaling domain thereof is CD3.epsilon., CD3.delta., CD3.zeta.,
pT.alpha., TCR.alpha., TCR.beta., FcR.alpha., FcR.beta.,
FcR.gamma., NKG2D, CD22, CD79A, or CD79B, or any combination
thereof.
[0033] In particular embodiments, the costimulatory domain is
selected from CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10,
4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or
any combination thereof.
[0034] In further embodiments, the non-natural cell further
overexpresses a costimulatory factor, an immunomodulatory factor,
an agonist for a costimulatory factor, an agonist for an
immunomodulatory factor, or any combination thereof.
[0035] In certain embodiments, the second nucleic acid molecule
further encodes a secretion signal such that the second fusion
protein is secreted from the non-natural cell when expressed, and
optionally further encodes an anchor domain.
[0036] In additional embodiments, the binding domain of the second
fusion protein is a single chain antibody variable region, a
receptor ectodomain, or a ligand.
[0037] In particular embodiments, the single chain antibody
variable region is a domain antibody, sFv, scFv, F(ab').sub.2, or
Fab.
[0038] In some embodiments, the binding domain of the second fusion
protein is amino terminal to the multimerization domain.
[0039] In additional embodiments, the binding domain of the second
fusion protein is carboxy terminal to the multimerization
domain.
[0040] In further embodiments, the second nucleic acid molecule
encoding the second fusion protein further comprises a sequence
encoding a linker disposed between the binding domain and the
second multimerization domain.
[0041] In particular embodiments, the fusion proteins comprising a
binding domain have one, two, three, or four binding domains.
[0042] In certain embodiments, the one, two, three, or four binding
domains are specific for one target or up to four different
targets.
[0043] In certain embodiments, the binding domain is specific for a
target that is an antigen associated with a cancer, an inflammatory
disease, an autoimmune disease, or a graft versus host disease.
[0044] In additional embodiments, the cancer is a solid malignancy
or a hematologic malignancy.
[0045] In particular embodiments, the hematologic malignancy
associated antigen target is CD19, CD20, CD22, CD33, or CD37.
[0046] In some embodiments, the binding domain specifically binds
to a target selected from .alpha.-folate receptor,
.alpha..sub.v.beta..sub.6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19,
CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70, CD123,
CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR, EGFR family
including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM, FAP, FBP,
fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3 (GPC3),
h5T4, IL-11R.alpha., IL13R-.alpha.2, KDR, .kappa. light chain,
.lamda. light chain, LeY, L1CAM, MAGE-A1 mesothelin, MHC presented
peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notch 1, Notch2/3,
NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs, TERT, VEGFR2,
and ROR1.
[0047] In further embodiments, the encoded first fusion protein
comprises a first multimerization domain of FRB T2098L, a
transmembrane domain, a costimulatory domain of 4-1BB, and actuator
domain of CD3.zeta.;wherein the second encoded fusion protein
comprises a binding domain of an scFv specific for CD19 and a
second multimerization domain of FKBP12 and a CD154 or a CD71
transmembrane domain; and wherein the first bridging factor that
promotes the formation of a polypeptide complex on the non-natural
cell surface is rapalog AP21967.
[0048] In particular embodiments, the encoded first fusion protein
comprises a first multimerization domain of FRB, a transmembrane
domain, a costimulatory domain of 4-1BB, and actuator domain of
CD3.zeta.; wherein the second encoded fusion protein comprises a
binding domain of an scFv specific for CD19 and a second
multimerization domain of FKBP12 and a CD154 or a CD71
transmembrane domain; and wherein the first bridging factor that
promotes the formation of a polypeptide complex on the non-natural
cell surface is Rapamycin, temsirolimus or everolimus.
[0049] In various embodiments, the present invention contemplates,
in part, a method for treating a hyperproliferative, inflammatory,
autoimmune, or graft-versus-host disease, comprising: administering
a non-natural cell according to any one of embodiments contemplated
herein; and administering a bridging factor, wherein the bridging
factor promotes the formation of a polypeptide complex on the
recombinant cell surface with the bridging factor associated with
and disposed between the multimerization domains of the first and
second fusion proteins; wherein the binding domain of the
polypeptide complex specifically binds a cell surface target on a
hyperproliferative, inflammatory, autoimmune, or graft-versus-host
disease cell to promote an immunomodulatory response and thereby
treats the hyperproliferative, inflammatory, autoimmune, or
graft-versus-host disease.
[0050] In certain embodiments, the method further comprises
administering an agent that antagonizes or blocks an inhibitor of
T-cell activation.
[0051] In additional embodiments, the agent antagonizes or blocks a
T-cell ligand.
[0052] In particular embodiments, the agent antagonizes or blocks a
T-cell receptor.
[0053] In particular embodiments, the agent that antagonizes or
blocks an inhibitor of T-cell activation is an anti-PD1 antibody or
antigen binding fragment thereof, anti-PD-L1 antibody or antigen
binding fragment thereof, or an anti-CTLA4 antibody or antigen
binding fragment thereof or an engineered homing endonuclease that
targets PD-1.
[0054] In some embodiments, the method further comprises
administering a cytokine agonist.
[0055] In various embodiments, the present invention contemplates,
in part, a fusion polypeptide heterocomplex, comprising: a first
fusion protein comprising a first multimerization domain, a first
hydrophobic domain, and an actuator domain; a second fusion protein
comprising an extracellular binding domain, a second
multimerization domain, and a second hydrophobic domain; and a
bridging factor; wherein the first fusion protein, second fusion
protein, and bridging factor associate to form a polypeptide
heterocomplex with the bridging factor associated with and disposed
between the multimerization domains of the first and second fusion
proteins.
[0056] In further embodiments, the binding domain is a single chain
antibody variable region, a receptor ectodomain, or a ligand.
[0057] In certain embodiments, the single chain antibody variable
region is a domain antibody, sFv, scFv, F(ab').sub.2, or Fab.
[0058] In certain embodiments, the binding domain is amino terminal
to the multimerization domain.
[0059] In some embodiments, the binding domain is carboxy terminal
to the multimerization domain.
[0060] In particular embodiments, the first multimerization domain
comprises a first FKBP polypeptide or variant thereof, and the
second multimerization domain comprises a first FRB polypeptide or
variant thereof.
[0061] In additional embodiments, the first multimerization domain
comprises a first FRB polypeptide or variant thereof, and the
second multimerization domain comprises a first FKBP polypeptide or
variant thereof.
[0062] In particular embodiments, the first hydrophobic domain is a
transmembrane domain.
[0063] In some embodiments, the second hydrophobic domain comprises
a CD154 transmembrane domain.
[0064] In certain embodiments, the second hydrophobic domain
comprises a CD71 transmembrane domain.
[0065] In particular embodiments, the first hydrophobic domain and
the second hydrophobic domain do not increase cytotoxic activity of
the non-natural cell in the absence of the bridging factor.
[0066] In further embodiments, the first hydrophobic domain and the
second hydrophobic domain increase cytotoxic activity of the
non-natural cell in the absence of the bridging factor, wherein the
increase in cytotoxic activity is less than the cytotoxic activity
in the presence of the bridging factor.
[0067] In certain embodiments, the actuator domain comprises a
lymphocyte receptor chain.
[0068] In particular embodiments, the bridging factor is rapamycin
or a rapalog thereof, coumermycin or a derivative thereof,
gibberellin or a derivative thereof, ABA or a derivative thereof,
methotrexate or a derivative thereof, cyclosporin A or a derivative
thereof, FKCsA or a derivative thereof, or Tmp-SLF or a derivative
thereof.
[0069] In additional embodiments, the second fusion protein further
comprises a sub-threshold signaling domain.
[0070] In some embodiments, the binding domain is specific for a
target that is an antigen associated with a cancer, an inflammatory
disease, an autoimmune disease, or a graft versus host disease.
[0071] In particular embodiments, the cancer is a hematologic
malignancy having an antigen target of CD19, CD20, CD22, CD33, or
CD37.
[0072] In various embodiments, the present invention contemplates,
in part, a nucleic acid molecule encoding any one or more of the
fusion proteins contemplated herein.
[0073] In certain embodiments, the nucleic acid molecule is
disposed between 5' and 3' polynucleotide sequences homologous to a
genomic locus.
[0074] In various embodiments, the present invention contemplates,
in part, an expression vector containing a nucleic acid encoding
any one or more of the fusion proteins contemplated herein.
[0075] In further embodiments, the first and second fusion proteins
are encoded as a polycistronic message or as a single protein
separated by a 2A peptide.
[0076] In additional embodiments, the polycistronic message
comprises an internal ribosome entry site (IRES) between the
nucleotide sequences that encode the fusion proteins.
[0077] In particular embodiments, the first protein is expressed
from a first promoter and the second fusion protein is expressed
from a second promoter.
[0078] In some embodiments, the first promoter is selected from the
group consisting of: a CMV promoter, an EF1.alpha. promoter, and an
MND promoter.
[0079] In particular embodiments, the second promoter is selected
from the group consisting of: a CMV promoter, an EF1.alpha.
promoter, and an MND promoter.
[0080] In further embodiments, the first promoter and the second
promoter are not the same promoter.
BRIEF DESCRIPTION THE DRAWINGS
[0081] FIGS. 1A-1M show schematics of various types of multipartite
signaling complexes of this disclosure.
[0082] FIG. 2 shows a schematic of an assay to detect specific cell
killing and cytokine secretion with a particular multipartite
signaling complex of this disclosure.
[0083] FIGS. 3A and 3B show the cytotoxic properties of human T
cells expressing a multipartite signaling complex of this
disclosure.
[0084] FIG. 4 shows the cytokine secretion profile of human T cells
expressing a multipartite signaling complex of this disclosure.
[0085] FIG. 5 shows that use of independent multimerization domains
having different specificities for bridging components allows for
directed cytotoxic activity of human T cells expressing a
multipartite signaling complex of this disclosure. In addition,
this figure shows that human T cells expressing a multipartite
signaling complex of this disclosure can be cytotoxic even when the
DARIC binding and signaling components are individually expressed
in separate cells.
[0086] FIG. 6 shows that bridging factors can function in the DARIC
system at clinically relevant concentrations.
[0087] FIG. 7 shows that a DARIC binding component can be released
from a cell or tethered to the cell surface and still functionally
associate with a DARIC signaling component to form a multipartite
signaling complex of this disclosure.
[0088] FIG. 8 shows that a DARIC binding component may be tethered
to the cell surface via GPI-anchor and still functionally associate
with a DARIC signaling component in the presence of a bridging
factor to form a multipartite signaling complex of this
disclosure.
[0089] FIG. 9 shows a DARIC system targeting an additional model
antigen, CD123, that may be used either to eradicate a myeloid
cancer, or in a conditioning regimen to ablate myeloid cells prior
to a bone marrow transplant.
[0090] FIG. 10 shows that the FRB and FKBP12 multimerization
domains may be appended to the DARIC binding component or signaling
component and still form a functional multipartite signaling
complex in the presence of a bridging factor.
[0091] FIG. 11 shows that the coupling of the DARIC binding and
signaling components can be deactivated by the addition of an
anti-bridging factor, a monovalent drug that binds only to one of
the multimerization domains and thereby blocks the activation of
the cell.
[0092] FIG. 12 shows that T cells harboring a dual vector promoter
that expresses both the DARIC binding component and the DARIC
signaling component mediates a target cell specific cytotoxic
response.
[0093] FIGS. 13A-C show that T cells expressing a DARIC signaling
component can mediate antigen specific cytotoxicity when a soluble
DARIC binding component that recognizes the target cell is provided
in trans, e.g., secreted into the culture medium or extracellular
milieu as a model for delivery of the DARIC binding component as a
separate biologic drug.
[0094] FIGS. 14A-B shows that a prototypical transmembrane DARIC
binding component harboring a CD4 transmembrane domain has residual
signaling activity in the absence of a bridging factor against
autologous B cells. The residual signaling activity is reduced or
eliminated when the CD4 transmembrane domain is replaced with
another transmembrane domain, e.g., a CD71 or CD154 transmembrane
domain.
[0095] FIG. 15A shows that T cells expressing DARIC complexes
comprising alternative transmembrane domains (CD154 TM) have
increased antigen specific cytotoxicity in the presence a bridging
factor and also show little or no basal cytotoxicity in the absence
of the bridging factor. FIG. 15B shows that T cells expressing
DARIC complexes comprising alternative transmembrane domains (CD71
TM) or transmembrane topology maintain antigen specific
cytotoxicity in the presence a bridging factor and also show
reduced basal cytotoxicity in the absence of the bridging
factor.
DETAILED DESCRIPTION
[0096] In one embodiment, multi-component fusion proteins for use
in modulating a biological response to immunotherapy, such as
adoptive immunotherapy, are provided. By way of background, signal
transduction by cell surface receptors converts extracellular
information into intracellular responses and requires machinery for
both ligand recognition and transmembrane signal transduction. Cell
surface receptors recognize ligands through the use of an
extracellular binding domain and, upon ligand binding, transduce
signals across the plasma membrane via membrane spanning domains
connected with intracellular signaling domains. These occur either
as single-chain units, where binding and signaling are linked
directly, or through multi-chain contacts whereby cell surface
binding of ligand allows intracellular interactions of signaling
domains with other proteins to mediate cell signal
transduction.
[0097] An advantage of the compositions and methods contemplated
herein is to provide both spatial and temporal control over such
signal transduction binding and signaling activities. Since the
binding component isexpressed on the surface, or delivered in a
recombinant form, it is then present in the extracellular
environment without being basally coupled to any cell signal
transduction machinery. The transmembrane signaling fusion protein
to be expressed by the cell of interest comprises one or more
intracellular signaling (actuator) domains fused via a
transmembrane domain to an extracellular multimerization domain,
such as a FRB or FKBP12 protein (whichever is not present on the
binding component).
[0098] In one embodiment, this disclosure provides a binding
component and a signaling component that are each expressed as
separate fusion proteins, but contain an extracellular
multimerization mechanism (bridging factor) for recoupling of the
two functional components on a cell surface--referred to herein as
DARIC binding and signaling components--which provides temporal
control. In particular embodiments, DARIC components have
surprisingly low or negligible recoupling in the absence of the
bridging factor but still maintain potent cell signaling properties
in the presence of bridging factor.
[0099] But, the temporal control achieved through the
multimerization mechanism described herein only primes the
machinery for signaling. The chemically induced multimerization
reconstitutes a signaling-potentiated receptor, but it does not
activate downstream signaling because there is no aggregation of
intracellular signaling components. Spatial control is, therefore,
achieved on the basis of the presence or absence of a target
recognized by the binding domain on the binding component. Since
the binding component fusion protein is displayed on the outside of
the cell, it only localizes to cells expressing the target antigen,
such that cells will only become activated when both target antigen
(e.g., cell surface antigen) and the bridging factor are
present.
[0100] In certain embodiments, a recombinant or non-natural cell
comprises a first nucleic acid molecule encoding a first fusion
protein comprising a first multimerization domain, a first
hydrophobic domain, and an actuator domain, wherein the first
multimerization domain localizes extracellularly when the first
fusion protein is expressed is administered to a subject having a
hyperproliferative disease (e.g., cancer), an inflammatory disease,
an autoimmune disease, or a graft-versus-host disease. Such a
fusion protein can be referred to as a DARIC signaling component,
which may be expressed as one or more transmembrane protein(s). A
DARIC signaling component may contain more than one multimerization
domain, including a multimerization domain that promotes
homodimerization in the presence of homo-bivalent bridging factor.
In such an embodiment (see FIG. 1c), the administration of a
bridging factor will promote some level of basal signaling in the
absence of binding to an extracellular target--for example, as a
way to drive cell proliferation in vitro or in vivo prior to
activation with a DARIC binding component (which in this context
functions like a drug). For T cells, it is known that lower level
activation promotes proliferation, whereas the higher order
multimerization (as would occur by high density of antigen on a
target cell and heterodimerization of the DARIC components with a
bridging component) would lead to activation of a cytotoxicity
response.
[0101] In further embodiments, a subject receiving a recombinant
(non-natural) cell (e.g., T cell) expressing a DARIC signaling
component and a fusion protein comprising a binding domain, a
multimerization domain, and a hydrophobic domain (e.g., CD154 or
CD71 transmembrane domain)--a DARIC binding component--and a
bridging factor (e.g., rapamycin or rapalog thereof) to promote the
formation of a polypeptide complex on the non-natural cell surface
with the bridging factor associated with and disposed between the
multimerization domains of the first and second fusion proteins
(DARIC signaling and binding components, respectively). In certain
embodiments, a nucleic acid molecule further encodes a fusion
protein comprising a secretion signal, a binding domain, a
multimerization domain, and a hydrophobic domain wherein the fusion
protein (DARIC binding component) is secreted from the non-natural
cell when expressed. In some embodiments, a nucleic acid molecule
further encodes a fusion protein comprising a secretion signal, a
binding domain, a multimerization domain, and a hydrophobic domain
wherein the expressed fusion protein (DARIC binding component) is
expressed on the cell surface of the non-natural cell (see FIG.
11-K). The DARIC binding component will specifically bind to a
target cell (e.g., cancer, autoimmune) either before or after
associating with the DARIC signaling component through the bridging
factor, wherein in the absence of the bridging factor the complex
will not elicit an appreciable cellular response, and wherein the
tripartite association of the two DARIC components and bridging
factor will trigger a cellular response that treats the
hyperproliferative, inflammatory, autoimmune, or graft-versus-host
disease. For example, the presence at least one DARIC binding
component and a cell surface target would lead to increasing
signals proportional to the density of target due to
multimerization.
[0102] In a further embodiment, the DARIC signaling component may
be created by leveraging existing activating receptors on the cell
(e.g., T cell) surface using a drug regulated bi-specific engager
(BiTE). In this instance, both DARIC components are secreted: a
binding component that binds to a target cell, and a signaling
component that binds to a receptor (e.g., the TCR/CD3 complex) on a
T cell. In one embodiment, a non-natural cell secretes both
components. In another embodiment, one or more non-natural cells
secretes one or more of the components.
[0103] In a particular embodiment, a non-natural cell further
comprises a deconstructed drug regulated bispecific T cell engager
(BiTE) expressed as separate fusion proteins is provided. The BiTE
comprises a DARIC signaling component comprising a binding agent
that binds a T cell receptor and a first multimerization domain;
and a DARIC binding component comprising a binding agent that binds
an antigen on a target cell and a second multimerization domain,
such as a FRB or FKBP12 protein (whichever is not present on the
binding component). Only upon the application of the FRB/FKBP12
coupling drug (e.g., rapamycin or a rapalog thereof) do the BiTE
components form a complex that is capable of initiating signal
transduction.
[0104] In particular preferred embodiments, DARIC signaling and
binding components are provided that exhibit potent antigen
specific cytotoxic responses in the presence of a bridging factor
and minimal or non-detectable cytotoxic activity in the absence of
the bridging factor, e.g., FIG. 15.
[0105] Prior to setting forth this disclosure in more detail, it
may be helpful to an understanding thereof to provide definitions
of certain terms to be used herein. Additional definitions are set
forth throughout this disclosure.
[0106] Reference throughout this specification to "one embodiment,"
"an embodiment," "a particular embodiment," "a related embodiment,"
"a certain embodiment," "an additional embodiment," or "a further
embodiment" or combinations thereof means that a particular
feature, structure or characteristic described in connection with
the embodiment is included in at least one embodiment of the
present invention. Thus, the appearances of the foregoing phrases
in various places throughout this specification are not necessarily
all referring to the same embodiment. Furthermore, the particular
features, structures, or characteristics may be combined in any
suitable manner in one or more embodiments. It is also understood
that the positive recitation of a feature in one embodiment, serves
as a basis for excluding the feature in a particular
embodiment.
[0107] In the present description, any concentration range,
percentage range, ratio range, or integer range is to be understood
to include the value of any integer within the recited range and,
when appropriate, fractions thereof (such as one tenth and one
hundredth of an integer), unless otherwise indicated. Also, any
number range recited herein relating to any physical feature, such
as polymer subunits, size or thickness, are to be understood to
include any integer within the recited range, unless otherwise
indicated. As used herein, the terms "about" means (1).+-.1%,
.+-.2%, .+-.3%, .+-.4%, .+-.5%, .+-.10%, .+-.15%, or .+-.20% of the
indicated range, value or structure; (2) a value includes the
inherent variation of error for the method being employed to
determine the value; or (3) a value includes the variation that
exists among replicate experiments, unless otherwise indicated. It
should be understood that the terms "a" and "an" as used herein
refer to "one or more" of the enumerated components. The use of the
alternative (e.g., "or") should be understood to mean either one,
both, or any combination thereof of the alternatives or enumerated
components. As used herein, the terms "include," "have" and
"comprise" are used synonymously, which terms and variants thereof
are intended to be construed as non-limiting.
[0108] As used herein, a protein or polypeptide "consists
essentially of" several domains (e.g., a binding domain, a linker
or spacer, a hydrophobic domain, a multimerization domain, an
actuator domain) when the portions outside of the several domains
(e.g., amino acids at the amino- or carboxy-terminus or between two
domains), in combination, contribute to at most 20% (e.g., at most
15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the
protein or polypeptide and do not substantially affect (i.e., do
not alter the activity by more than 50%, such as no more than 40%,
30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) the activities of one
or more of the various domains (e.g., the target binding affinity
of the binding domain, the capability of the multimerization domain
to facilitate complex formation, and the capability of the actuator
domain to transmit functional signals to a cell). In certain
embodiments, a protein (e.g., a single chain polypeptide) consists
essentially of a binding domain that specifically binds a target, a
linker, and a multimerization domain, wherein the protein may
comprise junction amino acids at the amino- and/or carboxy-terminus
of the protein or between two different domains (e.g., between the
binding domain and the multimerization domain, between the
multimerization domain and the linker).
[0109] A "fusion protein" or "chimeric protein," as used herein,
refers to a protein that includes polypeptide components derived
from one or more parental proteins or polypeptides and does not
naturally occur in a host cell. A fusion protein will contain two
or more naturally-occurring amino acid sequences that are linked
together in a way that does not occur naturally. For example, a
fusion protein may have two or more portions from the same protein
linked in a way not normally found in a cell, or a fusion protein
may have portions from two, three, four, five or more different
proteins linked in a way not normally found in a cell. A fusion
protein can be encoded by a nucleic acid molecule wherein a
nucleotide sequence encoding one protein or portion thereof is
appended in frame with, and optionally separated by nucleotides
that encode a linker, spacer or junction amino acids, a nucleic
acid molecule that encodes one or more different proteins or a
portion thereof. In certain embodiments, a nucleic acid molecule
encoding a fusion protein is introduced into a host cell and
expressed.
[0110] As used herein, the term "host" refers to a cell (e.g., T
cell) or microorganism that may be genetically modified with an
exogenous nucleic acid molecule to produce a polypeptide of
interest (e.g., DARIC binding or signaling components). In certain
embodiments, a host cell may optionally already possess or be
modified to include other genetic modifications that confer desired
properties related or unrelated to fusion protein biosynthesis
(e.g., deleted, altered or truncated TCR; increased costimulatory
factor expression). In certain embodiments, a host cell is a human
T cell or a human T cell with TCR.alpha., TCR.beta., or both
knocked out with a site-specific nuclease (e.g., a LAGLIDADG homing
endonuclease, LHE).
[0111] As used herein, "recombinant" or "non-natural" refers to an
organism, microorganism, cell, nucleic acid molecule, or vector
that has at least one engineered genetic alteration or has been
modified by the introduction of a heterologous nucleic acid
molecule, or refers to a cell that has been altered such that the
expression of an endogenous nucleic acid molecule or gene can be
controlled. Recombinant also refers to a cell that is derived from
a non-natural cell or is progeny of a non-natural cell having one
or more such modifications. Genetic alterations include, for
example, modifications introducing expressible nucleic acid
molecules encoding proteins, or other nucleic acid molecule
additions, deletions, substitutions or other functional alteration
of a cell's genetic material. For example, recombinant cells may
express genes or other nucleic acid molecules that are not found in
identical or homologous form within a native (wild-type) cell
(e.g., a fusion or chimeric protein), or may provide an altered
expression pattern of endogenous genes, such as being
over-expressed, under-expressed, minimally expressed, or not
expressed at all.
[0112] Recombinant methods for expression of exogenous or
heterologous nucleic acids in cells are well known in the art. Such
methods can be found described in, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring
Harbor Laboratory, N.Y. (2001); and Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.
(1999). Exemplary exogenous proteins or enzymes to be expressed
include scFv, CD3.zeta.,FKBP, FRB, cytokines, or any combination
thereof. Genetic modifications to nucleic acid molecules encoding
fusion proteins can confer a biochemical or metabolic capability to
a recombinant or non-natural cell that is altered from its
naturally occurring state.
[0113] As used herein, the term "endogenous" or "native" refers to
a gene, protein, compound or activity that is normally present in a
host cell. The term "homologous" or "homolog" refers to a molecule
or activity from an exogenous (non-native) source that is the same
or similar molecule or activity as that found in or derived from a
host cell, species or strain.
[0114] As used herein, "heterologous" nucleic acid molecule,
construct or sequence refers to a nucleic acid molecule or portion
of a nucleic acid molecule sequence that is not native to a cell in
which it is expressed, a nucleic acid molecule or portion of a
nucleic acid molecule native to a host cell that has been altered
or mutated, or a nucleic acid molecule with an altered expression
as compared to the native expression levels under similar
conditions. For example, a heterologous control sequence (e.g.,
promoter, enhancer) may be used to regulate expression of a gene or
a nucleic acid molecule in a way that is different than the gene or
a nucleic acid molecule that is normally expressed in nature or
culture. In certain embodiments, a heterologous nucleic acid
molecule may be homologous to a native host cell gene, but may have
an altered expression level or have a different sequence or both.
In other embodiments, heterologous or exogenous nucleic acid
molecules may not be endogenous to a host cell or host genome
(e.g., fusion protein), but instead may have been introduced into a
host cell by transformation (e.g., transfection, electroporation),
wherein the added molecule may integrate into the host genome or
can exist as extra-chromosomal genetic material either transiently
(e.g., mRNA) or stably for more than one generation (e.g., episomal
viral vector, plasmid or other self-replicating vector).
[0115] In certain embodiments, more than one heterologous or
exogenous nucleic acid molecule can be introduced into a host cell
as separate nucleic acid molecules, as a polycistronic nucleic acid
molecule, as a single nucleic acid molecule encoding a fusion
protein, or any combination thereof, and still be considered as
more than one heterologous or exogenous nucleic acid. When two or
more exogenous nucleic acid molecules are introduced into a host
cell, it is understood that the two more exogenous nucleic acid
molecules can be introduced as a single nucleic acid molecule
(e.g., on a single vector), on separate vectors, as single or
multiple mRNA molecules, integrated into the host chromosome at a
single site or multiple sites, and each of these embodiments is
still to be considered two or more exogenous nucleic acid
molecules. Thus, the number of referenced heterologous nucleic acid
molecules or protein activities refers to the number of encoding
nucleic acid molecules or the number of protein activities, not the
number of separate nucleic acid molecules introduced into a host
cell.
[0116] For example, a cell can be modified to express two or more
heterologous or exogenous nucleic acid molecules, which may be the
same or different, that encode one or more fusion proteins, as
disclosed herein. In certain embodiments, a host cell will contain
a first nucleic acid molecule encoding a first fusion protein and a
separate second nucleic acid molecule encoding a second fusion
protein, or a host cell will contain a single polycistronic nucleic
acid molecule that encodes a first fusion protein and second fusion
protein, or single nucleic acid molecule that encodes a first
fusion protein, a self-cleaving amino acid sequence and a second
fusion protein.
[0117] Suitable protease cleavages sites and self-cleaving peptides
are known to the skilled person (see, e.g., in Ryan et al., 1997.
J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature
Biotech. 5, 589-594). Exemplary protease cleavage sites include,
but are not limited to the cleavage sites of potyvirus NIa
proteases (e.g., tobacco etch virus protease), potyvirus HC
proteases, potyvirus P1 (P35) proteases, byovirus NIa proteases,
byovirus RNA-2-encoded proteases, aphthovirus L proteases,
enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3C
proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV
(rice tungro spherical virus) 3C-like protease, PYVF (parsnip
yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa
and enterokinase. Due to its high cleavage stringency, TEV (tobacco
etch virus) protease cleavage sites are preferred in one
embodiment, e.g., EXXYXQ(G/S), for example, ENLYFQG and ENLYFQS,
wherein X represents any amino acid (cleavage by TEV occurs between
Q and G or Q and S).
[0118] In certain embodiments, the self-cleaving polypeptide site
comprises a 2A or 2A-like site, sequence or domain (Donnelly et
al., 2001. J. Gen. Virol. 82:1027-1041). In a particular
embodiment, the viral 2A peptide is an aphthovirus 2A peptide, a
potyvirus 2A peptide, or a cardiovirus 2A peptide.
[0119] In one embodiment, the viral 2A peptide is selected from the
group consisting of: a foot-and-mouth disease virus (FMDV) 2A
peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea
asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A
peptide, a Theilovirus 2A peptide, and an encephalomyocarditis
virus 2A peptide.
[0120] A "polypeptide complex" or "protein complex," as used
herein, refers to a dimer, trimer, or higher order multimer formed
by at least two different single chain polypeptides, comprising at
least one chain having a binding domain specific for a target and
one chain having an actuator domain. This term does not include an
antibody formed from four single chain polypeptides (i.e., two
light chains and two heavy chains). A "dimer" refers to a
biological entity that contains two subunits associated with each
other, and a "polypeptide complex" refers to a biological entity
that includes at least two proteins subunits and a bridging factor
associated with each other, via one or more forms of intramolecular
forces, including covalent bonds (e.g., disulfide bonds) and other
interactions (e.g., electrostatic interactions, salt bridges,
hydrogen bonding, and hydrophobic interactions), and is stable
under appropriate conditions (e.g., under physiological conditions,
in an aqueous solution suitable for expressing, purifying, and/or
storing recombinant proteins, or under conditions for
non-denaturing and/or non-reducing electrophoresis).
[0121] A "single chain polypeptide" is a single, linear and
contiguous arrangement of covalently linked amino acids. It does
not include two polypeptide chains that link together in a
non-linear fashion, such as via an interchain disulfide bond (e.g.,
a half immunoglobulin molecule in which a light chain links with a
heavy chain via a disulfide bond). In certain embodiments, a single
chain polypeptide may have or form one or more intrachain disulfide
bonds. In certain other embodiments, two or more single chain
polypeptides (e.g., fusion proteins) may associate via an
interchain disulfide bond to provide a potentially active complex
provided the complex is made up of at least one non-natural
protein, such as fusion or chimeric proteins and is not a natural
antibody.
[0122] A "multimerization domain," as used herein, refers to a
polypeptide molecule that preferentially interacts or associates
with another different polypeptide molecule directly or via a
bridging molecule, wherein the interaction of the different
multimerization domains substantially contribute to or efficiently
promote multimerization (i.e., the formation of a dimer, trimer, or
multipartite complex, which may be a homodimer, heterodimer,
homotrimer, heterotrimer, homomultimer, heteromultimer).
Representative multimerization domains of the present disclosure
include an FKBP, FRB, calcineurin, cyclophilin, bacterial DHFR,
PYL1, ABI1, GIB1, GAI, or variants thereof, as provided herein.
[0123] In certain embodiments, a polypeptide complex comprises (i)
a first fusion protein having a first multimerization domain and
(ii) second fusion protein having a second multimerization domain
that is not the same as the first multimerization domain, wherein
the first and second multimerization domains substantially
contribute to or efficiently promote formation of the polypeptide
complex in the presence of a bridging factor. The interaction(s)
between the first and second multimerization domains substantially
contributes to or efficiently promotes the multimerization of the
first and second fusion proteins if there is a statistically
significant reduction in the association between the first and
second fusion proteins in the absence of the first multimerization
domain, the second multimerization domain, or the bridging factor.
In certain embodiments, when the first and second fusion proteins
are co-expressed, at least about 60%, for instance, at least about
60% to about 70%, at least about 70% to about 80%, at least about
80% to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100%, and at least about 90% to about 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% of the first and second single chain polypeptides form
multimers with each other in the presence of a bridging factor.
[0124] As used herein, "hydrophobic domain" refers to an amino acid
sequence having a three-dimensional structure that is
thermodynamically stable in a cell membrane. The structure of a
hydrophobic domain may comprise an alpha helix, a beta barrel, a
beta sheet, a beta helix, or any combination thereof. In certain
embodiments, a hydrophobic domain is a transmembrane domain, such
as one derived from an integral membrane protein (e.g., receptor,
cluster of differentiation (CD) molecule, enzyme, transporter, cell
adhesion molecule, or the like).
[0125] As used herein, "anchor domain" refers to an amino acid
sequence or other molecule that promotes tethering, anchoring or
association of a fusion protein of this disclosure with a cell
surface. Exemplary anchor domains include an amino acid sequence
with a structure that is stable in a cell membrane or an amino acid
sequence that promotes the addition of a glycolipid (also known as
glycosyl phosphatidylinositols or GPIs), or the like. By way of
background, a GPI molecule is post-translationally attached to a
protein target by a transamidation reaction, which results in the
cleavage of a carboxy-terminal GPI signal sequence (see, e.g.,
White et al., J. Cell Sci. 113:721, 2000) and the simultaneous
transfer of the already synthesized GPI anchor molecule to the
newly formed carboxy-terminal amino acid (see
www.ncbi.nlm.nih.gov/books/NBK20711 for exemplary GPI anchors,
which GPI anchors are incorporated by reference in their entirety.
In certain embodiments, an anchor domain is a hydrophobic domain
(e.g., transmembrane domain) or a GPI signal sequence. In some
embodiments, a nucleic acid molecule encoding a fusion protein of
this disclosure with an anchor domain results in a fusion protein
further comprising a GPI molecule.
[0126] An "actuator domain," as used herein, directly or
indirectly, promotes a biological or physiological response in a
cell when receiving the appropriate signal. In certain embodiments,
the actuator domain is part of a protein or protein complex that
receives a signal when bound or it binds to a target molecule and
the binding triggers a signal from the actuator domain. The
actuator domain may directly promote a cellular response when it
contains signaling domains or motifs, such as an immunoreceptor
tyrosine-based activation motif (ITAM). In other embodiments, an
actuator domain will indirectly promote a cellular response by
associating with one or more other proteins that directly promote a
cellular response. Exemplary actuator domains include CD2,
CD3.epsilon., CD3.delta., CD3.zeta., pT.alpha., TCR.alpha.,
TCR.beta., FcR.alpha., FcR.beta., FcR.gamma., NKG2D, CD79A, CD79B,
CD22, CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB,
CARD11, HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any
combination thereof.
[0127] In particular embodiments, a "transmembrane domain" refers
to a portion of the signaling component that fuses an extracellular
multimerization domain and one or more intracellular signaling
domains and anchors the signaling component to the plasma membrane
of the T cell. In one embodiment, the transmembrane domain may be
heterologous to other domains of the fusion polypeptides
contemplated herein. In certain embodiments, a "transmembrane
domain" refers to a portion of the binding component that is fused
to an extracellular multimerization domain and anchors the binding
component to the plasma membrane of the T cell. The transmembrane
domain may be derived either from a natural, synthetic,
semi-synthetic, or recombinant source. Illustrative transmembrane
domains may be derived from (i.e., comprise at least the
transmembrane region(s) of) the alpha, beta or zeta chain of the
T-cell receptor, CD3.epsilon., CD3.zeta., CD4, CD5, CD8.alpha.,
CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80,
CD86, CD 134, CD137, CD152, CD 154, AMN, and PD1. In various
embodiments, a transmembrane domain of a binding component and/or
signaling component is fused to a short oligo- or polypeptide
linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino
acids in length and that optionally links the transmembrane domain
and the intracellular signaling domain of the signaling component.
In particular embodiments, a fusion protein contemplated herein
comprises a type I transmembrane domain. In other embodiments, a
fusion protein contemplated herein comprises a type II
transmembrane domain. In certain embodiments, a fusion protein
contemplated herein comprises a type I transmembrane domain that
has been converted to a type I transmembrane domain from a type II
transmembrane domain. In other embodiments, a fusion protein
contemplated herein comprises a type II transmembrane domain that
has been converted to a type II transmembrane domain from a type I
transmembrane domain.
[0128] A "binding domain" (also referred to as a "binding region,"
"binding agent," or "binding moiety"), as used herein, refers to
one or more proteins, polypeptides, oligopeptides, or peptides that
possesses the ability to specifically recognize and bind to a
target (e.g., CD19, CD20). A binding domain includes any naturally
occurring, synthetic, semi-synthetic, or recombinantly produced
binding partner for a biological molecule or another target of
interest. Exemplary binding domains include single chain antibody
variable regions (e.g., domain antibodies, sFv, scFv, Fab),
receptor ectodomains (e.g., c-Met), or ligands (e.g., cytokines,
chemokines, or cell surface associated ligands). In particular
embodiments, a binding domain comprises an antibody or antigen
binding fragment thereof, including but not limited to a Camel Ig
(a camelid antibody (VHH)), Ig NAR, Fab fragments, Fab' fragments,
F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv antibody
("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody,
tetrabody, disulfide stabilized Fv protein ("dsFv"), and
single-domain antibody (sdAb, Nanobody). A variety of assays are
known for identifying binding domains of the present disclosure
that specifically bind a particular target, including Western blot,
ELISA, and Biacore analysis.
[0129] A binding domain and a fusion protein thereof "specifically
binds" a target if it binds the target with an affinity or K.sub.a
(i.e., an equilibrium association constant of a particular binding
interaction with units of 1/M) equal to or greater than 10.sup.5
M.sup.-1, while not significantly binding other components present
in a test sample. Binding domains (or fusion proteins thereof) may
be classified as "high affinity" binding domains (or fusion
proteins thereof) and "low affinity" binding domains (or fusion
proteins thereof). "High affinity" binding domains refer to those
binding domains with a K.sub.a of at least 10.sup.7 M.sup.-1, at
least 10.sup.8M.sup.-1, at least 10.sup.9M.sup.-1, at least
10.sup.10 M.sup.-1, at least 10.sup.11M.sup.-1, at least 10.sup.12
M.sup.-1, or at least 10.sup.13 M.sup.-1. "Low affinity" binding
domains refer to those binding domains with a K.sub.a of up to
10.sup.7 M.sup.-1, up to 10.sup.6 M.sup.-1, up to 10.sup.5
M.sup.-1. Alternatively, affinity may be defined as an equilibrium
dissociation constant (Ka) of a particular binding interaction with
units of M (e.g., 10.sup.-5 M to 10.sup.-13 M). Affinities of
binding domain polypeptides and fusion proteins according to the
present disclosure can be readily determined using conventional
techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci.
51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, or the
equivalent).
[0130] "T cell receptor" (TCR) is a molecule found on the surface
of T cells that, along with CD3, is generally responsible for
recognizing antigens bound to major histocompatibility complex
(MHC) molecules. It consists of a disulfide-linked heterodimer of
the highly variable a and .beta. chains in most T cells. In other T
cells, an alternative receptor made up of variable y and 6 chains
is expressed. Each chain of the TCR is a member of the
immunoglobulin superfamily and possesses one N-terminal
immunoglobulin variable domain, one immunoglobulin constant domain,
a transmembrane region, and a short cytoplasmic tail at the
C-terminal end (see, Abbas and Lichtman, Cellular and Molecular
Immunology (5th Ed.), Editor: Saunders, Philadelphia, 2003; Janeway
et al., Immunobiology: The Immune System in Health and Disease,
4.sup.th Ed., Current Biology Publications, p148, 149, and 172,
1999). TCR as used in the present disclosure may be from one or
various animal species, including human, mouse, rat, or other
mammals.
[0131] "CD3" is known in the art as a multi-protein complex of six
chains (see, Abbas and Lichtman, 2003; Janeway et al., p172 and
178, 1999). In mammals, the complex comprises a CD3.gamma. chain, a
CD3.delta. chain, two CD3.epsilon. chains, and a homodimer of
CD3.zeta. chains. The CD3.gamma., CD3.delta. and CD3.epsilon.
chains are highly related cell surface proteins of the
immunoglobulin superfamily containing a single immunoglobulin
domain. The transmembrane regions of the CD3.gamma., CD3.delta.,and
CD3.epsilon. chains are negatively charged, which is a
characteristic that allows these chains to associate with the
positively charged T cell receptor chains. The intracellular tails
of the CD3.gamma., CD3.delta., and CD3.epsilon. chains each contain
a single conserved motif known as an immunoreceptor tyrosine-based
activation motif or ITAM, whereas each CD3.zeta. chain has three.
It is believed the ITAMs are important for the signaling capacity
of a TCR complex. CD3 as used in the present disclosure may be from
one or various animal species, including human, mouse, rat, or
other mammals.
[0132] "TCR complex," as used herein, refers to a complex formed by
the association of CD3 with TCR. For example, a TCR complex can be
composed of a CD3.gamma. chain, a CD3.delta. chain, two
CD3.epsilon. chains, a homodimer of CD3.zeta. chains, a TCR.alpha.
chain, and a TCR.beta. chain. Alternatively, a TCR complex can be
composed of a CD3.gamma. chain, a CD3.delta. chain, two
CD3.epsilon. chains, a homodimer of CD3.zeta. chains, a TCR.gamma.
chain, and a TCR.delta. chain.
[0133] "A component of a TCR complex," as used herein, refers to a
TCR chain (i.e., TCR.alpha., TCR.beta., TCR.gamma. or TCR.delta.),
a CD3 chain (i.e., CD3.gamma., CD3.delta., CD3.epsilon. or
CD3.zeta.), or a complex formed by two or more TCR chains or CD3
chains (e.g., a complex of TCR.alpha. and TCR.beta., a complex of
TCR.gamma. and TCR.delta., a complex of CD3.epsilon. and
CD3.delta.,a complex of CD3.gamma. and CD3.epsilon., or a sub-TCR
complex of TCR.alpha., TCR.beta., CD3.gamma., CD3.delta., and two
CD3.epsilon. chains).
[0134] Terms understood by those in the art of antibody technology
are each given the meaning acquired in the art, unless expressly
defined differently herein. Antibodies are known to have variable
regions, a hinge region, and constant domains. Immunoglobulin
structure and function are reviewed, for example, in Harlow et al.,
Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring
Harbor Laboratory, Cold Spring Harbor, 1988).
[0135] For example, the terms "VL" and "VH" refer to the variable
binding region from an antibody light and heavy chain,
respectively. The variable binding regions are made up of discrete,
well-defined sub-regions known as "complementarity determining
regions" (CDRs) and "framework regions" (FRs). The term "CL" refers
to an "immunoglobulin light chain constant region" or a "light
chain constant region," i.e., a constant region from an antibody
light heavy chain. The term "CH" refers to an "immunoglobulin heavy
chain constant region" or a "heavy chain constant region," which is
further divisible, depending on the antibody isotype into CH1, CH2,
and CH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE,
IgM). A "Fab" (fragment antigen binding) is the part of an antibody
that binds to antigens and includes the variable region and CH1 of
the heavy chain linked to the light chain via an inter-chain
disulfide bond.
[0136] As used herein, "an Fc region constant domain portion" or
"Fc region portion" refers to the heavy chain constant region
segment of the Fc fragment (the "fragment crystallizable" region or
Fc region) from an antibody, which can include one or more constant
domains, such as CH2, CH3, CH4, or any combination thereof. In
certain embodiments, an Fc region portion includes the CH2 and CH3
domains of an IgG, IgA, or IgD antibody and any combination
thereof, or the CH3 and CH4 domains of an IgM or IgE antibody and
any combination thereof. In one embodiment, the CH2CH3 or the
CH3CH4 structures are from the same antibody isotype, such as IgG,
IgA, IgD, IgE, or IgM. By way of background, the Fc region is
responsible for the effector functions of an immunoglobulin, such
as ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP
(antibody-dependent cellular phagocytosis), CDC
(complement-dependent cytotoxicity) and complement fixation,
binding to Fc receptors (e.g., CD16, CD32, FcRn), greater half-life
in vivo relative to a polypeptide lacking an Fc region, protein A
binding, and perhaps even placental transfer (see Capon et al.,
Nature, 337:525 (1989)).
[0137] A "linker" or "spacer" refers to an amino acid sequence that
connects two proteins, polypeptides, peptides, domains, regions, or
motifs and may provide a spacer function compatible with
interaction of the two sub-binding (e.g., multimerization) domains
so that the resulting polypeptide retains a specific binding
affinity to a target molecule or retains signaling activity (e.g.,
actuator domain activity). In certain embodiments, a linker is
comprised of about two to about 35 amino acids, for instance, or
about four to about 20 amino acids or about eight to about 15 amino
acids or about 15 to about 25 amino acids. In other embodiments, a
spacer may have a particular structure, such as an antibody CH2CH3
domain, hinge domain or the like. In one embodiment, a spacer
comprises the CH2 and CH3 domains of IgG1 or IgG4.
[0138] The DARIC components may further comprise one or more "hinge
domains," which plays a role in positioning the domains to enable
proper cell/cell contact, antigen binding and activation. A DARIC
may comprises one or more hinge domains between the binding domain
and the multimerization domain and/or the transmembrane domain (TM)
or between the multimerization domain and the transmembrane domain.
The hinge domain may be derived either from a natural, synthetic,
semi-synthetic, or recombinant source. The hinge domain can include
the amino acid sequence of a naturally occurring immunoglobulin
hinge region or an altered immunoglobulin hinge region.
[0139] An "altered hinge region" refers to (a) a naturally
occurring hinge region with up to 30% amino acid changes (e.g., up
to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or
deletions), (b) a portion of a naturally occurring hinge region
that is at least 10 amino acids (e.g., at least 12, 13, 14 or 15
amino acids) in length with up to 30% amino acid changes (e.g., up
to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or
deletions), or (c) a portion of a naturally occurring hinge region
that comprises the core hinge region (which may be 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, or 15 amino acids in length). In certain embodiments,
one or more cysteine residues in a naturally occurring
immunoglobulin hinge region may be substituted by one or more other
amino acid residues (e.g., one or more serine residues). An altered
immunoglobulin hinge region may alternatively or additionally have
a proline residue of a wild type immunoglobulin hinge region
substituted by another amino acid residue (e.g., a serine
residue).
[0140] Other illustrative hinge domains suitable for use in the
DARICs described herein include the hinge region derived from the
extracellular regions of type 1 membrane proteins such as
CD8.alpha., CD4, CD28 and CD7, which may be wild-type hinge regions
from these molecules or may be altered. In another embodiment, the
hinge domain comprises a CD8.alpha. hinge region.
[0141] "Junction amino acids" or "junction amino acid residues"
refer to one or more (e.g., about 2-10) amino acid residues between
two adjacent motifs, regions or domains of a polypeptide, such as
between a binding domain and an adjacent multimerization domain or
between a hydrophobic region and an adjacent multimerization domain
or between a peptide linker or spacer that links two motifs,
regions or domains and an adjacent actuator domain. Junction amino
acids may result from the construct design of a fusion protein
(e.g., amino acid residues resulting from the use of a restriction
enzyme site during the construction of a nucleic acid molecule
encoding a fusion protein).
[0142] An "altered domain" or "altered protein" refers to a motif,
region, domain, peptide, polypeptide, or protein with a sequence
identity to a wild type motif, region, domain, peptide,
polypeptide, or protein (e.g., a wild type human FKBP12, FRP, ITAM,
CD3.zeta., TCR) of at least 75% (e.g., 80%, 82%, 84%, 86%, 88%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%). For
example, an "altered FKBP" refers to a FKBP with a sequence
identity to a wild type FKBP (e.g., a human FKBP) of at least 75%
(e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 99.5%). Similarly, an "altered CD3.zeta." refers
to a CD3.zeta. with a sequence identity to a wild type CD3.zeta.
(e.g., a human CD3.zeta.) of at least 75% (e.g., 80%, 82%, 84%,
86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
99.5%).
[0143] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to any of deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), oligonucleotides, fragments generated, for example, by the
polymerase chain reaction (PCR) or by in vitro translation, and
fragments generated by any of ligation, scission, endonuclease
action, or exonuclease action. In certain embodiments, the nucleic
acids of the present disclosure are produced by PCR. Nucleic acids
may be composed of monomers that are naturally occurring
nucleotides (such as deoxyribonucleotides and ribonucleotides),
analogs of naturally occurring nucleotides (e.g.,
.alpha.-enantiomeric enantiomeric forms of naturally-occurring
nucleotides), or a combination of both. Modified nucleotides can
have modifications in or replacement of sugar moieties, or
pyrimidine or purine base moieties. Nucleic acid monomers can be
linked by phosphodiester bonds or analogs of such linkages. Analogs
of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate,
morpholino, or the like. The term "nucleic acid molecule" also
includes "peptide nucleic acids" (PNAs), which comprise naturally
occurring or modified nucleic acid bases attached to a polyamide
backbone. Nucleic acid molecules can be either single stranded or
double stranded.
[0144] As used herein, "mutation" refers to a change in the
sequence of a nucleic acid molecule or polypeptide molecule as
compared to a reference or wild-type nucleic acid molecule or
polypeptide molecule, respectively. A mutation can result in
several different types of change in sequence, including
substitution, insertion or deletion of nucleotide(s) or amino
acid(s). In other embodiments, a mutation is a substitution of one
or more nucleotides or residues.
[0145] The term "construct" refers to any polynucleotide that
contains a recombinant nucleic acid. A construct may be present in
a vector (e.g., a bacterial vector, a viral vector) or may be
integrated into a genome. A "vector" is a nucleic acid molecule
that is capable of transporting another nucleic acid. Vectors may
be, for example, plasmids, cosmids, viruses, a RNA vector or a
linear or circular DNA or RNA molecule that may include
chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic
acids. Exemplary vectors are those capable of autonomous
replication (episomal vector) and/or expression of nucleic acids to
which they are linked (expression vectors).
[0146] Viral vectors include retrovirus, adenovirus, parvovirus
(e.g., adeno-associated viruses), coronavirus, negative strand RNA
viruses such as ortho-myxovirus (e.g., influenza virus),
rhabdovirus (e.g., rabies and vesicular stomatitis virus),
paramyxovirus (e.g., measles and Sendai), positive strand RNA
viruses such as picornavirus and alphavirus, and double-stranded
DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex
virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and
poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses
include Norwalk virus, togavirus, flavivirus, reoviruses,
papovavirus, hepadnavirus, and hepatitis virus, for example.
Examples of retroviruses include avian leukosis-sarcoma, mammalian
C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus,
spumavirus (Coffin, J. M., Retroviridae: The viruses and their
replication, In Fundamental Virology, Third Edition, B. N. Fields,
et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
[0147] "Lentiviral vector," as used herein, means HIV-based
lentiviral vectors that are very promising for gene delivery
because of their relatively large packaging capacity, reduced
immunogenicity and their ability to stably transduce with high
efficiency a large range of different cell types. Lentiviral
vectors are usually generated following transient transfection of
three (packaging, envelope and transfer) or more plasmids into
producer cells. Like HIV, lentiviral vectors enter the target cell
through the interaction of viral surface glycoproteins with
receptors on the cell surface. On entry, the viral RNA undergoes
reverse transcription, which is mediated by the viral reverse
transcriptase complex. The product of reverse transcription is a
double-stranded linear viral DNA, which is the substrate for viral
integration in the DNA of infected cells.
[0148] "Integrative lentiviral vectors (or LV)," as used herein,
means such vectors as examples of those that are able to integrate
into the genome of a target cell.
[0149] By "non-integrative lentiviral vectors" (or NILV) is meant
efficient gene delivery vectors that do not integrate into the
genome of a target cell through the action of the viral integrase.
In one embodiment, a NILV refers to a lentivirus having an
integrase protein mutated to specifically decrease its integrase
activity. Illustrative mutations in the HIV-1 pol gene suitable to
reduce integrase activity include, but are not limited to: H12N,
H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V,
E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E,
E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A,
R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A,
R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F,
W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and
K264H.
[0150] The term "operably-linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably-linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
the coding sequence is under the transcriptional control of the
promoter). "Unlinked" means that the associated genetic elements
are not closely associated with one another and the function of one
does not affect the other.
[0151] As used herein, "expression vector" refers to a DNA
construct containing a nucleic acid molecule that is
operably-linked to a suitable control sequence capable of effecting
the expression of the nucleic acid molecule in a suitable host.
Such control sequences include a promoter to effect transcription,
an optional operator sequence to control such transcription, a
sequence encoding suitable mRNA ribosome binding sites, and
sequences which control termination of transcription and
translation. The vector may be a plasmid, a phage particle, a
virus, or simply a potential genomic insert. Once transformed into
a suitable host, the vector may replicate and function
independently of the host genome, or may, in some instances,
integrate into the genome itself. In the present specification,
"plasmid," "expression plasmid," "virus" and "vector" are often
used interchangeably.
[0152] The polynucleotides of the present invention, regardless of
the length of the coding sequence itself, may be combined with
other DNA sequences, such as promoters and/or enhancers,
untranslated regions (UTRs), signal sequences, Kozak sequences,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, internal ribosomal entry sites (IRES),
recombinase recognition sites (e.g., LoxP, FRT, and Att sites),
termination codons, transcriptional termination signals, and
polynucleotides encoding self-cleaving polypeptides, epitope tags,
as disclosed elsewhere herein or as known in the art, such that
their overall length may vary considerably. It is therefore
contemplated that a polynucleotide fragment of almost any length
may be employed, with the total length preferably being limited by
the ease of preparation and use in the intended recombinant DNA
protocol.
[0153] In particular embodiments, a vector for use in practicing
the invention including, but not limited to expression vectors and
viral vectors, will include exogenous, endogenous, or heterologous
control sequences such as promoters and/or enhancers. An
"endogenous" control sequence is one which is naturally linked with
a given gene in the genome. An "exogenous" control sequence is one
which is placed in juxtaposition to a gene by means of genetic
manipulation (i.e., molecular biological techniques) such that
transcription of that gene is directed by the linked
enhancer/promoter. A "heterologous" control sequence is an
exogenous sequence that is from a different species than the cell
being genetically manipulated.
[0154] The term "promoter" as used herein refers to a recognition
site of a polynucleotide (DNA or RNA) to which an RNA polymerase
binds. An RNA polymerase initiates and transcribes polynucleotides
operably linked to the promoter. In particular embodiments,
promoters operative in mammalian cells comprise an AT-rich region
located approximately 25 to 30 bases upstream from the site where
transcription is initiated and/or another sequence found 70 to 80
bases upstream from the start of transcription, a CNCAAT region
where N may be any nucleotide.
[0155] The term "enhancer" refers to a segment of DNA which
contains sequences capable of providing enhanced transcription and
in some instances can function independent of their orientation
relative to another control sequence. An enhancer can function
cooperatively or additively with promoters and/or other enhancer
elements. The term "promoter/enhancer" refers to a segment of DNA
which contains sequences capable of providing both promoter and
enhancer functions.
[0156] Illustrative expression control sequences suitable for use
in particular embodiments of the invention include, but are not
limited to, a cytomegalovirus (CMV) immediate early promoter, a
viral simian virus 40 (SV40) (e.g., early or late), a Moloney
murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus
(RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase)
promoter, H5, P7.5, and P11 promoters from vaccinia virus, an
elongation factor 1-alpha (EF1a) promoter, early growth response 1
(EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde
3-phosphate dehydrogenase (GAPDH), eukaryotic translation
initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPAS),
heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock
protein 70kDa (HSP70), .beta.-kinesin (.beta.-KIN), the human ROSA
26 locus (Irions et al., Nature Biotechnology 25, 1477-1482
(2007)), a Ubiquitin C promoter (UBC), a phosphoglycerate kinase-1
(PGK) promoter, a cytomegalovirus enhancer/chicken (3-actin (CAG)
promoter, a (3-actin promoter and a myeloproliferative sarcoma
virus enhancer, negative control region deleted, d1587rev
primer-binding site substituted (MND) promoter (Challita et al., J
Virol. 69(2):748-55 (1995)).
[0157] In one embodiment, a vector of the invention comprises a MND
promoter.
[0158] In one embodiment, a vector of the invention comprises an
EF1a promoter comprising the first intron of the human EF1a
gene.
[0159] In one embodiment, a vector of the invention comprises an
EF1a promoter that lacks the first intron of the human EF1a
gene.
[0160] In one embodiment, a vector is a bicistronic vector
comprising at least two promoters. In a particular embodiment, a
bicistronic vector comprises two or more promoters selected from
the group consisting of: a CMV promoter, an SV40 promoter, an MoMLV
LTR promoter, an RSV LTR, an HSV-TK promoter, H5, P7.5, and P11
promoters from vaccinia virus, an EF1a promoter, a UBC promoter, a
PGK promoter, a CAG promoter, a .beta.-actin promoter and an MND
promoter.
[0161] The term "expression", as used herein, refers to the process
by which a polypeptide is produced based on the nucleic acid
sequence of a gene. The process includes both transcription and
translation.
[0162] The term "introduced" in the context of inserting a nucleic
acid sequence into a cell, means "transfection", or
"transformation" or "transduction" and includes reference to the
incorporation of a nucleic acid sequence into a eukaryotic or
prokaryotic cell wherein the nucleic acid sequence may be
incorporated into the genome of the cell (e.g., chromosome,
plasmid, plastid, or mitochondrial DNA), converted into an
autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0163] "Sequence identity," as used herein, refers to the
percentage of amino acid residues in one sequence that are
identical with the amino acid residues in another reference
polypeptide sequence after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. The percentage sequence identity
values are generated by the NCBI BLAST2.0 software as defined by
Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs," Nucleic Acids Res.
25:3389-3402, with the parameters set to default values.
[0164] In certain embodiments, an altered immunoglobulin domain
only contains conservative amino acid substitutions of a wild type
immunoglobulin domain. In certain other embodiments, an altered
immunoglobulin domain only contains non-conservative amino acid
substitutions of a wild type immunoglobulin domain. In yet other
embodiments, an altered immunoglobulin domain contains both
conservative and non-conservative amino acid substitutions.
[0165] A "conservative substitution" is recognized in the art as a
substitution of one amino acid for another amino acid that has
similar properties. Exemplary conservative substitutions are well
known in the art (see, e.g., WO 97/09433, page 10, published Mar.
13, 1997; Lehninger, Biochemistry, Second Edition; Worth
Publishers, Inc. NY:NY (1975), pp.71-77; Lewin, Genes IV, Oxford
University Press, NY and Cell Press, Cambridge, Mass. (1990), p.
8). In certain embodiments, a conservative substitution includes a
leucine to serine substitution.
[0166] As used herein, the term "derivative" refers to a
modification of one or more amino acid residues of a peptide by
chemical or biological means, either with or without an enzyme,
e.g., by glycosylation, alkylation, acylation, ester formation, or
amide formation. Generally, a "derivative" differs from an
"analogue" in that a parent polypeptide may be the starting
material to generate a "derivative," whereas the parent polypeptide
may not necessarily be used as the starting material to generate an
"analogue." A derivative may have different chemical, biological or
physical properties of the parent polypeptide. For example, a
derivative may be more hydrophilic or it may have altered
reactivity (e.g., a CDR having an amino acid change that alters its
affinity for a target, or FKBP having an amino acid change that
alters its affinity for rapamycin or a rapalog thereof) as compared
to the parent polypeptide.
[0167] A "receptor" is a protein present in the plasma membrane or
in the cytoplasm of a cell to which a signal molecule (i.e., a
ligand, such as a hormone, neurotransmitter, toxin, cytokine) may
bind or attach. The binding of the single molecule to the receptor
may result in a conformational change of the receptor, which can
initiate a cellular response. However, some ligands merely block
receptors without inducing any response (e.g., antagonists). Some
receptor proteins are peripheral membrane proteins, many hormone
and neurotransmitter receptors are transmembrane proteins that are
embedded in the phospholipid bilayer of cell membranes, and another
major class of receptors are intracellular proteins such as those
for steroid and intracrine peptide hormone receptors.
[0168] As used herein, the term "isolated" refers to a substance
that has been removed from the source in which it naturally occurs.
A substance need not be purified in order to be isolated. For
example, a protein produced in a host cell is considered isolated
when it is removed or released from the cell. A protein contained
within a crude cell lysate fraction is considered "isolated" for
purposes of the present disclosure. Further, an "isolated nucleic
acid molecule" refers to a polynucleotide molecule in the form of a
separate fragment or as a component of a larger nucleic acid
construct, which has been separated from its source cell, including
the chromosome it normally resides in, at least once. For example,
a DNA molecule that encodes a recombinant polypeptide, peptide, or
variant thereof, which has been separated from the genomic DNA of a
cell, is an isolated nucleic acid molecule. Another example of an
isolated nucleic acid molecule is a bacteriophage promoter (e.g.,
T5 or T7), or nucleic acid expression control sequence, which can
be cloned into a vector capable of replication in a suitable host
cell. Still another example of an isolated nucleic acid molecule is
a chemically synthesized or PCR synthesized nucleic acid
molecule.
[0169] As used herein, the term "purified" refers to a substance
that has been rendered at least partially free of contaminants and
other materials that typically accompany it. Substances can be
purified to varying degrees. A substance is "substantially pure"
when a preparation or composition of the substance contains less
than about 1% contaminants. A substance is "essentially pure" when
a preparation or composition of the substance contains less than
about 5% contaminants. A substance is "pure" when a preparation or
composition of the substance contains less than about 2%
contaminants. For substances that are "purified to homogeneity,"
contaminants cannot be detected with conventional analytical
methods.
[0170] "Treatment," "treating" or "ameliorating" refers to either a
therapeutic treatment or prophylactic/preventative treatment. A
treatment is therapeutic if at least one symptom of disease in an
individual receiving treatment improves or a treatment may delay
worsening of a progressive disease in an individual, or prevent
onset of additional associated diseases.
[0171] A "therapeutically effective amount (or dose)" or "effective
amount (or dose)" of a specific binding molecule or compound refers
to that amount of the compound sufficient to result in amelioration
of one or more symptoms of the disease being treated in a
statistically significant manner. When referring to an individual
active ingredient, administered alone, a therapeutically effective
dose refers to that ingredient alone. When referring to a
combination, a therapeutically effective dose refers to combined
amounts of the active ingredients that result in the therapeutic
effect, whether administered serially or simultaneously.
[0172] The term "pharmaceutically acceptable" refers to molecular
entities and compositions that do not produce allergic or other
serious adverse reactions when administered using routes well known
in the art.
[0173] A "subject in need" refers to a subject at risk of, or
suffering from, a disease, disorder or condition that is amenable
to treatment or amelioration with a non-natural cell, polypeptide
complex or a composition thereof provided herein. In certain
embodiments, a subject is a human.
[0174] Additional definitions are provided throughout the present
disclosure. In certain aspects, the instant disclosure is directed
to a non-natural cell, comprising (a) a first nucleic acid molecule
encoding a first fusion protein comprising a first multimerization
domain, a hydrophobic domain, and an actuator domain, wherein the
first multimerization domain localizes extracellularly when the
first fusion protein is expressed; and (b) a second nucleic acid
molecule encoding a second fusion protein comprising a binding
domain and a second multimerization domain, wherein the second
fusion protein localizes extracellularly, either secreted from the
cell or anchored to the cell surface, when expressed; wherein a
first bridging factor promotes the formation of a polypeptide
complex on the non-natural cell surface with the bridging factor
associated with and disposed between the multimerization domains of
the first and second fusion proteins. In certain embodiments, the
second fusion protein (e.g., DARIC binding component) further
comprises an anchor domain (e.g., transmembrane domain, GPI signal
sequence), wherein the extracellularly localized second fusion
protein is tethered or anchored to the surface of the non-natural
cell. In certain embodiments, a fusion protein is anchored to the
surface of a non-natural cell by a transmembrane domain, such as a
transmembrane domain from CD4, CD8, CD28, CD71, CD154, AlVIN, or
the like. In some embodiments, a fusion protein is anchored to the
surface of a non-natural cell by a GPI molecule.
[0175] In particular embodiments, a non-natural cell comprises a
multipartite signaling complex comprising a first fusion
polypeptide that comprises a first hydrophobic domain, e.g., a
transmembrane domain, and a second fusion polypeptide that
comprises a second hydrophobic domain, e.g., a transmembrane
domain, wherein the hydrophobic domains of the first and second
fusion polypeptides do not associate or interact in such a way as
to increase cytotoxic activity of the non-natural cell in the
absence of the bridging factor.
[0176] In other particular embodiments, a non-natural cell
comprises a multipartite signaling complex comprising a first
fusion polypeptide that comprises a first hydrophobic domain, e.g.,
a transmembrane domain, and a second fusion polypeptide that
comprises a second hydrophobic domain, e.g., a transmembrane
domain, wherein the hydrophobic domains of the first and second
fusion polypeptides associate or interact in such a way as to
increase cytotoxic activity of the non-natural cell in the absence
of the bridging factor , but wherein the increase is less than the
increase in or level of cytotoxic activity of the non-natural cell
in the presence of the bridging factor. In a further embodiment, a
first fusion protein, rather than comprising its own hydrophobic
and actuator domains, instead comprises a binding domain that binds
to a transmembrane protein expressed on the surface of a T cell
that comprises a hydrophobic and actuator domain (e.g., TCR/CD3 or
the like).
[0177] In further aspects, the instant disclosure is directed to a
first non-natural cell comprising a heterologous nucleic acid
molecule encoding a first fusion protein comprising a first
multimerization domain, a hydrophobic domain, and an actuator
domain, wherein the first multimerization domain localizes
extracellularly when the first fusion protein is expressed; and a
second non-natural cell comprising a heterologous a second nucleic
acid molecule encoding a second fusion protein comprising a binding
domain and a second multimerization domain, wherein the second
fusion protein is released extracellularly when expressed; wherein
a first bridging factor promotes the formation of a polypeptide
complex on the first non-natural cell surface with the bridging
factor associated with and disposed between the multimerization
domains of the first and second fusion proteins.
[0178] In certain embodiments, the first and second multimerization
domains are the same or different. Exemplary bridging factors that
associate with multimerization domains and are useful with the
fusion proteins of this disclosure include rapamycin (sirolimus) or
a rapalog thereof, coumermycin or a derivative thereof, gibberellin
or a derivative thereof, abscisic acid (ABA) or a derivative
thereof, methotrexate or a derivative thereof, cyclosporin A or a
derivative thereof, FKCsA or a derivative thereof, trimethoprim
(Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, or
any combination thereof.
[0179] Exemplary rapamycin analogs (rapalogs) include those
disclosed in U.S. Pat. No. 6,649,595, which rapalog structures are
incorporated herein by reference. In certain embodiments, a
bridging factor is a rapalog with substantially reduced
immunosuppressive effect as compared to rapamycin. A "substantially
reduced immunosuppressive effect" refers to a rapalog having at
least less than 0.1 to 0.005 times the immunosuppressive effect
observed or expected for an equimolar amount of rapamycin, as
measured either clinically or in an appropriate in vitro (e.g.,
inhibition of T cell proliferation) or in vivo surrogate of human
immunosuppressive activity. Alternatively, "substantially reduced
immunosuppressive effect" refers to a rapalog having an EC.sub.50
value in such an in vitro assay that is at least 10 to 250 times
larger than the EC.sub.50 value observed for rapamycin in the same
assay. Other exemplary rapalogs include everolimus, novolimus,
pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus,
and zotarolimus.
[0180] In certain embodiments, multimerization domains will
associate with a bridging factor being a rapamycin or rapalog
thereof. For example, the first and second multimerization domains
are a pair selected from FKBP and FRB. FRB domains are polypeptide
regions (protein "domains") that are capable of forming a
tripartite complex with an FKBP protein and rapamycin or rapalog
thereof. FRB domains are present in a number of naturally occurring
proteins, including mTOR proteins (also referred to in the
literature as FRAP, RAPT1, or RAFT) from human and other species;
yeast proteins including Tor1 and Tor2; and a Candida FRAP homolog.
Information concerning the nucleotide sequences, cloning, and other
aspects of these proteins is already known in the art. For example,
a protein sequence accession number for a human mTOR is GenBank
Accession No. L34075.1 (Brown et al., Nature 369:756, 1994).
[0181] FRB domains for use in the fusion proteins of this
disclosure generally contain at least about 85 to about 100 amino
acid residues. In certain embodiments, an FRB amino acid sequence
for use in fusion proteins of this disclosure will comprise a 93
amino acid sequence Ile-2021 through Lys -2113 and a mutation of
T2098L, based the amino acid sequence of GenBank Accession No.
L34075.1. A FRB domain for use in fusion proteins of this
disclosure will be capable of binding to a complex of an FKBP
protein bound to rapamycin or a rapalog thereof of this disclosure.
In certain embodiments, a peptide sequence of an FRB domain
comprises (a) a naturally occurring peptide sequence spanning at
least the indicated 93 amino acid region of human mTOR or
corresponding regions of homologous proteins; (b) a variant of a
naturally occurring FRB in which up to about ten amino acids, or
about 1 to about 5 amino acids or about 1 to about 3 amino acids,
or in some embodiments just one amino acid, of the
naturally-occurring peptide have been deleted, inserted, or
substituted; or (c) a peptide encoded by a nucleic acid molecule
capable of selectively hybridizing to a DNA molecule encoding a
naturally occurring FRB domain or by a DNA sequence which would be
capable, but for the degeneracy of the genetic code, of selectively
hybridizing to a DNA molecule encoding a naturally occurring FRB
domain.
[0182] FKBPs (FK506 binding proteins) are the cytosolic receptors
for macrolides, such as FK506, FK520 and rapamycin, and are highly
conserved across species lines. For the purpose of this disclosure,
FKBPs are proteins or protein domains that are capable of binding
to rapamycin or to a rapalog thereof and further forming a
tripartite complex with an FRB-containing protein or fusion
protein. An FKBP domain may also be referred to as a "rapamycin
binding domain". Information concerning the nucleotide sequences,
cloning, and other aspects of various FKBP species is known in the
art (see, e.g., Staendart et al., Nature 346:671, 1990 (human
FKBP12); Kay, Biochem. 1 3/4:361, 1996). Homologous FKBP proteins
in other mammalian species, in yeast, and in other organisms are
also known in the art and may be used in the fusion proteins
disclosed herein. The size of FKBP domains for use in this
invention varies, depending on which FKBP protein is employed. An
FKBP domain of a fusion protein of this disclosure will be capable
of binding to rapamycin or a rapalog thereof and participating in a
tripartite complex with an FRB-containing protein (as may be
determined by any means, direct or indirect, for detecting such
binding).
[0183] The peptide sequence of an FKBP domain of an FKBP fusion
protein of this invention comprises (a) a naturally occurring FKBP
peptide sequence, preferably derived from the human FKBP12 protein
(GenBank Accession No. AAA58476.1) or a peptide sequence derived
therefrom, from another human FKBP, from a murine or other
mammalian FKBP, or from some other animal, yeast or fungal FKBP;
(b) a variant of a naturally occurring FKBP sequence in which up to
about ten amino acids, or about 1 to about 5 amino acids or about 1
to about 3 amino acids, or in some embodiments just one amino acid,
of the naturally-occurring peptide have been deleted, inserted, or
substituted; or (c) a peptide sequence encoded by a nucleic acid
molecule capable of selectively hybridizing to a DNA molecule
encoding a naturally occurring FKBP or by a DNA sequence which
would be capable, but for the degeneracy of the genetic code, of
selectively hybridizing to a DNA molecule encoding a naturally
occurring FKBP.
[0184] Other multimerization domain pairs include FKBP and
calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR,
calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or
variants thereof.
[0185] In yet other embodiments, an anti-bridging factor blocks the
association of at least two first fusion proteins with the bridging
factor. For example, cyclosporin or FK506 could be used as
anti-bridging factors to titrate out rapamycin and, therefore, stop
signaling since only one multimerization domain is bound. In
certain embodiments, an anti-bridging factor (e.g., cyclosporine,
FK506) is an immunosuppressive agent. For example, an
immunosuppressive anti-bridging factor may be used to block or
minimize the function of the fusion proteins of the instant
disclosure and at the same time inhibit or block an unwanted or
pathological inflammatory response in a clinical setting.
[0186] In certain embodiments, a first fusion protein (e.g., DARIC
signaling component) has a first multimerization domain comprising
a first FKBP polypeptide or variant thereof, and a second fusion
protein (e.g., DARIC binding component) has a second
multimerization domain comprising a first FRB polypeptide or
variant thereof. In other embodiments, a first fusion protein
(e.g., DARIC signaling component) has a first multimerization
domain comprising a first FRB polypeptide or variant thereof, and a
second fusion protein (e.g., DARIC binding component) has a second
multimerization domain comprising a first FKBP polypeptide or
variant thereof. In any of these embodiments, the second fusion
protein further comprises an anchor domain (e.g., transmembrane
domain, GPI signal sequence) and optionally a sub-threshold
signaling domain. In some embodiments, a second fusion protein
contains a GPI molecule, wherein the GPI signal sequence has been
removed or altered to attach the GPI molecule.
[0187] In certain embodiments, a first nucleic acid molecule
encoding a first fusion protein comprising a first multimerization
domain, a third multimerization domain, a hydrophobic domain, and
an actuator domain, wherein the first and third multimerization
domains localize extracellularly when the first fusion protein is
expressed in a cell. In certain embodiments, the third
multimerization domain of the first fusion protein is a binding
domain for a bridging factor selected from rapamycin or a rapalog
thereof, coumermycin or a derivative thereof, gibberellin or a
derivative thereof, ABA or a derivative thereof, methotrexate or a
derivative thereof, cyclosporin A or a derivative thereof, FKCsA or
a derivative thereof, Tmp-SLF or a derivative thereof, or any
combination thereof.
[0188] In still further embodiments, a second bridging factor
promotes the association of at least two first fusion proteins with
the bridging factor associated with and disposed between the third
multimerization domains of the first fusion proteins. In certain
embodiments, a protein complex that is formed is a homocomplex
comprising at least two first fusion proteins, wherein the
multimerization domains may be DHFR (with the bridging molecule
being methotrexate) or GyrB (with the bridging molecule being
coumermycin) or FKBP (with the bridging molecule being AP1903 or
AP20187). In certain other embodiments, a protein complex is a
heterocomplex comprising one or more first fusion proteins and one
or more second fusion proteins.
[0189] In certain embodiments, a hydrophobic domain is a
transmembrane domain, such as a transmembrane domain from CD4, CD8,
CD28, CD71, CD154, AMN or the like. In some embodiments, a fusion
protein (e.g., DARIC binding component) comprises an anchor domain,
such as a transmembrane domain or GPI signal sequence. In certain
embodiments, the transmembrane domain is from CD4, CD8, CD28, CD71,
CD154, AMN or the like. In further embodiments, a fusion protein
(e.g., DARIC binding component) contains a GPI molecule, wherein
the GPI signal sequence has been removed or altered to attach the
GPI molecule.
[0190] In further embodiments, the actuator domain comprises a
lymphocyte receptor signaling domain or comprises an amino acid
sequences having one or a plurality of immunoreceptor
tyrosine-based activation motifs (ITAMs). In still further
embodiments, an actuator domain comprises a cytoplasmic portion
that associates with a cytoplasmic signaling protein, wherein the
cytoplasmic signaling protein is a lymphocyte receptor or signaling
domain thereof, a protein comprising a plurality of immunoreceptor
tyrosine-based activation motifs (ITAMs), a costimulatory domain,
an adhesion factor, or any combination thereof. Exemplary actuator
domains include, but are not limited to, CD2, CD3.epsilon.,
CD3.delta., CD3.zeta., pT.alpha., TCR.alpha., TCR.beta.,
FcR.alpha., FcR.beta., FcR.gamma., NKG2D, CD22, CD79A, and CD79B,
CD27, CD28, CD30, CD40, LAT, Zap70, ICOS, DAP10, 4-1BB, CARD11,
HVEM, LAG3, SLAMF1, Lck, Fyn, Slp76, TRIM, OX40, or any combination
thereof. In yet further embodiments, a first nucleic acid molecule
encodes the first fusion protein further comprising one or more
different actuator domains, costimulatory domains, adhesion
factors, or any combination thereof. As used herein, the term,
"costimulatory signaling domain," or "costimulatory domain", refers
to an intracellular signaling domain of a costimulatory factor.
Exemplary costimulatory domains include, but are not limited to
intracellular signaling domains from CD2, CD27, CD28, CD30, CD40,
LAT, Zap70, ICOS, DAP10, 4-1BB, CARD11, HVEM, LAG3, SLAMF1, Lck,
Fyn, Slp76, TRIM, and OX40.
[0191] In certain embodiments, a non-natural cell further
overexpresses a costimulatory factor, an immunomodulatory factor,
an agonist for a costimulatory factor, an agonist for an
immunomodulatory factor, or any combination thereof. In a related
embodiment, cofactor IL-12 is overexpressed or supplied to the
cell.
[0192] Fusion protein binding domains useful in the instant
invention include those known in the art or as described herein, or
those generated by a variety of methods known in the art (see,
e.g., U.S. Pat. Nos. 6,291,161 and 6,291,158). For example, fusion
protein binding domains may be identified by screening a Fab phage
library for Fab fragments that specifically bind to a target of
interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005).
Additionally, traditional strategies for hybridoma development,
such as using a target antigen as an immunogen in convenient
systems (e.g., mice, HuMAb mouse.RTM., TC mouse.TM., KM-mouse
.RTM., llamas, sheep, chicken, rats, hamsters, rabbits, etc.), can
be used to develop anti-target antibodies having target-specific
binding domains of interest.
[0193] Sources of further binding domains include target-specific
antibody variable domains from various species (which can be
formatted as antibodies, sFvs, scFvs, Fabs, or soluble VH domain or
domain antibodies), including human, rodent, avian, and ovine.
Additional sources of binding domains include variable domains of
antibodies from other species, such as camelid (from camels,
dromedaries, or llamas (Ghahroudi et al., FEBS Letters 414:521,
1997; Vincke et al., J. Biol. Chem. 284:3273, 2009; and
Hamers-Casterman et al., Nature 363:446, 1993; and Nguyen et al.,
J. Mol. Biol. 275:413, 1998), nurse sharks (Roux et al., Proc.
Nat'l. Acad. Sci. (USA) 95:11804, 1998), spotted ratfish (Nguyen et
al., Immunogenetics 54:39, 2002), or lamprey (Herrin et al., Proc.
Nat'l. Acad. Sci. (USA) 105:2040, 2008 and Alder et al., Nature
Immunol. 9:319, 2008). These antibodies can apparently form
antigen-binding regions using only heavy chain variable region,
i.e., these functional antibodies are homodimers of heavy chains
only (referred to as "heavy chain antibodies") (Jespers et al.,
Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res.
64:2853, 2004; Baral et al., Nature Med. 12:580, 2006, and
Barthelemy et al., J. Biol. Chem. 283:3639, 2008).
[0194] Other alternative sources of target-specific binding domains
includes sequences that encode random peptide libraries or
sequences that encode an engineered diversity of amino acids in
loop regions of alternative non-antibody scaffolds, such as
fibrinogen domains (see, e.g., Weisel et al. (1985) Science
230:1388), Kunitz domains (see, e.g., U.S. Pat. No. 6,423,498),
ankyrin repeat proteins (also known as DARPins; Binz et al., J.
Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575,
2004), fibronectin binding domains (also known as adnectins or
monobodies; Richards et al., J. Mol. Biol. 326:1475, 2003; Parker
et al., Protein Eng. Des. Sel. 18:435, 2005 and Hackel et al., J.
Mol. Biol. 381:1238, 2008), cysteine-knot miniproteins (Vita et
al., Proc. Nat'l. Acad. Sci. (USA) 92:6404, 1995; Martin et al.,
Nat. Biotechnol. 21:71, 2002 and Huang et al., Structure 13:755,
2005), tetratricopeptide repeat domains (Main et al., Structure
11:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008),
leucine-rich repeat domains (Stumpp et al., J. Mol. Biol. 332:471,
2003), anticalins (Skerra, FEBS J. 275:2677, 2008), lipocalin
domains (see, e.g., PCT Publication No. WO 2006/095164, Beste et
al., Proc. Nat'l. Acad. Sci. (USA) 96:1898, 1999 and Schonfeld et
al., Proc. Nat'l. Acad. Sci. (USA) 106:8198, 2009), armadillo
repeat proteins (ArmRPs; Varadamsetty et al., J. Mol. Biol. 424:68,
2012), diabodies (Manzke et al., Int. J. Cancer 82:700, 1999),
repebodies (Lee et al., Proc. Nat'l. Acad. Sci. U.S.A. 109: 3299,
2012), minibodies (Hu et al., Cancer Res. 56:3055, 1996),
cyclotides (Craik et al., J. Mol. Biol. 294:1327, 1999), V-like
domains (see, e.g., US Patent Application Publication No.
2007/0065431), C-type lectin domains (Zelensky and Gready, FEBS J.
272:6179, 2005; Beavil et al.I, Proc. Nat'l. Acad. Sci. (USA)
89:753, 1992 and Sato et al., Proc. Nat'l. Acad. Sci. (USA)
100:7779, 2003), mAb.sup.2 or Fcab.TM. (see, e.g., PCT Publication
Nos. WO 2007/098934; WO 2006/072620), or the like (Nord et al.,
Protein Eng. 8:601, 1995; Nord et al., Nat. Biotechnol. 15:772,
1997; Nord et al., Eur. J. Biochem. 268:4269, 2001; and Binz et al.
(2005) Nat. Biotechnol. 23:1257, 2005).
[0195] In certain embodiments, the binding domain of the second
fusion protein is a single chain antibody variable region, a
receptor ectodomain, or a ligand. In further embodiments, the
single chain antibody variable region is a domain antibody, sFv,
scFv, F(ab').sub.2, or Fab. In still further embodiments, the
binding domain of the second fusion protein is amino or carboxy
terminal to the multimerization domain.
[0196] In certain further aspects, a non-natural cell comprises a
nucleic acid molecule that encodes a fusion comprising a binding
domain and multimerization domain, and optionally an anchor domain
(e.g., transmembrane domain, GPI signal sequence) or an anchor
domain with a sub-threshold signaling domain, wherein the binding
domain specifically binds to a target located on a target cell
surface. In further embodiments, a binding domain is specific for a
target that is an antigen associated with a cancer (e.g., solid
malignancy, hematologic malignancy), an inflammatory disease, an
autoimmune disease, or a graft versus host disease. Exemplary
target antigens include, but are not limited to, .alpha.-folate
receptor, .alpha..sub.v.beta..sub.6 integrin, BCMA, B7-H3, B7-H6,
CAIX, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8,
CD70, CD123, CD138, CD171, CEA, DLL4, EGP-2, EGP-40, CSPG4, EGFR,
EGFR family including ErbB2 (HER2), EGFRvIII, EPCAM, EphA2, EpCAM,
FAP, FBP, fetal acetylcholine receptor, Fzd7, GD2, GD3, Glypican-3
(GPC3), h5T4, IL-11R.alpha., IL13R-.alpha.2, KDR, .kappa. light
chain, .lamda. light chain, LeY, L1CAM, MAGE-A1, mesothelin, MHC
presented peptides, MUC1, MUC16, NCAM, NKG2D ligands, Notchl,
Notch2/3, NY-ESO-1, PRAME, PSCA, PSMA, Survivin, TAG-72, TEMs,
TERT, VEGFR2, and ROR1.
[0197] In certain embodiments, such a binding fusion protein (DARIC
binding component) forms a tripartite complex with DARIC signaling
component and a bridging factor to form a polypeptide complex.
Exemplary bridging factors for such a complex include rapamycin or
a rapalog thereof, coumermycin or a derivative thereof, gibberellin
or a derivative thereof, ABA or a derivative thereof, methotrexate
or a derivative thereof, cyclosporin A or a derivative thereof,
FKCsA or a derivative thereof, or Tmp-SLF or a derivative
thereof.
[0198] In other embodiments, the instant disclosure is directed to
a non-natural cell comprising (a) a heterologous first nucleic acid
molecule encoding a first fusion protein comprising a first
multimerization domain, a hydrophobic domain, and an actuator
domain, wherein the first multimerization domain localizes
extracellularly when the first fusion protein is expressed; and (b)
a second nucleic acid molecule encoding a second fusion protein
comprising a binding domain, a second multimerization domain and an
anchor domain (e.g., transmembrane domain, GPI molecule), wherein
the second fusion protein localizes to the cell surface when
expressed; wherein a first bridging factor promotes the formation
of a polypeptide complex on the non-natural cell surface with the
bridging factor associated with and disposed between the
multimerization domains of the first and second fusion proteins. In
certain embodiments, the second fusion protein further comprises an
intracellularly localized sub-threshold signaling domain.
[0199] As used herein, a "sub-threshold signaling domain" is not
capable of inducing or activating a sufficiently robust signal
transduction cascade in the presence of one or more other
sub-threshold signaling domains, but can induce or activate a
signal transduction cascade or adjust a signal qualitatively in the
presence of an actuator domain. For example, a second fusion
protein tethered to a cell surface that associates with another
second fusion protein tethered to a cell surface will not induce or
will minimally activate signal transduction. Exemplary
sub-threshold signaling domains include costimulatory domains, such
as CD28, CD2, CD4, CD5, CD8, CD9, CD27, CD44, CD46, CD81, CD137,
LFA-1, ICAM-1, VLA-4, OX40, 4-1BB, LIGHT, SLAM, ICOS, CTLA-4, PD-1,
or the like.
[0200] In particular embodiments, an encoded first fusion protein
comprises a first multimerization domain of FRB T2098L, a
transmembrane domain, a costimulatory domain of 4-1BB, and actuator
domain of CD3.zeta. wherein the second encoded fusion protein
comprises a binding domain of an scFv specific for CD19 and a
second multimerization domain of FKBP12, and optionally an anchor
domain (e.g., transmembrane domain, GPI signal sequence) or an
anchor domain with a sub-threshold signaling domain; and wherein
the first bridging factor that promotes the formation of a
polypeptide complex on the non-natural cell surface is rapalog
AP21967. An exemplary first fusion protein has an amino acid
sequence as set forth in SEQ ID NO.:15 and an exemplary second
fusion protein has an amino acid sequence as set forth in SEQ ID
NO.:1 or 56.
[0201] In certain embodiments, a DARIC binding component may have
multiple binding domains. For example, a non-natural cell further
comprises a third nucleic acid molecule encoding a third fusion
protein comprising a binding domain and a second multimerization
domain, optionally an anchor domain (e.g., transmembrane domain,
GPI signal sequence) or an anchor domain with a sub-threshold
signaling domain, wherein the third fusion protein localizes
extracellularly when expressed. In related embodiments, the fusion
proteins comprise a binding domain have one, two, three, or four
binding domains, wherein the one, two, three, or four binding
domains are specific for one target or up to four different
targets.
[0202] In any of the aforementioned embodiments, a second nucleic
acid molecule encoding a second (binding) fusion protein may
further comprise a sequence encoding a linker, spacer or junction
amino acids disposed between the binding domain and the second
multimerization domain. In certain embodiments, a second nucleic
acid molecule encoding a second fusion protein (e.g., DARIC binding
component) further comprises an anchor domain (e.g., transmembrane
domain, GPI signal sequence) and optionally a sub-threshold
signaling domain. In further embodiments, a second fusion protein
(e.g., DARIC binding component) contains a GPI molecule, wherein
the GPI signal sequence has been removed or altered to attach the
GPI molecule.
[0203] Exemplary diseases or disorders associated with excess
receptor-mediated signal transduction include cancer (e.g., solid
malignancy and hematologic malignancy), autoimmune or inflammatory
diseases or conditions, sepsis resulting from bacterial infection,
and viral infection.
[0204] In one aspect, the present disclosure provides a method for
directing T cell activation, comprising administering to a subject
in need thereof an effective amount of a DARIC binding component or
a pharmaceutical composition thereof that specifically binds a
target, such as a cell surface target that is a tumor-specific
antigen or other antigen of choice at a site or cell where T cell
activation is desired.
[0205] Pharmaceutically acceptable carriers for therapeutic use are
also well known in the pharmaceutical art, and are described, for
example, in the Physicians Desk Reference, 62nd edition. Oradell,
N.J.: Medical Economics Co., 2008; Goodman & Gilman's The
Pharmacological Basis of Therapeutics, Eleventh Edition.
McGraw-Hill, 2005; Remington: The Science and Practice of Pharmacy,
20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins,
2000; and The Merck Index, Fourteenth Edition. Whitehouse Station,
N.J.: Merck Research Laboratories, 2006; each of which is hereby
incorporated by reference in relevant parts. Exemplary
pharmaceutically acceptable carriers include sterile saline and
phosphate buffered saline at physiological pH. Preservatives,
stabilizers, dyes and the like may be provided in the
pharmaceutical composition. In addition, antioxidants and
suspending agents may also be used.
[0206] Pharmaceutical compositions may also contain diluents such
as buffers, antioxidants such as ascorbic acid, low molecular
weight (less than about 10 residues) polypeptides, proteins, amino
acids, carbohydrates (e.g., glucose, sucrose, dextrins), chelating
agents (e.g., EDTA), glutathione and other stabilizers and
excipients. Neutral buffered saline or saline mixed with
nonspecific serum albumin are exemplary diluents.
[0207] In another aspect, the present disclosure provides a method
for inhibiting growth, metastasis or metastatic growth of a
malignancy (e.g., a solid malignancy or a hematologic malignancy),
comprising administering to a subject in need thereof an effective
amount of a cell encoding a polypeptide complex provided herein or
a composition thereof.
[0208] A wide variety of cancers, including solid malignancy and
hematologic malignancy, are amenable to the compositions and
methods disclosed herein. Types of cancer that may be treated
include adenocarcinoma of the breast, prostate, pancreas, colon and
rectum; all forms of bronchogenic carcinoma of the lung (including
squamous cell carcinoma, adenocarcinoma, small cell lung cancer and
non-small cell lung cancer); myeloid; melanoma; hepatoma;
neuroblastoma; papilloma; apudoma; choristoma; branchioma;
malignant carcinoid syndrome; carcinoid heart disease; and
carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce,
ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small
cell lung, oat cell, papillary, scirrhous, bronchiolar,
bronchogenic, squamous cell, and transitional cell). Additional
types of cancers that may be treated include: histiocytic
disorders; leukemia; histiocytosis malignant; Hodgkin's disease;
non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis;
melanoma; renal cell carcinoma; chondroblastoma; chondroma;
chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors;
histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;
myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma;
dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma;
ameloblastoma; cementoma; odontoma; teratoma; thymoma;
trophoblastic tumor.
[0209] Further, the following types of cancers are also
contemplated as amenable to treatment: adenoma; cholangioma;
cholesteatoma; cyclindroma; cystadenocarcinoma; cystadenoma;
granulosa cell tumor; gynandroblastoma; hepatoma; hidradenoma;
islet cell tumor; Leydig cell tumor; papilloma; sertoli cell tumor;
theca cell tumor; leimyoma; leiomyosarcoma; myoblastoma; myomma;
myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma;
ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma;
neuroblastoma; neuroepithelioma; neurofibroma; neuroma;
paraganglioma; paraganglioma nonchromaffin; and glioblastoma
multiforme. The types of cancers that may be treated also include
angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angioma
sclerosing; angiomatosis; glomangioma; hemangioendothelioma;
hemangioma; hemangiopericytoma; hemangiosarcoma; lymphangioma;
lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma;
chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma;
hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma;
lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma;
rhabdomyosarcoma; sarcoma; neoplasms; neurofibromatosis; and
cervical dysplasia.
[0210] Additional exemplary cancers that are also amenable to the
compositions and methods disclosed herein are B-cell cancers,
including B-cell lymphomas (such as various forms of Hodgkin's
disease, non-Hodgkins lymphoma (NHL) or central nervous system
lymphomas), leukemias (such as acute lymphoblastic leukemia (ALL),
chronic lymphocytic leukemia (CLL), Hairy cell leukemia and chronic
myoblastic leukemia) and myelomas (such as multiple myeloma).
Additional B cell cancers include small lymphocytic lymphoma,
B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic
marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma
of bone, extraosseous plasmacytoma, extra-nodal marginal zone
B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal
marginal zone B-cell lymphoma, follicular lymphoma, mantle cell
lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large
B-cell lymphoma, intravascular large B-cell lymphoma, primary
effusion lymphoma, Burkitt lymphoma/leukemia, B-cell proliferations
of uncertain malignant potential, lymphomatoid granulomatosis, and
post-transplant lymphoproliferative disorder.
[0211] In certain embodiments, cells encoding polypeptide complexes
useful for inhibiting growth of a solid malignancy or metastasis or
metastatic growth of a solid malignancy or a hematologic malignancy
include those that specifically bind to a tumor or cancer antigen
and a second target antigen on the cancer cell.
[0212] In another aspect, the present disclosure provides a method
for treating an autoimmune or inflammatory disease, disorder or
condition, comprising administering to a subject in need thereof an
effective amount of a cell encoding a polypeptide complex provided
herein or a composition thereof.
[0213] Exemplary autoimmune or inflammatory diseases, disorders or
conditions that may be treated by the fusion proteins and
compositions and unit dose forms thereof include inflammatory bowel
disease (e.g., Crohn's disease or ulcerative colitis), diabetes
mellitus (e.g., type I diabetes), dermatomyositis, polymyositis,
pernicious anaemia, primary biliary cirrhosis, acute disseminated
encephalomyelitis (ADEM), Addison's disease, ankylosing
spondylitis, antiphospholipid antibody syndrome (APS), autoimmune
hepatitis, Goodpasture's syndrome, Graves' disease, Guillain-Barre
syndrome (GB S), Hashimoto's disease, idiopathic thrombocytopenic
purpura, systemic lupus erythematosus, lupus nephritis,
neuropsychiatric lupus, multiple sclerosis (MS), myasthenia gravis,
pemphigus vulgaris, asthma, psoriatic arthritis, rheumatoid
arthritis, Sjogren's syndrome, temporal arteritis (also known as
"giant cell arteritis"), autoimmune hemolytic anemia, Bullous
pemphigoid, vasculitis, coeliac disease, chronic obstructive
pulmonary disease, endometriosis, Hidradenitis suppurativa,
interstitial cystitis, morphea, scleroderma, narcolepsy,
neuromyotonia, vitiligo, and autoimmune inner ear disease.
[0214] In certain embodiments, a method for treating a
hyperproliferative, inflammatory, autoimmune, or graft-versus-host
disease, comprises (a) administering a recombinant cell comprising
a first and a second nucleic acid molecule, wherein the first
nucleic acid molecule encodes a first fusion protein comprising a
first multimerization domain, a hydrophobic domain, and an actuator
domain, wherein the first multimerization domain localizes
extracellularly when the first fusion protein is expressed, and the
second nucleic acid molecule encodes a second fusion protein
comprising a binding domain and a second multimerization domain,
wherein the second fusion protein localizes extracellularly when
expressed; and (c) administering a bridging factor, wherein the
bridging factor promotes the formation of a polypeptide complex on
the recombinant cell surface with the bridging factor associated
with and disposed between the multimerization domains of the first
and second fusion proteins; wherein the binding domain of the
polypeptide complex specifically binds a cell surface target on a
hyperproliferative disease cell to promote an immunomodulatory
response and thereby treats the hyperproliferative disease.
[0215] In particular embodiments, a method for treating a
hyperproliferative, inflammatory, autoimmune, or graft-versus-host
disease, comprises (a) administering one or more recombinant cells
comprising a first nucleic acid molecule and a second nucleic acid
molecule, wherein the first nucleic acid molecule encodes a first
fusion protein comprising a binding agent that binds a receptor
expressed on a T cell and first multimerization domain, and the
second nucleic acid molecule encodes a second fusion protein
comprising a binding agent that binds a cell surface target on a
hyperproliferative disease cell and a second multimerization
domain, and (c) administering a bridging factor, wherein the
bridging factor promotes the formation of a polypeptide complex,
e.g., a BiTE, with the bridging factor associated with and disposed
between the multimerization domains of the first and second fusion
proteins; wherein the binding agent of the first fusion protein
binds a receptor on a T cell and the binding agent of the second
fusion protein binds a cell surface target on a hyperproliferative
disease cell to promote an immunomodulatory response and thereby
treats the hyperproliferative disease.
[0216] In other embodiments, a method for treating a
hyperproliferative, inflammatory, autoimmune, or graft-versus-host
disease, comprises (a) administering a non-natural cell comprising
a first nucleic acid molecule encoding a first fusion protein
comprising a first multimerization domain, a hydrophobic domain,
and an actuator domain, wherein the first multimerization domain
localizes extracellularly when the first fusion protein is
expressed; (b) administering a second fusion protein comprising a
binding domain and a second multimerization domain, optionally
comprising an anchor domain (e.g., transmembrane domain, GPI signal
sequence) or an anchor domain with a sub-threshold signaling
domain; and (c) administering a bridging factor, wherein the
bridging factor promotes the formation of a polypeptide
heterocomplex on the recombinant cell surface with the bridging
factor associated with and disposed between the multimerization
domains of the first and second fusion proteins; wherein the
binding domain of the polypeptide heterocomplex specifically binds
a cell surface target on a hyperproliferative disease cell to
promote an immunomodulatory response and thereby treats the
hyperproliferative disease.
[0217] Any of the aforementioned non-natural cells, fusion
proteins, bridging factors and other accessory molecules may be
used in the methods of treatment of this disclosure. In certain
embodiments, a method further comprises administering an agent that
antagonizes or blocks an inhibitor of T cell activation, such as an
agent that antagonizes or blocks a T cell ligand or a T cell
receptor. In certain embodiments, an agent that antagonizes or
blocks an inhibitor of T cell activation is an anti-PD1 antibody,
anti-PD-L1 antibody, or an anti-CTLA4 antibody or antigen binding
fragment thereof, or an engineered homing endonuclease that targets
PD-1. In further embodiments, the method further comprises
administering a cytokine agonist.
[0218] The cells, fusion proteins, bridging factors, other
accessory molecules or compositions thereof of the present
disclosure may be administered orally, topically, transdermally,
parenterally, by inhalation spray, vaginally, rectally, or by
intracranial injection, or any combination thereof. In certain
embodiments, fusion proteins, bridging factors, or compositions
thereof are administered parenterally. The term "parenteral," as
used herein, includes subcutaneous injections, intravenous,
intramuscular, intracisternal injection, or infusion techniques.
Administration by intravascular, intravenous, intraarterial,
intradermal, intramuscular, intramammary, intraperitoneal,
intrathecal, retrobulbar, intrapulmonary injection and/or surgical
implantation at a particular site is contemplated as well. In
certain embodiments, fusion proteins, bridging factors, or
compositions thereof are administered by injection, such as
intravenously.
[0219] Also contemplated is the administration of recombinant cells
with a bridging factor, recombinant cells with a fusion protein and
a bridging factor, or compositions thereof in combination with a
second agent. A second agent may be one accepted in the art as a
standard treatment for a particular disease state or disorder, such
as in cancer, inflammation, autoimmunity, and infection. Exemplary
second agents contemplated include recombinant cells with a
bridging factor, recombinant cells with a fusion protein and a
bridging factor, or compositions thereof that bind to targets
different from those that the primary protein complex binds,
polyclonal antibodies, monoclonal antibodies,
immunoglobulin-derived fusion proteins, chemotherapeutics, ionizing
radiation, steroids, NSAIDs, anti-infective agents, or other active
and ancillary agents, or any combination thereof.
[0220] Second agents useful in combination with recombinant cells
with a bridging factor, recombinant cells with a fusion protein and
a bridging factor, or compositions thereof provided herein may be
steroids, NSAIDs, mTOR inhibitors (e.g., rapamycin (sirolimus),
temsirolimus, deforolimus, everolimus, zotarolimus, curcumin,
farnesylthiosalicylic acid), calcineurin inhibitors (e.g.,
cyclosporine, tacrolimus), anti-metabolites (e.g., mycophenolic
acid, mycophenolate mofetil), polyclonal antibodies (e.g.,
anti-thymocyte globulin), monoclonal antibodies (e.g., daclizumab,
basiliximab), and CTLA4-Ig fusion proteins (e.g., abatacept or
belatacept).
[0221] Second agents useful for inhibiting growth of a solid
malignancy, inhibiting metastasis or metastatic growth of a solid
malignancy, or treating or ameliorating a hematologic malignancy
include chemotherapeutic agents, ionizing radiation, and other
anti-cancer drugs. Examples of chemotherapeutic agents contemplated
as further therapeutic agents include alkylating agents, such as
nitrogen mustards (e.g., mechlorethamine, cyclophosphamide,
ifosfamide, melphalan, and chlorambucil); bifunctional
chemotherapeutics (e.g., bendamustine); nitrosoureas (e.g.,
carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU));
ethyleneimines and methyl-melamines (e.g., triethylenemelamine
(TEM), tri ethylene thiophosphoramide (thiotepa), and
hexamethylmelamine (HIVIM, altretamine)); alkyl sulfonates (e.g.,
buslfan); and triazines (e.g., dacabazine (DTIC)); antimetabolites,
such as folic acid analogues (e.g., methotrexate, trimetrexate, and
pemetrexed (multi-targeted antifolate)); pyrimidine analogues (such
as 5-fluorouracil (5-FU), fluorodeoxyuridine, gemcitabine, cytosine
arabinoside (AraC, cytarabine), 5-azacytidine, and
2,2'-difluorodeoxycytidine); and purine analogues (e.g,
6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin
(pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine
phosphate, 2-chlorodeoxyadenosine (cladribine, 2-CdA)); Type I
topoisomerase inhibitors such as camptothecin (CPT), topotecan, and
irinotecan; natural products, such as epipodophylotoxins (e.g.,
etoposide and teniposide); and vinca alkaloids (e.g., vinblastine,
vincristine, and vinorelbine); anti-tumor antibiotics such as
actinomycin D, doxorubicin, and bleomycin; radiosensitizers such as
5-bromodeozyuridine, 5-iododeoxyuridine, and bromodeoxycytidine;
platinum coordination complexes such as cisplatin, carboplatin, and
oxaliplatin; substituted ureas, such as hydroxyurea; and
methylhydrazine derivatives such as N-methylhydrazine (MIH) and
procarbazine.
[0222] Further therapeutic agents contemplated by this disclosure
for treatment of autoimmune diseases are referred to as
immunosuppressive agents, which act to suppress or mask the immune
system of the individual being treated. Immunosuppressive agents
include, for example, non-steroidal anti-inflammatory drugs
(NSAIDs), analgesics, glucocorticoids, disease-modifying
antirheumatic drugs (DMARDs) for the treatment of arthritis, or
biologic response modifiers. Compositions in the DMARD description
are also useful in the treatment of many other autoimmune diseases
aside from rheumatoid arthritis.
[0223] Exemplary NSAIDs include ibuprofen, naproxen, naproxen
sodium, Cox-2 inhibitors (such as Vioxx or Celebrex), and
sialylates. Exemplary analgesics include acetaminophen, oxycodone,
tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids
include cortisone, dexamethasone, hydrocortisone,
methylprednisolone, prednisolone, or prednisone. Exemplary
biological response modifiers include molecules directed against
cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors,
such as the TNF antagonists (e.g. etanercept (Enbrel), adalimumab
(Humira) and infliximab (Remicade)), chemokine inhibitors and
adhesion molecule inhibitors. The biological response modifiers
include monoclonal antibodies as well as recombinant forms of
molecules. Exemplary DMARDs include azathioprine, cyclophosphamide,
cyclosporine, methotrexate, penicillamine, leflunomide,
sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and
intramuscular) and minocycline.
[0224] In still further aspects, the instant disclosure provides a
fusion polypeptide heterocomplex, comprising (a) a first fusion
protein comprising a first multimerization domain, a hydrophobic
domain, and an actuator domain; (b) a second fusion protein
comprising an extracellular binding domain and second
multimerization domain; and (c) a bridging factor; wherein the
first fusion protein, second fusion protein, and bridging factor
associate to form a polypeptide heterocomplex with the bridging
factor associated with and disposed between the multimerization
domains of the first and second fusion proteins. Any of the
aforementioned fusion protein components and bridging factors and
may be used in these embodiments.
[0225] In other aspects, the instant disclosure provides a nucleic
acid molecule encoding any one or more of the aforementioned fusion
proteins. Such nucleic acid molecules may be incorporated into an
expression vector (e.g., lentiviral vector), wherein the first and
second fusion proteins are encoded as a polycistronic message or as
a single protein separated by a 2A peptide. In certain embodiments,
the polycistronic message comprises an internal ribosome entry site
(IRES) between the nucleotide sequences that encode the fusion
proteins.
[0226] Illustrative examples of DARIC binding and signaling
components are provided in SEQ ID NOs: 1-100 and below in Table
1.
TABLE-US-00001 TABLE 1 Exemplary DARIC Binding and Signaling
Components SEQ ID NO. Construct Sequence 1 scFvCD19-FKBP
MGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDG protein
TVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQ
QGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGP
GLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS
ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY
YYGGSYAMDYWGQGTSVTVSSASGGGGSGVQVETISPGDGRTFP
KRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGW
EEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL KLEG 2
SS-scFvCD19-FKBP AUGCCCCUGGGCCUGCUGUGGCUGGGCCUGGCCCUGCUGGGC mRNA
GCCCUGCACGCCCAGGCCGGAUCCGAUAUCCAGAUGACCCAG
ACCACCAGCAGCCUGAGCGCCAGCCUGGGCGAUAGAGUGACC
AUCAGCUGCAGAGCCAGCCAGGACAUCAGCAAGUACCUGAA
CUGGUAUCAGCAGAAACCCGACGGCACCGUGAAGCUGCUGA
UCUACCACACCAGCAGACUGCACAGCGGCGUGCCCAGCAGAU
UUUCUGGCAGCGGCUCCGGCACCGACUACAGCCUGACCAUCU
CCAACCUGGAACAGGAAGAUAUCGCUACCUACUUCUGUCAG
CAAGGCAACACCCUGCCCUACACCUUCGGCGGAGGCACCAAG
CUGGAAAUCACCGGCAGCACAAGCGGCAGCGGCAAGCCUGG
AUCUGGCGAGGGAAGCACCAAGGGCGAAGUGAAACUGCAGG
AAAGCGGCCCUGGACUGGUGGCCCCAAGCCAGUCUCUGAGCG
UGACCUGUACCGUGUCCGGCGUGUCCCUGCCUGACUAUGGCG
UGUCCUGGAUCAGACAGCCCCCCAGAAAGGGCCUGGAAUGG
CUGGGAGUGAUCUGGGGCAGCGAGACAACCUACUACAACAG
CGCCCUGAAGUCCCGGCUGACCAUCAUCAAGGACAACUCCAA
GAGCCAGGUGUUCCUGAAGAUGAACAGCCUGCAGACCGACG
ACACCGCCAUCUACUACUGCGCCAAGCACUACUACUACGGCG
GCAGCUACGCCAUGGACUACUGGGGCCAGGGCACAAGCGUG
ACCGUGUCCAGCGCUAGCGGCGGAGGUGGGAGCGGAGUGCA
GGUGGAAACCAUCUCCCCAGGAGACGGGCGCACCUUCCCCAA
GCGCGGCCAGACCUGCGUGGUGCACUACACCGGGAUGCUUG
AAGAUGGAAAGAAAUUUGAUUCCUCCCGGGACAGAAACAAG
CCCUUUAAGUUUAUGCUAGGCAAGCAGGAGGUGAUCCGAGG
CUGGGAAGAAGGGGUUGCCCAGAUGAGUGUGGGUCAGAGAG
CCAAACUGACUAUAUCUCCAGAUUAUGCCUAUGGUGCCACU
GGGCACCCAGGCAUCAUCCCACCACAUGCCACUCUCGUCUUC
GAUGUGGAGCUUCUAAAACUGGAAGGCUGA 3 SS-scFvCD19-FKBP
ATGCCCCTGGGCCTGCTGTGGCTGGGCCTGGCCCTGCTGGGCG DNA
CCCTGCACGCCCAGGCCGGATCCGATATCCAGATGACCCAGAC
CACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCAT
CAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG
GTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTAC
CACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTG
GCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCT
GGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAAC
ACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCA
CCGGCAGCACAAGCGGCAGCGGCAAGCCTGGATCTGGCGAGG
GAAGCACCAAGGGCGAAGTGAAACTGCAGGAAAGCGGCCCTG
GACTGGTGGCCCCAAGCCAGTCTCTGAGCGTGACCTGTACCGT
GTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGA
CAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGG
GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG
CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGA
AGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTG
CGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTAC
TGGGGCCAGGGCACAAGCGTGACCGTGTCCAGCGCTAGCGGC
GGAGGTGGGAGCGGAGTGCAGGTGGAAACCATCTCCCCAGGA
GACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGC
ACTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTC
CCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCA
GGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAG
TGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCC
TATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCA
CTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGCTGA 4 scFvCD19-FKBP
MGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDG (F36V) protein
TVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQ
QGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGP
GLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS
ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY
YYGGSYAMDYWGQGTSVTVSSASGGGGSGVQVETISPGDGRTFP
KRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGW
EEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL KLEG 5
SS-scFvCD19-FKBP AUGCCCCUGGGCCUGCUGUGGCUGGGCCUGGCCCUGCUGGGC (F36V)
mRNA GCCCUGCACGCCCAGGCCGGAUCCGAUAUCCAGAUGACCCAG
ACCACCAGCAGCCUGAGCGCCAGCCUGGGCGAUAGAGUGACC
AUCAGCUGCAGAGCCAGCCAGGACAUCAGCAAGUACCUGAA
CUGGUAUCAGCAGAAACCCGACGGCACCGUGAAGCUGCUGA
UCUACCACACCAGCAGACUGCACAGCGGCGUGCCCAGCAGAU
UUUCUGGCAGCGGCUCCGGCACCGACUACAGCCUGACCAUCU
CCAACCUGGAACAGGAAGAUAUCGCUACCUACUUCUGUCAG
CAAGGCAACACCCUGCCCUACACCUUCGGCGGAGGCACCAAG
CUGGAAAUCACCGGCAGCACAAGCGGCAGCGGCAAGCCUGG
AUCUGGCGAGGGAAGCACCAAGGGCGAAGUGAAACUGCAGG
AAAGCGGCCCUGGACUGGUGGCCCCAAGCCAGUCUCUGAGCG
UGACCUGUACCGUGUCCGGCGUGUCCCUGCCUGACUAUGGCG
UGUCCUGGAUCAGACAGCCCCCCAGAAAGGGCCUGGAAUGG
CUGGGAGUGAUCUGGGGCAGCGAGACAACCUACUACAACAG
CGCCCUGAAGUCCCGGCUGACCAUCAUCAAGGACAACUCCAA
GAGCCAGGUGUUCCUGAAGAUGAACAGCCUGCAGACCGACG
ACACCGCCAUCUACUACUGCGCCAAGCACUACUACUACGGCG
GCAGCUACGCCAUGGACUACUGGGGCCAGGGCACAAGCGUG
ACCGUGUCCAGCGCUAGCGGCGGAGGUGGGAGCGGAGUGCA
GGUGGAAACCAUCUCCCCAGGAGACGGGCGCACCUUCCCCAA
GCGCGGCCAGACCUGCGUGGUGCACUACACCGGGAUGCUUG
AAGAUGGAAAGAAAGUUGAUUCCUCCCGGGACAGAAACAAG
CCCUUUAAGUUUAUGCUAGGCAAGCAGGAGGUGAUCCGAGG
CUGGGAAGAAGGGGUUGCCCAGAUGAGUGUGGGUCAGAGAG
CCAAACUGACUAUAUCUCCAGAUUAUGCCUAUGGUGCCACU
GGGCACCCAGGCAUCAUCCCACCACAUGCCACUCUCGUCUUC
GAUGUGGAGCUUCUAAAACUGGAAGGCUGA 6 SS-scFvCD19-FKBP
ATGCCCCTGGGCCTGCTGTGGCTGGGCCTGGCCCTGCTGGGCG (F36V) DNA
CCCTGCACGCCCAGGCCGGATCCGATATCCAGATGACCCAGAC
CACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCAT
CAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG
GTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTAC
CACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTG
GCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCT
GGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAAC
ACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCA
CCGGCAGCACAAGCGGCAGCGGCAAGCCTGGATCTGGCGAGG
GAAGCACCAAGGGCGAAGTGAAACTGCAGGAAAGCGGCCCTG
GACTGGTGGCCCCAAGCCAGTCTCTGAGCGTGACCTGTACCGT
GTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGA
CAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGG
GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG
CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGA
AGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTG
CGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTAC
TGGGGCCAGGGCACAAGCGTGACCGTGTCCAGCGCTAGCGGC
GGAGGTGGGAGCGGAGTGCAGGTGGAAACCATCTCCCCAGGA
GACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGC
ACTACACCGGGATGCTTGAAGATGGAAAGAAAGTTGATTCCTC
CCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCA
GGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAG
TGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCC
TATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCA
CTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGCTGA 7 scFvCD19-FRB
MGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDG (T2098L) protein
TVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQ
QGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGP
GLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS
ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY
YYGGSYAMDYWGQGTSVTVSSASGGGGSILWHEMWHEGLEEA
SRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDL
MEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKG 8 SS-scFvCD19-FRB
AUGCCCCUGGGCCUGCUGUGGCUGGGCCUGGCCCUGCUGGGC (T2098L) mRNA
GCCCUGCACGCCCAGGCCGGAUCCGAUAUCCAGAUGACCCAG
ACCACCAGCAGCCUGAGCGCCAGCCUGGGCGAUAGAGUGACC
AUCAGCUGCAGAGCCAGCCAGGACAUCAGCAAGUACCUGAA
CUGGUAUCAGCAGAAACCCGACGGCACCGUGAAGCUGCUGA
UCUACCACACCAGCAGACUGCACAGCGGCGUGCCCAGCAGAU
UUUCUGGCAGCGGCUCCGGCACCGACUACAGCCUGACCAUCU
CCAACCUGGAACAGGAAGAUAUCGCUACCUACUUCUGUCAG
CAAGGCAACACCCUGCCCUACACCUUCGGCGGAGGCACCAAG
CUGGAAAUCACCGGCAGCACAAGCGGCAGCGGCAAGCCUGG
AUCUGGCGAGGGAAGCACCAAGGGCGAAGUGAAACUGCAGG
AAAGCGGCCCUGGACUGGUGGCCCCAAGCCAGUCUCUGAGCG
UGACCUGUACCGUGUCCGGCGUGUCCCUGCCUGACUAUGGCG
UGUCCUGGAUCAGACAGCCCCCCAGAAAGGGCCUGGAAUGG
CUGGGAGUGAUCUGGGGCAGCGAGACAACCUACUACAACAG
CGCCCUGAAGUCCCGGCUGACCAUCAUCAAGGACAACUCCAA
GAGCCAGGUGUUCCUGAAGAUGAACAGCCUGCAGACCGACG
ACACCGCCAUCUACUACUGCGCCAAGCACUACUACUACGGCG
GCAGCUACGCCAUGGACUACUGGGGCCAGGGCACAAGCGUG
ACCGUGUCCAGCGCUAGCGGCGGAGGUGGGAGCAUCCUCUG
GCAUGAGAUGUGGCAUGAAGGCCUGGAAGAGGCAUCUCGUU
UGUACUUUGGGGAAAGGAACGUGAAAGGCAUGUUUGAGGUG
CUGGAGCCCUUGCAUGCUAUGAUGGAACGGGGCCCCCAGAC
UCUGAAGGAAACAUCCUUUAAUCAGGCCUAUGGUCGAGAUU
UAAUGGAGGCCCAAGAGUGGUGCAGGAAGUACAUGAAAUCA
GGGAAUGUCAAGGACCUCCUCCAAGCCUGGGACCUCUAUUA
UCAUGUGUUCCGACGAAUCUCAAAGGGCUGA 9 SS-scFvCD19-FRB
ATGCCCCTGGGCCTGCTGTGGCTGGGCCTGGCCCTGCTGGGCG (T2098L) DNA
CCCTGCACGCCCAGGCCGGATCCGATATCCAGATGACCCAGAC
CACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCAT
CAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG
GTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTAC
CACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTG
GCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCT
GGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAAC
ACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCA
CCGGCAGCACAAGCGGCAGCGGCAAGCCTGGATCTGGCGAGG
GAAGCACCAAGGGCGAAGTGAAACTGCAGGAAAGCGGCCCTG
GACTGGTGGCCCCAAGCCAGTCTCTGAGCGTGACCTGTACCGT
GTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGA
CAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGG
GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG
CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGA
AGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTG
CGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTAC
TGGGGCCAGGGCACAAGCGTGACCGTGTCCAGCGCTAGCGGC
GGAGGTGGGAGCATCCTCTGGCATGAGATGTGGCATGAAGGC
CTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAGGAACGTGA
AAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCTATGATGGA
ACGGGGCCCCCAGACTCTGAAGGAAACATCCTTTAATCAGGCC
TATGGTCGAGATTTAATGGAGGCCCAAGAGTGGTGCAGGAAG
TACATGAAATCAGGGAATGTCAAGGACCTCCTCCAAGCCTGGG
ACCTCTATTATCATGTGTTCCGACGAATCTCAAAGGGCTGA 13 scFvCD19-TM-41BB-
AUGGCUCUGCCUGUGACAGCUCUGCUGCUGCCUCUGGCCCUG CD3z-BFP mRNA
CUGCUCCAUGCCGCCAGACCCGGAUCCGAUAUCCAGAUGACC
CAGACCACCAGCAGCCUGAGCGCCAGCCUGGGCGAUAGAGUG
ACCAUCAGCUGCAGAGCCAGCCAGGACAUCAGCAAGUACCUG
AACUGGUAUCAGCAGAAACCCGACGGCACCGUGAAGCUGCU
GAUCUACCACACCAGCAGACUGCACAGCGGCGUGCCCAGCAG
AUUUUCUGGCAGCGGCUCCGGCACCGACUACAGCCUGACCAU
CUCCAACCUGGAACAGGAAGAUAUCGCUACCUACUUCUGUC
AGCAAGGCAACACCCUGCCCUACACCUUCGGCGGAGGCACCA
AGCUGGAAAUCACCGGCAGCACAAGCGGCAGCGGCAAGCCU
GGAUCUGGCGAGGGAAGCACCAAGGGCGAAGUGAAACUGCA
GGAAAGCGGCCCUGGACUGGUGGCCCCAAGCCAGUCUCUGA
GCGUGACCUGUACCGUGUCCGGCGUGUCCCUGCCUGACUAUG
GCGUGUCCUGGAUCAGACAGCCACCCAGAAAGGGCCUGGAA
UGGCUGGGAGUGAUCUGGGGCAGCGAGACAACCUACUACAA
CAGCGCCCUGAAGUCCCGGCUGACCAUCAUCAAGGACAACUC
CAAGAGCCAGGUGUUCCUGAAGAUGAACAGCCUGCAGACCG
ACGACACCGCCAUCUACUACUGCGCCAAGCACUACUACUACG
GCGGCAGCUACGCCAUGGACUACUGGGGCCAGGGCACAAGC
GUGACCGUGUCCAGCGCUAGCGCCAAGCCUACCACCACCCCU
GCCCCUAGACCUCCAACACCCGCCCCAACAAUCGCCAGCCAG
CCUCUGUCUCUGAGGCCCGAGGCUUGUAGACCAGCUGCUGGC
GGAGCCGUGCACACCAGAGGACUGGAUUUCGCCUGCGACAU
CUACAUCUGGGCCCCUCUGGCCGGCACAUGUGGCGUGCUGCU
GCUGAGCCUCGUGAUCACCAUGCAUAAACGGGGCAGAAAGA
AACUCCUGUAUAUAUUCAAACAACCAUUUAUGAGACCAGUA
CAAACUACUCAAGAGGAAGAUGGCUGUAGCUGCCGAUUUCC
AGAAGAAGAAGAAGGAGGAUGUGAACUGCGGGUGAAGUUCA
GCAGAAGCGCCGACGCCCCUGCCUACCAGCAGGGCCAGAAUC
AGCUGUACAACGAGCUGAACCUGGGCAGAAGGGAAGAGUAC
GACGUCCUGGAUAAGCGGAGAGGCCGGGACCCUGAGAUGGG
CGGCAAGCCUCGGCGGAAGAACCCCCAGGAAGGCCUGUAUA
ACGAACUGCAGAAAGACAAGAUGGCCGAGGCCUACAGCGAG
AUCGGCAUGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGA
CGGCCUGUAUCAGGGCCUGUCCACCGCCACCAAGGAUACCUA
CGACGCCCUGCACAUGCAGGCCCUGCCCCCAAGGGGCGGCCG
CUCCGGUGAGGGCAGAGGAAGUCUUCUAACAUGCGGUGACG
UGGAGGAGAAUCCGGGCCCCUCUAGAAGCGAGCUGAUUAAG
GAGAACAUGCACAUGAAGCUGUACAUGGAGGGCACCGUGGA
CAACCAUCACUUCAAGUGCACAUCCGAGGGCGAAGGCAAGCC
CUACGAGGGCACCCAGACCAUGAGAAUCAAGGUGGUCGAGG
GCGGCCCUCUCCCCUUCGCCUUCGACAUCCUGGCUACUAGCU
UCCUCUACGGCAGCAAGACCUUCAUCAACCACACCCAGGGCA
UCCCCGACUUCUUCAAGCAGUCCUUCCCUGAGGGCUUCACAU
GGGAGAGAGUCACCACAUACGAAGACGGGGGCGUGCUGACC
GCUACCCAGGACACCAGCCUCCAGGACGGCUGCCUCAUCUAC
AACGUCAAGAUCAGAGGGGUGAACUUCACAUCCAACGGCCC
UGUGAUGCAGAAGAAAACACUCGGCUGGGAGGCCUUCACCG
AGACGCUGUACCCCGCUGACGGCGGCCUGGAAGGCAGAAAC
GACAUGGCCCUGAAGCUCGUGGGCGGGAGCCAUCUGAUCGC
AAACAUCAAGACCACAUAUAGAUCCAAGAAACCCGCUAAGA
ACCUCAAGAUGCCUGGCGUCUACUAUGUGGACUACAGACUG
GAAAGAAUCAAGGAGGCCAACAACGAGACCUACGUCGAGCA
GCACGAGGUGGCAGUGGCCAGAUACUGCGACCUCCCUAGCA AACUGGGGCACAAGCUUAAUUGA
15 FRB(T2098L)-TM- MGSILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMER
41BB-CD3z protein GPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWD
LYYHVFRRISKASAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA
GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITMHKRGRKKL
LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP
AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPRG 16
SS-FRB(T2098L)-TM- AUGGCUCUGCCUGUGACAGCUCUGCUGCUGCCUCUGGCCCUG
41BB-CD3z mRNA CUGCUCCAUGCCGCCAGACCCGGAUCCAUCCUCUGGCAUGAG
AUGUGGCAUGAAGGCCUGGAAGAGGCAUCUCGUUUGUACUU
UGGGGAAAGGAACGUGAAAGGCAUGUUUGAGGUGCUGGAGC
CCUUGCAUGCUAUGAUGGAACGGGGCCCCCAGACUCUGAAG
GAAACAUCCUUUAAUCAGGCCUAUGGUCGAGAUUUAAUGGA
GGCCCAAGAGUGGUGCAGGAAGUACAUGAAAUCAGGGAAUG
UCAAGGACCUCCUCCAAGCCUGGGACCUCUAUUAUCAUGUG
UUCCGACGAAUCUCAAAGGCUAGCGCCAAGCCUACCACCACC
CCUGCCCCUAGACCUCCAACACCCGCCCCAACAAUCGCCAGC
CAGCCUCUGUCUCUGAGGCCCGAGGCUUGUAGACCAGCUGCU
GGCGGAGCCGUGCACACCAGAGGACUGGAUUUCGCCUGCGA
CAUCUACAUCUGGGCCCCUCUGGCCGGCACAUGUGGCGUGCU
GCUGCUGAGCCUCGUGAUCACCAUGCAUAAACGGGGCAGAA
AGAAACUCCUGUAUAUAUUCAAACAACCAUUUAUGAGACCA
GUACAAACUACUCAAGAGGAAGAUGGCUGUAGCUGCCGAUU
UCCAGAAGAAGAAGAAGGAGGAUGUGAACUGCGGGUGAAGU
UCAGCAGAAGCGCCGACGCCCCUGCCUACCAGCAGGGCCAGA
AUCAGCUGUACAACGAGCUGAACCUGGGCAGAAGGGAAGAG
UACGACGUCCUGGAUAAGCGGAGAGGCCGGGACCCUGAGAU
GGGCGGCAAGCCUCGGCGGAAGAACCCCCAGGAAGGCCUGU
AUAACGAACUGCAGAAAGACAAGAUGGCCGAGGCCUACAGC
GAGAUCGGCAUGAAGGGCGAGCGGAGGCGGGGCAAGGGCCA
CGACGGCCUGUAUCAGGGCCUGUCCACCGCCACCAAGGAUAC
CUACGACGCCCUGCACAUGCAGGCCCUGCCCCCAAGGGGC 17 SS-FRB(T2098L)-TM-
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCT 41BB-CD3z DNA
GCTCCATGCCGCCAGACCCGGATCCATCCTCTGGCATGAGATG
TGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGG
AAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGC
ATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAACAT
CCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGA
GTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCT
CCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCT
CAAAGGCTAGCGCCAAGCCTACCACCACCCCTGCCCCTAGACC
TCCAACACCCGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTG
AGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCAC
ACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCC
CTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGAT
CACCATGCATAAACGGGGCAGAAAGAAACTCCTGTATATATTC
AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA
GATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA
TGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCT
GCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAAC
CTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGA
GGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATG
GCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGG
CGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACC
GCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGC CCCCAAGGGGC 18
SS-FRB(T2098L)- ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCT
spacer-TM-41BB-CD3z GCTCCATGCCGCCAGACCCGGATCCATCCTCTGGCATGAGATG DNA
TGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGG
AAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGC
ATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAACAT
CCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGA
GTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCT
CCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCT
CAAAGGCTAGCGAGAGCAAGTACGGACCGCCCTGCCCACCTT
GCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTT
CCCACCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAAGATCCC
GAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCAC
AACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACC
TACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGC
TGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCC
TGCCCAGCAGCATCGAAAAGACCATCAGCAAGGCCAAGGGCC
AGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGA
AGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAA
GGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
CGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTG
GACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGG
ACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCG
TGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCC
TGAGCCTGTCCCTGGGCAAGATGCATAAACGGGGCAGAAAGA
AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACA
AACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAA
GAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGA
AGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGT
ACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCC
TGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGC
CTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGC
AGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGA
AGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATC
AGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCA
CATGCAGGCCCTGCCCCCAAGGGGC 19 FKBP(F36V)-TM-
MGSGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRD 41BB-CD3z protein
RNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGA
TGHPGIIPPHATLVFDVELLKLEASAKPTTTPAPRPPTPAPTIASQPL
SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVI
TMHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP
EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH
DGLYQGLSTATKDTYDALHMQALPPRG 20 SS-FKBP(F36V)-TM-
AUGGCUCUGCCUGUGACAGCUCUGCUGCUGCCUCUGGCCCUG 41BB-CD3z mRNA
CUGCUCCAUGCCGCCAGACCCGGAUCCGGAGUGCAGGUGGAA
ACCAUCUCCCCAGGAGACGGGCGCACCUUCCCCAAGCGCGGC
CAGACCUGCGUGGUGCACUACACCGGGAUGCUUGAAGAUGG
AAAGAAAGUUGAUUCCUCCCGGGACAGAAACAAGCCCUUUA
AGUUUAUGCUAGGCAAGCAGGAGGUGAUCCGAGGCUGGGAA
GAAGGGGUUGCCCAGAUGAGUGUGGGUCAGAGAGCCAAACU
GACUAUAUCUCCAGAUUAUGCCUAUGGUGCCACUGGGCACC
CAGGCAUCAUCCCACCACAUGCCACUCUCGUCUUCGAUGUGG
AGCUUCUAAAACUGGAAGCUAGCGCCAAGCCUACCACCACCC
CUGCCCCUAGACCUCCAACACCCGCCCCAACAAUCGCCAGCC
AGCCUCUGUCUCUGAGGCCCGAGGCUUGUAGACCAGCUGCU
GGCGGAGCCGUGCACACCAGAGGACUGGAUUUCGCCUGCGA
CAUCUACAUCUGGGCCCCUCUGGCCGGCACAUGUGGCGUGCU
GCUGCUGAGCCUCGUGAUCACCAUGCAUAAACGGGGCAGAA
AGAAACUCCUGUAUAUAUUCAAACAACCAUUUAUGAGACCA
GUACAAACUACUCAAGAGGAAGAUGGCUGUAGCUGCCGAUU
UCCAGAAGAAGAAGAAGGAGGAUGUGAACUGCGGGUGAAGU
UCAGCAGAAGCGCCGACGCCCCUGCCUACCAGCAGGGCCAGA
AUCAGCUGUACAACGAGCUGAACCUGGGCAGAAGGGAAGAG
UACGACGUCCUGGAUAAGCGGAGAGGCCGGGACCCUGAGAU
GGGCGGCAAGCCUCGGCGGAAGAACCCCCAGGAAGGCCUGU
AUAACGAACUGCAGAAAGACAAGAUGGCCGAGGCCUACAGC
GAGAUCGGCAUGAAGGGCGAGCGGAGGCGGGGCAAGGGCCA
CGACGGCCUGUAUCAGGGCCUGUCCACCGCCACCAAGGAUAC
CUACGACGCCCUGCACAUGCAGGCCCUGCCCCCAAGGGGC 21 SS-FKBP(F36V)-TM-
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCT 41BB-CD3z DNA
GCTCCATGCCGCCAGACCCGGATCCGGAGTGCAGGTGGAAAC
CATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAG
ACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAG
AAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTA
TGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGG
TTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATC
TCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATC
CCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAAC
TGGAAGCTAGCGCCAAGCCTACCACCACCCCTGCCCCTAGACC
TCCAACACCCGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTG
AGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCAC
ACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCC
CTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGAT
CACCATGCATAAACGGGGCAGAAAGAAACTCCTGTATATATTC
AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA
GATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA
TGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCT
GCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAAC
CTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGA
GGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATG
GCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGG
CGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACC
GCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGC CCCCAAGGGGC 22
SS-FKBP(F36V)- ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCT
spacer-TM-41BB-CD3z GCTCCATGCCGCCAGACCCGGATCCGGAGTGCAGGTGGAAAC DNA
CATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAG
ACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAG
AAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTA
TGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGG
TTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATC
TCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATC
CCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAAC
TGGAAGCTAGCGAGAGCAAGTACGGACCGCCCTGCCCACCTT
GCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTT
CCCACCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAAGATCCC
GAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCAC
AACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACC
TACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGC
TGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCC
TGCCCAGCAGCATCGAAAAGACCATCAGCAAGGCCAAGGGCC
AGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGA
AGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAA
GGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
CGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTG
GACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGG
ACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCG
TGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCC
TGAGCCTGTCCCTGGGCAAGATGCATAAACGGGGCAGAAAGA
AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACA
AACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAA
GAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGA
AGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGT
ACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCC
TGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGC
CTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGC
AGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGA
AGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATC
AGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCA
CATGCAGGCCCTGCCCCCAAGGGGC 23 FKBP-TM-41BB-CD3z
MGSGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRD protein
RNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGA
TGHPGIIPPHATLVFDVELLKLEASAKPTTTPAPRPPTPAPTIASQPL
SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVI
TMHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP
EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH
DGLYQGLSTATKDTYDALHMQALPPRG 24 FKBP-TM-41BB-CD3z
AUGGCUCUGCCUGUGACAGCUCUGCUGCUGCCUCUGGCCCUG mRNA
CUGCUCCAUGCCGCCAGACCCGGAUCCGGAGUGCAGGUGGAA
ACCAUCUCCCCAGGAGACGGGCGCACCUUCCCCAAGCGCGGC
CAGACCUGCGUGGUGCACUACACCGGGAUGCUUGAAGAUGG
AAAGAAAUUUGAUUCCUCCCGGGACAGAAACAAGCCCUUUA
AGUUUAUGCUAGGCAAGCAGGAGGUGAUCCGAGGCUGGGAA
GAAGGGGUUGCCCAGAUGAGUGUGGGUCAGAGAGCCAAACU
GACUAUAUCUCCAGAUUAUGCCUAUGGUGCCACUGGGCACC
CAGGCAUCAUCCCACCACAUGCCACUCUCGUCUUCGAUGUGG
AGCUUCUAAAACUGGAAGCUAGCGCCAAGCCUACCACCACCC
CUGCCCCUAGACCUCCAACACCCGCCCCAACAAUCGCCAGCC
AGCCUCUGUCUCUGAGGCCCGAGGCUUGUAGACCAGCUGCU
GGCGGAGCCGUGCACACCAGAGGACUGGAUUUCGCCUGCGA
CAUCUACAUCUGGGCCCCUCUGGCCGGCACAUGUGGCGUGCU
GCUGCUGAGCCUCGUGAUCACCAUGCAUAAACGGGGCAGAA
AGAAACUCCUGUAUAUAUUCAAACAACCAUUUAUGAGACCA
GUACAAACUACUCAAGAGGAAGAUGGCUGUAGCUGCCGAUU
UCCAGAAGAAGAAGAAGGAGGAUGUGAACUGCGGGUGAAGU
UCAGCAGAAGCGCCGACGCCCCUGCCUACCAGCAGGGCCAGA
AUCAGCUGUACAACGAGCUGAACCUGGGCAGAAGGGAAGAG
UACGACGUCCUGGAUAAGCGGAGAGGCCGGGACCCUGAGAU
GGGCGGCAAGCCUCGGCGGAAGAACCCCCAGGAAGGCCUGU
AUAACGAACUGCAGAAAGACAAGAUGGCCGAGGCCUACAGC
GAGAUCGGCAUGAAGGGCGAGCGGAGGCGGGGCAAGGGCCA
CGACGGCCUGUAUCAGGGCCUGUCCACCGCCACCAAGGAUAC
CUACGACGCCCUGCACAUGCAGGCCCUGCCCCCAAGGGGC 25 FKBP-TM-41BB-CD3z
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCT DNA
GCTCCATGCCGCCAGACCCGGATCCGGAGTGCAGGTGGAAAC
CATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAG
ACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAG
AAATTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTA
TGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGG
TTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATC
TCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATC
CCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAAC
TGGAAGCTAGCGCCAAGCCTACCACCACCCCTGCCCCTAGACC
TCCAACACCCGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTG
AGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCAC
ACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCC
CTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGAT
CACCATGCATAAACGGGGCAGAAAGAAACTCCTGTATATATTC
AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA
GATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA
TGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCT
GCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAAC
CTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGA
GGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATG
GCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGG
CGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACC
GCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGC CCCCAAGGGGC 26
FKBP-spacer-TM- ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCT
41BB-CD3z DNA GCTCCATGCCGCCAGACCCGGATCCGGAGTGCAGGTGGAAAC
CATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAG
ACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAG
AAATTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTA
TGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGG
TTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATC
TCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATC
CCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAAC
TGGAAGCTAGCGAGAGCAAGTACGGACCGCCCTGCCCACCTT
GCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTT
CCCACCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCC
GAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAAGATCCC
GAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCAC
AACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACC
TACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGC
TGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCC
TGCCCAGCAGCATCGAAAAGACCATCAGCAAGGCCAAGGGCC
AGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGA
AGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAA
GGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA
CGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTG
GACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGG
ACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCG
TGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCC
TGAGCCTGTCCCTGGGCAAGATGCATAAACGGGGCAGAAAGA
AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACA
AACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAA
GAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGA
AGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGT
ACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCC
TGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGC
CTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGC
AGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGA
AGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATC
AGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCA
CATGCAGGCCCTGCCCCCAAGGGGC 37 SS-2xDmrB-DmrC-
MALPVTALLLPLALLLHAARPGSGGVQVETISPGDGRTFPKRGQT TM-41BB-CD3z
CVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVA protein
QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEFLKLESGT
SGTSGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSR
DRNKPFKFMLGKQEVIRGWEEGV 38 SS-2xDmrB-DmrC-
AUGGCUCUGCCUGUGACAGCUCUGCUGCUGCCUCUGGCCCUG TM-41BB-CD3z
CUGCUCCAUGCCGCCAGACCCGGAUCCGGCGGUGUCCAAGUC mRNA
GAAACUAUAUCGCCUGGCGAUGGCAGAACGUUUCCCAAACG
UGGCCAGACCUGUGUCGUACACUAUACCGGCAUGCUAGAGG
AUGGGAAAAAGGUUGAUUCCAGUCGCGAUCGGAACAAACCG
UUUAAAUUCAUGUUGGGGAAGCAAGAGGUUAUCAGGGGAUG
GGAAGAGGGUGUCGCGCAAAUGUCGGUUGGGCAACGUGCGA
AACUCACAAUUUCCCCGGAUUACGCAUACGGAGCUACCGGAC
ACCCUGGGAUUAUCCCACCGCAUGCGACGCUAGUGUUUGAC
GUAGAGUUCUUGAAGCUCGAAUCAGGUACAAGCGGCACUUC
UGGCGUACAGGUUGAGACAAUUAGUCCCGGAGACGGACGUA
CAUUCCCAAAGAGAGGGCAAACUUGCGUAGUCCAUUACACU
GGAAUGUUGGAAGACGGCAAGAAAGUGGACAGUUCAAGAGA
CCGCAAUAAGCCUUUCAAGUUUAUGCUCGGAAAACAGGAAG
UCAUACGCGGUUGGGAGGAAGGCGUGGCUCAGAUGAGCGUC
GGACAGAGGGCAAAGUUGACCAUCAGUCCCGACUAUGCGUA
UGGCGCGACAGGCCAUCCCGGAAUCAUACCUCCCCACGCAAC
CUUGGUAUUCGAUGUCGAACUGCUCAAAUUAGAGGGUAGUA
GAUCCAUCCUCUGGCAUGAGAUGUGGCAUGAAGGCCUGGAA
GAGGCAUCUCGUUUGUACUUUGGGGAAAGGAACGUGAAAGG
CAUGUUUGAGGUGCUGGAGCCCUUGCAUGCUAUGAUGGAAC
GGGGCCCCCAGACUCUGAAGGAAACAUCCUUUAAUCAGGCC
UAUGGUCGAGAUUUAAUGGAGGCCCAAGAGUGGUGCAGGAA
GUACAUGAAAUCAGGGAAUGUCAAGGACCUCCUCCAAGCCU
GGGACCUCUAUUAUCAUGUGUUCCGACGAAUCUCAAAGGCU
AGCGCCAAGCCUACCACCACCCCUGCCCCUAGACCUCCAACA
CCCGCCCCAACAAUCGCCAGCCAGCCUCUGUCUCUGAGGCCC
GAGGCUUGUAGACCAGCUGCUGGCGGAGCCGUACACACCAG
AGGACUGGAUUUCGCCUGCGACAUCUACAUCUGGGCCCCUCU
GGCCGGCACAUGUGGCGUGCUGCUGCUGAGCCUCGUGAUCA
CCAUGCAUAAACGGGGCAGAAAGAAACUCCUGUAUAUAUUC
AAACAACCAUUUAUGAGACCAGUACAAACUACUCAAGAGGA
AGAUGGCUGUAGCUGCCGAUUUCCAGAAGAAGAAGAAGGAG
GAUGUGAACUGCGGGUGAAGUUCAGCAGAAGCGCCGACGCC
CCUGCCUACCAGCAGGGCCAGAAUCAGCUGUACAACGAGCUG
AACCUGGGCAGAAGGGAAGAGUACGACGUCCUGGAUAAGCG
GAGAGGCCGGGACCCUGAGAUGGGCGGCAAGCCUCGGCGGA
AGAACCCCCAGGAAGGCCUGUAUAACGAACUGCAGAAAGAC
AAGAUGGCCGAGGCCUACAGCGAGAUCGGCAUGAAGGGCGA
GCGGAGGCGGGGCAAGGGCCACGACGGCCUGUAUCAGGGCC
UGUCCACCGCCACCAAGGAUACCUACGACGCCCUGCACAUGC AGGCCCUGCCCCCAAGGGGC 39
SS-2xDmrB-DmrC- ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCT
TM-41BB-CD3z DNA GCTCCATGCCGCCAGACCCGGATCCGGCGGTGTCCAAGTCGAA
ACTATATCGCCTGGCGATGGCAGAACGTTTCCCAAACGTGGCC
AGACCTGTGTCGTACACTATACCGGCATGCTAGAGGATGGGAA
AAAGGTTGATTCCAGTCGCGATCGGAACAAACCGTTTAAATTC
ATGTTGGGGAAGCAAGAGGTTATCAGGGGATGGGAAGAGGGT
GTCGCGCAAATGTCGGTTGGGCAACGTGCGAAACTCACAATTT
CCCCGGATTACGCATACGGAGCTACCGGACACCCTGGGATTAT
CCCACCGCATGCGACGCTAGTGTTTGACGTAGAGTTCTTGAAG
CTCGAATCAGGTACAAGCGGCACTTCTGGCGTACAGGTTGAGA
CAATTAGTCCCGGAGACGGACGTACATTCCCAAAGAGAGGGC
AAACTTGCGTAGTCCATTACACTGGAATGTTGGAAGACGGCAA
GAAAGTGGACAGTTCAAGAGACCGCAATAAGCCTTTCAAGTTT
ATGCTCGGAAAACAGGAAGTCATACGCGGTTGGGAGGAAGGC
GTGGCTCAGATGAGCGTCGGACAGAGGGCAAAGTTGACCATC
AGTCCCGACTATGCGTATGGCGCGACAGGCCATCCCGGAATCA
TACCTCCCCACGCAACCTTGGTATTCGATGTCGAACTGCTCAA
ATTAGAGGGTAGTAGATCCATCCTCTGGCATGAGATGTGGCAT
GAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAGG
AACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCTA
TGATGGAACGGGGCCCCCAGACTCTGAAGGAAACATCCTTTAA
TCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGAGTGGTGC
AGGAAGTACATGAAATCAGGGAATGTCAAGGACCTCCTCCAA
GCCTGGGACCTCTATTATCATGTGTTCCGACGAATCTCAAAGG
CTAGCGCCAAGCCTACCACCACCCCTGCCCCTAGACCTCCAAC
ACCCGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCC
GAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTACACACCAGA
GGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGG
CCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCAT
GCATAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACA
ACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGG
CTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGA
ACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTAC
CAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGC
AGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGG
GACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAG
GAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAG
GCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGC
AAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCA
AGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAG GGGC 41
SS-scFvCD19-DmrA- METDTLLLWVLLLWVPGSTGDYKDEGSDIQMTQTTSSLSASLGD
fuP2A-DmrC-TM- RVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS
41BB-CD3z protein GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGS
TSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL
PDYGVSWIRQPPRKGLEWLGVIWGSE 42 SS-scFvCD19-DmrA-
ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGG fuP2A-DmrC-TM-
TTCCAGGTTCCACTGGTGACTACAAGGACGAGGGATCCGATAT 41BB-CD3z DNA
CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGG
CGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAG
CAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGT
GAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGT
GCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGC
CTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACT
TCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGG
CACCAAGCTGGAAATCACCGGCAGCACAAGCGGCAGCGGCAA
GCCTGGATCTGGCGAGGGAAGCACCAAGGGCGAAGTGAAACT
GCAGGAAAGCGGCCCTGGACTGGTGGCCCCAAGCCAGTCTCT
GAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTAT
GGCGTGTCCTGGATCAGACAGCCACCCAGAAAGGGCCTGGAA
TGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAAC
AGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCA
AGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACG
ACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG
CAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGAC
CGTGTCCAGCGCTAGCGGCTCAGGAGGAGTGCAGGTTGAAAC
CATCTCCCCAGGAGACGGGCGCACCTTCCCGAAGCGCGGACA
GACATGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAA
GAAATTCGATTCATCGCGGGACAGAAACAAGCCCTTTAAGTTT
ATGCTGGGCAAGCAGGAGGTCATCCGAGGCTGGGAAGAAGGG
GTTGCCCAGATGAGTGTCGGCCAGAGAGCCAAACTGACTATAT
CACCTGACTACGCCTATGGGGCCACTGGGCACCCTGGCATAAT
TCCGCCACACGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAA
CTGGAAGGCGGCCGCGCTCGTTACAAGCGAAGTGTCTCAGGAT
CTGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGT
TGAAGAAAACCCCGGGCCTTCAAGATCCATCCTCTGGCATGAG
ATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTG
GGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCT
TGCATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAA
CATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCA
AGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGA
CCTCCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGA
ATCTCAAAGGCTAGCGCCAAGCCTACCACCACCCCTGCCCCTA
GACCTCCAACACCCGCCCCAACAATCGCCAGCCAGCCTCTGTC
TCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTA
CACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGG
CCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGT
GATCACCATGCATAAACGGGGCAGAAAGAAACTCCTGTATAT
ATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAG
GAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA
GGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCC
CCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTG
AACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGG
AGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAG
AACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAG
ATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGG
AGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCC
ACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCC TGCCCCCAAGGGGC 44
SS-scFvCD19-DmrA- METDTLLLWVLLLWVPGSTGDYKDEGSDIQMTQTTSSLSASLGD
fuP2A-FRB-TM-41BB- RVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS
CD3z protein GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGS
TSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL
PDYGVSWIRQPPRKGLEWLGVIWGSE 45 SS-scFvCD19-DmrA-
ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGG fuP2A-FRB-TM-41BB-
TTCCAGGTTCCACTGGTGACTACAAGGACGAGGGATCCGATAT CD3z DNA
CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGG
CGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAG
CAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGT
GAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGT
GCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGC
CTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACT
TCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGG
CACCAAGCTGGAAATCACCGGCAGCACAAGCGGCAGCGGCAA
GCCTGGATCTGGCGAGGGAAGCACCAAGGGCGAAGTGAAACT
GCAGGAAAGCGGCCCTGGACTGGTGGCCCCAAGCCAGTCTCT
GAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTAT
GGCGTGTCCTGGATCAGACAGCCACCCAGAAAGGGCCTGGAA
TGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAAC
AGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCA
AGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACG
ACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG
CAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGAC
CGTGTCCAGCGCTAGCGGCTCAGGAGGAGTGCAGGTTGAAAC
CATCTCCCCAGGAGACGGGCGCACCTTCCCGAAGCGCGGACA
GACATGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAA
GAAATTCGATTCATCGCGGGACAGAAACAAGCCCTTTAAGTTT
ATGCTGGGCAAGCAGGAGGTCATCCGAGGCTGGGAAGAAGGG
GTTGCCCAGATGAGTGTCGGCCAGAGAGCCAAACTGACTATAT
CACCTGACTACGCCTATGGGGCCACTGGGCACCCTGGCATAAT
TCCGCCACACGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAA
CTGGAAGGCGGCCGCGCTCGTTACAAGCGAAGTGTCTCAGGAT
CTGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGT
TGAAGAAAACCCCGGGCCTTCAAGATCCATCCTCTGGCATGAG
ATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTG
GGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCT
TGCATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAA
CATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCA
AGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGA
CCTCACCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGA
ATCTCAAAGGCTAGCGCCAAGCCTACCACCACCCCTGCCCCTA
GACCTCCAACACCCGCCCCAACAATCGCCAGCCAGCCTCTGTC
TCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTA
CACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGG
CCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGT
GATCACCATGCATAAACGGGGCAGAAAGAAACTCCTGTATAT
ATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAG
GAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA
GGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCC
CCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTG
AACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGG
AGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAG
AACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAG
ATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGG
AGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCC
ACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCC TGCCCCCAAGGGGC 47
SS-scFvCD19-DmrA- METDTLLLWVLLLWVPGSTGDYKDEGSDIQMTQTTSSLSASLGD
fuP2A-2xDmrB-DmrC- RVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS
TM-41BB-CD3z GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGS protein
TSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL
PDYGVSWIRQPPRKGLEWLGVIWGSE 48 SS-scFvCD19-DmrA-
ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGG fuP2A-2xDmrB-DmrC-
TTCCAGGTTCCACTGGTGACTACAAGGACGAGGGATCCGATAT TM-41BB-CD3z DNA
CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGG
CGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAG
CAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGT
GAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGT
GCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGC
CTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACT
TCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGG
CACCAAGCTGGAAATCACCGGCAGCACAAGCGGCAGCGGCAA
GCCTGGATCTGGCGAGGGAAGCACCAAGGGCGAAGTGAAACT
GCAGGAAAGCGGCCCTGGACTGGTGGCCCCAAGCCAGTCTCT
GAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTAT
GGCGTGTCCTGGATCAGACAGCCACCCAGAAAGGGCCTGGAA
TGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAAC
AGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCA
AGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACG
ACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG
CAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGAC
CGTGTCCAGCGCTAGCGGCTCAGGAGGAGTGCAGGTTGAAAC
CATCTCCCCAGGAGACGGGCGCACCTTCCCGAAGCGCGGACA
GACATGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAA
GAAATTCGATTCATCGCGGGACAGAAACAAGCCCTTTAAGTTT
ATGCTGGGCAAGCAGGAGGTCATCCGAGGCTGGGAAGAAGGG
GTTGCCCAGATGAGTGTCGGCCAGAGAGCCAAACTGACTATAT
CACCTGACTACGCCTATGGGGCCACTGGGCACCCTGGCATAAT
TCCGCCACACGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAA
CTGGAAGGCGGCCGCGCTCGTTACAAGCGAAGTGTCTCAGGAT
CTGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGT
TGAAGAAAACCCCGGGCCTTCAAGATCCGGCGGTGTCCAAGTC
GAAACTATATCGCCTGGCGATGGCAGAACGTTTCCCAAACGTG
GCCAGACCTGTGTCGTACACTATACCGGCATGCTAGAGGATGG
GAAAAAGGTTGATTCCAGTCGCGATCGGAACAAACCGTTTAA
ATTCATGTTGGGGAAGCAAGAGGTTATCAGGGGATGGGAAGA
GGGTGTCGCGCAAATGTCGGTTGGGCAACGTGCGAAACTCAC
AATTTCCCCGGATTACGCATACGGAGCTACCGGACACCCTGGG
ATTATCCCACCGCATGCGACGCTAGTGTTTGACGTAGAGTTCT
TGAAGCTCGAATCAGGTACAAGCGGCACTTCTGGCGTACAGGT
TGAGACAATTAGTCCCGGAGACGGACGTACATTCCCAAAGAG
AGGGCAAACTTGCGTAGTCCATTACACTGGAATGTTGGAAGAC
GGCAAGAAAGTGGACAGTTCAAGAGACCGCAATAAGCCTTTC
AAGTTTATGCTCGGAAAACAGGAAGTCATACGCGGTTGGGAG
GAAGGCGTGGCTCAGATGAGCGTCGGACAGAGGGCAAAGTTG
ACCATCAGTCCCGACTATGCGTATGGCGCGACAGGCCATCCCG
GAATCATACCTCCCCACGCAACCTTGGTATTCGATGTCGAACT
GCTCAAATTAGAGGGTAGTAGATCCATCCTCTGGCATGAGATG
TGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGG
AAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGC
ATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAACAT
CCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGA
GTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCT
CCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCT
CAAAGGCTAGCGCCAAGCCTACCACCACCCCTGCCCCTAGACC
TCCAACACCCGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTG
AGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTACAC
ACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCC
CTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGAT
CACCATGCATAAACGGGGCAGAAAGAAACTCCTGTATATATTC
AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAA
GATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA
TGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCT
GCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAAC
CTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGA
GGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC
CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATG
GCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGG
CGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACC
GCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGC CCCCAAGGGGC 50
SS-CD19scFv-DmrA- MPLGLLWLGLALLGALHAQAGSDIQMTQTTSSLSASLGDRVTISC
CD4TM protein RASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG
TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG
KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV
SWIRQPPRKGLEWLGVIWGSETTYYN 51 SS-CD19scFv-DmrA-
ATGCCCCTGGGCCTGCTGTGGCTGGGCCTGGCCCTGCTGGGCG CD4TM DNA
CCCTGCACGCCCAGGCCGGATCCGATATCCAGATGACCCAGAC
CACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCAT
CAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG
GTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTAC
CACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTG
GCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCT
GGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAAC
ACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCA
CCGGCAGCACAAGCGGCAGCGGCAAGCCTGGATCTGGCGAGG
GAAGCACCAAGGGCGAAGTGAAACTGCAGGAAAGCGGCCCTG
GACTGGTGGCCCCAAGCCAGTCTCTGAGCGTGACCTGTACCGT
GTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGA
CAGCCACCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGG
GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG
CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGA
AGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTG
CGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTAC
TGGGGCCAGGGCACAAGCGTGACCGTGTCCAGCGCTAGCGGC
GGAGGTGGGAGCGGAGTGCAGGTGGAAACCATCTCCCCAGGA
GACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGC
ACTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTC
CCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCA
GGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAG
TGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCC
TATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCA
CTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGCGGCCG
CATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTT
TTCATTGGGCTAGGCATCTTCTTC 53 SS-CD19scFv-DmrA-
MPLGLLWLGLALLGALHAQAGSDIQMTQTTSSLSASLGDRVTISC CD8hingeTM protein
RASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG
TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG
KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV
SWIRQPPRKGLEWLGVIWGSETTYYN 54 SS-CD19scFv-DmrA-
ATGCCCCTGGGCCTGCTGTGGCTGGGCCTGGCCCTGCTGGGCG CD8hingeTM DNA
CCCTGCACGCCCAGGCCGGATCCGATATCCAGATGACCCAGAC
CACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCAT
CAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG
GTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTAC
CACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTG
GCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCT
GGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAAC
ACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCA
CCGGCAGCACAAGCGGCAGCGGCAAGCCTGGATCTGGCGAGG
GAAGCACCAAGGGCGAAGTGAAACTGCAGGAAAGCGGCCCTG
GACTGGTGGCCCCAAGCCAGTCTCTGAGCGTGACCTGTACCGT
GTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGA
CAGCCACCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGG
GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG
CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGA
AGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTG
CGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTAC
TGGGGCCAGGGCACAAGCGTGACCGTGTCCAGCGCTAGCGGC
GGAGGTGGGAGCGGAGTGCAGGTGGAAACCATCTCCCCAGGA
GACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGC
ACTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTC
CCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCA
GGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAG
TGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCC
TATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCA
CTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGCGGCCG
CGCCAAGCCTACCACCACCCCTGCCCCTAGACCTCCAACACCC
GCCCCAACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGG
CTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACT
GGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGC
ACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACC 56 SS-CD19scFv-DmrA-
MPLGLLWLGLALLGALHAQAGSDIQMTQTTSSLSASLGDRVTISC Spacer-CD4TM
RASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG protein
TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG
KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV
SWIRQPPRKGLEWLGVIWGSETTYYN 57 SS-CD19scFv-DmrA-
ATGCCCCTGGGCCTGCTGTGGCTGGGCCTGGCCCTGCTGGGCG Spacer-CD4TM DNA
CCCTGCACGCCCAGGCCGGATCCGATATCCAGATGACCCAGAC
CACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCAT
CAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG
GTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTAC
CACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTG
GCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCT
GGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAAC
ACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCA
CCGGCAGCACAAGCGGCAGCGGCAAGCCTGGATCTGGCGAGG
GAAGCACCAAGGGCGAAGTGAAACTGCAGGAAAGCGGCCCTG
GACTGGTGGCCCCAAGCCAGTCTCTGAGCGTGACCTGTACCGT
GTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGA
CAGCCACCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGG
GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG
CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGA
AGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTG
CGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTAC
TGGGGCCAGGGCACAAGCGTGACCGTGTCCAGCGCTAGCGGC
GGAGGTGGGAGCGGAGTGCAGGTGGAAACCATCTCCCCAGGA
GACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGC
ACTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTC
CCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCA
GGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAG
TGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCC
TATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCA
CTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGCGGCCG
CGAGAGCAAGTACGGACCGCCCTGCCCACCTTGCCCTGCCCCC
GAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCACCCAAGC
CCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCT
GCGTGGTGGTGGACGTGAGCCAGGAAGATCCCGAGGTCCAGT
TCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGA
CCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGG
TGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAA
AGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAG
CATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCTCGCGA
GCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATGACC
AAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTG
AGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACG
GCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCG
GTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACGA
GGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCC
CTGGGCAAGATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCC
TCCTGCTTTTCATTGGGCTAGGCATCTTCTTC 59 SS-CD19scFv-DmrA-
MPLGLLWLGLALLGALHAQAGSDIQMTQTTSSLSASLGDRVTISC CD52 GPI anchor
RASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG protein
TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG
KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV
SWIRQPPRKGLEWLGVIWGSETTYYN 60 SS-CD19scFv-DmrA-
ATGCCCCTGGGCCTGCTGTGGCTGGGCCTGGCCCTGCTGGGCG CD52 GPI anchor DNA
CCCTGCACGCCCAGGCCGGATCCGATATCCAGATGACCCAGAC
CACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCAT
CAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG
GTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTAC
CACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTG
GCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCT
GGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAAC
ACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCA
CCGGCAGCACAAGCGGCAGCGGCAAGCCTGGATCTGGCGAGG
GAAGCACCAAGGGCGAAGTGAAACTGCAGGAAAGCGGCCCTG
GACTGGTGGCCCCAAGCCAGTCTCTGAGCGTGACCTGTACCGT
GTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGA
CAGCCACCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGG
GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG
CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGA
AGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTG
CGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTAC
TGGGGCCAGGGCACAAGCGTGACCGTGTCCAGCGCTAGCGGC
GGAGGTGGGAGCGGAGTGCAGGTGGAAACCATCTCCCCAGGA
GACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGC
ACTACACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTC
CCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCA
GGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAG
TGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCC
TATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCA
CTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGCGGCCG
CACCAGCCAAACCAGCAGCCCCTCAGCATCCAGCAACATAAG
CGGAGGCATTTTCCTTTTCTTCGTGGCCAATGCCATAATCCACC TCTTCTGCTTCAGT 64
CD8ss-DmrC-CD8TM- MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE
41BB-CD3z-P2A- RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE
IgKss-CD19scFv- WCRKYMKSGNVKDLLQAWDLYYHVFRRISKASAGTGSDIYIWA
DmrA-CD4TM protein PLAGTCGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDG
CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGS
GATNFSLLKQAGDVEENPGPSMETDTLLLWVLLLWVPGSTGSDI
QMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL
IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL
PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPS
QSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY
NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSSASGGGGSGVQVETISPGDGRTFPKRGQT
CVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVA
QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGG
RMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQ 65 CD8ss-DmrC-CD8TM-
ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCT 41BB-CD3z-P2A-
GCTCCATGCCGCCAGACCCGGATCCATCCTCTGGCATGAGATG IgKss-CD19scFv-
TGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGG DmrA-CD4TM DNA
AAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGC
ATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGGAAACAT
CCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGA
GTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAAGGACCT
CCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGACGAATCT
CAAAGGCTAGCGCCGGCACTGGTTCCGACATCTACATCTGGGC
CCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTG
ATCACCATGCATAAACGGGGCAGAAAGAAACTCCTGTATATAT
TCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGG
AAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG
GATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCC
CTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGA
ACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGA
GAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGA
ACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGA
TGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGA
GGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCA
CCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCT
GCCCCCAAGGTCAGGATCTGGCGCCACGAACTTCTCTCTGTTA
AAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTTCAATG
GAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTC
CAGGTTCCACTGGTTCCGATATCCAGATGACCCAGACCACCAG
CAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTG
CAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCA
GCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACAC
CAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGC
GGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAAC
AGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCT
GCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGG
CAGCACAAGCGGCAGCGGCAAGCCTGGATCTGGCGAGGGAAG
CACCAAGGGCGAAGTGAAACTGCAGGAAAGCGGCCCTGGACT
GGTGGCCCCAAGCCAGTCTCTGAGCGTGACCTGTACCGTGTCC
GGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGC
CACCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCA
GCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGA
CCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGAT
GAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCC
AAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGG
GCCAGGGCACAAGCGTGACCGTGTCCAGCGCTAGCGGCGGAG
GTGGGAGCGGAGTGCAGGTGGAAACCATCTCCCCAGGAGACG
GGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTA
CACCGGGATGCTTGAAGATGGAAAGAAATTTGATTCCTCCCGG
GACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAG
GTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTG
GGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATG
GTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCT
CGTCTTCGATGTGGAGCTTCTAAAACTGGAAGGCGGCCGCATG
GCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCAT
TGGGCTAGGCATCTTCTTCTGTGTCAGGTGCCGGCACCGAAGG CGCCAATAA 67
CD8ss-DmrC-CD8TM- MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE
41BB-CD3z-P2A- RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE
IgKss-CD19scFv- WCRKYMKSGNVKDLLQAWDLYYHVFRRISKASAGTGSDIYIWA
DmrA-CD4TM codon PLAGTCGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDG
optimized protein CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGS
GATNFSLLKQAGDVEENPGPSMETDTLLLWVLLLWVPGSTGDIQ
MTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI
YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL
PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPS
QSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY
NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSSPRGGGGSGVQVETISPGDGRTFPKRGQT
CVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVA
QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGG
RMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQ 68 CD8ss-DmrC-CD8TM-
ATGGCCCTCCCTGTGACCGCCCTGCTGCTCCCCCTCGCCCTGTT 41BB-CD3z-P2A-
GCTCCATGCTGCCCGACCTGGATCCATCCTTTGGCACGAGATG IgKss-CD19scFv-
TGGCACGAGGGACTCGAAGAAGCGTCCCGGCTGTACTTCGGA DmrA-CD4TM codon
GAGCGGAACGTGAAGGGGATGTTCGAAGTGCTGGAACCCCTG optimized DNA
CACGCCATGATGGAGCGGGGTCCTCAGACCCTTAAAGAAACA
AGCTTCAACCAGGCGTACGGGCGCGACCTGATGGAAGCCCAG
GAGTGGTGCCGCAAGTACATGAAGTCCGGAAACGTGAAGGAT
CTGCTGCAAGCCTGGGATCTGTACTACCACGTGTTCAGAAGGA
TCTCAAAGGCTAGCGCCGGCACTGGTTCGGATATCTACATTTG
GGCACCGCTCGCCGGCACTTGTGGAGTGCTGTTGCTGTCCCTC
GTGATCACCATGCATAAGAGGGGACGGAAGAAGCTGCTGTAC
ATTTTCAAGCAGCCATTCATGCGGCCTGTGCAAACCACCCAGG
AGGAGGACGGGTGCAGCTGCCGGTTCCCTGAGGAAGAGGAGG
GCGGATGCGAACTGCGCGTGAAGTTCAGCCGGAGCGCAGATG
CTCCCGCATACCAACAGGGACAGAACCAGCTGTATAACGAGC
TGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTCGACAAGC
GGCGGGGACGCGACCCAGAAATGGGAGGAAAGCCCCGCCGGA
AGAACCCGCAGGAAGGCCTGTACAACGAGTTGCAGAAAGACA
AGATGGCTGAAGCTTACTCGGAGATTGGCATGAAGGGGGAGA
GAAGAAGAGGGAAGGGCCACGACGGCCTTTACCAAGGACTGA
GCACTGCCACCAAGGACACCTACGATGCGCTGCACATGCAGG
CCCTGCCCCCGCGGTCCGGTTCGGGCGCGACTAACTTCAGCCT
GCTGAAGCAGGCCGGAGATGTGGAGGAAAACCCTGGACCGTC
CATGGAGACTGATACCCTGCTTCTGTGGGTCCTGCTCCTCTGG
GTGCCGGGCTCCACCGGTGACATCCAGATGACCCAGACCACCT
CATCCCTGAGCGCCTCTCTGGGTGATCGCGTGACTATCTCCTGC
CGGGCGTCGCAGGATATCTCCAAGTACCTGAACTGGTACCAGC
AAAAACCGGACGGGACCGTGAAACTGCTGATCTACCATACTTC
CCGCCTTCATTCCGGAGTGCCCTCCCGGTTTTCCGGCTCGGGTT
CAGGGACTGATTATTCGCTGACCATTTCCAACCTGGAGCAGGA
GGACATTGCGACCTACTTCTGCCAACAAGGAAACACCCTGCCC
TACACTTTCGGTGGTGGAACCAAGCTCGAGATCACCGGATCAA
CCTCGGGCAGCGGGAAGCCGGGCAGCGGAGAGGGATCGACGA
AAGGAGAAGTCAAGCTGCAGGAATCCGGCCCGGGACTGGTGG
CCCCGAGCCAGTCGCTCTCCGTCACTTGCACCGTGTCGGGAGT
GTCCTTGCCCGACTACGGAGTGTCATGGATTCGGCAGCCACCT
CGCAAGGGCCTGGAATGGCTCGGCGTGATTTGGGGCTCAGAA
ACCACATACTACAACAGCGCCCTGAAGTCTCGGCTCACCATCA
TCAAGGACAATTCCAAGTCCCAAGTGTTCCTGAAGATGAATAG
CTTGCAGACTGACGACACCGCGATCTACTACTGTGCCAAGCAC
TACTACTACGGCGGTTCCTACGCCATGGACTACTGGGGACAAG
GAACTTCCGTGACTGTCTCCTCCCCTAGGGGGGGTGGTGGTTC
GGGGGTCCAGGTGGAAACCATTTCCCCCGGCGACGGGCGCAC
CTTCCCGAAGCGCGGACAGACCTGTGTGGTGCACTATACCGGA
ATGCTCGAAGATGGAAAGAAGTTTGACAGCTCCAGGGACCGC
AACAAGCCTTTCAAGTTTATGCTTGGAAAGCAGGAAGTCATCC
GGGGCTGGGAAGAGGGAGTCGCCCAGATGAGCGTCGGCCAGC
GGGCCAAGCTGACGATCTCCCCTGACTATGCCTACGGCGCTAC
CGGCCATCCCGGAATCATTCCGCCGCACGCAACCCTCGTGTTC
GACGTGGAATTGCTCAAGCTGGAAGGCGGCCGCATGGCGCTG
ATAGTGCTCGGCGGAGTGGCCGGACTGCTGCTGTTCATCGGCC
TGGGCATCTTCTTCTGCGTGAGATGCCGCCATAGAAGGCGGCA ATGA 70 SS-DmrC-CD8TM-
MRPTWAWWLFLVLLLALWAPARGGSILWHEMWHEGLEEASRL 41BB-CD3z-P2A-SS-
YFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLME CD123scFv-DmrA-
AQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKASAGTGSDIY CD4TM protein
IWAPLAGTCGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQE
EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNL
GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE
AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
GGRSGSGATNFSLLKQAGDVEENPGPSLWWRLWWLLLLLLLLW
PMVWAPRADYKDIVMTQSHKFMSTSVGDRVNITCKASQNVDSA
VAWYQQKPGQSPKALIYSASYRYSGVPDRFTGRGSGTDFTLTISS
VQAEDLAVYYCQQYYSTPWTFGGGTKLEIKRGGGGSGGGGSGG
GGSGGGGSEVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSW
VRQPPGKALEWLALIRSKADGYTTEYSASVKGRFTLSRDDSQSIL
YLQMNALRPEDSATYYCARDAAYYSYYSPEGAMDYWGQGTSV
TVSSSASGGGGSGVQVETISPGDGRTFPKRGQTCVVHYTGMLED
GKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI
SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGRMALIVLGGVAG LLLFIGLGIFFCVRCRHRRRQ
71 SS-DmrC-CD8TM- ATGCGCCCCACCTGGGCCTGGTGGCTGTTCCTGGTGCTGCTGC
41BB-CD3z-P2A-SS- TGGCCCTGTGGGCACCCGCTCGCGGCGGATCCATCCTCTGGCA
CD123scFv-DmrA- TGAGATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTAC CD4TM
DNA TTTGGGGAAAGGAACGTGAAAGGCATGTTTGAGGTGCTGGAG
CCCTTGCATGCTATGATGGAACGGGGCCCCCAGACTCTGAAGG
AAACATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGC
CCAAGAGTGGTGCAGGAAGTACATGAAATCAGGGAATGTCAA
GGACCTCCTCCAAGCCTGGGACCTCTATTATCATGTGTTCCGA
CGAATCTCAAAGGCTAGCGCCGGCACTGGTTCCGACATCTACA
TCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAG
CCTCGTGATCACCATGCATAAACGGGGCAGAAAGAAACTCCT
GTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACT
CAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA
GAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCC
GACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAAC
GAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGAT
AAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGG
CGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAA
GACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGC
GAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGC
CTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGC
AGGCCCTGCCCCCAAGGGGCGGCCGCTCAGGATCTGGCGCCA
CGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAA
CCCCGGGCCTTCACTGTGGTGGCGCCTGTGGTGGCTGCTCCTG
CTTCTGTTGCTCCTGTGGCCCATGGTGTGGGCCCCTAGGGCGG
ACTACAAAGATATTGTGATGACCCAGTCTCACAAATTCATGTC
CACATCAGTAGGAGACAGGGTCAACATCACCTGCAAGGCCAG
TCAGAATGTGGATAGTGCTGTAGCCTGGTATCAACAGAAACCA
GGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGT
ACAGTGGAGTCCCTGATCGCTTCACAGGCAGGGGATCTGGGAC
AGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTG
GCAGTTTATTACTGTCAGCAATATTATAGCACTCCGTGGACGT
TCGGTGGAGGCACCAAGCTGGAAATCAAACGTGGTGGTGGTG
GTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGG
TGGATCCGAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTTGGT
ACAGCCTGGGGGTTCTCTGAGTCTCTCCTGTGCAGCTTCTGGAT
TCACCTTCACTGATTACTACATGAGCTGGGTCCGCCAGCCTCC
AGGGAAGGCACTTGAGTGGTTGGCTTTGATTAGAAGCAAAGCT
GATGGTTACACAACAGAATACAGTGCATCTGTGAAGGGTCGGT
TCACCCTCTCCAGAGATGATTCCCAAAGCATCCTCTATCTTCAA
ATGAATGCCCTGAGACCTGAAGACAGTGCCACTTATTACTGTG
CAAGAGATGCGGCCTACTATAGTTACTATAGTCCCGAGGGGGC
TATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCG
AGCGCTAGCGGCGGAGGTGGGAGCGGAGTGCAGGTGGAAACC
ATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAG
ACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAG
AAATTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTA
TGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGG
TTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATC
TCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATC
CCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAAC
TGGAAGGCGGCCGCATGGCCCTGATTGTGCTGGGGGGCGTCGC
CGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTCTGTGTCA
GGTGCCGGCACCGAAGGCGCCAATAA 78 CD8ss.DmrC.CD8TM.
MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE 41BB.Zeta.P2A.IgKss.
RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE CD19scFv.DmrA.CD1
WCRKYMKSGNVKDLLQAWDLYYHVFRRISKASAGTGSDIYIWA 54TM codon optimized
PLAGTCGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDG protein
CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGS
GATNFSLLKQAGDVEENPGPSMETDTLLLWVLLLWVPGSTGDIQ
MTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI
YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL
PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPS
QSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY
NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSSPRGGGGSGVQVETISPGDGRTFPKRGQT
CVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVA
QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGG
RMKIFMYLLTVFLITQMIGSALFAVYLHRR 80 CD8ss.DmrC.CD8hinge-
MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE TM.41BB.Zeta
RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE protein
WCRKYMKSGNVKDLLQAWDLYYHVFRRISKASAKPTTTPAPRPP
TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT
CGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF
PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV
LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 82 hScnSS.CD19scFv.
MPLGLLWLGLALLGALHAQAGSDIQMTQTTSSLSASLGDRVTISC DmrA protein
RASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG
TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG
KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV
SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL
KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAS
GGGGSGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSS
RDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAY
GATGHPGIIPPHATLVFDVELLKLEG 84 hScnSS.CD20scFv.
MPLGLLWLGLALLGALHAQAGSEVQLQQSGAELVKPGASVKMS DmrA protein
CKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKF
KGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWF
FDVWGAGTTVTVSSGSTSGGGSGGGSGGGGSSDIVLTQSPAILSA
SPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGV
PARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTK
LEIKASGGGGSGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDG
KKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTIS
PDYAYGATGHPGIIPPHATLVFDVELLKLEG 86 CD8ss.FRB.CD8TM.
MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE 41BB.Zeta.P2A.IgKss.
RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE CD19scFv.DmrA.CD4TM
WCRKYMKSGNVKDLTQAWDLYYHVFRRISKASAGTGSDIYIWA codon optimized
PLAGTCGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDG protein
CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGS
GATNFSLLKQAGDVEENPGPSMETDTLLLWVLLLWVPGSTGDIQ
MTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI
YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL
PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPS
QSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY
NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSSPRGGGGSGVQVETISPGDGRTFPKRGQT
CVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVA
QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGG
RMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQ 88 CD8ss.FRB.CD8TM.
MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE 41BB.Zeta.P2A.IgKss.
RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE CD19scFv.DmrA.CD154TM
WCRKYMKSGNVKDLTQAWDLYYHVFRRISKASAGTGSDIYIWA codon optimized
PLAGTCGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDG protein
CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGS
GATNFSLLKQAGDVEENPGPSMETDTLLLWVLLLWVPGSTGDIQ
MTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI
YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL
PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPS
QSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY
NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSSPRGGGGSGVQVETISPGDGRTFPKRGQT
CVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVA
QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGG
RMKIFMYLLTVFLITQMIGSALFAVYLHRR 90 CD8ss.FRB.CD8hinge-
MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE TM.41BB.Zeta.P2A.
RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE IgKss.CD19scFv.DmrA.
WCRKYMKSGNVKDLTQAWDLYYHVFRRISKASAKPTTTPAPRPP CD154TM codon
TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT optimized protein
CGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF
PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV
LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGSGATNFS
LLKQAGDVEENPGPSMETDTLLLWVLLLWVPGSTGDIQMTQTTS
SLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRL
HSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG
GTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVT
CTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKS
RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY
WGQGTSVTVSSPRGGGGSGVQVETISPGDGRTFPKRGQTCVVHY
TGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVG
QRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGRMKIF
MYLLTVFLITQMIGSALFAVYLHRR 92 CD8ss.FRB.AMN.
MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE 41BB.Zeta.P2A.IgKss.
RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE CD19scFv.DmrA.CD154TM
WCRKYMKSGNVKDLTQAWDLYYHVFRRISKASVWGSSAAGLA codon optimized
GGVAAAVLLALLVLLVAPPLLMHKRGRKKLLYIFKQPFMRPVQT protein
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNE
LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK
MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL
PPRSGSGATNFSLLKQAGDVEENPGPSMETDTLLLWVLLLWVPG
STGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDG
TVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQ
QGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGP
GLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS
ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY
YYGGSYAMDYWGQGTSVTVSSPRGGGGSGVQVETISPGDGRTFP
KRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGW
EEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
KLEGGRMKIFMYLLTVFLITQMIGSALFAVYLHRR 96 CD8ss.DmrC.CD8TM.
MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE 41BB.Zeta.P2A.IgKss.
RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE CD19scFv.DmrA.CD71TM
WCRKYMKSGNVKDLLQAWDLYYHVFRRISKASAGTGSDIYIWA codon optimized
PLAGTCGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDG protein
CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGS
GATNFSLLKQAGDVEENPGPSMETDTLLLWVLLLWVPGSTGDIQ
MTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI
YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL
PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPS
QSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY
NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSSPRGGGGSGVQVETISPGDGRTFPKRGQT
CVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVA
QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGG
RRCSGSICYGTIAVIVFFLIGFMIGYLGY 98 CD8ss.DmrC.CD8TM.
MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE 41BB.Zeta.P2A.CD71TM.
RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE DmrA.CD19scFv
WCRKYMKSGNVKDLLQAWDLYYHVFRRISKASAGTGSDIYIWA codon optimized
PLAGTCGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDG protein
CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGS
GATNFSLLKQAGDVEENPGPSRCSGSICYGTIAVIVFFLIGFMIGYL
GYTGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSR
DRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYG
ATGHPGIIPPHATLVFDVELLKLEGGGGSPRDIQMTQTTSSLSASL
GDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS
RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI
TGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSG
VSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIK
DNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSS 100
CD8ss.FRB.CD8Hinge. MALPVTALLLPLALLLHAARPGSILWHEMWHEGLEEASRLYFGE
CD8TM.41BB.Zeta. RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE
P2A.IgKss.CD19scFv. WCRKYMKSGNVKDLTQAWDLYYHVFRRISKASAKPTTTPAPRPP
DmrA codon optimized TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT
protein CGVLLLSLVITMHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF
PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV
LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGSGATNFS
LLKQAGDVEENPGPSMETDTLLLWVLLLWVPGSTGDIQMTQTTS
SLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRL
HSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG
GTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVT
CTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKS
RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY
WGQGTSVTVSSPRGGGGSGVQVETISPGDGRTFPKRGQTCVVHY
TGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVG
QRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE
EXAMPLES
Example 1
Construction of DARIC Building and Signaling Components
[0227] The DARIC binding and signaling components were each
separately cloned into a plasmid vector containing a T7 promoter, a
hScn or hCD8 secretion signal, respectively, and a downstream
linearization site. Linearized plasmids were then used as templates
for in vitro transcription reactions, followed by
3'-polyadenylation and 5'-capping steps to create mature in vitro
transcribed mRNA (IVT-mRNA) to be electroporated into primary human
T cells. Human T cells were isolated from PBMCs by negative
selection using paramagnetic beads and expanded with
anti-CD.sup.3/anti-CD28 beads for 48 hours prior to
electroporation. Control electroporations using IVT-mRNA encoding
fluorescent proteins were performed in parallel to confirm
transfection efficiency, or 2A protein-linked fluorescent proteins
were incorporated directly into the DARIC component mRNA
species.
[0228] Exemplary IVT-mRNA encoding binding components (scFv
specific for CD19 and multimerization domain FKBP12 ("DmrA"),
FKBP12 F36V ("DmrB"), FRB (2021-2113) T2098L ("DmrC")) are provided
in SEQ ID NOs.:2, 5, and 8 (scFv specific for CD19 and
multimerization domain FKBP12, FKBP12 F36V, or FRB (2021-2113)
T2098L, respectively). Exemplary IVT-mRNA encoding signaling
components are provided in SEQ ID NOs.:16, 20, and 24
(multimerization domain FRB (2021-2113) T2098L, FKBP12 F36V, or
FKBP12, respectively, transmembrane domain, 4-1BB, and
CD3.zeta.).
[0229] Multimerization is promoted with a bridging factor, such as
rapamycin or rapalogs thereof, or gibberellin or derivatives
thereof. Rapamycin and its derivatives (e.g., AP21967, also known
as C-16-(S)-7-methylindolerapamycin, ICd.sub.50=10nM, a chemically
modified non-immunosuppressive rapamycin analogue) can induce
heterodimerization of FKBP12 and FRB-containing fusion proteins.
AP1903 or AP20187 are homo-bivalent drugs based on the
FKBP12-interacting component of rapamycin, which can be used in
homodimerization scenarios described herein.
Example 2
Cytotoxicity of T Cells Encoding DARIC Components
[0230] Recombinant T cells expressing the two DARIC components were
incubated with K562 target cells (a human myeloid leukemia cell
line), which were modified to express either CD19 or CD20 antigen,
to examine target cell lysis. Briefly, T cells were co-incubated
with a 50:50 mixture of K562-CD19 and K562-CD20 target cell lines,
at 3:1 or 10:1 T cell to target cell ratios. In experimental
samples, 500 nM final concentration of the hetero-bivalent rapalog
AP21967 was added. The relative percentage of each of the target
cell lines was monitored by flow cytometry staining for the CD19
and CD20 antigens to evaluate cell lysis (see FIG. 3).
[0231] Four samples of primary human T cells were prepared by
electroporation with IVT-mRNA encoding (i) an extensively validated
single-chain chimeric antigen receptor (CAR) (CD19-CAR, SEQ ID
NO.:14, positive control); (ii) the DARIC signaling component only
(DSC, SEQ ID NO.:16, negative control); iii) the DARIC binding
component only (DBC-CD19, SEQ ID NO.:2, negative control); and (iv)
both DARIC binding and signaling components (DSC, SEQ ID NO.:16
plus DBC-CD19, SEQ ID NO.:2). The relative percentages of each of
the target cell lines were monitored by flow cytometry staining for
the CD19 and CD20 antigens (FIG. 3A).
[0232] The percent specific cytotoxicity was calculated for each
condition as the percentage change relative the input
K562-CD19:K562-CD20 ratio. T cells expressing the validated
CD19-CAR (SEQ ID NO.:14) showed substantial cytotoxicity and
skewing of the ratio of CD19 versus CD20 cells in the live cell
gate, particularly at a 10:1 T cell to target cell ratio. The T
cells expressing the DARIC binding component alone, DARIC signaling
component alone, or both DARIC components but without the addition
of the hetero-bivalent rapalog AP21967, showed no significant
cytotoxicity. In the presence of AP21967, a substantial specific
cytotoxicity and loss of the K562-CD19 target cells was observed
upon co-incubation with T cells expressing both DARIC components
(FIG. 3B).
[0233] These results indicate that the DARIC mechanism can
reconstitute antigen-specific target cell lysis. Furthermore, the
DARIC design enables pharmacological control of antigen-specific T
cell cytotoxicity.
Example 3
Cytokine Secretion Profile of T Cells Encoding DARIC Components
[0234] Recombinant T cells expressing the two DARIC components were
incubated with K562 target cells (a human myeloid leukemia cell
line), which were modified to express either CD19 or CD20 antigen,
to examine cytokine expression. Briefly, IVT-mRNA transfected T
cells were co-incubated with either the K562-CD19 or K562-CD20 cell
lines using T cell to target ratios of 1:1, with or without the
addition of 500nM AP21967. Supernatants were isolated for analysis
of cytokine production (see FIG. 4).
[0235] Two samples of primary human T cells were isolated,
expanded, and then prepared by electroporation with IVT-mRNA
encoding either (i) the validated single-chain CAR (CD19-CAR, SEQ
ID NO.:13, positive control); or (ii) both DARIC binding and
signaling components (DSC, SEQ ID NO.:16 plus DBC-CD19, SEQ ID
NO.:2). After extensively washing the expanded and electroporated T
cells to remove residual cytokines from the growth media, the T
cells were co-incubated with K562 cell lines expressing either the
human CD19 antigen (left panels) or the CD20 antigen (right panels)
at 1:1 T cell to target cell ratios and in the presence or absence
of the AP21967 rapalog. The supernatants were then collected and
assayed for analyte concentrations using cytokine capture
antibody-labeled beads (Becton Dickenson Cytokine Bead Array, human
Th1/Th2 kit). Comparison with recombinant protein standards enabled
calculation of absolute concentrations of each of the six cytokines
encompassed by the bead array.
[0236] Consistent with previous cytotoxicity findings, T cells
expressing the positive control CD19-CAR produced substantial
amounts of interferon-gamma (IFN.gamma.) and interleukin-2 (IL-2)
when co-incubated with CD19 expressing K562 target cells. T cells
expressing the DARIC components in the absence of bridging factor
AP21967 showed no significant cytokine production, but in the
presence of AP21967 produced IFNy and IL-2 at levels equivalent, or
superior, to the single chain CD19-CAR positive control.
Example 4
Lentiviral Delivery of DARIC Components
[0237] Primary human T cells were isolated, activated, and then
transduced with lentiviral vectors encoding DARIC binding and
signaling components (SEQ ID NOS.:44 and 47). The transduced T
cells were then co-incubated with about a 50:50 mixture of the K562
target cells expressing either CD19 (K562-CD19) or CD20 (K562-CD20)
to evaluate antigen-specific cytotoxicity. The overall ratio of T
cells to K562 cells was 5:1 in all samples. In control samples, no
bridging factor was added, whereas in experimental samples either
rapamycin (10 nM) or AP21967 (100 nM) were applied as the bridging
factor for the secreted antigen binding component and the signaling
component (see, e.g., FIG. 1B). The DARIC antigen binding component
includes a CD19 antigen binding scFv domain and a FKBP12
multimerization domain, which was linked to a mCherry fluorescent
protein. Two independent multimerization domains having different
specificities for bridging components were tested on the DARIC
signaling component: FRB, which is responsive to rapamycin, and the
FRB (2021-2113) T2098L variant, which is responsive to both
rapamycin and AP21967, each linked to the blue fluorescent protein
(BFP).
[0238] Flow cytometric analysis of the lentivirus-transduced T
cells demonstrated expression of both mCherry and BFP proteins
simultaneously, indicating both DARIC components were being
expressed within the same cells (see FIG. 5, first column for each
treatment). Flow cytometric analysis of the K562 cells demonstrated
rapamycin and AP21967-dependent elimination of the CD19 expressing
K562 cells in the sample expressing variant FRB (2021-2113) T2098L
multimerization domain, whereas no addition of a bridging factor
had no effect on cell survival (see FIG. 5, top row of second
column for each treatment). But, only rapamycin was able to
activate the elimination of the K562-CD19 cells by T cells
expressing the FRB dimerization domain, while AP21967 or no
addition of a bridging factor had no effect on cell survival (see
FIG. 5, second row of second column for each treatment). These data
show the specificity of cytotoxic activity that can be achieved
with the DARIC multipartite component system.
[0239] In addition, two distinct T cell populations were mixed,
wherein one population was expressing a DARIC antigen binding
component and the other population was expressing a DARIC signaling
component. This mixed cell population, when co-cultured with the
CD19 and CD20 expressing K562 cells, showed a rapamycin-dependent
cytotoxicity response against K562-CD19 cells, while the absence of
a bridging factor had no effect on target cell survival (see FIG.
5, bottom row). These data indicate that a DARIC antigen binding
component expressed by one T cell population can act in trans with
a different population of T cells that express a DARIC signaling
component and attack the target cells.
[0240] The flexibility of the DARIC system was validated by
swapping the multimerization domains such that the DARIC binding
component targeting CD19 comprised the FRB based DmrC domain and
the DARIC signaling component comprised the FKBP12 based DmrA
domain (SEQ ID NOs.:12, 31). Primary human T cells were made to
express the `swapped` DARIC components and then co-incubated with
50:50 mixtures of the K562-CD19 and C562-CD20 target cells either
in the absence or presence of the indicated concentrations of
rapamycin (FIG. 10). Antigen specific cytotoxicity was observed in
the experimental samples containing the bridging factor, but absent
from the control sample lacking rapamycin. These data demonstrate
that the architecture of the DARIC system is flexible and amenable
to a variety of multimerization domain orientations.
Example 5
Titration of Bridging Factors to Sub-Therapeutic Levels
[0241] A broad range of bridging factor (rapamycin and everolimus)
concentrations were tested to determine whether a DARIC system can
function at clinically relevant concentrations. As in the Example
4, primary human T cells were isolated, activated, and then
transduced with lentiviral vectors expressing a DARIC binding
component (SEQ ID NOS.:1, 4, 7) and a DARIC signaling component
(SEQ ID NOS.:15, 19, 23). The DARIC expressing T cells were then
co-incubated with 50:50 mixtures of the K562-CD19 and K562-CD20
target cells to evaluate antigen-specific cytotoxicity. The overall
ratio of T cells to K562 cells was 5:1 in all samples.
[0242] The indicated concentrations of rapamycin and everolimus
were added to the co-culture samples and then the cytotoxicity
responses were evaluated by flow cytometry (FIG. 6). Cytotoxicity
responses were maintained to sub-nanomolar drug concentrations,
well below the steady state concentrations of rapamycin and
everolimus that are presently achieved when these drugs are
administered to patients in the clinic.
Example 6
Use of a Tethered DARIC Binding Component
[0243] A series of additional DARIC molecules, in which the antigen
binding component was maintained on the T cell surface rather than
released into the extracellular space, were tested (see, e.g., FIG.
1I). Several protein regions and transmembrane domains were used to
anchor the binding domain to the T cell surface (SEQ ID NOS.:50,
53, 56, 59), each altering the spacing or steric parameters
governing multimerization of the DARIC binding and signaling
components. As in the previous examples, antigen-specific
cytotoxicity responses using lentivirus-transduced T cells and
50:50 mixtures of the K562-CD19 and K562-CD20 target cells were
used to evaluate the tethered DARIC binding component. The overall
ratio of T cells to K562 cells was 5:1 in all samples, with the
indicated concentrations of a bridging factor used in experimental
samples.
[0244] Each design had the property of bridging factor-responsive,
antigen-specific cytotoxicity against the K562-CD19 cells. The
tethered DARIC binding component containing the CD8 hinge/CD8
transmembrane domain (SEQ ID NO.:53) showed a measurable level of
activity in the absence of a bridging factor. The tethered DARIC
binding component comprising the IgG4 CH2CH3 spacer with CD4
transmembrane domain (SEQ ID NO.:56) provided the strongest
cytotoxic response upon addition of the rapamycin (bridging
factor), while the tethered DARIC binding component comprising only
the CD4 transmembrane domain (SEQ ID NO.:50) were moderately active
(FIG. 7). A DARIC binding components comprising a GPI signal
sequence from the CD52 protein (see schematic in FIG. 1K) were also
tested. The GPI anchored DARIC produced an antigen specific
cytotoxicity response only in the presence of an appropriate
bridging factor (FIG. 8). These data demonstrate that a DARIC
binding component can be either released or tethered to the cell
surface and still function with a DARIC signaling component.
[0245] Additional lentiviral constructs comprising tethered DARIC
binding components were generated and similarly tested in human T
cells, including a modified CD4 transmembrane domain with improved
activity over other transmembrane tethered DARIC binding components
(SEQ ID NOs.:64-69). Additionally, the DARIC signaling and binding
components were integrated into a single open reading frame
comprising a 2A peptide situated between the two components (such
as that used in FIG. 11), thus validating a simplified DARIC
delivery scheme using a single lentiviral vector (SEQ ID NOs.:66,
69, 72).
[0246] For any of the DARIC componentry designs, si
References