U.S. patent application number 15/555951 was filed with the patent office on 2018-02-15 for immunomodulatory fusion proteins and uses thereof.
The applicant listed for this patent is Fred Hutchinson Cancer Research Center. Invention is credited to Philip D. Greenberg, Shannon K. Oda, Thomas M. Schmitt.
Application Number | 20180044404 15/555951 |
Document ID | / |
Family ID | 55587371 |
Filed Date | 2018-02-15 |
United States Patent
Application |
20180044404 |
Kind Code |
A1 |
Oda; Shannon K. ; et
al. |
February 15, 2018 |
IMMUNOMODULATORY FUSION PROTEINS AND USES THEREOF
Abstract
The present disclosure relates to immunomodulatory fusion
proteins containing an extracellular binding domain and an
intracellular signaling domain, wherein binding of a target can
generate a modulatory signal in a host cell, such as a T cell. The
present disclosure also relates to uses of immune cells expressing
such immunomodulatory fusion proteins to treat certain diseases,
such as cancer or infectious disease.
Inventors: |
Oda; Shannon K.; (Lake
Forest Park, WA) ; Greenberg; Philip D.; (Mercer
Island, WA) ; Schmitt; Thomas M.; (Seattle,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fred Hutchinson Cancer Research Center |
Seattle |
WA |
US |
|
|
Family ID: |
55587371 |
Appl. No.: |
15/555951 |
Filed: |
March 4, 2016 |
PCT Filed: |
March 4, 2016 |
PCT NO: |
PCT/US2016/021064 |
371 Date: |
September 5, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62128979 |
Mar 5, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/02 20180101;
A61P 37/06 20180101; A61P 35/00 20180101; C07K 14/70517 20130101;
C07K 14/70521 20130101; A61K 38/00 20130101; C12N 5/0638 20130101;
C12N 2510/00 20130101; C07K 14/70507 20130101; A61K 35/17 20130101;
C07K 14/70578 20130101; C12N 5/0636 20130101; C12N 5/0637 20130101;
C07K 14/7051 20130101; C07K 14/70503 20130101; A61P 31/12 20180101;
C07K 2319/03 20130101; C07K 14/70596 20130101; A61P 31/04 20180101;
A61K 48/00 20130101; C07K 2319/74 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; A61K 35/17 20060101 A61K035/17; C12N 5/0783 20060101
C12N005/0783; C07K 14/725 20060101 C07K014/725 |
Claims
1. A fusion protein, comprising (a) an extracellular component
comprised of a binding domain that specifically binds a target, (b)
an intracellular component comprised of an intracellular signaling
domain, and (c) a hydrophobic component connecting the
extracellular and intracellular components, wherein the
extracellular portion of a complex formed by specific binding of
the fusion protein to the target (fusion protein::target complex)
is of a size, or spans a distance, of (i) up to about a distance
between two cell membranes of an immunological synapse, (ii) up to
about or substantially the same as a distance spanned by the
extracellular portion of a complex between a T cell receptor (TCR)
and an MHC-peptide complex specifically bound by the TCR, (iii) up
to about or substantially the same as a distance spanned by the
extracellular portion of a complex between a natural molecule
comprising the binding domain and its cognate binding partner;
(iii) less than or up to about 40 nm, 25 nm, 20 nm, 15 nm, or 14
nm; or (iv) any combination thereof.
2. The fusion protein according to claim 1, wherein the fusion
protein::target complex localizes to a supramolecular activation
cluster (SMAC).
3. The fusion protein according to claim 1 or 2, wherein the fusion
protein::target complex localizes to a central region
supramolecular activation cluster (cSMAC).
4. The fusion protein according to claim 2 or claim 3, wherein the
SMAC has a width of a T cell receptor (TCR) associated with an
antigen::human leukocyte antigen (HLA) complex.
5. The fusion protein according to any one of claims 1-4, wherein
the extracellular component is associated with a negative signal
and the intracellular component is associated with a positive
signal.
6. A fusion protein, comprising (a) an extracellular component
comprised of a binding domain that specifically binds a target, (b)
an intracellular component comprised of an intracellular signaling
domain, and (c) a hydrophobic component connecting the
extracellular and intracellular components, wherein the binding
domain is, or has at least 95% identity to, an inhibitory molecule
binding domain and the intracellular signaling domain is, or
contains at least 95% identity to, a costimulatory or stimulatory
molecule binding domain, and wherein the inhibitory molecule is not
a B7-CD28 superfamily member, is not CTLA4, is not PD1, does not
bind to B7, is a glycoprotein, or any combination thereof.
7. The fusion protein according to any of claims 1-6, wherein the
expression of the fusion protein in a T cell comprising a TCR or
chimeric antigen receptor specific for an antigen results in at
least about a 1.5-fold, 2-fold, or 3-fold increase in survival,
expansion, cytotoxicity, cytokine secretion, and/or response to
multiple rounds of stimulation, by the T cell, in response to
binding of the antigen and/or following administration to a
subject, and/or results in at least about a 1.5-fold, 2-fold, or
3-fold increase in time of survival, disease-free survival, or
amelioration of one or more disease symptom, of a subject to which
the cell is administered, as compared to a cell substantially the
same as the T cell but not containing the fusion protein.
8. The fusion protein according to any of claims 1-7, wherein the
fusion protein is capable, when expressed in a T cell, of
co-localizing with a TCR or CAR expressed by the T cell.
9. The fusion protein according to any of claims 6-8, wherein the
binding domain and/or the inhibitory molecule specifically binds to
a CD200 or a CD47.
10. The fusion protein according to any of claims 6-9, wherein the
binding domain is derived from, and/or the inhibitory molecule is,
a CD200R, a SIRP.alpha., a TIM3, a CD2, a CD95 (Fas), a CD223
(LAG3), an A2aR, a KIR, TIM3, a CD300, or a LPA5.
11. The fusion protein according to any of claims 6-9, wherein the
inhibitory molecule is or comprises a CD200R.
12. The fusion protein according to any of claims 6-9, wherein the
inhibitory molecule is or comprises a SIRP.alpha..
13. The fusion protein according to any one of claims 1-12, wherein
the binding domain is an antibody binding fragment, a receptor
ectodomain, a cytokine, or a ligand.
14. The fusion protein according to any one of claims 1-13, wherein
the extracellular component comprises an extracellular portion of a
CD200R, SIRP.alpha., CD279 (PD-1), CD2, CD95 (Fas), CD152 (CTLA4),
CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5.
15. The fusion protein according to any of claims 1-14, wherein the
extracellular component further comprises an additional
extracellular portion, wherein the additional extracellular portion
optionally is from or shares identity with an extracellular portion
of a molecule that is distinct from the binding domain source
molecule or does not contain the binding domain.
16. The fusion protein according to any one of claims 1-15, wherein
the extracellular component further comprises an extracellular
portion from a the hydrophobic component, or that contains the
hydrophobic component or a portion thereof.
17. The fusion protein according to claim 15 or 16, wherein the
additional extracellular portion comprises a multimerization domain
and/or a spacer.
18. The fusion protein according to any one of claims 1-17, wherein
the extracellular component comprises an extracellular portion of a
CD200R or wherein the binding domain comprises a CD200-binding
portion of a CD200R or binding variant thereof.
19. The fusion protein according to any one of claims 1-17, wherein
the extracellular component comprises an extracellular portion of a
SIRP.alpha. or wherein the binding domain comprises a CD47 binding
portion of a SIRP.alpha. or binding variant thereof.
20. The fusion protein according to any one of claims 1-19, wherein
the extracellular component or the additional extracellular portion
comprises a multimerization domain.
21. The fusion protein according to claim 20, wherein the
multimerization domain comprises a cysteine residue.
22. The fusion protein according to claim 20, wherein the
multimerization domain comprises an extracellular component
modified to contain a cysteine residue within about 2 to about 15
amino acids from the hydrophobic component.
23. The fusion protein according to any one of claims 1-22, wherein
the hydrophobic component comprises a transmembrane domain of a
CD2, CD3.epsilon., CD3.delta., CD3.zeta., CD25, CD27, CD28, CD40,
CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB),
CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM),
CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279
(PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcR.alpha., FcR.beta.,
FcR.gamma., Fyn, GALS, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2,
NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRP.alpha., pT.alpha.,
TCR.alpha., TCR.beta., TIM3, TRIM, LPA5, or Zap70.
24. The fusion protein according to any one of claims 1-23, wherein
the hydrophobic component comprises a transmembrane domain of a
CD28.
25. The fusion protein according to any one of claims 1-23, wherein
the hydrophobic component comprises a transmembrane domain of a
4-1BB.
26. The fusion protein according to any one of claims 1-25, wherein
the intracellular signaling domain comprises an intracellular
signaling domain of a costimulatory molecule.
27. The fusion protein of claim 26, wherein the costimulatory
molecule comprises CD28, CD137 (4-1BB), or ICOS.
28. The fusion protein of any of claims 1-27, wherein the
intracellular signaling domain comprises an intracellular signaling
domain of a CD3.epsilon., CD3.delta., CD3.zeta., CD25, CD27, CD28,
CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150
(SLAMF1), CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12,
FcR.alpha., FcR.beta., FcR.gamma., Fyn, Lck, LAT, LRP, NKG2D,
NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pT.alpha.,
TCR.alpha., TCR.beta., TRIM, Zap70, PTCH2, or any combination
thereof.
29. The fusion protein according to any one of claims 1-28, wherein
the intracellular signaling domain comprises a costimulatory domain
of a CD137 (4-1BB), CD27, CD28, ICOS, OX40 (CD134), or any
combination thereof.
30. The fusion protein according to any one of claims 1-29, wherein
the intracellular signaling domain comprises a costimulatory domain
of a CD137 (4-1BB) or CD28, or any combination thereof.
31. The fusion protein according to any one of claims 1-30, wherein
the intracellular signaling domain comprises a costimulatory domain
of a CD28.
32. The fusion protein according to any one of claims 1-31, wherein
the intracellular signaling domain comprises a costimulatory domain
of a CD137 (4-1BB).
33. The fusion protein according to any one of claims 1-32, wherein
the target is an immunosupressive ligand.
34. The fusion protein according to any one of claims 1-32, wherein
the target is selected from a CD47, a CD58, a CD95L (FasL), a
CD200, a CD270 (HVEM), a CD274 (PD-L1), and a GAL9.
35. The fusion protein according to any of claims 1-34, wherein (a)
the extracellular component comprises an extracellular portion of a
CD200R, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28 or a 41BB.
36. The fusion protein according to claim 35, wherein the
extracellular portion of the CD200R comprises the entire
extracellular domain of CD200R.
37. The fusion protein according to claim 35, wherein the
extracellular portion of the CD200R comprises at least 200 amino
acids from the N-terminus of CD200R.
38. The fusion protein according to claim 35, wherein the
extracellular portion of the CD200R comprises at least about 225
amino acids to at least about 235 amino acids from the N-terminus
of CD200R.
39. The fusion protein according to claim 35, wherein the
extracellular portion of the CD200R comprises at least about 234
amino acids from the N-terminus of CD200R.
40. The fusion protein according to any of claims 35-39, wherein
the CD200R is a human CD200R.
41. The fusion protein according to any one of claims 35-40,
wherein the intracellular component comprises a second
intracellular signaling domain.
42. The fusion protein according to claim 41, wherein the second
intracellular signaling domain comprises an intracellular signaling
domain of a CD137 (4-1BB).
43. The fusion protein according to any one of claims 35-42,
wherein the extracellular component comprises an extracellular
portion of a CD28 extending from the CD28 transmembrane domain.
44. The fusion protein according to claim 43, wherein the
extracellular portion of the CD28 comprises a cysteine residue.
45. The fusion protein according to any of claims 1-44, wherein the
intracellular signaling component does not contain a CD3.zeta.
signaling domain and/or does not contain a signaling domain capable
of delivering a primary signal to a T cell and/or does not contain
a signaling domain from a molecule naturally capable of delivering
a primary signal to a T cell.
46. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:2, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:3, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
47. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:2, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
48. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:8, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises the amino acid sequence encoded
by a nucleic acid molecule as set forth in SEQ ID NO.:5.
49. The fusion protein according to claim 41, wherein the
extracellular component further comprises a multimerization domain
having an amino acid sequence encoded by a nucleic acid molecule as
set forth in SEQ ID NO.:9.
50. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:11, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
51. The fusion protein according to claim 50, wherein the
extracellular component further comprises an amino acid sequence
encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:9.
52. The fusion protein according to claim 51, wherein the
intracellular component further comprises a second intracellular
signaling domain having an amino acid sequence encoded by a nucleic
acid molecule as set forth in SEQ ID NO.:13.
53. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an extracellular portion of a
CD200R, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD137 (4-1BB).
54. The fusion protein according to claim 53, wherein the
extracellular portion of the CD200R comprises the entire
extracellular domain of CD200R.
55. The fusion protein according to claim 53, wherein the
extracellular portion of the CD200R comprises at least 200 amino
acids from the N-terminus of CD200R.
56. The fusion protein according to claim 53, wherein the
extracellular portion of the CD200R comprises at least about 225
amino acids to at least about 235 amino acids from the N-terminus
of CD200R.
57. The fusion protein according to any one of claims 53-56,
wherein the extracellular component comprises an extracellular
portion of a CD28 extending from the CD28 transmembrane domain.
58. The fusion protein according to claim 57, wherein the
extracellular portion of the CD28 comprises a cysteine residue.
59. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an extracellular portion of a
CD200R, (b) the hydrophobic component comprises a transmembrane
domain of a CD137 (4-1BB), and (c) the intracellular component
comprises an intracellular signaling domain of a CD137 (4-1BB).
60. The fusion protein according to claim 59, wherein the
extracellular portion of the CD200R comprises the entire
extracellular domain of CD200R.
61. The fusion protein according to claim 59, wherein the
extracellular portion of the CD200R comprises at least a portion of
the extracellular domain at least 230 amino acids from the
N-terminus of CD200R.
62. The fusion protein according to any one of claims 59-61,
wherein the extracellular component comprises an extracellular
portion of a CD137 (4-1BB) extending from the CD137 (4-1BB)
transmembrane domain.
63. The fusion protein according to claim 62, wherein the
extracellular portion of the CD137 (4-1BB) comprises a cysteine
residue.
64. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises a binding domain with an amino
acid sequence encoded by a nucleic acid molecule as set forth in
SEQ ID NO.:8 and a multimerization domain with an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:9, (b) the hydrophobic component comprises an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:4, and (c) the intracellular component comprises an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:13.
65. The fusion protein according to claim 64, wherein the
intracellular component further comprises a second intracellular
signaling domain having an amino acid sequence encoded by a nucleic
acid molecule as set forth in SEQ ID NO.:5.
66. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises a binding domain with an amino
acid sequence encoded by a nucleic acid molecule as set forth in
SEQ ID NO.:11 and a multimerization domain with an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:9, (b) the hydrophobic component comprises an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:4, and (c) the intracellular component comprises an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:13.
67. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an extracellular portion of a
SIRP.alpha., (b) the hydrophobic component comprises a
transmembrane domain of a CD28, and (c) the intracellular component
comprises an intracellular signaling domain of a CD28.
68. The fusion protein according to claim 67, wherein the
extracellular portion of the SIRP.alpha. comprises the entire
extracellular domain of SIRP.alpha..
69. The fusion protein according to claim 67, wherein the
extracellular portion of the SIRP.alpha. comprises at least 361
amino acids from the N-terminus of SIRP.alpha..
70. The fusion protein according to any one of claims 67-69,
wherein the intracellular component comprises a second
intracellular signaling domain.
71. The fusion protein according to claim 70, wherein the second
intracellular signaling domain comprises an intracellular signaling
domain of a CD137 (4-1BB).
72. The fusion protein according to any one of claims 67-71,
wherein the extracellular component comprises an extracellular
portion of a CD28 extending from the CD28 transmembrane domain.
73. The fusion protein according to claim 72, wherein the
extracellular portion of the CD28 comprises a cysteine residue.
74. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:17, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:18, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
75. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:17, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
76. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises a binding domain with an amino
acid sequence encoded by a nucleic acid molecule as set forth in
SEQ ID NO.:21 and a multimerization domain with an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:9, (b) the hydrophobic component comprises the amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:4, and (c) the intracellular component comprises the amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:5.
77. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises a binding domain with an amino
acid sequence encoded by a nucleic acid molecule as set forth in
SEQ ID NO.:21 and a multimerization domain with an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:9, (b) the hydrophobic component comprises the amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:4, and (c) the intracellular component comprises the amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:13.
78. The fusion protein according to claim 70, wherein the
intracellular component further comprises the amino acid sequence
encoded by a nucleic acid molecule as set forth in SEQ. ID.
NO:5.
79. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an extracellular portion of a
CD279 (PD-1), (b) the hydrophobic component comprises a
transmembrane domain of a CD28, and (c) the intracellular component
comprises an intracellular signaling domain of a CD28.
80. The fusion protein according to claim 79, wherein the
extracellular portion of the CD279 (PD-1) comprises the entire
extracellular domain of CD279 (PD-1).
81. The fusion protein according to claim 79, wherein the
extracellular portion of the CD279 (PD-1) comprises a portion of
the extracellular domain at least 100 amino acids from the
N-terminus of CD279 (PD-1).
82. The fusion protein according to claim 79, wherein the
extracellular portion of the CD279 (PD-1) comprises a portion of
the extracellular domain at least 149 amino acids from the
N-terminus of CD279 (PD-1).
83. The fusion protein according to any one of claims 79-82,
wherein the extracellular component comprises an extracellular
portion of a CD28 extending from the CD28 transmembrane domain.
84. The fusion protein according to claim 83, wherein the
extracellular portion of the CD28 comprises a cysteine residue.
85. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:89 and a
multimerization domain with an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:9, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
86. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:91 and a
multimerization domain with an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:9, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
87. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:95 and a
multimerization domain with an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:9, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
88. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an extracellular portion of a
CD95 (Fas), (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
89. The fusion protein according to claim 88, wherein the
extracellular portion of the CD95 (Fas) comprises the entire
extracellular domain of CD95 (Fas).
90. The fusion protein according to claim 88, wherein the
extracellular portion of the CD95 (Fas) comprises at least a
portion of the extracellular domain of at least 175 amino acids
from the N-terminus of CD95 (Fas).
91. The fusion protein according to claim 88, wherein the
extracellular portion of the CD95 (Fas) comprises at least a
portion of the extracellular domain of at least 173 amino acids
from the N-terminus of CD95 (Fas).
92. The fusion protein according to claim 88, wherein the
extracellular portion of the CD95 (Fas) comprises at least a
portion of the extracellular domain of at least 166 amino acids
from the N-terminus of CD95 (Fas).
93. The fusion protein according to claim 88, wherein the
extracellular portion of the CD95 (Fas) comprises at least a
portion of the extracellular domain of at least 161 amino acids
from the N-terminus of CD95 (Fas).
94. The fusion protein according to any one of claims 88-93,
wherein the extracellular component comprises an extracellular
portion of a CD28 extending from the CD28 transmembrane domain.
95. The fusion protein according to claim 94, wherein the
extracellular portion of the CD28 comprises a cysteine residue.
96. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:71 and a
multimerization domain with an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:9, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
97. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:73 and a
multimerization domain with an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:9, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
98. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:75 and a
multimerization domain with an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:9, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
99. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an extracellular portion of a
CD2, (b) the hydrophobic component comprises a transmembrane domain
of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
100. The fusion protein according to claim 99, wherein the
extracellular portion of the CD2 comprises the entire extracellular
domain of CD2.
101. The fusion protein according to claim 99, wherein the
extracellular portion of the CD2 comprises at least a portion of
the extracellular domain of at least 175 amino acids from the
N-terminus of CD2.
102. The fusion protein according to any one of claims 99-101,
wherein the extracellular component comprises an extracellular
portion of a CD28 extending from the CD28 transmembrane domain.
103. The fusion protein according to claim 102, wherein the
extracellular portion of the CD28 comprises a cysteine residue.
104. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:61 and a
multimerization domain with an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:9, (b) the
hydrophobic component comprises an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:4, and (c) the
intracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:5.
105. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an extracellular portion of a
TIM3, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
106. The fusion protein according to claim 105, wherein the
extracellular portion of the TIM3 comprises the entire
extracellular domain of TIM3.
107. The fusion protein according to claim 105, wherein the
extracellular portion of the TIM3 comprises at least a portion of
the extracellular domain of at least 180 amino acids from the
N-terminus of TIM3.
108. The fusion protein according to any one of claims 105-107,
wherein the extracellular component comprises an extracellular
portion of a CD28 extending from the CD28 transmembrane domain.
109. The fusion protein according to claim 108, wherein the
extracellular portion of the CD28 comprises a cysteine residue.
110. The fusion protein according to claim 1, wherein (a) the
extracellular component comprises an extracellular portion of a
LAG3, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
111. The fusion protein according to claim 110, wherein the
extracellular portion of the LAG3 comprises the entire
extracellular domain of LAG3.
112. The fusion protein according to claim 110, wherein the
extracellular portion of the LAG3 comprises at least a portion of
the extracellular domain of at least 410 amino acids from the
N-terminus of LAG3.
113. The fusion protein according to any one of claims 110-112,
wherein the extracellular component comprises an extracellular
portion of a LAG3 extending from the CD28 transmembrane domain.
114. The fusion protein according to claim 113, wherein the
extracellular portion of the CD28 comprises a cysteine residue.
115. A fusion protein according to claim 5, wherein the
extracellular component comprises a binding domain with an amino
acid sequence as set forth in SEQ ID NO.:31 and a multimerization
domain with an amino acid sequence as set forth in SEQ ID NO.:32,
(b) the hydrophobic component comprises an amino acid sequence as
set forth in SEQ ID NO.:27, and (c) the intracellular component
comprises an amino acid as set forth in SEQ ID NO.:36.
116. The fusion protein according to claim 115, wherein the
intracellular component further comprises a second intracellular
signaling domain having an amino acid sequence as set forth in SEQ
ID NO.:28.
117. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises a binding domain with an amino
acid sequence as set forth in SEQ ID NO.:34 and a multimerization
domain with an amino acid sequence as set forth in SEQ ID NO.:32,
(b) the hydrophobic component comprises an amino acid sequence as
set forth in SEQ ID NO.:27, and (c) the intracellular component
comprises an amino acid sequence as set forth in SEQ ID NO.:36.
118. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:40, (b) the hydrophobic component comprises an
amino acid sequence as set forth in SEQ ID NO.:41, and (c) the
intracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:28.
119. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:40, (b) the hydrophobic component comprises an
amino acid sequence as set forth in SEQ ID NO.:27, and (c) the
intracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:28.
120. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises a binding domain with an amino
acid sequence as set forth in SEQ ID NO.:44 and a multimerization
domain with an amino acid sequence as set forth in SEQ ID NO.:32,
(b) the hydrophobic component comprises the amino acid sequence as
set forth in SEQ ID NO.:27, and (c) the intracellular component
comprises the amino acid sequence as set forth in SEQ ID
NO.:28.
121. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises a binding domain with an amino
acid sequence as set forth in SEQ ID NO.:44 and a multimerization
domain with an amino acid sequence as set forth in SEQ ID NO.:32,
(b) the hydrophobic component comprises the amino acid sequence as
set forth in SEQ ID NO.:27, and (c) the intracellular component
comprises the amino acid sequence as set forth in SEQ ID
NO.:36.
122. The fusion protein according to claim 121, wherein the
intracellular component further comprises the amino acid sequence
as set forth in SEQ. ID. NO:28.
123. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:90 and a multimerization domain with an amino
acid sequence as set forth in SEQ ID NO.:32, (b) the hydrophobic
component comprises an amino acid sequence as set forth in SEQ ID
NO.:27, and (c) the intracellular component comprises an amino acid
sequence as set forth in SEQ ID NO.:28.
124. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:92 and a
multimerization domain with an amino acid sequence as set forth in
SEQ ID NO.:32, (b) the hydrophobic component comprises an amino
acid sequence as set forth in SEQ ID NO.:27, and (c) the
intracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:28.
125. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:96 and a
multimerization domain with an amino acid sequence as set forth in
SEQ ID NO.:32, (b) the hydrophobic component comprises an amino
acid sequence as set forth in SEQ ID NO.:27, and (c) the
intracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:28.
126. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:72 and a multimerization domain with an amino
acid sequence as set forth in SEQ ID NO.:32, (b) the hydrophobic
component comprises an amino acid sequence as set forth in SEQ ID
NO.:27, and (c) the intracellular component comprises an amino acid
sequence as set forth in SEQ ID NO.:28.
127. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:74 and a multimerization domain with an amino
acid sequence as set forth in SEQ ID NO.:32, (b) the hydrophobic
component comprises an amino acid sequence as set forth in SEQ ID
NO.:27, and (c) the intracellular component comprises an amino acid
sequence as set forth in SEQ ID NO.:28.
128. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:76 and a multimerization domain with an amino
acid sequence as set forth in SEQ ID NO.:32, (b) the hydrophobic
component comprises an amino acid sequence as set forth in SEQ ID
NO.:27, and (c) the intracellular component comprises an amino acid
sequence as set forth in SEQ ID NO.:28.
129. The fusion protein according to claim 5, wherein (a) the
extracellular component comprises an amino acid sequence as set
forth in SEQ ID NO.:62 and a multimerization domain with an amino
acid sequence as set forth in SEQ ID NO.:32, (b) the hydrophobic
component comprises an amino acid sequence as set forth in SEQ ID
NO.:27, and (c) the intracellular component comprises an amino acid
sequence as set forth in SEQ ID NO.:28.
130. A nucleic acid molecule encoding a fusion protein according to
any one of claims 1-129.
131. A vector comprising a nucleic acid molecule according to claim
130.
132. The vector according to claim 131, wherein the vector is a
viral vector.
133. The vector according to claim 132, wherein the viral vector is
a lentiviral or retroviral vector.
134. The vector according to claim 133, wherein the viral vector is
a lentiviral vector.
135. The vector according to any one of claims 131-134, further
encoding an antigen-specific TCR.
136. The vector according to claim 135, wherein the TCR is
exogenous to a host cell.
137. The vector according to claim 135 or claim 136, wherein the
TCR is specific to a HLA class I restricted antigen.
138. The vector according to any one of claims 135-137, wherein the
antigen is a cancer-specific antigen.
139. The vector according to claim 138, wherein the cancer-specific
antigen comprises WT-1, mesothelin, or cyclin-A1.
140. The vector according to any one of claims 131-139, further
encoding a ligand.
141. The vector according to claim 140, wherein the ligand is
CD200, CD47, PD-L1, or CD58.
142. The vector according to any one of claims 131-141, further
encoding an siRNA for reducing the expression of an endogenous
receptor.
143. The vector according to claim 142, wherein the endogenous
receptor comprises CD200R, SIRP.alpha., CD279 (PD-1), CD95 (Fas),
or CD2 or is a TCR or portion thereof.
144. A host cell, comprising a fusion protein according to any one
of claims 1-129.
145. A host cell, comprising at least two different fusion proteins
according to any one of claims 1-129.
146. A host cell according to claim 145, comprising: a fusion
protein according to claim 79; and a fusion protein according to
claim 105.
147. A host cell, comprising a nucleic acid molecule according to
claim 130.
148. A host cell, comprising a vector according to any one of
claims 131-143.
149. The host cell according to any one of claims 144-148, wherein
the host cell is an immune system cell.
150. The host cell according to claim 149, wherein the immune
system cell is a T cell.
151. The host cell according to claim 150, wherein the T cell is a
CD4+ T cell.
152. The host cell according to claim 150, wherein the T cell is a
CD8+ T cell.
153. The host cell according to any one of claims 144-152, further
comprising an antigen receptor, which optionally is an
antigen-specific TCR.
154. The host cell according to any one of claim 153, wherein the
antigen-specific TCR is exogenous to the cell or the host.
155. The host cell according to claim 153 or claim 154, wherein the
TCR binds to an antigen::HLA complex with high affinity.
156. The host cell according to claim 155, wherein the high
affinity binding has a K.sub.a equal to or greater than 10.sup.7
M.sup.-1.
157. The host cell according to any one of claims 153-156, wherein
the TCR is specific to a HLA class I restricted antigen.
158. The host cell according to any one of claims 153-156, wherein
the antigen is a cancer-specific antigen.
159. The host cell according to claim 158, wherein the
cancer-specific antigen comprises WT-1, mesothelin, or
cyclin-A1.
160. The host cell according to any one of claims 153-156, wherein
the antigen is a viral antigen.
161. The host cell according to claim 153, wherein the antigen
receptor is a chimeric antigen receptor.
162. The host cell according to claim 161, wherein the chimeric
antigen receptor comprises an extracellular antigen binding domain
and an intracellular signaling domain capable of delivering a
primary signal to a T cell and optionally a costimulatory
domain.
163. The host cell according to any one of claims 144-162, further
encoding a ligand.
164. The host cell according to claim 163, wherein the ligand is
CD200, CD47, PD-L1, or CD58.
165. The host cell according to any one of claims 144-164, further
encoding an siRNA for reducing the expression of an endogenous
receptor.
166. The host cell according to claim 165, wherein the endogenous
receptor comprises CD200R, SIRP.alpha., CD279 (PD-1), CD95 (Fas) or
CD2.
167. A method of treating a disease in a subject comprising
administering a fusion protein according to any one of claims 1-129
to the subject.
168. A method of treating a disease in a subject comprising
administering a vector according to any one of claims 131-143 to
the subject.
169. A method of treating a disease in a subject comprising
administering a host cell according to any one of claims 144-166 to
the subject.
170. The method according to any one of claims 167-169, wherein the
disease is selected from the group consisting of viral infection,
bacterial infection, cancer, and autoimmune disease.
171. The method according to any one of claims 167-170, wherein the
subject is human.
172. A fusion protein according to any one of claims 1-129, for use
in treating cancer.
Description
STATEMENT REGARDING SEQUENCE LISTING
[0001] 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
360056_433WO_SEQUENCE_LISTING.txt. The text file is 286 KB, was
created on Mar. 4, 2016, and is being submitted electronically via
EFS-Web.
BACKGROUND
[0002] T cell-based immunotherapies began to be developed when
tumor-reactive T cells were found among a population of
tumor-infiltrating lymphocytes (TILs) (Clark et al., Cancer Res.
29:705, 1969). One strategy, known as adoptive T cell transfer, in
some contexts involves the isolation of tumor infiltrating
lymphocytes pre-selected for tumor-reactivity, clonal expansion of
the tumor-reactive T cells induced by anti-CD3 and anti-CD28
antibodies in the presence of IL-2, and finally infusing the
expanded cell population back to the tumor-bearing patient
(together with chemotherapy and repetitive administration of IL-2)
(Dudley et al., Science 298:850, 2002). This form of adoptive T
cell therapy with tumor infiltrating lymphocytes can be technically
cumbersome and leads to complete remission in only a minor fraction
of patients with melanoma and is rarely effective in other cancers
(Besser et al., Clin. Cancer Res. 16:2646, 2010).
[0003] Isolation of tumor-reactive T cell clones led to the
development of another immunotherapeutic approach--the generation
of recombinant T cell receptors (TCRs) specific for particular
antigens, which may be introduced into T cells, e.g., using a
vector delivery system, to confer specificity for a desired target
such as a tumor-associated peptide presented by a major
histocompatibility complex (MEW) molecule expressed on a tumor cell
(known as human leukocyte antigen (HLA) molecule in humans).
Another approach introduces a synthetic receptor, termed a chimeric
antigen receptor (CAR), which generally contains an antigen-binding
domain, which, e.g., in the context of anti-tumor therapy can bind
to a tumor-specific or associated antigen, linked to one or more
intracellular component comprising an effector domains, such as a
primary signaling domain such as a TCR signaling domain or in some
contexts costimulatory signaling domains. Unlike administration of
TILs, the basic procedure for engineered TCR or CAR T cell
immunotherapy is generally to genetically modify human T cells with
a transgene encoding a tumor targeting moiety, ex vivo expansion of
the recombinant T cells, and transfusing the expanded recombinant T
cells back into patients.
[0004] Adoptive T cell therapy using T cells expressing recombinant
TCRs has been shown to have a promising clinical benefit,
especially in certain B cell cancers. However, effective T cell
activation often requires or is enhanced by a concurrent
co-stimulatory signal (Chen and Flies, Nat. Rev. Immunol. 13:
227-242, 2013). In the tumor microenvironment, co-stimulatory
molecules are generally downregulated. As a result, exogenous
stimulus via IL-2 is typically needed for T cells that express
recombinant TCRs specific for cancer antigens.
[0005] Activation of T cells is initiated when the TCR engages a
specific peptide presented in MHC on an antigen-presenting cell
(APC) (Rossy et al., Frontiers in Immunol. 3: 1-12, 2012). The
point of interaction of the T cell and the APC becomes the
immunological synapse, which is comprised of three concentric
supramolecular activation clusters (SMACs), including the central
cSMAC, peripheral pSMAC, and the distal dSMAC (Rossy et al.,
Frontiers in Immunol. 3: 1-12, 2012). Within the cSMAC,
co-stimulatory receptors can recruit signaling molecules to amplify
the TCR signal. Such co-stimulatory receptors can include CD28, and
in some contexts form microclusters with the TCR to lower the
threshold of activation (Chen and Flies, Nat. Rev. Immunol. 13:
227-242, 2013). Access to the cSMAC by transmembrane proteins
expressed by T cells may be restricted by the size of the
extracellular domain. For example, CD45 has a large ectodomain and
is generally excluded from the immunological synapse, thereby
preventing its ability to inhibit TCR signaling (James and Vale,
Nature 487:64-69, 2012).
[0006] There remains a need in the immunotherapy field for
alternative compositions and methods that provide immunomodulatory
signals to host cells for treating various diseases, such as cancer
or infections. Presently disclosed embodiments address these needs
and provide other related advantages.
BRIEF SUMMARY
[0007] In certain aspects, the present disclosure is directed to a
fusion protein, comprising an extracellular component that contains
a binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein::target complex spans a distance similar to a distance
between membranes in an immunological synapse.
[0008] In some embodiments, a length or spatial distance of a
complex formed between the fusion protein and the target or a
portion of such fusion protein::target complex (generally the
extracellular portion of such complex) is or spans a particular
distance, e.g., in some embodiments, is a distance that is less
than or less than about a certain distance. In some aspects, a
distance of the fusion protein::target complex (or, typically, the
extracellular portion thereof) is less than at or about 50 nm, less
than at or about 40 nm, less than at or about 30 nm, or less than
at or about 20 nm or equal to or less than at or about 15 nm. In
some embodiments, it is at or about 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 nm, such as at or about 14 or 15 nm. In some aspects,
the distance is one that is similar to a distance between membranes
in an immunological synapse or is a distance that is the same,
about the same, or substantially the same, as a distance between
the membrane proximal-most portion, e.g., residue, of the
extracellular domain of a TCR and the membrane proximal-most
portion, e.g., residue, of an MHC (e.g., HLA, such as an MHCI or
MHCII) molecule, with respect to a TCR-peptide/MHC complex or the
distance spanned by the extracellular portions of such a complex
(or spatial distance spanned by the extracellular portion known to
be contained within a synapse, such as a complex containing CD8,
CD4, CD28, and the respective binding partner or ligand thereof).
In some embodiments, spatial distances of complexes refer to a
distance between membranes of two different cells, wherein a first
cell and a second cell each express on their surface a binding
partner that can form a complex between the membranes when the
cells are in proximity to each other. In some aspects, the distance
is a distance that is the same, about the same, or substantially
the same, as a distance spanned by the extracellular portions of a
complex formed between a TCR and cognate interaction with an MEW
molecule. In some aspects, such as where a fusion protein comprises
a binding domain from a molecule ordinarily capable of entering an
immunological synapse or co-localizing with an antigen receptor,
the distance is similar to or the same as that spanned by a complex
formed between the molecule (having the binding domain used in the
fusion protein), and a natural binding partner thereof. In some
aspects, such as where the fusion protein comprises a binding
domain from a molecule ordinarily not capable of entering an
immunological synapse or ordinarily not capable of co-localizing
with an antigen receptor, the distance is different than, e.g.,
less than or substantially less than, that spanned by a complex
formed between the molecule (having the binding domain or
functional portion thereof used in the fusion protein), and a
natural binding partner thereof.
[0009] In some embodiments, a binding domain within the
extracellular component of a fusion protein of this disclosure
contains a target-binding portion of a molecule capable of
delivering an inhibitory signal, such as of an inhibitory molecule,
e.g., an immunoinhibitory molecule, such as an immunoinhibitory
receptor or immune checkpoint molecule. In some aspects, such a
molecule is a glycoprotein, checkpoint family member. In certain
embodiments, the fusion protein comprising a binding domain from a
glycoprotein, checkpoint family member or is not a B7 or B7-binding
molecule or is not a CD28-B7-superfamily member (e.g., is not a
CD28, CTLA4, ICOS, or other B7 family binding molecule) Exemplary
glycoprotein, checkpoint family members include CD200R,
SIRP.alpha., CD279 (PD-1), CD2, CD95 (Fas), CTLA4 (CD152), CD223
(LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5, or a binding
variant of any such molecule. In some embodiments, a binding domain
within the extracellular component of a fusion protein of this
disclosure comprises a binding partner of any of the foregoing, or
a binding variant of any such molecule. In some aspects of such
embodiments, the intracellular portion of a fusion protein includes
a signaling domain capable of delivering a stimulatory, such as a
costimulatory, signal to a lymphocyte, such as a T cell, such as a
costimulatory region of CD28, 41BB, ICOS, or other costimulatory
molecule. In some aspects, the intracellular portion of the fusion
protein does not include an intracellular signaling domain of the
inhibitory molecule, such as of a checkpoint or immunoinhibitory
molecule, when the extracellular binding portion is from a
checkpoint or immunoinhibitory molecule. In some aspects, a fusion
protein does not include a primary signaling domain such as a
CD3.zeta. signaling domain or other domain capable of delivering a
primary signal to a T cell.
[0010] In certain aspects, the extracellular component or the
binding portion thereof contains or is a binding domain of a
molecule or ectodomain capable of specifically binding to CD200,
such as a binding portion of a CD200R or variant thereof. In some
embodiments, the binding domain is or includes a binding region of
a molecule or of an ectodomain that is capable of specifically
binding to a CD47, such as a SIRP ectodomain or CD47-binding region
thereof, such as a SIRP.alpha. ectodomain or CD47-binding region
thereof. In some embodiments, the binding domain is capable of
binding to a PD-L1 or a PD-L2 or a LAG3 molecule. Exemplary targets
may be one or more proteins whose expression is increased or
upregulated in certain cells or tissues associated with or of a
disease or condition to be treated or ameliorated with the fusion
proteins and compositions provided herein, such as a tumor cell or
tumor microenvironment, or is bound by a receptor generally
upregulated on immune cells such as lymphocytes infiltrating a
diseased tissue, such as a tumor.
[0011] In some embodiments, the extracellular component further
includes one or more additional regions or domains, for example,
from a molecule other than that from which the binding domain is
derived or other than the molecule with which the binding domain
shares identity. The one or more additional extracellular domain(s)
may include a spacer region, such as one from an immunoglobulin
molecule, which may contain all or a portion of a hinge, or
constant region domain such as CH2 or CH3 domain, or from another
cell surface molecule such as a costimulatory receptor, such as
CD28. The additional extracellular domain(s) may include, in some
aspects, a multimerization domain, e.g., a dimerization domain or
sequence that may promote homo- or heterodimerization with another
molecule, such as multimerization of two or more of the fusion
proteins. In some embodiments, such a domain includes a portion of
an extracellular domain of a CD28 molecule including at least the
transmembrane-proximal-most cysteine, and generally an
extracellular portion between such cysteine and the membrane, or
modified variant thereof. In some aspects, such a domain includes
an amino acid sequence as set forth in SEQ ID NO: 32, or portion
thereof, or variant thereof such as having at least 90%, 95%, or
99% identity thereto. In some aspects, such a domain may be
included in order to facilitate or promote multimerization. In some
embodiments, a fusion protein contains an extracellular component
including a CD200-binding domain, such as an extracellular portion
(or portion thereof, such as a binding domain thereof) of a CD200R,
such as an extracellular portion of CD200R having an amino acid
sequence as set forth in SEQ ID NO: 25 or encoded by a nucleic acid
molecule as set forth in SEQ ID NO: 2, or a CD200-binding portion
thereof or variant thereof or binding portion thereof. In some
aspects of such embodiments, the extracellular portion of the
fusion protein further includes a portion of an extracellular
region of CD28, such as up to about 9 to about 12 amino acids
thereof (e.g., 9 amino acids or 12 amino acids), and in some
aspects including a membrane-proximal-most cysteine residue of a
CD28 extracellular region. In some such embodiments, the length of
the CD200R portion of the extracellular region is reduced in length
corresponding to the number of additional residues in the
CD28-derived portion, such as by about 9 to about 12 amino acids
(e.g., 9 amino acids or 12 amino acids), or by a sufficient number
of amino acids that the distance spanned by the extracellular
portion of a complex between the fusion protein and a CD200
molecule is similar to, substantially similar to, or the same as
that spanned by the extracellular portion of a complex between a
human CD200R, e.g., a CD200R, and CD200; or that spanned by the
extracellular portion of a complex between a TCR in cognate
interaction with an MHC molecule (e.g., MHC I or MHCII) in binding
to a cognate peptide-MHC complex; or that of an immunological
synapse. In some aspects, the fusion protein further includes a
transmembrane domain, such as a CD28 transmembrane, such as a
transmembrane domain encoded by the sequence set forth as SEQ ID
NO: 4 or portion thereof, or a modified version thereof, such as a
variant modified to contain additional charged regions or residues
or hydrophilic residues to facilitate intermolecular interactions.
In some embodiments, the protein further includes a CD28
intracellular signaling domain, such as a costimulatory domain of
CD28, such as one that is capable of recruiting one or more adapter
molecules to a CD28 in response to ligation. In some aspects, the
CD28 intracellular domain includes or is a sequence encoded by the
nucleotide sequence of SEQ ID NO: 5 or a portion or functional
variant thereof.
[0012] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein:target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
CD200R, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
[0013] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein:target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
CD200R, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28 and an intracellular
signaling domain of a CD137 (4-1BB).
[0014] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein::target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
CD200R, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD137 (4-1BB).
[0015] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein:target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
SIRPa, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
[0016] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein::target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
CD279 (PD-1), (b) the hydrophobic component comprises a
transmembrane domain of a CD28, and (c) the intracellular component
comprises an intracellular signaling domain of a CD28.
[0017] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein::target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
CD95 (Fas), (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
[0018] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein:target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
TIM3, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
[0019] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein:target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
LAG3, (b) the hydrophobic component comprises a transmembrane
domain of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
[0020] In some embodiments, the present disclosure is directed to a
fusion protein comprising an extracellular component comprised of a
binding domain that specifically binds a target, an intracellular
component comprised of an intracellular signaling domain, and a
hydrophobic component connecting the extracellular and
intracellular components, provided that the length of a fusion
protein::target complex spans a distance similar to a distance
between membranes in an immunological synapse, wherein (a) the
extracellular component comprises an extracellular portion of a
CD2, (b) the hydrophobic component comprises a transmembrane domain
of a CD28, and (c) the intracellular component comprises an
intracellular signaling domain of a CD28.
[0021] In certain aspects, the present disclosure is directed to a
nucleic acid molecule encoding a fusion protein as described
herein.
[0022] In certain aspects, the present disclosure is directed to a
vector comprising a nucleic molecule that encodes a fusion protein
as described herein.
[0023] In certain other aspects, the present disclosure is directed
to a host cell comprising a fusion protein, nucleic acid, or vector
as described herein.
[0024] In certain other aspects, a method of increasing the
activity of an immune cell is provided, comprising administering to
a subject in need of increased immune cell activity an effective
amount of a host cell as described herein.
[0025] In other aspects, the present disclosure is directed to a
method of enhancing or prolonging an immune response, comprising
administering to a subject in need of enhanced or prolonged immune
cell activity an effective amount of a host cell as described
herein.
[0026] In still other aspects, the present disclosure provides a
method of stimulating an antigen-specific T cell response,
comprising administering to a subject in need of increased immune
cell activity an effective amount of a host cell as described
herein.
[0027] In other aspects, the present disclosure is directed to a
method of inhibiting an immunosuppressive signaling pathway,
comprising administering to a subject in need thereof an effective
amount of a host cell as described herein.
[0028] In other aspects, the present disclosure is directed to a
method of treating cancer, comprising administering to a subject
having cancer a therapeutically effective amount of a host cell as
described herein.
[0029] In other aspects, the present disclosure is directed to a
method of inhibiting immune resistance of cancer cells, comprising
administering to a subject in need thereof an effective amount of a
host cell as described herein.
[0030] In still other aspects, the present disclosure provides a
method for treating a tumor, comprising administering to a subject
having a tumor a therapeutically effective amount of a host cell as
described herein, wherein the administered host cell is capable of
proliferating in an immunosuppressive tumor microenvironment.
[0031] A method of treating an infection, comprising administering
to a subject having the infection a therapeutically effective
amount of a host cell as described herein, is also provided by the
present disclosure.
[0032] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIGS. 1A and 1B show CD200R-CD28 constructs expressed at
high levels on primary murine CD8.sup.+ T cells. (A) Schematic
representation of exemplary CD200R-CD28 constructs. Construct "I"
contains CD200R extracellular ("EC") and transmembrane ("TM")
domains and a CD28 intracellular ("IC") signaling domain
(CD200Rtm-CD28). Construct "II" contains the extracellular domain
of CD200R and the transmembrane and intracellular domains of CD28
(CD200R-CD28tm). Constructs "III-V" also incorporate a portion of
the extracellular domain of CD28 to the transmembrane-proximal
cysteine to promote multimerization and enhance CD28 signaling. To
account for any extra extracellular amino acids (e.g., from one to
about 50 amino acids; such as exemplary murine constructs disclosed
here contain an extra nine (9) amino acids and exemplary human
constructs disclosed here contain twelve (12) amino acids), some
constructs have a truncated portion of an extracellular or
intracellular domain (e.g., a CD200R that preserves an N linked
glycosylation site). For example, construct IV has a truncated
portion of CD200R that is truncated by 3 amino acids. Construct V
has a truncated portion of CD200R that is truncated 9 amino acids.
Constructs "I", "II", and "V" maintain the short spatial distance
between the cells (e.g., between a T cell and an antigen presenting
cell) and may co-localize with the TCR within the cSMAC and deliver
a strong co-stimulatory signal. (B) Transgenic expression of murine
CD200R-CD28 constructs on TCR.sub.gag T cells as detected by
anti-CD200R antibody. The control vector contains green fluorescent
protein (GFP).
[0034] FIGS. 2A to 2G show that CD200R-CD28 constructs promote
proliferation, accumulation, and effector function in response to
CD200.sup.+ tumor target cells in vitro, and accumulate in the
immunological synapse. Splenocytes from naive TCR.sub.gag mice were
stimulated in vitro with anti-CD3, anti-CD28, and recombinant human
IL-2 (100 U/ml) and transduced with retroviral supernatant for 2
days. Cells were restimulated every 7 days with irradiated FBL and
splenocytes and cultured with rhIL-2 (50 U/mL) for up to three
stimulations. T cells were used for assays 5-7 days after the last
stimulation. (A) Proliferation of CD200R-CD28 and GFP control
TCR.sub.gag T cells as measured by CellTrace Violet dilution. T
cells were stimulated with CD200.sup.- FBL (upper panels) or
CD200.sup.+ FBL (lower panels) for 3 days. (B) Preferential
expansion/survival of transduced TCR.sub.gag T cells during
co-culture with non-transduced TCR.sub.gag T cells during weekly
cycles of stimulation with irradiated CD200.sup.+ FBL and
splenocytes. (C) Enrichment of transduced T cells. Repeated
restimulation with irradiated CD200.sup.+ tumor cells enriched the
cells transduced with CD200R-9aas-CD28Cys compared to wild-type T
cells transduced with an empty GFP control vector. (D) Increased
CD200R and CD200 signal intensity at T cell:FBL synapse. Lipid
rafts are increased at the immunological synapse (I).
CD200R-9aas-CD28Cys fusion proteins co-localized with lipid rafts,
indicating that the fusion proteins concentrate within the
immunological synapse (III, IV). (E) CD200R-CD28.sup.+ CD8.sup.+ T
cells display enhanced ability to lyse CD200.sup.+ FBL cells in
vitro. Target tumor cells were labeled with different dilutions of
the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl
ester (CFSE), as indicated. Effector TCR.sub.gag T cells transduced
with the indicated CD200R-CD28 fusion protein or an empty vector
control were incubated at the indicated effector to target ratio
with a 1:1 mix of CD200.sup.+ FBL (CFSE.sup.hi) and non-specific
EL4 (CFSE.sup.lo) control targets for 5 hours. The percentage of
FBL of the sum of FBL and control tumor cells was determined by
flow cytometry. The percentage lysis was determined by dividing the
percent of FBL incubated with T cells by the percent of FBL
incubated without T cells. (F) Target tumor cells for CFSE assay in
(G). Target tumor cells were labeled with different dilutions of
the fluorescent dyes CellTrace Violet (CTV) or CFSE. A 1:1:1 mix of
EL4 cells (CTV+), CD200.sup.+ FBL (CFSE.sup.hi) and non-specific
EL4 (CFSE.sup.lo) control targets was generated. (G) CFSE
cytotoxicity assay. TCR.sub.gag T cells were transduced with
CD200R-CD28 receptor or GFP control vector. Effector TCR.sub.gag T
cells were incubated at the indicated effector to target ratio with
a 1:1 mix of CD200.sup.- FBL or CD200.sup.+ FBL and non-specific
EL4 control targets for 4 hours. The percentage of FBL of the sum
of FBL and control tumor cells was determined by flow cytometry.
The percentage lysis was determined by dividing the percent of FBL
incubated with T cells by the percent of FBL incubated without T
cells.
[0035] FIGS. 3A to 3D show that T cells transduced with
CD200R-9aas-CD28Cys preferentially accumulate in response to tumor
challenge in vivo and express surface proteins consistent with an
effector phenotype after injection into Cytoxan-treated,
FBL-bearing mice. Transduced TCR.sub.gag T cells were generated as
described in Example 2. (A) Experimental schematic. C57BL/6 mice
were injected with 4.times.10.sup.6 CD200.sup.+ FBL cells. Five
days later, CD200R-9aas-CD28Cys (Thy1.1 homozygous) and eGFP
control (Thy1.1 heterozygous) TCR.sub.gag T cells were co-injected
into Cytoxan-treated FBL-bearing B6 mice at 4.times.10.sup.6
cells/mouse. IL-2 was administered every 2 days (2.times.10.sup.4
U/dose). On day 8 post-T cell transfer, mice were euthanized and
spleens and inguinal lymph nodes harvested. (B) CD200R-9aas-CD28Cys
TCR.sub.gag T cells accumulate in the spleen in response to FBL.
(LN=lymph node; Spl=spleen). (C) Comparison of surface proteins 3
days post-transfer for T cells transduced to express
CD200R-9aas-CD28Cys, T cells transduced with an empty vector, and
endogenous T cells. CD200R-9aas-CD28Cys TCR.sub.gag T cells
expressed reduced CD62L compared to control TCR.sub.gag T cells,
suggesting an effector T cell phenotype. (D) Comparison of surface
proteins 15 days post-transfer for cells transduced to express
CD200R-9aas-CD28Cys.sup.+ T cells, T cells transduced with an empty
vector, and endogenous T cells. CD200R-9aas-CD28Cys TCR.sub.gag T
cells express similar levels of cell surface proteins compared to
control TCR.sub.gag T cells.
[0036] FIGS. 4A to 4D show that adoptive immunotherapy with
CD200R-CD28-transduced T cells can eradicate disseminated leukemia.
(A) Experiment schematic. C57BL/6 mice were injected with
4.times.10.sup.6 CD200.sup.+ FBL cells. Five days later,
CD200R-CD28tm, CD200R-CD28Cys, CD200R-9aas-CD28Cys, or eGFP
TCR.sub.gag T cells were injected i.p. into Cy-treated FBL-bearing
mice at 10.sup.5 cells/mouse. IL-2 was administered every 2 days
(2.times.10.sup.4 U/dose) in a cohort of mice as indicated. (B)
Representative example of expression of cell surface proteins in
CD200R-CD28tm transduced T cells and non-transduced T cells on day
of injection with IL-2, as determined by flow cytometry. (C)
Survival of mice treated in the presence of IL-2 injections. (D)
Survival of mice treated in the absence of IL-2 injections.
Transfer of CD200R-9aas-CD28Cys TCR.sub.gag T cells significantly
improved survival in the absence of IL-2 injections (p<0.05,
log-rank Mantel-Cox test).
[0037] FIGS. 5A to 5C show that T cells expressing
CD200R-9aas-CD28Cys do not induce detectable autoimmune liver
damage or infiltrate normal tissues. (A) Experiment schematic.
Cytoxan-treated Alb/Gag mice were injected with 4.times.10.sup.6
CD200.sup.+ FBL cells. Five days later, CD200R-9aas-CD28Cys, and
eGFP TCR.sub.gag T cells were injected i.p. into the
Cytoxan-treated FBL-bearing mice at 10.sup.5 cells/mouse. IL-2 was
administered every 2 days (2.times.10.sup.4 U/dose) in a cohort of
mice as indicated. Three and 7 days post-transfer, liver damage was
assessed by quantification of serum levels of liver enzymes
aspartate aminotransferase (AST) and alanine aminotransferase
(ALT). (B) AST and ALT levels measured at 3 and 7 days
post-transfer for mice receiving no T cells, control T cells
expressing GFP, or T cells expressing CD200R-9aas-CD28Cys did not
vary by treatment. (C) Assessment of T cell infiltration of normal
tissue. Limited presence of T cells in liver tissue was observed
using antibodies specific to the T cell marker CD3 (left panel),
with no significant difference between recipients of
CD200R-9aas-CD28Cys TCR.sub.gag or control TCR.sub.gag T cells
(right panel).
[0038] FIGS. 6A to 6D show that 4-1BB co-stimulatory signaling
domains promote accumulation and effector function of transduced T
cells in vitro and promote survival of tumor-bearing recipients of
transduced T cell in response to CD200.sup.+ tumor target cells.
(A) Schematic representation of CD200R-CD28 ("V"), -4-1BB ("VI"),
and -CD28-4-1BB ("VII") constructs. (B) Expansion of transduced
TCR.sub.gag T cells relative to non-transduced TCR.sub.gag T cells
after weekly stimulation with irradiated CD200.sup.+ FBL and
splenocytes. CD200R-4-1BB and CD200R-CD28-4-1BB also promote
accumulation of transduced T cells in vitro. (C)
CD200R-9aas-4-1BB.sup.+ CD8.sup.+ T cells displayed an enhanced
ability to lyse CD200.sup.+ FBL cells in vitro relative to
controls, using a standard CFSE-based cytotoxicity assay. The
percentage of FBL of the sum of FBL and control tumor cells was
determined by flow cytometry. The percentage lysis was determined
by dividing the percent of FBL incubated with T cells by the
percent of FBL incubated without T cells. (D)
CD200R-41BB-transduced T cells also promote survival relative to
controls. C57BL/6 mice were injected with 4.times.10.sup.6
CD200.sup.+ FBL cells. Five days later, CD200R-9aas-CD28,
CD200R-9aas-4-1BB, CD200R-9aas-CD28-4-1BB, or eGFP TCR.sub.gag T
cells were injected i.p. into Cytoxan-treated FBL-bearing mice at
10.sup.5 cells/mouse.
[0039] FIGS. 7A to 7D show that human primary T cells transduced to
express a WT1-specific TCR and a CD200Rtm-CD28 fusion protein
exhibit enhanced proliferation to target cells that express CD200
and increased cytokine production in response to tumor cells that
express CD200. (A) Expression of the WT1.sub.126-specific TCR, C4,
and CD200Rtm-CD28. (B) Expression of CD200 in T2 and K562 cells. T2
cells exhibit low-level endogenous CD200 expression. (C)
Proliferation of T cells as indicated by CFSE. Cells that
proliferate in response to antigen show reduced CFSE fluorescence
intensity. T cells transduced with both C4 and the IFP show
enhanced proliferation to target cells expressing low levels of
CD200 relative to T cells transduced with C4 only. (D) Cytokine
production in response to exposure to CD200dim tumor cells, as
measured by flow cytometry. Relative to control T cells transduced
with the TCR C4 alone, T cells transduced with both C4 and the IFP
CD200Rtm-CD28 show increased cytokine production.
[0040] FIGS. 8A to 8E show that fusion proteins comprising
SIRP.alpha. extracellular components and CD28 co-stimulatory
signaling domains promote accumulation and proliferation of
transduced T cells in vitro. (A) Schematic representation of
exemplary SIRP.alpha.-CD28 constructs. Construct "I" contains
SIRP.alpha. extracellular ("EC") and transmembrane ("TM") domains
and a CD28 intracellular ("IC") signaling domain
(SIRP.alpha.tm-CD28). Construct "II" contains the extracellular
domain of SIRP.alpha. and the transmembrane and intracellular
domains of CD28 (SIRP.alpha.-CD28tm). Constructs "III-VI" also
incorporate a portion of the extracellular domain of CD28 to the
transmembrane-proximal cysteine to promote multimerization and
enhance CD28 signaling. To account for the extra extracellular
amino acids (e.g., extra nine (9) amino acids for murine
constructs, or twelve (12) amino acids for human constructs), some
constructs have a truncated portion of an extracellular or
intracellular domain (e.g., a SIRP.alpha. that preserves an N
linked glycosylation site). Construct IV has a truncated portion of
SIRP.alpha. that is truncated 6 amino acids to preserve an N linked
glycosylation site. Construct V has a truncated portion of
SIRP.alpha. that is truncated 9 amino acids. Construct VI has a
truncated portion of SIRP.alpha. that is truncated 23 amino acids.
Constructs "I", "II", and "V" maintain the short spatial distance
between the cells (e.g., between a T cell and an antigen presenting
cell) and may co-localize with the TCR within the cSMAC and deliver
a strong co-stimulatory signal. (B) Expansion of transduced
TCR.sub.gag T cells relative to non-transduced TCR.sub.gag T cells
after weekly stimulation with irradiated SIRP.alpha..sup.+ FBL and
splenocytes. SIRP.alpha.-CD28 constructs promote accumulation of
transduced T cells in vitro, with SIRP.alpha.-9aas-CD28Cys
exhibiting enhanced accumulation. (C) Proliferation of T cells
transduced with SIRP.alpha.-CD28 constructs in a CellTrace Violet
(CTV) dilution proliferation assay. T cells expressing
SIRP.alpha.-CD28 constructs engineered to maintain T cell-tumor
cell distance exhibited enhanced proliferation relative to
nontransduced T cells. (D) CD47.sup.+ tumor cells were killed after
co-culture with SIRP.alpha.-CD28.sup.+ T cells transduced to
express SIRP.alpha.tm-CD28 or SIRP.alpha.-9aas-CD28Cys constructs.
In contrast, tumor cells were not eradicated when cultured with T
cells receiving empty vector, or a truncated SIRP.alpha. lacking
its intracellular domain. (E) Results of an IncuCyte assay used to
quantify killing of CD47.sup.+ tumor cells. CD47.sup.+ FBL tumor
cells were transduced with mCherry. Loss of red signal indicates
killing of tumor cells. Killing of tumor cells was tested at the
effector:target ratios of 10:1, 2:1, and 0.4:1.
SIRP.alpha.-CD28.sup.+ T cells killed CD47.sup.+ tumor cells, even
at the lowest effector-to-target ratio tested.
[0041] FIGS. 9A and 9B show that fusion proteins comprising PD-1
extracellular components and CD28 co-stimulatory signaling domains
promote cytokine production in vitro. (A) Schematic representation
of exemplary PD-1-CD28 constructs. Construct "I" contains PD-1
extracellular ("EC") and transmembrane ("TM") domains and a CD28
intracellular ("IC") signaling domain (PD1tm-CD28). Construct "II"
contains the extracellular domain of PD-1 and the transmembrane and
intracellular domains of CD28 (PD1-CD28tm). Constructs "III-VII"
also incorporate a portion of the extracellular domain of CD28
adjacent to the transmembrane-proximal cysteine to promote
multimerization and enhance CD28 signaling. To account for the
extra extracellular amino acids (e.g., extra nine (9) amino acids
for murine constructs, or twelve (12) amino acids for human
constructs), constructs IV-VII have a truncated portion of PD-1.
Construct IV has a truncated portion of PD-1 that is truncated 9
amino acids. Construct V has a truncated portion of PD-1 that is
truncated 12 amino acids. Construct VI has a truncated portion of
PD-1 that is truncated 15 amino acids. Construct VII has a
truncated portion of PD-1 that is truncated 21 amino acids.
Constructs "I", "II", and "V" maintain the short spatial distance
between the cells (e.g., between a T cell and an antigen presenting
cell) and may co-localize with the TCR within the cSMAC and deliver
a strong co-stimulatory signal. (B) PD1-CD28.sup.+ T cells
exhibited increased cytokine production in response to stimulation
for 5 hours in the presence of Brefeldin A with FBL cells that
endogenously express the PD-1 ligands, PD-L1 and PD-L2. Stimulated
T cells were assessed for intracellular expression of the effector
cytokines, IFN.gamma. and TNF.alpha., by flow cytometry.
[0042] FIG. 10 shows co-expression of the TCR C4 and a PD-1 IFP
(PD1-12aas-CD28Cys, PD1-15aas-CD28Cys, or PD1-21aas-CD28Cys). T
cells transduced with C4 and PD1-12aas-CD28Cys or PD1-15aas-CD28Cys
exhibited high transduction efficiencies and expression of both
proteins.
[0043] FIGS. 11A to 11C show that fusion proteins comprising Fas
extracellular components and CD28 co-stimulatory signaling domains
accumulate in vitro upon stimulation with irradiated FBL cells. (A)
Schematic representation of exemplary Fas-CD28 constructs.
Construct "I" contains Fas extracellular ("EC") and transmembrane
("TM") domains and a CD28 intracellular ("IC") signaling domain
(Fastm-CD28). Construct "II" contains the extracellular domain of
Fas and the transmembrane and intracellular domains of CD28
(Fas-CD28tm). Constructs "III" and "IV" also incorporate a portion
of the extracellular domain of CD28 adjacent to the
transmembrane-proximal cysteine to promote multimerization and
enhance CD28 signaling. To account for the extra extracellular
amino acids (e.g., extra nine (9) amino acids for murine
constructs, or twelve (12) amino acids for human constructs),
construct IV has a truncated portion of Fas, wherein the Fas
extracellular domain is truncated 9 amino acids. Constructs "I",
"II", and "IV" maintain the short spatial distance between the
cells (e.g., between a T cell and an antigen presenting cell) and
may co-localize with the TCR within the cSMAC and deliver a strong
co-stimulatory signal. (B) Accumulation of TCR.sub.gag T cells
transduced with Fas constructs over multiple stimulations with
irradiated FBL cells. All of the constructs promoted accumulation
of T cells relative to control T cells. (C) Expression of Fas-CD28
constructs but not full-length (FL) Fas promoted survival or
expansion of T cells upon multiple stimulations in vitro.
[0044] FIGS. 12A and 12B show the structure and expression of
fusion proteins comprising LAG3 extracellular components and CD28
co-stimulatory signaling domains. (A) Schematic representation of
exemplary LAG3-CD28 constructs. Construct "I" contains LAG3
extracellular ("EC") and transmembrane ("TM") domains and a CD28
intracellular ("IC") signaling domain (LAG3tm-CD28). Construct "II"
contains the extracellular domain of LAG3 and the transmembrane and
intracellular domains of CD28 (LAG3-CD28tm). Constructs "III" and
"IV" also incorporate a portion of the extracellular domain of CD28
adjacent to the transmembrane-proximal cysteine to promote
multimerization and enhance CD28 signaling. To account for the
extra extracellular amino acids (e.g., extra nine (9) amino acids
for murine constructs, or twelve (12) amino acids for human
constructs), construct IV has a truncated portion of LAG3, wherein
the LAG3 extracellular domain is truncated 9 amino acids.
Constructs "I", "II", and "IV" maintain the short spatial distance
between the cells (e.g., between a T cell and an antigen presenting
cell) and may co-localize with the TCR within the cSMAC and deliver
a strong co-stimulatory signal. (B) Expression of LAG3-CD28
constructs by murine CD8.sup.+ T cells, as determined by anti-LAG3
antibody staining and flow cytometry. T cells transduced to express
LAG3-CD28 constructs (LAG3tm-CD28; LAG3-CD28tm; LAG3-CD28Cys;
LAG3-9aas-CD28Cys) exhibited expression of the constructs, in
contrast with control T cells that received empty vector.
[0045] FIGS. 13A and 13B show the structure and expression of
fusion proteins comprising TIM3 extracellular components and CD28
co-stimulatory signaling domains. (A) Schematic representation of
exemplary TIM3-CD28 constructs. Construct "I" contains TIM3
extracellular ("EC") and transmembrane ("TM") domains and a CD28
intracellular ("IC") signaling domain (TIM3tm-CD28). Construct "II"
contains the extracellular domain of TIM3 and the transmembrane and
intracellular domains of CD28 (TIM3-CD28tm). Constructs "III" and
"IV" also incorporate a portion of the extracellular domain of CD28
adjacent to the transmembrane-proximal cysteine to promote
multimerization and enhance CD28 signaling. To account for the
extra extracellular amino acids (e.g., extra nine (9) amino acids
for murine constructs, or twelve (12) amino acids for human
constructs), construct IV has a truncated portion of TIM3, wherein
the TIM3 extracellular domain is truncated 9 amino acids.
Constructs "I", "II", and "IV" maintain the short spatial distance
between the cells (e.g., between a T cell and an antigen presenting
cell) and may co-localize with the TCR within the cSMAC and deliver
a strong co-stimulatory signal. (B) Expression of TIM3-CD28
constructs by murine CD8.sup.+ T cells, as determined by anti-TIM3
antibody staining and flow cytometry. T cells transduced to express
TIM3-CD28 constructs (TIM3tm-CD28; TIM3-CD28tm; TIM3-CD28Cys;
TIM3-9aas-CD28Cys) typically exhibited expression of the
constructs, in contrast with control T cells that received empty
vector.
DETAILED DESCRIPTION
[0046] The instant disclosure provides fusion proteins that
modulate signaling in a host cell, such as an immune cell. For
example, fusion proteins of this disclosure can provide an
activation or co-stimulatory signal in a human T cell, wherein the
T cell may optionally be engineered to have a preferred
antigen-specific TCR. These immunomodulatory fusion proteins (IFPs)
can interact with ubiquitously expressed targets or with targets
that are commonly upregulated or overexpressed in non-normal cells
(e.g., a cancer cell). Such IFPs have an extracellular binding
domain and an intracellular signaling domain. By transducing T
cells with engineered TCRs (e.g., high affinity TCRs) and fusion
proteins of this disclosure that generate activation signals,
certain embodiments of T cells may no longer require exogenous
co-stimulation upon interaction with, for example, a tumor
cell.
[0047] In certain aspects, the present disclosure provides host
cells (e.g., immune cells such as T cells, dendritic cells, NK
cells or the like) comprising an IFP, vectors encoding IFPs, and
methods of activating T cells comprising an IFP for various
therapeutic applications, including the treatment of a disease in
subject (e.g., cancer, infectious disease).
[0048] 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.
[0049] 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 term "about" means .+-.20% of the
indicated range, value, or structure, 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. 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.
[0050] The term "consisting essentially of" limits the scope of a
claim to the specified materials or steps, or to those that do not
materially affect the basic characteristics of a claimed invention.
For example, a protein domain, region, or module (e.g., a binding
domain, hinge region, linker module) or a protein (which may have
one or more domains, regions, or modules) "consists essentially of"
a particular amino acid sequence when the amino acid sequence of a
domain, region, or module or protein includes extensions,
deletions, mutations, or any combination thereof (e.g., amino acids
at the amino- or carboxy-terminus or between domains) that, 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 a domain, region, or
module or protein and do not substantially affect (i.e., do not
reduce the activity by more than 50%, such as no more than 40%,
30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s),
region(s), module(s), or protein (e.g., the target binding affinity
of a binding protein).
[0051] As used herein, "heterologous" or "non-endogenous" or
"exogenous" refers to any gene, protein, compound, molecule, or
activity that is not native to a host cell or a subject, or is any
gene, protein, compound, molecule, or activity native to a host or
host cell that has been altered or mutated such that the structure,
activity or both is different as between the native and mutated
molecules. In certain embodiments, heterologous, non-endogenous or
exogenous molecules (e.g., receptors, ligands) may not be
endogenous to a host cell or subject, but instead nucleic acids
encoding such molecules may have been added to a host cell by
conjugation, transformation, transfection, electroporation, or the
like, wherein the added nucleic acid molecule may integrate into a
host cell genome or can exist as extra-chromosomal genetic material
(e.g., as a plasmid or other self-replicating vector). The term
"homologous" or "homolog" refers to a molecule or activity found in
or derived from a host cell, species, or strain. For example, a
heterologous or exogenous molecule or gene encoding the molecule
may be homologous to a native host or host cell molecule or gene
that encodes the molecule, respectively, but may have an altered
structure, sequence, expression level or combinations thereof. A
non-endogenous molecule may be from the same species, a different
species, or a combination thereof.
[0052] As used herein, the term "endogenous" or "native" refers to
a gene, protein, compound, molecule, or activity that is normally
present in a host or host cell and has no engineered
alterations.
[0053] A "binding domain" (also referred to as a "binding region"
or "binding moiety"), as used herein, refers to a molecule, such as
a peptide, oligopeptide, polypeptide or protein, that possesses the
ability to specifically and non-covalently associate, unite, or
combine with a target molecule (e.g., CD200, CD47, CD19, CD20,
CD22, ROR1, mesothelin, PD-L1, PD-L2, PSMA, WT-1, cyclin-A1). A
binding domain includes any naturally occurring, synthetic,
semi-synthetic, or recombinantly produced binding partner for a
biological molecule or other target of interest or binding protein
thereof. In some embodiments, the binding domain is an
antigen-binding domain, such as an antibody or T cell receptor
(TCR) or functional binding domain or antigen-binding fragment
thereof. Exemplary binding domains include receptor ectodomains
(e.g., those of CD200R, PD-1, CTLA4, BTLA, CD2, Fas) or binding
portions thereof, ligands (e.g., cytokines such as IL35,
chemokines) or binding portions thereof, single chain antibody
variable regions (e.g., domain antibodies, sFv, scFv, Fab) or
binding portions thereof, antigen-binding regions of T cell
receptors (TCRs), such as single chain TCRs (scTCRs), or synthetic
polypeptides selected for the specific ability to bind to a
biological molecule.
[0054] In some embodiments, "specifically binds" refers to an
association or union of a binding domain, or a fusion protein
thereof, to a target molecule 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, or binds to such target molecule while not significantly
associating or uniting with any other molecules or components in a
sample. Binding domains (or fusion proteins thereof) may be
classified as "high affinity" binding domains (or fusion proteins
thereof) or "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.8 M.sup.-1, at least 10.sup.9 M.sup.-1, at least 10.sup.10
M.sup.-1, at least 10.sup.11 M.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 (K.sub.d) of a particular binding interaction
with units of M (e.g., 10.sup.-5 M to 10.sup.-13M). In certain
embodiments, a binding domain may have "enhanced affinity," which
refers to a selected or engineered binding domain with stronger
binding to a target antigen than a wild type (or parent) binding
domain. For example, enhanced affinity may be due to a Ka
(equilibrium association constant) for the target antigen that is
higher than the wild type binding domain, or due to a K.sub.d
(dissociation constant) for the target antigen that is less than
that of the wild type binding domain, or due to an off-rate
(K.sub.off) for the target antigen that is less than that of the
wild type binding domain. A variety of assays are known for
identifying binding domains of the present disclosure that
specifically bind a particular target, as well as determining
binding domain or fusion protein affinities, such as Western blot,
ELISA, and Biacore.RTM. analysis (see also, e.g., Scatchard et al.,
Ann. N.Y. Acad. Sci. 51:660, 1949; and U.S. Pat. Nos. 5,283,173,
5,468,614, or the equivalent).
[0055] As used herein, a "fusion protein" refers to a polypeptide
that, in a single chain, has at least two distinct domains, wherein
the domains are not naturally found together in a protein. A
nucleic acid molecule encoding a fusion protein may be constructed
using PCR, recombinantly engineered, or the like, or such fusion
proteins can be made using methods of protein synthesis. A fusion
protein may further contain other components (e.g., covalently
bound), such as a tag or bioactive molecule. In certain
embodiments, a fusion protein expressed or produced by a host cell
(e.g., T cell) locates to the cell surface, where the fusion
protein is anchored to the cell membrane with a portion of the
fusion protein located extracellularly (e.g., containing a binding
domain) and a portion of the fusion protein located intracellularly
(e.g., containing a signaling domain).
[0056] A "hydrophobic component," as used herein, means any amino
acid sequence having a three-dimensional structure that is
thermodynamically stable in a cell membrane, and generally ranges
in length from about 15 amino acids to about 30 amino acids. The
structure of a hydrophobic component may comprise an alpha helix, a
beta barrel, a beta sheet, a beta helix, or any combination
thereof. In certain embodiments, a hydrophobic component is
comprised of a "transmembrane domain" from a known transmembrane
protein, which is a portion of a transmembrane protein that can
insert into or span a cell membrane. In further embodiments, a
hydrophobic component or transmembrane domain can be disposed
between and connect the extracellular and intracellular portions of
a fusion protein. Additionally, the hydrophobic component may be
modified to contain charged regions or hydrophilic residues to
facilitate intermolecular interactions.
[0057] As used herein, an "intracellular signaling domain" is an
intracellular portion of molecule, such as one used in a fusion
protein of this disclosure, that can directly or indirectly promote
a response such as a co-stimulatory, positive, or activating
biological or physiological response in a cell when receiving the
appropriate signal. In certain embodiments, an intracellular
signaling domain is part of a protein or protein complex that
receives a signal when bound, or itself can bind directly to a
target molecule to transmit a signal to other components in the
cell. An intracellular signaling domain may directly promote a
cellular response when it contains one or more signaling domains or
motifs, such as an immunoreceptor tyrosine-based activation motif
(ITAM), a kinase domain, a co-stimulatory domain, or the like. In
other embodiments, an intracellular signaling domain will
indirectly promote a cellular response by associating with one or
more other proteins that in turn directly promote a cellular
response. In some embodiments, an intracellular signaling domain or
functional fragment thereof may be from a CD3.epsilon., CD3.delta.,
CD3.zeta., CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134
(OX40), CD137 (4 1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR),
CARD11, DAP10, DAP12, FcR.alpha., FcR.beta., FcR.gamma., Fyn, Lck,
LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76,
pT.alpha., TCR.alpha., TCR.beta., TRIM, Zap70, PTCH2, or any
combination thereof. In some embodiments, an intracellular
signaling domain or functional fragment thereof does not comprise a
CD3.zeta..
[0058] A "multimerization domain," as used herein, refers to a
polypeptide molecule or region that preferentially interacts or
associates with another polypeptide molecule or region, directly or
indirectly, wherein the interaction of multimerization domains
substantially contribute to or efficiently promote multimerization
(i.e., the formation of a dimer, trimer, tetramer, or higher order
multimers, which may be a homodimer, heterodimer, homotrimer,
heterotrimer, homomultimer, heteromultimer, or the like). For
example, multimerization may be due to one or more types of
molecular forces, including covalent bonds (e.g., disulfide bonds
or bridges), ionic bonds, metallic bonds, electrostatic
interactions, salt bridges, dipole-dipole forces, hydrogen bonding,
Van der Waals forces, hydrophobic interactions, or any combination
thereof. A multimer is stable under appropriate conditions (e.g.,
physiological conditions, in an aqueous solution suitable for
expressing, purifying, or storing recombinant or engineered
proteins, or under conditions for non-denaturing or non-reducing
electrophoresis). Exemplary multimerization domains may comprise
one or more disulfide bonds, zinc finger motif, a leucine zipper
motif, helix-turn-helix, helix-loop-helix, or the like.
[0059] In certain embodiments, a fusion protein may contain a
"linker," which can provide a spacer function to facilitate the
interaction of two single chain fusion proteins, or positioning of
one or more binding domains, so that the resulting polypeptide
structure maintains a specific binding affinity to a target
molecule or maintains signaling activity (e.g., effector domain
activity) or both. Exemplary linkers include from one to about ten
repeats of Gly.sub.xSer.sub.y, wherein x and y are independently an
integer from 1 to 5.
[0060] "Junction amino acids" or "junction amino acid residues"
refer to one or more (e.g., about 2-20) amino acid residues between
two adjacent motifs, regions, or domains of a fusion protein, such
as between a binding domain and an adjacent hydrophobic component,
or on one or both ends of a hydrophobic component. 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). In certain embodiments, junction amino
acids form a linker, such as those having from one to about ten
repeats of Gly.sub.xSer.sub.y, wherein x and y are independently an
integer from 1 to 5.
[0061] As used herein, an "immune system cell" means any cell of
the immune system that originates from a hematopoietic stem cell in
the bone marrow, which gives rise to two major lineages, a myeloid
progenitor cell (which give rise to myeloid cells such as
monocytes, macrophages, dendritic cells, megakaryocytes and
granulocytes) and a lymphoid progenitor cell (which give rise to
lymphoid cells such as T cells, B cells and natural killer (NK)
cells). Exemplary immune system cells include a CD4+ T cell, a CD8+
T cell, a CD4- CD8- double negative T cell, a .gamma..delta. T
cell, a regulatory T cell, a natural killer cell, and a dendritic
cell. Macrophages and dendritic cells may be referred to as
"antigen presenting cells" or "APCs," which are specialized cells
that can activate T cells when a major histocompatibility complex
(MEW) receptor on the surface of the APC complexed with a peptide
interacts with a TCR on the surface of a T cell.
[0062] A "T cell" is an immune system cell that matures in the
thymus and produces T cell receptors (TCRs). T cells can be naive
(not exposed to antigen; increased expression of CD62L, CCR7, CD28,
CD3, CD127, and CD45RA, and decreased expression of CD45RO as
compared to T.sub.CM), memory T cells (T.sub.M)
(antigen-experienced and long-lived), and effector cells
(antigen-experienced, cytotoxic). T.sub.M can be further divided
into subsets of central memory T cells (T.sub.CM, increased
expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and
decreased expression of CD54RA as compared to naive T cells) and
effector memory T cells (T.sub.EM, decreased expression of CD62L,
CCR7, CD28, CD45RA, and increased expression of CD127 as compared
to naive T cells or T.sub.CM). Effector T cells (T.sub.E) refers to
antigen-experienced CD8+ cytotoxic T lymphocytes that have
decreased expression of CD62L, CCR7, CD28, and are positive for
granzyme and perforin as compared to T.sub.CM. Other exemplary T
cells include regulatory T cells, such as CD4+ CD25+ (Foxp3+)
regulatory T cells and Treg17 cells, as well as Tr1, Th3,
CD8+CD28-, and Qa-1 restricted T cells.
[0063] "T cell receptor" (TCR) refers to a molecule found on the
surface of T cells (or T lymphocytes) that, in association with
CD3, is generally responsible for recognizing antigens bound to
major histocompatibility complex (MEW) molecules. The TCR has a
disulfide-linked heterodimer of the highly variable .alpha. and
.beta. chains (also known as TCR.alpha. and TCR.beta.,
respectively) in most T cells. In a small subset of T cells, the
TCR is made up of a heterodimer of variable .gamma. and .delta.
chains (also known as TCR.gamma. and TCR.delta., respectively).
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 Janeway et al.,
Immunobiology: The Immune System in Health and Disease, 3.sup.rd
Ed., Current Biology Publications, p. 4:33, 1997). TCR, as used in
the present disclosure, may be from various animal species,
including human, mouse, rat, cat, dog, goat, horse, or other
mammals. TCRs may be cell-bound (i.e., have a transmembrane region
or domain) or in soluble form.
[0064] "Major histocompatibility complex molecules" (MHC
molecules), which is used interchangeably and is understood to also
refer to the human counterpart human leukocyte antigen (HLA
molecules), refer to glycoproteins that deliver peptide antigens to
a cell surface. MHC class I molecules are heterodimers consisting
of a membrane spanning .alpha. chain (with three a domains) and a
non-covalently associated .beta.2 microglobulin. MHC class II
molecules are composed of two transmembrane glycoproteins, .alpha.
and .beta., both of which span the membrane. Each chain has two
domains. MHC (HLA) class I molecules deliver peptides originating
in the cytosol to the cell surface, where peptide:MHC (or
peptide:HLA in humans) complex is recognized by CD8.sup.+ T cells.
MHC (HLA) class II molecules deliver peptides originating in the
vesicular system to the cell surface, where they are recognized by
CD4.sup.+ T cells. An MHC molecule may be from various animal
species, including human, mouse, rat, or other mammals.
[0065] "Nucleic acid molecule", or polynucleotide, may be in the
form of RNA or DNA, which includes cDNA, genomic DNA, and synthetic
DNA. A nucleic acid molecule may be double stranded or single
stranded, and if single stranded, may be the coding strand or
non-coding (anti-sense strand). A coding molecule may have a coding
sequence identical to a coding sequence known in the art or may
have a different coding sequence, which, as the result of the
redundancy or degeneracy of the genetic code, or by splicing, can
encode the same polypeptide.
[0066] Variants of the nucleic acid molecules or polynucleotides of
this disclosure are also contemplated. Variant polynucleotides are
at least 90%, and preferably 95%, 99%, or 99.9% identical to one of
the polynucleotides of defined sequence as described herein, or
that hybridizes to one of those polynucleotides of defined sequence
under stringent hybridization conditions of 0.015M sodium chloride,
0.0015M sodium citrate at about 65-68.degree. C. or 0.015M sodium
chloride, 0.0015M sodium citrate, and 50% formamide at about
42.degree. C. The polynucleotide variants retain the capacity to
encode a binding domain or fusion protein thereof having the
functionality described herein.
[0067] The term "stringent" is used to refer to conditions that are
commonly understood in the art as stringent. Hybridization
stringency is principally determined by temperature, ionic
strength, and the concentration of denaturing agents such as
formamide. Examples of stringent conditions for hybridization and
washing are 0.015M sodium chloride, 0.0015M sodium citrate at about
65-68.degree. C. or 0.015M sodium chloride, 0.0015M sodium citrate,
and 50% formamide at about 42.degree. C. (see Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989).
[0068] More stringent conditions (such as higher temperature, lower
ionic strength, higher formamide, or other denaturing agent) may
also be used; however, the rate of hybridization will be affected.
In instances wherein hybridization of deoxyoligonucleotides is
concerned, additional exemplary stringent hybridization conditions
include washing in 6.times.SSC, 0.05% sodium pyrophosphate at
37.degree. C. (for 14-base oligonucleotides), 48.degree. C. (for
17-base oligonucleotides), 55.degree. C. (for 20-base
oligonucleotides), and 60.degree. C. (for 23-base
oligonucleotides).
[0069] A "vector" is a nucleic acid molecule that is capable of
transporting another nucleic acid. Vectors may be, for example,
plasmids, cosmids, viruses, or phage. An "expression vector" is a
vector that is capable of directing the expression of a protein
encoded by one or more genes carried by the vector when it is
present in the appropriate environment.
[0070] "Retroviruses" are viruses having an RNA genome.
"Gammaretrovirus" refers to a genus of the retroviridae family.
Exemplary gammaretroviruses include mouse stem cell virus, murine
leukemia virus, feline leukemia virus, feline sarcoma virus, and
avian reticuloendotheliosis viruses.
[0071] "Lentivirus" refers to a genus of retroviruses that are
capable of infecting dividing and non-dividing cells. Several
examples of lentiviruses include HIV (human immunodeficiency virus:
including HIV type 1, and HIV type 2); equine infectious anemia
virus; feline immunodeficiency virus (Hy); bovine immune deficiency
virus (BIV); and simian immunodeficiency virus (SIV).
[0072] The terms "identical" or "percent identity," in the context
of two or more polypeptide or nucleic acid molecule sequences,
means two or more sequences or subsequences that are the same or
have a specified percentage of amino acid residues or nucleotides
that are the same over a specified region (e.g., 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% identity), when compared and aligned for maximum
correspondence over a comparison window, or designated region, as
measured using methods known in the art, such as a sequence
comparison algorithm, by manual alignment, or by visual inspection.
For example, preferred algorithms suitable for determining percent
sequence identity and sequence similarity are the BLAST and BLAST
2.0 algorithms, which are described in Altschul et al. (1977)
Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol.
215:403, respectively.
[0073] "Treat" or "treatment" or "ameliorate" refers to medical
management of a disease, disorder, or condition of a subject (e.g.,
a human or non-human mammal, such as a primate, horse, dog, mouse,
or rat). In general, an appropriate dose or treatment regimen
comprising a host cell expressing a fusion protein of this
disclosure, and optionally an adjuvant or adjunctive therapy, is
administered in an amount sufficient to elicit a therapeutic or
prophylactic benefit. Therapeutic or prophylactic/preventive
benefit includes improved clinical outcome; lessening or
alleviation of symptoms associated with a disease; decreased
occurrence of symptoms; improved quality of life; longer
disease-free status; diminishment of extent of disease,
stabilization of disease state; delay of disease progression;
remission; survival; prolonged survival; or any combination
thereof.
[0074] A "therapeutically effective amount" or "effective amount"
of a fusion protein or cell expressing a fusion protein of this
disclosure (e.g., CD200R-CD28, SIRP.alpha.-CD28, CD200R-41BB,
SIRP.alpha.-41BB, CD200R-CD28-41BB, SIRP.alpha.-CD28-4-1BB or other
such fusion proteins), in the context of a disease or condition
being treated, refers to that amount of fusion protein or number of
cells sufficient to result in amelioration of one or more symptoms
of the disease being treated in a statistically significant manner
(e.g., reducing infection, reducing tumor size, inhibiting cancer
growth or the like).
Immunomodulatory Fusion Proteins (IFPs)
[0075] In certain aspects, the present disclosure provides a fusion
protein, comprising an extracellular component, a hydrophobic
component, and an intracellular component. In some embodiments, the
extracellular component includes a binding domain such as one that
specifically binds to a target. In some embodiments, the binding
domain is from a molecule that ordinarily, e.g., in its natural
setting, is capable of delivering a negative or inhibitory signal
when bound to its binding partner or ligand or receptor, such as an
immunoinhibitory receptor or checkpoint molecule, or the target is
an inhibitory receptor or ligand or checkpoint molecule or other
inhibitory ligand. In some embodiments, the intracellular component
includes a signaling domain, such as a costimulatory signaling
domain or signaling region of a molecule generally capable of
delivering a costimulatory or positive signal, e.g., to an immune
cell. Thus, in some aspects, the fusion proteins are capable of
delivering a positive or costimulatory signal in response to a
binding event that in a natural setting would result in an
inhibitory signal.
[0076] In some embodiments, the fusion protein is such that a
particular distance is achieved. For example, in some embodiments,
a fusion protein::target complex (such as one comprised of an
extracellular portion of a complex formed between the fusion
protein and the target by specific binding thereto) is of a
particular length or spans a particular distance, such as a
distance of up to a distance between membranes in an immunological
synapse, or that spanned by the extracellular portion of a cognate
complex between a TCR and WIC molecule, e.g., following specific
recognition thereof by a TCR, or the distance spanned by the
extracellular portion of a complex formed between the natural
molecule and its natural binding partner. In some embodiments, the
distance or length is sufficient to promote the colocalization of a
fusion protein with antigen receptor or other signaling molecule
when expressed in an immune cell, such as a T cell, or entry into
an immunologic synapse.
[0077] By way of background, an immunological synapse is an
interface between cells, which can form between a variety of cells,
such as between immune cells (Rossy et al., Frontiers in Immunol.
3: 1-12, 2012; Hatherley et al., Structure 21:820, 2013). For
example, in the case of a T cell contacting an antigen-presenting
cell (APC), an immunological synapse can be formed by the binding
of a TCR (found on the surface of a T cell) with an HLA-peptide
(MHC-peptide for non-human) complex (found on the surface of, for
example, APCs; HLA class I molecules can be found on the surface of
all nucleated cells, while HLA class II can conditionally be
expressed on all cell types but are regularly found on APCs). In
addition, an immunological synapse may be organized into
supramolecular activation clusters (SMACs), which can affect
lymphocyte activation, direct antigen-HLA (or antigen-MHC) complex
presentation to lymphocytes, and direct secretion of cytokines or
lytic granules between cells. A SMAC can be comprised of three
structures arranged in concentric circles: a central region (cSMAC)
containing a high number of TCRs as well as co-stimulatory and
inhibitory molecules, a peripheral region (pSMAC) where LFA-1 and
talins are clustered, and a distal region (dSMAC) that is enriched
for CD43 and CD45 molecules. In certain embodiments, an
immunological synapse will span from about 10 nm to about 15 nm.
For example, protein interactions found within the immunological
synapse, such as the TCR::HLA-peptide interaction or a fusion
protein-target interaction, generally span about 14 nm between
membranes. In certain embodiments, the width of a SMAC in an
immunological synapse does not exceed 15 nm.
[0078] In some embodiments, the extracellular span of a fusion
protein::target complex is such that it can localize to a
particular compartment of an immunological synapse. Some complexes
thought to localize to various compartments of the immunological
synapse are well-characterized with regard to the length of their
extracellular span. For example, the MHC-TCR complex is thought to
have an extracellular span of approximately 10-15 nm and more
integrin-based complexes are thought to have extracellular spans on
the order of approximately 40 nm (Alakoskela et al., Biophys J
100:2865, 2011). Additional exemplary complexes include the
CD2-CD48 complex, which is thought to have an extracellular span of
approximately 12.8 nm (Milstein et al., J Biol Chem 283:34414,
2008). Additionally, exemplary ligand-binding molecules thought to
localize to the cSMAC include the TCR and MHC complexes, CD2, CD4,
CD8, CD28, and ligands thereof (Dustin et al., CSH Perspectives in
Biology 2:a002311, 2010); thus, it is contemplated that these
molecules complexed with their natural ligands are of an
appropriate size to localize to the cSMAC.
[0079] In some aspects, the length or distance or approximate
length or distance of a particular construct or engineered
extracellular portion thereof such as an extracellular portion of a
fusion protein, or complex of any of the foregoing such as with a
binding partner thereof, may be determined or modeled by known
methods. In some exemplary models, a protein's tertiary structure,
binding domains, and other characteristics may be approximated
using an input amino acid or nucleic acid sequence. The tertiary
structure of a protein may be used to approximate extracellular
portion size, flexibility, and other characteristics useful for
determining the approximate length of the extracellular portion of
the protein or complex thereof. In general, methods for modeling or
approximating the length of the extracellular portion of a protein
are known. For example, molbiol-tools.ca and Swiss-Model contain
multiple tools useful for predicting protein structure (see also
Schwede, T., Structure 21:1531, 2013).
[0080] In certain embodiments, a fusion protein of this disclosure
complexed, associated or interacting with a target is capable of
residing within an immunological synapse. In some embodiments, the
extracellular portion of a fusion protein::target complex spans an
immunological synapse. In other embodiments, a fusion
protein::target complex is localized in a supramolecular activation
cluster (SMAC), such as a cSMAC. In further embodiments, the
extracellular portion of a fusion protein::target complex spans an
immunological synapse defined by the extracellular portion of a
TCR::HLA-peptide interaction. In still further embodiments, the
length of the extracellular portion of a fusion protein::target
complex is about 12 nm to about 15 nm, or is about 14 nm.
[0081] The distance between the cell membranes of cells interacting
in an immunological synapse may be measured by any method known in
the art. For example, in particular embodiments, the distance may
be measured by a subdiffraction-resolution method or electron
microscopy (James and Vale, Nature 487:64-69, 2012).
[0082] In particular embodiments, a fusion protein as disclosed
herein comprises an extracellular portion that extends less than 40
nm from the cell membrane. In some embodiments, a fusion protein as
disclosed herein comprises an extracellular portion that extends
less than 30 nm from the cell membrane. In some embodiments, a
fusion protein as disclosed herein comprises an extracellular
portion that extends less than 20 nm from the cell membrane. In
some embodiments, a fusion protein as disclosed herein comprises an
extracellular portion that extends less than 15 nm from the cell
membrane.
[0083] In some embodiments, the provided fusion proteins provide
the advantage of having an extracellular length or spatial distance
as compared to the distance between cell membrane(s) that allows
for entry into a synapse or co-localization with antigen receptor,
or that mimic a distance or length present in the natural proteins.
In some embodiments, where the extracellular portion of the fusion
protein includes domain(s) from an additional molecule, which is
from a different molecule from which a binding domain is obtained,
the length of the extracellular component containing the binding
domain is reduced, e.g., truncated, as compared to the
extracellular region of the natural molecule, to provide for such
similar length or distance. In some embodiments, a fusion protein
as described herein comprises an extracellular component comprising
an extracellular domain of a cell-surface receptor and a second
domain (e.g., a linker or an extracellular domain of a second
cell-surface receptor). In some such embodiments, to maintain an
extracellular component capable of residing within an immunological
synapse or spanning an immunological synapse when complexed with a
target molecule, one or more domains of the extracellular component
may be truncated.
[0084] In some diseases (e.g., cancer), the amplitude and quality
of a T cell response resulting from antigen recognition by a T cell
receptor (TCR) can be dysregulated (e.g., reduced) due to an
imbalance between co-stimulatory and inhibitory signals, which can
result in immune resistance. One advantage of certain fusion
proteins of the instant disclosure is that a first signal can be
converted into a qualitatively different second signal. For
example, in some embodiments, the fusion proteins are such that a
negative or inhibitory signal can effectively be converted into a
positive or co-stimulatory signal to thereby relieve or minimize
immune resistance associated with a disease, such as cancer. For
example, upon binding to a target that, if bound by its natural
binding partner, would result in inhibition or delivery of a
negative signal, a fusion protein as provided herein, in some
embodiments, is capable of instead delivering a positive, e.g.,
costimulatory signal, to a cell in which it is expressed, such as
in a T cell. In certain embodiments, a fusion protein of this
disclosure comprises an extracellular component associated with a
negative signal and an intracellular component associated with a
positive signal. An exemplary receptor found on the surface of T
cells, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4 or
CD152), can receive an inhibitory signal when bound by one of its
ligands, CD80 or CD86, found on APCs. CTLA4 regulates the amplitude
of early stage T cell activation by counteracting the T cell
co-stimulatory receptor CD28 (see Rudd et al., Immunol. Rev.
229:12, 2009). Another exemplary receptor found on the surface of T
cells, programmed cell death protein 1 (PD-1 or CD279), can receive
an inhibitory signal when bound by one of its ligands, PD-L1
(B7-H1, CD274) or PD-L2 (B7-DC, CD73), found on APCs. PD-1 limits
the activity of T cells in peripheral tissues during inflammation
and to minimize autoimmunity (see Keir et al., Annu. Rev. Immunol.
26:677, 2008). Representative fusion proteins of this disclosure
comprising an extracellular component associated with a negative
signal (e.g., CTLA4 or PD-1) and an intracellular component
associated with a positive signal (e.g., CD28, CD137) include a
CTLA4-CD28 fusion protein, a CTLA4-CD137 fusion protein, a
CTLA4-CD28-CD137 fusion protein, a PD1-CD28 fusion protein, a
PD1-CD137 fusion protein, or a PD1-CD28-CD137 fusion protein.
[0085] Fusion proteins of the instant disclosure may block or
reduce the number of inhibitory signals received by an immune cell.
For example, in some embodiments, a fusion protein as disclosed
herein converts an inhibitory signal into a positive signal,
thereby reducing the total number of inhibitory signals received by
an immune cell or converting an ordinarily negative or inhibitory
signal to a positive one. In other embodiments, a fusion protein as
disclosed herein blocks the signaling of a wild-type receptor. For
example, dominant negative fusion proteins are included within the
scope of the disclosure. In some embodiments, a fusion protein as
disclosed herein binds to a wild-type receptor and blocks signaling
of the wild-type receptor by forming an oligomer with the wild-type
receptor.
[0086] Yet another advantage of certain fusion proteins of the
instant disclosure is that more than one such fusion protein may be
expressed by a cell, providing multiple stimulatory signals. It has
been observed that recombinant TCRs possessing multiple
co-stimulatory domains may not produce adequate co-stimulatory
signaling. Co-expressing multiple immunomodulatory fusion proteins,
especially those capable of residing within an immunological
synapse, may provide the co-stimulatory signaling necessary for T
cells to avoid anergy and proliferate.
[0087] In some embodiments, a fusion protein of the instant
disclosure operates in trans relative to a TCR or chimeric antigen
receptor (CAR) or other antigen receptor. In some embodiments, a
fusion protein as disclosed herein operates outside of the
immunological synapse.
[0088] In yet another aspect, a fusion protein of the instant
disclosure allows for enrichment of transduced T cells by
restimulation with tumor cells expressing a ligand that binds to
the fusion protein, without the need for sorting.
[0089] In one exemplary embodiment, a fusion protein comprising (a)
an extracellular portion of a CD200R, (b) a transmembrane domain of
a CD28, and (c) an intracellular signaling domain of a CD28 is
provided. In some embodiments, the extracellular portion further
comprises an extracellular portion of a CD28 extending from the
CD28 transmembrane domain. In further embodiments, the
extracellular portion of the CD200R comprises at least about 231
amino acids from the N-terminus of CD200R. In still further
embodiments, the fusion protein further comprises an intracellular
signaling domain of a CD137 (4-1BB).
[0090] In another exemplary embodiment, the present disclosure
provides a fusion protein comprising (a) an extracellular portion
of a SIRP.alpha., (b) a transmembrane domain of a CD28, and (c) an
intracellular signaling domain of a CD28. In some embodiments, the
fusion protein further comprises an extracellular portion of a CD28
extending from the CD28 transmembrane domain. In further
embodiments, the extracellular portion of the SIRP.alpha. comprises
at least about 361 amino acids from the N-terminus of SIRP.alpha..
In still further embodiments, the fusion protein further comprises
an intracellular signaling domain of a CD137 (4-1BB).
[0091] Component parts of the fusion proteins of the present
disclosure are further described in detail herein.
[0092] Extracellular Component
[0093] As described herein, a fusion protein of the present
disclosure generally comprises an extracellular component
comprising a binding domain that specifically binds a target.
Binding of a target by the fusion protein binding domain may (1)
block the interaction of target with another molecule (e.g., block
or interfere with a receptor-ligand interaction), (2) interfere,
reduce or eliminate certain functions of the target (e.g.,
inhibitory signal transduction), (3) induce certain biological
pathways not normally induced when the target is bound (e.g.,
converting an inhibitory or negative signal into a stimulatory or
positive signal), such as in a cell in which the fusion protein is
expressed, or any combination thereof. In some embodiments, the
fusion proteins as described herein comprise an extracellular
portion, wherein the extracellular portion comprises an
extracellular portion of protein associated with a negative
signal.
[0094] Exemplary binding domains of this disclosure may be
ectodomains of cell-surface receptors, or binding portions thereof,
ectodomains of cell-surface ligands, cytokines (e.g., IL35),
chemokines, antibody-based binding domains, TCR-based binding
domains, non-conventional binding domains, or any combination
thereof. For example, binding domains comprising an ectodomain of
CD200R, SIRP.alpha., CD279 (PD-1), CD2, CD95 (Fas), CTLA4 (CD152),
CD223 (LAG3), CD272 (BTLA), A2aR, KIR, TIM3, CD300, or LPA5 are
within the scope of this disclosure. As used herein, an
"ectodomain" from a cell-surface receptor or ligand includes a
complete extracellular domain or a functional (binding) fragment
thereof. In certain embodiments, an ectodomain comprises a mutated
extracellular domain or a functional (binding) fragment thereof
that has a higher avidity for target as compared to a wild-type or
reference protein. In certain embodiments, an ectodomain comprises
a variable-like domain or a CDR of a variable-like domain.
[0095] In some embodiments, a fusion protein contains an
extracellular component comprising a CD200-binding domain, such as
a CD200R ectodomain or CD200-binding portion thereof. By way of
background, CD200R is a receptor that binds to CD200, a type-1
membrane protein of the immunoglobulin superfamily (Tonks et al.,
Leukemia 21:566-568, 2007). CD200 has been reported to be
upregulated on various malignancies, including leukemias, multiple
myeloma, and various solid tumors (e.g., melanoma, breast, and
squamous cell carcinoma). In fact, high levels of CD200 expression
have been linked with poor prognosis for acute myeloid leukemia
(AML), and CD200R signaling has been shown to have an inhibitory
effect on T cells (Coles et al., Leukemia 26: 2148-2151, 2012). In
certain embodiments, a CD200R ectodomain includes a full length
extracellular portion of a CD200R protein, a full length mature
extracellular portion of a CD200R protein, a binding fragment of an
extracellular portion of a CD200R protein, or a binding fragment of
an extracellular portion of a CD200R protein along with a portion
of the transmembrane domain of CD200R, or any combination
thereof.
[0096] In further embodiments, a CD200R is encoded by a nucleic
acid molecule as set forth in SEQ ID NO.:2. In certain other
embodiments, a CD200R ectodomain comprises at least 200 amino acids
from the N-terminus of CD200R. In some other embodiments, a CD200R
is encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:11. In yet other embodiments, an extracellular portion of the
CD200R comprises at least 180, 190, 200, 210, 220, 230, 231, 234,
or 243 amino acids from the N-terminus of CD200R. For example, in
certain embodiments, a CD200R is encoded by the nucleic acid
molecule as set forth in SEQ ID NO.:8. In any of the aforementioned
embodiments, a CD200R, a CD200R ectodomain, or any CD200R fragment
thereof used in a fusion protein of this disclosure is a human
CD200R. In further embodiments, there are provided CD200R
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:2.
[0097] In some embodiments, a CD200R comprises an amino acid
sequence as set forth in SEQ ID NO.:25. In some embodiments, a
CD200R comprises an amino acid sequence as set forth in SEQ ID
NO.:34. In certain embodiments, a CD200R comprises an amino acid
sequence as set forth in SEQ ID NO.:31. In any of the
aforementioned embodiments, a CD200R, a CD200R ectodomain, or any
CD200R fragment thereof used in a fusion protein of this disclosure
is a human CD200R. In further embodiments, there are provided
CD200R ectodomains that have a sequence that is at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence as set forth
in SEQ ID NO.:25.
[0098] In some embodiments, a fusion protein contains an
extracellular component comprising a CD47-binding domain such as a
SIRP.alpha. ectodomain or binding portion thereof. By way of
background, CD47 is a widely expressed transmembrane protein that
plays a role in protecting cells from phagocytosis (Willingham et
al., PNAS 109: 6662-6667, 2012). Binding of CD47 to SIRP.alpha.
initiates SIRP.alpha. signaling, which inhibits phagocytosis by
macrophages. Accordingly, downregulation of SIRP.alpha. will result
in increased phagocytosis by macrophages. SIRP.alpha. is expressed
on multiple human tumor types including AML, chronic myelogenous
leukemia (CML), acute lymphoblastic leukemia (ALL), Non-Hodgkin
lymphoma (NHL), multiple myeloma (MM), lung, bladder, and other
solid tumors. In certain embodiments, a SIRP.alpha. ectodomain
includes a full length extracellular portion of a SIRP.alpha.
protein, a full length mature extracellular portion of a
SIRP.alpha. protein, a binding fragment of an extracellular portion
of a SIRP.alpha. protein, and a binding fragment of an
extracellular portion of a SIRP.alpha. protein along with a portion
of the transmembrane domain of SIRP.alpha., or any combination
thereof.
[0099] In further embodiments, a SIRP.alpha. ectodomain or binding
portion thereof is encoded by a nucleic acid molecule as set forth
in SEQ ID NO.:17. In certain embodiments, a SIRP.alpha. ectodomain
comprises at least 300, 310, 320, 330, 340, 350, 360, 361, 370,
373, or more amino acids from the N-terminus of SIRP.alpha.. In
some other embodiments, a SIRP.alpha. is encoded by a nucleic acid
molecule as set forth in SEQ ID NO.:21. In any of the
aforementioned embodiments, a SIRP.alpha., a SIRP.alpha.
ectodomain, or any SIRP.alpha. fragment thereof used in a fusion
protein of this disclosure is a human SIRP.alpha.. In further
embodiments, there are provided SIRP.alpha. ectodomains that have a
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, at least
99.5%, or at least 100% identical to an ectodomain of a molecule
having an amino acid sequence encoded by a nucleic acid molecule as
set forth in SEQ ID NO.:17.
[0100] In further embodiments, a SIRP.alpha. ectodomain comprises
an amino acid sequence as set forth in SEQ ID NO.:40. In some
embodiments, a SIRP.alpha. comprises an amino acid sequence as set
forth in SEQ ID NO.:44. In any of the aforementioned embodiments, a
SIRP.alpha., a SIRP.alpha. ectodomain, or any SIRP.alpha. fragment
thereof used in a fusion protein of this disclosure is a human
SIRP.alpha.. In further embodiments, there are provided SIRP.alpha.
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence as set forth
in SEQ ID NO.:40.
[0101] In some embodiments, a fusion protein contains an
extracellular component comprising a binding domain that binds to
PD-L1, PD-L2, or both. In some embodiments, a fusion protein
contains an extracellular component comprising a PD-1 ectodomain or
ligand-binding portion thereof. In certain embodiments, a PD-1
ectodomain includes a full length extracellular portion of a PD-1
protein, a full length mature extracellular portion of a PD-1
protein, a binding fragment of an extracellular portion of a PD-1
protein, or a binding fragment of an extracellular portion of a
PD-1 protein along with a portion of the transmembrane domain of
PD-1, or any combination thereof. In certain embodiments, a PD-1
ectodomain comprises at least 80, 90, 100, 110, 120, 125, 130, 132,
135, 137, 140, 149, 150, 155, 158, 160, or 170 amino acids from the
N-terminus of PD-1. For example, in certain embodiments, a PD-1
ectodomain is encoded by the nucleic acid molecule as set forth in
SEQ ID NO.:91, 93, or 95. In further embodiments, a PD-1 ectodomain
comprises at least from about 90 amino acids to at least about 130
amino acids from a PD-1 as set forth in SEQ ID NO.:60. In still
further embodiments, a PD-1 ectodomain comprises 170 amino acids
from the N-terminus of a PD-1 ectodomain, as set forth in SEQ ID
NO.:90. In some embodiments, a PD-1 is encoded by a nucleic acid
molecule as set forth in SEQ ID NO.:89. In any of the
aforementioned embodiments, a PD-1, a PD-1 ectodomain, or any PD-1
fragment thereof used in a fusion protein of this disclosure is a
human PD-1. In further embodiments, there are provided PD-1
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence as set forth
in SEQ ID NO.:60. In further embodiments, there are provided PD-1
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence as set forth
in SEQ ID NO.:90. In still further embodiments, there are provided
PD-1 binding domains that have a sequence that is at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:89.
[0102] In certain embodiments, a PD-1 ectodomain comprises the
amino acid sequence as set forth in SEQ ID NO.:92, 94, or 96. In
further embodiments, there are provided PD-1 ectodomains that have
a sequence that is at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, at least
99.5%, or at least 100% identical to an ectodomain of a molecule
having an amino acid sequence as set forth in SEQ ID NO.: 92, 94,
or 96. In any of the aforementioned embodiments, a PD-1, a PD-1
ectodomain, or any PD-1 fragment thereof used in a fusion protein
of this disclosure is a human PD-1.
[0103] In some embodiments, a fusion protein contains an
extracellular component comprising a CD2 ectodomain. In certain
embodiments, a CD2 ectodomain is encoded by a nucleic acid molecule
as set forth in SEQ ID NO.:61. In certain embodiments, a CD2
ectodomain includes a full length extracellular portion of a CD2
protein, a full length mature extracellular portion of a CD2
protein, a binding fragment of an extracellular portion of a CD2
protein, or a binding fragment of an extracellular portion of a CD2
protein along with a portion of the transmembrane domain of CD2, or
any combination thereof. In any of the aforementioned embodiments,
a CD2, a CD2 ectodomain, or any CD2 fragment thereof used in a
fusion protein of this disclosure is a human CD2. In further
embodiments, there are provided CD2 ectodomains that have a
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, at least
99.5%, or at least 100% identical to an ectodomain of a molecule
having an amino acid sequence encoded by a nucleic acid molecule as
set forth in GenBank Accession No. NM_001767.3. In further
embodiments, there are provided CD2 ectodomains s that have a
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, at least
99.5%, or at least 100% identical to an ectodomain of a molecule
having an amino acid sequence encoded by a nucleic acid molecule as
set forth in SEQ ID NO.:61.
[0104] In some embodiments, a CD2 ectodomain comprises an amino
acid sequence as set forth in SEQ ID NO.:62. In further
embodiments, there are provided CD2 ectodomains that have a
sequence that is at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, at least
99.5%, or at least 100% identical to an ectodomain of a molecule
having an amino acid sequence as set forth in SEQ ID NO.:62. In any
of the aforementioned embodiments, a CD2, a CD2 ectodomain, or any
CD2 fragment thereof used in a fusion protein of this disclosure is
a human CD2.
[0105] In some embodiments, a fusion protein contains an
extracellular component comprising a binding domain that binds to
FasL. In some embodiments, a fusion protein contains an
extracellular component comprising a Fas (CD95) ectodomain. Fas is
expressed on tumor-associated vasculature and prevents CD8 cell
infiltration by inducing cell death. In certain embodiments, a Fas
ectodomain includes a full length extracellular portion of a Fas
protein, a full length mature extracellular portion of a Fas
protein, a binding fragment of an extracellular portion of a Fas
protein, and a binding fragment of an extracellular portion of a
Fas protein along with a portion of the transmembrane domain of
Fas, or any combination thereof. In some embodiments, a Fas
ectodomain is encoded by a nucleic acid molecule as set forth in
SEQ ID NO.:71. In yet other embodiments, a Fas ectodomain comprises
at least 150, 160, 161, 166, 170, or 173 amino acids from the
N-terminus of Fas. For example, in certain embodiments, a Fas is
encoded by the nucleic acid molecule as set forth in SEQ ID NO.:73.
In certain other embodiments, a Fas is encoded by the nucleic acid
molecule as set forth in SEQ ID NO.:75. In any of the
aforementioned embodiments, a Fas, a Fas ectodomain, or any Fas
fragment thereof used in a fusion protein of this disclosure is a
human Fas. In further embodiments, there are provided Fas
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence encoded by a
nucleic acid molecule as set forth in GenBank Accession No.
NM_000043.4. In still further embodiments, there are provided Fas
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:71.
[0106] In some embodiments, a Fas ectodomain comprises an amino
acid sequence as set forth in SEQ ID NO.:72. In certain
embodiments, a Fas ectodomain comprises the amino acid sequence as
set forth in SEQ ID NO.:74. In certain other embodiments, a Fas
ectodomain comprises the amino acid sequence as set forth in SEQ ID
NO.:76. In any of the aforementioned embodiments, a Fas, a Fas
ectodomain, or any Fas fragment thereof used in a fusion protein of
this disclosure is a human Fas. In further embodiments, there are
provided Fas ectodomains that have a sequence that is at least 80%,
at least 85%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, at least 99.5%, or at least 100% identical
to an ectodomain of a molecule having an amino acid sequence as set
forth in SEQ ID NO.:72.
[0107] In some embodiments, a fusion protein contains an
extracellular component comprising a LAG3 (CD223) ectodomain. In
certain embodiments, a LAG3 ectodomain includes a full length
extracellular portion of a LAG3 protein, a full length mature
extracellular portion of a LAG3 protein, a binding fragment of an
extracellular portion of a LAG3 protein, and a binding fragment of
an extracellular portion of a LAG3 protein along with a portion of
the transmembrane domain of LAG3, or any combination thereof. For
example, in some embodiments, a LAG3 ectodomain comprises about
420, 416, 415, 413, or 410 amino acids from the N terminus of LAG3.
In any of the aforementioned embodiments, a LAG3, a LAG3
ectodomain, or any LAG3 fragment thereof used in a fusion protein
of this disclosure is a human LAG3. In further embodiments, there
are provided LAG3 ectodomains that have a sequence that is at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, at least 99.5%, or at least 100%
identical to an ectodomain of a molecule having an amino acid
sequence encoded by a nucleic acid molecule as set forth in GenBank
Accession No. NM_002286.5.
[0108] In further embodiments, a LAG3 is encoded by a nucleic acid
molecule as set forth in SEQ ID NO.:153. In certain other
embodiments, a LAG3 ectodomain comprises at least 430, 435, 438,
440, 445, or 450 amino acids from the N-terminus of LAG3. For
example, in certain embodiments, a LAG3 is encoded by the nucleic
acid molecule as set forth in SEQ ID NO.:161. In any of the
aforementioned embodiments, a LAG3, LAG3 ectodomain, or any LAG3
fragment thereof used in a fusion protein of this disclosure is a
human LAG3. In further embodiments, there are provided LAG3
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:153.
[0109] In some embodiments, a LAG3 comprises an amino acid sequence
as set forth in SEQ ID NO.:154. In some embodiments, a LAG3
comprises an amino acid sequence as set forth in SEQ ID NO.:162. In
any of the aforementioned embodiments, a LAG3, a LAG3 ectodomain,
or any LAG3 fragment thereof used in a fusion protein of this
disclosure is a human LAG3. In further embodiments, there are
provided LAG3 ectodomains that have a sequence that is at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, at least 99.5%, or at least 100%
identical to an ectodomain of a molecule having an amino acid
sequence as set forth in SEQ ID NO.:154.
[0110] In some embodiments, a fusion protein contains an
extracellular component comprising a TIM3 ectodomain. In certain
embodiments, a TIM3 ectodomain includes a full length extracellular
portion of a TIM3 protein, a full length mature extracellular
portion of a TIM3 protein, a binding fragment of an extracellular
portion of a TIM3 protein, and a binding fragment of an
extracellular portion of a TIM3 protein along with a portion of the
transmembrane domain of TIM3, or any combination thereof. In any of
the aforementioned embodiments, a TIM3, a TIM3 ectodomain, or any
TIM3 fragment thereof used in a fusion protein of this disclosure
is a human TIM3. In further embodiments, there are provided TIM3
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence encoded by a
nucleic acid molecule as set forth in GenBank Accession No.
NM_032782.4.
[0111] In further embodiments, a TIM3 is encoded by a nucleic acid
molecule as set forth in SEQ ID NO.:167. In certain other
embodiments, a TIM3 ectodomain comprises at least 180, 185, 190,
195, or 200 amino acids from the N-terminus of TIM3. For example,
in certain embodiments, a TIM3 is encoded by the nucleic acid
molecule as set forth in SEQ ID NO.:177. In any of the
aforementioned embodiments, a TIM3, TIM3 ectodomain, or any TIM3
fragment thereof used in a fusion protein of this disclosure is a
human TIM3. In further embodiments, there are provided TIM3
ectodomains that have a sequence that is at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, at least 99.5%, or at least 100% identical to an
ectodomain of a molecule having an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:167.
[0112] In some embodiments, a TIM3 comprises an amino acid sequence
as set forth in SEQ ID NO.:168. In some embodiments, a TIM3
comprises an amino acid sequence as set forth in SEQ ID NO.:178. In
any of the aforementioned embodiments, a TIM3, a TIM3 ectodomain,
or any TIM3 fragment thereof used in a fusion protein of this
disclosure is a human TIM3. In further embodiments, there are
provided TIM3 ectodomains that have a sequence that is at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, at least 99.5%, or at least 100%
identical to an ectodomain of a molecule having an amino acid
sequence as set forth in SEQ ID NO.:168.
[0113] A binding domain may be any peptide that specifically binds
a target of interest. Sources of binding domains include antibody
variable regions from various species (which can be in the form of
antibodies, sFvs, scFvs, Fabs, scFv-based grababody, or soluble VH
domain or domain antibodies), including human, rodent, avian, or
ovine. Additional sources of binding domains include variable
regions of antibodies from other species, such as camelid (from
camels, dromedaries, or llamas; Ghahroudi et al., FEBS Lett.
414:521, 1997; Vincke et al., J. Biol. Chem. 284:3273, 2009;
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 raffish (Nguyen et al.,
Immunogen. 54:39, 2002), or lamprey (Herrin et al., Proc. Nat'l.
Acad. Sci. (USA) 105:2040, 2008 and Alder et al. Nat. Immunol.
9:319, 2008). These antibodies can form antigen-binding regions
using only a 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).
[0114] An alternative source of non-conventional binding domains of
this disclosure 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 scTCR (see, e.g., Lake et al., Int. Immunol. 11:745, 1999;
Maynard et al., J. Immunol. Methods 306:51, 2005; U.S. Pat. No.
8,361,794), fibrinogen domains (see, e.g., Weisel et al., Science
230:1388, 1985), Kunitz domains (see, e.g., U.S. Pat. No.
6,423,498), designed ankyrin repeat proteins (DARPins) (Binz et
al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol.
22:575, 2004), fibronectin binding domains (adnectins or
monobodies) (Richards et al., J. Mol. Biol. 326:1475, 2003; Parker
et al., Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al.
(2008) J. Mol. Biol. 381:1238-1252), cysteine-knot miniproteins
(Vita et al. (1995) Proc. Nat'l. Acad. Sci. (USA) 92:6404-6408;
Martin et al. (2002) Nat. Biotechnol. 21:71, 2002 and Huang et al.
(2005) 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), lipocalin domains (see, e.g., 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), 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., 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 Patent
Application Publication Nos. WO 2007/098934; WO 2006/072620),
armadillo repeat proteins (see, e.g., Madhurantakam et al., Protein
Sci. 21: 1015, 2012; PCT Patent Application Publication No. WO
2009/040338), affilin (Ebersbach et al., J. Mol. Biol. 372: 172,
2007), affibody, avimers, knottins, fynomers, atrimers, cytotoxic
T-lymphocyte associated protein-4 (Weidle et al., Cancer Gen.
Proteo. 10:155, 2013) or the like (Nord et al., Protein Eng. 8:601,
1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al.,
Euro. J. Biochem. 268:4269, 2001; Binz et al., Nat. Biotechnol.
23:1257, 2005; Boersma and Pluckthun, Curr. Opin. Biotechnol.
22:849, 2011).
[0115] In some embodiments, a binding domain is a single chain T
cell receptor (scTCR) comprising V.sub..alpha./.beta. and
C.sub..alpha./.beta. chains (e.g., V.sub..alpha.-C.sub..alpha.,
V.sub..beta.-C.sub..beta., V.sub..alpha.-V.sub..beta.) or
comprising V.sub..alpha.-C.sub..alpha., V.sub..beta.-C.sub..beta.,
V.sub..alpha.-V.sub..beta. pair specific for a target of interest
(e.g., peptide-MHC complex or peptide-HLA complex).
[0116] In certain embodiments, a binding domain comprises or is a
sequence that is at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, at least 99.5%, or 100% identical to an
ectodomain of a molecule having an amino acid sequence of a TCR
V.sub..alpha., V.sub..beta., C.sub..alpha., or C.sub..beta.,
wherein each CDR comprises zero changes or at most one, two, or
three changes, from a TCR or fragment or derivative thereof that
specifically binds to a target of interest.
[0117] In certain embodiments, a binding domain V.sub..alpha.,
V.sub..beta., C.sub..alpha., or C.sub..beta. region of the present
disclosure can be derived from or based on a V.sub..alpha.,
V.sub..beta., C.sub..alpha., or C.sub..beta. of a known TCR (e.g.,
a high-affinity TCR) and contains one or more (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino
acid substitutions (e.g., conservative amino acid substitutions or
non-conservative amino acid substitutions), or a combination of the
above-noted changes, when compared with the V.sub..alpha.,
V.sub..beta., C.sub..alpha., or C.sub..beta. of a known TCR. An
insertion, deletion or substitution may be anywhere in a
V.sub..alpha., V.sub..beta., C.sub..alpha., or C.sub..beta. region,
including at the amino- or carboxy-terminus or both ends of these
regions, provided that each CDR comprises zero changes or at most
one, two, or three changes and provided a binding domain containing
a modified V.sub..alpha., V.sub..beta., C.sub..alpha., or
C.sub..beta. region can still specifically bind its target with an
affinity similar to wild type. In certain embodiments, a TCR has an
affinity for a peptide-HLA complex ranging from about 10 .mu.M to
about 500 .mu.M. In further embodiments, a TCR has a high affinity
for a peptide-HLA complex ranging from about 10 nM to about 200
pM.
[0118] In certain aspects, a fusion protein according to the
present disclosure has an extracellular component comprised of a
binding domain that specifically binds a target (e.g., a ligand or
receptor), wherein the extracellular component optionally includes
one or more other functional subcomponents or domains, such as a
multimerization domain, a linker, junction amino acids, or any
combination thereof.
[0119] In certain embodiments, a fusion protein disclosed herein
further comprises an additional extracellular region in addition to
the binding domain or in addition to the portion derived from the
molecule from which the binding domain is derived, such as a spacer
or a multimerization domain. For example, in some aspects a
multimerization domain is contained in or is a part of the
extracellular component of the fusion protein. For example, a
multimerization domain may be created by altering (e.g., mutating)
the extracellular component, or a multimerization domain may be
created by adding 1 to about 50 amino acid residues to the
extracellular component. A multimerization domain may be located
between the binding domain of the extracellular component and
hydrophobic component of a fusion protein of this disclosure. In
certain embodiments, a fusion protein expressed on a cell surface
comprises a multimerization domain within the extracellular
component and is proximal to the cell membrane, within one to 50
amino acids from the hydrophobic component. For example, a fusion
protein multimerization domain may comprise one or more cysteine
residues located within 30, 25, 20, 15, 14, 13, 12, 11, 10, 9 8, 7,
6, 5, 4, 3, 2, 1 or 0 amino acids from the fusion protein
hydrophobic component, wherein such one or more cysteine residues
from one fusion protein can form one or more disulfide bridges with
one or more other fusion proteins. In some embodiments, the
additional extracellular portion is derived from the same molecule
from which a transmembrane or stimulatory region of the fusion
protein is derived.
[0120] In further embodiments, interaction(s) between
multimerization domains of two or more fusion proteins
substantially contribute to or efficiently promote signal
transduction (e.g., immune cell stimulation or activation) as
compared to a fusion protein monomer. In certain embodiments,
multimerization of fusion proteins promote signal transduction in a
host cell in a statistically significant manner over fusion protein
monomers. In further embodiments, multimerization of fusion
proteins that promotes or enhances signal transduction in a host
cell is via a disulfide bridge.
[0121] An exemplary multimer is a "dimer," which refers to a
biological entity containing two molecules, such as two fusion
proteins, associated with each other. Such a dimer is considered a
"homodimer" when the two associated fusion proteins have
substantially similar or identical amino acid sequences. Similarly,
multimerization of three substantially or fully identical fusion
proteins is referred to as a "homotrimer." In some embodiments, a
multimerization domain comprises at least one cysteine residue,
wherein a multimerization domain cysteine residue from a first
fusion protein can form a disulfide bridge with a multimerization
domain cysteine residue from a second fusion protein. In certain
embodiments, a fusion protein dimer forms via a disulfide bridge.
In other embodiments, a fusion protein trimer forms via two or more
disulfide bridges. Alternatively, a dimer, homodimer, trimer or
homotrimer may multimerize via a zinc finger motif or a leucine
zipper motif. In still further embodiments, a fusion protein
comprises a plurality of multimerization domains, which can be
located extracellularly, intracellularly or both.
[0122] In some embodiments, a multimerization domain contained in
the extracellular component of a fusion protein comprises an
extracellular portion extending from the hydrophobic component. For
example, in some embodiments, a multimerization domain contained in
the extracellular component of a fusion protein comprises an
extracellular portion of a CD28 extending from a CD28 transmembrane
domain. In some embodiments, an extracellular portion of the CD28
comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or up to
about 25 amino acids adjacent to the transmembrane domain. In some
embodiments, the extracellular portion of the CD28 comprises 9
amino acids or 12 amino acids adjacent to the transmembrane domain.
In some embodiments, the extracellular portion of a CD28 comprises
the amino acid sequence encoded by a nucleic acid molecule as set
forth in SEQ ID NO.:9. In some embodiments, the extracellular
portion of a CD28 comprises the amino acid sequence as set forth in
SEQ ID NO.:32. In yet another exemplary embodiment, a
multimerization domain contained in the extracellular component of
a fusion protein comprises an extracellular portion of a CD137
(4-1BB) (e.g., ranging from one to about 50 amino acids) extending
from a CD137 (4-1BB) transmembrane domain. In certain embodiments,
the multimerization domain and the hydrophobic component are from
different proteins. For example, a multimerization domain contained
in the extracellular component of a fusion protein comprises an
extracellular portion of a CD28 extending from a CD137
transmembrane domain, or comprises an extracellular portion of a
CD137 extending from a CD28 transmembrane domain. In any of the
aforementioned embodiments, a multimerization domain may further
comprise a glycosylation site.
[0123] In some embodiments, a fusion protein may contain a linker
or junction amino acids connecting, for example, an extracellular
component with a multimerization domain or connecting an
extracellular component with a hydrophobic component or connecting
a hydrophobic component with an intracellular component. In some
embodiments, the linker is a Gly.sub.xSer.sub.y, wherein x and y
are independently an integer from 1 to 5.
[0124] A target molecule, which is specifically bound by a binding
domain contained in a fusion protein of the present disclosure, may
be found on or in association with a cell of interest ("target
cell"). Exemplary target cells include an immune cell, a cancer
cell, a cell associated with an autoimmune disease or disorder or
with an inflammatory disease or disorder, and an infectious
organism or cell (e.g., bacteria, virus, virus-infected cell), or
any cell presenting antigen complexed with a MHC or human leukocyte
antigen (HLA). A cell of an infectious organism, such as a
mammalian parasite, is also contemplated as a target cell. In some
embodiments, the target is an immunosuppressive ligand. In some
embodiments, the target is selected from a CD47, CD58, CD80, CD86,
CD95L (FasL), CD200, CD270 (HVEM), CD274 (PD-L1), or GAL9.
[0125] Intracellular Component
[0126] An intracellular component contained in a fusion protein of
the present disclosure will have an intracellular signaling domain,
such as an activating domain or a co-stimulatory domain, capable of
transmitting functional signals to a cell. In certain embodiments,
an intracellular signaling domain will indirectly promote a
cellular response by associating with one or more other proteins
that directly promote a cellular response. An intracellular
signaling domain may include one, two, three or more receptor
signaling domains, costimulatory domains, or combinations thereof.
Any intracellular component comprising an activating domain,
co-stimulatory domain, or both from any of a variety of signaling
molecules (e.g., signal transduction receptors) may be used in the
fusion proteins of this disclosure.
[0127] As used herein, an "intracellular signaling domain" from a
cell-surface receptor or ligand includes a complete intracellular
domain, a portion comprising an intracellular signaling domain, or
a functional (signaling) fragment thereof. In certain embodiments,
an intracellular signaling domain comprises a mutated intracellular
domain or a functional (signaling) fragment thereof that has
increased signaling activity as compared to a wild-type or
reference intracellular signaling domain.
[0128] A "co-stimulatory molecule" as used herein refers to a
receptor or cell-surface molecule that can transduce signals into T
cells to positively modulate T cell activation (Chen and Flies,
Nat. Rev. Immunol. 13: 227-242, 2013). By way of background, T cell
activation and proliferation requires two signals mediated through
engagement of the T cell antigen-specific receptor (TCR) and a
co-stimulatory signal, most typically binding of CD28 by CD80 and
CD86 (Ledbetter et al., Blood 75:1531, 1990).
[0129] An intracellular signaling domain or functional fragment
thereof useful in the fusion proteins of this disclosure may be
from a CD3.epsilon., CD3.delta., CD3.zeta., CD25, CD27, CD28, CD40,
CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1),
CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12, FcR.alpha.,
FcR.beta., FcR.gamma., Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2,
NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pT.alpha., TCR.alpha., TCR.beta.,
TRIM, Zap70, PTCH2, or any combination thereof. In some
embodiments, an intracellular signaling domain or functional
fragment thereof does not comprise a primary signal. In some
embodiments, an intracellular signaling domain does not comprise a
CD3.zeta..
[0130] In some embodiments, an intracellular signaling domain of a
fusion protein of this disclosure comprises a CD28. CD28 signaling
promotes proliferation of T cells stimulated via the TCR (Chen and
Flies, Nat. Rev. Immunol. 13: 227-242, 2013). CD28 forms
disulfide-linked homodimers, as a result of the cysteine residue
proximal to the transmembrane domain (Lazar-Molnar et al., Cell
Immunol. 244: 125-129, 2006). In certain embodiments, a CD28
signaling domain includes a full length intracellular portion of a
CD28 protein, a full length mature intracellular portion of a CD28
protein, a signaling fragment of an intracellular portion of a CD28
protein, and a signaling fragment of an intracellular portion of a
CD28 protein along with a transmembrane domain or fragment thereof
of CD28, or any combination thereof.
[0131] In some embodiments, an intracellular signaling domain of a
fusion protein contains an intracellular signaling domain of a
CD137 (4-1BB). CD137 is a co-stimulatory molecule, wherein binding
of CD137 to its ligand (4-1BBL or CD137L) is associated with T cell
activation and proliferation (Cheuk et al., Cancer Gene Therapy 11:
215-226, 2004). In certain embodiments, a CD137 signaling domain
includes a full length intracellular portion of a CD137 protein, a
full length mature intracellular portion of a CD137 protein, a
signaling fragment of an intracellular portion of a CD137 protein,
and a signaling fragment of an intracellular portion of a CD137
protein along with a transmembrane domain or fragment thereof of
CD137, or any combination thereof.
[0132] In certain embodiments, an intracellular signaling 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 intracellular signaling 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 ITAMs, a costimulatory factor, or any combination
thereof.
[0133] In some exemplary embodiments, the present disclosure
provides a fusion protein having an extracellular component
comprising an extracellular portion of a CD200R that specifically
binds CD200, an intracellular component comprising an intracellular
portion of CD28, and a hydrophobic component connecting the
extracellular and intracellular components, provided that a fusion
protein::target complex spans a distance similar to a distance
between membranes in an immunological synapse.
[0134] In particular embodiments, an intracellular component of a
fusion protein of the instant disclosure comprises a CD28, a CD137
(4-1BB) or both. For example, in some embodiments, an intracellular
component comprises the amino acid sequence encoded by a nucleic
acid molecule as set forth in in SEQ ID NO.:5. In some other
embodiments, an intracellular component comprises an amino acid
sequence encoded by a nucleic acid molecule as set forth in SEQ ID
NO.:13. In some embodiments, an intracellular component comprises
two intracellular signaling domains, for example, a CD28 and a
CD137 (4-1BB). In some embodiments, an intracellular component
comprises an amino acid sequence encoded by a nucleic acid molecule
as set forth in SEQ ID NO.:5 and the amino acid sequence encoded by
the nucleotide sequence as set SEQ ID NO.:13.
Hydrophobic Component
[0135] A hydrophobic portion contained in a single chain fusion
protein of the present disclosure will allow a fusion protein of
this disclosure to associate with a cellular membrane such that a
portion of the fusion protein will be located extracellularly and a
portion will be located intracellularly (e.g., intracellular
signaling domain). A hydrophobic component will generally be
disposed within the cellular membrane phospholipid bilayer. In
certain embodiments, one or more junction amino acids may be
disposed between and connecting a hydrophobic portion with an
intracellular signaling domain.
[0136] 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). In some
embodiments, the hydrophobic domain comprises a transmembrane
domain found in or derived from an integral membrane protein,
wherein the transmembrane domain has been modified by the addition,
removal, or replacement of one or more amino acids with at least
one different amino acid, or any combination thereof, such as
charged or hydrophilic residues that facilitate intermolecular
interactions. Thus, the term "hydrophobic domain" includes
transmembrane domains having, for example, modifications that may
reduce hydrophobicity.
[0137] In some embodiments, the hydrophobic component comprises a
transmembrane domain of a CD2, CD3.epsilon., CD3.delta., CD3.zeta.,
CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD80, CD86, CD95 (Fas),
CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R,
CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274
(PD-L1), CD278 (ICOS), CD279 (PD-1), TIM3, CD300, CD357 (GITR),
A2aR, DAP10, FcR.alpha., FcR.beta., FcR.gamma., Fyn, GALS, KIR,
Lck, LAT, LPA5, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2,
ROR2, Ryk, Slp76, SIRP.alpha., pT.alpha., TCR.alpha., TCR.beta.,
TIM3, TRIM, or Zap70. In particular embodiments, a hydrophobic
portion is a transmembrane domain from CD28, CD4, CD8, CD27, or
CD137 (4-1BB). In certain embodiments, a transmembrane domain is a
CD28 transmembrane domain having an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:4. In certain
other embodiments, a transmembrane domain is a CD200R transmembrane
domain having an amino acid sequence encoded by a nucleic acid
molecule as set forth in SEQ ID NO.:3. In still other embodiments,
a transmembrane domain is a SIRP.alpha. transmembrane domain having
an amino acid sequence encoded by a nucleic acid molecule as set
forth in SEQ ID NO.:18. In further embodiments, a transmembrane
domain is a CD2 transmembrane domain having an amino acid sequence
encoded by a nucleic acid molecule as set forth in SEQ ID NO.:63.
In still further embodiments, a transmembrane domain is a Fas
transmembrane domain having an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:77. In still
further embodiments, a transmembrane domain is a TIM3 transmembrane
domain. In still further embodiments, a transmembrane domain is a
TIM3 transmembrane domain having an amino acid sequence encoded by
a nucleic acid molecule as set forth in SEQ ID NO.:169. In still
further embodiments, a transmembrane domain is a LAG3 transmembrane
domain. In some embodiments, a transmembrane domain is a LAG3
transmembrane domain having an amino acid sequence encoded by a
nucleic acid molecule as set forth in SEQ ID NO.:155.
Nucleic Acids and Host Cells
[0138] In certain aspects, the present disclosure provides nucleic
acid molecules that encode any one or more of the fusion proteins
described herein, which may be immunomodulatory fusion proteins
(IFPs). Such nucleic acid molecules can be inserted into an
appropriate vector (e.g., viral vector or non-viral plasmid vector)
for introduction in a host cell of interest (e.g., hematopoietic
progenitor cell, T cell).
[0139] As used herein, the term "recombinant" or "non-natural"
refers to an organism, microorganism, cell, nucleic acid molecule,
or vector that includes at least one genetic alteration or has been
modified by introduction of an exogenous nucleic acid molecule,
wherein such alterations or modifications are introduced by genetic
engineering. Genetic alterations include, for example,
modifications introducing expressible nucleic acid molecules
encoding proteins, fusion proteins or enzymes, or other nucleic
acid molecule additions, deletions, substitutions or other
functional disruption of a cell's genetic material. Additional
modifications include, for example, non-coding regulatory regions
in which the modifications alter expression of a gene or operon. In
certain embodiments, a cell, such as a T cell, obtained from a
subject may be converted into a non-natural or recombinant cell
(e.g., a non-natural or recombinant T cell) by introducing a
nucleic acid that encodes a fusion protein as described herein and
whereby the cell expresses a fusion protein.
[0140] In certain embodiments, nucleic acid molecules encoding
fusion proteins may be codon optimized to enhance or maximize
expression in certain types of cells, such as T cells (Scholten et
al., Clin. Immunol. 119: 135-145, 2006).
[0141] In one exemplary embodiment, the present disclosure provides
a nucleic acid molecule that encodes a CD200R-CD28 construct
(huCD200Rtm-CD28), wherein the extracellular component comprises a
CD200R ectodomain, the hydrophobic component comprises the
transmembrane domain of a CD200R, and the intracellular component
comprises the intracellular signaling domain of a CD28. For
example, in one embodiment, a nucleic acid molecule as set forth in
SEQ ID NO.:1 is provided.
[0142] In another exemplary embodiment, the present disclosure
provides a nucleic acid molecule that encodes a CD200R-CD28
construct (huCD200R-CD28tm), wherein the hydrophobic component
comprises the transmembrane domain of a CD28. For example, in one
embodiment, the disclosure provides a nucleic acid molecule as set
forth in SEQ ID NO.:6.
[0143] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a CD200R-CD28
construct, wherein the extracellular comprises a truncated
extracellular domain of CD200R and an extracellular portion of
CD28. For example, the CD200R extracellular domain may be truncated
by 9 amino acids (e.g., huCD200R-9aas-CD28Cys, SEQ ID NO.:7) or by
12 amino acids (e.g., huCD200R-12aas-CD28Cys, SEQ ID NO.:10).
[0144] In one exemplary embodiment, the present disclosure provides
a nucleic acid molecule that encodes a CD200R-CD28-4-1BB construct
(huCD200R-9aas-CD28Cystm-41BBic or
huCD200R-12aas-CD28Cystm-41BBic), wherein the intracellular
component comprises the intracellular signaling domain of CD137
(4-1BB). For example, in one embodiment, the nucleic acid molecule
has the nucleotide sequence as set forth in SEQ ID NO.:12 or SEQ ID
NO.:14.
[0145] In another exemplary embodiment, the present disclosure
provides a nucleic acid molecule that encodes a CD200R-CD28-4-1BB
construct (huCD200R-9aas-CD28Cys tm is 41BBic or
huCD200R-12aas-CD28Cys tm ic-41BBic), wherein the intracellular
component comprises the intracellular signaling domain of CD28 and
of CD137 (4-1BB). In one embodiment, for example, the nucleic acid
of the present disclosure has the nucleotide sequence as set forth
in SEQ ID NO.:9 or SEQ ID NO.:15.
[0146] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a SIRP.alpha.-CD28
construct. For example, the present disclosure includes a nucleic
acid molecule as set forth in SEQ ID NO.:16 (huSIRP.alpha.tm-CD28)
or SEQ ID NO.:19 (huSIRP.alpha.-CD28tm).
[0147] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a SIRP.alpha.-CD28
construct, wherein the extracellular component comprises a
truncated extracellular domain of SIRP.alpha. and an extracellular
portion of CD28. For example, the SIRP.alpha. extracellular domain
may be truncated by 12 amino acids (e.g.,
huSIRP.alpha.-12aas-CD28Cys, SEQ ID NO.:20).
[0148] In one exemplary embodiment, the present disclosure provides
a nucleic acid molecule that encodes a SIRP.alpha.-CD28-4-1BB
construct (huSIRP.alpha.-12aas-CD28Cystm-41BBic), wherein the
intracellular component comprises the intracellular signaling
domain of CD137 (4-1BB). For example, in one embodiment, the
nucleic acid of the present disclosure has the nucleotide sequence
as set forth in SEQ ID NO.:22.
[0149] In another exemplary embodiment, the present disclosure
provides a nucleic acid molecule that encodes a
SIRP.alpha.-CD28-4-1BB construct (huSIRP.alpha.-12aas-CD28Cys tm
ic-41BBic), wherein the intracellular component comprises the
intracellular signaling domain of CD28 and of CD137 (4-1BB). In one
embodiment, for example, the nucleic acid of the present disclosure
has the nucleotide sequence as set forth in SEQ ID NO.:23.
[0150] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a PD-1-CD28
construct. For example, the present disclosure includes a nucleic
acid molecule as set forth in SEQ ID NO.:97 (huPD1-CD28Cys).
[0151] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a PD-1-CD28
construct, wherein the extracellular component comprises a
truncated extracellular domain of PD-1 and an extracellular portion
of CD28. For example, the PD-1 extracellular domain may be
truncated by 12 amino acids (e.g., huPD1-12aas-CD28Cys, SEQ ID
NO.:99), 15 amino acids (e.g., huPD1-15aas-CD28Cys, SEQ ID
NO.:101), or 21 amino acids (e.g., huPD1-21aas-CD28Cys, SEQ ID
NO.:103).
[0152] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a CD2-CD28 construct.
For example, the present disclosure includes a nucleic acid
molecule as set forth in SEQ ID NO.:69 (huCD2-CD28Cys).
[0153] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a Fas-CD28 construct.
For example, the present disclosure includes a nucleic acid
molecule as set forth in SEQ ID NO.:83 (huFas-CD28Cys).
[0154] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a Fas-CD28 construct,
wherein the extracellular component comprises a truncated
extracellular domain of Fas and an extracellular portion of CD28.
For example, the Fas extracellular domain may be truncated by 7
amino acids (e.g., huFas-7aas-CD28Cys, SEQ ID NO.:85) or 12 amino
acids (e.g., huFas-12aas-CD28Cys, SEQ ID NO.:87).
[0155] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a TIM3-CD28
construct. For example, the present disclosure includes a nucleic
acid molecule as set forth in SEQ ID NO.:173 (huTIM3-CD28Cys). Also
included within the scope of the disclosure is a TIM3-CD28 fusion
protein, wherein the extracellular component comprises a truncated
extracellular domain of TIM3 and an extracellular portion of CD28.
For example, the TIM3 extracellular domain may be truncated by 12
amino acids (e.g., huTIM3-12aas-CD28Cys, SEQ ID NO.:175).
[0156] In other exemplary embodiments, the present disclosure
provides a nucleic acid molecule that encodes a LAG3-CD28
construct. For example, the present disclosure includes a nucleic
acid molecule as set forth in SEQ ID NO.:163 (huLAG3-CD28Cys). Also
included within the scope of the disclosure is a LAG3-CD28 fusion
protein, wherein the extracellular component comprises a truncated
extracellular domain of LAG3 and an extracellular portion of CD28.
For example, the LAG3 extracellular domain may be truncated by 12
amino acids (e.g., huLAG3-12aas-CD28Cys, SEQ ID NO.:159).
[0157] A vector that encodes a core virus is referred to herein as
a "viral vector." There are a large number of available viral
vectors suitable for use with the compositions of the instant
disclosure, including those identified for human gene therapy
applications (see Pfeifer and Verma, Ann. Rev. Genomics Hum. Genet.
2:177, 2001). Suitable viral vectors include vectors based on RNA
viruses, such as retrovirus-derived vectors, e.g., Moloney murine
leukemia virus (MLV)-derived vectors, and include more complex
retrovirus-derived vectors, e.g., lentivirus-derived vectors.
HIV-1-derived vectors belong to this category. Other examples
include lentivirus vectors derived from HIV-2, FIV, equine
infectious anemia virus, SIV, and Maedi-Visna virus (ovine
lentivirus). Methods of using retroviral and lentiviral viral
vectors and packaging cells for transducing mammalian host cells
with viral particles containing chimeric antigen receptor
transgenes are known in the art and have been previous described,
for example, in U.S. Pat. No. 8,119,772; Walchli et al., PLoS One
6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et
al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther.
18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506:97, 2009.
Retroviral and lentiviral vector constructs and expression systems
are also commercially available.
[0158] In certain embodiments, a viral vector is used to introduce
a non-endogenous nucleic acid sequence encoding a fusion protein or
a non-endogenous nucleic acid sequence encoding a fusion protein
specific for a target. A viral vector may be a retroviral vector or
a lentiviral vector. A viral vector may also include nucleic acid
sequences encoding a marker for transduction. Transduction markers
for viral vectors are known in the art and include selection
markers, which may confer drug resistance, or detectable markers,
such as fluorescent markers or cell surface proteins that can be
detected by methods such as flow cytometry. In particular
embodiments, a viral vector further comprises a gene marker for
transduction comprising green fluorescent protein (GFP), an
extracellular domain of human CD2, or a truncated human EGFR
(huEGFRt; see Wang et al., Blood 118:1255, 2011). When a viral
vector genome comprises a plurality of nucleic acid sequences to be
expressed in a host cell as separate transcripts, the viral vector
may also comprise additional sequences between the two (or more)
transcripts allowing bicistronic or multicistronic expression.
Examples of such sequences used in viral vectors include internal
ribosome entry sites (IRES), furin cleavage sites, viral 2A
peptide, or any combination thereof.
[0159] Other vectors also can be used for polynucleotide delivery
including DNA viral vectors, including, for example
adenovirus-based vectors and adeno-associated virus (AAV)-based
vectors; vectors derived from herpes simplex viruses (HSVs),
including amplicon vectors, replication-defective HSV and
attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).
[0160] Other vectors recently developed for gene therapy uses can
also be used with the compositions and methods of this disclosure.
Such vectors include those derived from baculoviruses and
.alpha.-viruses (Jolly, D J. 1999. Emerging Viral Vectors. pp
209-40 in Friedmann T. ed. The Development of Human Gene Therapy.
New York: Cold Spring Harbor Lab), or plasmid vectors (such as
sleeping beauty or other transposon vectors). In some embodiments,
a viral or plasmid vector further comprises a gene marker for
transduction (e.g. green fluorescent protein, huEGFRt).
[0161] In some embodiments, a vector encoding a fusion protein as
disclosed herein may encode more than one fusion protein. For
example, a vector may encode two different fusion proteins (e.g., a
first fusion protein comprising a PD-1 ectodomain and a second
fusion protein comprising a TIM3 ectodomain).
[0162] In some embodiments, a vector encoding a fusion protein as
disclosed herein may further comprise an antigen-specific TCR. In
some embodiments, the antigen-specific TCR is exogenous. In some
embodiments, the antigen-specific TCR is specific to a HLA (MHC)
class I restricted antigen. In some embodiments, the antigen is a
cancer-specific antigen. Embodiments wherein the cancer-specific
antigen comprises WT-1, mesothelin, or cyclin-A1 are also within
the scope of the disclosure. In still other embodiments, a vector
that encodes a fusion protein as disclosed herein further encodes a
ligand, which may be CD200, CD47, PD-L1, or CD58. In yet further
embodiments, a vector that encodes a fusion protein as disclosed
herein further encodes an siRNA for reducing the expression of an
endogenous receptor. In some particular embodiments, the endogenous
receptor is CD200R, SIRP.alpha., CD279 (PD-1), CD95 (Fas) or
CD2.
[0163] In some embodiments, host cells capable of expressing a
fusion protein of this disclosure on the cell surface are immune
cells. In some embodiments, host cells capable of expressing a
fusion protein of this disclosure on the cell surface are T cells,
including primary cells or cell lines derived from human, mouse,
rat, or other mammals. If obtained from a mammal, a T cell can be
obtained from numerous sources, including blood, bone marrow, lymph
node, thymus, or other tissues or fluids. A T cell may be enriched
or purified. T cell lines are well known in the art, some of which
are described in Sandberg et al., Leukemia 21:230, 2000. In certain
embodiments, T cells that lack endogenous expression of TCR.alpha.
and .beta. chains are used. Such T cells may naturally lack
endogenous expression of TCR.alpha. and .beta. chains or may have
been modified to block expression (e.g., T cells from a transgenic
mouse that does not express TCR .alpha. and .beta. chains or cells
that have been manipulated to inhibit expression of TCR .alpha. and
.beta. chains) or to knockout TCR.alpha. chain, TCR.beta. chain, or
both genes. In some embodiments, T cells may be engineered to
express a TCR specific to a particular antigen.
[0164] In certain embodiments, a host cell transfected to express a
fusion protein of this disclosure is a functional T cell, such as a
virus-specific T cell, a tumor antigen specific cytotoxic T cell, a
naive T cell, a memory stem T cell, a central or effector memory T
cell, .gamma..delta. T cells, or a CD4+ CD25+ regulatory T cell. In
further embodiments, a nucleic acid molecule encoding a fusion
protein of this disclosure is introduced into bulk CD8+ T cells,
naive CD8+ T cells, CD8+ T.sub.CM cells, CD8+ T.sub.EM cells, or
any combination thereof. In still further embodiments, a nucleic
acid molecule encoding a fusion protein of this disclosure is
introduced into bulk CD4+ T cells, naive CD4+ T cells, CD4+
T.sub.CM cells, CD4+ T.sub.EM cells, or any combination thereof. In
other embodiments, a nucleic acid molecule encoding a fusion
protein of this disclosure is introduced into a population of T
cells enriched for naive CD8+ T cells and CD8+ T.sub.CM cells. In
still other embodiments, a nucleic acid molecule encoding a fusion
protein of this disclosure is introduced into a population of T
cells enriched for naive CD4+ T cells and CD4+ T.sub.CM cells. In
any of the aforementioned embodiments, the T cells further contain
a nucleic acid molecule encoding an engineered antigen-specific T
cell receptor (TCR), an engineered antigen-specific high affinity
TCR, an exogenous co-stimulatory molecule, a chimeric antigen
receptor (CAR), or any combination thereof.
[0165] In certain embodiments, a host cell transfected to express a
fusion protein of this disclosure is a functional natural killer
cell.
[0166] One or more growth factor cytokines that promote
proliferation of T cells expressing a fusion protein of this
disclosure may be added to the culture used to expand T cells. The
cytokines may be human or non-human. Exemplary growth factor
cytokines that may be used promote T cell proliferation include
IL2, IL15, or the like.
[0167] In certain embodiments, a host T cell transfected to express
a fusion protein of this disclosure is a CD4.sup.+ T cell that also
expresses an antigen-specific high-affinity TCR specific to a HLA
(MHC) class I restricted antigen (see Soto et al., Cancer Immunol
Immunother. 62: 359-369, 2013).
[0168] In certain embodiments, a host T cell transfected to express
a fusion protein of this disclosure also expresses a recombinant
TCR specific to a cancer antigen. In some embodiments, the cancer
antigen is a WT1. "WT1" refers to Wilm's tumor 1, a transcription
factor that contains four zinc-finger motifs at the C-terminus and
a proline/glutamine-rich DNA binding domain at the N-terminus. WT1
has an essential role in the normal development of the urogenital
system and is mutated in a small subset of patients with Wilm's
tumors. High expression of WT1 has been observed in various
cancers, including, breast cancer, ovarian cancer, acute leukemias,
vascular neoplasms, melanomas, colon cancer, lung cancer, thyroid
cancer, bone and soft tissue sarcoma, and esophageal cancer.
Alternative splicing has been noted for WT1.
[0169] In certain embodiments, a host T cell transfected to express
a fusion protein of this disclosure also expresses a recombinant
TCR specific to mesothelin. "Mesothelin" (MSLN) refers to a gene
that encodes a precursor protein that is cleaved into two products,
megakaryocyte potentiating factor and mesothelin. Megakaryocyte
potentiation factor functions as a cytokine that can stimulate
colony formation in bone marrow megakaryocytes. Mesothelin is a
glycosylphosphatidylinositol-anchored cell-surface protein that may
function as a cell adhesion protein. This protein is overexpressed
in epithelial mesotheliomas, ovarian cancers and in specific
squamous cell carcinomas. Alternative splicing results in multiple
transcript variants.
[0170] In certain embodiments, a host T cell transfected to express
a fusion protein of this disclosure also expresses a recombinant
TCR specific to cyclin-A1.
[0171] In certain embodiments, a host T cell transfected to express
a fusion protein of this disclosure also expresses a CAR.
[0172] In still other embodiments, a host cell that expresses a
fusion protein as disclosed herein further comprises a ligand,
which may be CD200, CD47, PD-L1, or CD58. In yet further
embodiments, a host cell that expresses a fusion protein as
disclosed herein further expresses an siRNA for reducing the
expression of an endogenous receptor. In some particular
embodiments, the endogenous receptor is CD200R, SIRP.alpha., CD279
(PD-1), CD95 (Fas), or CD2.
[0173] In some embodiments, a host cell that expresses a fusion
protein as disclosed herein may express more than one fusion
protein. For example, the host cell may express two different
fusion proteins (e.g., a first fusion protein comprising a PD-1
ectodomain and a second fusion protein comprising a TIM3
ectodomain).
Uses
[0174] Diseases that may be treated with cells expressing fusion
proteins as described in the present disclosure include cancer,
infectious diseases (viral, bacterial, protozoan infections),
immune diseases (e.g., autoimmune), or aging-related diseases
(e.g., senescence). Adoptive immune and gene therapy are promising
treatments for various types of cancer (Morgan et al., Science
314:126, 2006; Schmitt et al., Hum. Gene Ther. 20:1240, 2009; June,
J. Clin. Invest. 117:1466, 2007) and infectious disease (Kitchen et
al., PLoS One 4:38208, 2009; Rossi et al., Nat. Biotechnol.
25:1444, 2007; Zhang et al., PLoS Pathog. 6:e1001018, 2010; Luo et
al., J. Mol. Med. 89:903, 2011).
[0175] A wide variety of cancers, including solid tumors and
leukemias are amenable to the compositions and methods disclosed
herein. Exemplary types of cancer that may be treated include
adenocarcinoma of the breast, prostate, and colon; all forms of
bronchogenic carcinoma of the lung; myeloid leukemia; 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; malignant histiocytosis; leukemia; Hodgkin's disease;
immunoproliferative small; non-Hodgkin's lymphoma; plasmacytoma;
reticuloendotheliosis; melanoma; 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. 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. 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; nerofibromatosis;
and cervical dysplasia.
[0176] Exemplifying the variety of hyperproliferative disorders
amenable to a fusion protein T cell therapy 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, B cell
blast transformation of chronic myeloid 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's lymphoma/leukemia, B-cell proliferations of uncertain
malignant potential, lymphomatoid granulomatosis, and
post-transplant lymphoproliferative disorder.
[0177] Inflammatory and autoimmune diseases include arthritis,
rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, polychondritis, psoriatic arthritis, psoriasis,
dermatitis, polymyositis/dermatomyositis, inclusion body myositis,
inflammatory myositis, toxic epidermal necrolysis, systemic
scleroderma and sclerosis, CREST syndrome, inflammatory bowel
disease, Crohn's disease, ulcerative colitis, respiratory distress
syndrome, adult respiratory distress syndrome (ARDS), meningitis,
encephalitis, uveitis, colitis, glomerulonephritis, allergic
conditions, eczema, asthma, conditions involving infiltration of T
cells and chronic inflammatory responses, atherosclerosis,
autoimmune myocarditis, leukocyte adhesion deficiency, systemic
lupus erythematosus (SLE), subacute cutaneous lupus erythematosus,
discoid lupus, lupus myelitis, lupus cerebritis, juvenile onset
diabetes, multiple sclerosis, allergic encephalomyelitis,
neuromyelitis optica, rheumatic fever, Sydenham's chorea, immune
responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including Wegener's granulomatosis and Churg-Strauss
disease, agranulocytosis, vasculitis (including hypersensitivity
vasculitis/angiitis, ANCA and rheumatoid vasculitis), aplastic
anemia, Diamond Blackfan anemia, immune hemolytic anemia including
autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red
cell aplasia (PRCA), Factor VIII deficiency, hemophilia A,
autoimmune neutropenia, pancytopenia, leukopenia, diseases
involving leukocyte diapedesis, central nervous system (CNS)
inflammatory disorders, multiple organ injury syndrome, myasthenia
gravis, antigen-antibody complex mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Behcet disease, Castleman's syndrome,
Goodpasture's syndrome, Lambert-Eaton Myasthenic Syndrome,
Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome,
solid organ transplant rejection, graft versus host disease (GVHD),
bullous pemphigoid, pemphigus, autoimmune polyendocrinopathies,
seronegative spondyloarthropathies, Reiter's disease, stiff-man
syndrome, giant cell arteritis, immune complex nephritis, IgA
nephropathy, IgM polyneuropathies or IgM mediated neuropathy,
idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmune
thrombocytopenia, autoimmune disease of the testis and ovary
including autoimmune orchitis and oophoritis, primary
hypothyroidism; autoimmune endocrine diseases including autoimmune
thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis),
subacute thyroiditis, idiopathic hypothyroidism, Addison's disease,
Grave's disease, autoimmune polyglandular syndromes (or
polyglandular endocrinopathy syndromes), Type I diabetes also
referred to as insulin-dependent diabetes mellitus (IDDM) and
Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial
pneumonitis (HIV), bronchiolitis obliterans (non-transplant),
non-specific interstitial pneumonia (NSIP), Guillain-Barre
Syndrome, large vessel vasculitis (including polymyalgia rheumatica
and giant cell (Takayasu's) arteritis), medium vessel vasculitis
(including Kawasaki's disease and polyarteritis nodosa),
polyarteritis nodosa (PAN) ankylosing spondylitis, Berger's disease
(IgA nephropathy), rapidly progressive glomerulonephritis, primary
biliary cirrhosis, Celiac sprue (gluten enteropathy),
cryoglobulinemia, cryoglobulinemia associated with hepatitis,
amyotrophic lateral sclerosis (ALS), coronary artery disease,
familial Mediterranean fever, microscopic polyangiitis, Cogan's
syndrome, Whiskott-Aldrich syndrome and thromboangiitis
obliterans.
[0178] In particular embodiments, a method of treating a subject
with the fusion protein as disclosed herein include acute
myelocytic leukemia, acute lymphocytic leukemia, and chronic
myelocytic leukemia.
[0179] Infectious diseases include those associated with infectious
agents and include any of a variety of bacteria (e.g., pathogenic
E. coli, S. typhimurium, P. aeruginosa, B. anthracis, C. botulinum,
C. difficile, C. perfringens, H. pylori, V. cholerae, Listeria
spp., Rickettsia spp., Chlamydia spp., and the like), mycobacteria,
and parasites (including any known parasitic member of the
Protozoa). Infectious viruses include eukaryotic viruses, such as
adenovirus, bunyavirus, herpesvirus, papovavirus, papillomavirus
(e.g., HPV), paramyxovirus, picornavirus, rhabdovirus (e.g.,
Rabies), orthomyxovirus (e.g., influenza), poxvirus (e.g.,
Vaccinia), reovirus, retrovirus, lentivirus (e.g., HIV), flavivirus
(e.g., HCV, HBV) or the like. In certain embodiments, infection
with cytosolic pathogens whose antigens are processed and displayed
with HLA (MHC) Class I molecules, are treated with fusion proteins
of this disclosure.
[0180] A fusion protein of this disclosure may be administered to a
subject in cell-bound form (e.g., gene therapy of target cell
population (mature T cells (e.g., CD8.sup.+ or CD4.sup.+ T cells)
or other cells of T cell lineage)). In a particular embodiment,
cells of T cell lineage expressing fusion proteins administered to
a subject are syngeneic, allogeneic, or autologous cells.
[0181] Pharmaceutical compositions including fusion proteins of
this disclosure may be administered in a manner appropriate to the
disease or condition to be treated (or prevented) as determined by
persons skilled in the medical art. An appropriate dose, suitable
duration, and frequency of administration of the compositions will
be determined by such factors as the condition of the patient,
size, type and severity of the disease, particular form of the
active ingredient, and the method of administration. The present
disclosure provides pharmaceutical compositions comprising cells
expressing a fusion protein as disclosed herein and a
pharmaceutically acceptable carrier, diluents, or excipient.
Suitable excipients include water, saline, dextrose, glycerol, or
the like and combinations thereof.
[0182] In some embodiments, the disclosure is directed to a method
of increasing the activity of an immune cell, enhancing or
prolonging an immune response, stimulating an antigen-specific T
cell response, inhibiting an immunosuppressive signaling pathway,
treating cancer or a tumor, inhibiting immune resistance of cancer
cells, or treating an infection, comprising administering to a
subject in need thereof an effective amount of a host cell
expressing a fusion protein as described herein. In further
embodiments, a host cell for use in any of the aforementioned
methods further expresses an engineered antigen-specific TCR, an
engineered antigen-specific high affinity TCR, a CAR, a
co-stimulatory molecule, or any combination thereof. In particular
embodiments, methods of treating leukemia are provided, comprising
co-expressing a fusion protein as disclosed herein and a
recombinant, antigen-specific TCR.
[0183] In some embodiments, there are provided methods of inducing
or enhancing a Class I HLA response by a CD4+ T cell, comprising
administering to a subject in need thereof an effective amount of a
CD4+ T cell expressing a fusion protein as described herein. In
further embodiments, a host cell for use in inducing or enhancing a
Class I HLA response by a CD4+ T cell further expresses an
engineered antigen-specific TCR, an engineered antigen-specific
high affinity TCR, a CAR, a co-stimulatory molecule, or any
combination thereof.
[0184] In any of the aforementioned embodiments, the methods are
effective in the absence of administering exogenous IL-2.
[0185] In still other embodiments, a subject of any of the
aforementioned methods is further treated with an adjunctive
therapy, such as a chemotherapy. Exemplary chemotherapeutic agents
include, for example, alkylating agents such as thiotepa and
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide and
trimethylolomelamine; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,
chromomycins, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine, 5-FU; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.TM.; razoxane; sizofiran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (Taxol.TM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel
(Taxotere.TM., Rhone-Poulenc Rorer, Antony, France); chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
esperamicins, capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0186] In some embodiments, the adjunctive therapy is a vaccine, an
inhibitor of an immunosuppression signal, a B-Raf inhibitor, a MEK
inhibitor, a tyrosine kinase inhibitor, a cytotoxic agent, a
chemotherapeutic, or any combination thereof. In some embodiments,
the inhibitor of an immunosuppression signal is an antibody or
siRNA. In some embodiments, the antibody or siRNA is specific for
PD-1, PD-L1, PD-L2, CTLA4, LAG3, KIR, CD244, B7-H3, B7-H4, BTLA,
HVEM, GALS, TIM3, A2aR, or any combination thereof.
EXAMPLES
Example 1
CD200R-CD28 Fusion Protein Constructions
[0187] Exemplary fusion proteins as described herein are
illustrated using schematic representations in FIG. 1A. Exemplary
fusion proteins include immunomodulatory fusion proteins (IFPs)
comprised of the extracellular domain of CD200R or a portion
thereof, and an intracellular signaling domain of CD28 or a portion
thereof (FIG. 1A, constructs I-V). The hydrophobic component may be
comprised of the transmembrane domain of either CD200R (FIG. 1A,
construct I) or CD28 (FIG. 1A, constructs II-V), or portions
thereof. In some exemplary CD200R-CD28 fusion proteins, the
hydrophobic component comprises the transmembrane domain of CD28
and the extracellular component further comprises an extracellular
portion of CD28, particularly an extracellular cysteine residue
adjacent to the hydrophobic component (e.g., FIG. 1A construct III,
CD200R-CD28Cys; construct IV, CD200R-3aas-CD28Cys; and construct V,
CD200R-9aas-CD28Cys). The extracellular component may comprise all
or a portion of the extracellular domain of CD200R. In some
embodiments, the extracellular component comprises the entire
extracellular domain of CD200R (FIG. 1A, constructs I-III). In
other examples, the extracellular component comprises the first 235
amino acids (preserving an N-linked glycosylation site) (e.g., FIG.
1A, construct IV, CD200R-3aas-CD28Cys) or the first 229 amino acids
(e.g., FIG. 1A, construct V, CD200R-9aas-CD28Cys) from the
N-terminus of CD200R. The size of the extracellular component,
which may be manipulated by adjusting the fusion protein construct,
may affect the ability of the fusion protein to enter the
immunological synapse and co-localize with the TCR within the cSMAC
to deliver a strong co-stimulatory signal. Additionally, the
CD200R-CD28 construct has the capacity to convert what would
typically be an inhibitory signal from the binding of CD200R to its
target into a positive signal generated by the CD28 intracellular
signaling domain.
[0188] An exemplary nucleic acid molecule encoding a CD200R-CD28
fusion protein comprises the following elements (5' to 3'):
Extracellular Component (CD200R)-Multimerization Domain (CD28
Cysteine)-Hydrophobic Component (CD28 transmembrane)-Intracellular
Component (CD28 intracellular). In some embodiments, a nucleic acid
molecule encoding a CD200R-CD28 fusion protein comprises a nucleic
acid molecule as set forth in any one of SEQ ID NOS.:47-51 or 1, 6,
7, 10, 12, 14, or 15.
[0189] Nucleic acids encoding the constructs were ordered from
Invitrogen or generated in-house by PCR then directionally
TOPO-cloned into the pENTR.TM./D-TOPO.RTM. vector (Invitrogen), and
transferred into the retroviral vector pMP71-attR using
Gateway.RTM. technology (Invitrogen). In certain embodiments, the
nucleic acid molecules encoding IFPs of the instant disclosure were
codon optimized before cloning into the pMP71-attR retroviral
vector.
Example 2
Transgenic Expression of CD200R-CD28 Constructs
[0190] A preclinical mouse model for disseminated leukemia, based
on the murine C57BL/6 Friend virus-induced erythroleukemia (FBL)
and TCR.sub.gag transgenic mice, was used to determine if
CD200R-CD28 chimeric receptors can improve T cell function.
[0191] TCR transgenic mice were generated to produce CD8.sup.+ T
cells specific for the gag epitope (TCR.sub.gag). C57BL/6 (B6) mice
were purchased from the Jackson Laboratory. TCR.sub.gag transgenic
mice express a TCR transgene specific for the Friend virus gag
epitope in CD8.sup.+ T cell (Ohlen et al., J. Immunol. 166:
2863-2870, 2001). All animal studies performed were approved under
the University of Washington Institutional Animal Care and Use
Committee protocol (Protocol #2013-01). The murine B6 Friend virus
induced erythroleukemia (FBL) expresses the F-MuLV encoded gag
epitope (peptide CCLCLTVFL).
[0192] CD200R-CD28 chimeric constructs based on murine genes were
inserted into the pMP71 retroviral vector and used to transduce
primary mouse splenocytes stimulated with anti-CD3 and anti-CD28
antibodies. Constructs were designed as described in Example 1, and
ordered from Invitrogen or generated in-house by PCR. The
constructs were then directionally TOPO-cloned into the
pENTR.TM./D-TOPO.RTM. vector (Invitrogen), and transferred into the
retroviral vector pMP71-attR using Gateway.RTM. technology
(Invitrogen). The retroviral packaging cell line Plat-E (Morita et
al., 2000, Gene Therapy 7:1063-1066, 2000; Cell Biolabs, Inc.) was
transduced with the retroviral vector using effectene transduction
reagent (Qiagen). Viral supernatant was collected on days 2 and 3
and then used to transduce TCR.sub.gag T cells.
[0193] One day prior to the transfection, TCR.sub.gag T cells were
stimulated with anti-CD3/CD28 and 100 U/mL rhIL-2. Transduction of
TCR.sub.gag T cells was performed in 12 well plates in the presence
of IL-2 and polybrene by spinfection for 90 minutes at 1000 g. FBL
cells were transduced with CD200 with polybrene spinfection,
similar to T cell transduction, and subsequently sorted to generate
a homogenous population.
[0194] Five days after transduction, CD8.sup.+ T cells were
analyzed for construct expression by anti-CD200R antibody staining
and flow cytometry (FIG. 1B). A vector encoding green fluorescent
protein (GFP) was used as a control. Transduction efficiency ranged
from 4-36% and the mean fluorescent intensity (MFI) of the
transduced cells was similar between constructs.
Example 3
CD200R-CD28 Constructs Promote In Vitro Proliferation,
Accumulation, and Effector Function of Transduced T Cells
[0195] The CD200R-CD28 constructs described in Examples 1 and 2
were assessed for their abilities to promote proliferation,
accumulation, and effector function of TCR.sub.gag T cells.
[0196] Expansion of Effector Cells In Vitro
[0197] TCR.sub.gag effector cells were generated in vitro as
previously described (Stromnes et al., J. Clin. Invest. 120:
3722-34, 2010). Irradiated antigen presenting splenocytes
(5.times.10.sup.6), irradiated FBL (3.times.10.sup.6), and
TCR.sub.gag tg cells (10.sup.6) were cultured together with IL-2
(50 U/mL) in 10 mL of culture media (IMDM supplemented with
non-essential amino acids, 2 .mu.M glutamine, 100 U/mL
penicillin/streptomycin, 10% FBS, and 50 .mu.M 2-mercapatoethanol).
T cells were restimulated weekly and assessed by flow cytometry 5-7
days after the last stimulation.
[0198] In Vitro T Cell Proliferation Assay
[0199] TCR.sub.gag T cells were transduced as in Example 2. To
assess T cell proliferation in vitro, TCR.sub.gag T cells were
stained with CellTrace Violet (CTV, Life Technologies) according to
the manufacturer's protocol. CTV-labeled Tg T cells (10.sup.5) and
GFP control T cells were stimulated with titrating numbers of
CD200.sup.- FBL or CD200.sup.+ FBL cells. After 3 days, CTV
dilution of TCR.sub.gag T cells was assessed by flow cytometry.
[0200] Flow cytometry results indicating the number of TCR.sub.gag
T cells after stimulation with titrating numbers of CD200- FBL
cells (upper) or CD200.sup.+ FBL (lower) are shown in FIG. 2A. Four
of the five CD200R-CD28 constructs tested dramatically improved
proliferation of TCR.sub.gag T cells in response to CD200.sup.+ FBL
(blue lines) compared to GFP control-transduced T cells (red
lines).
[0201] In Vitro T Cell Accumulation Assay
[0202] To determine if the enhanced proliferation also resulted in
increased accumulation of transduced cells, the proportion of
transduced cells in the total TCR.sub.gag population over multiple
cycles of stimulation with irradiated CD200.sup.+ FBL was
measured.
[0203] Several of the constructs promoted accumulation of
transduced T cells, including CD200R-CD28tm, CD200R-CD28Cys,
CD200R-3aas-CD28Cys, and CD200R-9aas-CD28Cys (FIG. 2B). Of these
constructs, CD200R-9aas-CD28Cys exhibited the greatest increase in
transduced T cells over multiple stimulations, resulting in more
than a 3-fold expansion over 3 stimulations.
[0204] In Vitro T Cell Enrichment Assay
[0205] A mixed population of transduced and nontransduced CD8.sup.+
T cells were restimulated with CD200.sup.+ or CD200.sup.-
irradiated FBL cells to determine if restimulation would enrich the
population for the transduced CD200R-9aas-CD28Cys IFP.sup.+ T
cells. Repeated restimulation with irradiated CD200.sup.+ tumor
cells enriched the cells transduced with the IFP compared to wild
type T cells, demonstrating that recognition of a target expressing
the ligand for the CD200R-9aas-CD28Cys IRP enhances the response
(FIG. 2C).
[0206] In Vitro Colocalization Assay
[0207] Transduced T cells were imaged by microscopy to determine if
the CD200R-9aas-CD28Cys IFP colocalized with the cognate ligand in
the immunological synapse (IS) during T cell activation. CTxB was
used to stain lipids within the cell membrane, which are enriched
at the synapse (FIG. 2D, panel I). Labeled antibodies that target
CD200 expressed by the FBL cell (FIG. 2D, panel II) or CD200R
expressed by the T cell (FIG. 2D, panel III) were used to visualize
the location of the molecules in relation to the IS. CD200 ligand
and CD200R colocalized within the IS (FIG. 2D, panel IV),
demonstrating that the construct is sized appropriately to be
accommodated by the immunologic synapse.
[0208] CFSE-Based Cytotoxicity Assay
[0209] Increased CD28 signaling also promotes effector function
(Chen and Flies, Nat. Rev. Immunol. 13: 227-242, 2013). CD200R-CD28
fusion protein-transduced T cells were tested for increased killing
of target tumor cells. FBL and control EL4 tumors were incubated
for 10 minutes at room temperature with 2.5 .mu.M (CFSE.sub.hi) or
0.25 .mu.M (CFSE.sup.lo) CFSE in PBS, respectively. Excess dye was
removed by washing tumor cells in serum-containing media. A 1:1
mixture of EL4 and FBL tumor cells was incubated with titrated
numbers of CD200R-CD28 or GFP vector transduced TCR.sub.gag in
vitro expanded effector T cells for 4 hours in 96-well,
round-bottom plates at 37.degree. C. and 5% CO.sub.2. Specific FBL
lysis was determined by flow cytometric analyses of the %
CFSE.sub.hi (FBL) of total CFSE positive cells (FBL+EL4) remaining
in the well.
[0210] TCR.sub.gag T cells transduced with CD200R-CD28 constructs
displayed an enhanced ability to lyse FBL tumor in vitro compared
to TCR.sub.gag T cells transduced with an empty vector (FIGS. 2E,
2G). Target tumor cells were labeled with different dilutions of
the fluorescent dyes CellTrace Violet (CTV) or CFSE to generate a
1:1:1 mix of EL4 cells (CTV+), CD200.sup.+ FBL (CFSE.sup.hi) and
non-specific EL4)(CFSE.sup.lo) control targets (FIG. 2F).
Additionally, control GFP-transduced TCR.sub.gag T cells lysed
CD200.sup.- FBL and CD200.sup.+ FBL at equal efficiencies (FIG.
2G). By contrast, TCR.sub.gag T cells transduced with
CD200R-9aas-CD28Cys exhibited increased killing of CD200.sup.+ FBL
cells compared to control T cells, lysing over 40% of CD200.sup.+
FBL at the lowest E:T ratio tested (FIG. 2G).
[0211] Taken together, these data show that CD200R-CD28 constructs
function to increase accumulation and the lytic activity of
transduced T cells in response to tumor cell stimulation.
Example 4
T Cells Transduced with CD200R-9aas-CD28Cys Exhibit Enhanced
Accumulation In Vivo in Response to Recognition of FBL
[0212] B6 mice were injected with 4.times.10.sup.6 live FBL
leukemia intraperitoneal (i.p.) as previously described (Stromnes
et al., J. Clin. Invest. 120: 3722-34, 2010). After allowing 5 days
for the FBL to disseminate, mice received 180 mg/kg
cyclophosphamide (Cy, "Cytoxan") i.p. at least 6 hours before
transfer of the effector T cells. For survival studies, 10.sup.5
TCR.sub.gag T cells which previously underwent 1-3 stimulations in
vitro were transferred into tumor-bearing mice. To assess
short-term proliferation and accumulation, 2.times.10.sup.6 of each
of fusion protein-transduced and a GFP-control-transduced T cells
were co-injected into tumor-bearing mice and the mice euthanized
for analysis 8 days later. Mice were regularly monitored for tumor
burden and euthanized if evidence of tumor progression predicted
mortality would occur within 24-48 hours.
[0213] To assess whether CD200R-9aas-CD28Cys fusion
protein-transduced T cells exhibited greater proliferation and
accumulation in vivo in response to recognition of FBL, a mixed
population of fusion protein-transduced and control cells were
transferred into tumor-bearing mice and the ratio of cells by ex
vivo analysis were compared 8 days after transfer (FIG. 3A). By use
of congenic markers, transduced T cells were detected at a
1.2-1.4-fold greater ratio over control cells in both the spleen
and lymph nodes relative to the ratio that was injected (FIG. 3B).
Transduced CD200R-9aas-CD28Cys.sup.+ TCR.sub.gag T cells exhibited
reduced CD62L expression 3 days post-transfer to tumor-bearing
mice, suggesting an effector T cell phenotype (FIG. 3C). By day 15,
transduced and control T cells exhibited similar phenotypes,
including a lack of exhaustion markers (FIG. 3D). Similar to the in
vitro findings, T cells that expressed CD200R-9aas-CD28Cys
displayed increased accumulation in response to tumor stimulation
in vivo. Furthermore, they exhibited protein expression patterns
consistent with an effector T cell phenotype for at least 3 days
following transfer to tumor-bearing mice.
Example 5
Adoptive Immunotherapy with CD200R-CD28.sup.+ T Cells Exhibits
Greater Activity in Therapy of Disseminated Leukemia
[0214] Adoptive immunotherapy with T cells transduced with
CD200R-CD28 mediated increased therapeutic activity in the
preclinical mouse model of disseminated leukemia.
[0215] Mice were injected with a lethal dose of CD200.sup.+ FBL
leukemia and five days later, cohorts of Cy-treated mice received
additional therapy with 10.sup.5 T cells (FIG. 4A). The
contribution of the CD28 cysteine bond to efficacy mediated by the
CD200R-CD28 construct was assessed by comparing T cells transduced
with CD200R-CD28tm, CD200R-9aas-CD28Cys, and GFP control constructs
as shown in FIG. 1A. IL-2 was administered for 10 days as an
additional therapeutic reagent to a cohort of mice to promote the
activity of the T cells (Stromnes et al., J. Clin. Invest. 120:
3722-34, 2010). Before injection, T cells were assessed for various
surface proteins by flow cytometry. Transduced and control
TCR.sub.gag T cells displayed similar phenotypes, indicating that
transduction did not alter the phenotype of the cells prior to
injection (FIG. 4B).
[0216] In the small cohort of mice that received IL-2 injections, T
cells improved survival but a significant difference in the
survival of mice that received the different groups of T cells
could not be detected (FIG. 4C). However, in the cohort of mice
that did not receive IL-2 injections, there was a significant
improvement in the survival of mice that received T cells
transduced with CD200R-CD28 constructs appropriately sized to fit
within the immunological synapse (FIG. 4D). The majority of the
mice not receiving T cells, receiving T cells transduced with the
GFP control vector or T cells transduced with the largest
ectodomain (CD200R-CD28Cys IFP) did not survive beyond day 30
(FIGS. 4C and 4D, black solid, dashed, and orange lines,
respectively). In contrast, 71% of mice that received
CD200R-CD28tm.sup.+ T cells and 83% of mice that received
CD200R-9aas-CD28Cys.sup.+ T cells survived more than 100 days
post-therapy (FIGS. 4C and 4D, green and red lines, respectively).
These data suggest that transduction of T cells with CD200R-CD28
constructs that span a distance similar to a distance between
membranes in an immunological synapse provides sufficient
costimulation to overcome the dependence of T cell immunotherapy on
injection of exogenous IL-2. Furthermore, although there were
differences in proliferation and accumulation between the
CD200Rtm-CD28 and CD200R-9aas-CD28Cys constructs tested in mice
that did not receive injections of exogenous IL-2, both IFPs
effectively enhanced T cell immunotherapy to significantly improve
the clinical outcome from otherwise progressive leukemia.
Example 6
CD200R-9aas-CD28Cys.sup.+ T Cells do not Cause Autoreactivity with
Endogenous Tissues and do not Exhibit Infiltration of Normal
Tissues In Vivo
[0217] To determine if transduction of TCR.sub.gag T cells lowered
the threshold of activation sufficiently to result in
autoreactivity with endogenous tissues, autoimmune toxicity was
assessed in transgenic mice engineered to express the FBL gag tumor
Ag as a self-antigen in hepatocytes, under control of the albumin
promoter (FIG. 5A). TCR.sub.gag effectors were generated in vitro
and 10.sup.6 were transferred into Cytoxan-treated Alb:Gag mice
with disseminated leukemia. At 3 and 7 days post-transfer, liver
damage was assessed by quantification of serum levels of the liver
enzymes aspartate aminotransferase (AST) and alanine
aminotransferase (ALT). Adoptive therapy with control or
CD200R-9aas-CD28Cys.sup.+ TCR.sub.gag cells in mice did not affect
serum levels of AST or ALT at days 3 or 7 post-transfer, indicating
that CD200R-9aas-CD28Cys does not induce detectable autoimmune
liver damage in Alb:Gag mice (FIG. 5B).
[0218] T cells transduced with IFP do not exhibit increased
infiltration of normal tissues compared to control T cells. Mice
were euthanized 7 days post-transfer and liver sections were
stained with an antibody to the T cell marker CD3 to quantify T
cell infiltration. Limited presence of T cells in liver tissue was
observed, with no significant difference between recipients of
CD200R-9aas-CD28Cys.sup.+ or control TCR.sub.gag, indicating no
increased lymphocytic cellular infiltration as a result of IFP
expression (FIG. 5C).
Example 7
4-1BB Co-Stimulatory Signaling Domain Promotes Accumulation of
Transduced T Cells In Vitro
[0219] Co-stimulatory receptor 4-1BB is upregulated on activated T
cells, which promotes T cell survival and cytokine production (Chen
and Flies, Nat. Rev. Immunol. 13: 227-242, 2013). To assess if the
intracellular signaling domain of 4-1BB, with or without the
intracellular signaling domain of CD28, could induce increased T
cell proliferation and accumulation, IFPs using 4-1BB
(CD200R-9aas-4-1BB) or combining 4-1BB with CD28
(CD200R-9aas-CD28-4-1BB) were generated (FIG. 6A) using the methods
described in Example 2. TCR.sub.gag T cells were transduced as in
Example 2, and TCR.sub.gag effector cells were generated in vitro
as in Example 3.
[0220] As was observed with CD200R-9aas-CD28Cys, T cells transduced
with the 4-1BB constructs accumulated over multiple rounds of
stimulation in vitro (FIG. 6B). These data indicate that 4-1BB IFPs
also promote proliferation and survival of T cells.
[0221] TCR.sub.gag T cells transduced with a CD200R-4-1BB displayed
an enhanced ability to lyse FBL tumor in vitro using the CFSE-based
cytotoxicity assay described in Example 3 (FIG. 6C).
CD200R-41BB-transduced T cells also promote survival (FIG. 6D).
Example 8
[0222] Co-Expression of CD200Rtm-CD28 Enhances Function in
WT1-Specific TCR Primary T Cells
[0223] A human CD200Rtm-CD28 construct (SEQ ID NO.:1) was generated
to determine if IFP expression enhanced T cell function of human
primary T cells. The construct was combined with the beta and alpha
chains of the HLA-A2-restricted WT1.sub.126-specific TCR "C4" by
linking the genes with P2A elements (FIG. 7A). The first P2A
sequence was codon optimized to prevent genetic recombination with
the second P2A sequence. To generate lentiviruses, 293 T/17 cells
(3.times.10.sup.6 cells/plate) were transduced with human
constructs in the pRRLSIN and the packaging vectors pMDLg/pRRE,
pMD2-G, and pRSV-REV using Effectene (Qiagen). Culture media was
changed on day 1 post-transfection and virus-containing supernatant
collected on days 2 and 3, and aliquots frozen for future use.
[0224] The Jurkat human T cell subline, which lacks an endogenous
TCR, was used to test expression of the IFP and TCRs. These Jurkat
T cells were transduced by spinfection of 2.times.10.sup.6 cells
with 2 ml of retroviral supernatant at 1000 g for 90 min at
32.degree. C. Transduction of the Jurkat human T cell line with the
three-gene construct resulted in high expression of the IFP and
expression of the TCR at a similar MFI as T cells transduced with
the TCR only (FIG. 7A).
[0225] To transduce primary human T cells, peripheral blood
mononuclear cells (PBMC) were harvested from HLA-A2+ donors.
CD8.sup.+ T cells were purified using Miltenyi magnetic beads and
stimulated with Human T cell Expander CD3/CD28 Dynabeads (Life
Technologies) and 50 IU/ml IL-2. Four hours following stimulation,
T cells were transduced as described above for Jurkat T cells. T
cells were restimulated every 10-14 days with a rapid expansion
protocol (REP), as has been previously described (Ho et al., J
Immunol Methods 310:40-52, 2006).
[0226] The human cell line T2 was used as an APC, because it is
deficient in TAP and thus cannot present endogenous peptides, while
low level MHCI expression allows presentation of exogenously loaded
peptides. Expression of CD200 by the T2 cells was assessed by flow
cytometry (FIG. 7B). T2 cells exhibited a low level of endogenous
CD200 expression (FIG. 7B).
[0227] Transduced T cells were stimulated with WT1.sub.126-pulsed
T2 cells. Despite a low level of CD200 expression on the target
cells, CD200Rtm-CD28-transduced T cells exhibited enhanced
proliferation as compared to T cells transduced with the C4 TCR
alone (FIG. 7C). In addition, stimulated CD200Rtm-CD28-transduced T
cells (i.e., IFP.sup.+ T cells) produced increased levels of
IFN.gamma. and IL-2 compared to control T cells when exposed to
CD200dim tumor cells (FIG. 7D).
[0228] Overall, these results showed that primary T cells
transduced to express a human CD200Rtm-CD28 construct and the beta
and alpha chains of a WT1.sub.126-specific TCR exhibited enhanced
proliferation and increased cytokine production relative to T cells
transduced with the TCR construct alone.
Example 9
SIRP.alpha.-CD28 Fusion Protein Constructs Promote Accumulation of
Transduced T Cells In Vitro
[0229] Exemplary fusion proteins as described herein also include
IFPs comprised of the extracellular domain of SIRP.alpha., or
portions thereof, and an intracellular signaling domain of CD28
(FIG. 8A). The hydrophobic component may be comprised of the
transmembrane domain of either SIRP.alpha. or CD28, or portions
thereof. In some exemplary SIRP.alpha.-CD28 fusion proteins, the
hydrophobic component comprises the transmembrane domain of CD28
and the extracellular component further comprises an extracellular
portion of CD28, particularly an extracellular cysteine residue
adjacent to the hydrophobic component (e.g., SIRP.alpha.-CD28Cys,
SIRP.alpha.-6aas-CD28Cys, SIRP.alpha.-9aas-CD28Cys, and
SIRP.alpha.-9aas-CD28Cys). The extracellular component may comprise
all or a portion of the extracellular domain of SIRP.alpha.. In
some embodiments, the extracellular component comprises the entire
extracellular domain of SIRP.alpha.. In other examples, the
extracellular component comprises the first 367 amino acids (e.g.,
SIRP.alpha.-6aas-CD28Cys), the first 364 amino acids (e.g.,
SIRP.alpha.-9aas-CD28Cys), or the first 350
(SIRP.alpha.-23aas-CD28Cys) amino acids from the N-terminus of
SIRP.alpha.. The size of the extracellular component may affect the
ability of the fusion protein to enter the immunological synapse
and co-localize with the TCR within the cSMAC to deliver a strong
co-stimulatory signal. In some examples, the extracellular
component comprises a truncated SIRP.alpha., which may alter the
size of the extracellular component. For example, to account for
the additional extracellular amino acids of the extracellular
domain of the fusion protein (e.g., an additional 9 or 12 amino
acids), SIRP.alpha.-6aas-CD28 has a truncated portion of
SIRP.alpha. that preserves a natural N-linked glycosylation site.
In another example, SIRP.alpha.-23aas-CD28 has a truncated portion
of SIRP.alpha. that lacks the entire stem region of the SIRP.alpha.
extracellular domain. Additionally, a SIRP.alpha.-CD28 construct
has the capacity to convert a signal initiated by the binding of
SIRP.alpha. to its target into a positive (e.g., costimulatory)
signal generated by the CD28 intracellular signaling domain.
[0230] IFPs using SIRP.alpha. extracellular components were
generated (FIG. 8A) using the methods described in Example 2.
TCR.sub.gag T cells were transduced as in Example 2, and
TCR.sub.gag effector cells were generated in vitro as in Example 3.
FBL cells were transduced with CD47 or mCherry with polybrene
spinfection, similar to T cell transduction, and subsequently
sorted to generate a homogenous population.
[0231] As was observed with CD200R-9aas-CD28Cys, T cells transduced
with the SIRP.alpha. constructs accumulated over multiple rounds of
stimulation in vitro (FIG. 8B). These data suggest that
SIRP.alpha.-CD28 IFPs also promote proliferation and survival of T
cells.
[0232] To assess T cell proliferation in vitro, a CTV Dilution
Proliferation assay was performed as described in Example 2. As was
observed with CD200R-9aas-CD28Cys, T cells transduced with the
SIRP.alpha. constructs engineered to maintain the T cell-tumor cell
synapse distance exhibited enhanced proliferation as compared to
control T cells (FIG. 8C). In addition, CD47.sup.+ tumor cells were
efficiently killed after 3 days of co-culture with
SIRP.alpha.-CD28.sup.+ T cells but not control T cells or T cells
transduced with a SIRP.alpha. construct that lacked an
intracellular signaling domain (FIG. 8D). To further assess the
lytic capacity of SIRP.alpha.-CD28.sup.+ T cells, an IncuCyte assay
was used to quantify killing of CD47.sup.+ FBL. A total of 10.sup.5
mCherry.sup.+ CD47.sup.+ FBL were co-cultured in 24-well plates
with a titration of human T cells transduced with SIRP.alpha.-CD28
constructs. The plate was incubated in an IncuCyte (Essen
BioScience) inside a cell culture incubator for 70 hours. Images
were captured every hour to monitor killing of tumor cells, as
determined by loss of red signal. SIRP.alpha.-CD28.sup.+ T cells
killed CD47.sup.+ tumor cells, even at the lowest
effector-to-target ratio tested (0.4:1; FIG. 8E).
Example 10
PD-1-CD28 Fusion Protein Constructs Promote Cytokine Production in
Transduced T Cells In Vitro
[0233] Exemplary fusion proteins as described herein also include
IFPs comprised of the extracellular domain of PD-1, or portions
thereof, and an intracellular signaling domain of CD28 (FIG. 9A).
The transmembrane component may be comprised of the transmembrane
domain of either PD-1 or CD28, or portions thereof. In some
exemplary PD1-CD28 fusion proteins, the transmembrane component
comprises the transmembrane domain of CD28 and the extracellular
component further comprises an extracellular portion of CD28,
particularly an extracellular cysteine residue adjacent to the
transmembrane component (e.g., PD1-CD28Cys, PD1-9aas-CD28Cys, and
PD1-21aas-CD28Cys) to promote inter-chain dimerization. The
extracellular component may comprise all or a portion of the
extracellular domain of PD-1, or may be truncated (e.g., -9aas in
murine constructs, -12aas or -15aas in human constructs; lacking
the stem region of PD-1, -21aas) to maintain the short spatial
distance between the cells to facilitate access of the liganded
receptor to the immunologic synapse. Additionally, a PD1-CD28
construct has the capacity to convert what would typically be an
inhibitory signal from the binding of PD1 to its target into a
positive (e.g., costimulatory) signal generated by the CD28
intracellular signaling domain.
[0234] IFPs comprising PD-1 extracellular components were generated
(FIG. 9A) using the methods described in Example 2. TCR.sub.gag T
cells were transduced as in Example 2, and TCR.sub.gag effector
cells were generated in vitro as in Example 3.
[0235] Murine PD1-CD28 IFPs were generated using constructs I-IV
and VII (FIG. 9A). PD1-CD28.sup.+ T cells were restimulated in the
presence of Brefeldin A (to retain produced cytokines) with FBL
cells endogenously expressing the PD-1 ligands, PD-L1 and PD-L2.
After 5 hours, cells were fixed and treated with the BD
Cytofix/Cytoperm kit, to allow intracellular staining of the
effector cytokines, IFN.gamma. and TNF.alpha.. Transduction with
each of the five PD1-CD28 constructs enhanced production of
intracellular cytokines compared to control T cells (FIG. 9B).
[0236] Human PD1-CD28 IFPs were generated using constructs I-III
and V-VII (FIG. 9A). Vectors containing the PD1-CD28 IFP and C4 TCR
were generated as described above. Jurkat T cells were transduced
as described above. T cells transduced with the TCR and
PD1-12aas-CD28Cys or PD1-15aas-CD28Cys exhibited high transduction
efficiencies and expression of both proteins (FIG. 10).
Example 11
Fas-CD28 Fusion Protein Constructs Promote Accumulation and
Enhanced Function in Transduced T Cells In Vitro
[0237] Exemplary fusion proteins as described herein also include
IFPs comprised of the extracellular domain of Fas, or portions
thereof, and an intracellular signaling domain of CD28 (FIG. 11A).
The transmembrane component may be comprised of the domain of
either Fas or CD28, or portions thereof. In some exemplary Fas-CD28
fusion proteins, the transmembrane component comprises the
transmembrane domain of CD28 and the extracellular component
further comprises an extracellular portion of CD28, particularly an
extracellular cysteine residue adjacent to the transmembrane
component (e.g., Fas-CD28Cys and Fas-9aas-CD28Cys). The
extracellular component may comprise all or a portion of the
extracellular domain of Fas or may be truncated to preserve
maintain a short spatial distance between the cells (-9aas) upon
receptor-ligand interaction. Additionally, a Fas-CD28 construct has
the capacity to convert a signal initiated by the binding of Fas to
its target into a positive (e.g., costimulatory) signal generated
by the CD28 intracellular signaling domain.
[0238] IFPs comprising Fas extracellular components were generated
(FIG. 11A) using the methods described in Example 2. TCR.sub.gag T
cells were transduced as in Example 2, and TCR.sub.gag effector
cells were generated in vitro as in Example 3.
[0239] To determine if expression of the Fas-CD28 IFP results in
increased accumulation of transduced cells, the proportion of
transduced cells from the mixed population in the total TCR.sub.gag
population was measured over multiple cycles of stimulation with
irradiated FBL, as described in Example 3. All of the constructs
promoted accumulation of transduced T cells compared to control T
cells (FIG. 11B). In addition, expression of Fas-CD28 constructs
but not full-length (FL) Fas promoted survival or expansion of T
cells upon multiple stimulations in vitro FIG. 11C).
Example 12
LAG3-CD28 Fusion Protein Constructs
[0240] Exemplary fusion proteins as described herein also include
IFPs comprised of the extracellular domain of LAG3, or portions
thereof, and an intracellular signaling domain of CD28 (FIG. 12A).
The transmembrane component may be comprised of the domain of
either LAG3 or CD28, or portions thereof. In some exemplary
LAG3-CD28 fusion proteins, the transmembrane component comprises
the transmembrane domain of CD28 and the extracellular component
further comprises an extracellular portion of CD28, particularly an
extracellular cysteine residue adjacent to the transmembrane
component (e.g., LAG3-CD28Cys and LAG3-9aas-CD28Cys). The
extracellular component may comprise all or a portion of the
extracellular domain of LAG3 or may be truncated to maintain a
short spatial distance between the cells (e.g., -9aas) upon
receptor-ligand interaction. Additionally, a LAG3-CD28 construct
has the capacity to convert what would typically be an inhibitory
signal from the binding of LAG3 to its target into a positive
(e.g., costimulatory) signal generated by the CD28 intracellular
signaling domain.
[0241] IFPs using LAG3 extracellular components were generated
(FIG. 12A) using the methods described in Example 2. T cells were
transduced with LAG3-eGFP constructs as described. Five days after
transduction, CD8.sup.+ T cells were analyzed for construct
expression by anti-LAG3 antibody staining and flow cytometry (FIG.
12B). A vector encoding only green fluorescent protein (GFP) was
used as a control. All constructs exhibited expression of LAG3
(FIG. 12B).
Example 13
TIM3-CD28 Fusion Protein Constructs
[0242] Exemplary fusion proteins as described herein also include
IFPs comprised of the extracellular domain of TIM3, or portions
thereof, and an intracellular signaling domain of CD28 (FIG. 13A).
The transmembrane component may be comprised of the domain of
either TIM3 or CD28, or portions thereof. In some exemplary
TIM3-CD28 fusion proteins, the transmembrane component comprises
the transmembrane domain of CD28 and the extracellular component
further comprises an extracellular portion of CD28, particularly an
extracellular cysteine residue adjacent to the transmembrane
component (e.g., TIM3-CD28Cys and TIM3-9aas-CD28Cys). The
extracellular component may comprise all or a portion of the
extracellular domain of TIM3 or may be truncated to maintain the
short spatial distance between the cells (e.g., -9aas).
Additionally, a TIM3-CD28 construct has the capacity to convert
what would typically be an inhibitory signal from the binding of
TIM3 to its target into a positive signal generated by the CD28
intracellular signaling domain.
[0243] New IFPs using TIM3 extracellular components were generated
(FIG. 13A) using the methods described in Example 2. T cells were
transduced with GFP-TIM3 constructs as described. Five days after
transduction, CD8.sup.+ T cells were analyzed for construct
expression by anti-TIM3 antibody staining and flow cytometry (FIG.
13B). A vector encoding only green fluorescent protein (GFP) was
used as a control. Most constructs exhibited similar expression of
TIM3 (FIG. 13B).
[0244] While specific embodiments of the invention have been
illustrated and described, it will be readily appreciated that the
various embodiments described above can be combined to provide
further embodiments, and that various changes can be made therein
without departing from the spirit and scope of the invention.
[0245] All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications, and non-patent publications referred to in
this specification or listed in the Application Data Sheet,
including but not limited to U.S. Provisional Patent Application
No. 62/128,979, are incorporated herein by reference, in their
entirety. Aspects of the embodiments can be modified, if necessary
to employ concepts of the various patents, applications and
publications to provide yet further embodiments.
[0246] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
Sequence CWU 1
1
1781915DNAArtificial SequencehuCD200Rtm-CD28 construct 1atgctgtgcc
cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60gtggccgcca
gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag
120gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc
cgtgctgtgc 180tgccctccta tcgccctgcg gaacctgatc atcatcacct
gggagatcat cctgcggggc 240cagcccagct gtaccaaggc ctaccggaaa
gagacaaacg agacaaaaga aacaaactgc 300accgacgagc ggatcacatg
ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360cccgtggcca
tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac
420ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct
gttccagaac 480cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac
ctgccgccca gatctcttgg 540atccccgagg gcgattgcgc caccaagcag
gaatactggt ccaacggcac cgtgaccgtg 600aagtccacct gtcactggga
ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660ctgaccggca
acaagagcct gtacatcgag ctgctgcctg tgcctggcgc caagaagtcc
720gccaagctgt acatccccta catcatcctg acaatcatca ttctgaccat
cgtgggcttc 780atctggctgc tgcgcagcaa gcggagcaga ggcggccaca
gcgactacat gaacatgacc 840cctagacggc ctggccccac cagaaagcac
taccagccct acgcccctcc ccgggacttt 900gccgcctaca gaagc
9152728DNAArtificial SequencehuCD200R entire extracellular domain
2tgctgtgccc ttggagaacc gccaacctgg gcctgctgct gatcctgacc atcttcctgg
60tggccgccag cagcagcctg tgcatggacg agaagcagat cacccagaac tacagcaagg
120tgctggccga agtgaacacc agctggcccg tgaagatggc caccaacgcc
gtgctgtgct 180gccctcctat cgccctgcgg aacctgatca tcatcacctg
ggagatcatc ctgcggggcc 240agcccagctg taccaaggcc taccggaaag
agacaaacga gacaaaagaa acaaactgca 300ccgacgagcg gatcacatgg
gtgtccagac ccgaccagaa cagcgacctg cagatcagac 360ccgtggccat
cacccacgac ggctactacc ggtgcatcat ggtcaccccc gatggcaact
420tccaccgggg ataccatctg caggtgctcg tgacccccga agtgaccctg
ttccagaacc 480ggaacagaac cgccgtgtgc aaggccgtgg ccggaaaacc
tgccgcccag atctcttgga 540tccccgaggg cgattgcgcc accaagcagg
aatactggtc caacggcacc gtgaccgtga 600agtccacctg tcactgggag
gtgcacaacg tgtccaccgt gacatgccac gtgtcccacc 660tgaccggcaa
caagagcctg tacatcgagc tgctgcctgt gcctggcgcc aagaagtccg 720ccaagctg
728363DNAArtificial SequencehuCD200R transmembrane domain
3tacatcccct acatcatcct gacaatcatc attctgacca tcgtgggctt catctggctg
60ctg 63481DNAArtificial SequenceCD28 transmembrane domain
4ttctgggtgc tggtggtggt cggaggcgtg ctggcctgct acagcctgct ggtcaccgtg
60gccttcatca tcttttgggt c 815123DNAArtificial SequenceCD28
intracellular domain 5cgcagcaagc ggagcagagg cggccacagc gactacatga
acatgacccc tagacggcct 60ggccccacca gaaagcacta ccagccctac gcccctcccc
gggactttgc cgcctacaga 120agc 1236933DNAArtificial
SequencehuCD200R-CD28tm construct 6atgctgtgcc cttggagaac cgccaacctg
ggcctgctgc tgatcctgac catcttcctg 60gtggccgcca gcagcagcct gtgcatggac
gagaagcaga tcacccagaa ctacagcaag 120gtgctggccg aagtgaacac
cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180tgccctccta
tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc
240cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga
aacaaactgc 300accgacgagc ggatcacatg ggtgtccaga cccgaccaga
acagcgacct gcagatcaga 360cccgtggcca tcacccacga cggctactac
cggtgcatca tggtcacccc cgatggcaac 420ttccaccggg gataccatct
gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480cggaacagaa
ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg
540atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac
cgtgaccgtg 600aagtccacct gtcactggga ggtgcacaac gtgtccaccg
tgacatgcca cgtgtcccac 660ctgaccggca acaagagcct gtacatcgag
ctgctgcctg tgcctggcgc caagaagtcc 720gccaagctgt tctgggtgct
ggtggtggtc ggaggcgtgc tggcctgcta cagcctgctg 780gtcaccgtgg
ccttcatcat cttttgggtc cgcagcaagc ggagcagagg cggccacagc
840gactacatga acatgacccc tagacggcct ggccccacca gaaagcacta
ccagccctac 900gcccctcccc gggactttgc cgcctacaga agc
9337942DNAArtificial SequencehuCD200R-9aas-CD28Cys construct
7atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg
60gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag
120gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc
cgtgctgtgc 180tgccctccta tcgccctgcg gaacctgatc atcatcacct
gggagatcat cctgcggggc 240cagcccagct gtaccaaggc ctaccggaaa
gagacaaacg agacaaaaga aacaaactgc 300accgacgagc ggatcacatg
ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360cccgtggcca
tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac
420ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct
gttccagaac 480cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac
ctgccgccca gatctcttgg 540atccccgagg gcgattgcgc caccaagcag
gaatactggt ccaacggcac cgtgaccgtg 600aagtccacct gtcactggga
ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660ctgaccggca
acaagagcct gtacatcgag ctgctgcctg tgtgtcccag ccctctgttt
720cccggcccta gcaagccttt ctgggtgctg gtggtggtcg gaggcgtgct
ggcctgctac 780agcctgctgg tcaccgtggc cttcatcatc ttttgggtcc
gcagcaagcg gagcagaggc 840ggccacagcg actacatgaa catgacccct
agacggcctg gccccaccag aaagcactac 900cagccctacg cccctccccg
ggactttgcc gcctacagaa gc 9428702DNAArtificial SequencehuCD200R-9aas
portion of extracellular domain 8atgctgtgcc cttggagaac cgccaacctg
ggcctgctgc tgatcctgac catcttcctg 60gtggccgcca gcagcagcct gtgcatggac
gagaagcaga tcacccagaa ctacagcaag 120gtgctggccg aagtgaacac
cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180tgccctccta
tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc
240cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga
aacaaactgc 300accgacgagc ggatcacatg ggtgtccaga cccgaccaga
acagcgacct gcagatcaga 360cccgtggcca tcacccacga cggctactac
cggtgcatca tggtcacccc cgatggcaac 420ttccaccggg gataccatct
gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480cggaacagaa
ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg
540atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac
cgtgaccgtg 600aagtccacct gtcactggga ggtgcacaac gtgtccaccg
tgacatgcca cgtgtcccac 660ctgaccggca acaagagcct gtacatcgag
ctgctgcctg tg 702936DNAArtificial SequenceCD28Cys multimerization
domain 9tgtcccagcc ctctgtttcc cggccctagc aagcct
3610933DNAArtificial SequencehuCD200R-12aas-CD28Cys construct
10atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg
60gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag
120gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc
cgtgctgtgc 180tgccctccta tcgccctgcg gaacctgatc atcatcacct
gggagatcat cctgcggggc 240cagcccagct gtaccaaggc ctaccggaaa
gagacaaacg agacaaaaga aacaaactgc 300accgacgagc ggatcacatg
ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360cccgtggcca
tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac
420ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct
gttccagaac 480cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac
ctgccgccca gatctcttgg 540atccccgagg gcgattgcgc caccaagcag
gaatactggt ccaacggcac cgtgaccgtg 600aagtccacct gtcactggga
ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660ctgaccggca
acaagagcct gtacatcgag ctgtgtccca gccctctgtt tcccggccct
720agcaagcctt tctgggtgct ggtggtggtc ggaggcgtgc tggcctgcta
cagcctgctg 780gtcaccgtgg ccttcatcat cttttgggtc cgcagcaagc
ggagcagagg cggccacagc 840gactacatga acatgacccc tagacggcct
ggccccacca gaaagcacta ccagccctac 900gcccctcccc gggactttgc
cgcctacaga agc 93311693DNAArtificial SequencehuCD200R-12aas portion
of extracellular domain 11atgctgtgcc cttggagaac cgccaacctg
ggcctgctgc tgatcctgac catcttcctg 60gtggccgcca gcagcagcct gtgcatggac
gagaagcaga tcacccagaa ctacagcaag 120gtgctggccg aagtgaacac
cagctggccc gtgaagatgg ccaccaacgc cgtgctgtgc 180tgccctccta
tcgccctgcg gaacctgatc atcatcacct gggagatcat cctgcggggc
240cagcccagct gtaccaaggc ctaccggaaa gagacaaacg agacaaaaga
aacaaactgc 300accgacgagc ggatcacatg ggtgtccaga cccgaccaga
acagcgacct gcagatcaga 360cccgtggcca tcacccacga cggctactac
cggtgcatca tggtcacccc cgatggcaac 420ttccaccggg gataccatct
gcaggtgctc gtgacccccg aagtgaccct gttccagaac 480cggaacagaa
ccgccgtgtg caaggccgtg gccggaaaac ctgccgccca gatctcttgg
540atccccgagg gcgattgcgc caccaagcag gaatactggt ccaacggcac
cgtgaccgtg 600aagtccacct gtcactggga ggtgcacaac gtgtccaccg
tgacatgcca cgtgtcccac 660ctgaccggca acaagagcct gtacatcgag ctg
69312945DNAArtificial SequencehuCD200R-9aas-CD28Cys tm-41BBic
construct 12atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac
catcttcctg 60gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa
ctacagcaag 120gtgctggccg aagtgaacac cagctggccc gtgaagatgg
ccaccaacgc cgtgctgtgc 180tgccctccta tcgccctgcg gaacctgatc
atcatcacct gggagatcat cctgcggggc 240cagcccagct gtaccaaggc
ctaccggaaa gagacaaacg agacaaaaga aacaaactgc 300accgacgagc
ggatcacatg ggtgtccaga cccgaccaga acagcgacct gcagatcaga
360cccgtggcca tcacccacga cggctactac cggtgcatca tggtcacccc
cgatggcaac 420ttccaccggg gataccatct gcaggtgctc gtgacccccg
aagtgaccct gttccagaac 480cggaacagaa ccgccgtgtg caaggccgtg
gccggaaaac ctgccgccca gatctcttgg 540atccccgagg gcgattgcgc
caccaagcag gaatactggt ccaacggcac cgtgaccgtg 600aagtccacct
gtcactggga ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac
660ctgaccggca acaagagcct gtacatcgag ctgctgcctg tgtgtcccag
ccctctgttt 720cccggcccta gcaagccttt ctgggtgctg gtggtggtcg
gaggcgtgct ggcctgctac 780agcctgctgg tcaccgtggc cttcatcatc
ttttgggtca agcggggcag aaagaagctg 840ctgtacatct tcaagcagcc
tttcatgcgg cccgtgcaga ccacccagga agaggacggc 900tgctcctgca
gattccccga ggaagaagaa ggcggctgcg agctg 94513126DNAArtificial
Sequence4-1BB intracellular component 13aagcggggca gaaagaagct
gctgtacatc ttcaagcagc ctttcatgcg gcccgtgcag 60accacccagg aagaggacgg
ctgctcctgc agattccccg aggaagaaga aggcggctgc 120gagctg
12614936DNAArtificial SequencehuCD200R-12aas-CD28Cys tm-41BBic
construct 14atgctgtgcc cttggagaac cgccaacctg ggcctgctgc tgatcctgac
catcttcctg 60gtggccgcca gcagcagcct gtgcatggac gagaagcaga tcacccagaa
ctacagcaag 120gtgctggccg aagtgaacac cagctggccc gtgaagatgg
ccaccaacgc cgtgctgtgc 180tgccctccta tcgccctgcg gaacctgatc
atcatcacct gggagatcat cctgcggggc 240cagcccagct gtaccaaggc
ctaccggaaa gagacaaacg agacaaaaga aacaaactgc 300accgacgagc
ggatcacatg ggtgtccaga cccgaccaga acagcgacct gcagatcaga
360cccgtggcca tcacccacga cggctactac cggtgcatca tggtcacccc
cgatggcaac 420ttccaccggg gataccatct gcaggtgctc gtgacccccg
aagtgaccct gttccagaac 480cggaacagaa ccgccgtgtg caaggccgtg
gccggaaaac ctgccgccca gatctcttgg 540atccccgagg gcgattgcgc
caccaagcag gaatactggt ccaacggcac cgtgaccgtg 600aagtccacct
gtcactggga ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac
660ctgaccggca acaagagcct gtacatcgag ctgtgtccca gccctctgtt
tcccggccct 720agcaagcctt tctgggtgct ggtggtggtc ggaggcgtgc
tggcctgcta cagcctgctg 780gtcaccgtgg ccttcatcat cttttgggtc
aagcggggca gaaagaagct gctgtacatc 840ttcaagcagc ctttcatgcg
gcccgtgcag accacccagg aagaggacgg ctgctcctgc 900agattccccg
aggaagaaga aggcggctgc gagctg 936151059DNAArtificial
SequencehuCD200R-12aas-CD28Cys tm ic-41BBic construct 15atgctgtgcc
cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60gtggccgcca
gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag
120gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc
cgtgctgtgc 180tgccctccta tcgccctgcg gaacctgatc atcatcacct
gggagatcat cctgcggggc 240cagcccagct gtaccaaggc ctaccggaaa
gagacaaacg agacaaaaga aacaaactgc 300accgacgagc ggatcacatg
ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360cccgtggcca
tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac
420ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct
gttccagaac 480cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac
ctgccgccca gatctcttgg 540atccccgagg gcgattgcgc caccaagcag
gaatactggt ccaacggcac cgtgaccgtg 600aagtccacct gtcactggga
ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660ctgaccggca
acaagagcct gtacatcgag ctgtgtccca gccctctgtt tcccggccct
720agcaagcctt tctgggtgct ggtggtggtc ggaggcgtgc tggcctgcta
cagcctgctg 780gtcaccgtgg ccttcatcat cttttgggtc cgcagcaagc
ggagcagagg cggccacagc 840gactacatga acatgacccc tagacggcct
ggccccacca gaaagcacta ccagccctac 900gcccctcccc gggactttgc
cgcctacaga agcaagcggg gcagaaagaa gctgctgtac 960atcttcaagc
agcctttcat gcggcccgtg cagaccaccc aggaagagga cggctgctcc
1020tgcagattcc ccgaggaaga agaaggcggc tgcgagctg
1059161305DNAArtificial SequencehuSIRPalphatm-CD28 construct
16atggaacctg ccggacctgc tcctggcaga ctgggacctc tgctgtgtct gctgctggcc
60gcctcttgtg cttggagcgg agtggctggc gaagaggaac tgcaagtgat ccagcccgac
120aagagcgtgc tggtggccgc tggcgaaacc gccaccctga gatgtacagc
caccagcctg 180atccccgtgg gccccatcca gtggtttaga ggcgctggcc
ctggcagaga gctgatctac 240aaccagaaag agggccactt ccccagagtg
accaccgtgt ccgacctgac caagcggaac 300aacatggact tcagcatccg
gatcggcaac atcacccctg ccgatgccgg cacctactac 360tgcgtgaagt
tccggaaggg cagccccgac gacgtggaat tcaaaagcgg agccggcacc
420gagctgagcg tgcgggctaa accttctgcc cctgtggtgt ctggacctgc
cgccagagct 480acacctcagc acaccgtgtc ttttacctgc gagagccacg
gcttcagccc cagagacatc 540accctgaagt ggttcaagaa cggcaacgag
ctgtccgact tccagaccaa cgtggaccct 600gtgggcgaga gcgtgtccta
cagcatccac agcaccgcca aggtggtgct gacccgcgaa 660gatgtgcaca
gccaagtgat ctgcgaggtg gcccacgtga cactgcaggg cgatcctctg
720agaggaaccg ccaacctgtc cgagacaatc agagtgcccc ccaccctgga
agtgacccag 780cagcctgtgc gggccgagaa ccaagtgaac gtgacctgcc
aagtgcggaa gttctacccc 840cagcggctgc agctgacctg gctggaaaac
ggcaatgtgt cccggaccga gacagccagc 900accgtgaccg agaacaagga
tggcacctac aattggatga gctggctgct cgtgaacgtg 960tccgcccacc
gggacgatgt gaagctgaca tgccaggtgg aacacgacgg ccagcctgcc
1020gtgtccaaga gccacgatct gaaggtgtca gcccatccca aagagcaggg
ctccaacaca 1080gccgccgaga acaccggcag caacgagcgg aacatctaca
tcgtcgtggg cgtcgtgtgc 1140accctgctgg tggcactgct gatggccgct
ctgtacctcg tgcgcagcaa gcggagcaga 1200ggcggccaca gcgactacat
gaacatgacc cctagacggc ctggccccac cagaaagcac 1260taccagccct
acgcccctcc ccgggacttt gccgcctaca gaagc 1305171119DNAArtificial
SequencehuSIRP alpha entire extracellular domain 17atggaacctg
ccggacctgc tcctggcaga ctgggacctc tgctgtgtct gctgctggcc 60gcctcttgtg
cttggagcgg agtggctggc gaagaggaac tgcaagtgat ccagcccgac
120aagagcgtgc tggtggccgc tggcgaaacc gccaccctga gatgtacagc
caccagcctg 180atccccgtgg gccccatcca gtggtttaga ggcgctggcc
ctggcagaga gctgatctac 240aaccagaaag agggccactt ccccagagtg
accaccgtgt ccgacctgac caagcggaac 300aacatggact tcagcatccg
gatcggcaac atcacccctg ccgatgccgg cacctactac 360tgcgtgaagt
tccggaaggg cagccccgac gacgtggaat tcaaaagcgg agccggcacc
420gagctgagcg tgcgggctaa accttctgcc cctgtggtgt ctggacctgc
cgccagagct 480acacctcagc acaccgtgtc ttttacctgc gagagccacg
gcttcagccc cagagacatc 540accctgaagt ggttcaagaa cggcaacgag
ctgtccgact tccagaccaa cgtggaccct 600gtgggcgaga gcgtgtccta
cagcatccac agcaccgcca aggtggtgct gacccgcgaa 660gatgtgcaca
gccaagtgat ctgcgaggtg gcccacgtga cactgcaggg cgatcctctg
720agaggaaccg ccaacctgtc cgagacaatc agagtgcccc ccaccctgga
agtgacccag 780cagcctgtgc gggccgagaa ccaagtgaac gtgacctgcc
aagtgcggaa gttctacccc 840cagcggctgc agctgacctg gctggaaaac
ggcaatgtgt cccggaccga gacagccagc 900accgtgaccg agaacaagga
tggcacctac aattggatga gctggctgct cgtgaacgtg 960tccgcccacc
gggacgatgt gaagctgaca tgccaggtgg aacacgacgg ccagcctgcc
1020gtgtccaaga gccacgatct gaaggtgtca gcccatccca aagagcaggg
ctccaacaca 1080gccgccgaga acaccggcag caacgagcgg aacatctac
11191863DNAArtificial SequencehuSIRP alpha transmembrane domain
18atcgtcgtgg gcgtcgtgtg caccctgctg gtggcactgc tgatggccgc tctgtacctc
60gtg 63191323DNAArtificial SequencehuSIRP alpha-CD28tm construct
19atggaacctg ccggacctgc tcctggcaga ctgggacctc tgctgtgtct gctgctggcc
60gcctcttgtg cttggagcgg agtggctggc gaagaggaac tgcaagtgat ccagcccgac
120aagagcgtgc tggtggccgc tggcgaaacc gccaccctga gatgtacagc
caccagcctg 180atccccgtgg gccccatcca gtggtttaga ggcgctggcc
ctggcagaga gctgatctac 240aaccagaaag agggccactt ccccagagtg
accaccgtgt ccgacctgac caagcggaac 300aacatggact tcagcatccg
gatcggcaac atcacccctg ccgatgccgg cacctactac 360tgcgtgaagt
tccggaaggg cagccccgac gacgtggaat tcaaaagcgg agccggcacc
420gagctgagcg tgcgggctaa accttctgcc cctgtggtgt ctggacctgc
cgccagagct 480acacctcagc acaccgtgtc ttttacctgc gagagccacg
gcttcagccc cagagacatc 540accctgaagt ggttcaagaa cggcaacgag
ctgtccgact tccagaccaa cgtggaccct 600gtgggcgaga gcgtgtccta
cagcatccac agcaccgcca aggtggtgct gacccgcgaa 660gatgtgcaca
gccaagtgat ctgcgaggtg gcccacgtga cactgcaggg cgatcctctg
720agaggaaccg ccaacctgtc cgagacaatc agagtgcccc ccaccctgga
agtgacccag 780cagcctgtgc gggccgagaa ccaagtgaac gtgacctgcc
aagtgcggaa gttctacccc 840cagcggctgc agctgacctg gctggaaaac
ggcaatgtgt cccggaccga gacagccagc 900accgtgaccg agaacaagga
tggcacctac aattggatga gctggctgct cgtgaacgtg 960tccgcccacc
gggacgatgt gaagctgaca tgccaggtgg aacacgacgg ccagcctgcc
1020gtgtccaaga gccacgatct gaaggtgtca gcccatccca aagagcaggg
ctccaacaca 1080gccgccgaga acaccggcag caacgagcgg aacatctact
tctgggtgct ggtggtggtc 1140ggaggcgtgc tggcctgcta cagcctgctg
gtcaccgtgg ccttcatcat cttttgggtc 1200cgcagcaagc ggagcagagg
cggccacagc gactacatga acatgacccc tagacggcct 1260ggccccacca
gaaagcacta ccagccctac gcccctcccc gggactttgc cgcctacaga 1320agc
1323201323DNAArtificial SequencehuSIRP alpha 12aas-CD28Cys
construct 20atggaacctg ccggacctgc tcctggcaga ctgggacctc tgctgtgtct
gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc gaagaggaac tgcaagtgat
ccagcccgac 120aagagcgtgc tggtggccgc tggcgaaacc gccaccctga
gatgtacagc caccagcctg
180atccccgtgg gccccatcca gtggtttaga ggcgctggcc ctggcagaga
gctgatctac 240aaccagaaag agggccactt ccccagagtg accaccgtgt
ccgacctgac caagcggaac 300aacatggact tcagcatccg gatcggcaac
atcacccctg ccgatgccgg cacctactac 360tgcgtgaagt tccggaaggg
cagccccgac gacgtggaat tcaaaagcgg agccggcacc 420gagctgagcg
tgcgggctaa accttctgcc cctgtggtgt ctggacctgc cgccagagct
480acacctcagc acaccgtgtc ttttacctgc gagagccacg gcttcagccc
cagagacatc 540accctgaagt ggttcaagaa cggcaacgag ctgtccgact
tccagaccaa cgtggaccct 600gtgggcgaga gcgtgtccta cagcatccac
agcaccgcca aggtggtgct gacccgcgaa 660gatgtgcaca gccaagtgat
ctgcgaggtg gcccacgtga cactgcaggg cgatcctctg 720agaggaaccg
ccaacctgtc cgagacaatc agagtgcccc ccaccctgga agtgacccag
780cagcctgtgc gggccgagaa ccaagtgaac gtgacctgcc aagtgcggaa
gttctacccc 840cagcggctgc agctgacctg gctggaaaac ggcaatgtgt
cccggaccga gacagccagc 900accgtgaccg agaacaagga tggcacctac
aattggatga gctggctgct cgtgaacgtg 960tccgcccacc gggacgatgt
gaagctgaca tgccaggtgg aacacgacgg ccagcctgcc 1020gtgtccaaga
gccacgatct gaaggtgtca gcccatccca aagagcaggg ctccaacaca
1080gcctgtccca gccctctgtt tcccggccct agcaagcctt tctgggtgct
ggtggtggtc 1140ggaggcgtgc tggcctgcta cagcctgctg gtcaccgtgg
ccttcatcat cttttgggtc 1200cgcagcaagc ggagcagagg cggccacagc
gactacatga acatgacccc tagacggcct 1260ggccccacca gaaagcacta
ccagccctac gcccctcccc gggactttgc cgcctacaga 1320agc
1323211083DNAArtificial SequencehuSIRP alpha 12aas portion of
extracellular domain 21atggaacctg ccggacctgc tcctggcaga ctgggacctc
tgctgtgtct gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc gaagaggaac
tgcaagtgat ccagcccgac 120aagagcgtgc tggtggccgc tggcgaaacc
gccaccctga gatgtacagc caccagcctg 180atccccgtgg gccccatcca
gtggtttaga ggcgctggcc ctggcagaga gctgatctac 240aaccagaaag
agggccactt ccccagagtg accaccgtgt ccgacctgac caagcggaac
300aacatggact tcagcatccg gatcggcaac atcacccctg ccgatgccgg
cacctactac 360tgcgtgaagt tccggaaggg cagccccgac gacgtggaat
tcaaaagcgg agccggcacc 420gagctgagcg tgcgggctaa accttctgcc
cctgtggtgt ctggacctgc cgccagagct 480acacctcagc acaccgtgtc
ttttacctgc gagagccacg gcttcagccc cagagacatc 540accctgaagt
ggttcaagaa cggcaacgag ctgtccgact tccagaccaa cgtggaccct
600gtgggcgaga gcgtgtccta cagcatccac agcaccgcca aggtggtgct
gacccgcgaa 660gatgtgcaca gccaagtgat ctgcgaggtg gcccacgtga
cactgcaggg cgatcctctg 720agaggaaccg ccaacctgtc cgagacaatc
agagtgcccc ccaccctgga agtgacccag 780cagcctgtgc gggccgagaa
ccaagtgaac gtgacctgcc aagtgcggaa gttctacccc 840cagcggctgc
agctgacctg gctggaaaac ggcaatgtgt cccggaccga gacagccagc
900accgtgaccg agaacaagga tggcacctac aattggatga gctggctgct
cgtgaacgtg 960tccgcccacc gggacgatgt gaagctgaca tgccaggtgg
aacacgacgg ccagcctgcc 1020gtgtccaaga gccacgatct gaaggtgtca
gcccatccca aagagcaggg ctccaacaca 1080gcc 1083221326DNAArtificial
SequencehuSIRP alpha-12aas-CD28Cys tm-41BBic construct 22atggaacctg
ccggacctgc tcctggcaga ctgggacctc tgctgtgtct gctgctggcc 60gcctcttgtg
cttggagcgg agtggctggc gaagaggaac tgcaagtgat ccagcccgac
120aagagcgtgc tggtggccgc tggcgaaacc gccaccctga gatgtacagc
caccagcctg 180atccccgtgg gccccatcca gtggtttaga ggcgctggcc
ctggcagaga gctgatctac 240aaccagaaag agggccactt ccccagagtg
accaccgtgt ccgacctgac caagcggaac 300aacatggact tcagcatccg
gatcggcaac atcacccctg ccgatgccgg cacctactac 360tgcgtgaagt
tccggaaggg cagccccgac gacgtggaat tcaaaagcgg agccggcacc
420gagctgagcg tgcgggctaa accttctgcc cctgtggtgt ctggacctgc
cgccagagct 480acacctcagc acaccgtgtc ttttacctgc gagagccacg
gcttcagccc cagagacatc 540accctgaagt ggttcaagaa cggcaacgag
ctgtccgact tccagaccaa cgtggaccct 600gtgggcgaga gcgtgtccta
cagcatccac agcaccgcca aggtggtgct gacccgcgaa 660gatgtgcaca
gccaagtgat ctgcgaggtg gcccacgtga cactgcaggg cgatcctctg
720agaggaaccg ccaacctgtc cgagacaatc agagtgcccc ccaccctgga
agtgacccag 780cagcctgtgc gggccgagaa ccaagtgaac gtgacctgcc
aagtgcggaa gttctacccc 840cagcggctgc agctgacctg gctggaaaac
ggcaatgtgt cccggaccga gacagccagc 900accgtgaccg agaacaagga
tggcacctac aattggatga gctggctgct cgtgaacgtg 960tccgcccacc
gggacgatgt gaagctgaca tgccaggtgg aacacgacgg ccagcctgcc
1020gtgtccaaga gccacgatct gaaggtgtca gcccatccca aagagcaggg
ctccaacaca 1080gcctgtccca gccctctgtt tcccggccct agcaagcctt
tctgggtgct ggtggtggtc 1140ggaggcgtgc tggcctgcta cagcctgctg
gtcaccgtgg ccttcatcat cttttgggtc 1200aagcggggca gaaagaagct
gctgtacatc ttcaagcagc ctttcatgcg gcccgtgcag 1260accacccagg
aagaggacgg ctgctcctgc agattccccg aggaagaaga aggcggctgc 1320gagctg
1326231449DNAArtificial SequencehuSIRP alpha-12aas-CD28Cys tm
ic-41BBic construct 23atggaacctg ccggacctgc tcctggcaga ctgggacctc
tgctgtgtct gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc gaagaggaac
tgcaagtgat ccagcccgac 120aagagcgtgc tggtggccgc tggcgaaacc
gccaccctga gatgtacagc caccagcctg 180atccccgtgg gccccatcca
gtggtttaga ggcgctggcc ctggcagaga gctgatctac 240aaccagaaag
agggccactt ccccagagtg accaccgtgt ccgacctgac caagcggaac
300aacatggact tcagcatccg gatcggcaac atcacccctg ccgatgccgg
cacctactac 360tgcgtgaagt tccggaaggg cagccccgac gacgtggaat
tcaaaagcgg agccggcacc 420gagctgagcg tgcgggctaa accttctgcc
cctgtggtgt ctggacctgc cgccagagct 480acacctcagc acaccgtgtc
ttttacctgc gagagccacg gcttcagccc cagagacatc 540accctgaagt
ggttcaagaa cggcaacgag ctgtccgact tccagaccaa cgtggaccct
600gtgggcgaga gcgtgtccta cagcatccac agcaccgcca aggtggtgct
gacccgcgaa 660gatgtgcaca gccaagtgat ctgcgaggtg gcccacgtga
cactgcaggg cgatcctctg 720agaggaaccg ccaacctgtc cgagacaatc
agagtgcccc ccaccctgga agtgacccag 780cagcctgtgc gggccgagaa
ccaagtgaac gtgacctgcc aagtgcggaa gttctacccc 840cagcggctgc
agctgacctg gctggaaaac ggcaatgtgt cccggaccga gacagccagc
900accgtgaccg agaacaagga tggcacctac aattggatga gctggctgct
cgtgaacgtg 960tccgcccacc gggacgatgt gaagctgaca tgccaggtgg
aacacgacgg ccagcctgcc 1020gtgtccaaga gccacgatct gaaggtgtca
gcccatccca aagagcaggg ctccaacaca 1080gcctgtccca gccctctgtt
tcccggccct agcaagcctt tctgggtgct ggtggtggtc 1140ggaggcgtgc
tggcctgcta cagcctgctg gtcaccgtgg ccttcatcat cttttgggtc
1200cgcagcaagc ggagcagagg cggccacagc gactacatga acatgacccc
tagacggcct 1260ggccccacca gaaagcacta ccagccctac gcccctcccc
gggactttgc cgcctacaga 1320agcaagcggg gcagaaagaa gctgctgtac
atcttcaagc agcctttcat gcggcccgtg 1380cagaccaccc aggaagagga
cggctgctcc tgcagattcc ccgaggaaga agaaggcggc 1440tgcgagctg
144924305PRTArtificial SequencehuCD200Rtm-CD28 protein 24Met Leu
Cys Pro Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr
Ile Phe Leu Val Ala Ala Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25
30Gln Ile Thr Gln Asn Tyr Ser Lys Val Leu Ala Glu Val Asn Thr Ser
35 40 45Trp Pro Val Lys Met Ala Thr Asn Ala Val Leu Cys Cys Pro Pro
Ile 50 55 60Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu
Arg Gly65 70 75 80Gln Pro Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr
Asn Glu Thr Lys 85 90 95Glu Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp
Val Ser Arg Pro Asp 100 105 110Gln Asn Ser Asp Leu Gln Ile Arg Pro
Val Ala Ile Thr His Asp Gly 115 120 125Tyr Tyr Arg Cys Ile Met Val
Thr Pro Asp Gly Asn Phe His Arg Gly 130 135 140Tyr His Leu Gln Val
Leu Val Thr Pro Glu Val Thr Leu Phe Gln Asn145 150 155 160Arg Asn
Arg Thr Ala Val Cys Lys Ala Val Ala Gly Lys Pro Ala Ala 165 170
175Gln Ile Ser Trp Ile Pro Glu Gly Asp Cys Ala Thr Lys Gln Glu Tyr
180 185 190Trp Ser Asn Gly Thr Val Thr Val Lys Ser Thr Cys His Trp
Glu Val 195 200 205His Asn Val Ser Thr Val Thr Cys His Val Ser His
Leu Thr Gly Asn 210 215 220Lys Ser Leu Tyr Ile Glu Leu Leu Pro Val
Pro Gly Ala Lys Lys Ser225 230 235 240Ala Lys Leu Tyr Ile Pro Tyr
Ile Ile Leu Thr Ile Ile Ile Leu Thr 245 250 255Ile Val Gly Phe Ile
Trp Leu Leu Arg Ser Lys Arg Ser Arg Gly Gly 260 265 270His Ser Asp
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 275 280 285Lys
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 290 295
300Ser30525243PRTArtificial SequencehuCD200R entire extracellular
domain 25Met Leu Cys Pro Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu
Ile Leu1 5 10 15Thr Ile Phe Leu Val Ala Ala Ser Ser Ser Leu Cys Met
Asp Glu Lys 20 25 30Gln Ile Thr Gln Asn Tyr Ser Lys Val Leu Ala Glu
Val Asn Thr Ser 35 40 45Trp Pro Val Lys Met Ala Thr Asn Ala Val Leu
Cys Cys Pro Pro Ile 50 55 60Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp
Glu Ile Ile Leu Arg Gly65 70 75 80Gln Pro Ser Cys Thr Lys Ala Tyr
Arg Lys Glu Thr Asn Glu Thr Lys 85 90 95Glu Thr Asn Cys Thr Asp Glu
Arg Ile Thr Trp Val Ser Arg Pro Asp 100 105 110Gln Asn Ser Asp Leu
Gln Ile Arg Pro Val Ala Ile Thr His Asp Gly 115 120 125Tyr Tyr Arg
Cys Ile Met Val Thr Pro Asp Gly Asn Phe His Arg Gly 130 135 140Tyr
His Leu Gln Val Leu Val Thr Pro Glu Val Thr Leu Phe Gln Asn145 150
155 160Arg Asn Arg Thr Ala Val Cys Lys Ala Val Ala Gly Lys Pro Ala
Ala 165 170 175Gln Ile Ser Trp Ile Pro Glu Gly Asp Cys Ala Thr Lys
Gln Glu Tyr 180 185 190Trp Ser Asn Gly Thr Val Thr Val Lys Ser Thr
Cys His Trp Glu Val 195 200 205His Asn Val Ser Thr Val Thr Cys His
Val Ser His Leu Thr Gly Asn 210 215 220Lys Ser Leu Tyr Ile Glu Leu
Leu Pro Val Pro Gly Ala Lys Lys Ser225 230 235 240Ala Lys
Leu2621PRTArtificial SequencehuCD200R transmembrane domain 26Tyr
Ile Pro Tyr Ile Ile Leu Thr Ile Ile Ile Leu Thr Ile Val Gly1 5 10
15Phe Ile Trp Leu Leu 202727PRTArtificial SequenceCD28
transmembrane domain protein 27Phe Trp Val Leu Val Val Val Gly Gly
Val Leu Ala Cys Tyr Ser Leu1 5 10 15Leu Val Thr Val Ala Phe Ile Ile
Phe Trp Val 20 252841PRTArtificial SequenceCD28 intracellular
domain protein 28Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr
Met Asn Met Thr1 5 10 15Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
Gln Pro Tyr Ala Pro 20 25 30Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35
4029311PRTArtificial SequencehuCD200R-CD28tm protein 29Met Leu Cys
Pro Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile
Phe Leu Val Ala Ala Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln
Ile Thr Gln Asn Tyr Ser Lys Val Leu Ala Glu Val Asn Thr Ser 35 40
45Trp Pro Val Lys Met Ala Thr Asn Ala Val Leu Cys Cys Pro Pro Ile
50 55 60Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg
Gly65 70 75 80Gln Pro Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn
Glu Thr Lys 85 90 95Glu Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp Val
Ser Arg Pro Asp 100 105 110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val
Ala Ile Thr His Asp Gly 115 120 125Tyr Tyr Arg Cys Ile Met Val Thr
Pro Asp Gly Asn Phe His Arg Gly 130 135 140Tyr His Leu Gln Val Leu
Val Thr Pro Glu Val Thr Leu Phe Gln Asn145 150 155 160Arg Asn Arg
Thr Ala Val Cys Lys Ala Val Ala Gly Lys Pro Ala Ala 165 170 175Gln
Ile Ser Trp Ile Pro Glu Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185
190Trp Ser Asn Gly Thr Val Thr Val Lys Ser Thr Cys His Trp Glu Val
195 200 205His Asn Val Ser Thr Val Thr Cys His Val Ser His Leu Thr
Gly Asn 210 215 220Lys Ser Leu Tyr Ile Glu Leu Leu Pro Val Pro Gly
Ala Lys Lys Ser225 230 235 240Ala Lys Leu Phe Trp Val Leu Val Val
Val Gly Gly Val Leu Ala Cys 245 250 255Tyr Ser Leu Leu Val Thr Val
Ala Phe Ile Ile Phe Trp Val Arg Ser 260 265 270Lys Arg Ser Arg Gly
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg 275 280 285Arg Pro Gly
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 290 295 300Asp
Phe Ala Ala Tyr Arg Ser305 31030314PRTArtificial
SequencehuCD200R-9aas-CD28Cys protein 30Met Leu Cys Pro Trp Arg Thr
Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile Phe Leu Val Ala
Ala Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln Ile Thr Gln Asn
Tyr Ser Lys Val Leu Ala Glu Val Asn Thr Ser 35 40 45Trp Pro Val Lys
Met Ala Thr Asn Ala Val Leu Cys Cys Pro Pro Ile 50 55 60Ala Leu Arg
Asn Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg Gly65 70 75 80Gln
Pro Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn Glu Thr Lys 85 90
95Glu Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp Val Ser Arg Pro Asp
100 105 110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val Ala Ile Thr His
Asp Gly 115 120 125Tyr Tyr Arg Cys Ile Met Val Thr Pro Asp Gly Asn
Phe His Arg Gly 130 135 140Tyr His Leu Gln Val Leu Val Thr Pro Glu
Val Thr Leu Phe Gln Asn145 150 155 160Arg Asn Arg Thr Ala Val Cys
Lys Ala Val Ala Gly Lys Pro Ala Ala 165 170 175Gln Ile Ser Trp Ile
Pro Glu Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185 190Trp Ser Asn
Gly Thr Val Thr Val Lys Ser Thr Cys His Trp Glu Val 195 200 205His
Asn Val Ser Thr Val Thr Cys His Val Ser His Leu Thr Gly Asn 210 215
220Lys Ser Leu Tyr Ile Glu Leu Leu Pro Val Cys Pro Ser Pro Leu
Phe225 230 235 240Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val
Val Gly Gly Val 245 250 255Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
Ala Phe Ile Ile Phe Trp 260 265 270Val Arg Ser Lys Arg Ser Arg Gly
Gly His Ser Asp Tyr Met Asn Met 275 280 285Thr Pro Arg Arg Pro Gly
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 290 295 300Pro Pro Arg Asp
Phe Ala Ala Tyr Arg Ser305 31031234PRTArtificial
SequencehuCD200R-9aas protein 31Met Leu Cys Pro Trp Arg Thr Ala Asn
Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile Phe Leu Val Ala Ala Ser
Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln Ile Thr Gln Asn Tyr Ser
Lys Val Leu Ala Glu Val Asn Thr Ser 35 40 45Trp Pro Val Lys Met Ala
Thr Asn Ala Val Leu Cys Cys Pro Pro Ile 50 55 60Ala Leu Arg Asn Leu
Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg Gly65 70 75 80Gln Pro Ser
Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn Glu Thr Lys 85 90 95Glu Thr
Asn Cys Thr Asp Glu Arg Ile Thr Trp Val Ser Arg Pro Asp 100 105
110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val Ala Ile Thr His Asp Gly
115 120 125Tyr Tyr Arg Cys Ile Met Val Thr Pro Asp Gly Asn Phe His
Arg Gly 130 135 140Tyr His Leu Gln Val Leu Val Thr Pro Glu Val Thr
Leu Phe Gln Asn145 150 155 160Arg Asn Arg Thr Ala Val Cys Lys Ala
Val Ala Gly Lys Pro Ala Ala 165 170 175Gln Ile Ser Trp Ile Pro Glu
Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185 190Trp Ser Asn Gly Thr
Val Thr Val Lys Ser Thr Cys His Trp Glu Val 195 200 205His Asn Val
Ser Thr Val Thr Cys His Val Ser His Leu Thr Gly Asn 210 215 220Lys
Ser Leu Tyr Ile Glu Leu Leu Pro Val225 2303212PRTArtificial
SequenceCD28Cys (extracellular portion) protein 32Cys Pro Ser Pro
Leu Phe Pro Gly Pro Ser Lys Pro1 5 1033311PRTArtificial
SequencehuCD200R-12aas-CD28Cys protein 33Met Leu Cys
Pro Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile
Phe Leu Val Ala Ala Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln
Ile Thr Gln Asn Tyr Ser Lys Val Leu Ala Glu Val Asn Thr Ser 35 40
45Trp Pro Val Lys Met Ala Thr Asn Ala Val Leu Cys Cys Pro Pro Ile
50 55 60Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg
Gly65 70 75 80Gln Pro Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn
Glu Thr Lys 85 90 95Glu Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp Val
Ser Arg Pro Asp 100 105 110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val
Ala Ile Thr His Asp Gly 115 120 125Tyr Tyr Arg Cys Ile Met Val Thr
Pro Asp Gly Asn Phe His Arg Gly 130 135 140Tyr His Leu Gln Val Leu
Val Thr Pro Glu Val Thr Leu Phe Gln Asn145 150 155 160Arg Asn Arg
Thr Ala Val Cys Lys Ala Val Ala Gly Lys Pro Ala Ala 165 170 175Gln
Ile Ser Trp Ile Pro Glu Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185
190Trp Ser Asn Gly Thr Val Thr Val Lys Ser Thr Cys His Trp Glu Val
195 200 205His Asn Val Ser Thr Val Thr Cys His Val Ser His Leu Thr
Gly Asn 210 215 220Lys Ser Leu Tyr Ile Glu Leu Cys Pro Ser Pro Leu
Phe Pro Gly Pro225 230 235 240Ser Lys Pro Phe Trp Val Leu Val Val
Val Gly Gly Val Leu Ala Cys 245 250 255Tyr Ser Leu Leu Val Thr Val
Ala Phe Ile Ile Phe Trp Val Arg Ser 260 265 270Lys Arg Ser Arg Gly
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg 275 280 285Arg Pro Gly
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 290 295 300Asp
Phe Ala Ala Tyr Arg Ser305 31034231PRTArtificial
SequencehuCD200R-12aas protein 34Met Leu Cys Pro Trp Arg Thr Ala
Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile Phe Leu Val Ala Ala
Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln Ile Thr Gln Asn Tyr
Ser Lys Val Leu Ala Glu Val Asn Thr Ser 35 40 45Trp Pro Val Lys Met
Ala Thr Asn Ala Val Leu Cys Cys Pro Pro Ile 50 55 60Ala Leu Arg Asn
Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg Gly65 70 75 80Gln Pro
Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn Glu Thr Lys 85 90 95Glu
Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp Val Ser Arg Pro Asp 100 105
110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val Ala Ile Thr His Asp Gly
115 120 125Tyr Tyr Arg Cys Ile Met Val Thr Pro Asp Gly Asn Phe His
Arg Gly 130 135 140Tyr His Leu Gln Val Leu Val Thr Pro Glu Val Thr
Leu Phe Gln Asn145 150 155 160Arg Asn Arg Thr Ala Val Cys Lys Ala
Val Ala Gly Lys Pro Ala Ala 165 170 175Gln Ile Ser Trp Ile Pro Glu
Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185 190Trp Ser Asn Gly Thr
Val Thr Val Lys Ser Thr Cys His Trp Glu Val 195 200 205His Asn Val
Ser Thr Val Thr Cys His Val Ser His Leu Thr Gly Asn 210 215 220Lys
Ser Leu Tyr Ile Glu Leu225 23035315PRTArtificial
SequencehuCD200R-9aas-CD28Cys tm-41BBic protein 35Met Leu Cys Pro
Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile Phe
Leu Val Ala Ala Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln Ile
Thr Gln Asn Tyr Ser Lys Val Leu Ala Glu Val Asn Thr Ser 35 40 45Trp
Pro Val Lys Met Ala Thr Asn Ala Val Leu Cys Cys Pro Pro Ile 50 55
60Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg Gly65
70 75 80Gln Pro Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn Glu Thr
Lys 85 90 95Glu Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp Val Ser Arg
Pro Asp 100 105 110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val Ala Ile
Thr His Asp Gly 115 120 125Tyr Tyr Arg Cys Ile Met Val Thr Pro Asp
Gly Asn Phe His Arg Gly 130 135 140Tyr His Leu Gln Val Leu Val Thr
Pro Glu Val Thr Leu Phe Gln Asn145 150 155 160Arg Asn Arg Thr Ala
Val Cys Lys Ala Val Ala Gly Lys Pro Ala Ala 165 170 175Gln Ile Ser
Trp Ile Pro Glu Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185 190Trp
Ser Asn Gly Thr Val Thr Val Lys Ser Thr Cys His Trp Glu Val 195 200
205His Asn Val Ser Thr Val Thr Cys His Val Ser His Leu Thr Gly Asn
210 215 220Lys Ser Leu Tyr Ile Glu Leu Leu Pro Val Cys Pro Ser Pro
Leu Phe225 230 235 240Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val
Val Val Gly Gly Val 245 250 255Leu Ala Cys Tyr Ser Leu Leu Val Thr
Val Ala Phe Ile Ile Phe Trp 260 265 270Val Lys Arg Gly Arg Lys Lys
Leu Leu Tyr Ile Phe Lys Gln Pro Phe 275 280 285Met Arg Pro Val Gln
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 290 295 300Phe Pro Glu
Glu Glu Glu Gly Gly Cys Glu Leu305 310 3153642PRTArtificial
Sequence4-1BB intracellular component protein 36Lys Arg Gly Arg Lys
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg Pro Val Gln
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30Pro Glu Glu
Glu Glu Gly Gly Cys Glu Leu 35 4037312PRTArtificial
SequencehuCD200R-12aas-CD28Cys tm-41BBic protein 37Met Leu Cys Pro
Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile Phe
Leu Val Ala Ala Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln Ile
Thr Gln Asn Tyr Ser Lys Val Leu Ala Glu Val Asn Thr Ser 35 40 45Trp
Pro Val Lys Met Ala Thr Asn Ala Val Leu Cys Cys Pro Pro Ile 50 55
60Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg Gly65
70 75 80Gln Pro Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn Glu Thr
Lys 85 90 95Glu Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp Val Ser Arg
Pro Asp 100 105 110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val Ala Ile
Thr His Asp Gly 115 120 125Tyr Tyr Arg Cys Ile Met Val Thr Pro Asp
Gly Asn Phe His Arg Gly 130 135 140Tyr His Leu Gln Val Leu Val Thr
Pro Glu Val Thr Leu Phe Gln Asn145 150 155 160Arg Asn Arg Thr Ala
Val Cys Lys Ala Val Ala Gly Lys Pro Ala Ala 165 170 175Gln Ile Ser
Trp Ile Pro Glu Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185 190Trp
Ser Asn Gly Thr Val Thr Val Lys Ser Thr Cys His Trp Glu Val 195 200
205His Asn Val Ser Thr Val Thr Cys His Val Ser His Leu Thr Gly Asn
210 215 220Lys Ser Leu Tyr Ile Glu Leu Cys Pro Ser Pro Leu Phe Pro
Gly Pro225 230 235 240Ser Lys Pro Phe Trp Val Leu Val Val Val Gly
Gly Val Leu Ala Cys 245 250 255Tyr Ser Leu Leu Val Thr Val Ala Phe
Ile Ile Phe Trp Val Lys Arg 260 265 270Gly Arg Lys Lys Leu Leu Tyr
Ile Phe Lys Gln Pro Phe Met Arg Pro 275 280 285Val Gln Thr Thr Gln
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 290 295 300Glu Glu Glu
Gly Gly Cys Glu Leu305 31038353PRTArtificial
SequencehuCD200R-12aas-CD28Cys tm ic-41BBic protein 38Met Leu Cys
Pro Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile
Phe Leu Val Ala Ala Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln
Ile Thr Gln Asn Tyr Ser Lys Val Leu Ala Glu Val Asn Thr Ser 35 40
45Trp Pro Val Lys Met Ala Thr Asn Ala Val Leu Cys Cys Pro Pro Ile
50 55 60Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg
Gly65 70 75 80Gln Pro Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn
Glu Thr Lys 85 90 95Glu Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp Val
Ser Arg Pro Asp 100 105 110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val
Ala Ile Thr His Asp Gly 115 120 125Tyr Tyr Arg Cys Ile Met Val Thr
Pro Asp Gly Asn Phe His Arg Gly 130 135 140Tyr His Leu Gln Val Leu
Val Thr Pro Glu Val Thr Leu Phe Gln Asn145 150 155 160Arg Asn Arg
Thr Ala Val Cys Lys Ala Val Ala Gly Lys Pro Ala Ala 165 170 175Gln
Ile Ser Trp Ile Pro Glu Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185
190Trp Ser Asn Gly Thr Val Thr Val Lys Ser Thr Cys His Trp Glu Val
195 200 205His Asn Val Ser Thr Val Thr Cys His Val Ser His Leu Thr
Gly Asn 210 215 220Lys Ser Leu Tyr Ile Glu Leu Cys Pro Ser Pro Leu
Phe Pro Gly Pro225 230 235 240Ser Lys Pro Phe Trp Val Leu Val Val
Val Gly Gly Val Leu Ala Cys 245 250 255Tyr Ser Leu Leu Val Thr Val
Ala Phe Ile Ile Phe Trp Val Arg Ser 260 265 270Lys Arg Ser Arg Gly
Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg 275 280 285Arg Pro Gly
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 290 295 300Asp
Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr305 310
315 320Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
Glu 325 330 335Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
Gly Cys Glu 340 345 350Leu 39435PRTArtificial SequencehuSIRP alpha
tm-CD28 protein 39Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly
Pro Leu Leu Cys1 5 10 15Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly
Val Ala Gly Glu Glu 20 25 30Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
Val Leu Val Ala Ala Gly 35 40 45Glu Thr Ala Thr Leu Arg Cys Thr Ala
Thr Ser Leu Ile Pro Val Gly 50 55 60Pro Ile Gln Trp Phe Arg Gly Ala
Gly Pro Gly Arg Glu Leu Ile Tyr65 70 75 80Asn Gln Lys Glu Gly His
Phe Pro Arg Val Thr Thr Val Ser Asp Leu 85 90 95Thr Lys Arg Asn Asn
Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr 100 105 110Pro Ala Asp
Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 115 120 125Pro
Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 130 135
140Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg
Ala145 150 155 160Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser
His Gly Phe Ser 165 170 175Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys
Asn Gly Asn Glu Leu Ser 180 185 190Asp Phe Gln Thr Asn Val Asp Pro
Val Gly Glu Ser Val Ser Tyr Ser 195 200 205Ile His Ser Thr Ala Lys
Val Val Leu Thr Arg Glu Asp Val His Ser 210 215 220Gln Val Ile Cys
Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu225 230 235 240Arg
Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu 245 250
255Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
260 265 270Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
Trp Leu 275 280 285Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser
Thr Val Thr Glu 290 295 300Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser
Trp Leu Leu Val Asn Val305 310 315 320Ser Ala His Arg Asp Asp Val
Lys Leu Thr Cys Gln Val Glu His Asp 325 330 335Gly Gln Pro Ala Val
Ser Lys Ser His Asp Leu Lys Val Ser Ala His 340 345 350Pro Lys Glu
Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn 355 360 365Glu
Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val 370 375
380Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ser Lys Arg Ser
Arg385 390 395 400Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg
Arg Pro Gly Pro 405 410 415Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
Pro Arg Asp Phe Ala Ala 420 425 430Tyr Arg Ser
43540373PRTArtificial SequencehuSIRP alpha entire extracellular
domain protein 40Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly
Pro Leu Leu Cys1 5 10 15Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly
Val Ala Gly Glu Glu 20 25 30Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
Val Leu Val Ala Ala Gly 35 40 45Glu Thr Ala Thr Leu Arg Cys Thr Ala
Thr Ser Leu Ile Pro Val Gly 50 55 60Pro Ile Gln Trp Phe Arg Gly Ala
Gly Pro Gly Arg Glu Leu Ile Tyr65 70 75 80Asn Gln Lys Glu Gly His
Phe Pro Arg Val Thr Thr Val Ser Asp Leu 85 90 95Thr Lys Arg Asn Asn
Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr 100 105 110Pro Ala Asp
Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 115 120 125Pro
Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 130 135
140Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg
Ala145 150 155 160Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser
His Gly Phe Ser 165 170 175Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys
Asn Gly Asn Glu Leu Ser 180 185 190Asp Phe Gln Thr Asn Val Asp Pro
Val Gly Glu Ser Val Ser Tyr Ser 195 200 205Ile His Ser Thr Ala Lys
Val Val Leu Thr Arg Glu Asp Val His Ser 210 215 220Gln Val Ile Cys
Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu225 230 235 240Arg
Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu 245 250
255Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
260 265 270Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
Trp Leu 275 280 285Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser
Thr Val Thr Glu 290 295 300Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser
Trp Leu Leu Val Asn Val305 310 315 320Ser Ala His Arg Asp Asp Val
Lys Leu Thr Cys Gln Val Glu His Asp 325 330 335Gly Gln Pro Ala Val
Ser Lys Ser His Asp Leu Lys Val Ser Ala His 340 345 350Pro Lys Glu
Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn 355 360 365Glu
Arg Asn Ile Tyr 3704121PRTArtificial SequencehuSIRP alpha
transmembrane domain protein 41Ile Val Val Gly Val Val Cys Thr Leu
Leu Val Ala Leu Leu Met Ala1 5 10 15Ala Leu Tyr Leu Val
2042441PRTArtificial SequencehuSIRP alpha -CD28tm protein 42Met Glu
Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys1 5 10
15Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu
20 25 30Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala
Gly 35 40 45Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
Val Gly 50 55 60Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu
Leu Ile Tyr65 70 75 80Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr
Thr Val Ser Asp Leu 85 90 95Thr Lys Arg Asn Asn Met Asp Phe Ser Ile
Arg Ile Gly Asn Ile Thr 100 105 110Pro Ala Asp Ala Gly Thr Tyr Tyr
Cys Val Lys Phe Arg Lys Gly Ser 115 120 125Pro Asp Asp Val Glu Phe
Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 130 135 140Arg Ala Lys Pro
Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala145 150 155 160Thr
Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser 165 170
175Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
180 185 190Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
Tyr Ser 195 200 205Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu
Asp Val His Ser 210 215 220Gln Val Ile Cys Glu Val Ala His Val Thr
Leu Gln Gly Asp Pro Leu225 230 235 240Arg Gly Thr Ala Asn Leu Ser
Glu Thr Ile Arg Val Pro Pro Thr Leu 245 250 255Glu Val Thr Gln Gln
Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr 260 265 270Cys Gln Val
Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu 275 280 285Glu
Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu 290 295
300Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn
Val305 310 315 320Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln
Val Glu His Asp 325 330 335Gly Gln Pro Ala Val Ser Lys Ser His Asp
Leu Lys Val Ser Ala His 340 345 350Pro Lys Glu Gln Gly Ser Asn Thr
Ala Ala Glu Asn Thr Gly Ser Asn 355 360 365Glu Arg Asn Ile Tyr Phe
Trp Val Leu Val Val Val Gly Gly Val Leu 370 375 380Ala Cys Tyr Ser
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val385 390 395 400Arg
Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 405 410
415Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
420 425 430Pro Arg Asp Phe Ala Ala Tyr Arg Ser 435
44043441PRTArtificial SequencehuSIRP alpha-12aas-CD28Cys protein
43Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys1
5 10 15Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu
Glu 20 25 30Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
Ala Gly 35 40 45Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile
Pro Val Gly 50 55 60Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg
Glu Leu Ile Tyr65 70 75 80Asn Gln Lys Glu Gly His Phe Pro Arg Val
Thr Thr Val Ser Asp Leu 85 90 95Thr Lys Arg Asn Asn Met Asp Phe Ser
Ile Arg Ile Gly Asn Ile Thr 100 105 110Pro Ala Asp Ala Gly Thr Tyr
Tyr Cys Val Lys Phe Arg Lys Gly Ser 115 120 125Pro Asp Asp Val Glu
Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 130 135 140Arg Ala Lys
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala145 150 155
160Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser
165 170 175Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu
Leu Ser 180 185 190Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser
Val Ser Tyr Ser 195 200 205Ile His Ser Thr Ala Lys Val Val Leu Thr
Arg Glu Asp Val His Ser 210 215 220Gln Val Ile Cys Glu Val Ala His
Val Thr Leu Gln Gly Asp Pro Leu225 230 235 240Arg Gly Thr Ala Asn
Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu 245 250 255Glu Val Thr
Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr 260 265 270Cys
Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu 275 280
285Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu
290 295 300Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
Asn Val305 310 315 320Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys
Gln Val Glu His Asp 325 330 335Gly Gln Pro Ala Val Ser Lys Ser His
Asp Leu Lys Val Ser Ala His 340 345 350Pro Lys Glu Gln Gly Ser Asn
Thr Ala Cys Pro Ser Pro Leu Phe Pro 355 360 365Gly Pro Ser Lys Pro
Phe Trp Val Leu Val Val Val Gly Gly Val Leu 370 375 380Ala Cys Tyr
Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val385 390 395
400Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
405 410 415Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
Ala Pro 420 425 430Pro Arg Asp Phe Ala Ala Tyr Arg Ser 435
44044361PRTArtificial SequencehuSIRP alpha-12aas protein 44Met Glu
Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys1 5 10 15Leu
Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu 20 25
30Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly
35 40 45Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val
Gly 50 55 60Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
Ile Tyr65 70 75 80Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr
Val Ser Asp Leu 85 90 95Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg
Ile Gly Asn Ile Thr 100 105 110Pro Ala Asp Ala Gly Thr Tyr Tyr Cys
Val Lys Phe Arg Lys Gly Ser 115 120 125Pro Asp Asp Val Glu Phe Lys
Ser Gly Ala Gly Thr Glu Leu Ser Val 130 135 140Arg Ala Lys Pro Ser
Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala145 150 155 160Thr Pro
Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser 165 170
175Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser
180 185 190Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser
Tyr Ser 195 200 205Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu
Asp Val His Ser 210 215 220Gln Val Ile Cys Glu Val Ala His Val Thr
Leu Gln Gly Asp Pro Leu225 230 235 240Arg Gly Thr Ala Asn Leu Ser
Glu Thr Ile Arg Val Pro Pro Thr Leu 245 250 255Glu Val Thr Gln Gln
Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr 260 265 270Cys Gln Val
Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu 275 280 285Glu
Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu 290 295
300Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn
Val305 310 315 320Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln
Val Glu His Asp 325 330 335Gly Gln Pro Ala Val Ser Lys Ser His Asp
Leu Lys Val Ser Ala His 340 345 350Pro Lys Glu Gln Gly Ser Asn Thr
Ala 355 36045442PRTArtificial SequencehuSIRP alpha-12aas-CD28Cys
tm-41BBic protein 45Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly
Pro Leu Leu Cys1 5 10 15Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly
Val Ala Gly Glu Glu 20 25 30Glu Leu Gln Val Ile Gln Pro Asp Lys Ser
Val Leu Val Ala Ala Gly 35 40 45Glu Thr Ala Thr Leu Arg Cys Thr Ala
Thr Ser Leu Ile Pro Val Gly 50 55 60Pro Ile Gln Trp Phe Arg Gly Ala
Gly Pro Gly Arg Glu Leu Ile Tyr65 70 75 80Asn Gln Lys Glu Gly His
Phe Pro Arg Val Thr Thr Val Ser Asp Leu 85 90 95Thr Lys Arg Asn Asn
Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr 100 105 110Pro Ala Asp
Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 115 120 125Pro
Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 130 135
140Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg
Ala145 150 155 160Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser
His Gly Phe Ser 165 170 175Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys
Asn Gly Asn Glu Leu Ser 180 185 190Asp Phe Gln Thr Asn Val Asp Pro
Val Gly Glu Ser Val Ser Tyr Ser 195 200 205Ile His Ser Thr Ala Lys
Val Val Leu Thr Arg Glu Asp Val His Ser 210 215 220Gln Val Ile Cys
Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu225 230 235 240Arg
Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu 245 250
255Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr
260 265 270Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
Trp Leu 275 280 285Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser
Thr Val Thr Glu 290 295 300Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser
Trp Leu Leu Val Asn Val305 310 315 320Ser Ala His Arg Asp Asp Val
Lys Leu Thr Cys Gln Val Glu His Asp 325 330 335Gly Gln Pro Ala Val
Ser Lys Ser His Asp Leu Lys Val Ser Ala His 340 345 350Pro Lys Glu
Gln Gly Ser Asn Thr Ala Cys Pro Ser Pro Leu Phe Pro 355 360 365Gly
Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu 370 375
380Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
Val385 390 395 400Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
Gln Pro Phe Met 405 410 415Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
Gly Cys Ser Cys Arg Phe 420 425 430Pro Glu Glu Glu Glu Gly Gly Cys
Glu Leu 435 44046483PRTArtificial SequencehuSIRP
alpha-12aas-CD28Cys tm ic-41BBic protein 46Met Glu Pro Ala Gly Pro
Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys1 5 10 15Leu Leu Leu Ala Ala
Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu 20 25 30Glu Leu Gln Val
Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 35 40 45Glu Thr Ala
Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly 50 55 60Pro Ile
Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr65 70 75
80Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu
85 90 95Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile
Thr 100 105 110Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg
Lys Gly Ser 115 120 125Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly
Thr Glu Leu Ser Val 130 135 140Arg Ala Lys Pro Ser Ala Pro Val Val
Ser Gly Pro Ala Ala Arg Ala145 150 155 160Thr Pro Gln His Thr Val
Ser Phe Thr Cys Glu Ser His Gly Phe Ser 165 170 175Pro Arg Asp Ile
Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser 180 185 190Asp Phe
Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser 195 200
205Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser
210 215 220Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp
Pro Leu225 230 235 240Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg
Val Pro Pro Thr Leu 245 250 255Glu Val Thr Gln Gln Pro Val Arg Ala
Glu Asn Gln Val Asn Val Thr 260 265 270Cys Gln Val Arg Lys Phe Tyr
Pro Gln Arg Leu Gln Leu Thr Trp Leu 275 280 285Glu Asn Gly Asn Val
Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu 290 295 300Asn Lys Asp
Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val305 310 315
320Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp
325 330 335Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser
Ala His 340 345 350Pro Lys Glu Gln Gly Ser Asn Thr Ala Cys Pro Ser
Pro Leu Phe Pro 355 360 365Gly Pro Ser Lys Pro Phe Trp Val Leu Val
Val Val Gly Gly Val Leu 370 375 380Ala Cys Tyr Ser Leu Leu Val Thr
Val Ala Phe Ile Ile Phe Trp Val385 390 395 400Arg Ser Lys Arg Ser
Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 405 410 415Pro Arg Arg
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 420 425 430Pro
Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu 435 440
445Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
450 455 460Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
Gly Gly465 470 475 480Cys Glu Leu47900DNAArtificial
SequencemuCD200Rtm-CD28 47atgttctgct tctggcggac aagcgccctg
gccgtgctgc tgatctgggg agtgtttgtg 60gccggcagca gctgcaccga caagaaccag
accacccaga acaacagcag cagccccctg 120acccaagtga acaccaccgt
gtccgtgcag atcggcacca aggccctgct gtgctgtttc 180agcatccctc
tgaccaaggc tgtgctgatc acctggatca tcaagctgag aggcctgccc
240agctgcacaa tcgcctacaa ggtggacacc aagaccaacg agacaagctg
cctgggcaga 300aacatcacct gggccagcac cccagaccac agccctgagc
tgcagatcag cgccgtgaca 360ctgcagcacg agggcaccta cacatgcgag
acagtgaccc ccgagggcaa cttcgagaag 420aactacgatc tgcaggtgct
ggtgcccccc gaagtgacct acttccccga gaagaataga 480agcgccgtgt
gcgaggccat ggctggcaaa cctgccgccc agatctcttg gagccctgac
540ggcgactgtg tgaccaccag cgagagccac agcaacggca cagtgaccgt
gcggagcacc 600tgtcactggg agcagaacaa cgtgtccgac gtgtcctgca
tcgtgtccca cctgaccggc 660aaccagagcc tgagcatcga gctgagcaga
ggcggaaacc agtccctgag gccctacatc 720ccttacatca tccccagcat
catcatcctg atcatcatcg gctgcatctg cctgctgaac 780agcagaagaa
acagaggcgg ccagagcgac tacatgaaca tgacccccag aaggcctggc
840ctgaccagaa agccctacca gccttacgcc cctgccagag acttcgccgc
ctacagacct 90048918DNAArtificial SequencemuCD200R-CD28tm
48atgttctgct tctggcggac aagcgccctg gccgtgctgc tgatctgggg agtgtttgtg
60gccggcagca gctgcaccga caagaaccag accacccaga acaacagcag cagccccctg
120acccaagtga acaccaccgt gtccgtgcag atcggcacca aggccctgct
gtgctgtttc 180agcatccctc tgaccaaggc tgtgctgatc acctggatca
tcaagctgag aggcctgccc 240agctgcacaa tcgcctacaa ggtggacacc
aagaccaacg agacaagctg cctgggcaga 300aacatcacct gggccagcac
cccagaccac agccctgagc tgcagatcag cgccgtgaca 360ctgcagcacg
agggcaccta cacatgcgag acagtgaccc ccgagggcaa cttcgagaag
420aactacgatc tgcaggtgct ggtgcccccc gaagtgacct acttccccga
gaagaataga 480agcgccgtgt gcgaggccat ggctggcaaa cctgccgccc
agatctcttg gagccctgac 540ggcgactgtg tgaccaccag cgagagccac
agcaacggca cagtgaccgt gcggagcacc 600tgtcactggg agcagaacaa
cgtgtccgac gtgtcctgca tcgtgtccca cctgaccggc 660aaccagagcc
tgagcatcga gctgagcaga ggcggaaacc agtccctgag gcccttctgg
720gccctggtgg tggtggccgg cgtgctgttt tgttacggcc
tgctcgtgac cgtggccctg 780tgcgtgatct ggaccaacag cagaagaaac
agaggcggcc agagcgacta catgaacatg 840acccccagaa ggcctggcct
gaccagaaag ccctaccagc cttacgcccc tgccagagac 900ttcgccgcct acagacct
91849945DNAArtificial SequencemuCD200R-CD28Cys 49atgttctgct
tctggcggac aagcgccctg gccgtgctgc tgatctgggg agtgtttgtg 60gccggcagca
gctgcaccga caagaaccag accacccaga acaacagcag cagccccctg
120acccaagtga acaccaccgt gtccgtgcag atcggcacca aggccctgct
gtgctgtttc 180agcatccctc tgaccaaggc tgtgctgatc acctggatca
tcaagctgag aggcctgccc 240agctgcacaa tcgcctacaa ggtggacacc
aagaccaacg agacaagctg cctgggcaga 300aacatcacct gggccagcac
cccagaccac agccctgagc tgcagatcag cgccgtgaca 360ctgcagcacg
agggcaccta cacatgcgag acagtgaccc ccgagggcaa cttcgagaag
420aactacgatc tgcaggtgct ggtgcccccc gaagtgacct acttccccga
gaagaataga 480agcgccgtgt gcgaggccat ggctggcaaa cctgccgccc
agatctcttg gagccctgac 540ggcgactgtg tgaccaccag cgagagccac
agcaacggca cagtgaccgt gcggagcacc 600tgtcactggg agcagaacaa
cgtgtccgac gtgtcctgca tcgtgtccca cctgaccggc 660aaccagagcc
tgagcatcga gctgagcaga ggcggaaacc agtccctgag gccctgccac
720acccagagca gccccaagct gttctgggcc ctggtggtgg tggccggcgt
gctgttttgt 780tacggcctgc tcgtgaccgt ggccctgtgc gtgatctgga
ccaacagcag aagaaacaga 840ggcggccaga gcgactacat gaacatgacc
cccagaaggc ctggcctgac cagaaagccc 900taccagcctt acgcccctgc
cagagacttc gccgcctaca gacct 94550936DNAArtificial
SequencemuCD200R-3aas-CD28Cys 50atgttctgct tctggcggac aagcgccctg
gccgtgctgc tgatctgggg agtgtttgtg 60gccggcagca gctgcaccga caagaaccag
accacccaga acaacagcag cagccccctg 120acccaagtga acaccaccgt
gtccgtgcag atcggcacca aggccctgct gtgctgtttc 180agcatccctc
tgaccaaggc tgtgctgatc acctggatca tcaagctgag aggcctgccc
240agctgcacaa tcgcctacaa ggtggacacc aagaccaacg agacaagctg
cctgggcaga 300aacatcacct gggccagcac cccagaccac agccctgagc
tgcagatcag cgccgtgaca 360ctgcagcacg agggcaccta cacatgcgag
acagtgaccc ccgagggcaa cttcgagaag 420aactacgatc tgcaggtgct
ggtgcccccc gaagtgacct acttccccga gaagaataga 480agcgccgtgt
gcgaggccat ggctggcaaa cctgccgccc agatctcttg gagccctgac
540ggcgactgtg tgaccaccag cgagagccac agcaacggca cagtgaccgt
gcggagcacc 600tgtcactggg agcagaacaa cgtgtccgac gtgtcctgca
tcgtgtccca cctgaccggc 660aaccagagcc tgagcatcga gctgagcaga
ggcggaaacc agtcctgcca cacccagagc 720agccccaagc tgttctgggc
cctggtggtg gtggccggcg tgctgttttg ttacggcctg 780ctcgtgaccg
tggccctgtg cgtgatctgg accaacagca gaagaaacag aggcggccag
840agcgactaca tgaacatgac ccccagaagg cctggcctga ccagaaagcc
ctaccagcct 900tacgcccctg ccagagactt cgccgcctac agacct
93651918DNAArtificial SequencemuCD200R-9aas-CD28Cys 51atgttctgct
tctggcggac aagcgccctg gccgtgctgc tgatctgggg agtgtttgtg 60gccggcagca
gctgcaccga caagaaccag accacccaga acaacagcag cagccccctg
120acccaagtga acaccaccgt gtccgtgcag atcggcacca aggccctgct
gtgctgtttc 180agcatccctc tgaccaaggc tgtgctgatc acctggatca
tcaagctgag aggcctgccc 240agctgcacaa tcgcctacaa ggtggacacc
aagaccaacg agacaagctg cctgggcaga 300aacatcacct gggccagcac
cccagaccac agccctgagc tgcagatcag cgccgtgaca 360ctgcagcacg
agggcaccta cacatgcgag acagtgaccc ccgagggcaa cttcgagaag
420aactacgatc tgcaggtgct ggtgcccccc gaagtgacct acttccccga
gaagaataga 480agcgccgtgt gcgaggccat ggctggcaaa cctgccgccc
agatctcttg gagccctgac 540ggcgactgtg tgaccaccag cgagagccac
agcaacggca cagtgaccgt gcggagcacc 600tgtcactggg agcagaacaa
cgtgtccgac gtgtcctgca tcgtgtccca cctgaccggc 660aaccagagcc
tgagcatcga gctgagctgc cacacccaga gcagccccaa gctgttctgg
720gccctggtgg tggtggccgg cgtgctgttt tgttacggcc tgctcgtgac
cgtggccctg 780tgcgtgatct ggaccaacag cagaagaaac agaggcggcc
agagcgacta catgaacatg 840acccccagaa ggcctggcct gaccagaaag
ccctaccagc cttacgcccc tgccagagac 900ttcgccgcct acagacct
91852939DNAArtificial SequencemuCD200R-9aas-CD28Cys tm-41BBic
52atgttctgct tctggcggac aagcgccctg gccgtgctgc tgatctgggg agtgtttgtg
60gccggcagca gctgcaccga caagaaccag accacccaga acaacagcag cagccccctg
120acccaagtga acaccaccgt gtccgtgcag atcggcacca aggccctgct
gtgctgtttc 180agcatccctc tgaccaaggc tgtgctgatc acctggatca
tcaagctgag aggcctgccc 240agctgcacaa tcgcctacaa ggtggacacc
aagaccaacg agacaagctg cctgggcaga 300aacatcacct gggccagcac
cccagaccac agccctgagc tgcagatcag cgccgtgaca 360ctgcagcacg
agggcaccta cacatgcgag acagtgaccc ccgagggcaa cttcgagaag
420aactacgatc tgcaggtgct ggtgcccccc gaagtgacct acttccccga
gaagaataga 480agcgccgtgt gcgaggccat ggctggcaaa cctgccgccc
agatctcttg gagccctgac 540ggcgactgtg tgaccaccag cgagagccac
agcaacggca cagtgaccgt gcggagcacc 600tgtcactggg agcagaacaa
cgtgtccgac gtgtcctgca tcgtgtccca cctgaccggc 660aaccagagcc
tgagcatcga gctgagctgc cacacccaga gcagccccaa gctgttctgg
720gccctggtgg tggtggccgg cgtgctgttt tgttacggcc tgctcgtgac
cgtggccctg 780tgcgtgatct ggaccagcgt gctgaagtgg atcagaaaga
agttccccca catcttcaag 840cagcccttca agaaaaccac cggcgctgcc
caggaagagg acgcctgcag ctgtagatgc 900cctcaggaag aagaaggcgg
cggaggcggc tacgagctg 939531062DNAArtificial
SequencemuCD200R-9aas-CD28Cys tm ic-41BBic 53atgttctgct tctggcggac
aagcgccctg gccgtgctgc tgatctgggg agtgtttgtg 60gccggcagca gctgcaccga
caagaaccag accacccaga acaacagcag cagccccctg 120acccaagtga
acaccaccgt gtccgtgcag atcggcacca aggccctgct gtgctgtttc
180agcatccctc tgaccaaggc tgtgctgatc acctggatca tcaagctgag
aggcctgccc 240agctgcacaa tcgcctacaa ggtggacacc aagaccaacg
agacaagctg cctgggcaga 300aacatcacct gggccagcac cccagaccac
agccctgagc tgcagatcag cgccgtgaca 360ctgcagcacg agggcaccta
cacatgcgag acagtgaccc ccgagggcaa cttcgagaag 420aactacgatc
tgcaggtgct ggtgcccccc gaagtgacct acttccccga gaagaataga
480agcgccgtgt gcgaggccat ggctggcaaa cctgccgccc agatctcttg
gagccctgac 540ggcgactgtg tgaccaccag cgagagccac agcaacggca
cagtgaccgt gcggagcacc 600tgtcactggg agcagaacaa cgtgtccgac
gtgtcctgca tcgtgtccca cctgaccggc 660aaccagagcc tgagcatcga
gctgagctgc cacacccaga gcagccccaa gctgttctgg 720gccctggtgg
tggtggccgg cgtgctgttt tgttacggcc tgctcgtgac cgtggccctg
780tgcgtgatct ggaccaacag cagaagaaac agaggcggcc agagcgacta
catgaacatg 840acccccagaa ggcctggcct gaccagaaag ccctaccagc
cttacgcccc tgccagagac 900ttcgccgcct acagacctag cgtgctgaag
tggatcagaa agaagttccc ccacatcttc 960aagcagccct tcaagaaaac
caccggcgct gcccaggaag aggacgcctg cagctgtaga 1020tgccctcagg
aagaagaagg cggcggaggc ggctacgagc tg 1062541305DNAArtificial
SequencemuSIRP alpha tm-CD28 54atggaacctg ctggacctgc ccctggcaga
ctgggacctc tgctgctgtg cctgctgctg 60agcgccagct gtttctgtac cggcgccacc
ggcaaagaac tgaaagtgac ccagcccgag 120aagtccgtgt ctgtggccgc
tggcgacagc accgtgctga actgtaccct gaccagcctg 180ctgcccgtgg
gccccatcag atggtataga ggcgtgggcc ctagcagact gctgatctac
240agcttcgctg gcgagtacgt gcccagaatc agaaacgtgt ccgacaccac
caagcggaac 300aacatggact tcagcatcag gatcagcaac gtgacccctg
ccgacgccgg catctactac 360tgcgtgaagt tccagaaggg cagcagcgag
cccgacaccg agattcagtc tggcggcgga 420accgaggtgt acgtgctggc
taagcccagc cctcctgagg tgtccggccc tgctgataga 480ggcatccccg
accagaaagt gaacttcaca tgcaagagcc acggcttcag ccccagaaac
540atcaccctga agtggttcaa ggacggccag gaactgcacc ccctggaaac
caccgtgaac 600cccagcggca agaacgtgtc ctacaacatc agctccaccg
tgcgggtggt gctgaacagc 660atggacgtga acagcaaagt gatctgcgag
gtggcccaca tcacactgga cagaagcccc 720ctgagaggaa tcgccaacct
gagcaacttc atcagagtgt ccccaaccgt gaaagtgaca 780cagcagagcc
ccaccagcat gaaccaagtg aacctgacct gcagagccga gagattctac
840cccgaggacc tgcagctgat ctggctggaa aacggcaacg tgtccagaaa
cgacaccccc 900aagaacctga caaagaacac cgacggcacc tacaactaca
cctccctgtt tctcgtgaac 960tcctccgccc accgcgagga cgtggtgttc
acgtgccaag tgaagcacga ccagcagccc 1020gccatcacca gaaaccacac
agtgctgggc ttcgcccaca gcagcgacca gggcagcatg 1080cagaccttcc
ccgacaacaa cgccacccac aactggaacg tgttcatcgg cgtgggcgtg
1140gcctgtgctc tgctggtggt gctgctgatg gccgccctgt ataacagcag
aagaaacaga 1200ggcggccaga gcgactacat gaacatgacc cccagaaggc
ctggcctgac cagaaagccc 1260taccagcctt acgcccctgc cagagacttc
gccgcctaca gacct 1305551323DNAArtificial SequencemuSIRP
alpha-CD28tm 55atggaacctg ctggacctgc ccctggcaga ctgggacctc
tgctgctgtg cctgctgctg 60agcgccagct gtttctgtac cggcgccacc ggcaaagaac
tgaaagtgac ccagcccgag 120aagtccgtgt ctgtggccgc tggcgacagc
accgtgctga actgtaccct gaccagcctg 180ctgcccgtgg gccccatcag
atggtataga ggcgtgggcc ctagcagact gctgatctac 240agcttcgctg
gcgagtacgt gcccagaatc agaaacgtgt ccgacaccac caagcggaac
300aacatggact tcagcatcag gatcagcaac gtgacccctg ccgacgccgg
catctactac 360tgcgtgaagt tccagaaggg cagcagcgag cccgacaccg
agattcagtc tggcggcgga 420accgaggtgt acgtgctggc taagcccagc
cctcctgagg tgtccggccc tgctgataga 480ggcatccccg accagaaagt
gaacttcaca tgcaagagcc acggcttcag ccccagaaac 540atcaccctga
agtggttcaa ggacggccag gaactgcacc ccctggaaac caccgtgaac
600cccagcggca agaacgtgtc ctacaacatc agctccaccg tgcgggtggt
gctgaacagc 660atggacgtga acagcaaagt gatctgcgag gtggcccaca
tcacactgga cagaagcccc 720ctgagaggaa tcgccaacct gagcaacttc
atcagagtgt ccccaaccgt gaaagtgaca 780cagcagagcc ccaccagcat
gaaccaagtg aacctgacct gcagagccga gagattctac 840cccgaggacc
tgcagctgat ctggctggaa aacggcaacg tgtccagaaa cgacaccccc
900aagaacctga caaagaacac cgacggcacc tacaactaca cctccctgtt
tctcgtgaac 960tcctccgccc accgcgagga cgtggtgttc acgtgccaag
tgaagcacga ccagcagccc 1020gccatcacca gaaaccacac agtgctgggc
ttcgcccaca gcagcgacca gggcagcatg 1080cagaccttcc ccgacaacaa
cgccacccac aactggaact tctgggccct ggtggtggtg 1140gccggcgtgc
tgttttgtta cggcctgctc gtgaccgtgg ccctgtgcgt gatctggacc
1200aacagcagaa gaaacagagg cggccagagc gactacatga acatgacccc
cagaaggcct 1260ggcctgacca gaaagcccta ccagccttac gcccctgcca
gagacttcgc cgcctacaga 1320cct 1323561350DNAArtificial
SequencemuSIRP alpha-CD28cys 56atggaacctg ctggacctgc ccctggcaga
ctgggacctc tgctgctgtg cctgctgctg 60agcgccagct gtttctgtac cggcgccacc
ggcaaagaac tgaaagtgac ccagcccgag 120aagtccgtgt ctgtggccgc
tggcgacagc accgtgctga actgtaccct gaccagcctg 180ctgcccgtgg
gccccatcag atggtataga ggcgtgggcc ctagcagact gctgatctac
240agcttcgctg gcgagtacgt gcccagaatc agaaacgtgt ccgacaccac
caagcggaac 300aacatggact tcagcatcag gatcagcaac gtgacccctg
ccgacgccgg catctactac 360tgcgtgaagt tccagaaggg cagcagcgag
cccgacaccg agattcagtc tggcggcgga 420accgaggtgt acgtgctggc
taagcccagc cctcctgagg tgtccggccc tgctgataga 480ggcatccccg
accagaaagt gaacttcaca tgcaagagcc acggcttcag ccccagaaac
540atcaccctga agtggttcaa ggacggccag gaactgcacc ccctggaaac
caccgtgaac 600cccagcggca agaacgtgtc ctacaacatc agctccaccg
tgcgggtggt gctgaacagc 660atggacgtga acagcaaagt gatctgcgag
gtggcccaca tcacactgga cagaagcccc 720ctgagaggaa tcgccaacct
gagcaacttc atcagagtgt ccccaaccgt gaaagtgaca 780cagcagagcc
ccaccagcat gaaccaagtg aacctgacct gcagagccga gagattctac
840cccgaggacc tgcagctgat ctggctggaa aacggcaacg tgtccagaaa
cgacaccccc 900aagaacctga caaagaacac cgacggcacc tacaactaca
cctccctgtt tctcgtgaac 960tcctccgccc accgcgagga cgtggtgttc
acgtgccaag tgaagcacga ccagcagccc 1020gccatcacca gaaaccacac
agtgctgggc ttcgcccaca gcagcgacca gggcagcatg 1080cagaccttcc
ccgacaacaa cgccacccac aactggaact gccacaccca gagcagcccc
1140aagctgttct gggctctggt ggtggtggcc ggcgtgctgt tttgttacgg
cctgctcgtg 1200accgtggccc tgtgcgtgat ctggaccaac agcagaagaa
acagaggcgg ccagagcgac 1260tacatgaaca tgacccccag aaggcctggc
ctgacccgga agccttacca gccttacgcc 1320cctgccagag acttcgccgc
ctacagacct 1350571332DNAArtificial SequencemuSIRP alpha
-6aas-CD28cys 57atggaacctg ctggacctgc ccctggcaga ctgggacctc
tgctgctgtg cctgctgctg 60agcgccagct gtttctgtac cggcgccacc ggcaaagaac
tgaaagtgac ccagcccgag 120aagtccgtgt ctgtggccgc tggcgacagc
accgtgctga actgtaccct gaccagcctg 180ctgcccgtgg gccccatcag
atggtataga ggcgtgggcc ctagcagact gctgatctac 240agcttcgctg
gcgagtacgt gcccagaatc agaaacgtgt ccgacaccac caagcggaac
300aacatggact tcagcatcag gatcagcaac gtgacccctg ccgacgccgg
catctactac 360tgcgtgaagt tccagaaggg cagcagcgag cccgacaccg
agattcagtc tggcggcgga 420accgaggtgt acgtgctggc taagcccagc
cctcctgagg tgtccggccc tgctgataga 480ggcatccccg accagaaagt
gaacttcaca tgcaagagcc acggcttcag ccccagaaac 540atcaccctga
agtggttcaa ggacggccag gaactgcacc ccctggaaac caccgtgaac
600cccagcggca agaacgtgtc ctacaacatc agctccaccg tgcgggtggt
gctgaacagc 660atggacgtga acagcaaagt gatctgcgag gtggcccaca
tcacactgga cagaagcccc 720ctgagaggaa tcgccaacct gagcaacttc
atcagagtgt ccccaaccgt gaaagtgaca 780cagcagagcc ccaccagcat
gaaccaagtg aacctgacct gcagagccga gagattctac 840cccgaggacc
tgcagctgat ctggctggaa aacggcaacg tgtccagaaa cgacaccccc
900aagaacctga caaagaacac cgacggcacc tacaactaca cctccctgtt
tctcgtgaac 960tcctccgccc accgcgagga cgtggtgttc acgtgccaag
tgaagcacga ccagcagccc 1020gccatcacca gaaaccacac agtgctgggc
ttcgcccaca gcagcgacca gggcagcatg 1080cagaccttcc ccgacaacaa
ctgccacacc cagagcagcc ccaagctgtt ctgggctctg 1140gtggtggtgg
ccggcgtgct gttttgttac ggcctgctcg tgaccgtggc cctgtgcgtg
1200atctggacca acagcagaag aaacagaggc ggccagagcg actacatgaa
catgaccccc 1260agaaggcctg gcctgacccg gaagccttac cagccttacg
cccctgccag agacttcgcc 1320gcctacagac ct 1332581323DNAArtificial
SequencemuSIRP alpha -9aas-CD28cys 58atggaacctg ctggacctgc
ccctggcaga ctgggacctc tgctgctgtg cctgctgctg 60agcgccagct gtttctgtac
cggcgccacc ggcaaagaac tgaaagtgac ccagcccgag 120aagtccgtgt
ctgtggccgc tggcgacagc accgtgctga actgtaccct gaccagcctg
180ctgcccgtgg gccccatcag atggtataga ggcgtgggcc ctagcagact
gctgatctac 240agcttcgctg gcgagtacgt gcccagaatc agaaacgtgt
ccgacaccac caagcggaac 300aacatggact tcagcatcag gatcagcaac
gtgacccctg ccgacgccgg catctactac 360tgcgtgaagt tccagaaggg
cagcagcgag cccgacaccg agattcagtc tggcggcgga 420accgaggtgt
acgtgctggc taagcccagc cctcctgagg tgtccggccc tgctgataga
480ggcatccccg accagaaagt gaacttcaca tgcaagagcc acggcttcag
ccccagaaac 540atcaccctga agtggttcaa ggacggccag gaactgcacc
ccctggaaac caccgtgaac 600cccagcggca agaacgtgtc ctacaacatc
agctccaccg tgcgggtggt gctgaacagc 660atggacgtga acagcaaagt
gatctgcgag gtggcccaca tcacactgga cagaagcccc 720ctgagaggaa
tcgccaacct gagcaacttc atcagagtgt ccccaaccgt gaaagtgaca
780cagcagagcc ccaccagcat gaaccaagtg aacctgacct gcagagccga
gagattctac 840cccgaggacc tgcagctgat ctggctggaa aacggcaacg
tgtccagaaa cgacaccccc 900aagaacctga caaagaacac cgacggcacc
tacaactaca cctccctgtt tctcgtgaac 960tcctccgccc accgcgagga
cgtggtgttc acgtgccaag tgaagcacga ccagcagccc 1020gccatcacca
gaaaccacac agtgctgggc ttcgcccaca gcagcgacca gggcagcatg
1080cagaccttcc cctgccacac ccagagcagc cccaagctgt tctgggctct
ggtggtggtg 1140gccggcgtgc tgttttgtta cggcctgctc gtgaccgtgg
ccctgtgcgt gatctggacc 1200aacagcagaa gaaacagagg cggccagagc
gactacatga acatgacccc cagaaggcct 1260ggcctgaccc ggaagcctta
ccagccttac gcccctgcca gagacttcgc cgcctacaga 1320cct
1323591281DNAArtificial SequencemuSIRP alpha-23aas-CD28cys
59atggaacctg ctggacctgc ccctggcaga ctgggacctc tgctgctgtg cctgctgctg
60agcgccagct gtttctgtac cggcgccacc ggcaaagaac tgaaagtgac ccagcccgag
120aagtccgtgt ctgtggccgc tggcgacagc accgtgctga actgtaccct
gaccagcctg 180ctgcccgtgg gccccatcag atggtataga ggcgtgggcc
ctagcagact gctgatctac 240agcttcgctg gcgagtacgt gcccagaatc
agaaacgtgt ccgacaccac caagcggaac 300aacatggact tcagcatcag
gatcagcaac gtgacccctg ccgacgccgg catctactac 360tgcgtgaagt
tccagaaggg cagcagcgag cccgacaccg agattcagtc tggcggcgga
420accgaggtgt acgtgctggc taagcccagc cctcctgagg tgtccggccc
tgctgataga 480ggcatccccg accagaaagt gaacttcaca tgcaagagcc
acggcttcag ccccagaaac 540atcaccctga agtggttcaa ggacggccag
gaactgcacc ccctggaaac caccgtgaac 600cccagcggca agaacgtgtc
ctacaacatc agctccaccg tgcgggtggt gctgaacagc 660atggacgtga
acagcaaagt gatctgcgag gtggcccaca tcacactgga cagaagcccc
720ctgagaggaa tcgccaacct gagcaacttc atcagagtgt ccccaaccgt
gaaagtgaca 780cagcagagcc ccaccagcat gaaccaagtg aacctgacct
gcagagccga gagattctac 840cccgaggacc tgcagctgat ctggctggaa
aacggcaacg tgtccagaaa cgacaccccc 900aagaacctga caaagaacac
cgacggcacc tacaactaca cctccctgtt tctcgtgaac 960tcctccgccc
accgcgagga cgtggtgttc acgtgccaag tgaagcacga ccagcagccc
1020gccatcacca gaaaccacac agtgctgggc tgccacaccc agagcagccc
caagctgttc 1080tgggctctgg tggtggtggc cggcgtgctg ttttgttacg
gcctgctcgt gaccgtggcc 1140ctgtgcgtga tctggaccaa cagcagaaga
aacagaggcg gccagagcga ctacatgaac 1200atgaccccca gaaggcctgg
cctgacccgg aagccttacc agccttacgc ccctgccaga 1260gacttcgccg
cctacagacc t 128160137PRTArtificial SequencehuPD-1 ectodomain 60Pro
Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn1 5 10
15Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu
20 25 30Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
Ala 35 40 45Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe
Arg Val 50 55 60Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val
Val Arg Ala65 70 75 80Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly
Ala Ile Ser Leu Ala 85 90 95Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val Thr 100 105 110Glu Arg Arg Ala Glu Val Pro Thr
Ala His Pro Ser Pro Ser Pro Arg 115 120 125Ser Ala Gly Gln Phe Gln
Thr Leu Val 130 13561627DNAArtificial SequencehuCD2 entire
extracellular domain 61atgagctttc catgtaaatt tgtagccagc ttccttctga
ttttcaatgt ttcttccaaa 60ggtgcagtct ccaaagagat tacgaatgcc ttggaaacct
ggggtgcctt gggtcaggac 120atcaacttgg acattcctag ttttcaaatg
agtgatgata ttgacgatat aaaatgggaa 180aaaacttcag acaagaaaaa
gattgcacaa ttcagaaaag
agaaagagac tttcaaggaa 240aaagatacat ataagctatt taaaaatgga
actctgaaaa ttaagcatct gaagaccgat 300gatcaggata tctacaaggt
atcaatatat gatacaaaag gaaaaaatgt gttggaaaaa 360atatttgatt
tgaagattca agagagggtc tcaaaaccaa agatctcctg gacttgtatc
420aacacaaccc tgacctgtga ggtaatgaat ggaactgacc ccgaattaaa
cctgtatcaa 480gatgggaaac atctaaaact ttctcagagg gtcatcacac
acaagtggac caccagcctg 540agtgcaaaat tcaagtgcac agcagggaac
aaagtcagca aggaatccag tgtcgagcct 600gtcagctgtc cagagaaagg tctggac
62762209PRTArtificial SequencehuCD2 entire extracellular domain
62Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn1
5 10 15Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu
Glu 20 25 30Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro
Ser Phe 35 40 45Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys
Thr Ser Asp 50 55 60Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu
Thr Phe Lys Glu65 70 75 80Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly
Thr Leu Lys Ile Lys His 85 90 95Leu Lys Thr Asp Asp Gln Asp Ile Tyr
Lys Val Ser Ile Tyr Asp Thr 100 105 110Lys Gly Lys Asn Val Leu Glu
Lys Ile Phe Asp Leu Lys Ile Gln Glu 115 120 125Arg Val Ser Lys Pro
Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu 130 135 140Thr Cys Glu
Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln145 150 155
160Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp
165 170 175Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn
Lys Val 180 185 190Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro
Glu Lys Gly Leu 195 200 205Asp 6378DNAArtificial SequencehuCD2
transmembrane domain 63atctatctca tcattggcat atgtggagga ggcagcctct
tgatggtctt tgtggcactg 60ctcgttttct atatcacc 786426PRTArtificial
SequencehuCD2 transmembrane domain 64Ile Tyr Leu Ile Ile Gly Ile
Cys Gly Gly Gly Ser Leu Leu Met Val1 5 10 15Phe Val Ala Leu Leu Val
Phe Tyr Ile Thr 20 2565828DNAArtificial SequencehuCD2tm-CD28 DNA
65atgagctttc catgtaaatt tgtagccagc ttccttctga ttttcaatgt ttcttccaaa
60ggtgcagtct ccaaagagat tacgaatgcc ttggaaacct ggggtgcctt gggtcaggac
120atcaacttgg acattcctag ttttcaaatg agtgatgata ttgacgatat
aaaatgggaa 180aaaacttcag acaagaaaaa gattgcacaa ttcagaaaag
agaaagagac tttcaaggaa 240aaagatacat ataagctatt taaaaatgga
actctgaaaa ttaagcatct gaagaccgat 300gatcaggata tctacaaggt
atcaatatat gatacaaaag gaaaaaatgt gttggaaaaa 360atatttgatt
tgaagattca agagagggtc tcaaaaccaa agatctcctg gacttgtatc
420aacacaaccc tgacctgtga ggtaatgaat ggaactgacc ccgaattaaa
cctgtatcaa 480gatgggaaac atctaaaact ttctcagagg gtcatcacac
acaagtggac caccagcctg 540agtgcaaaat tcaagtgcac agcagggaac
aaagtcagca aggaatccag tgtcgagcct 600gtcagctgtc cagagaaagg
tctggacatc tatctcatca ttggcatatg tggaggaggc 660agcctcttga
tggtctttgt ggcactgctc gttttctata tcacccgcag caagcggagc
720agaggcggcc acagcgacta catgaacatg acccctagac ggcctggccc
caccagaaag 780cactaccagc cctacgcccc tccccgggac tttgccgcct acagaagc
82866276PRTArtificial SequencehuCD2tm-CD28 66Met Ser Phe Pro Cys
Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn1 5 10 15Val Ser Ser Lys
Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu 20 25 30Thr Trp Gly
Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe 35 40 45Gln Met
Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp 50 55 60Lys
Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu65 70 75
80Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His
85 90 95Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp
Thr 100 105 110Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys
Ile Gln Glu 115 120 125Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys
Ile Asn Thr Thr Leu 130 135 140Thr Cys Glu Val Met Asn Gly Thr Asp
Pro Glu Leu Asn Leu Tyr Gln145 150 155 160Asp Gly Lys His Leu Lys
Leu Ser Gln Arg Val Ile Thr His Lys Trp 165 170 175Thr Thr Ser Leu
Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val 180 185 190Ser Lys
Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu 195 200
205Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met
210 215 220Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Arg Ser Lys
Arg Ser225 230 235 240Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
Pro Arg Arg Pro Gly 245 250 255Pro Thr Arg Lys His Tyr Gln Pro Tyr
Ala Pro Pro Arg Asp Phe Ala 260 265 270Ala Tyr Arg Ser
27567831DNAArtificial SequencehuCD2-CD28tm 67atgagctttc catgtaaatt
tgtagccagc ttccttctga ttttcaatgt ttcttccaaa 60ggtgcagtct ccaaagagat
tacgaatgcc ttggaaacct ggggtgcctt gggtcaggac 120atcaacttgg
acattcctag ttttcaaatg agtgatgata ttgacgatat aaaatgggaa
180aaaacttcag acaagaaaaa gattgcacaa ttcagaaaag agaaagagac
tttcaaggaa 240aaagatacat ataagctatt taaaaatgga actctgaaaa
ttaagcatct gaagaccgat 300gatcaggata tctacaaggt atcaatatat
gatacaaaag gaaaaaatgt gttggaaaaa 360atatttgatt tgaagattca
agagagggtc tcaaaaccaa agatctcctg gacttgtatc 420aacacaaccc
tgacctgtga ggtaatgaat ggaactgacc ccgaattaaa cctgtatcaa
480gatgggaaac atctaaaact ttctcagagg gtcatcacac acaagtggac
caccagcctg 540agtgcaaaat tcaagtgcac agcagggaac aaagtcagca
aggaatccag tgtcgagcct 600gtcagctgtc cagagaaagg tctggacttc
tgggtgctgg tggtggtcgg aggcgtgctg 660gcctgctaca gcctgctggt
caccgtggcc ttcatcatct tttgggtccg cagcaagcgg 720agcagaggcg
gccacagcga ctacatgaac atgaccccta gacggcctgg ccccaccaga
780aagcactacc agccctacgc ccctccccgg gactttgccg cctacagaag c
83168277PRTArtificial SequencehuCD2-CD28tm 68Met Ser Phe Pro Cys
Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn1 5 10 15Val Ser Ser Lys
Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu 20 25 30Thr Trp Gly
Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe 35 40 45Gln Met
Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp 50 55 60Lys
Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu65 70 75
80Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His
85 90 95Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp
Thr 100 105 110Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys
Ile Gln Glu 115 120 125Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys
Ile Asn Thr Thr Leu 130 135 140Thr Cys Glu Val Met Asn Gly Thr Asp
Pro Glu Leu Asn Leu Tyr Gln145 150 155 160Asp Gly Lys His Leu Lys
Leu Ser Gln Arg Val Ile Thr His Lys Trp 165 170 175Thr Thr Ser Leu
Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val 180 185 190Ser Lys
Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu 195 200
205Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
210 215 220Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
Lys Arg225 230 235 240Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met
Thr Pro Arg Arg Pro 245 250 255Gly Pro Thr Arg Lys His Tyr Gln Pro
Tyr Ala Pro Pro Arg Asp Phe 260 265 270Ala Ala Tyr Arg Ser
27569969DNAArtificial SequencehuCD200R-CD28Cys 69atgctgtgcc
cttggagaac cgccaacctg ggcctgctgc tgatcctgac catcttcctg 60gtggccgcca
gcagcagcct gtgcatggac gagaagcaga tcacccagaa ctacagcaag
120gtgctggccg aagtgaacac cagctggccc gtgaagatgg ccaccaacgc
cgtgctgtgc 180tgccctccta tcgccctgcg gaacctgatc atcatcacct
gggagatcat cctgcggggc 240cagcccagct gtaccaaggc ctaccggaaa
gagacaaacg agacaaaaga aacaaactgc 300accgacgagc ggatcacatg
ggtgtccaga cccgaccaga acagcgacct gcagatcaga 360cccgtggcca
tcacccacga cggctactac cggtgcatca tggtcacccc cgatggcaac
420ttccaccggg gataccatct gcaggtgctc gtgacccccg aagtgaccct
gttccagaac 480cggaacagaa ccgccgtgtg caaggccgtg gccggaaaac
ctgccgccca gatctcttgg 540atccccgagg gcgattgcgc caccaagcag
gaatactggt ccaacggcac cgtgaccgtg 600aagtccacct gtcactggga
ggtgcacaac gtgtccaccg tgacatgcca cgtgtcccac 660ctgaccggca
acaagagcct gtacatcgag ctgctgcctg tgcctggcgc caagaagtcc
720gccaagctgt gtcccagccc tctgtttccc ggccctagca agcctttctg
ggtgctggtg 780gtggtcggag gcgtgctggc ctgctacagc ctgctggtca
ccgtggcctt catcatcttt 840tgggtccgca gcaagcggag cagaggcggc
cacagcgact acatgaacat gacccctaga 900cggcctggcc ccaccagaaa
gcactaccag ccctacgccc ctccccggga ctttgccgcc 960tacagaagc
96970323PRTArtificial SequencehuCD200R-CD28Cys 70Met Leu Cys Pro
Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu1 5 10 15Thr Ile Phe
Leu Val Ala Ala Ser Ser Ser Leu Cys Met Asp Glu Lys 20 25 30Gln Ile
Thr Gln Asn Tyr Ser Lys Val Leu Ala Glu Val Asn Thr Ser 35 40 45Trp
Pro Val Lys Met Ala Thr Asn Ala Val Leu Cys Cys Pro Pro Ile 50 55
60Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp Glu Ile Ile Leu Arg Gly65
70 75 80Gln Pro Ser Cys Thr Lys Ala Tyr Arg Lys Glu Thr Asn Glu Thr
Lys 85 90 95Glu Thr Asn Cys Thr Asp Glu Arg Ile Thr Trp Val Ser Arg
Pro Asp 100 105 110Gln Asn Ser Asp Leu Gln Ile Arg Pro Val Ala Ile
Thr His Asp Gly 115 120 125Tyr Tyr Arg Cys Ile Met Val Thr Pro Asp
Gly Asn Phe His Arg Gly 130 135 140Tyr His Leu Gln Val Leu Val Thr
Pro Glu Val Thr Leu Phe Gln Asn145 150 155 160Arg Asn Arg Thr Ala
Val Cys Lys Ala Val Ala Gly Lys Pro Ala Ala 165 170 175Gln Ile Ser
Trp Ile Pro Glu Gly Asp Cys Ala Thr Lys Gln Glu Tyr 180 185 190Trp
Ser Asn Gly Thr Val Thr Val Lys Ser Thr Cys His Trp Glu Val 195 200
205His Asn Val Ser Thr Val Thr Cys His Val Ser His Leu Thr Gly Asn
210 215 220Lys Ser Leu Tyr Ile Glu Leu Leu Pro Val Pro Gly Ala Lys
Lys Ser225 230 235 240Ala Lys Leu Cys Pro Ser Pro Leu Phe Pro Gly
Pro Ser Lys Pro Phe 245 250 255Trp Val Leu Val Val Val Gly Gly Val
Leu Ala Cys Tyr Ser Leu Leu 260 265 270Val Thr Val Ala Phe Ile Ile
Phe Trp Val Arg Ser Lys Arg Ser Arg 275 280 285Gly Gly His Ser Asp
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 290 295 300Thr Arg Lys
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala305 310 315
320Tyr Arg Ser71519DNAArtificial SequencehuFas entire extracellular
domain 71atgctgggca tctggaccct gctgcctctg gtgctgacaa gcgtggccag
actgagcagc 60aagagcgtga acgcccaagt gaccgacatc aacagcaagg gcctggaact
gagaaagacc 120gtgaccaccg tggaaaccca gaacctggaa ggcctgcacc
acgacggcca gttctgccac 180aagccttgtc cccctggcga gcggaaggcc
agagactgta ctgtgaacgg cgacgagccc 240gactgcgtgc cctgtcagga
aggcaaagag tacaccgaca aggcccactt cagcagcaag 300tgccggcggt
gcagactgtg tgatgagggc cacggcctgg aagtggaaat caactgcacc
360cggacccaga acaccaagtg cagatgcaag cccaacttct tctgcaacag
caccgtgtgc 420gagcactgcg acccctgtac caagtgcgaa cacggcatca
tcaaagagtg caccctgacc 480tccaacacaa agtgcaaaga ggaaggcagc agaagcaac
51972173PRTArtificial SequencehuFas entire extracellular domain
72Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala1
5 10 15Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn
Ser 20 25 30Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr
Gln Asn 35 40 45Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys
Pro Cys Pro 50 55 60Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn
Gly Asp Glu Pro65 70 75 80Asp Cys Val Pro Cys Gln Glu Gly Lys Glu
Tyr Thr Asp Lys Ala His 85 90 95Phe Ser Ser Lys Cys Arg Arg Cys Arg
Leu Cys Asp Glu Gly His Gly 100 105 110Leu Glu Val Glu Ile Asn Cys
Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120 125Cys Lys Pro Asn Phe
Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 130 135 140Pro Cys Thr
Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr145 150 155
160Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn 165
17073498DNAArtificial SequencehuFas extracellular domain -7aas
73atgctgggca tctggaccct gctgcctctg gtgctgacaa gcgtggccag actgagcagc
60aagagcgtga acgcccaagt gaccgacatc aacagcaagg gcctggaact gagaaagacc
120gtgaccaccg tggaaaccca gaacctggaa ggcctgcacc acgacggcca
gttctgccac 180aagccttgtc cccctggcga gcggaaggcc agagactgta
ctgtgaacgg cgacgagccc 240gactgcgtgc cctgtcagga aggcaaagag
tacaccgaca aggcccactt cagcagcaag 300tgccggcggt gcagactgtg
tgatgagggc cacggcctgg aagtggaaat caactgcacc 360cggacccaga
acaccaagtg cagatgcaag cccaacttct tctgcaacag caccgtgtgc
420gagcactgcg acccctgtac caagtgcgaa cacggcatca tcaaagagtg
caccctgacc 480tccaacacaa agtgcaaa 49874166PRTArtificial
SequencehuFas extracellular domain -7aas 74Met Leu Gly Ile Trp Thr
Leu Leu Pro Leu Val Leu Thr Ser Val Ala1 5 10 15Arg Leu Ser Ser Lys
Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser 20 25 30Lys Gly Leu Glu
Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn 35 40 45Leu Glu Gly
Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro 50 55 60Pro Gly
Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro65 70 75
80Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His
85 90 95Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His
Gly 100 105 110Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr
Lys Cys Arg 115 120 125Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val
Cys Glu His Cys Asp 130 135 140Pro Cys Thr Lys Cys Glu His Gly Ile
Ile Lys Glu Cys Thr Leu Thr145 150 155 160Ser Asn Thr Lys Cys Lys
16575483DNAArtificial SequencehuFas extracellular domain -12aas
75atgctgggca tctggaccct gctgcctctg gtgctgacaa gcgtggccag actgagcagc
60aagagcgtga acgcccaagt gaccgacatc aacagcaagg gcctggaact gagaaagacc
120gtgaccaccg tggaaaccca gaacctggaa ggcctgcacc acgacggcca
gttctgccac 180aagccttgtc cccctggcga gcggaaggcc agagactgta
ctgtgaacgg cgacgagccc 240gactgcgtgc cctgtcagga aggcaaagag
tacaccgaca aggcccactt cagcagcaag 300tgccggcggt gcagactgtg
tgatgagggc cacggcctgg aagtggaaat caactgcacc 360cggacccaga
acaccaagtg cagatgcaag cccaacttct tctgcaacag caccgtgtgc
420gagcactgcg acccctgtac caagtgcgaa cacggcatca tcaaagagtg
caccctgacc 480tcc 48376161PRTArtificial SequencehuFas extracellular
domain -12aas 76Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr
Ser Val Ala1 5 10 15Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr
Asp Ile Asn Ser 20 25 30Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr
Val Glu Thr Gln Asn 35 40 45Leu Glu Gly Leu His His Asp Gly Gln Phe
Cys His Lys Pro Cys Pro 50 55 60Pro Gly Glu Arg Lys Ala Arg Asp Cys
Thr Val Asn Gly Asp Glu Pro65 70 75 80Asp Cys Val Pro Cys Gln Glu
Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95Phe Ser Ser Lys Cys Arg
Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100
105 110Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys
Arg 115 120 125Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu
His Cys Asp 130 135 140Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys
Glu Cys Thr Leu Thr145 150 155 160Ser7751DNAArtificial
SequencehuFas transmembrane domain 77ctgggctggc tgtgcctcct
gctgctgccc atccctctga tcgtgtgggt c 517817PRTArtificial
SequencehuFas transmembrane domain 78Leu Gly Trp Leu Cys Leu Leu
Leu Leu Pro Ile Pro Leu Ile Val Trp1 5 10 15Val79693DNAArtificial
SequencehuFAStm-CD28 79atgctgggca tctggaccct gctgcctctg gtgctgacaa
gcgtggccag actgagcagc 60aagagcgtga acgcccaagt gaccgacatc aacagcaagg
gcctggaact gagaaagacc 120gtgaccaccg tggaaaccca gaacctggaa
ggcctgcacc acgacggcca gttctgccac 180aagccttgtc cccctggcga
gcggaaggcc agagactgta ctgtgaacgg cgacgagccc 240gactgcgtgc
cctgtcagga aggcaaagag tacaccgaca aggcccactt cagcagcaag
300tgccggcggt gcagactgtg tgatgagggc cacggcctgg aagtggaaat
caactgcacc 360cggacccaga acaccaagtg cagatgcaag cccaacttct
tctgcaacag caccgtgtgc 420gagcactgcg acccctgtac caagtgcgaa
cacggcatca tcaaagagtg caccctgacc 480tccaacacaa agtgcaaaga
ggaaggcagc agaagcaacc tgggctggct gtgcctcctg 540ctgctgccca
tccctctgat cgtgtgggtc cgcagcaagc ggagcagagg cggccacagc
600gactacatga acatgacccc tagacggcct ggccccacca gaaagcacta
ccagccctac 660gcccctcccc gggactttgc cgcctacaga agc
69380231PRTArtificial SequencehuFAStm-CD28 80Met Leu Gly Ile Trp
Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala1 5 10 15Arg Leu Ser Ser
Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser 20 25 30Lys Gly Leu
Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn 35 40 45Leu Glu
Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro 50 55 60Pro
Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro65 70 75
80Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His
85 90 95Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His
Gly 100 105 110Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr
Lys Cys Arg 115 120 125Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val
Cys Glu His Cys Asp 130 135 140Pro Cys Thr Lys Cys Glu His Gly Ile
Ile Lys Glu Cys Thr Leu Thr145 150 155 160Ser Asn Thr Lys Cys Lys
Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp 165 170 175Leu Cys Leu Leu
Leu Leu Pro Ile Pro Leu Ile Val Trp Val Arg Ser 180 185 190Lys Arg
Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg 195 200
205Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
210 215 220Asp Phe Ala Ala Tyr Arg Ser225 23081723DNAArtificial
SequencehuFAS-CD28tm 81atgctgggca tctggaccct gctgcctctg gtgctgacaa
gcgtggccag actgagcagc 60aagagcgtga acgcccaagt gaccgacatc aacagcaagg
gcctggaact gagaaagacc 120gtgaccaccg tggaaaccca gaacctggaa
ggcctgcacc acgacggcca gttctgccac 180aagccttgtc cccctggcga
gcggaaggcc agagactgta ctgtgaacgg cgacgagccc 240gactgcgtgc
cctgtcagga aggcaaagag tacaccgaca aggcccactt cagcagcaag
300tgccggcggt gcagactgtg tgatgagggc cacggcctgg aagtggaaat
caactgcacc 360cggacccaga acaccaagtg cagatgcaag cccaacttct
tctgcaacag caccgtgtgc 420gagcactgcg acccctgtac caagtgcgaa
cacggcatca tcaaagagtg caccctgacc 480tccaacacaa agtgcaaaga
ggaaggcagc agaagcaact tctgggtgct ggtggtggtc 540ggaggcgtgc
tggcctgcta cagcctgctg gtcaccgtgg ccttcatcat cttttgggtc
600cgcagcaagc ggagcagagg cggccacagc gactacatga acatgacccc
tagacggcct 660ggccccacca gaaagcacta ccagccctac gcccctcccc
gggactttgc cgcctacaga 720agc 72382241PRTArtificial
SequencehuFAS-CD28tm 82Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val
Leu Thr Ser Val Ala1 5 10 15Arg Leu Ser Ser Lys Ser Val Asn Ala Gln
Val Thr Asp Ile Asn Ser 20 25 30Lys Gly Leu Glu Leu Arg Lys Thr Val
Thr Thr Val Glu Thr Gln Asn 35 40 45Leu Glu Gly Leu His His Asp Gly
Gln Phe Cys His Lys Pro Cys Pro 50 55 60Pro Gly Glu Arg Lys Ala Arg
Asp Cys Thr Val Asn Gly Asp Glu Pro65 70 75 80Asp Cys Val Pro Cys
Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95Phe Ser Ser Lys
Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110Leu Glu
Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120
125Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp
130 135 140Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr
Leu Thr145 150 155 160Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg
Ser Asn Phe Trp Val 165 170 175Leu Val Val Val Gly Gly Val Leu Ala
Cys Tyr Ser Leu Leu Val Thr 180 185 190Val Ala Phe Ile Ile Phe Trp
Val Arg Ser Lys Arg Ser Arg Gly Gly 195 200 205His Ser Asp Tyr Met
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 210 215 220Lys His Tyr
Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg225 230 235
240Ser83759DNAArtificial SequencehuFAS-CD28Cys 83atgctgggca
tctggaccct gctgcctctg gtgctgacaa gcgtggccag actgagcagc 60aagagcgtga
acgcccaagt gaccgacatc aacagcaagg gcctggaact gagaaagacc
120gtgaccaccg tggaaaccca gaacctggaa ggcctgcacc acgacggcca
gttctgccac 180aagccttgtc cccctggcga gcggaaggcc agagactgta
ctgtgaacgg cgacgagccc 240gactgcgtgc cctgtcagga aggcaaagag
tacaccgaca aggcccactt cagcagcaag 300tgccggcggt gcagactgtg
tgatgagggc cacggcctgg aagtggaaat caactgcacc 360cggacccaga
acaccaagtg cagatgcaag cccaacttct tctgcaacag caccgtgtgc
420gagcactgcg acccctgtac caagtgcgaa cacggcatca tcaaagagtg
caccctgacc 480tccaacacaa agtgcaaaga ggaaggcagc agaagcaact
gtcccagccc tctgtttccc 540ggccctagca agcctttctg ggtgctggtg
gtggtcggag gcgtgctggc ctgctacagc 600ctgctggtca ccgtggcctt
catcatcttt tgggtccgca gcaagcggag cagaggcggc 660cacagcgact
acatgaacat gacccctaga cggcctggcc ccaccagaaa gcactaccag
720ccctacgccc ctccccggga ctttgccgcc tacagaagc 75984253PRTArtificial
SequencehuFAS-CD28Cys 84Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val
Leu Thr Ser Val Ala1 5 10 15Arg Leu Ser Ser Lys Ser Val Asn Ala Gln
Val Thr Asp Ile Asn Ser 20 25 30Lys Gly Leu Glu Leu Arg Lys Thr Val
Thr Thr Val Glu Thr Gln Asn 35 40 45Leu Glu Gly Leu His His Asp Gly
Gln Phe Cys His Lys Pro Cys Pro 50 55 60Pro Gly Glu Arg Lys Ala Arg
Asp Cys Thr Val Asn Gly Asp Glu Pro65 70 75 80Asp Cys Val Pro Cys
Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95Phe Ser Ser Lys
Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110Leu Glu
Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120
125Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp
130 135 140Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr
Leu Thr145 150 155 160Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg
Ser Asn Cys Pro Ser 165 170 175Pro Leu Phe Pro Gly Pro Ser Lys Pro
Phe Trp Val Leu Val Val Val 180 185 190Gly Gly Val Leu Ala Cys Tyr
Ser Leu Leu Val Thr Val Ala Phe Ile 195 200 205Ile Phe Trp Val Arg
Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr 210 215 220Met Asn Met
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln225 230 235
240Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 245
25085738DNAArtificial SequencehuFAS-7aas-CD28Cys 85atgctgggca
tctggaccct gctgcctctg gtgctgacaa gcgtggccag actgagcagc 60aagagcgtga
acgcccaagt gaccgacatc aacagcaagg gcctggaact gagaaagacc
120gtgaccaccg tggaaaccca gaacctggaa ggcctgcacc acgacggcca
gttctgccac 180aagccttgtc cccctggcga gcggaaggcc agagactgta
ctgtgaacgg cgacgagccc 240gactgcgtgc cctgtcagga aggcaaagag
tacaccgaca aggcccactt cagcagcaag 300tgccggcggt gcagactgtg
tgatgagggc cacggcctgg aagtggaaat caactgcacc 360cggacccaga
acaccaagtg cagatgcaag cccaacttct tctgcaacag caccgtgtgc
420gagcactgcg acccctgtac caagtgcgaa cacggcatca tcaaagagtg
caccctgacc 480tccaacacaa agtgcaaatg tcccagccct ctgtttcccg
gccctagcaa gcctttctgg 540gtgctggtgg tggtcggagg cgtgctggcc
tgctacagcc tgctggtcac cgtggccttc 600atcatctttt gggtccgcag
caagcggagc agaggcggcc acagcgacta catgaacatg 660acccctagac
ggcctggccc caccagaaag cactaccagc cctacgcccc tccccgggac
720tttgccgcct acagaagc 73886246PRTArtificial
SequencehuFAS-7aas-CD28Cys 86Met Leu Gly Ile Trp Thr Leu Leu Pro
Leu Val Leu Thr Ser Val Ala1 5 10 15Arg Leu Ser Ser Lys Ser Val Asn
Ala Gln Val Thr Asp Ile Asn Ser 20 25 30Lys Gly Leu Glu Leu Arg Lys
Thr Val Thr Thr Val Glu Thr Gln Asn 35 40 45Leu Glu Gly Leu His His
Asp Gly Gln Phe Cys His Lys Pro Cys Pro 50 55 60Pro Gly Glu Arg Lys
Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro65 70 75 80Asp Cys Val
Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95Phe Ser
Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105
110Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg
115 120 125Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His
Cys Asp 130 135 140Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu
Cys Thr Leu Thr145 150 155 160Ser Asn Thr Lys Cys Lys Cys Pro Ser
Pro Leu Phe Pro Gly Pro Ser 165 170 175Lys Pro Phe Trp Val Leu Val
Val Val Gly Gly Val Leu Ala Cys Tyr 180 185 190Ser Leu Leu Val Thr
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 195 200 205Arg Ser Arg
Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 210 215 220Pro
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp225 230
235 240Phe Ala Ala Tyr Arg Ser 24587723DNAArtificial
SequenceFAS-12aas-CD28Cys 87atgctgggca tctggaccct gctgcctctg
gtgctgacaa gcgtggccag actgagcagc 60aagagcgtga acgcccaagt gaccgacatc
aacagcaagg gcctggaact gagaaagacc 120gtgaccaccg tggaaaccca
gaacctggaa ggcctgcacc acgacggcca gttctgccac 180aagccttgtc
cccctggcga gcggaaggcc agagactgta ctgtgaacgg cgacgagccc
240gactgcgtgc cctgtcagga aggcaaagag tacaccgaca aggcccactt
cagcagcaag 300tgccggcggt gcagactgtg tgatgagggc cacggcctgg
aagtggaaat caactgcacc 360cggacccaga acaccaagtg cagatgcaag
cccaacttct tctgcaacag caccgtgtgc 420gagcactgcg acccctgtac
caagtgcgaa cacggcatca tcaaagagtg caccctgacc 480tcctgtccca
gccctctgtt tcccggccct agcaagcctt tctgggtgct ggtggtggtc
540ggaggcgtgc tggcctgcta cagcctgctg gtcaccgtgg ccttcatcat
cttttgggtc 600cgcagcaagc ggagcagagg cggccacagc gactacatga
acatgacccc tagacggcct 660ggccccacca gaaagcacta ccagccctac
gcccctcccc gggactttgc cgcctacaga 720agc 72388241PRTArtificial
SequenceFAS-12aas-CD28Cys 88Met Leu Gly Ile Trp Thr Leu Leu Pro Leu
Val Leu Thr Ser Val Ala1 5 10 15Arg Leu Ser Ser Lys Ser Val Asn Ala
Gln Val Thr Asp Ile Asn Ser 20 25 30Lys Gly Leu Glu Leu Arg Lys Thr
Val Thr Thr Val Glu Thr Gln Asn 35 40 45Leu Glu Gly Leu His His Asp
Gly Gln Phe Cys His Lys Pro Cys Pro 50 55 60Pro Gly Glu Arg Lys Ala
Arg Asp Cys Thr Val Asn Gly Asp Glu Pro65 70 75 80Asp Cys Val Pro
Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95Phe Ser Ser
Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110Leu
Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120
125Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp
130 135 140Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr
Leu Thr145 150 155 160Ser Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser
Lys Pro Phe Trp Val 165 170 175Leu Val Val Val Gly Gly Val Leu Ala
Cys Tyr Ser Leu Leu Val Thr 180 185 190Val Ala Phe Ile Ile Phe Trp
Val Arg Ser Lys Arg Ser Arg Gly Gly 195 200 205His Ser Asp Tyr Met
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 210 215 220Lys His Tyr
Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg225 230 235
240Ser89510DNAArtificial SequencehuPD1 entire extracellular domain
2 89atgcagatcc ctcaggcccc ttggcctgtc gtgtgggctg tgctgcagct
gggatggcgg 60cctggctggt ttctggacag ccccgacaga ccctggaacc cccctacatt
ttcccctgcc 120ctgctggtcg tgaccgaggg cgacaatgcc accttcacct
gtagcttcag caacaccagc 180gagagcttcg tgctgaactg gtacagaatg
agccccagca accagaccga caagctggcc 240gccttccccg aggatagatc
tcagcccggc caggactgcc ggttcagagt gacccagctg 300cccaacggcc
gggacttcca catgtctgtc gtgcgggcca gacggaacga cagcggcaca
360tatctgtgcg gcgccatcag cctggccccc aaggcccaga tcaaagagag
cctgagagcc 420gagctgagag tgaccgagag aagggccgaa gtgcctaccg
cccaccctag cccatctcca 480agacctgccg gccagttcca gacactggtc
51090170PRTArtificial SequencehuPD1 entire extracellular domain 2
90Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1
5 10 15Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro
Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu
Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu
Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr
Asp Lys Leu Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly
Gln Asp Cys Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp
Phe His Met Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly
Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln
Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg
Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro145 150 155
160Arg Pro Ala Gly Gln Phe Gln Thr Leu Val 165
17091474DNAArtificial SequencehuPD1 2 -12aas 91atgcagatcc
ctcaggcccc ttggcctgtc gtgtgggctg tgctgcagct gggatggcgg 60cctggctggt
ttctggacag ccccgacaga ccctggaacc cccctacatt ttcccctgcc
120ctgctggtcg tgaccgaggg cgacaatgcc accttcacct gtagcttcag
caacaccagc 180gagagcttcg tgctgaactg gtacagaatg agccccagca
accagaccga caagctggcc 240gccttccccg aggatagatc tcagcccggc
caggactgcc ggttcagagt gacccagctg 300cccaacggcc gggacttcca
catgtctgtc gtgcgggcca gacggaacga cagcggcaca 360tatctgtgcg
gcgccatcag cctggccccc aaggcccaga tcaaagagag cctgagagcc
420gagctgagag tgaccgagag aagggccgaa gtgcctaccg cccaccctag ccca
47492158PRTArtificial SequencehuPD1 2 -12aas 92Met Gln Ile Pro Gln
Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg
Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro
Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala
Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75
80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val
Arg 100 105
110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu
Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro
Ser Pro145 150 15593465DNAArtificial SequencehuPD1 2 -15aas
93atgcagatcc ctcaggcccc ttggcctgtc gtgtgggctg tgctgcagct gggatggcgg
60cctggctggt ttctggacag ccccgacaga ccctggaacc cccctacatt ttcccctgcc
120ctgctggtcg tgaccgaggg cgacaatgcc accttcacct gtagcttcag
caacaccagc 180gagagcttcg tgctgaactg gtacagaatg agccccagca
accagaccga caagctggcc 240gccttccccg aggatagatc tcagcccggc
caggactgcc ggttcagagt gacccagctg 300cccaacggcc gggacttcca
catgtctgtc gtgcgggcca gacggaacga cagcggcaca 360tatctgtgcg
gcgccatcag cctggccccc aaggcccaga tcaaagagag cctgagagcc
420gagctgagag tgaccgagag aagggccgaa gtgcctaccg cccac
46594155PRTArtificial SequencehuPD1 2 -15aas 94Met Gln Ile Pro Gln
Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg
Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro
Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala
Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75
80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val
Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala
Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr
Ala His145 150 15595447DNAArtificial SequencehuPD1 2 -21aas
95atgcagatcc ctcaggcccc ttggcctgtc gtgtgggctg tgctgcagct gggatggcgg
60cctggctggt ttctggacag ccccgacaga ccctggaacc cccctacatt ttcccctgcc
120ctgctggtcg tgaccgaggg cgacaatgcc accttcacct gtagcttcag
caacaccagc 180gagagcttcg tgctgaactg gtacagaatg agccccagca
accagaccga caagctggcc 240gccttccccg aggatagatc tcagcccggc
caggactgcc ggttcagagt gacccagctg 300cccaacggcc gggacttcca
catgtctgtc gtgcgggcca gacggaacga cagcggcaca 360tatctgtgcg
gcgccatcag cctggccccc aaggcccaga tcaaagagag cctgagagcc
420gagctgagag tgaccgagag aagggcc 44796149PRTArtificial
SequencehuPD1 2 -21aas 96Met Gln Ile Pro Gln Ala Pro Trp Pro Val
Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro Gly Trp Phe Leu
Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro Ala
Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys Ser
Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg Met
Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75 80Ala Phe Pro Glu
Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95Val Thr Gln
Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110Ala
Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120
125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140Thr Glu Arg Arg Ala14597750DNAArtificial
SequencehuPD1-CD28Cys 97atgcagatcc ctcaggcccc ttggcctgtc gtgtgggctg
tgctgcagct gggatggcgg 60cctggctggt ttctggacag ccccgacaga ccctggaacc
cccctacatt ttcccctgcc 120ctgctggtcg tgaccgaggg cgacaatgcc
accttcacct gtagcttcag caacaccagc 180gagagcttcg tgctgaactg
gtacagaatg agccccagca accagaccga caagctggcc 240gccttccccg
aggatagatc tcagcccggc caggactgcc ggttcagagt gacccagctg
300cccaacggcc gggacttcca catgtctgtc gtgcgggcca gacggaacga
cagcggcaca 360tatctgtgcg gcgccatcag cctggccccc aaggcccaga
tcaaagagag cctgagagcc 420gagctgagag tgaccgagag aagggccgaa
gtgcctaccg cccaccctag cccatctcca 480agacctgccg gccagttcca
gacactggtc tgtcccagcc ctctgtttcc cggccctagc 540aagcctttct
gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc
600accgtggcct tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg
ccacagcgac 660tacatgaaca tgacccctag acggcctggc cccaccagaa
agcactacca gccctacgcc 720cctccccggg actttgccgc ctacagaagc
75098250PRTArtificial SequencehuPD1-CD28Cys 98Met Gln Ile Pro Gln
Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg
Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro
Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala
Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75
80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val
Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala
Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg
Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr
Ala His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe
Gln Thr Leu Val Cys Pro Ser Pro Leu Phe 165 170 175Pro Gly Pro Ser
Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val 180 185 190Leu Ala
Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp 195 200
205Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met
210 215 220Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
Tyr Ala225 230 235 240Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 245
25099714DNAArtificial SequencehuPD1-12aas-CD28Cys 99atgcagatcc
ctcaggcccc ttggcctgtc gtgtgggctg tgctgcagct gggatggcgg 60cctggctggt
ttctggacag ccccgacaga ccctggaacc cccctacatt ttcccctgcc
120ctgctggtcg tgaccgaggg cgacaatgcc accttcacct gtagcttcag
caacaccagc 180gagagcttcg tgctgaactg gtacagaatg agccccagca
accagaccga caagctggcc 240gccttccccg aggatagatc tcagcccggc
caggactgcc ggttcagagt gacccagctg 300cccaacggcc gggacttcca
catgtctgtc gtgcgggcca gacggaacga cagcggcaca 360tatctgtgcg
gcgccatcag cctggccccc aaggcccaga tcaaagagag cctgagagcc
420gagctgagag tgaccgagag aagggccgaa gtgcctaccg cccaccctag
cccatgtccc 480agccctctgt ttcccggccc tagcaagcct ttctgggtgc
tggtggtggt cggaggcgtg 540ctggcctgct acagcctgct ggtcaccgtg
gccttcatca tcttttgggt ccgcagcaag 600cggagcagag gcggccacag
cgactacatg aacatgaccc ctagacggcc tggccccacc 660agaaagcact
accagcccta cgcccctccc cgggactttg ccgcctacag aagc
714100238PRTArtificial SequencehuPD1-12aas-CD28Cys 100Met Gln Ile
Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly
Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40
45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu
Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys
Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met
Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu
Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile Lys Glu
Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu
Val Pro Thr Ala His Pro Ser Pro Cys Pro145 150 155 160Ser Pro Leu
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val 165 170 175Val
Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 180 185
190Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp
195 200 205Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
His Tyr 210 215 220Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
Arg Ser225 230 235101705DNAArtificial SequencehuPD1-15aas-CD28Cys
101atgcagatcc ctcaggcccc ttggcctgtc gtgtgggctg tgctgcagct
gggatggcgg 60cctggctggt ttctggacag ccccgacaga ccctggaacc cccctacatt
ttcccctgcc 120ctgctggtcg tgaccgaggg cgacaatgcc accttcacct
gtagcttcag caacaccagc 180gagagcttcg tgctgaactg gtacagaatg
agccccagca accagaccga caagctggcc 240gccttccccg aggatagatc
tcagcccggc caggactgcc ggttcagagt gacccagctg 300cccaacggcc
gggacttcca catgtctgtc gtgcgggcca gacggaacga cagcggcaca
360tatctgtgcg gcgccatcag cctggccccc aaggcccaga tcaaagagag
cctgagagcc 420gagctgagag tgaccgagag aagggccgaa gtgcctaccg
cccactgtcc cagccctctg 480tttcccggcc ctagcaagcc tttctgggtg
ctggtggtgg tcggaggcgt gctggcctgc 540tacagcctgc tggtcaccgt
ggccttcatc atcttttggg tccgcagcaa gcggagcaga 600ggcggccaca
gcgactacat gaacatgacc cctagacggc ctggccccac cagaaagcac
660taccagccct acgcccctcc ccgggacttt gccgcctaca gaagc
705102235PRTArtificial SequencehuPD1-15aas-CD28Cys 102Met Gln Ile
Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly
Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40
45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu
Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys
Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met
Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu
Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile Lys Glu
Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu
Val Pro Thr Ala His Cys Pro Ser Pro Leu145 150 155 160Phe Pro Gly
Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 165 170 175Val
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 180 185
190Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn
195 200 205Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
Pro Tyr 210 215 220Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser225
230 235103687DNAArtificial SequencehuPD1-21aas-CD28Cys
103atgcagatcc ctcaggcccc ttggcctgtc gtgtgggctg tgctgcagct
gggatggcgg 60cctggctggt ttctggacag ccccgacaga ccctggaacc cccctacatt
ttcccctgcc 120ctgctggtcg tgaccgaggg cgacaatgcc accttcacct
gtagcttcag caacaccagc 180gagagcttcg tgctgaactg gtacagaatg
agccccagca accagaccga caagctggcc 240gccttccccg aggatagatc
tcagcccggc caggactgcc ggttcagagt gacccagctg 300cccaacggcc
gggacttcca catgtctgtc gtgcgggcca gacggaacga cagcggcaca
360tatctgtgcg gcgccatcag cctggccccc aaggcccaga tcaaagagag
cctgagagcc 420gagctgagag tgaccgagag aagggcctgt cccagccctc
tgtttcccgg ccctagcaag 480cctttctggg tgctggtggt ggtcggaggc
gtgctggcct gctacagcct gctggtcacc 540gtggccttca tcatcttttg
ggtccgcagc aagcggagca gaggcggcca cagcgactac 600atgaacatga
cccctagacg gcctggcccc accagaaagc actaccagcc ctacgcccct
660ccccgggact ttgccgccta cagaagc 687104229PRTArtificial
SequencehuPD1-21aas-CD28Cys 104Met Gln Ile Pro Gln Ala Pro Trp Pro
Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro Gly Trp Phe
Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr Phe Ser Pro
Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala Thr Phe Thr Cys
Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu Asn Trp Tyr Arg
Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75 80Ala Phe Pro
Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95Val Thr
Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105
110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu
Arg Val 130 135 140Thr Glu Arg Arg Ala Cys Pro Ser Pro Leu Phe Pro
Gly Pro Ser Lys145 150 155 160Pro Phe Trp Val Leu Val Val Val Gly
Gly Val Leu Ala Cys Tyr Ser 165 170 175Leu Leu Val Thr Val Ala Phe
Ile Ile Phe Trp Val Arg Ser Lys Arg 180 185 190Ser Arg Gly Gly His
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 195 200 205Gly Pro Thr
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 210 215 220Ala
Ala Tyr Arg Ser225105810DNAArtificial SequencemuCD2tm-CD28
105atgaagtgca agttcctggg ctcattcttc ctgctgttca gcctgagcgg
caagggcgcc 60gactgcagag acaacgagac aatctggggc gtgctgggcc acggcatcac
cctgaacatc 120cccaacttcc agatgaccga cgacatcgac gaagtgcgct
gggtgcgaag aggcacactg 180gtggccgagt tcaagagaaa gaagccccca
ttcctgatca gcgagacata cgaggtgctg 240gccaacggca gcctgaagat
caagaaaccc atgatgagaa acgacagcgg cacctacaac 300gtgatggtgt
acggcaccaa cggcatgacc agactggaaa aggacctgga cgtgcggatc
360ctggaaaggg tgtccaagcc catgatccac tgggagtgcc ccaacaccac
cctgacctgt 420gctgtgctgc agggcaccga cttcgagctg aagctgtacc
agggcgagac actgctgaac 480tccctgcccc agaaaaacat gagctaccag
tggaccaacc tgaacgcccc cttcaagtgc 540gaggccatca accccgtgtc
caaagaaagc aagatggaag tcgtgaactg ccccgagaag 600ggcctgagct
tctacgtgac agtgggcgtg ggagctggcg gactgctgct ggtgctgctg
660gtggccctgt tcatcttctg catctgcaac agcagacgga acagaggcgg
ccagagcgac 720tacatgaaca tgacccccag aaggcctggc ctgaccagaa
agccctacca gccttacgcc 780cctgccagag acttcgccgc ctacagacct
810106270PRTArtificial SequencemuCD2tm-CD28 106Met Lys Cys Lys Phe
Leu Gly Ser Phe Phe Leu Leu Phe Ser Leu Ser1 5 10 15Gly Lys Gly Ala
Asp Cys Arg Asp Asn Glu Thr Ile Trp Gly Val Leu 20 25 30Gly His Gly
Ile Thr Leu Asn Ile Pro Asn Phe Gln Met Thr Asp Asp 35 40 45Ile Asp
Glu Val Arg Trp Val Arg Arg Gly Thr Leu Val Ala Glu Phe 50 55 60Lys
Arg Lys Lys Pro Pro Phe Leu Ile Ser Glu Thr Tyr Glu Val Leu65 70 75
80Ala Asn Gly Ser Leu Lys Ile Lys Lys Pro Met Met Arg Asn Asp Ser
85 90 95Gly Thr Tyr Asn Val Met Val Tyr Gly Thr Asn Gly Met Thr Arg
Leu 100 105 110Glu Lys Asp Leu Asp Val Arg Ile Leu Glu Arg Val Ser
Lys Pro Met 115 120 125Ile His Trp Glu Cys Pro Asn Thr Thr Leu Thr
Cys Ala Val Leu Gln 130 135 140Gly Thr Asp Phe Glu Leu Lys Leu Tyr
Gln Gly Glu Thr Leu Leu Asn145 150 155 160Ser Leu Pro Gln Lys Asn
Met Ser Tyr Gln Trp Thr Asn Leu Asn Ala 165 170 175Pro Phe Lys Cys
Glu Ala Ile Asn Pro Val Ser Lys Glu Ser Lys Met 180 185 190Glu Val
Val Asn Cys Pro Glu Lys Gly Leu Ser Phe Tyr Val Thr Val 195 200
205Gly Val Gly Ala Gly Gly Leu Leu Leu Val Leu Leu Val Ala Leu Phe
210 215 220Ile Phe Cys Ile Cys Asn Ser Arg Arg Asn Arg Gly Gly Gln
Ser Asp225 230 235 240Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Leu
Thr Arg Lys Pro Tyr 245 250 255Gln Pro Tyr Ala Pro Ala Arg Asp Phe
Ala Ala Tyr Arg Pro 260 265 270107813DNAArtificial
SequencemuCD2-CD28tm 107atgaagtgca agttcctggg ctcattcttc ctgctgttca
gcctgagcgg caagggcgcc 60gactgcagag acaacgagac aatctggggc gtgctgggcc
acggcatcac cctgaacatc 120cccaacttcc agatgaccga cgacatcgac
gaagtgcgct gggtgcgaag aggcacactg
180gtggccgagt tcaagagaaa gaagccccca ttcctgatca gcgagacata
cgaggtgctg 240gccaacggca gcctgaagat caagaaaccc atgatgagaa
acgacagcgg cacctacaac 300gtgatggtgt acggcaccaa cggcatgacc
agactggaaa aggacctgga cgtgcggatc 360ctggaaaggg tgtccaagcc
catgatccac tgggagtgcc ccaacaccac cctgacctgt 420gctgtgctgc
agggcaccga cttcgagctg aagctgtacc agggcgagac actgctgaac
480tccctgcccc agaaaaacat gagctaccag tggaccaacc tgaacgcccc
cttcaagtgc 540gaggccatca accccgtgtc caaagaaagc aagatggaag
tcgtgaactg ccccgagaag 600ggcctgagct tctgggccct ggtggtggtg
gccggcgtgc tgttttgtta cggcctgctc 660gtgaccgtgg ccctgtgcgt
gatctggacc aacagcagaa gaaacagagg cggccagagc 720gactacatga
acatgacccc cagaaggcct ggcctgacca gaaagcccta ccagccttac
780gcccctgcca gagacttcgc cgcctacaga ccc 813108271PRTArtificial
SequencemuCD2-CD28tm 108Met Lys Cys Lys Phe Leu Gly Ser Phe Phe Leu
Leu Phe Ser Leu Ser1 5 10 15Gly Lys Gly Ala Asp Cys Arg Asp Asn Glu
Thr Ile Trp Gly Val Leu 20 25 30Gly His Gly Ile Thr Leu Asn Ile Pro
Asn Phe Gln Met Thr Asp Asp 35 40 45Ile Asp Glu Val Arg Trp Val Arg
Arg Gly Thr Leu Val Ala Glu Phe 50 55 60Lys Arg Lys Lys Pro Pro Phe
Leu Ile Ser Glu Thr Tyr Glu Val Leu65 70 75 80Ala Asn Gly Ser Leu
Lys Ile Lys Lys Pro Met Met Arg Asn Asp Ser 85 90 95Gly Thr Tyr Asn
Val Met Val Tyr Gly Thr Asn Gly Met Thr Arg Leu 100 105 110Glu Lys
Asp Leu Asp Val Arg Ile Leu Glu Arg Val Ser Lys Pro Met 115 120
125Ile His Trp Glu Cys Pro Asn Thr Thr Leu Thr Cys Ala Val Leu Gln
130 135 140Gly Thr Asp Phe Glu Leu Lys Leu Tyr Gln Gly Glu Thr Leu
Leu Asn145 150 155 160Ser Leu Pro Gln Lys Asn Met Ser Tyr Gln Trp
Thr Asn Leu Asn Ala 165 170 175Pro Phe Lys Cys Glu Ala Ile Asn Pro
Val Ser Lys Glu Ser Lys Met 180 185 190Glu Val Val Asn Cys Pro Glu
Lys Gly Leu Ser Phe Trp Ala Leu Val 195 200 205Val Val Ala Gly Val
Leu Phe Cys Tyr Gly Leu Leu Val Thr Val Ala 210 215 220Leu Cys Val
Ile Trp Thr Asn Ser Arg Arg Asn Arg Gly Gly Gln Ser225 230 235
240Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro
245 250 255Tyr Gln Pro Tyr Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg
Pro 260 265 270109840DNAArtificial SequencemuCD2-CD28Cys
109atgaagtgca agttcctggg ctcattcttc ctgctgttca gcctgagcgg
caagggcgcc 60gactgcagag acaacgagac aatctggggc gtgctgggcc acggcatcac
cctgaacatc 120cccaacttcc agatgaccga cgacatcgac gaagtgcgct
gggtgcgaag aggcacactg 180gtggccgagt tcaagagaaa gaagccccca
ttcctgatca gcgagacata cgaggtgctg 240gccaacggca gcctgaagat
caagaaaccc atgatgagaa acgacagcgg cacctacaac 300gtgatggtgt
acggcaccaa cggcatgacc agactggaaa aggacctgga cgtgcggatc
360ctggaaaggg tgtccaagcc catgatccac tgggagtgcc ccaacaccac
cctgacctgt 420gctgtgctgc agggcaccga cttcgagctg aagctgtacc
agggcgagac actgctgaac 480tccctgcccc agaaaaacat gagctaccag
tggaccaacc tgaacgcccc cttcaagtgc 540gaggccatca accccgtgtc
caaagaaagc aagatggaag tcgtgaactg ccccgagaag 600ggcctgagct
gccacaccca gagcagcccc aagctgttct gggccctggt ggtggtggcc
660ggcgtgctgt tttgttacgg cctgctcgtg accgtggccc tgtgcgtgat
ctggaccaac 720agcagaagaa acagaggcgg ccagagcgac tacatgaaca
tgacccccag aaggcctggc 780ctgaccagaa agccctacca gccttacgcc
cctgccagag acttcgccgc ctacagacct 840110280PRTArtificial
SequencemuCD2-CD28Cys 110Met Lys Cys Lys Phe Leu Gly Ser Phe Phe
Leu Leu Phe Ser Leu Ser1 5 10 15Gly Lys Gly Ala Asp Cys Arg Asp Asn
Glu Thr Ile Trp Gly Val Leu 20 25 30Gly His Gly Ile Thr Leu Asn Ile
Pro Asn Phe Gln Met Thr Asp Asp 35 40 45Ile Asp Glu Val Arg Trp Val
Arg Arg Gly Thr Leu Val Ala Glu Phe 50 55 60Lys Arg Lys Lys Pro Pro
Phe Leu Ile Ser Glu Thr Tyr Glu Val Leu65 70 75 80Ala Asn Gly Ser
Leu Lys Ile Lys Lys Pro Met Met Arg Asn Asp Ser 85 90 95Gly Thr Tyr
Asn Val Met Val Tyr Gly Thr Asn Gly Met Thr Arg Leu 100 105 110Glu
Lys Asp Leu Asp Val Arg Ile Leu Glu Arg Val Ser Lys Pro Met 115 120
125Ile His Trp Glu Cys Pro Asn Thr Thr Leu Thr Cys Ala Val Leu Gln
130 135 140Gly Thr Asp Phe Glu Leu Lys Leu Tyr Gln Gly Glu Thr Leu
Leu Asn145 150 155 160Ser Leu Pro Gln Lys Asn Met Ser Tyr Gln Trp
Thr Asn Leu Asn Ala 165 170 175Pro Phe Lys Cys Glu Ala Ile Asn Pro
Val Ser Lys Glu Ser Lys Met 180 185 190Glu Val Val Asn Cys Pro Glu
Lys Gly Leu Ser Cys His Thr Gln Ser 195 200 205Ser Pro Lys Leu Phe
Trp Ala Leu Val Val Val Ala Gly Val Leu Phe 210 215 220Cys Tyr Gly
Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp Thr Asn225 230 235
240Ser Arg Arg Asn Arg Gly Gly Gln Ser Asp Tyr Met Asn Met Thr Pro
245 250 255Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala
Pro Ala 260 265 270Arg Asp Phe Ala Ala Tyr Arg Pro 275
280111861DNAArtificial SequencemuCD2-CD28Cys-41BBic 111atgaagtgca
agttcctggg ctcattcttc ctgctgttca gcctgagcgg caagggcgcc 60gactgcagag
acaacgagac aatctggggc gtgctgggcc acggcatcac cctgaacatc
120cccaacttcc agatgaccga cgacatcgac gaagtgcgct gggtgcgaag
aggcacactg 180gtggccgagt tcaagagaaa gaagccccca ttcctgatca
gcgagacata cgaggtgctg 240gccaacggca gcctgaagat caagaaaccc
atgatgagaa acgacagcgg cacctacaac 300gtgatggtgt acggcaccaa
cggcatgacc agactggaaa aggacctgga cgtgcggatc 360ctggaaaggg
tgtccaagcc catgatccac tgggagtgcc ccaacaccac cctgacctgt
420gctgtgctgc agggcaccga cttcgagctg aagctgtacc agggcgagac
actgctgaac 480tccctgcccc agaaaaacat gagctaccag tggaccaacc
tgaacgcccc cttcaagtgc 540gaggccatca accccgtgtc caaagaaagc
aagatggaag tcgtgaactg ccccgagaag 600ggcctgagct gccacaccca
gagcagcccc aagctgttct gggccctggt ggtggtggcc 660ggcgtgctgt
tttgttacgg cctgctcgtg accgtggccc tgtgcgtgat ctggaccagc
720gtgctgaagt ggatcagaaa gaagttcccc cacatcttca agcagccctt
caagaaaacc 780accggcgctg cccaggaaga ggacgcctgc agctgtagat
gccctcagga agaagaaggc 840ggcggaggcg gctacgagct g
861112287PRTArtificial SequencemuCD2-CD28Cys-41BBic 112Met Lys Cys
Lys Phe Leu Gly Ser Phe Phe Leu Leu Phe Ser Leu Ser1 5 10 15Gly Lys
Gly Ala Asp Cys Arg Asp Asn Glu Thr Ile Trp Gly Val Leu 20 25 30Gly
His Gly Ile Thr Leu Asn Ile Pro Asn Phe Gln Met Thr Asp Asp 35 40
45Ile Asp Glu Val Arg Trp Val Arg Arg Gly Thr Leu Val Ala Glu Phe
50 55 60Lys Arg Lys Lys Pro Pro Phe Leu Ile Ser Glu Thr Tyr Glu Val
Leu65 70 75 80Ala Asn Gly Ser Leu Lys Ile Lys Lys Pro Met Met Arg
Asn Asp Ser 85 90 95Gly Thr Tyr Asn Val Met Val Tyr Gly Thr Asn Gly
Met Thr Arg Leu 100 105 110Glu Lys Asp Leu Asp Val Arg Ile Leu Glu
Arg Val Ser Lys Pro Met 115 120 125Ile His Trp Glu Cys Pro Asn Thr
Thr Leu Thr Cys Ala Val Leu Gln 130 135 140Gly Thr Asp Phe Glu Leu
Lys Leu Tyr Gln Gly Glu Thr Leu Leu Asn145 150 155 160Ser Leu Pro
Gln Lys Asn Met Ser Tyr Gln Trp Thr Asn Leu Asn Ala 165 170 175Pro
Phe Lys Cys Glu Ala Ile Asn Pro Val Ser Lys Glu Ser Lys Met 180 185
190Glu Val Val Asn Cys Pro Glu Lys Gly Leu Ser Cys His Thr Gln Ser
195 200 205Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val Ala Gly Val
Leu Phe 210 215 220Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys Val
Ile Trp Thr Ser225 230 235 240Val Leu Lys Trp Ile Arg Lys Lys Phe
Pro His Ile Phe Lys Gln Pro 245 250 255Phe Lys Lys Thr Thr Gly Ala
Ala Gln Glu Glu Asp Ala Cys Ser Cys 260 265 270Arg Cys Pro Gln Glu
Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu 275 280
2851131080DNAArtificial SequencemuCD200R-3aas-CD28Cys tm ic-41BB
113atgttctgct tctggcggac aagcgccctg gccgtgctgc tgatctgggg
agtgtttgtg 60gccggcagca gctgcaccga caagaaccag accacccaga acaacagcag
cagccccctg 120acccaagtga acaccaccgt gtccgtgcag atcggcacca
aggccctgct gtgctgtttc 180agcatccctc tgaccaaggc tgtgctgatc
acctggatca tcaagctgag aggcctgccc 240agctgcacaa tcgcctacaa
ggtggacacc aagaccaacg agacaagctg cctgggcaga 300aacatcacct
gggccagcac cccagaccac agccctgagc tgcagatcag cgccgtgaca
360ctgcagcacg agggcaccta cacatgcgag acagtgaccc ccgagggcaa
cttcgagaag 420aactacgatc tgcaggtgct ggtgcccccc gaagtgacct
acttccccga gaagaataga 480agcgccgtgt gcgaggccat ggctggcaaa
cctgccgccc agatctcttg gagccctgac 540ggcgactgtg tgaccaccag
cgagagccac agcaacggca cagtgaccgt gcggagcacc 600tgtcactggg
agcagaacaa cgtgtccgac gtgtcctgca tcgtgtccca cctgaccggc
660aaccagagcc tgagcatcga gctgagcaga ggcggaaacc agtcctgcca
cacccagagc 720agccccaagc tgttctgggc cctggtggtg gtggccggcg
tgctgttttg ttacggcctg 780ctcgtgaccg tggccctgtg cgtgatctgg
accaacagca gaagaaacag aggcggccag 840agcgactaca tgaacatgac
ccccagaagg cctggcctga ccagaaagcc ctaccagcct 900tacgcccctg
ccagagactt cgccgcctac agacctagcg tgctgaagtg gatcagaaag
960aagttccccc acatcttcaa gcagcccttc aagaaaacca ccggcgctgc
ccaggaagag 1020gacgcctgca gctgtagatg ccctcaggaa gaagaaggcg
gcggaggcgg ctacgagctg 1080114360PRTArtificial
SequencemuCD200R-3aas-CD28Cys tm ic-41BB 114Met Phe Cys Phe Trp Arg
Thr Ser Ala Leu Ala Val Leu Leu Ile Trp1 5 10 15Gly Val Phe Val Ala
Gly Ser Ser Cys Thr Asp Lys Asn Gln Thr Thr 20 25 30Gln Asn Asn Ser
Ser Ser Pro Leu Thr Gln Val Asn Thr Thr Val Ser 35 40 45 Val Gln
Ile Gly Thr Lys Ala Leu Leu Cys Cys Phe Ser Ile Pro Leu 50 55 60Thr
Lys Ala Val Leu Ile Thr Trp Ile Ile Lys Leu Arg Gly Leu Pro65 70 75
80Ser Cys Thr Ile Ala Tyr Lys Val Asp Thr Lys Thr Asn Glu Thr Ser
85 90 95Cys Leu Gly Arg Asn Ile Thr Trp Ala Ser Thr Pro Asp His Ser
Pro 100 105 110Glu Leu Gln Ile Ser Ala Val Thr Leu Gln His Glu Gly
Thr Tyr Thr 115 120 125Cys Glu Thr Val Thr Pro Glu Gly Asn Phe Glu
Lys Asn Tyr Asp Leu 130 135 140Gln Val Leu Val Pro Pro Glu Val Thr
Tyr Phe Pro Glu Lys Asn Arg145 150 155 160Ser Ala Val Cys Glu Ala
Met Ala Gly Lys Pro Ala Ala Gln Ile Ser 165 170 175Trp Ser Pro Asp
Gly Asp Cys Val Thr Thr Ser Glu Ser His Ser Asn 180 185 190Gly Thr
Val Thr Val Arg Ser Thr Cys His Trp Glu Gln Asn Asn Val 195 200
205Ser Asp Val Ser Cys Ile Val Ser His Leu Thr Gly Asn Gln Ser Leu
210 215 220Ser Ile Glu Leu Ser Arg Gly Gly Asn Gln Ser Cys His Thr
Gln Ser225 230 235 240Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val
Ala Gly Val Leu Phe 245 250 255Cys Tyr Gly Leu Leu Val Thr Val Ala
Leu Cys Val Ile Trp Thr Asn 260 265 270Ser Arg Arg Asn Arg Gly Gly
Gln Ser Asp Tyr Met Asn Met Thr Pro 275 280 285Arg Arg Pro Gly Leu
Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro Ala 290 295 300Arg Asp Phe
Ala Ala Tyr Arg Pro Ser Val Leu Lys Trp Ile Arg Lys305 310 315
320Lys Phe Pro His Ile Phe Lys Gln Pro Phe Lys Lys Thr Thr Gly Ala
325 330 335Ala Gln Glu Glu Asp Ala Cys Ser Cys Arg Cys Pro Gln Glu
Glu Glu 340 345 350Gly Gly Gly Gly Gly Tyr Glu Leu 355
360115966DNAArtificial SequencemuCD200R-CD28Cys tm ic-41BB
115atgttctgct tctggcggac aagcgccctg gccgtgctgc tgatctgggg
agtgtttgtg 60gccggcagca gctgcaccga caagaaccag accacccaga acaacagcag
cagccccctg 120acccaagtga acaccaccgt gtccgtgcag atcggcacca
aggccctgct gtgctgtttc 180agcatccctc tgaccaaggc tgtgctgatc
acctggatca tcaagctgag aggcctgccc 240agctgcacaa tcgcctacaa
ggtggacacc aagaccaacg agacaagctg cctgggcaga 300aacatcacct
gggccagcac cccagaccac agccctgagc tgcagatcag cgccgtgaca
360ctgcagcacg agggcaccta cacatgcgag acagtgaccc ccgagggcaa
cttcgagaag 420aactacgatc tgcaggtgct ggtgcccccc gaagtgacct
acttccccga gaagaataga 480agcgccgtgt gcgaggccat ggctggcaaa
cctgccgccc agatctcttg gagccctgac 540ggcgactgtg tgaccaccag
cgagagccac agcaacggca cagtgaccgt gcggagcacc 600tgtcactggg
agcagaacaa cgtgtccgac gtgtcctgca tcgtgtccca cctgaccggc
660aaccagagcc tgagcatcga gctgagcaga ggcggaaacc agtccctgag
gccctgccac 720acccagagca gccccaagct gttctgggcc ctggtggtgg
tggccggcgt gctgttttgt 780tacggcctgc tcgtgaccgt ggccctgtgc
gtgatctgga ccagcgtgct gaagtggatc 840agaaagaagt tcccccacat
cttcaagcag cccttcaaga aaaccaccgg cgctgcccag 900gaagaggacg
cctgcagctg tagatgccct caggaagaag aaggcggcgg aggcggctac 960gagctg
966116322PRTArtificial SequencemuCD200R-CD28Cys tm ic-41BB 116Met
Phe Cys Phe Trp Arg Thr Ser Ala Leu Ala Val Leu Leu Ile Trp1 5 10
15Gly Val Phe Val Ala Gly Ser Ser Cys Thr Asp Lys Asn Gln Thr Thr
20 25 30Gln Asn Asn Ser Ser Ser Pro Leu Thr Gln Val Asn Thr Thr Val
Ser 35 40 45Val Gln Ile Gly Thr Lys Ala Leu Leu Cys Cys Phe Ser Ile
Pro Leu 50 55 60Thr Lys Ala Val Leu Ile Thr Trp Ile Ile Lys Leu Arg
Gly Leu Pro65 70 75 80Ser Cys Thr Ile Ala Tyr Lys Val Asp Thr Lys
Thr Asn Glu Thr Ser 85 90 95Cys Leu Gly Arg Asn Ile Thr Trp Ala Ser
Thr Pro Asp His Ser Pro 100 105 110Glu Leu Gln Ile Ser Ala Val Thr
Leu Gln His Glu Gly Thr Tyr Thr 115 120 125Cys Glu Thr Val Thr Pro
Glu Gly Asn Phe Glu Lys Asn Tyr Asp Leu 130 135 140Gln Val Leu Val
Pro Pro Glu Val Thr Tyr Phe Pro Glu Lys Asn Arg145 150 155 160Ser
Ala Val Cys Glu Ala Met Ala Gly Lys Pro Ala Ala Gln Ile Ser 165 170
175Trp Ser Pro Asp Gly Asp Cys Val Thr Thr Ser Glu Ser His Ser Asn
180 185 190Gly Thr Val Thr Val Arg Ser Thr Cys His Trp Glu Gln Asn
Asn Val 195 200 205Ser Asp Val Ser Cys Ile Val Ser His Leu Thr Gly
Asn Gln Ser Leu 210 215 220Ser Ile Glu Leu Ser Arg Gly Gly Asn Gln
Ser Leu Arg Pro Cys His225 230 235 240Thr Gln Ser Ser Pro Lys Leu
Phe Trp Ala Leu Val Val Val Ala Gly 245 250 255Val Leu Phe Cys Tyr
Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile 260 265 270Trp Thr Ser
Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe 275 280 285Lys
Gln Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala 290 295
300Cys Ser Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly
Tyr305 310 315 320Glu Leu117681DNAArtificial SequencemuFas tm-CD28
117atgctgtgga tctgggccgt gctgcctctg gtgctggctg gatcacagct
gagagtgcac 60acccagggca ccaacagcat cagcgagagc ctgaagctga gaagaagagt
gcgcgagaca 120gacaagaact gcagcgaggg cctgtaccag ggcggaccct
tctgctgtca gccttgccag 180cccggcaaga aaaaggtgga agattgcaag
atgaacggcg gcacccctac ctgcgcccct 240tgtacagagg gcaaagagta
catggacaag aaccactacg ccgacaagtg cagacggtgc 300accctgtgcg
acgaggaaca cggcctggaa gtggaaacaa actgcaccct gacccagaac
360accaagtgca agtgcaaacc cgacttctac tgcgacagcc ccggctgcga
gcactgcgtc 420agatgtgcct cttgcgagca cggcaccctg gaaccttgta
ccgccaccag caacaccaac 480tgccggaagc agagccccag aaacagactg
tggctgctga ccatcctggt gctgctgatc 540cccctggtgt tcatctacaa
cagcagaaga aacagaggcg gccagagcga ctacatgaac 600atgaccccca
gaaggcctgg cctgaccaga aagccctacc agccttacgc ccctgccaga
660gacttcgccg cctacagacc t 681118227PRTArtificial SequencemuFas
tm-CD28 118Met Leu Trp Ile Trp Ala Val Leu Pro Leu Val Leu
Ala Gly Ser Gln1 5 10 15Leu Arg Val His Thr Gln Gly Thr Asn Ser Ile
Ser Glu Ser Leu Lys 20 25 30Leu Arg Arg Arg Val Arg Glu Thr Asp Lys
Asn Cys Ser Glu Gly Leu 35 40 45Tyr Gln Gly Gly Pro Phe Cys Cys Gln
Pro Cys Gln Pro Gly Lys Lys 50 55 60Lys Val Glu Asp Cys Lys Met Asn
Gly Gly Thr Pro Thr Cys Ala Pro65 70 75 80Cys Thr Glu Gly Lys Glu
Tyr Met Asp Lys Asn His Tyr Ala Asp Lys 85 90 95Cys Arg Arg Cys Thr
Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu 100 105 110Thr Asn Cys
Thr Leu Thr Gln Asn Thr Lys Cys Lys Cys Lys Pro Asp 115 120 125Phe
Tyr Cys Asp Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser 130 135
140Cys Glu His Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr
Asn145 150 155 160Cys Arg Lys Gln Ser Pro Arg Asn Arg Leu Trp Leu
Leu Thr Ile Leu 165 170 175Val Leu Leu Ile Pro Leu Val Phe Ile Tyr
Asn Ser Arg Arg Asn Arg 180 185 190Gly Gly Gln Ser Asp Tyr Met Asn
Met Thr Pro Arg Arg Pro Gly Leu 195 200 205Thr Arg Lys Pro Tyr Gln
Pro Tyr Ala Pro Ala Arg Asp Phe Ala Ala 210 215 220Tyr Arg
Pro225119711DNAArtificial SequencemuFas-CD28tm 119atgctgtgga
tctgggccgt gctgcctctg gtgctggctg gatcacagct gagagtgcac 60acccagggca
ccaacagcat cagcgagagc ctgaagctga gaagaagagt gcgcgagaca
120gacaagaact gcagcgaggg cctgtaccag ggcggaccct tctgctgtca
gccttgccag 180cccggcaaga aaaaggtgga agattgcaag atgaacggcg
gcacccctac ctgcgcccct 240tgtacagagg gcaaagagta catggacaag
aaccactacg ccgacaagtg cagacggtgc 300accctgtgcg acgaggaaca
cggcctggaa gtggaaacaa actgcaccct gacccagaac 360accaagtgca
agtgcaaacc cgacttctac tgcgacagcc ccggctgcga gcactgcgtc
420agatgtgcct cttgcgagca cggcaccctg gaaccttgta ccgccaccag
caacaccaac 480tgccggaagc agagccccag aaacagattc tgggccctgg
tggtggtggc cggcgtgctg 540ttttgttacg gcctgctcgt gaccgtggcc
ctgtgcgtga tctggaccaa cagcagaaga 600aacagaggcg gccagagcga
ctacatgaac atgaccccca gaaggcctgg cctgaccaga 660aagccctacc
agccttacgc ccctgccaga gacttcgccg cctacagacc t
711120237PRTArtificial SequencemuFas-CD28tm 120Met Leu Trp Ile Trp
Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gln1 5 10 15Leu Arg Val His
Thr Gln Gly Thr Asn Ser Ile Ser Glu Ser Leu Lys 20 25 30Leu Arg Arg
Arg Val Arg Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu 35 40 45Tyr Gln
Gly Gly Pro Phe Cys Cys Gln Pro Cys Gln Pro Gly Lys Lys 50 55 60Lys
Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro65 70 75
80Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys
85 90 95Cys Arg Arg Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val
Glu 100 105 110Thr Asn Cys Thr Leu Thr Gln Asn Thr Lys Cys Lys Cys
Lys Pro Asp 115 120 125Phe Tyr Cys Asp Ser Pro Gly Cys Glu His Cys
Val Arg Cys Ala Ser 130 135 140Cys Glu His Gly Thr Leu Glu Pro Cys
Thr Ala Thr Ser Asn Thr Asn145 150 155 160Cys Arg Lys Gln Ser Pro
Arg Asn Arg Phe Trp Ala Leu Val Val Val 165 170 175Ala Gly Val Leu
Phe Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys 180 185 190Val Ile
Trp Thr Asn Ser Arg Arg Asn Arg Gly Gly Gln Ser Asp Tyr 195 200
205Met Asn Met Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln
210 215 220Pro Tyr Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro225
230 235121738DNAArtificial SequencemuFas-CD28Cys 121atgctgtgga
tctgggccgt gctgcctctg gtgctggctg gatcacagct gagagtgcac 60acccagggca
ccaacagcat cagcgagagc ctgaagctga gaagaagagt gcgcgagaca
120gacaagaact gcagcgaggg cctgtaccag ggcggaccct tctgctgtca
gccttgccag 180cccggcaaga aaaaggtgga agattgcaag atgaacggcg
gcacccctac ctgcgcccct 240tgtacagagg gcaaagagta catggacaag
aaccactacg ccgacaagtg cagacggtgc 300accctgtgcg acgaggaaca
cggcctggaa gtggaaacaa actgcaccct gacccagaac 360accaagtgca
agtgcaaacc cgacttctac tgcgacagcc ccggctgcga gcactgcgtc
420agatgtgcct cttgcgagca cggcaccctg gaaccttgta ccgccaccag
caacaccaac 480tgccggaagc agagccccag aaacagatgc cacacccaga
gcagccccaa gctgttctgg 540gccctggtgg tggtggccgg cgtgctgttt
tgttacggcc tgctcgtgac cgtggccctg 600tgcgtgatct ggaccaacag
cagaagaaac agaggcggcc agagcgacta catgaacatg 660acccccagaa
ggcctggcct gaccagaaag ccctaccagc cttacgcccc tgccagagac
720ttcgccgcct acagacct 738122246PRTArtificial SequencemuFas-CD28Cys
122Met Leu Trp Ile Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gln1
5 10 15Leu Arg Val His Thr Gln Gly Thr Asn Ser Ile Ser Glu Ser Leu
Lys 20 25 30Leu Arg Arg Arg Val Arg Glu Thr Asp Lys Asn Cys Ser Glu
Gly Leu 35 40 45Tyr Gln Gly Gly Pro Phe Cys Cys Gln Pro Cys Gln Pro
Gly Lys Lys 50 55 60Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro
Thr Cys Ala Pro65 70 75 80Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys
Asn His Tyr Ala Asp Lys 85 90 95Cys Arg Arg Cys Thr Leu Cys Asp Glu
Glu His Gly Leu Glu Val Glu 100 105 110Thr Asn Cys Thr Leu Thr Gln
Asn Thr Lys Cys Lys Cys Lys Pro Asp 115 120 125Phe Tyr Cys Asp Ser
Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser 130 135 140Cys Glu His
Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn145 150 155
160Cys Arg Lys Gln Ser Pro Arg Asn Arg Cys His Thr Gln Ser Ser Pro
165 170 175Lys Leu Phe Trp Ala Leu Val Val Val Ala Gly Val Leu Phe
Cys Tyr 180 185 190Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp
Thr Asn Ser Arg 195 200 205Arg Asn Arg Gly Gly Gln Ser Asp Tyr Met
Asn Met Thr Pro Arg Arg 210 215 220Pro Gly Leu Thr Arg Lys Pro Tyr
Gln Pro Tyr Ala Pro Ala Arg Asp225 230 235 240Phe Ala Ala Tyr Arg
Pro 245123711DNAArtificial SequencemuFas-9aas-CD28Cys 123atgctgtgga
tctgggccgt gctgcctctg gtgctggctg gatcacagct gagagtgcac 60acccagggca
ccaacagcat cagcgagagc ctgaagctga gaagaagagt gcgcgagaca
120gacaagaact gcagcgaggg cctgtaccag ggcggaccct tctgctgtca
gccttgccag 180cccggcaaga aaaaggtgga agattgcaag atgaacggcg
gcacccctac ctgcgcccct 240tgtacagagg gcaaagagta catggacaag
aaccactacg ccgacaagtg cagacggtgc 300accctgtgcg acgaggaaca
cggcctggaa gtggaaacaa actgcaccct gacccagaac 360accaagtgca
agtgcaaacc cgacttctac tgcgacagcc ccggctgcga gcactgcgtc
420agatgtgcct cttgcgagca cggcaccctg gaaccttgta ccgccaccag
caacaccaac 480tgccacaccc agagcagccc caagctgttc tgggccctgg
tggtggtggc cggcgtgctg 540ttttgttacg gcctgctcgt gaccgtggcc
ctgtgcgtga tctggaccaa cagcagaaga 600aacagaggcg gccagagcga
ctacatgaac atgaccccca gaaggcctgg cctgaccaga 660aagccctacc
agccttacgc ccctgccaga gacttcgccg cctacagacc t
711124237PRTArtificial SequencemuFas-9aas-CD28Cys 124Met Leu Trp
Ile Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gln1 5 10 15Leu Arg
Val His Thr Gln Gly Thr Asn Ser Ile Ser Glu Ser Leu Lys 20 25 30Leu
Arg Arg Arg Val Arg Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu 35 40
45Tyr Gln Gly Gly Pro Phe Cys Cys Gln Pro Cys Gln Pro Gly Lys Lys
50 55 60Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala
Pro65 70 75 80Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr
Ala Asp Lys 85 90 95Cys Arg Arg Cys Thr Leu Cys Asp Glu Glu His Gly
Leu Glu Val Glu 100 105 110Thr Asn Cys Thr Leu Thr Gln Asn Thr Lys
Cys Lys Cys Lys Pro Asp 115 120 125Phe Tyr Cys Asp Ser Pro Gly Cys
Glu His Cys Val Arg Cys Ala Ser 130 135 140Cys Glu His Gly Thr Leu
Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn145 150 155 160Cys His Thr
Gln Ser Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val 165 170 175Ala
Gly Val Leu Phe Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys 180 185
190Val Ile Trp Thr Asn Ser Arg Arg Asn Arg Gly Gly Gln Ser Asp Tyr
195 200 205Met Asn Met Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro
Tyr Gln 210 215 220Pro Tyr Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg
Pro225 230 235125693DNAArtificial SequencemuPD1tm-CD28
125atgtgggtgc gacaggtgcc ctggtctttc acctgggctg tgctgcagct
gagctggcag 60tctggctggc tgctggaagt gcctaacggc ccttggagaa gcctgacctt
ctaccccgct 120tggctgaccg tgtctgaggg cgccaacgcc accttcacct
gtagcctgag caattggagc 180gaggacctga tgctgaactg gaacagactg
agccccagca accagaccga gaagcaggcc 240gccttctgca acggcctgtc
tcagcctgtg caggacgcca gattccagat catccagctg 300cccaacagac
acgacttcca catgaacatc ctggacacca gaagaaacga cagcggcatc
360tacctgtgcg gcgccatcag cctgcacccc aaggccaaga tcgaggaatc
tcctggcgcc 420gagctggtcg tgaccgagag aatcctggaa acctccacca
gataccccag ccccagccct 480aagcccgagg gcagatttca gggcatggtc
atcggcatca tgagcgccct cgtgggcatc 540ccagtgttgc tgctgctggc
ctgggccctg aacagcagaa gaaacagagg cggccagagc 600gactacatga
acatgacccc cagaaggcct ggcctgacca gaaagcccta ccagccttac
660gcccctgcca gagacttcgc cgcctacaga cct 693126231PRTArtificial
SequencemuPD1tm-CD28 126Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr
Trp Ala Val Leu Gln1 5 10 15Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu
Val Pro Asn Gly Pro Trp 20 25 30Arg Ser Leu Thr Phe Tyr Pro Ala Trp
Leu Thr Val Ser Glu Gly Ala 35 40 45Asn Ala Thr Phe Thr Cys Ser Leu
Ser Asn Trp Ser Glu Asp Leu Met 50 55 60Leu Asn Trp Asn Arg Leu Ser
Pro Ser Asn Gln Thr Glu Lys Gln Ala65 70 75 80Ala Phe Cys Asn Gly
Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln 85 90 95Ile Ile Gln Leu
Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp 100 105 110Thr Arg
Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120
125His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val
130 135 140Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro
Ser Pro145 150 155 160Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile
Gly Ile Met Ser Ala 165 170 175Leu Val Gly Ile Pro Val Leu Leu Leu
Leu Ala Trp Ala Leu Asn Ser 180 185 190Arg Arg Asn Arg Gly Gly Gln
Ser Asp Tyr Met Asn Met Thr Pro Arg 195 200 205Arg Pro Gly Leu Thr
Arg Lys Pro Tyr Gln Pro Tyr Ala Pro Ala Arg 210 215 220Asp Phe Ala
Ala Tyr Arg Pro225 230127711DNAArtificial SequencemuPD1-CD28tm
127atgtgggtgc gacaggtgcc ctggtctttc acctgggctg tgctgcagct
gagctggcag 60tctggctggc tgctggaagt gcctaacggc ccttggagaa gcctgacctt
ctaccccgct 120tggctgaccg tgtctgaggg cgccaacgcc accttcacct
gtagcctgag caattggagc 180gaggacctga tgctgaactg gaacagactg
agccccagca accagaccga gaagcaggcc 240gccttctgca acggcctgtc
tcagcctgtg caggacgcca gattccagat catccagctg 300cccaacagac
acgacttcca catgaacatc ctggacacca gaagaaacga cagcggcatc
360tacctgtgcg gcgccatcag cctgcacccc aaggccaaga tcgaggaatc
tcctggcgcc 420gagctggtcg tgaccgagag aatcctggaa acctccacca
gataccccag ccccagccct 480aagcccgagg gcagatttca gggcatgttc
tgggccctgg tggtggtggc cggcgtgctg 540ttttgttacg gcctgctcgt
gaccgtggcc ctgtgcgtga tctggaccaa cagcagaaga 600aacagaggcg
gccagagcga ctacatgaac atgaccccca gaaggcctgg cctgaccaga
660aagccctacc agccttacgc ccctgccaga gacttcgccg cctacagacc t
711128237PRTArtificial SequencemuPD1-CD28tm 128Met Trp Val Arg Gln
Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln1 5 10 15Leu Ser Trp Gln
Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp 20 25 30Arg Ser Leu
Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala 35 40 45Asn Ala
Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met 50 55 60Leu
Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala65 70 75
80Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln
85 90 95Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu
Asp 100 105 110Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala
Ile Ser Leu 115 120 125His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly
Ala Glu Leu Val Val 130 135 140Thr Glu Arg Ile Leu Glu Thr Ser Thr
Arg Tyr Pro Ser Pro Ser Pro145 150 155 160Lys Pro Glu Gly Arg Phe
Gln Gly Met Phe Trp Ala Leu Val Val Val 165 170 175Ala Gly Val Leu
Phe Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys 180 185 190Val Ile
Trp Thr Asn Ser Arg Arg Asn Arg Gly Gly Gln Ser Asp Tyr 195 200
205Met Asn Met Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln
210 215 220Pro Tyr Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro225
230 235129738DNAArtificial SequencemuPD1-CD28Cys 129atgtgggtgc
gacaggtgcc ctggtctttc acctgggctg tgctgcagct gagctggcag 60tctggctggc
tgctggaagt gcctaacggc ccttggagaa gcctgacctt ctaccccgct
120tggctgaccg tgtctgaggg cgccaacgcc accttcacct gtagcctgag
caattggagc 180gaggacctga tgctgaactg gaacagactg agccccagca
accagaccga gaagcaggcc 240gccttctgca acggcctgtc tcagcctgtg
caggacgcca gattccagat catccagctg 300cccaacagac acgacttcca
catgaacatc ctggacacca gaagaaacga cagcggcatc 360tacctgtgcg
gcgccatcag cctgcacccc aaggccaaga tcgaggaatc tcctggcgcc
420gagctggtcg tgaccgagag aatcctggaa acctccacca gataccccag
ccccagccct 480aagcccgagg gcagatttca gggcatgtgc cacacccaga
gcagccccaa gctgttctgg 540gctctggtgg tggtggccgg cgtgctgttt
tgttacggcc tgctcgtgac cgtggccctg 600tgcgtgatct ggaccaacag
cagacggaac agaggcggcc agagcgacta catgaatatg 660acccccagaa
ggcctggcct gaccagaaag ccctaccagc cttacgcccc tgccagagac
720ttcgccgcct acagacct 738130246PRTArtificial SequencemuPD1-CD28Cys
130Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln1
5 10 15Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro
Trp 20 25 30Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu
Gly Ala 35 40 45Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu
Asp Leu Met 50 55 60Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr
Glu Lys Gln Ala65 70 75 80Ala Phe Cys Asn Gly Leu Ser Gln Pro Val
Gln Asp Ala Arg Phe Gln 85 90 95Ile Ile Gln Leu Pro Asn Arg His Asp
Phe His Met Asn Ile Leu Asp 100 105 110Thr Arg Arg Asn Asp Ser Gly
Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125His Pro Lys Ala Lys
Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val 130 135 140Thr Glu Arg
Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro145 150 155
160Lys Pro Glu Gly Arg Phe Gln Gly Met Cys His Thr Gln Ser Ser Pro
165 170 175Lys Leu Phe Trp Ala Leu Val Val Val Ala Gly Val Leu Phe
Cys Tyr 180 185 190Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp
Thr Asn Ser Arg 195 200 205Arg Asn Arg Gly Gly Gln Ser Asp Tyr Met
Asn Met Thr Pro Arg Arg 210 215 220Pro Gly Leu Thr Arg Lys Pro Tyr
Gln Pro Tyr Ala Pro Ala Arg Asp225 230 235 240Phe Ala Ala Tyr Arg
Pro 245131711DNAArtificial SequencemuPD1-9aas-CD28Cys 131atgtgggtgc
gacaggtgcc ctggtctttc acctgggctg tgctgcagct gagctggcag 60tctggctggc
tgctggaagt gcctaacggc ccttggagaa
gcctgacctt ctaccccgct 120tggctgaccg tgtctgaggg cgccaacgcc
accttcacct gtagcctgag caattggagc 180gaggacctga tgctgaactg
gaacagactg agccccagca accagaccga gaagcaggcc 240gccttctgca
acggcctgtc tcagcctgtg caggacgcca gattccagat catccagctg
300cccaacagac acgacttcca catgaacatc ctggacacca gaagaaacga
cagcggcatc 360tacctgtgcg gcgccatcag cctgcacccc aaggccaaga
tcgaggaatc tcctggcgcc 420gagctggtcg tgaccgagag aatcctggaa
acctccacca gataccccag ccccagccct 480tgccacaccc agagcagccc
caagctgttc tgggctctgg tggtggtggc cggcgtgctg 540ttttgttacg
gcctgctcgt gaccgtggcc ctgtgcgtga tctggaccaa cagcagacgg
600aacagaggcg gccagagcga ctacatgaat atgaccccca gaaggcctgg
cctgaccaga 660aagccctacc agccttacgc ccctgccaga gacttcgccg
cctacagacc t 711132237PRTArtificial SequencemuPD1-9aas-CD28Cys
132Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln1
5 10 15Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro
Trp 20 25 30Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu
Gly Ala 35 40 45Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu
Asp Leu Met 50 55 60Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr
Glu Lys Gln Ala65 70 75 80Ala Phe Cys Asn Gly Leu Ser Gln Pro Val
Gln Asp Ala Arg Phe Gln 85 90 95Ile Ile Gln Leu Pro Asn Arg His Asp
Phe His Met Asn Ile Leu Asp 100 105 110Thr Arg Arg Asn Asp Ser Gly
Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125His Pro Lys Ala Lys
Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val 130 135 140Thr Glu Arg
Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro145 150 155
160Cys His Thr Gln Ser Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val
165 170 175Ala Gly Val Leu Phe Cys Tyr Gly Leu Leu Val Thr Val Ala
Leu Cys 180 185 190Val Ile Trp Thr Asn Ser Arg Arg Asn Arg Gly Gly
Gln Ser Asp Tyr 195 200 205Met Asn Met Thr Pro Arg Arg Pro Gly Leu
Thr Arg Lys Pro Tyr Gln 210 215 220Pro Tyr Ala Pro Ala Arg Asp Phe
Ala Ala Tyr Arg Pro225 230 235133675DNAArtificial
SequencemuPD1-21aas-CD28Cys 133atgtgggtgc gacaggtgcc ctggtctttc
acctgggctg tgctgcagct gagctggcag 60tctggctggc tgctggaagt gcctaacggc
ccttggagaa gcctgacctt ctaccccgct 120tggctgaccg tgtctgaggg
cgccaacgcc accttcacct gtagcctgag caattggagc 180gaggacctga
tgctgaactg gaacagactg agccccagca accagaccga gaagcaggcc
240gccttctgca acggcctgtc tcagcctgtg caggacgcca gattccagat
catccagctg 300cccaacagac acgacttcca catgaacatc ctggacacca
gaagaaacga cagcggcatc 360tacctgtgcg gcgccatcag cctgcacccc
aaggccaaga tcgaggaatc tcctggcgcc 420gagctggtcg tgaccgagag
aatctgccac acccagagca gccccaagct gttctgggct 480ctggtggtgg
tggccggcgt gctgttttgt tacggcctgc tcgtgaccgt ggccctgtgc
540gtgatctgga ccaacagcag acggaacaga ggcggccaga gcgactacat
gaatatgacc 600cccagaaggc ctggcctgac cagaaagccc taccagcctt
acgcccctgc cagagacttc 660gccgcctaca gacct 675134225PRTArtificial
SequencemuPD1-21aas-CD28Cys 134Met Trp Val Arg Gln Val Pro Trp Ser
Phe Thr Trp Ala Val Leu Gln1 5 10 15Leu Ser Trp Gln Ser Gly Trp Leu
Leu Glu Val Pro Asn Gly Pro Trp 20 25 30Arg Ser Leu Thr Phe Tyr Pro
Ala Trp Leu Thr Val Ser Glu Gly Ala 35 40 45Asn Ala Thr Phe Thr Cys
Ser Leu Ser Asn Trp Ser Glu Asp Leu Met 50 55 60Leu Asn Trp Asn Arg
Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala65 70 75 80Ala Phe Cys
Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln 85 90 95Ile Ile
Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp 100 105
110Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu
Val Val 130 135 140Thr Glu Arg Ile Cys His Thr Gln Ser Ser Pro Lys
Leu Phe Trp Ala145 150 155 160Leu Val Val Val Ala Gly Val Leu Phe
Cys Tyr Gly Leu Leu Val Thr 165 170 175Val Ala Leu Cys Val Ile Trp
Thr Asn Ser Arg Arg Asn Arg Gly Gly 180 185 190Gln Ser Asp Tyr Met
Asn Met Thr Pro Arg Arg Pro Gly Leu Thr Arg 195 200 205Lys Pro Tyr
Gln Pro Tyr Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg 210 215
220Pro2251351512DNAArtificial SequencemuLag3tm-CD28 135atgagagagg
acctgctgct gggctttctg ctgctgggac tgctgtggga ggcccctgtg 60gtgtcatctg
gccctggcaa agaactgccc gtcgtgtggg ctcaggaagg cgctcctgtg
120catctgccct gcagcctgaa gtcccccaac ctggacccca acttcctgag
aagaggcggc 180gtgatctggc agcaccagcc tgattctggc cagcccacac
ctatccctgc cctggatctg 240caccagggca tgcctagccc tagacagcct
gcccctggca gatacaccgt gctgtctgtg 300gctcctggcg gcctgagaag
tggcagacag cctctgcacc ctcacgtgca gctggaagag 360aggggactgc
agaggggcga cttcagcctg tggctgaggc ctgccctgag aacagatgcc
420ggcgagtacc acgctaccgt gcggctgcct aacagagccc tgagctgctc
cctgagactg 480agagtgggcc aggccagcat gatcgcctct ccatctggcg
tgctgaagct gagcgactgg 540gtgctgctga actgcagctt ctccagaccc
gacagacccg tgtccgtgca ctggttccag 600ggacagaaca gagtgcccgt
gtacaacagc cccagacact tcctggccga gacattcctg 660ctgctgcccc
aggtgtcccc tctggactct ggcacatggg gctgcgtgct gacatacagg
720gacggcttca acgtgtccat cacctacaac ctgaaggtgc tgggcctgga
acccgtggct 780cctctgacag tgtacgccgc cgagggcagc agagtggaac
tgccttgtca tctgccaccc 840ggcgtgggca caccttctct gctgatcgcc
aagtggaccc ctccaggcgg aggacctgaa 900ctgccagtgg ctggcaagag
cggcaacttc accctgcacc tggaagcagt gggcctggct 960caggccggca
cctacacctg tagcatccat ctgcagggcc agcagctgaa cgccaccgtg
1020acactggccg tgatcaccgt gacccccaag agctttggcc tgcctggctc
cagaggcaag 1080ctgctgtgtg aagtgacccc cgccagcggc aaagaaagat
tcgtgtggcg gcctctgaac 1140aacctgagca gatcctgccc aggccccgtg
ctggaaatcc aggaagccag actgctggcc 1200gagcggtggc agtgccagct
gtatgaggga cagcgactgc tgggcgccac tgtgtacgct 1260gctgagtcta
gctctggcgc ccacagcgcc agaagaatca gcggcgatct gaagggcggc
1320cacctggtgc tggtgctgat cctgggcgct ctgagcctgt tcctgctggt
ggctggcgct 1380ttcggcttta acagcagaag aaacagaggc ggccagagcg
actacatgaa catgaccccc 1440agaaggcctg gcctgaccag aaagccctac
cagccttacg cccctgccag agacttcgcc 1500gcctacagac ct
1512136504PRTArtificial SequencemuLag3tm-CD28 136Met Arg Glu Asp
Leu Leu Leu Gly Phe Leu Leu Leu Gly Leu Leu Trp1 5 10 15Glu Ala Pro
Val Val Ser Ser Gly Pro Gly Lys Glu Leu Pro Val Val 20 25 30Trp Ala
Gln Glu Gly Ala Pro Val His Leu Pro Cys Ser Leu Lys Ser 35 40 45Pro
Asn Leu Asp Pro Asn Phe Leu Arg Arg Gly Gly Val Ile Trp Gln 50 55
60His Gln Pro Asp Ser Gly Gln Pro Thr Pro Ile Pro Ala Leu Asp Leu65
70 75 80His Gln Gly Met Pro Ser Pro Arg Gln Pro Ala Pro Gly Arg Tyr
Thr 85 90 95Val Leu Ser Val Ala Pro Gly Gly Leu Arg Ser Gly Arg Gln
Pro Leu 100 105 110His Pro His Val Gln Leu Glu Glu Arg Gly Leu Gln
Arg Gly Asp Phe 115 120 125Ser Leu Trp Leu Arg Pro Ala Leu Arg Thr
Asp Ala Gly Glu Tyr His 130 135 140Ala Thr Val Arg Leu Pro Asn Arg
Ala Leu Ser Cys Ser Leu Arg Leu145 150 155 160Arg Val Gly Gln Ala
Ser Met Ile Ala Ser Pro Ser Gly Val Leu Lys 165 170 175Leu Ser Asp
Trp Val Leu Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg 180 185 190Pro
Val Ser Val His Trp Phe Gln Gly Gln Asn Arg Val Pro Val Tyr 195 200
205Asn Ser Pro Arg His Phe Leu Ala Glu Thr Phe Leu Leu Leu Pro Gln
210 215 220Val Ser Pro Leu Asp Ser Gly Thr Trp Gly Cys Val Leu Thr
Tyr Arg225 230 235 240Asp Gly Phe Asn Val Ser Ile Thr Tyr Asn Leu
Lys Val Leu Gly Leu 245 250 255Glu Pro Val Ala Pro Leu Thr Val Tyr
Ala Ala Glu Gly Ser Arg Val 260 265 270Glu Leu Pro Cys His Leu Pro
Pro Gly Val Gly Thr Pro Ser Leu Leu 275 280 285Ile Ala Lys Trp Thr
Pro Pro Gly Gly Gly Pro Glu Leu Pro Val Ala 290 295 300Gly Lys Ser
Gly Asn Phe Thr Leu His Leu Glu Ala Val Gly Leu Ala305 310 315
320Gln Ala Gly Thr Tyr Thr Cys Ser Ile His Leu Gln Gly Gln Gln Leu
325 330 335Asn Ala Thr Val Thr Leu Ala Val Ile Thr Val Thr Pro Lys
Ser Phe 340 345 350Gly Leu Pro Gly Ser Arg Gly Lys Leu Leu Cys Glu
Val Thr Pro Ala 355 360 365Ser Gly Lys Glu Arg Phe Val Trp Arg Pro
Leu Asn Asn Leu Ser Arg 370 375 380Ser Cys Pro Gly Pro Val Leu Glu
Ile Gln Glu Ala Arg Leu Leu Ala385 390 395 400Glu Arg Trp Gln Cys
Gln Leu Tyr Glu Gly Gln Arg Leu Leu Gly Ala 405 410 415Thr Val Tyr
Ala Ala Glu Ser Ser Ser Gly Ala His Ser Ala Arg Arg 420 425 430Ile
Ser Gly Asp Leu Lys Gly Gly His Leu Val Leu Val Leu Ile Leu 435 440
445Gly Ala Leu Ser Leu Phe Leu Leu Val Ala Gly Ala Phe Gly Phe Asn
450 455 460Ser Arg Arg Asn Arg Gly Gly Gln Ser Asp Tyr Met Asn Met
Thr Pro465 470 475 480Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln
Pro Tyr Ala Pro Ala 485 490 495Arg Asp Phe Ala Ala Tyr Arg Pro
5001371530DNAArtificial SequencemuLag3-CD28tm 137atgagagagg
acctgctgct gggctttctg ctgctgggac tgctgtggga ggcccctgtg 60gtgtcatctg
gccctggcaa agaactgccc gtcgtgtggg ctcaggaagg cgctcctgtg
120catctgccct gcagcctgaa gtcccccaac ctggacccca acttcctgag
aagaggcggc 180gtgatctggc agcaccagcc tgattctggc cagcccacac
ctatccctgc cctggatctg 240caccagggca tgcctagccc tagacagcct
gcccctggca gatacaccgt gctgtctgtg 300gctcctggcg gcctgagaag
tggcagacag cctctgcacc ctcacgtgca gctggaagag 360aggggactgc
agaggggcga cttcagcctg tggctgaggc ctgccctgag aacagatgcc
420ggcgagtacc acgctaccgt gcggctgcct aacagagccc tgagctgctc
cctgagactg 480agagtgggcc aggccagcat gatcgcctct ccatctggcg
tgctgaagct gagcgactgg 540gtgctgctga actgcagctt ctccagaccc
gacagacccg tgtccgtgca ctggttccag 600ggacagaaca gagtgcccgt
gtacaacagc cccagacact tcctggccga gacattcctg 660ctgctgcccc
aggtgtcccc tctggactct ggcacatggg gctgcgtgct gacatacagg
720gacggcttca acgtgtccat cacctacaac ctgaaggtgc tgggcctgga
acccgtggct 780cctctgacag tgtacgccgc cgagggcagc agagtggaac
tgccttgtca tctgccaccc 840ggcgtgggca caccttctct gctgatcgcc
aagtggaccc ctccaggcgg aggacctgaa 900ctgccagtgg ctggcaagag
cggcaacttc accctgcacc tggaagcagt gggcctggct 960caggccggca
cctacacctg tagcatccat ctgcagggcc agcagctgaa cgccaccgtg
1020acactggccg tgatcaccgt gacccccaag agctttggcc tgcctggctc
cagaggcaag 1080ctgctgtgtg aagtgacccc cgccagcggc aaagaaagat
tcgtgtggcg gcctctgaac 1140aacctgagca gatcctgccc aggccccgtg
ctggaaatcc aggaagccag actgctggcc 1200gagcggtggc agtgccagct
gtatgaggga cagcgactgc tgggcgccac tgtgtacgct 1260gctgagtcta
gctctggcgc ccacagcgcc agaagaatca gcggcgatct gaagggcggc
1320cacctgttct gggccctggt ggtggtggcc ggcgtgctgt tttgttacgg
cctgctcgtg 1380accgtggccc tgtgcgtgat ctggaccaac agcagaagaa
acagaggcgg ccagagcgac 1440tacatgaaca tgacccccag aaggcctggc
ctgaccagaa agccctacca gccttacgcc 1500cctgccagag acttcgccgc
ctacagacct 1530138510PRTArtificial SequencemuLag3-CD28tm 138Met Arg
Glu Asp Leu Leu Leu Gly Phe Leu Leu Leu Gly Leu Leu Trp1 5 10 15Glu
Ala Pro Val Val Ser Ser Gly Pro Gly Lys Glu Leu Pro Val Val 20 25
30Trp Ala Gln Glu Gly Ala Pro Val His Leu Pro Cys Ser Leu Lys Ser
35 40 45Pro Asn Leu Asp Pro Asn Phe Leu Arg Arg Gly Gly Val Ile Trp
Gln 50 55 60His Gln Pro Asp Ser Gly Gln Pro Thr Pro Ile Pro Ala Leu
Asp Leu65 70 75 80His Gln Gly Met Pro Ser Pro Arg Gln Pro Ala Pro
Gly Arg Tyr Thr 85 90 95Val Leu Ser Val Ala Pro Gly Gly Leu Arg Ser
Gly Arg Gln Pro Leu 100 105 110His Pro His Val Gln Leu Glu Glu Arg
Gly Leu Gln Arg Gly Asp Phe 115 120 125Ser Leu Trp Leu Arg Pro Ala
Leu Arg Thr Asp Ala Gly Glu Tyr His 130 135 140Ala Thr Val Arg Leu
Pro Asn Arg Ala Leu Ser Cys Ser Leu Arg Leu145 150 155 160Arg Val
Gly Gln Ala Ser Met Ile Ala Ser Pro Ser Gly Val Leu Lys 165 170
175Leu Ser Asp Trp Val Leu Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg
180 185 190Pro Val Ser Val His Trp Phe Gln Gly Gln Asn Arg Val Pro
Val Tyr 195 200 205Asn Ser Pro Arg His Phe Leu Ala Glu Thr Phe Leu
Leu Leu Pro Gln 210 215 220Val Ser Pro Leu Asp Ser Gly Thr Trp Gly
Cys Val Leu Thr Tyr Arg225 230 235 240Asp Gly Phe Asn Val Ser Ile
Thr Tyr Asn Leu Lys Val Leu Gly Leu 245 250 255Glu Pro Val Ala Pro
Leu Thr Val Tyr Ala Ala Glu Gly Ser Arg Val 260 265 270Glu Leu Pro
Cys His Leu Pro Pro Gly Val Gly Thr Pro Ser Leu Leu 275 280 285Ile
Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Glu Leu Pro Val Ala 290 295
300Gly Lys Ser Gly Asn Phe Thr Leu His Leu Glu Ala Val Gly Leu
Ala305 310 315 320Gln Ala Gly Thr Tyr Thr Cys Ser Ile His Leu Gln
Gly Gln Gln Leu 325 330 335Asn Ala Thr Val Thr Leu Ala Val Ile Thr
Val Thr Pro Lys Ser Phe 340 345 350Gly Leu Pro Gly Ser Arg Gly Lys
Leu Leu Cys Glu Val Thr Pro Ala 355 360 365Ser Gly Lys Glu Arg Phe
Val Trp Arg Pro Leu Asn Asn Leu Ser Arg 370 375 380Ser Cys Pro Gly
Pro Val Leu Glu Ile Gln Glu Ala Arg Leu Leu Ala385 390 395 400Glu
Arg Trp Gln Cys Gln Leu Tyr Glu Gly Gln Arg Leu Leu Gly Ala 405 410
415Thr Val Tyr Ala Ala Glu Ser Ser Ser Gly Ala His Ser Ala Arg Arg
420 425 430Ile Ser Gly Asp Leu Lys Gly Gly His Leu Phe Trp Ala Leu
Val Val 435 440 445Val Ala Gly Val Leu Phe Cys Tyr Gly Leu Leu Val
Thr Val Ala Leu 450 455 460Cys Val Ile Trp Thr Asn Ser Arg Arg Asn
Arg Gly Gly Gln Ser Asp465 470 475 480Tyr Met Asn Met Thr Pro Arg
Arg Pro Gly Leu Thr Arg Lys Pro Tyr 485 490 495Gln Pro Tyr Ala Pro
Ala Arg Asp Phe Ala Ala Tyr Arg Pro 500 505 5101391557DNAArtificial
SequencemuLag3-CD28Cys 139atgagagagg acctgctgct gggctttctg
ctgctgggac tgctgtggga ggcccctgtg 60gtgtcatctg gccctggcaa agaactgccc
gtcgtgtggg ctcaggaagg cgctcctgtg 120catctgccct gcagcctgaa
gtcccccaac ctggacccca acttcctgag aagaggcggc 180gtgatctggc
agcaccagcc tgattctggc cagcccacac ctatccctgc cctggatctg
240caccagggca tgcctagccc tagacagcct gcccctggca gatacaccgt
gctgtctgtg 300gctcctggcg gcctgagaag tggcagacag cctctgcacc
ctcacgtgca gctggaagag 360aggggactgc agaggggcga cttcagcctg
tggctgaggc ctgccctgag aacagatgcc 420ggcgagtacc acgctaccgt
gcggctgcct aacagagccc tgagctgctc cctgagactg 480agagtgggcc
aggccagcat gatcgcctct ccatctggcg tgctgaagct gagcgactgg
540gtgctgctga actgcagctt ctccagaccc gacagacccg tgtccgtgca
ctggttccag 600ggacagaaca gagtgcccgt gtacaacagc cccagacact
tcctggccga gacattcctg 660ctgctgcccc aggtgtcccc tctggactct
ggcacatggg gctgcgtgct gacatacagg 720gacggcttca acgtgtccat
cacctacaac ctgaaggtgc tgggcctgga acccgtggct 780cctctgacag
tgtacgccgc cgagggcagc agagtggaac tgccttgtca tctgccaccc
840ggcgtgggca caccttctct gctgatcgcc aagtggaccc ctccaggcgg
aggacctgaa 900ctgccagtgg ctggcaagag cggcaacttc accctgcacc
tggaagcagt gggcctggct 960caggccggca cctacacctg tagcatccat
ctgcagggcc agcagctgaa cgccaccgtg 1020acactggccg tgatcaccgt
gacccccaag agctttggcc tgcctggctc cagaggcaag 1080ctgctgtgtg
aagtgacccc cgccagcggc aaagaaagat tcgtgtggcg gcctctgaac
1140aacctgagca gatcctgccc aggccccgtg ctggaaatcc aggaagccag
actgctggcc 1200gagcggtggc agtgccagct gtatgaggga cagcgactgc
tgggcgccac tgtgtacgct 1260gctgagtcta gctctggcgc
ccacagcgcc agaagaatca gcggcgatct gaagggcggc 1320cacctgtgcc
acacccagag cagccccaag ctgttctggg ccctggtggt ggtggccggc
1380gtgctgtttt gttacggcct gctcgtgacc gtggccctgt gcgtgatctg
gaccaacagc 1440agaagaaaca gaggcggcca gagcgactac atgaacatga
cccccagaag gcctggcctg 1500accagaaagc cctaccagcc ttacgcccct
gccagagact tcgccgccta cagacct 1557140519PRTArtificial
SequencemuLag3-CD28Cys 140Met Arg Glu Asp Leu Leu Leu Gly Phe Leu
Leu Leu Gly Leu Leu Trp1 5 10 15Glu Ala Pro Val Val Ser Ser Gly Pro
Gly Lys Glu Leu Pro Val Val 20 25 30Trp Ala Gln Glu Gly Ala Pro Val
His Leu Pro Cys Ser Leu Lys Ser 35 40 45Pro Asn Leu Asp Pro Asn Phe
Leu Arg Arg Gly Gly Val Ile Trp Gln 50 55 60His Gln Pro Asp Ser Gly
Gln Pro Thr Pro Ile Pro Ala Leu Asp Leu65 70 75 80His Gln Gly Met
Pro Ser Pro Arg Gln Pro Ala Pro Gly Arg Tyr Thr 85 90 95Val Leu Ser
Val Ala Pro Gly Gly Leu Arg Ser Gly Arg Gln Pro Leu 100 105 110His
Pro His Val Gln Leu Glu Glu Arg Gly Leu Gln Arg Gly Asp Phe 115 120
125Ser Leu Trp Leu Arg Pro Ala Leu Arg Thr Asp Ala Gly Glu Tyr His
130 135 140Ala Thr Val Arg Leu Pro Asn Arg Ala Leu Ser Cys Ser Leu
Arg Leu145 150 155 160Arg Val Gly Gln Ala Ser Met Ile Ala Ser Pro
Ser Gly Val Leu Lys 165 170 175Leu Ser Asp Trp Val Leu Leu Asn Cys
Ser Phe Ser Arg Pro Asp Arg 180 185 190Pro Val Ser Val His Trp Phe
Gln Gly Gln Asn Arg Val Pro Val Tyr 195 200 205Asn Ser Pro Arg His
Phe Leu Ala Glu Thr Phe Leu Leu Leu Pro Gln 210 215 220Val Ser Pro
Leu Asp Ser Gly Thr Trp Gly Cys Val Leu Thr Tyr Arg225 230 235
240Asp Gly Phe Asn Val Ser Ile Thr Tyr Asn Leu Lys Val Leu Gly Leu
245 250 255Glu Pro Val Ala Pro Leu Thr Val Tyr Ala Ala Glu Gly Ser
Arg Val 260 265 270Glu Leu Pro Cys His Leu Pro Pro Gly Val Gly Thr
Pro Ser Leu Leu 275 280 285Ile Ala Lys Trp Thr Pro Pro Gly Gly Gly
Pro Glu Leu Pro Val Ala 290 295 300Gly Lys Ser Gly Asn Phe Thr Leu
His Leu Glu Ala Val Gly Leu Ala305 310 315 320Gln Ala Gly Thr Tyr
Thr Cys Ser Ile His Leu Gln Gly Gln Gln Leu 325 330 335Asn Ala Thr
Val Thr Leu Ala Val Ile Thr Val Thr Pro Lys Ser Phe 340 345 350Gly
Leu Pro Gly Ser Arg Gly Lys Leu Leu Cys Glu Val Thr Pro Ala 355 360
365Ser Gly Lys Glu Arg Phe Val Trp Arg Pro Leu Asn Asn Leu Ser Arg
370 375 380Ser Cys Pro Gly Pro Val Leu Glu Ile Gln Glu Ala Arg Leu
Leu Ala385 390 395 400Glu Arg Trp Gln Cys Gln Leu Tyr Glu Gly Gln
Arg Leu Leu Gly Ala 405 410 415Thr Val Tyr Ala Ala Glu Ser Ser Ser
Gly Ala His Ser Ala Arg Arg 420 425 430Ile Ser Gly Asp Leu Lys Gly
Gly His Leu Cys His Thr Gln Ser Ser 435 440 445Pro Lys Leu Phe Trp
Ala Leu Val Val Val Ala Gly Val Leu Phe Cys 450 455 460Tyr Gly Leu
Leu Val Thr Val Ala Leu Cys Val Ile Trp Thr Asn Ser465 470 475
480Arg Arg Asn Arg Gly Gly Gln Ser Asp Tyr Met Asn Met Thr Pro Arg
485 490 495Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro
Ala Arg 500 505 510Asp Phe Ala Ala Tyr Arg Pro
5151411530DNAArtificial SequencemuLag3-9aas-CD28Cys 141atgagagagg
acctgctgct gggctttctg ctgctgggac tgctgtggga ggcccctgtg 60gtgtcatctg
gccctggcaa agaactgccc gtcgtgtggg ctcaggaagg cgctcctgtg
120catctgccct gcagcctgaa gtcccccaac ctggacccca acttcctgag
aagaggcggc 180gtgatctggc agcaccagcc tgattctggc cagcccacac
ctatccctgc cctggatctg 240caccagggca tgcctagccc tagacagcct
gcccctggca gatacaccgt gctgtctgtg 300gctcctggcg gcctgagaag
tggcagacag cctctgcacc ctcacgtgca gctggaagag 360aggggactgc
agaggggcga cttcagcctg tggctgaggc ctgccctgag aacagatgcc
420ggcgagtacc acgctaccgt gcggctgcct aacagagccc tgagctgctc
cctgagactg 480agagtgggcc aggccagcat gatcgcctct ccatctggcg
tgctgaagct gagcgactgg 540gtgctgctga actgcagctt ctccagaccc
gacagacccg tgtccgtgca ctggttccag 600ggacagaaca gagtgcccgt
gtacaacagc cccagacact tcctggccga gacattcctg 660ctgctgcccc
aggtgtcccc tctggactct ggcacatggg gctgcgtgct gacatacagg
720gacggcttca acgtgtccat cacctacaac ctgaaggtgc tgggcctgga
acccgtggct 780cctctgacag tgtacgccgc cgagggcagc agagtggaac
tgccttgtca tctgccaccc 840ggcgtgggca caccttctct gctgatcgcc
aagtggaccc ctccaggcgg aggacctgaa 900ctgccagtgg ctggcaagag
cggcaacttc accctgcacc tggaagcagt gggcctggct 960caggccggca
cctacacctg tagcatccat ctgcagggcc agcagctgaa cgccaccgtg
1020acactggccg tgatcaccgt gacccccaag agctttggcc tgcctggctc
cagaggcaag 1080ctgctgtgtg aagtgacccc cgccagcggc aaagaaagat
tcgtgtggcg gcctctgaac 1140aacctgagca gatcctgccc aggccccgtg
ctggaaatcc aggaagccag actgctggcc 1200gagcggtggc agtgccagct
gtatgaggga cagcgactgc tgggcgccac tgtgtacgct 1260gctgagtcta
gctctggcgc ccacagcgcc agaagaatct gccacaccca gagcagcccc
1320aagctgttct gggccctggt ggtggtggcc ggcgtgctgt tttgttacgg
cctgctcgtg 1380accgtggccc tgtgcgtgat ctggaccaac agcagaagaa
acagaggcgg ccagagcgac 1440tacatgaaca tgacccccag aaggcctggc
ctgaccagaa agccctacca gccttacgcc 1500cctgccagag acttcgccgc
ctacagacct 1530142510PRTArtificial SequencemuLag3-9aas-CD28Cys
142Met Arg Glu Asp Leu Leu Leu Gly Phe Leu Leu Leu Gly Leu Leu Trp1
5 10 15Glu Ala Pro Val Val Ser Ser Gly Pro Gly Lys Glu Leu Pro Val
Val 20 25 30Trp Ala Gln Glu Gly Ala Pro Val His Leu Pro Cys Ser Leu
Lys Ser 35 40 45Pro Asn Leu Asp Pro Asn Phe Leu Arg Arg Gly Gly Val
Ile Trp Gln 50 55 60His Gln Pro Asp Ser Gly Gln Pro Thr Pro Ile Pro
Ala Leu Asp Leu65 70 75 80His Gln Gly Met Pro Ser Pro Arg Gln Pro
Ala Pro Gly Arg Tyr Thr 85 90 95Val Leu Ser Val Ala Pro Gly Gly Leu
Arg Ser Gly Arg Gln Pro Leu 100 105 110His Pro His Val Gln Leu Glu
Glu Arg Gly Leu Gln Arg Gly Asp Phe 115 120 125Ser Leu Trp Leu Arg
Pro Ala Leu Arg Thr Asp Ala Gly Glu Tyr His 130 135 140Ala Thr Val
Arg Leu Pro Asn Arg Ala Leu Ser Cys Ser Leu Arg Leu145 150 155
160Arg Val Gly Gln Ala Ser Met Ile Ala Ser Pro Ser Gly Val Leu Lys
165 170 175Leu Ser Asp Trp Val Leu Leu Asn Cys Ser Phe Ser Arg Pro
Asp Arg 180 185 190Pro Val Ser Val His Trp Phe Gln Gly Gln Asn Arg
Val Pro Val Tyr 195 200 205Asn Ser Pro Arg His Phe Leu Ala Glu Thr
Phe Leu Leu Leu Pro Gln 210 215 220Val Ser Pro Leu Asp Ser Gly Thr
Trp Gly Cys Val Leu Thr Tyr Arg225 230 235 240Asp Gly Phe Asn Val
Ser Ile Thr Tyr Asn Leu Lys Val Leu Gly Leu 245 250 255Glu Pro Val
Ala Pro Leu Thr Val Tyr Ala Ala Glu Gly Ser Arg Val 260 265 270Glu
Leu Pro Cys His Leu Pro Pro Gly Val Gly Thr Pro Ser Leu Leu 275 280
285Ile Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Glu Leu Pro Val Ala
290 295 300Gly Lys Ser Gly Asn Phe Thr Leu His Leu Glu Ala Val Gly
Leu Ala305 310 315 320Gln Ala Gly Thr Tyr Thr Cys Ser Ile His Leu
Gln Gly Gln Gln Leu 325 330 335Asn Ala Thr Val Thr Leu Ala Val Ile
Thr Val Thr Pro Lys Ser Phe 340 345 350Gly Leu Pro Gly Ser Arg Gly
Lys Leu Leu Cys Glu Val Thr Pro Ala 355 360 365Ser Gly Lys Glu Arg
Phe Val Trp Arg Pro Leu Asn Asn Leu Ser Arg 370 375 380Ser Cys Pro
Gly Pro Val Leu Glu Ile Gln Glu Ala Arg Leu Leu Ala385 390 395
400Glu Arg Trp Gln Cys Gln Leu Tyr Glu Gly Gln Arg Leu Leu Gly Ala
405 410 415Thr Val Tyr Ala Ala Glu Ser Ser Ser Gly Ala His Ser Ala
Arg Arg 420 425 430Ile Cys His Thr Gln Ser Ser Pro Lys Leu Phe Trp
Ala Leu Val Val 435 440 445Val Ala Gly Val Leu Phe Cys Tyr Gly Leu
Leu Val Thr Val Ala Leu 450 455 460Cys Val Ile Trp Thr Asn Ser Arg
Arg Asn Arg Gly Gly Gln Ser Asp465 470 475 480Tyr Met Asn Met Thr
Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr 485 490 495Gln Pro Tyr
Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro 500 505
510143765DNAArtificial SequencemuTim3tm-CD28 143atgttcagcg
gcctgaccct gaactgcgtg ctcctgctgc tgcagctgct gctggccaga 60agcctggaaa
acgcctacgt gttcgaagtg ggcaagaacg cctacctgcc ctgcagctac
120accctgtcta cacctggcgc cctggtgcct atgtgttggg gcaagggctt
ctgcccttgg 180agccagtgca ccaacgagct gctgagaacc gacgagagaa
acgtgaccta ccagaagtcc 240agcagatacc agctgaaggg cgacctgaac
aagggcgacg tgtccctgat catcaagaac 300gtgaccctgg acgaccacgg
cacctactgc tgcagaatcc agttccccgg cctgatgaac 360gacaagaagc
tggaactgaa gctggacatc aaggccgcca aagtgacccc tgcccagaca
420gcccacggcg actctacaac agccagcccc agaaccctga ccaccgagag
gaacggcagc 480gagacacaga ccctcgtgac actgcacaac aacaacggca
ccaagatcag cacctgggcc 540gacgagatca aggacagcgg cgagacaatc
agaaccgcca tccacatcgg cgtgggcgtg 600tccgctggac tgacactggc
tctgatcatc ggagtgctga tcaacagcag aagaaacaga 660ggcggccaga
gcgactacat gaacatgacc cccagaaggc ctggcctgac cagaaagccc
720taccagcctt acgcccctgc cagagacttc gccgcctaca gacct
765144255PRTArtificial SequencemuTim3tm-CD28 144Met Phe Ser Gly Leu
Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Leu1 5 10 15Leu Leu Ala Arg
Ser Leu Glu Asn Ala Tyr Val Phe Glu Val Gly Lys 20 25 30Asn Ala Tyr
Leu Pro Cys Ser Tyr Thr Leu Ser Thr Pro Gly Ala Leu 35 40 45Val Pro
Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys Thr 50 55 60Asn
Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys Ser65 70 75
80Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly Asp Val Ser Leu
85 90 95Ile Ile Lys Asn Val Thr Leu Asp Asp His Gly Thr Tyr Cys Cys
Arg 100 105 110Ile Gln Phe Pro Gly Leu Met Asn Asp Lys Lys Leu Glu
Leu Lys Leu 115 120 125Asp Ile Lys Ala Ala Lys Val Thr Pro Ala Gln
Thr Ala His Gly Asp 130 135 140Ser Thr Thr Ala Ser Pro Arg Thr Leu
Thr Thr Glu Arg Asn Gly Ser145 150 155 160Glu Thr Gln Thr Leu Val
Thr Leu His Asn Asn Asn Gly Thr Lys Ile 165 170 175Ser Thr Trp Ala
Asp Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg Thr 180 185 190Ala Ile
His Ile Gly Val Gly Val Ser Ala Gly Leu Thr Leu Ala Leu 195 200
205Ile Ile Gly Val Leu Ile Asn Ser Arg Arg Asn Arg Gly Gly Gln Ser
210 215 220Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Leu Thr Arg
Lys Pro225 230 235 240Tyr Gln Pro Tyr Ala Pro Ala Arg Asp Phe Ala
Ala Tyr Arg Pro 245 250 255145783DNAArtificial
SequencemuTim3-CD28tm 145atgttcagcg gcctgaccct gaactgcgtg
ctcctgctgc tgcagctgct gctggccaga 60agcctggaaa acgcctacgt gttcgaagtg
ggcaagaacg cctacctgcc ctgcagctac 120accctgtcta cacctggcgc
cctggtgcct atgtgttggg gcaagggctt ctgcccttgg 180agccagtgca
ccaacgagct gctgagaacc gacgagagaa acgtgaccta ccagaagtcc
240agcagatacc agctgaaggg cgacctgaac aagggcgacg tgtccctgat
catcaagaac 300gtgaccctgg acgaccacgg cacctactgc tgcagaatcc
agttccccgg cctgatgaac 360gacaagaagc tggaactgaa gctggacatc
aaggccgcca aagtgacccc tgcccagaca 420gcccacggcg actctacaac
agccagcccc agaaccctga ccaccgagag gaacggcagc 480gagacacaga
ccctcgtgac actgcacaac aacaacggca ccaagatcag cacctgggcc
540gacgagatca aggacagcgg cgagacaatc agaaccgcct tctgggccct
ggtggtggtg 600gccggcgtgc tgttttgtta cggcctgctc gtgaccgtgg
ccctgtgcgt gatctggacc 660aacagcagaa gaaacagagg cggccagagc
gactacatga acatgacccc cagaaggcct 720ggcctgacca gaaagcccta
ccagccttac gcccctgcca gagacttcgc cgcctacaga 780cct
783146261PRTArtificial SequencemuTim3-CD28tm 146Met Phe Ser Gly Leu
Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Leu1 5 10 15Leu Leu Ala Arg
Ser Leu Glu Asn Ala Tyr Val Phe Glu Val Gly Lys 20 25 30Asn Ala Tyr
Leu Pro Cys Ser Tyr Thr Leu Ser Thr Pro Gly Ala Leu 35 40 45Val Pro
Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys Thr 50 55 60Asn
Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys Ser65 70 75
80Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly Asp Val Ser Leu
85 90 95Ile Ile Lys Asn Val Thr Leu Asp Asp His Gly Thr Tyr Cys Cys
Arg 100 105 110Ile Gln Phe Pro Gly Leu Met Asn Asp Lys Lys Leu Glu
Leu Lys Leu 115 120 125Asp Ile Lys Ala Ala Lys Val Thr Pro Ala Gln
Thr Ala His Gly Asp 130 135 140Ser Thr Thr Ala Ser Pro Arg Thr Leu
Thr Thr Glu Arg Asn Gly Ser145 150 155 160Glu Thr Gln Thr Leu Val
Thr Leu His Asn Asn Asn Gly Thr Lys Ile 165 170 175Ser Thr Trp Ala
Asp Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg Thr 180 185 190Ala Phe
Trp Ala Leu Val Val Val Ala Gly Val Leu Phe Cys Tyr Gly 195 200
205Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp Thr Asn Ser Arg Arg
210 215 220Asn Arg Gly Gly Gln Ser Asp Tyr Met Asn Met Thr Pro Arg
Arg Pro225 230 235 240Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala
Pro Ala Arg Asp Phe 245 250 255Ala Ala Tyr Arg Pro
260147810DNAArtificial SequencemuTim3-CD28Cys 147atgttcagcg
gcctgaccct gaactgcgtg ctcctgctgc tgcagctgct gctggccaga 60agcctggaaa
acgcctacgt gttcgaagtg ggcaagaacg cctacctgcc ctgcagctac
120accctgtcta cacctggcgc cctggtgcct atgtgttggg gcaagggctt
ctgcccttgg 180agccagtgca ccaacgagct gctgagaacc gacgagagaa
acgtgaccta ccagaagtcc 240agcagatacc agctgaaggg cgacctgaac
aagggcgacg tgtccctgat catcaagaac 300gtgaccctgg acgaccacgg
cacctactgc tgcagaatcc agttccccgg cctgatgaac 360gacaagaagc
tggaactgaa gctggacatc aaggccgcca aagtgacccc tgcccagaca
420gcccacggcg actctacaac agccagcccc agaaccctga ccaccgagag
gaacggcagc 480gagacacaga ccctcgtgac actgcacaac aacaacggca
ccaagatcag cacctgggcc 540gacgagatca aggacagcgg cgagacaatc
agaaccgcct gccacaccca gagcagcccc 600aagctgttct gggccctggt
ggtggtggcc ggcgtgctgt tttgttacgg cctgctcgtg 660accgtggccc
tgtgcgtgat ctggaccaac agcagaagaa acagaggcgg ccagagcgac
720tacatgaaca tgacccccag aaggcctggc ctgaccagaa agccctacca
gccttacgcc 780cctgccagag acttcgccgc ctacagacct
810148270PRTArtificial SequencemuTim3-CD28Cys 148Met Phe Ser Gly
Leu Thr Leu Asn Cys Val Leu Leu Leu Leu Gln Leu1 5 10 15Leu Leu Ala
Arg Ser Leu Glu Asn Ala Tyr Val Phe Glu Val Gly Lys 20 25 30Asn Ala
Tyr Leu Pro Cys Ser Tyr Thr Leu Ser Thr Pro Gly Ala Leu 35 40 45Val
Pro Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys Thr 50 55
60Asn Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys Ser65
70 75 80Ser Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly Asp Val Ser
Leu 85 90 95Ile Ile Lys Asn Val Thr Leu Asp Asp His Gly Thr Tyr Cys
Cys Arg 100 105 110Ile Gln Phe Pro Gly Leu Met Asn Asp Lys Lys Leu
Glu Leu Lys Leu 115 120 125Asp Ile Lys Ala Ala Lys Val Thr Pro Ala
Gln Thr Ala His Gly Asp 130 135 140Ser Thr Thr Ala Ser Pro Arg Thr
Leu Thr Thr Glu Arg Asn Gly Ser145 150
155 160Glu Thr Gln Thr Leu Val Thr Leu His Asn Asn Asn Gly Thr Lys
Ile 165 170 175Ser Thr Trp Ala Asp Glu Ile Lys Asp Ser Gly Glu Thr
Ile Arg Thr 180 185 190Ala Cys His Thr Gln Ser Ser Pro Lys Leu Phe
Trp Ala Leu Val Val 195 200 205Val Ala Gly Val Leu Phe Cys Tyr Gly
Leu Leu Val Thr Val Ala Leu 210 215 220Cys Val Ile Trp Thr Asn Ser
Arg Arg Asn Arg Gly Gly Gln Ser Asp225 230 235 240Tyr Met Asn Met
Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr 245 250 255Gln Pro
Tyr Ala Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro 260 265
270149783DNAArtificial SequencemuTim3-9aas-CD28Cys 149atgttcagcg
gcctgaccct gaactgcgtg ctcctgctgc tgcagctgct gctggccaga 60agcctggaaa
acgcctacgt gttcgaagtg ggcaagaacg cctacctgcc ctgcagctac
120accctgtcta cacctggcgc cctggtgcct atgtgttggg gcaagggctt
ctgcccttgg 180agccagtgca ccaacgagct gctgagaacc gacgagagaa
acgtgaccta ccagaagtcc 240agcagatacc agctgaaggg cgacctgaac
aagggcgacg tgtccctgat catcaagaac 300gtgaccctgg acgaccacgg
cacctactgc tgcagaatcc agttccccgg cctgatgaac 360gacaagaagc
tggaactgaa gctggacatc aaggccgcca aagtgacccc tgcccagaca
420gcccacggcg actctacaac agccagcccc agaaccctga ccaccgagag
gaacggcagc 480gagacacaga ccctcgtgac actgcacaac aacaacggca
ccaagatcag cacctgggcc 540gacgagatca agtgccacac ccagagcagc
cccaagctgt tctgggccct ggtggtggtg 600gccggcgtgc tgttttgtta
cggcctgctc gtgaccgtgg ccctgtgcgt gatctggacc 660aacagcagaa
gaaacagagg cggccagagc gactacatga acatgacccc cagaaggcct
720ggcctgacca gaaagcccta ccagccttac gcccctgcca gagacttcgc
cgcctacaga 780cct 783150261PRTArtificial
SequencemuTim3-9aas-CD28Cys 150Met Phe Ser Gly Leu Thr Leu Asn Cys
Val Leu Leu Leu Leu Gln Leu1 5 10 15Leu Leu Ala Arg Ser Leu Glu Asn
Ala Tyr Val Phe Glu Val Gly Lys 20 25 30Asn Ala Tyr Leu Pro Cys Ser
Tyr Thr Leu Ser Thr Pro Gly Ala Leu 35 40 45Val Pro Met Cys Trp Gly
Lys Gly Phe Cys Pro Trp Ser Gln Cys Thr 50 55 60Asn Glu Leu Leu Arg
Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys Ser65 70 75 80Ser Arg Tyr
Gln Leu Lys Gly Asp Leu Asn Lys Gly Asp Val Ser Leu 85 90 95Ile Ile
Lys Asn Val Thr Leu Asp Asp His Gly Thr Tyr Cys Cys Arg 100 105
110Ile Gln Phe Pro Gly Leu Met Asn Asp Lys Lys Leu Glu Leu Lys Leu
115 120 125Asp Ile Lys Ala Ala Lys Val Thr Pro Ala Gln Thr Ala His
Gly Asp 130 135 140Ser Thr Thr Ala Ser Pro Arg Thr Leu Thr Thr Glu
Arg Asn Gly Ser145 150 155 160Glu Thr Gln Thr Leu Val Thr Leu His
Asn Asn Asn Gly Thr Lys Ile 165 170 175Ser Thr Trp Ala Asp Glu Ile
Lys Cys His Thr Gln Ser Ser Pro Lys 180 185 190Leu Phe Trp Ala Leu
Val Val Val Ala Gly Val Leu Phe Cys Tyr Gly 195 200 205Leu Leu Val
Thr Val Ala Leu Cys Val Ile Trp Thr Asn Ser Arg Arg 210 215 220Asn
Arg Gly Gly Gln Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro225 230
235 240Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro Ala Arg Asp
Phe 245 250 255Ala Ala Tyr Arg Pro 2601511536DNAArtificial
SequencehuLag3tm-CD28 151atgtgggaag cgcagtttct tggacttctt
tttctccagc cgctgtgggt tgcgccagta 60aagccgctcc aacccggtgc agaggttccg
gtagtgtggg cgcaagaggg tgcaccagcg 120cagctcccct gcagtccgac
gattccgctg caagatttgt cactgcttag aagggcgggc 180gtaacgtggc
agcaccaacc ggatagtggc cctccggctg cagcaccagg gcacccactc
240gcccccggcc ctcatcccgc agcaccgagc agctggggtc ctagaccacg
cagatataca 300gtactctcag taggtcccgg cggcctgcgg tccggtcgct
tgccccttca acctagagta 360cagctggatg aaagaggtcg acaacggggt
gatttctccc tctggttgag gcctgcacga 420cgagcagatg ctggggagta
tagggctgcc gtacacctgc gagaccgcgc acttagttgt 480agactccggc
tccggctggg acaggcctct atgacagcgt ccccccctgg gtccctgcga
540gcctctgatt gggtaatact caactgctca ttttctcggc cagatcgccc
cgctagtgtt 600cattggttcc gaaatcgcgg ccaaggtcgc gtgcctgttc
gagaatctcc acaccaccat 660ttggcggagt cttttctttt tctgcctcag
gtctccccta tggactctgg accgtggggc 720tgtattttga catatcggga
tgggtttaac gtgagtataa tgtataatct cactgtcttg 780ggtcttgagc
cacctacgcc gctgacggtg tacgcgggag ccggcagccg ggttggtctg
840ccctgcaggc tgcctgcagg agtcgggaca aggtcattcc ttacagcaaa
gtggaccccg 900ccaggtgggg ggcccgacct ccttgtaacg ggagataatg
gagatttcac tctgagactt 960gaggatgtct ctcaagctca ggctgggact
tatacatgtc acattcactt gcaagaacag 1020cagttgaatg cgacggttac
cctggctatc ataacagtaa cacctaaatc tttcggtagt 1080ccgggtagcc
tgggcaaact gttgtgtgag gtaacccccg tgtcaggtca agagcggttc
1140gtctggagct cattggacac tccctcacag cgatccttta gcggaccctg
gctcgaagcc 1200caagaagccc agctgctttc ccaaccatgg cagtgtcaac
tctatcaggg tgagcgcctt 1260ctcggtgcgg ctgtctactt caccgaattg
tcctctccgg gagcgcaaag aagtggacgc 1320gccccagggg ccctcccggc
aggacacctt ctgctgtttt tgattttggg ggtacttagt 1380ttgctgctgc
ttgtcacagg cgctttcggt ttccgcagca agcggagcag aggcggccac
1440agcgactaca tgaacatgac ccctagacgg cctggcccca ccagaaagca
ctaccagccc 1500tacgcccctc cccgggactt tgccgcctac agaagc
1536152512PRTArtificial SequencehuLag3tm-CD28 152Met Trp Glu Ala
Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp1 5 10 15Val Ala Pro
Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val 20 25 30Trp Ala
Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45Pro
Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55
60His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu65
70 75 80Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg
Pro 85 90 95Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg
Ser Gly 100 105 110Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu
Arg Gly Arg Gln 115 120 125Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro
Ala Arg Arg Ala Asp Ala 130 135 140Gly Glu Tyr Arg Ala Ala Val His
Leu Arg Asp Arg Ala Leu Ser Cys145 150 155 160Arg Leu Arg Leu Arg
Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165 170 175Gly Ser Leu
Arg Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser 180 185 190Arg
Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln 195 200
205Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser
210 215 220Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro
Trp Gly225 230 235 240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val
Ser Ile Met Tyr Asn 245 250 255Leu Thr Val Leu Gly Leu Glu Pro Pro
Thr Pro Leu Thr Val Tyr Ala 260 265 270Gly Ala Gly Ser Arg Val Gly
Leu Pro Cys Arg Leu Pro Ala Gly Val 275 280 285Gly Thr Arg Ser Phe
Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly 290 295 300Pro Asp Leu
Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu305 310 315
320Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His
325 330 335Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile
Ile Thr 340 345 350Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu
Gly Lys Leu Leu 355 360 365Cys Glu Val Thr Pro Val Ser Gly Gln Glu
Arg Phe Val Trp Ser Ser 370 375 380Leu Asp Thr Pro Ser Gln Arg Ser
Phe Ser Gly Pro Trp Leu Glu Ala385 390 395 400Gln Glu Ala Gln Leu
Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln 405 410 415Gly Glu Arg
Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser 420 425 430Pro
Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly 435 440
445His Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu
450 455 460Val Thr Gly Ala Phe Gly Phe Arg Ser Lys Arg Ser Arg Gly
Gly His465 470 475 480Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
Gly Pro Thr Arg Lys 485 490 495His Tyr Gln Pro Tyr Ala Pro Pro Arg
Asp Phe Ala Ala Tyr Arg Ser 500 505 5101531350DNAArtificial
SequencehuLag3 ectodomain 153atgtgggaag cgcagtttct tggacttctt
tttctccagc cgctgtgggt tgcgccagta 60aagccgctcc aacccggtgc agaggttccg
gtagtgtggg cgcaagaggg tgcaccagcg 120cagctcccct gcagtccgac
gattccgctg caagatttgt cactgcttag aagggcgggc 180gtaacgtggc
agcaccaacc ggatagtggc cctccggctg cagcaccagg gcacccactc
240gcccccggcc ctcatcccgc agcaccgagc agctggggtc ctagaccacg
cagatataca 300gtactctcag taggtcccgg cggcctgcgg tccggtcgct
tgccccttca acctagagta 360cagctggatg aaagaggtcg acaacggggt
gatttctccc tctggttgag gcctgcacga 420cgagcagatg ctggggagta
tagggctgcc gtacacctgc gagaccgcgc acttagttgt 480agactccggc
tccggctggg acaggcctct atgacagcgt ccccccctgg gtccctgcga
540gcctctgatt gggtaatact caactgctca ttttctcggc cagatcgccc
cgctagtgtt 600cattggttcc gaaatcgcgg ccaaggtcgc gtgcctgttc
gagaatctcc acaccaccat 660ttggcggagt cttttctttt tctgcctcag
gtctccccta tggactctgg accgtggggc 720tgtattttga catatcggga
tgggtttaac gtgagtataa tgtataatct cactgtcttg 780ggtcttgagc
cacctacgcc gctgacggtg tacgcgggag ccggcagccg ggttggtctg
840ccctgcaggc tgcctgcagg agtcgggaca aggtcattcc ttacagcaaa
gtggaccccg 900ccaggtgggg ggcccgacct ccttgtaacg ggagataatg
gagatttcac tctgagactt 960gaggatgtct ctcaagctca ggctgggact
tatacatgtc acattcactt gcaagaacag 1020cagttgaatg cgacggttac
cctggctatc ataacagtaa cacctaaatc tttcggtagt 1080ccgggtagcc
tgggcaaact gttgtgtgag gtaacccccg tgtcaggtca agagcggttc
1140gtctggagct cattggacac tccctcacag cgatccttta gcggaccctg
gctcgaagcc 1200caagaagccc agctgctttc ccaaccatgg cagtgtcaac
tctatcaggg tgagcgcctt 1260ctcggtgcgg ctgtctactt caccgaattg
tcctctccgg gagcgcaaag aagtggacgc 1320gccccagggg ccctcccggc
aggacacctt 1350154450PRTArtificial SequencehuLag3 ectodomain 154Met
Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp1 5 10
15Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val
20 25 30Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr
Ile 35 40 45Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr
Trp Gln 50 55 60His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly
His Pro Leu65 70 75 80Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser
Trp Gly Pro Arg Pro 85 90 95Arg Arg Tyr Thr Val Leu Ser Val Gly Pro
Gly Gly Leu Arg Ser Gly 100 105 110Arg Leu Pro Leu Gln Pro Arg Val
Gln Leu Asp Glu Arg Gly Arg Gln 115 120 125Arg Gly Asp Phe Ser Leu
Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala 130 135 140Gly Glu Tyr Arg
Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser Cys145 150 155 160Arg
Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165 170
175Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser
180 185 190Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg
Gly Gln 195 200 205Gly Arg Val Pro Val Arg Glu Ser Pro His His His
Leu Ala Glu Ser 210 215 220Phe Leu Phe Leu Pro Gln Val Ser Pro Met
Asp Ser Gly Pro Trp Gly225 230 235 240Cys Ile Leu Thr Tyr Arg Asp
Gly Phe Asn Val Ser Ile Met Tyr Asn 245 250 255Leu Thr Val Leu Gly
Leu Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala 260 265 270Gly Ala Gly
Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala Gly Val 275 280 285Gly
Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly 290 295
300Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg
Leu305 310 315 320Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr
Cys His Ile His 325 330 335Leu Gln Glu Gln Gln Leu Asn Ala Thr Val
Thr Leu Ala Ile Ile Thr 340 345 350Val Thr Pro Lys Ser Phe Gly Ser
Pro Gly Ser Leu Gly Lys Leu Leu 355 360 365Cys Glu Val Thr Pro Val
Ser Gly Gln Glu Arg Phe Val Trp Ser Ser 370 375 380Leu Asp Thr Pro
Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala385 390 395 400Gln
Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln 405 410
415Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser
420 425 430Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro
Ala Gly 435 440 445His Leu 45015563DNAArtificial SequencehuLag3
transmembrane domain 155ctgctgtttt tgattttggg ggtacttagt ttgctgctgc
ttgtcacagg cgctttcggt 60ttc 6315621PRTArtificial SequencehuLag3
transmembrane domain 156Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu
Leu Leu Leu Val Thr1 5 10 15Gly Ala Phe Gly Phe
201571554DNAArtificial SequencehuLag3-CD28tm 157atgtgggaag
cgcagtttct tggacttctt tttctccagc cgctgtgggt tgcgccagta 60aagccgctcc
aacccggtgc agaggttccg gtagtgtggg cgcaagaggg tgcaccagcg
120cagctcccct gcagtccgac gattccgctg caagatttgt cactgcttag
aagggcgggc 180gtaacgtggc agcaccaacc ggatagtggc cctccggctg
cagcaccagg gcacccactc 240gcccccggcc ctcatcccgc agcaccgagc
agctggggtc ctagaccacg cagatataca 300gtactctcag taggtcccgg
cggcctgcgg tccggtcgct tgccccttca acctagagta 360cagctggatg
aaagaggtcg acaacggggt gatttctccc tctggttgag gcctgcacga
420cgagcagatg ctggggagta tagggctgcc gtacacctgc gagaccgcgc
acttagttgt 480agactccggc tccggctggg acaggcctct atgacagcgt
ccccccctgg gtccctgcga 540gcctctgatt gggtaatact caactgctca
ttttctcggc cagatcgccc cgctagtgtt 600cattggttcc gaaatcgcgg
ccaaggtcgc gtgcctgttc gagaatctcc acaccaccat 660ttggcggagt
cttttctttt tctgcctcag gtctccccta tggactctgg accgtggggc
720tgtattttga catatcggga tgggtttaac gtgagtataa tgtataatct
cactgtcttg 780ggtcttgagc cacctacgcc gctgacggtg tacgcgggag
ccggcagccg ggttggtctg 840ccctgcaggc tgcctgcagg agtcgggaca
aggtcattcc ttacagcaaa gtggaccccg 900ccaggtgggg ggcccgacct
ccttgtaacg ggagataatg gagatttcac tctgagactt 960gaggatgtct
ctcaagctca ggctgggact tatacatgtc acattcactt gcaagaacag
1020cagttgaatg cgacggttac cctggctatc ataacagtaa cacctaaatc
tttcggtagt 1080ccgggtagcc tgggcaaact gttgtgtgag gtaacccccg
tgtcaggtca agagcggttc 1140gtctggagct cattggacac tccctcacag
cgatccttta gcggaccctg gctcgaagcc 1200caagaagccc agctgctttc
ccaaccatgg cagtgtcaac tctatcaggg tgagcgcctt 1260ctcggtgcgg
ctgtctactt caccgaattg tcctctccgg gagcgcaaag aagtggacgc
1320gccccagggg ccctcccggc aggacacctt ttctgggtgc tggtggtggt
cggaggcgtg 1380ctggcctgct acagcctgct ggtcaccgtg gccttcatca
tcttttgggt ccgcagcaag 1440cggagcagag gcggccacag cgactacatg
aacatgaccc ctagacggcc tggccccacc 1500agaaagcact accagcccta
cgcccctccc cgggactttg ccgcctacag aagc 1554158518PRTArtificial
SequencehuLag3-CD28tm 158Met Trp Glu Ala Gln Phe Leu Gly Leu Leu
Phe Leu Gln Pro Leu Trp1 5 10 15Val Ala Pro Val Lys Pro Leu Gln Pro
Gly Ala Glu Val Pro Val Val 20 25 30Trp Ala Gln Glu Gly Ala Pro Ala
Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45Pro Leu Gln Asp Leu Ser Leu
Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60His Gln Pro Asp Ser Gly
Pro Pro Ala Ala Ala Pro Gly His Pro Leu65 70 75 80Ala Pro Gly Pro
His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro 85 90 95Arg Arg Tyr
Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100 105 110Arg
Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln 115 120
125Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala
130 135
140Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser
Cys145 150 155 160Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr
Ala Ser Pro Pro 165 170 175Gly Ser Leu Arg Ala Ser Asp Trp Val Ile
Leu Asn Cys Ser Phe Ser 180 185 190Arg Pro Asp Arg Pro Ala Ser Val
His Trp Phe Arg Asn Arg Gly Gln 195 200 205Gly Arg Val Pro Val Arg
Glu Ser Pro His His His Leu Ala Glu Ser 210 215 220Phe Leu Phe Leu
Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly225 230 235 240Cys
Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn 245 250
255Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala
260 265 270Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala
Gly Val 275 280 285Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro
Pro Gly Gly Gly 290 295 300Pro Asp Leu Leu Val Thr Gly Asp Asn Gly
Asp Phe Thr Leu Arg Leu305 310 315 320Glu Asp Val Ser Gln Ala Gln
Ala Gly Thr Tyr Thr Cys His Ile His 325 330 335Leu Gln Glu Gln Gln
Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr 340 345 350Val Thr Pro
Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu 355 360 365Cys
Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser 370 375
380Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu
Ala385 390 395 400Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys
Gln Leu Tyr Gln 405 410 415Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr
Phe Thr Glu Leu Ser Ser 420 425 430Pro Gly Ala Gln Arg Ser Gly Arg
Ala Pro Gly Ala Leu Pro Ala Gly 435 440 445His Leu Phe Trp Val Leu
Val Val Val Gly Gly Val Leu Ala Cys Tyr 450 455 460Ser Leu Leu Val
Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys465 470 475 480Arg
Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 485 490
495Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
500 505 510Phe Ala Ala Tyr Arg Ser 5151591554DNAArtificial
SequencehuLag3-12aas-CD28Cys 159atgtgggaag cgcagtttct tggacttctt
tttctccagc cgctgtgggt tgcgccagta 60aagccgctcc aacccggtgc agaggttccg
gtagtgtggg cgcaagaggg tgcaccagcg 120cagctcccct gcagtccgac
gattccgctg caagatttgt cactgcttag aagggcgggc 180gtaacgtggc
agcaccaacc ggatagtggc cctccggctg cagcaccagg gcacccactc
240gcccccggcc ctcatcccgc agcaccgagc agctggggtc ctagaccacg
cagatataca 300gtactctcag taggtcccgg cggcctgcgg tccggtcgct
tgccccttca acctagagta 360cagctggatg aaagaggtcg acaacggggt
gatttctccc tctggttgag gcctgcacga 420cgagcagatg ctggggagta
tagggctgcc gtacacctgc gagaccgcgc acttagttgt 480agactccggc
tccggctggg acaggcctct atgacagcgt ccccccctgg gtccctgcga
540gcctctgatt gggtaatact caactgctca ttttctcggc cagatcgccc
cgctagtgtt 600cattggttcc gaaatcgcgg ccaaggtcgc gtgcctgttc
gagaatctcc acaccaccat 660ttggcggagt cttttctttt tctgcctcag
gtctccccta tggactctgg accgtggggc 720tgtattttga catatcggga
tgggtttaac gtgagtataa tgtataatct cactgtcttg 780ggtcttgagc
cacctacgcc gctgacggtg tacgcgggag ccggcagccg ggttggtctg
840ccctgcaggc tgcctgcagg agtcgggaca aggtcattcc ttacagcaaa
gtggaccccg 900ccaggtgggg ggcccgacct ccttgtaacg ggagataatg
gagatttcac tctgagactt 960gaggatgtct ctcaagctca ggctgggact
tatacatgtc acattcactt gcaagaacag 1020cagttgaatg cgacggttac
cctggctatc ataacagtaa cacctaaatc tttcggtagt 1080ccgggtagcc
tgggcaaact gttgtgtgag gtaacccccg tgtcaggtca agagcggttc
1140gtctggagct cattggacac tccctcacag cgatccttta gcggaccctg
gctcgaagcc 1200caagaagccc agctgctttc ccaaccatgg cagtgtcaac
tctatcaggg tgagcgcctt 1260ctcggtgcgg ctgtctactt caccgaattg
tcctctccgg gagcgcaaag aagttgtccc 1320agccctctgt ttcccggccc
tagcaagcct ttctgggtgc tggtggtggt cggaggcgtg 1380ctggcctgct
acagcctgct ggtcaccgtg gccttcatca tcttttgggt ccgcagcaag
1440cggagcagag gcggccacag cgactacatg aacatgaccc ctagacggcc
tggccccacc 1500agaaagcact accagcccta cgcccctccc cgggactttg
ccgcctacag aagc 1554160518PRTArtificial
SequencehuLag3-12aas-CD28Cys 160Met Trp Glu Ala Gln Phe Leu Gly Leu
Leu Phe Leu Gln Pro Leu Trp1 5 10 15Val Ala Pro Val Lys Pro Leu Gln
Pro Gly Ala Glu Val Pro Val Val 20 25 30Trp Ala Gln Glu Gly Ala Pro
Ala Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45Pro Leu Gln Asp Leu Ser
Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60His Gln Pro Asp Ser
Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu65 70 75 80Ala Pro Gly
Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro 85 90 95Arg Arg
Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100 105
110Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln
115 120 125Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala
Asp Ala 130 135 140Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg
Ala Leu Ser Cys145 150 155 160Arg Leu Arg Leu Arg Leu Gly Gln Ala
Ser Met Thr Ala Ser Pro Pro 165 170 175Gly Ser Leu Arg Ala Ser Asp
Trp Val Ile Leu Asn Cys Ser Phe Ser 180 185 190Arg Pro Asp Arg Pro
Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln 195 200 205Gly Arg Val
Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser 210 215 220Phe
Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly225 230
235 240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr
Asn 245 250 255Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr
Val Tyr Ala 260 265 270Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg
Leu Pro Ala Gly Val 275 280 285Gly Thr Arg Ser Phe Leu Thr Ala Lys
Trp Thr Pro Pro Gly Gly Gly 290 295 300Pro Asp Leu Leu Val Thr Gly
Asp Asn Gly Asp Phe Thr Leu Arg Leu305 310 315 320Glu Asp Val Ser
Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His 325 330 335Leu Gln
Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr 340 345
350Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu
355 360 365Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp
Ser Ser 370 375 380Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro
Trp Leu Glu Ala385 390 395 400Gln Glu Ala Gln Leu Leu Ser Gln Pro
Trp Gln Cys Gln Leu Tyr Gln 405 410 415Gly Glu Arg Leu Leu Gly Ala
Ala Val Tyr Phe Thr Glu Leu Ser Ser 420 425 430Pro Gly Ala Gln Arg
Ser Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser 435 440 445Lys Pro Phe
Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 450 455 460Ser
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys465 470
475 480Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg
Arg 485 490 495Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
Pro Arg Asp 500 505 510Phe Ala Ala Tyr Arg Ser
5151611314DNAArtificial SequencehuLag3-12aas 161atgtgggaag
cgcagtttct tggacttctt tttctccagc cgctgtgggt tgcgccagta 60aagccgctcc
aacccggtgc agaggttccg gtagtgtggg cgcaagaggg tgcaccagcg
120cagctcccct gcagtccgac gattccgctg caagatttgt cactgcttag
aagggcgggc 180gtaacgtggc agcaccaacc ggatagtggc cctccggctg
cagcaccagg gcacccactc 240gcccccggcc ctcatcccgc agcaccgagc
agctggggtc ctagaccacg cagatataca 300gtactctcag taggtcccgg
cggcctgcgg tccggtcgct tgccccttca acctagagta 360cagctggatg
aaagaggtcg acaacggggt gatttctccc tctggttgag gcctgcacga
420cgagcagatg ctggggagta tagggctgcc gtacacctgc gagaccgcgc
acttagttgt 480agactccggc tccggctggg acaggcctct atgacagcgt
ccccccctgg gtccctgcga 540gcctctgatt gggtaatact caactgctca
ttttctcggc cagatcgccc cgctagtgtt 600cattggttcc gaaatcgcgg
ccaaggtcgc gtgcctgttc gagaatctcc acaccaccat 660ttggcggagt
cttttctttt tctgcctcag gtctccccta tggactctgg accgtggggc
720tgtattttga catatcggga tgggtttaac gtgagtataa tgtataatct
cactgtcttg 780ggtcttgagc cacctacgcc gctgacggtg tacgcgggag
ccggcagccg ggttggtctg 840ccctgcaggc tgcctgcagg agtcgggaca
aggtcattcc ttacagcaaa gtggaccccg 900ccaggtgggg ggcccgacct
ccttgtaacg ggagataatg gagatttcac tctgagactt 960gaggatgtct
ctcaagctca ggctgggact tatacatgtc acattcactt gcaagaacag
1020cagttgaatg cgacggttac cctggctatc ataacagtaa cacctaaatc
tttcggtagt 1080ccgggtagcc tgggcaaact gttgtgtgag gtaacccccg
tgtcaggtca agagcggttc 1140gtctggagct cattggacac tccctcacag
cgatccttta gcggaccctg gctcgaagcc 1200caagaagccc agctgctttc
ccaaccatgg cagtgtcaac tctatcaggg tgagcgcctt 1260ctcggtgcgg
ctgtctactt caccgaattg tcctctccgg gagcgcaaag aagt
1314162438PRTArtificial SequencehuLag3-12aas 162Met Trp Glu Ala Gln
Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp1 5 10 15Val Ala Pro Val
Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val 20 25 30Trp Ala Gln
Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45Pro Leu
Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60His
Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu65 70 75
80Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro
85 90 95Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser
Gly 100 105 110Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg
Gly Arg Gln 115 120 125Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala
Arg Arg Ala Asp Ala 130 135 140Gly Glu Tyr Arg Ala Ala Val His Leu
Arg Asp Arg Ala Leu Ser Cys145 150 155 160Arg Leu Arg Leu Arg Leu
Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165 170 175Gly Ser Leu Arg
Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser 180 185 190Arg Pro
Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln 195 200
205Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser
210 215 220Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro
Trp Gly225 230 235 240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val
Ser Ile Met Tyr Asn 245 250 255Leu Thr Val Leu Gly Leu Glu Pro Pro
Thr Pro Leu Thr Val Tyr Ala 260 265 270Gly Ala Gly Ser Arg Val Gly
Leu Pro Cys Arg Leu Pro Ala Gly Val 275 280 285Gly Thr Arg Ser Phe
Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly 290 295 300Pro Asp Leu
Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu305 310 315
320Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His
325 330 335Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile
Ile Thr 340 345 350Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu
Gly Lys Leu Leu 355 360 365Cys Glu Val Thr Pro Val Ser Gly Gln Glu
Arg Phe Val Trp Ser Ser 370 375 380Leu Asp Thr Pro Ser Gln Arg Ser
Phe Ser Gly Pro Trp Leu Glu Ala385 390 395 400Gln Glu Ala Gln Leu
Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln 405 410 415Gly Glu Arg
Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser 420 425 430Pro
Gly Ala Gln Arg Ser 4351631590DNAArtificial SequencehuLag3-CD28Cys
163atgtgggaag cgcagtttct tggacttctt tttctccagc cgctgtgggt
tgcgccagta 60aagccgctcc aacccggtgc agaggttccg gtagtgtggg cgcaagaggg
tgcaccagcg 120cagctcccct gcagtccgac gattccgctg caagatttgt
cactgcttag aagggcgggc 180gtaacgtggc agcaccaacc ggatagtggc
cctccggctg cagcaccagg gcacccactc 240gcccccggcc ctcatcccgc
agcaccgagc agctggggtc ctagaccacg cagatataca 300gtactctcag
taggtcccgg cggcctgcgg tccggtcgct tgccccttca acctagagta
360cagctggatg aaagaggtcg acaacggggt gatttctccc tctggttgag
gcctgcacga 420cgagcagatg ctggggagta tagggctgcc gtacacctgc
gagaccgcgc acttagttgt 480agactccggc tccggctggg acaggcctct
atgacagcgt ccccccctgg gtccctgcga 540gcctctgatt gggtaatact
caactgctca ttttctcggc cagatcgccc cgctagtgtt 600cattggttcc
gaaatcgcgg ccaaggtcgc gtgcctgttc gagaatctcc acaccaccat
660ttggcggagt cttttctttt tctgcctcag gtctccccta tggactctgg
accgtggggc 720tgtattttga catatcggga tgggtttaac gtgagtataa
tgtataatct cactgtcttg 780ggtcttgagc cacctacgcc gctgacggtg
tacgcgggag ccggcagccg ggttggtctg 840ccctgcaggc tgcctgcagg
agtcgggaca aggtcattcc ttacagcaaa gtggaccccg 900ccaggtgggg
ggcccgacct ccttgtaacg ggagataatg gagatttcac tctgagactt
960gaggatgtct ctcaagctca ggctgggact tatacatgtc acattcactt
gcaagaacag 1020cagttgaatg cgacggttac cctggctatc ataacagtaa
cacctaaatc tttcggtagt 1080ccgggtagcc tgggcaaact gttgtgtgag
gtaacccccg tgtcaggtca agagcggttc 1140gtctggagct cattggacac
tccctcacag cgatccttta gcggaccctg gctcgaagcc 1200caagaagccc
agctgctttc ccaaccatgg cagtgtcaac tctatcaggg tgagcgcctt
1260ctcggtgcgg ctgtctactt caccgaattg tcctctccgg gagcgcaaag
aagtggacgc 1320gccccagggg ccctcccggc aggacacctt tgtcccagcc
ctctgtttcc cggccctagc 1380aagcctttct gggtgctggt ggtggtcgga
ggcgtgctgg cctgctacag cctgctggtc 1440accgtggcct tcatcatctt
ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac 1500tacatgaaca
tgacccctag acggcctggc cccaccagaa agcactacca gccctacgcc
1560cctccccggg actttgccgc ctacagaagc 1590164530PRTArtificial
SequencehuLag3-CD28Cys 164Met Trp Glu Ala Gln Phe Leu Gly Leu Leu
Phe Leu Gln Pro Leu Trp1 5 10 15Val Ala Pro Val Lys Pro Leu Gln Pro
Gly Ala Glu Val Pro Val Val 20 25 30Trp Ala Gln Glu Gly Ala Pro Ala
Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45Pro Leu Gln Asp Leu Ser Leu
Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60His Gln Pro Asp Ser Gly
Pro Pro Ala Ala Ala Pro Gly His Pro Leu65 70 75 80Ala Pro Gly Pro
His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro 85 90 95Arg Arg Tyr
Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100 105 110Arg
Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln 115 120
125Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala
130 135 140Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu
Ser Cys145 150 155 160Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met
Thr Ala Ser Pro Pro 165 170 175Gly Ser Leu Arg Ala Ser Asp Trp Val
Ile Leu Asn Cys Ser Phe Ser 180 185 190Arg Pro Asp Arg Pro Ala Ser
Val His Trp Phe Arg Asn Arg Gly Gln 195 200 205Gly Arg Val Pro Val
Arg Glu Ser Pro His His His Leu Ala Glu Ser 210 215 220Phe Leu Phe
Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly225 230 235
240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn
245 250 255Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val
Tyr Ala 260 265 270Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu
Pro Ala Gly Val 275 280 285Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp
Thr Pro Pro Gly Gly Gly 290 295 300Pro Asp Leu Leu Val Thr Gly Asp
Asn Gly Asp Phe Thr Leu Arg Leu305 310 315 320Glu Asp Val Ser Gln
Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His 325 330 335Leu Gln Glu
Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr 340 345 350Val
Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu 355
360
365Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser
370 375 380Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu
Glu Ala385 390 395 400Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln
Cys Gln Leu Tyr Gln 405 410 415Gly Glu Arg Leu Leu Gly Ala Ala Val
Tyr Phe Thr Glu Leu Ser Ser 420 425 430Pro Gly Ala Gln Arg Ser Gly
Arg Ala Pro Gly Ala Leu Pro Ala Gly 435 440 445His Leu Cys Pro Ser
Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp 450 455 460Val Leu Val
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val465 470 475
480Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
485 490 495Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
Pro Thr 500 505 510Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
Phe Ala Ala Tyr 515 520 525Arg Ser 530165792DNAArtificial
SequencehuTim3tm-CD28 165atgttctccc atcttccctt cgactgtgtg
ttgctccttc tcctcctgct tctcacccgg 60tcaagcgaag tagagtaccg ggcggaagta
ggtcagaacg catatctccc ctgtttttac 120acacccgctg cgccgggaaa
cctggttccc gtgtgttggg gaaagggggc atgccctgtt 180ttcgagtgtg
gcaacgtggt cctccggacg gatgagcgag acgtgaatta ttggacgagc
240agatattggt tgaatggcga ttttagaaag ggtgatgtga gcttgaccat
tgagaatgta 300acgcttgctg atagcgggat atattgctgt agaattcaaa
tccctggtat aatgaacgac 360gaaaaattca atctgaagct ggtaattaag
ccggccaagg tgacacccgc cccgacacga 420cagcgcgact tcacggctgc
ctttccacgc atgttgacca caaggggaca tggtccagcg 480gagacccaga
cacttggtag cctcccggac ataaacctca cacaaatatc cacgttggcg
540aacgagctcc gagattccag gcttgcgaat gacctgaggg attctggagc
taccatcaga 600atcggtatct acataggtgc cgggatatgc gccggtctcg
cacttgcctt gattttcggg 660gcactgattc gcagcaagcg gagcagaggc
ggccacagcg actacatgaa catgacccct 720agacggcctg gccccaccag
aaagcactac cagccctacg cccctccccg ggactttgcc 780gcctacagaa gc
792166264PRTArtificial SequencehuTim3tm-CD28 166Met Phe Ser His Leu
Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Thr Arg
Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln 20 25 30Asn Ala Tyr
Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu 35 40 45Val Pro
Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly 50 55 60Asn
Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser65 70 75
80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr
85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg
Ile 100 105 110Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu
Lys Leu Val 115 120 125Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr
Arg Gln Arg Asp Phe 130 135 140Thr Ala Ala Phe Pro Arg Met Leu Thr
Thr Arg Gly His Gly Pro Ala145 150 155 160Glu Thr Gln Thr Leu Gly
Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile 165 170 175Ser Thr Leu Ala
Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu 180 185 190Arg Asp
Ser Gly Ala Thr Ile Arg Ile Gly Ile Tyr Ile Gly Ala Gly 195 200
205Ile Cys Ala Gly Leu Ala Leu Ala Leu Ile Phe Gly Ala Leu Ile Arg
210 215 220Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met
Thr Pro225 230 235 240Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
Pro Tyr Ala Pro Pro 245 250 255Arg Asp Phe Ala Ala Tyr Arg Ser
260167606DNAArtificial SequencehuTim3 ectodomain 167atgttctccc
atcttccctt cgactgtgtg ttgctccttc tcctcctgct tctcacccgg 60tcaagcgaag
tagagtaccg ggcggaagta ggtcagaacg catatctccc ctgtttttac
120acacccgctg cgccgggaaa cctggttccc gtgtgttggg gaaagggggc
atgccctgtt 180ttcgagtgtg gcaacgtggt cctccggacg gatgagcgag
acgtgaatta ttggacgagc 240agatattggt tgaatggcga ttttagaaag
ggtgatgtga gcttgaccat tgagaatgta 300acgcttgctg atagcgggat
atattgctgt agaattcaaa tccctggtat aatgaacgac 360gaaaaattca
atctgaagct ggtaattaag ccggccaagg tgacacccgc cccgacacga
420cagcgcgact tcacggctgc ctttccacgc atgttgacca caaggggaca
tggtccagcg 480gagacccaga cacttggtag cctcccggac ataaacctca
cacaaatatc cacgttggcg 540aacgagctcc gagattccag gcttgcgaat
gacctgaggg attctggagc taccatcaga 600atcggt 606168202PRTArtificial
SequencehuTim3 ectodomain 168Met Phe Ser His Leu Pro Phe Asp Cys
Val Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Thr Arg Ser Ser Glu Val
Glu Tyr Arg Ala Glu Val Gly Gln 20 25 30Asn Ala Tyr Leu Pro Cys Phe
Tyr Thr Pro Ala Ala Pro Gly Asn Leu 35 40 45Val Pro Val Cys Trp Gly
Lys Gly Ala Cys Pro Val Phe Glu Cys Gly 50 55 60Asn Val Val Leu Arg
Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser65 70 75 80Arg Tyr Trp
Leu Asn Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr 85 90 95Ile Glu
Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile 100 105
110Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val
115 120 125Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln Arg
Asp Phe 130 135 140Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly
His Gly Pro Ala145 150 155 160Glu Thr Gln Thr Leu Gly Ser Leu Pro
Asp Ile Asn Leu Thr Gln Ile 165 170 175Ser Thr Leu Ala Asn Glu Leu
Arg Asp Ser Arg Leu Ala Asn Asp Leu 180 185 190Arg Asp Ser Gly Ala
Thr Ile Arg Ile Gly 195 20016963DNAArtificial SequencehuTim3
transmembrane 169atctacatag gtgccgggat atgcgccggt ctcgcacttg
ccttgatttt cggggcactg 60att 6317021PRTArtificial SequencehuTim3
transmembrane 170Ile Tyr Ile Gly Ala Gly Ile Cys Ala Gly Leu Ala
Leu Ala Leu Ile1 5 10 15Phe Gly Ala Leu Ile 20171810DNAArtificial
SequencehuTim3-CD28tm 171atgttctccc atcttccctt cgactgtgtg
ttgctccttc tcctcctgct tctcacccgg 60tcaagcgaag tagagtaccg ggcggaagta
ggtcagaacg catatctccc ctgtttttac 120acacccgctg cgccgggaaa
cctggttccc gtgtgttggg gaaagggggc atgccctgtt 180ttcgagtgtg
gcaacgtggt cctccggacg gatgagcgag acgtgaatta ttggacgagc
240agatattggt tgaatggcga ttttagaaag ggtgatgtga gcttgaccat
tgagaatgta 300acgcttgctg atagcgggat atattgctgt agaattcaaa
tccctggtat aatgaacgac 360gaaaaattca atctgaagct ggtaattaag
ccggccaagg tgacacccgc cccgacacga 420cagcgcgact tcacggctgc
ctttccacgc atgttgacca caaggggaca tggtccagcg 480gagacccaga
cacttggtag cctcccggac ataaacctca cacaaatatc cacgttggcg
540aacgagctcc gagattccag gcttgcgaat gacctgaggg attctggagc
taccatcaga 600atcggtttct gggtgctggt ggtggtcgga ggcgtgctgg
cctgctacag cctgctggtc 660accgtggcct tcatcatctt ttgggtccgc
agcaagcgga gcagaggcgg ccacagcgac 720tacatgaaca tgacccctag
acggcctggc cccaccagaa agcactacca gccctacgcc 780cctccccggg
actttgccgc ctacagaagc 810172270PRTArtificial SequencehuTim3-CD28tm
172Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1
5 10 15Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly
Gln 20 25 30Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly
Asn Leu 35 40 45Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe
Glu Cys Gly 50 55 60Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn
Tyr Trp Thr Ser65 70 75 80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys
Gly Asp Val Ser Leu Thr 85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser
Gly Ile Tyr Cys Cys Arg Ile 100 105 110Gln Ile Pro Gly Ile Met Asn
Asp Glu Lys Phe Asn Leu Lys Leu Val 115 120 125Ile Lys Pro Ala Lys
Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe 130 135 140Thr Ala Ala
Phe Pro Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala145 150 155
160Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile
165 170 175Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn
Asp Leu 180 185 190Arg Asp Ser Gly Ala Thr Ile Arg Ile Gly Phe Trp
Val Leu Val Val 195 200 205Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
Leu Val Thr Val Ala Phe 210 215 220Ile Ile Phe Trp Val Arg Ser Lys
Arg Ser Arg Gly Gly His Ser Asp225 230 235 240Tyr Met Asn Met Thr
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 245 250 255Gln Pro Tyr
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 260 265
270173846DNAArtificial SequencehuTim3-CD28Cys 173atgttctccc
atcttccctt cgactgtgtg ttgctccttc tcctcctgct tctcacccgg 60tcaagcgaag
tagagtaccg ggcggaagta ggtcagaacg catatctccc ctgtttttac
120acacccgctg cgccgggaaa cctggttccc gtgtgttggg gaaagggggc
atgccctgtt 180ttcgagtgtg gcaacgtggt cctccggacg gatgagcgag
acgtgaatta ttggacgagc 240agatattggt tgaatggcga ttttagaaag
ggtgatgtga gcttgaccat tgagaatgta 300acgcttgctg atagcgggat
atattgctgt agaattcaaa tccctggtat aatgaacgac 360gaaaaattca
atctgaagct ggtaattaag ccggccaagg tgacacccgc cccgacacga
420cagcgcgact tcacggctgc ctttccacgc atgttgacca caaggggaca
tggtccagcg 480gagacccaga cacttggtag cctcccggac ataaacctca
cacaaatatc cacgttggcg 540aacgagctcc gagattccag gcttgcgaat
gacctgaggg attctggagc taccatcaga 600atcggttgtc ccagccctct
gtttcccggc cctagcaagc ctttctgggt gctggtggtg 660gtcggaggcg
tgctggcctg ctacagcctg ctggtcaccg tggccttcat catcttttgg
720gtccgcagca agcggagcag aggcggccac agcgactaca tgaacatgac
ccctagacgg 780cctggcccca ccagaaagca ctaccagccc tacgcccctc
cccgggactt tgccgcctac 840agaagc 846174282PRTArtificial
SequencehuTim3-CD28Cys 174Met Phe Ser His Leu Pro Phe Asp Cys Val
Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu Thr Arg Ser Ser Glu Val Glu
Tyr Arg Ala Glu Val Gly Gln 20 25 30Asn Ala Tyr Leu Pro Cys Phe Tyr
Thr Pro Ala Ala Pro Gly Asn Leu 35 40 45Val Pro Val Cys Trp Gly Lys
Gly Ala Cys Pro Val Phe Glu Cys Gly 50 55 60Asn Val Val Leu Arg Thr
Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser65 70 75 80Arg Tyr Trp Leu
Asn Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr 85 90 95Ile Glu Asn
Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile 100 105 110Gln
Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val 115 120
125Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe
130 135 140Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly His Gly
Pro Ala145 150 155 160Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile
Asn Leu Thr Gln Ile 165 170 175Ser Thr Leu Ala Asn Glu Leu Arg Asp
Ser Arg Leu Ala Asn Asp Leu 180 185 190Arg Asp Ser Gly Ala Thr Ile
Arg Ile Gly Cys Pro Ser Pro Leu Phe 195 200 205Pro Gly Pro Ser Lys
Pro Phe Trp Val Leu Val Val Val Gly Gly Val 210 215 220Leu Ala Cys
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp225 230 235
240Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met
245 250 255Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
Tyr Ala 260 265 270Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 275
280175810DNAArtificial SequencehuTim3-12aas-CD28Cys 175atgttctccc
atcttccctt cgactgtgtg ttgctccttc tcctcctgct tctcacccgg 60tcaagcgaag
tagagtaccg ggcggaagta ggtcagaacg catatctccc ctgtttttac
120acacccgctg cgccgggaaa cctggttccc gtgtgttggg gaaagggggc
atgccctgtt 180ttcgagtgtg gcaacgtggt cctccggacg gatgagcgag
acgtgaatta ttggacgagc 240agatattggt tgaatggcga ttttagaaag
ggtgatgtga gcttgaccat tgagaatgta 300acgcttgctg atagcgggat
atattgctgt agaattcaaa tccctggtat aatgaacgac 360gaaaaattca
atctgaagct ggtaattaag ccggccaagg tgacacccgc cccgacacga
420cagcgcgact tcacggctgc ctttccacgc atgttgacca caaggggaca
tggtccagcg 480gagacccaga cacttggtag cctcccggac ataaacctca
cacaaatatc cacgttggcg 540aacgagctcc gagattccag gcttgcgaat
tgtcccagcc ctctgtttcc cggccctagc 600aagcctttct gggtgctggt
ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc 660accgtggcct
tcatcatctt ttgggtccgc agcaagcgga gcagaggcgg ccacagcgac
720tacatgaaca tgacccctag acggcctggc cccaccagaa agcactacca
gccctacgcc 780cctccccggg actttgccgc ctacagaagc
810176270PRTArtificial SequencehuTim3-12aas-CD28Cys 176Met Phe Ser
His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1 5 10 15Leu Leu
Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln 20 25 30Asn
Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu 35 40
45Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly
50 55 60Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr
Ser65 70 75 80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val
Ser Leu Thr 85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr
Cys Cys Arg Ile 100 105 110Gln Ile Pro Gly Ile Met Asn Asp Glu Lys
Phe Asn Leu Lys Leu Val 115 120 125Ile Lys Pro Ala Lys Val Thr Pro
Ala Pro Thr Arg Gln Arg Asp Phe 130 135 140Thr Ala Ala Phe Pro Arg
Met Leu Thr Thr Arg Gly His Gly Pro Ala145 150 155 160Glu Thr Gln
Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile 165 170 175Ser
Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn Cys Pro 180 185
190Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val
195 200 205Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
Ala Phe 210 215 220Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly
Gly His Ser Asp225 230 235 240Tyr Met Asn Met Thr Pro Arg Arg Pro
Gly Pro Thr Arg Lys His Tyr 245 250 255Gln Pro Tyr Ala Pro Pro Arg
Asp Phe Ala Ala Tyr Arg Ser 260 265 270177570DNAArtificial
SequencehuTim3-12aas 177atgttctccc atcttccctt cgactgtgtg ttgctccttc
tcctcctgct tctcacccgg 60tcaagcgaag tagagtaccg ggcggaagta ggtcagaacg
catatctccc ctgtttttac 120acacccgctg cgccgggaaa cctggttccc
gtgtgttggg gaaagggggc atgccctgtt 180ttcgagtgtg gcaacgtggt
cctccggacg gatgagcgag acgtgaatta ttggacgagc 240agatattggt
tgaatggcga ttttagaaag ggtgatgtga gcttgaccat tgagaatgta
300acgcttgctg atagcgggat atattgctgt agaattcaaa tccctggtat
aatgaacgac 360gaaaaattca atctgaagct ggtaattaag ccggccaagg
tgacacccgc cccgacacga 420cagcgcgact tcacggctgc ctttccacgc
atgttgacca caaggggaca tggtccagcg 480gagacccaga cacttggtag
cctcccggac ataaacctca cacaaatatc cacgttggcg 540aacgagctcc
gagattccag gcttgcgaat 570178190PRTArtificial SequencehuTim3-12aas
178Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu1
5 10 15Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly
Gln 20 25 30Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly
Asn Leu 35 40 45Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe
Glu Cys Gly 50 55 60Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn
Tyr Trp Thr Ser65 70 75 80Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys
Gly Asp Val Ser Leu Thr 85 90 95Ile Glu Asn Val Thr Leu Ala Asp Ser
Gly Ile Tyr Cys Cys Arg Ile 100 105
110Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val
115 120 125Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln Arg
Asp Phe 130 135 140Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly
His Gly Pro Ala145 150 155 160Glu Thr Gln Thr Leu Gly Ser Leu Pro
Asp Ile Asn Leu Thr Gln Ile 165 170 175Ser Thr Leu Ala Asn Glu Leu
Arg Asp Ser Arg Leu Ala Asn 180 185 190
* * * * *