U.S. patent application number 17/576109 was filed with the patent office on 2022-05-05 for anti-sirp alpha antibodies.
This patent application is currently assigned to SAIROPA B.V.. The applicant listed for this patent is SAIROPA B.V.. Invention is credited to David Lutje HULSIK, Hans VAN EENENNAAM, Andrea VAN ELSAS, Paul VINK, Erik VOETS.
Application Number | 20220135677 17/576109 |
Document ID | / |
Family ID | 1000006082869 |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220135677 |
Kind Code |
A1 |
VAN EENENNAAM; Hans ; et
al. |
May 5, 2022 |
ANTI-SIRP ALPHA ANTIBODIES
Abstract
The present invention relates to anti-SIRP.alpha. antibodies, as
well as use of these antibodies in the treatment of diseases such
as cancer and infectious disease.
Inventors: |
VAN EENENNAAM; Hans;
(ROTTERDAM, NL) ; VAN ELSAS; Andrea; (ROTTERDAM,
NL) ; VOETS; Erik; (ROTTERDAM, NL) ; VINK;
Paul; (ROTTERDAM, NL) ; HULSIK; David Lutje;
(ROTTERDAM, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SAIROPA B.V. |
ROTTERDAM |
|
NL |
|
|
Assignee: |
SAIROPA B.V.
ROTTERDAM
NL
|
Family ID: |
1000006082869 |
Appl. No.: |
17/576109 |
Filed: |
January 14, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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17107334 |
Nov 30, 2020 |
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17576109 |
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15953201 |
Apr 13, 2018 |
10851164 |
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17107334 |
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Current U.S.
Class: |
424/133.1 |
Current CPC
Class: |
C07K 2317/92 20130101;
G01N 2333/70503 20130101; G01N 33/6854 20130101; C07K 2317/24
20130101; C07K 2317/76 20130101; C07K 2317/33 20130101; C07K
2317/70 20130101; G01N 33/57484 20130101; C07K 16/2803 20130101;
C07K 2317/34 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/68 20060101 G01N033/68; G01N 33/574 20060101
G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 13, 2017 |
NL |
2018708 |
Jul 3, 2017 |
NL |
2019166 |
Claims
1. An isolated therapeutic antibody, comprising: a variable region
that binds to human SIRP.alpha.V1 protein having the sequence of
SEQ ID NO: 34 with an EC.sub.50 of 10 nM or less, wherein the
variable region discriminates between human SIRP isoforms
comprising a proline in the SIRP IgV domain at a position
corresponding to Pro.sub.74 of SEQ ID NO: 34 and human SIRP
isoforms in which the proline corresponding to residue 74 of SEQ ID
NO: 34 is replaced by alanine.
2. An isolated therapeutic antibody according to claim 1, wherein
the antibody inhibits binding of human SIRP.alpha. having the
sequence of SEQ ID NO: 34 to human CD47 with an IC.sub.50 of of 10
nM or less.
3. An isolated therapeutic antibody according to claim 1, wherein
the antibody binds to a cell expressing human SIRP.alpha.V2 protein
with an EC.sub.50 of 10 nM or less.
4. An isolated therapeutic antibody according to claim 3, wherein
the antibody binds to a cell expressing human SIRP.alpha.V1 protein
having the sequence of SEQ ID NO: 34 with an EC.sub.50 of 1 nM or
less, and binds to a cell expressing human SIRP.alpha.V2 protein
with an EC.sub.50 of 1 nM or less.
5. An isolated therapeutic antibody of claim 1, wherein the
antibody is an IgG.
6. An isolated therapeutic antibody of claim 5, wherein each of the
two variable regions of the IgG binds to human SIRP.alpha.V1
protein having the sequence of SEQ ID NO: 34 with an EC.sub.50 of
10 nM or less and discriminates between human SIRP isoforms
comprising a proline in the SIRP IgV domain at a position
corresponding to Pro.sub.74 of SEQ ID NO: 34 and human SIRP
isoforms in which the proline corresponding to residue 74 of SEQ ID
NO: 34 is replaced by alanine.
7. An isolated therapeutic antibody of claim 5, wherein the
antibody exhibits a T20 "humanness" score of at least 79.
8. An isolated therapeutic antibody of claim 6, wherein the
antibody exhibits a T20 "humanness" score of at least 79.
9. An isolated therapeutic antibody of claim 4, wherein the
antibody is an IgG.
10. An isolated therapeutic antibody of claim 9, wherein each of
the two variable regions of the IgG binds to human SIRP.alpha.V1
protein having the sequence of SEQ ID NO: 34 with an EC.sub.50 of
10 nM or less and discriminates between human SIRP isoforms
comprising a proline in the SIRP IgV domain at a position
corresponding to Pro.sub.74 of SEQ ID NO: 34 and human SIRP
isoforms in which the proline corresponding to residue 74 of SEQ ID
NO: 34 is replaced by alanine.
11. An isolated therapeutic antibody of claim 9, wherein the
antibody exhibits a T20 "humanness" score of at least 79.
12. An isolated therapeutic antibody of claim 10, wherein the
antibody exhibits a T20 "humanness" score of at least 79.
13. A composition comprising: the therapeutic antibody of claim 1
and a pharmaceutically acceptable carrier or diluent.
14. A composition comprising: the therapeutic antibody of claim 4
and a pharmaceutically acceptable carrier or diluent.
15. A composition comprising: the therapeutic antibody of claim 5
and a pharmaceutically acceptable carrier or diluent.
16. A composition comprising: the therapeutic antibody of claim 6
and a pharmaceutically acceptable carrier or diluent.
17. A composition comprising: the therapeutic antibody of claim 7
and a pharmaceutically acceptable carrier or diluent.
18. A composition comprising: the therapeutic antibody of claim 8
and a pharmaceutically acceptable carrier or diluent.
19. A composition comprising: the therapeutic antibody of claim 9
and a pharmaceutically acceptable carrier or diluent.
20. A composition comprising: the therapeutic antibody of claim 10
and a pharmaceutically acceptable carrier or diluent.
21. A composition comprising: the therapeutic antibody of claim 11
and a pharmaceutically acceptable carrier or diluent.
22. A composition comprising: the therapeutic antibody of claim 12
and a pharmaceutically acceptable carrier or diluent.
Description
[0001] The present application is a divisional of U.S. patent
application Ser. No. 17/107,334, filed on Nov. 30, 2020, which is a
divisional of U.S. patent application Ser. No. 15/953,201, filed
Apr. 13, 2018, now U.S. Pat. No. 10,851,164, which claims the
benefit of Netherlands Patent Application No. 2018708, filed Apr.
13, 2017, and of Netherlands Patent Application No. 2019166, filed
Jul. 3, 2017, each of which is hereby incorporated by reference in
its entirety including all tables, figures, and claims.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] This application includes an electronically submitted
sequence listing in .txt format. The .txt file contains a sequence
listing entitled "ABE-0007-DV2_SeqListing.txt" created on Jan. 14,
2022, and is 233 kilobytes in size. The sequence listing contained
in this .txt file is part of the specification and is hereby
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to anti-SIRP.alpha.
antibodies, as well as use of these antibodies in the treatment of
diseases.
BACKGROUND OF THE INVENTION
[0004] Signal regulatory protein alpha (SIRP.alpha.) is membrane
glycoprotein from the SIRP family. Members of the SIRP family share
certain common structural motifs. These include a transmembrane
segment and an N-terminal extracellular domain that contains three
Ig-like loops connected by three pairs of disulfide bonds. The
C-terminal intracellular domain, however, differs between SIRP
family members. SIRP.alpha. has an extended intracellular domain
containing four tyrosine residues that form two immunoreceptor
tyrosine-based inhibitory motifs (ITIMs), while SIRP.beta.1
contains a lysine residue in the transmembrane domain followed by a
short intracellular tail lacking ITIMs serving as a receptor for
DAP12. Eight SIRP.alpha. single nucleotide polymorphisms have been
identified, with the most prevalent being SIRP.alpha.V1 and
SIRP.alpha.V2 (Takenaka et al., Nat. Immunol. 2007, 8:1313-23).
[0005] "Eat-me" signals (i.e. "altered self') are extracellular
players specifically produced by and displayed on the surface of
apoptotic cells, but not healthy cells, and are key to the
initiation of phagocytosis by activating phagocytic receptors and
subsequent signaling cascades. Eat-me signals require extracellular
trafficking in order to be displayed on apoptotic cells. A
particular category of eat-me signals is provided by
membrane-anchored proteins such as phosphatidylserine (PtdSer) and
calreticulin (CRT). Externalized PtdSer binds to its receptors on
phagocytes to facilitate clearance of apoptotic cells (a process
known as efferocytosis). Likewise, CRT is upregulated on the
surface of apoptotic cells and binds to LDL-receptor-related
protein 1 (LRP1) on the phagocyte thereby mediating engulfment.
[0006] SIRP.alpha. is broadly expressed on phagocytes (e.g.,
macrophages, granulocytes, and dendritic cells) and acts as an
inhibitory receptor through its interaction with a transmembrane
protein CD47. This interaction mediates a response referred to as
the "don't eat me" signal. This interaction negatively regulates
effector function of innate immune cells such as host cell
phagocytosis. As CD47 is often present on tumor cells, this "don't
eat me" signal is thought to contribute to the resistance of tumors
to phagocyte-dependent clearance. Despite the similarities in the
extracellular domains of SIRP.alpha. and SIRP.beta.1 functional
differences exist among the SIRP family members. For example,
SIRP.beta.1 does not bind CD47 at detectable levels and so does not
mediate the "don't eat me" signal. Instead, SIRP.beta.1 is involved
in the activation of myeloid cells.
[0007] Disruption of CD47-SIRP.alpha. signalling (e.g., by
antagonistic monoclonal antibodies that bind to either CD47 or
SIRP.alpha.) reportedly results in enhanced phagocytosis of both
solid and hematopoietic tumor cells, including increased
phagocytosis of glioblastoma cells in vitro and significant
anti-tumor activity in vivo.
SUMMARY OF THE INVENTION
[0008] In a first aspect, the invention provides anti-SIRP.alpha.
antibodies and antigen binding fragments thereof comprising the
structural and functional features specified below.
[0009] In various embodiments, the invention provides an antibody
or antigen binding fragment thereof that binds to human SIRP.alpha.
comprising one, two, or all three of (i), (ii) and (iii): (i) a
heavy chain variable region CDR1 comprising the amino acid sequence
of SEQ ID NO: 1 or an amino acid sequence differing from SEQ ID NO:
1 by 1, 2, 3, or more conservative substitutions; (ii) a heavy
chain variable region CDR2 comprising the amino acid sequence of
SEQ ID NO: 2 or an amino acid sequence differing from SEQ ID NO: 2
by 1, 2, 3, or more conservative substitutions; and/or (iii) a
heavy chain variable region CDR3 comprising the amino acid sequence
of SEQ ID NO: 3 or an amino acid sequence differing from SEQ ID NO:
3 by 1, 2, 3, or more conservative substitutions.
[0010] In various other embodiments, the invention provides an
antibody or antigen binding fragment thereof that binds to human
SIRP.alpha. comprising one, two, or all three of (i), (ii) and
(iii): (i) a heavy chain variable region CDR1 comprising the amino
acid sequence of SEQ ID NO: 69 or an amino acid sequence differing
from SEQ ID NO: 1 by 1, 2, 3, or more conservative substitutions;
(ii) a heavy chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 70 or an amino acid sequence differing from
SEQ ID NO: 2 by 1, 2, 3, or more conservative substitutions; and/or
(iii) a heavy chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 71 or an amino acid sequence differing from
SEQ ID NO: 3 by 1, 2, 3, or more conservative substitutions.
[0011] In certain embodiments, the antibody or antigen binding
fragment thereof comprises a heavy chain variable region comprising
an amino acid sequence selected from the group consisting of:
[0012] SEQ ID NO: 75 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0013] SEQ ID NO: 78
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0014] SEQ ID NO: 80 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0015] SEQ ID NO: 82 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0016] SEQ
ID NO: 84 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, [0017] SEQ ID NO: 86 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, [0018] SEQ ID NO: 88 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% identical thereto, [0019] SEQ
ID NO: 102 or an amino acid sequence at least 90%, 95%, 97%, 98%,
or 99% identical thereto, [0020] SEQ ID NO: 7 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0021] SEQ ID NO: 10 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0022] SEQ
ID NO: 12 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, [0023] SEQ ID NO: 14 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, [0024] SEQ ID NO: 16 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0025] SEQ ID NO: 18 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, and [0026] SEQ ID
NO: 30 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto.
[0027] In various embodiments, the invention also provides an
antibody or antigen binding fragment thereof that binds to human
SIRP.alpha. comprising one, two, or all three of (i), (ii) and
(iii): (i) a light chain variable region CDR1 comprising the amino
acid sequence of SEQ ID NO: 4 or an amino acid sequence differing
from SEQ ID NO: 4 by 1, 2, 3, or more conservative substitutions;
(ii) a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 5 or an amino acid sequence differing from
SEQ ID NO: 5 by 1, 2, 3, or more conservative substitutions; and/or
(iii) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 6 or an amino acid sequence differing from
SEQ ID NO: 6 by 1, 2, 3, or more conservative substitutions.
[0028] In various other embodiments, the invention also provides an
antibody or antigen binding fragment thereof that binds to human
SIRP.alpha. comprising one, two, or all three of (i), (ii) and
(iii): (i) a light chain variable region CDR1 comprising the amino
acid sequence of SEQ ID NO: 72 or an amino acid sequence differing
from SEQ ID NO: 4 by 1, 2, 3, or more conservative substitutions;
(ii) a light chain variable region CDR2 comprising the amino acid
sequence of SEQ ID NO: 73 or an amino acid sequence differing from
SEQ ID NO: 5 by 1, 2, 3, or more conservative substitutions; and/or
(iii) a light chain variable region CDR3 comprising the amino acid
sequence of SEQ ID NO: 74 or an amino acid sequence differing from
SEQ ID NO: 6 by 1, 2, 3, or more conservative substitutions.
[0029] In certain embodiments, the antibody or antigen binding
fragment thereof comprises a light chain variable region comprising
an amino acid sequence selected from the group consisting of:
[0030] SEQ ID NO: 76 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0031] SEQ ID NO: 90
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0032] SEQ ID NO: 92 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0033] SEQ ID NO: 94 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0034] SEQ
ID NO: 96 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, [0035] SEQ ID NO: 98 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, [0036] SEQ ID NO: 100 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0037] SEQ ID NO: 104 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% identical thereto, [0038] SEQ ID NO: 8 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, [0039] SEQ ID NO: 20 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0040] SEQ ID NO: 22 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0041] SEQ ID NO: 24
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0042] SEQ ID NO: 26 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0043] SEQ ID NO: 28 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, and [0044]
SEQ ID NO: 32 or an amino acid sequence at least 90%, 95%, 97%,
98%, or 99% similar or identical thereto.
[0045] In various embodiments, the invention provides an antibody
or antigen binding fragment thereof that binds to human SIRP.alpha.
comprising: [0046] (i) a heavy chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid
sequence differing from SEQ ID NO: 1 by 1, 2, 3, or more
conservative substitutions; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid
sequence differing from SEQ ID NO: 2 by 1, 2, 3, or more
conservative substitutions; and/or (iii) a heavy chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or
an amino acid sequence differing from SEQ ID NO: 3 by 1, 2, 3, or
more conservative substitutions; [0047] and [0048] (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 4 or an amino acid sequence differing from SEQ ID NO: 4
by 1, 2, 3, or more conservative substitutions; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 5 or an amino acid sequence differing from SEQ ID NO: 5 by 1,
2, 3, or more conservative substitutions; and/or (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 6 or an amino acid sequence differing from SEQ ID NO: 6 by 1,
2, 3, or more conservative substitutions.
[0049] In various other embodiments, the invention provides an
antibody or antigen binding fragment thereof that binds to human
SIRP.alpha. comprising: [0050] (i) a heavy chain variable region
CDR1 comprising the amino acid sequence of SEQ ID NO: 69 or an
amino acid sequence differing from SEQ ID NO: 1 by 1, 2, 3, or more
conservative substitutions; (ii) a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO: 70 or an amino
acid sequence differing from SEQ ID NO: 2 by 1, 2, 3, or more
conservative substitutions; and/or (iii) a heavy chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO: 71 or
an amino acid sequence differing from SEQ ID NO: 3 by 1, 2, 3, or
more conservative substitutions; [0051] and [0052] (iv) a light
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO: 72 or an amino acid sequence differing from SEQ ID NO: 4
by 1, 2, 3, or more conservative substitutions; (v) a light chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO: 73 or an amino acid sequence differing from SEQ ID NO: 5 by 1,
2, 3, or more conservative substitutions; and/or (vi) a light chain
variable region CDR3 comprising the amino acid sequence of SEQ ID
NO: 6 or an amino acid sequence differing from SEQ ID NO: 74 by 1,
2, 3, or more conservative substitutions.
[0053] In still other embodiments, the invention provides an
antibody or antigen binding fragment thereof that binds to human
SIRP.alpha. comprising:
a heavy chain variable region comprising an amino acid sequence
selected from the group consisting of: [0054] SEQ ID NO: 7 or an
amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, [0055] SEQ ID NO: 10 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0056] SEQ ID NO: 12 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0057] SEQ ID NO: 14
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0058] SEQ ID NO: 16 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0059] SEQ ID NO: 18 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, and [0060]
SEQ ID NO: 30 or an amino acid sequence at least 90%, 95%, 97%,
98%, or 99% similar or identical thereto; and a light chain
variable region comprising an amino acid sequence selected from the
group consisting of: [0061] SEQ ID NO: 8 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0062] SEQ ID NO: 20 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0063] SEQ ID NO: 22
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0064] SEQ ID NO: 24 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0065] SEQ ID NO: 26 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, and [0066]
SEQ ID NO: 28 or an amino acid sequence at least 90%, 95%, 97%,
98%, or 99% similar or identical thereto, and [0067] SEQ ID NO: 32
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto.
[0068] In still other embodiments, the invention provides an
antibody or antigen binding fragment thereof that binds to human
SIRP.alpha. comprising: [0069] a heavy chain variable region
comprising an amino acid sequence selected from the group
consisting of: [0070] SEQ ID NO: 75 or an amino acid sequence at
least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0071] SEQ ID NO: 78 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0072] SEQ ID NO: 80
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0073] SEQ ID NO: 82 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0074] SEQ ID NO: 84 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0075] SEQ
ID NO: 86 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, [0076] SEQ ID NO: 88 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto; and [0077] SEQ ID NO: 102 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto, and
[0078] a light chain variable region comprising an amino acid
sequence selected from the group consisting of: [0079] SEQ ID NO:
76 or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0080] SEQ ID NO: 90 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0081] SEQ ID NO: 92 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0082] SEQ
ID NO: 94 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, [0083] SEQ ID NO: 96 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, [0084] SEQ ID NO: 98 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0085] SEQ ID NO: 100 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, and [0086] SEQ ID
NO: 104 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% identical thereto.
[0087] In this context, "sequence similarity" is based on the
extent of identity combined with the extent of conservative
changes. The percentage of "sequence similarity" is the percentage
of amino acids or nucleotides which is either identical or
conservatively changed viz. "sequence similarity"=percent sequence
identity)+percent conservative changes). Thus, for the purpose of
this invention "conservative changes" and "identity" are considered
to be species of the broader term "similarity". Thus, whenever the
term sequence "similarity" is used it embraces sequence "identity"
and "conservative changes". According to certain embodiments the
conservative changes are disregarded and the percent sequence
similarity refers to percent sequence identity. In certain
embodiments, the changes in a sequence permitted by the referenced
percent sequence identity are all or nearly all conservative
changes; that is, when a sequence is 90% identical, the remaining
10% are all or nearly all conservative changes. The term "nearly
all" in this context refers to at least 75% of the permitted
sequence changes are conservative changes, more preferably at least
85%, still more preferably at least 90%, and most preferably at
least 95%. In certain embodiments of antibody heavy and/or light
chains, the permitted sequence changes are within the framework
regions and not in the CDRs.
[0088] Preferably said antibody has a heavy chain according to SEQ
ID NO: 7. Further preferably said antibody has a light chain
according to SEQ ID NO: 8. More preferably, the heavy chain is
chosen from any of SEQ ID NO: 10, 12, 14, 16, 18, or 30. More
preferably, the light chain is chosen from any of SEQ ID NO: 20,
22, 24, 26, 28, or 32.
[0089] Alternatively, said antibody has a heavy chain according to
SEQ ID NO: 75. Further preferably said antibody has a light chain
according to SEQ ID NO: 76. More preferably, the heavy chain is
chosen from any of SEQ ID NO: 78, 80, 82, 84, 86, 88 or 102. More
preferably, the light chain is chosen from any of SEQ ID NO: 90,
92, 94, 96, 98, 100 or 104.
[0090] In any of the above embodiments, the antibody or antigen
binding fragment thereof may be isolated, as that term is defined
herein.
[0091] In any of the above embodiments, the antibody or antigen
binding fragment thereof is a recombinant antibody, as that term is
defined herein.
[0092] In any of the above embodiments, the antibody or antigen
binding fragment thereof is a full-length antibody, as that term is
defined herein.
[0093] Antibodies or antigen binding fragments of the present
invention may be obtained from a variety of species. For example,
the antibodies of the present invention may comprise immunoglobulin
sequences which are rabbit, mouse, rat, guinea pig, chicken, goat,
sheep, donkey, human, llama or camelid sequences, or combinations
of such sequences (so-called chimeric antibodies). Most preferably,
the antibodies or antigen binding fragments are human or humanized
antibodies or antigen binding fragments.
[0094] The term antibody includes antigen-binding portions, i.e.,
"antigen binding sites," (e.g., fragments, subsequences,
complementarity determining regions (CDRs)) that retain capacity to
bind antigen, including (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains;
(ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region;
(iii) a Fd fragment consisting of the V.sub.H and C.sub.H1 domains;
(iv) a Fv fragment consisting of the V.sub.L and V.sub.H domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). Single chain
antibodies are also included by reference in the term "antibody."
Preferred therapeutic antibodies are intact IgG antibodies. The
term "intact IgG" as used herein is meant as a polypeptide
belonging to the class of antibodies that are substantially encoded
by a recognized immunoglobulin gamma gene. In humans this class
comprises IgG1, IgG2, IgG3, and IgG4. In mice this class comprises
IgG1, IgG2a, IgG2b, and IgG3. The known Ig domains in the IgG class
of antibodies are V.sub.H, C.gamma.1, C.gamma.2, C.gamma.3,
V.sub.L, and C.sub.L.
[0095] In any of the above embodiments, the antibody or antigen
binding fragment thereof is a human or humanized antibody
comprising two heavy chains and two light chains. In one
embodiment, the antibody is an IgG. In preferred embodiments,
antibody is an IgG1, IgG2, or IgG4, and preferably a human IgG1,
IgG2, or IgG4.
[0096] In any of the above-mentioned embodiments, the antibody or
antigen binding fragment thereof of the invention can comprise any
of the light chain variable regions described above and a human
kappa or lambda light chain constant domain and an IgG1, IgG2, or
IgG4 heavy chain constant domain. Exemplary light (kappa) and heavy
(IgG2 and IgG4) constant region sequences which may be used in
accordance with the invention are recited in SEQ ID NOs: 63, 65, 67
(each a nucleotide sequence), 64, 66, and 68 (each a polypeptide
sequence).
[0097] By way of example only, in various embodiments such antibody
or antigen binding fragment thereof comprises one of the following
combinations of heavy chain sequence/light chain variable region
sequences:
SEQ ID NO: 10/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H1L1) SEQ ID NO: 10/SEQ ID NO: 22 (referred to
herein as hSIRP.alpha..50A.H1L2) SEQ ID NO: 10/SEQ ID NO: 24
(referred to herein as hSIRP.alpha..50A.H1L3) SEQ ID NO: 10/SEQ ID
NO: 26 (referred to herein as hSIRP.alpha..50A.H1L4) SEQ ID NO:
10/SEQ ID NO: 28 (referred to herein as hSIRP.alpha..50A.H1L5) SEQ
ID NO: 12/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H2L1) SEQ ID NO: 12/SEQ ID NO: 22 (referred to
herein as hSIRP.alpha..50A.H2L2) SEQ ID NO: 12/SEQ ID NO: 24
(referred to herein as hSIRP.alpha..50A.H2L3) SEQ ID NO: 12/SEQ ID
NO: 26 (referred to herein as hSIRP.alpha..50A.H2L4) SEQ ID NO:
12/SEQ ID NO: 28 (referred to herein as hSIRP.alpha..50A.H2L5) SEQ
ID NO: 14/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H3L1) SEQ ID NO: 14/SEQ ID NO: 22 (referred to
herein as hSIRP.alpha..50A.H3L2) SEQ ID NO: 14/SEQ ID NO: 24
(referred to herein as hSIRP.alpha..50A.H3L3) SEQ ID NO: 14/SEQ ID
NO: 26 (referred to herein as hSIRP.alpha..50A.H3L4) SEQ ID NO:
14/SEQ ID NO: 28 (referred to herein as hSIRP.alpha..50A.H3L5) SEQ
ID NO: 16/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H4L1) SEQ ID NO: 16/SEQ ID NO: 22 (referred to
herein as hSIRP.alpha..50A.H4L2) SEQ ID NO: 16/SEQ ID NO: 24
(referred to herein as hSIRP.alpha..50A.H4L3) SEQ ID NO: 16/SEQ ID
NO: 26 (referred to herein as hSIRP.alpha..50A.H4L4) SEQ ID NO:
16/SEQ ID NO: 28 (referred to herein as hSIRP.alpha..50A.H4L5) SEQ
ID NO: 18/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H5L1) SEQ ID NO: 18/SEQ ID NO: 22 (referred to
herein as hSIRP.alpha..50A.H5L2) SEQ ID NO: 18/SEQ ID NO: 24
(referred to herein as hSIRP.alpha..50A.H5L3) SEQ ID NO: 18/SEQ ID
NO: 26 (referred to herein as hSIRP.alpha..50A.H5L4) SEQ ID NO:
18/SEQ ID NO: 28 (referred to herein as hSIRP.alpha..50A.H5L5) SEQ
ID NO: 78/SEQ ID NO: 90 (referred to herein as
hSIRP.alpha..40A.H1L1) SEQ ID NO: 78/SEQ ID NO: 92 (referred to
herein as hSIRP.alpha..40A.H1L2) SEQ ID NO: 78/SEQ ID NO: 94
(referred to herein as hSIRP.alpha..40A.H1L3) SEQ ID NO: 78/SEQ ID
NO: 96 (referred to herein as hSIRP.alpha..40A.H1L4) SEQ ID NO:
78/SEQ ID NO: 98 (referred to herein as hSIRP.alpha..40A.H1L5) SEQ
ID NO: 78/SEQ ID NO: 100 (referred to herein as
hSIRP.alpha..40A.H1L6) SEQ ID NO: 80/SEQ ID NO: 90 (referred to
herein as hSIRP.alpha..40A.H2L1) SEQ ID NO: 80/SEQ ID NO: 92
(referred to herein as hSIRP.alpha..40A.H2L2) SEQ ID NO: 80/SEQ ID
NO: 94 (referred to herein as hSIRP.alpha..40A.H2L3) SEQ ID NO:
80/SEQ ID NO: 96 (referred to herein as hSIRP.alpha..40A.H2L4) SEQ
ID NO: 80/SEQ ID NO: 98 (referred to herein as
hSIRP.alpha..40A.H2L5) SEQ ID NO: 80/SEQ ID NO: 100 (referred to
herein as hSIRP.alpha..40A.H2L6) SEQ ID NO: 82/SEQ ID NO: 90
(referred to herein as hSIRP.alpha..40A.H3L1) SEQ ID NO: 82/SEQ ID
NO: 92 (referred to herein as hSIRP.alpha..40A.H3L2) SEQ ID NO:
82/SEQ ID NO: 94 (referred to herein as hSIRP.alpha..40A.H3L3) SEQ
ID NO: 82/SEQ ID NO: 96 (referred to herein as
hSIRP.alpha..40A.H3L4) SEQ ID NO: 82/SEQ ID NO: 98 (referred to
herein as hSIRP.alpha..40A.H3L5) SEQ ID NO: 82/SEQ ID NO: 100
(referred to herein as hSIRP.alpha..40A.H3L6) SEQ ID NO: 84/SEQ ID
NO: 90 (referred to herein as hSIRP.alpha..40A.H4L1) SEQ ID NO:
84/SEQ ID NO: 92 (referred to herein as hSIRP.alpha..40A.H4L2) SEQ
ID NO: 84/SEQ ID NO: 94 (referred to herein as
hSIRP.alpha..40A.H4L3) SEQ ID NO: 84/SEQ ID NO: 96 (referred to
herein as hSIRP.alpha..40A.H4L4) SEQ ID NO: 84/SEQ ID NO: 98
(referred to herein as hSIRP.alpha..40A.H4L5) SEQ ID NO: 84/SEQ ID
NO: 100 (referred to herein as hSIRP.alpha..40A.H4L6) SEQ ID NO:
86/SEQ ID NO: 90 (referred to herein as hSIRP.alpha..40A.H5L1) SEQ
ID NO: 86/SEQ ID NO: 92 (referred to herein as
hSIRP.alpha..40A.H5L2) SEQ ID NO: 86/SEQ ID NO: 94 (referred to
herein as hSIRP.alpha..40A.H5L3) SEQ ID NO: 86/SEQ ID NO: 96
(referred to herein as hSIRP.alpha..40A.H5L4) SEQ ID NO: 86/SEQ ID
NO: 98 (referred to herein as hSIRP.alpha..40A.H5L5) SEQ ID NO:
86/SEQ ID NO: 100 (referred to herein as hSIRP.alpha..40A.H5L6) SEQ
ID NO: 88/SEQ ID NO: 90 (referred to herein as
hSIRP.alpha..40A.H6L1) SEQ ID NO: 88/SEQ ID NO: 92 (referred to
herein as hSIRP.alpha..40A.H6L2) SEQ ID NO: 88/SEQ ID NO: 94
(referred to herein as hSIRP.alpha..40A.H6L3) SEQ ID NO: 88/SEQ ID
NO: 96 (referred to herein as hSIRP.alpha..40A.H6L4) SEQ ID NO:
88/SEQ ID NO: 98 (referred to herein as hSIRP.alpha..40A.H6L5) SEQ
ID NO: 88/SEQ ID NO: 100 (referred to herein as
hSIRP.alpha..40A.H6L6) or, in each case, at least 90%, 95%, 97%,
98%, or 99% similar or identical to a respective SEQ ID NO.
[0098] In some preferred embodiments, the antibody or antigen
binding fragment is a humanized antibody that comprises two heavy
chains and two light chains, wherein each heavy chain comprises SEQ
ID NO: 10 and each light chain comprises SEQ ID NO: 20, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% similar or identical to a
respective SEQ ID NO, and most preferably each light chain
comprises a human kappa light chain or a human lambda light chain
constant domain; and each heavy chain comprises a human IgG1, IgG2,
or IgG4 constant region.
[0099] In other preferred embodiments, the antibody or antigen
binding fragment is a humanized antibody that comprises two heavy
chains and two light chains, wherein each heavy chain comprises SEQ
ID NO: 16 and each light chain comprises SEQ ID NO: 28, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% similar or identical to a
respective SEQ ID NO, and most preferably each light chain
comprises a human kappa light chain or a human lambda light chain
constant domain; and each heavy chain comprises a human IgG1, IgG2,
or IgG4 constant region.
[0100] In still other preferred embodiments, the antibody or
antigen binding fragment is a humanized antibody that comprises two
heavy chains and two light chains, wherein each heavy chain
comprises SEQ ID NO: 18 and each light chain comprises SEQ ID NO:
20, or, in each case, at least 90%, 95%, 97%, 98%, or 99% similar
or identical to a respective SEQ ID NO, and most preferably each
light chain comprises a human kappa light chain or a human lambda
light chain constant domain; and each heavy chain comprises a human
IgG1, IgG2, or IgG4 constant region.
[0101] In some preferred embodiments, the antibody or antigen
binding fragment is a humanized antibody that comprises two heavy
chains and two light chains, wherein each heavy chain comprises SEQ
ID NO: 80 and each light chain comprises SEQ ID NO: 90, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% similar or identical to a
respective SEQ ID NO, and most preferably each light chain
comprises a human kappa light chain or a human lambda light chain
constant domain; and each heavy chain comprises a human IgG1, IgG2,
or IgG4 constant region.
[0102] In some preferred embodiments, the antibody or antigen
binding fragment is a humanized antibody that comprises two heavy
chains and two light chains, wherein each heavy chain comprises SEQ
ID NO: 80 and each light chain comprises SEQ ID NO: 92, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% similar or identical to a
respective SEQ ID NO, and most preferably each light chain
comprises a human kappa light chain or a human lambda light chain
constant domain; and each heavy chain comprises a human IgG1, IgG2,
or IgG4 constant region.
[0103] In some preferred embodiments, the antibody or antigen
binding fragment is a humanized antibody that comprises two heavy
chains and two light chains, wherein each heavy chain comprises SEQ
ID NO: 80 and each light chain comprises SEQ ID NO: 96, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% similar or identical to a
respective SEQ ID NO, and most preferably each light chain
comprises a human kappa light chain or a human lambda light chain
constant domain; and each heavy chain comprises a human IgG1, IgG2,
or IgG4 constant region.
[0104] In one embodiment, the anti-SIRP.alpha. antibody of the
invention comprises a full length antibody structure having two
light chains and two heavy chains as recited above, wherein each
light chain comprises a human kappa light chain or a human lambda
light chain constant domain; and each heavy chain comprises a human
IgG1 constant region.
[0105] In one embodiment, the anti-SIRP.alpha. antibody of the
invention comprises a full length antibody structure having two
light chains and two heavy chains as recited above, wherein each
light chain comprises a human kappa light chain or a human lambda
light chain constant domain; and each heavy chain comprises a human
IgG2 constant region.
[0106] In one embodiment, the anti-SIRP.alpha. antibody of the
invention comprises a full-length antibody structure having two
light chains and two heavy chains as recited above, wherein each
light chain comprises a human kappa light chain or a human lambda
light chain constant domain; and each heavy chain comprises a human
IgG4 constant region.
[0107] In certain embodiments, the antibodies or antigen binding
fragments of the present invention have one, two, three, four, or
more, and preferably each of, the following functional
characteristics:
[0108] binds human SIRP.alpha.V1 protein having the sequence of SEQ
ID NO: 34 with an EC.sub.50<1 nM; and exhibits at least a
100-fold higher EC.sub.50 for SIRP.alpha.V1(P74A) having the
sequence of SEQ ID NO: 62; and optionally also at least a 100-fold
higher EC.sub.50 for human SIRP.beta.1 protein having the sequence
of SEQ ID NO: 38 (in each case wherein the reduced EC.sub.50 is
relative to the EC.sub.50 for human SIRP.alpha.V1 protein having
the sequence of [0109] SEQ ID NO: 34, and in each case preferably
when measured by cellular ELISA (CELISA) as described hereinafter;
[0110] binds to a cell expressing human SIRP.alpha.V1 protein with
an EC.sub.50<10 nM, preferably <5 nM, more preferably <1.5
nM, still more preferably <1.0 nM, even more preferably <0.5
nM, and most preferably about 0.3 nM or less; [0111] binds to a
cell expressing human SIRP.alpha.V2 protein with an EC.sub.50<10
nM, preferably <5 nM, more preferably <1.5 nM, still more
preferably <1.0 nM, even more preferably <0.5 nM, and most
preferably about 0.3 nM or less; [0112] does not appreciably bind
to SIRP.beta.1 protein at an antibody concentration of 50 nM,
preferably 67 nM, and more preferably 100 nM; or alternatively at a
concentration that is 10-fold greater, preferably 50-fold greater,
more preferably 100-fold greater, and still more preferably
200-fold greater than the antibody's EC.sub.50 for SIRP.alpha.V1 or
SIRP.alpha.V2; [0113] inhibits binding between human SIRP.alpha.
and CD47 with an IC.sub.50<10.0 nM, more preferably <5.0 nM,
still more preferably <2.5 nM, and most preferably about 1.0 nM
or less; and [0114] exhibits a T20 "humanness" score of at least
79, and more preferably 85.
[0115] Preferably, the anti-SIRP.alpha. antibodies or antigen
binding fragments of the invention do not appreciably bind to one
or both of SIRP.alpha.V1(P74A) and SIRP.beta.1 protein at an
antibody concentration of 100 nM or alternatively at an antibody
concentration that is 200-fold greater than the antibody's EC50 for
SIRP.alpha.V1 or SIRP.alpha.V2, while binding to a cell expressing
human SIRP.alpha.V1 protein with an EC50<10 nM. Most preferably,
each light chain comprises a human kappa light chain or a human
lambda light chain constant domain; and each heavy chain comprises
a human IgG1, IgG2, or IgG4 constant region.
[0116] In certain embodiments, the anti-SIRP.alpha. antibody or
antigen binding fragment thereof of the invention can be conjugated
to at least one therapeutic agent. In one embodiment, the
therapeutic agent is a second antibody or fragment thereof, an
immunomodulator, a hormone, a cytotoxic agent, an enzyme, a
radionuclide, or a second antibody conjugated to at least one
immunomodulator, enzyme, radioactive label, hormone, antisense
oligonucleotide, or cytotoxic agent, or a combination thereof.
[0117] The invention also provides isolated polypeptides comprising
the amino acid sequence of any one of SEQ ID NOs: 75, 78, 80, 82,
84, 86, 88, 76, 90, 92, 94, 96, 98, 100, 102, 104, 7, 10, 12, 14,
16, 18, 30, 8, 20, 22, 24, 26, 28, and 32 or a fragment of any said
sequences, or an amino acid sequence at least 90%, 95%, 97%, 98%,
or 99% identical thereto.
[0118] The invention also provides isolated nucleic acids encoding
anyone of the anti-SIRP.alpha. antibodies or antigen binding
fragments of the invention.
[0119] In one embodiment, the invention provides an isolated
nucleic acid which encodes an amino acid sequence selected from the
group consisting of: [0120] SEQ ID NO: 75 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0121] SEQ ID NO: 78 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0122] SEQ ID NO: 80
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0123] SEQ ID NO: 82 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0124] SEQ ID NO: 84 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0125] SEQ
ID NO: 86 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, [0126] SEQ ID NO: 88 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, [0127] SEQ ID NO: 102 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0128] SEQ ID NO: 10 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0129] SEQ ID NO: 12
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0130] SEQ ID NO: 14 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0131] SEQ ID NO: 16 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0132] SEQ
ID NO: 18 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, and [0133] SEQ ID NO: 30 or an
amino acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto.
[0134] In certain embodiments, the amino acid sequence of SEQ ID
NO: 10 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 9 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0135] In certain embodiments, the amino acid sequence of SEQ ID
NO: 12 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 11 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0136] In certain embodiments, the amino acid sequence of SEQ ID
NO: 14 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 13 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0137] In certain embodiments, the amino acid sequence of SEQ ID
NO: 16 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 15 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0138] In certain embodiments, the amino acid sequence of SEQ ID
NO: 18 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 17 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0139] In certain embodiments, the amino acid sequence of SEQ ID
NO: 30 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 29 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0140] In certain embodiments, the amino acid sequence of SEQ ID
NO: 78 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 77 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0141] In certain embodiments, the amino acid sequence of SEQ ID
NO: 80 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 79 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0142] In certain embodiments, the amino acid sequence of SEQ ID
NO: 82 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 81 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0143] In certain embodiments, the amino acid sequence of SEQ ID
NO: 84 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 83 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0144] In certain embodiments, the amino acid sequence of SEQ ID
NO: 86 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 85 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0145] In certain embodiments, the amino acid sequence of SEQ ID
NO: 88 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 87 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0146] In certain embodiments, the amino acid sequence of SEQ ID
NO: 102 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 101 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0147] In one embodiment, the invention provides an isolated
nucleic acid which encodes an amino acid sequence selected from the
group consisting of: [0148] SEQ ID NO: 76 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0149] SEQ ID NO: 90 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0150] SEQ ID NO: 92
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0151] SEQ ID NO: 94 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0152] SEQ ID NO: 96 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0153] SEQ
ID NO: 98 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, [0154] SEQ ID NO: 100 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, [0155] SEQ ID NO: 104 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% similar or identical thereto,
[0156] SEQ ID NO: 8 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% similar or identical thereto, [0157] SEQ ID NO: 20
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
similar or identical thereto, [0158] SEQ ID NO: 22 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto, [0159] SEQ ID NO: 24 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% similar or identical thereto, [0160] SEQ
ID NO: 26 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto, [0161] SEQ ID NO: 28 or an amino
acid sequence at least 90%, 95%, 97%, 98%, or 99% similar or
identical thereto, and [0162] SEQ ID NO: 32 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% similar or identical
thereto.
[0163] In certain embodiments, the amino acid sequence of SEQ ID
NO: 20 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 19 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0164] In certain embodiments, the amino acid sequence of SEQ ID
NO: 22 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 21 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0165] In certain embodiments, the amino acid sequence of SEQ ID
NO: 24 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 23 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0166] In certain embodiments, the amino acid sequence of SEQ ID
NO: 26 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 25 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0167] In certain embodiments, the amino acid sequence of SEQ ID
NO: 28 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 27 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0168] In certain embodiments, the amino acid sequence of SEQ ID
NO: 32 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 31 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0169] In certain embodiments, the amino acid sequence of SEQ ID
NO: 90 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 89 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0170] In certain embodiments, the amino acid sequence of SEQ ID
NO: 92 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 91 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0171] In certain embodiments, the amino acid sequence of SEQ ID
NO: 94 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 93 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0172] In certain embodiments, the amino acid sequence of SEQ ID
NO: 96 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 95 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0173] In certain embodiments, the amino acid sequence of SEQ ID
NO: 98 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 97 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0174] In certain embodiments, the amino acid sequence of SEQ ID
NO: 100 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 99 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0175] In certain embodiments, the amino acid sequence of SEQ ID
NO: 104 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% similar or identical thereto is encoded by a nucleic acid
sequence of SEQ ID NO: 103 or a nucleic acid sequence at least 90%,
95%, 97%, 98%, or 99% identical thereto.
[0176] In certain embodiments, the isolated nucleic acids of the
present invention can optionally comprise a leader sequence.
[0177] Such nucleic acids can comprise one or more of the following
nucleic acid sequences: [0178] a nucleic acid sequence of SEQ ID
NO: 77 or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or
99% identical thereto, [0179] a nucleic acid sequence of SEQ ID NO:
79 or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0180] a nucleic acid sequence of SEQ ID NO: 81
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0181] a nucleic acid sequence of SEQ ID NO: 83
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0182] a nucleic acid sequence of SEQ ID NO: 85
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0183] a nucleic acid sequence of SEQ ID NO: 87
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0184] a nucleic acid sequence of SEQ ID NO: 101
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0185] a nucleic acid sequence of SEQ ID NO: 89
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0186] a nucleic acid sequence of SEQ ID NO: 91
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0187] a nucleic acid sequence of SEQ ID NO: 93
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0188] a nucleic acid sequence of SEQ ID NO: 95
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0189] a nucleic acid sequence of SEQ ID NO: 97
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0190] a nucleic acid sequence of SEQ ID NO: 99
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0191] a nucleic acid sequence of SEQ ID NO: 101
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0192] a nucleic acid sequence of SEQ ID NO: 103
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0193] a nucleic acid sequence of SEQ ID NO: 9
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0194] a nucleic acid sequence of SEQ ID NO: 11
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0195] a nucleic acid sequence of SEQ ID NO: 13
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0196] a nucleic acid sequence of SEQ ID NO: 15
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0197] a nucleic acid sequence of SEQ ID NO: 17
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0198] a nucleic acid sequence of SEQ ID NO: 29
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0199] a nucleic acid sequence of SEQ ID NO: 19
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0200] a nucleic acid sequence of SEQ ID NO: 21
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0201] a nucleic acid sequence of SEQ ID NO: 23
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0202] a nucleic acid sequence of SEQ ID NO: 25
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0203] a nucleic acid sequence of SEQ ID NO: 27
or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, and/or [0204] a nucleic acid sequence of SEQ ID
NO: 31 or a nucleic acid sequence at least 90%, 95%, 97%, 98%, or
99% identical thereto.
[0205] In certain embodiments, the nucleic acid can encode a human
or humanized antibody, and includes nucleic acid sequences for both
heavy and light chains. In one embodiment, the antibody is an IgG.
In preferred embodiments, antibody is an IgG1, IgG2, or IgG4, and
preferably a human IgG1, IgG2, or IgG4. In certain embodiments, the
light chain sequence comprises a human kappa light chain or a human
lambda light chain constant domain sequence; and each heavy chain
sequence comprises a human IgG1, IgG2, or IgG4 constant region
sequence.
[0206] Preferably, such nucleic acids comprise the following
combination heavy chain and light chain variable region nucleic
acid sequences:
SEQ ID NO: 9/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H1L1) SEQ ID NO: 9/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H1L2) SEQ ID NO: 9/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H1L3) SEQ ID NO: 9/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H1L4) SEQ ID NO:
9/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H1L5) SEQ
ID NO: 11/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H2L1) SEQ ID NO: 11/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H2L2) SEQ ID NO: 11/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H2L3) SEQ ID NO: 11/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H2L4) SEQ ID NO:
11/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H2L5) SEQ
ID NO: 13/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H3L1) SEQ ID NO: 13/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H3L2) SEQ ID NO: 13/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H3L3) SEQ ID NO: 13/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H3L4) SEQ ID NO:
13/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H3L5) SEQ
ID NO: 15/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H4L1) SEQ ID NO: 15/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H4L2) SEQ ID NO: 15/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H4L3) SEQ ID NO: 15/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H4L4) SEQ ID NO:
15/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H4L5) SEQ
ID NO: 17/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H5L1) SEQ ID NO: 17/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H5L2) SEQ ID NO: 17/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H5L3) SEQ ID NO: 17/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H5L4) SEQ ID NO:
17/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H5L5) SEQ
ID NO: 77/SEQ ID NO: 89 (referred to herein as
hSIRP.alpha..40A.H1L1) SEQ ID NO: 77/SEQ ID NO: 91 (referred to
herein as hSIRP.alpha..40A.H1L2) SEQ ID NO: 77/SEQ ID NO: 93
(referred to herein as hSIRP.alpha..40A.H1L3) SEQ ID NO: 77/SEQ ID
NO: 95 (referred to herein as hSIRP.alpha..40A.H1L4) SEQ ID NO:
77/SEQ ID NO: 97 (referred to herein as hSIRP.alpha..40A.H1L5) SEQ
ID NO: 77/SEQ ID NO: 99 (referred to herein as
hSIRP.alpha..40A.H1L6) SEQ ID NO: 79/SEQ ID NO: 89 (referred to
herein as hSIRP.alpha..40A.H2L1) SEQ ID NO: 79/SEQ ID NO: 91
(referred to herein as hSIRP.alpha..40A.H2L2) SEQ ID NO: 79/SEQ ID
NO: 93 (referred to herein as hSIRP.alpha..40A.H2L3) SEQ ID NO:
79/SEQ ID NO: 95 (referred to herein as hSIRP.alpha..40A.H2L4) SEQ
ID NO: 79/SEQ ID NO: 97 (referred to herein as
hSIRP.alpha..40A.H2L5) SEQ ID NO: 79/SEQ ID NO: 99 (referred to
herein as hSIRP.alpha..40A.H2L6) SEQ ID NO: 81/SEQ ID NO: 89
(referred to herein as hSIRP.alpha..40A.H3L1) SEQ ID NO: 81/SEQ ID
NO: 91 (referred to herein as hSIRP.alpha..40A.H3L2) SEQ ID NO:
81/SEQ ID NO: 93 (referred to herein as hSIRP.alpha..40A.H3L3) SEQ
ID NO: 81/SEQ ID NO: 95 (referred to herein as
hSIRP.alpha..40A.H3L4) SEQ ID NO: 81/SEQ ID NO: 97 (referred to
herein as hSIRP.alpha..40A.H3L5) SEQ ID NO: 81/SEQ ID NO: 99
(referred to herein as hSIRP.alpha..40A.H3L6) SEQ ID NO: 83/SEQ ID
NO: 89 (referred to herein as hSIRP.alpha..40A.H4L1) SEQ ID NO:
83/SEQ ID NO: 91 (referred to herein as hSIRP.alpha..40A.H4L2) SEQ
ID NO: 83/SEQ ID NO: 93 (referred to herein as
hSIRP.alpha..40A.H4L3) SEQ ID NO: 83/SEQ ID NO: 95 (referred to
herein as hSIRP.alpha..40A.H4L4) SEQ ID NO: 83/SEQ ID NO: 97
(referred to herein as hSIRP.alpha..40A.H4L5) SEQ ID NO: 83/SEQ ID
NO: 99 (referred to herein as hSIRP.alpha..40A.H4L6) SEQ ID NO:
85/SEQ ID NO: 89 (referred to herein as hSIRP.alpha..40A.H5L1) SEQ
ID NO: 85/SEQ ID NO: 91 (referred to herein as
hSIRP.alpha..40A.H5L2) SEQ ID NO: 85/SEQ ID NO: 93 (referred to
herein as hSIRP.alpha..40A.H5L3) SEQ ID NO: 85/SEQ ID NO: 95
(referred to herein as hSIRP.alpha..40A.H5L4) SEQ ID NO: 85/SEQ ID
NO: 97 (referred to herein as hSIRP.alpha..40A.H5L5) SEQ ID NO:
85/SEQ ID NO: 99 (referred to herein as hSIRP.alpha..40A.H5L6) SEQ
ID NO: 87/SEQ ID NO: 89 (referred to herein as
hSIRP.alpha..40A.H6L1) SEQ ID NO: 87/SEQ ID NO: 91 (referred to
herein as hSIRP.alpha..40A.H6L2) SEQ ID NO: 87/SEQ ID NO: 93
(referred to herein as hSIRP.alpha..40A.H6L3) SEQ ID NO: 87/SEQ ID
NO: 95 (referred to herein as hSIRP.alpha..40A.H6L4) SEQ ID NO:
87/SEQ ID NO: 97 (referred to herein as hSIRP.alpha..40A.H6L5) SEQ
ID NO: 87/SEQ ID NO: 99 (referred to herein as
hSIRP.alpha..40A.H6L6) or, in each case, at least 90%, 95%, 97%,
98%, or 99% identical to a respective SEQ ID NO.
[0207] In some preferred embodiments, the nucleic acid comprises
SEQ ID NO: 9 and SEQ ID NO: 19 or, in each case, at least 90%, 95%,
97%, 98%, or 99% identical to a respective SEQ ID NO.
[0208] In some preferred embodiments, the nucleic acid comprises
SEQ ID NO: 15 and SEQ ID NO: 27 or, in each case, at least 90%,
95%, 97%, 98%, or 99% identical to a respective SEQ ID NO.
[0209] In some preferred embodiments, the nucleic acid comprises
SEQ ID NO: 17 and SEQ ID NO: 19 or, in each case, at least 90%,
95%, 97%, 98%, or 99% identical to a respective SEQ ID NO.
[0210] In some preferred embodiments, the nucleic acid comprises
SEQ ID NO: 79 and SEQ ID NO: 89 or, in each case, at least 90%,
95%, 97%, 98%, or 99% identical to a respective SEQ ID NO.
[0211] In some preferred embodiments, the nucleic acid comprises
SEQ ID NO: 79 and SEQ ID NO: 91 or, in each case, at least 90%,
95%, 97%, 98%, or 99% identical to a respective SEQ ID NO.
[0212] In some preferred embodiments, the nucleic acid comprises
SEQ ID NO: 79 and SEQ ID NO: 95 or, in each case, at least 90%,
95%, 97%, 98%, or 99% identical to a respective SEQ ID NO.
[0213] The invention also provides expression vectors comprising
one or more nucleic acids of the present invention. An expression
vector is a DNA molecule comprising the regulatory elements
necessary for transcription of a target nucleic acid in a host
cell. Typically, the target nucleic acid is placed under the
control of certain regulatory elements including constitutive or
inducible promoters, tissue-specific regulatory elements, and
enhancer elements. Such a target nucleic acid is said to be
"operably linked to" the regulatory elements when the regulating
element controls the expression of the gene.
[0214] These isolated nucleic acids and the expression vectors
comprising them may be used to express the antibodies of the
invention or antigen binding fragments thereof in recombinant host
cells. Thus, the invention also provides host cells comprising an
expression vector of the present invention.
[0215] Such expression vectors can comprise one or more of the
following nucleic acid sequences operably linked to regulatory
elements: [0216] a nucleic acid sequence of SEQ ID NO: 77 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0217] a nucleic acid sequence of SEQ ID NO: 79 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0218] a nucleic acid sequence of SEQ ID NO: 81 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0219] a nucleic acid sequence of SEQ ID NO: 83 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0220] a nucleic acid sequence of SEQ ID NO: 85 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0221] a nucleic acid sequence of SEQ ID NO: 87 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0222] a nucleic acid sequence of SEQ ID NO: 101 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0223] a nucleic acid sequence of SEQ ID NO: 89 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0224] a nucleic acid sequence of SEQ ID NO: 91 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0225] a nucleic acid sequence of SEQ ID NO: 93 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0226] a nucleic acid sequence of SEQ ID NO: 95 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0227] a nucleic acid sequence of SEQ ID NO: 97 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0228] a nucleic acid sequence of SEQ ID NO: 99 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0229] a nucleic acid sequence of SEQ ID NO: 103 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0230] a nucleic acid sequence of SEQ ID NO: 11 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0231] a nucleic acid sequence of SEQ ID NO: 13 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0232] a nucleic acid sequence of SEQ ID NO: 15 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0233] a nucleic acid sequence of SEQ ID NO: 17 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0234] a nucleic acid sequence of SEQ ID NO: 29 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0235] a nucleic acid sequence of SEQ ID NO: 19 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0236] a nucleic acid sequence of SEQ ID NO: 21 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0237] a nucleic acid sequence of SEQ ID NO: 23 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0238] a nucleic acid sequence of SEQ ID NO: 25 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0239] a nucleic acid sequence of SEQ ID NO: 27 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, and/or [0240] a nucleic acid sequence of SEQ ID NO: 31 or
a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto.
[0241] In certain embodiments, the expression vector comprises
nucleic acid sequences encoding both a heavy chain sequence and a
light chain sequence of an anti-SIRP.alpha. antibody of the present
invention. Preferably, such expression vectors comprise the
following combination heavy chain and light chain variable region
nucleic acid sequences:
SEQ ID NO: 9/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H1L1) SEQ ID NO: 9/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H1L2) SEQ ID NO: 9/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H1L3) SEQ ID NO: 9/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H1L4) SEQ ID NO:
9/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H1L5) SEQ
ID NO: 11/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H2L1) SEQ ID NO: 11/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H2L2) SEQ ID NO: 11/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H2L3) SEQ ID NO: 11/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H2L4) SEQ ID NO:
11/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H2L5) SEQ
ID NO: 13/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H3L1) SEQ ID NO: 13/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H3L2) SEQ ID NO: 13/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H3L3) SEQ ID NO: 13/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H3L4) SEQ ID NO:
13/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H3L5) SEQ
ID NO: 15/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H4L1) SEQ ID NO: 15/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H4L2) SEQ ID NO: 15/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H4L3) SEQ ID NO: 15/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H4L4) SEQ ID NO:
15/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H4L5) SEQ
ID NO: 17/SEQ ID NO: 19 (referred to herein as
hSIRP.alpha..50A.H5L1) SEQ ID NO: 17/SEQ ID NO: 21 (referred to
herein as hSIRP.alpha..50A.H5L2) SEQ ID NO: 17/SEQ ID NO: 23
(referred to herein as hSIRP.alpha..50A.H5L3) SEQ ID NO: 17/SEQ ID
NO: 25 (referred to herein as hSIRP.alpha..50A.H5L4) SEQ ID NO:
17/SEQ ID NO: 27 (referred to herein as hSIRP.alpha..50A.H5L5) SEQ
ID NO: 77/SEQ ID NO: 89 (referred to herein as
hSIRP.alpha..40A.H1L1) SEQ ID NO: 77/SEQ ID NO: 91 (referred to
herein as hSIRP.alpha..40A.H1L2) SEQ ID NO: 77/SEQ ID NO: 93
(referred to herein as hSIRP.alpha..40A.H1L3) SEQ ID NO: 77/SEQ ID
NO: 95 (referred to herein as hSIRP.alpha..40A.H1L4) SEQ ID NO:
77/SEQ ID NO: 97 (referred to herein as hSIRP.alpha..40A.H1L5) SEQ
ID NO: 77/SEQ ID NO: 99 (referred to herein as
hSIRP.alpha..40A.H1L6) SEQ ID NO: 79/SEQ ID NO: 89 (referred to
herein as hSIRP.alpha..40A.H2L1) SEQ ID NO: 79/SEQ ID NO: 91
(referred to herein as hSIRP.alpha..40A.H2L2) SEQ ID NO: 79/SEQ ID
NO: 93 (referred to herein as hSIRP.alpha..40A.H2L3) SEQ ID NO:
79/SEQ ID NO: 95 (referred to herein as hSIRP.alpha..40A.H2L4) SEQ
ID NO: 79/SEQ ID NO: 97 (referred to herein as
hSIRP.alpha..40A.H2L5) SEQ ID NO: 79/SEQ ID NO: 99 (referred to
herein as hSIRP.alpha..40A.H2L6) SEQ ID NO: 81/SEQ ID NO: 89
(referred to herein as hSIRP.alpha..40A.H3L1) SEQ ID NO: 81/SEQ ID
NO: 91 (referred to herein as hSIRP.alpha..40A.H3L2) SEQ ID NO:
81/SEQ ID NO: 93 (referred to herein as hSIRP.alpha..40A.H3L3) SEQ
ID NO: 81/SEQ ID NO: 95 (referred to herein as
hSIRP.alpha..40A.H3L4) SEQ ID NO: 81/SEQ ID NO: 97 (referred to
herein as hSIRP.alpha..40A.H3L5) SEQ ID NO: 81/SEQ ID NO: 99
(referred to herein as hSIRP.alpha..40A.H3L6) SEQ ID NO: 83/SEQ ID
NO: 89 (referred to herein as hSIRP.alpha..40A.H4L1) SEQ ID NO:
83/SEQ ID NO: 91 (referred to herein as hSIRP.alpha..40A.H4L2) SEQ
ID NO: 83/SEQ ID NO: 93 (referred to herein as
hSIRP.alpha..40A.H4L3) SEQ ID NO: 83/SEQ ID NO: 95 (referred to
herein as hSIRP.alpha..40A.H4L4) SEQ ID NO: 83/SEQ ID NO: 97
(referred to herein as hSIRP.alpha..40A.H4L5) SEQ ID NO: 83/SEQ ID
NO: 99 (referred to herein as hSIRP.alpha..40A.H4L6) SEQ ID NO:
85/SEQ ID NO: 89 (referred to herein as hSIRP.alpha..40A.H5L1) SEQ
ID NO: 85/SEQ ID NO: 91 (referred to herein as
hSIRP.alpha..40A.H5L2) SEQ ID NO: 85/SEQ ID NO: 93 (referred to
herein as hSIRP.alpha..40A.H5L3) SEQ ID NO: 85/SEQ ID NO: 95
(referred to herein as hSIRP.alpha..40A.H5L4) SEQ ID NO: 85/SEQ ID
NO: 97 (referred to herein as hSIRP.alpha..40A.H5L5) SEQ ID NO:
85/SEQ ID NO: 99 (referred to herein as hSIRP.alpha..40A.H5L6) SEQ
ID NO: 87/SEQ ID NO: 89 (referred to herein as
hSIRP.alpha..40A.H6L1) SEQ ID NO: 87/SEQ ID NO: 91 (referred to
herein as hSIRP.alpha..40A.H6L2) SEQ ID NO: 87/SEQ ID NO: 93
(referred to herein as hSIRP.alpha..40A.H6L3) SEQ ID NO: 87/SEQ ID
NO: 95 (referred to herein as hSIRP.alpha..40A.H6L4) SEQ ID NO:
87/SEQ ID NO: 97 (referred to herein as hSIRP.alpha..40A.H6L5) SEQ
ID NO: 87/SEQ ID NO: 99 (referred to herein as
hSIRP.alpha..40A.H6L6) or, in each case, at least 90%, 95%, 97%,
98%, or 99% identical to a respective SEQ ID NO.
[0242] In any of the above embodiments, the expression vector can
encode for expression a human or humanized antibody, and includes
nucleic acid sequences for both heavy and light chains. In one
embodiment, the antibody is an IgG. In preferred embodiments,
antibody is an IgG1, IgG2, or IgG4, and preferably a human IgG1,
IgG2, or IgG4. In certain embodiments, the light chain sequence
comprises a human kappa light chain or a human lambda light chain
constant domain sequence; and each heavy chain sequence comprises a
human IgG4 constant region sequence.
[0243] In some preferred embodiments, the expression vector encodes
for expression a human or humanized antibody, wherein the heavy
chain nucleic acid sequence comprises SEQ ID NO: 9 and the light
chain nucleic acid sequence comprises SEQ ID NO: 19, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% identical to a respective
SEQ ID NO, and is most preferably an IgG1, IgG2, or IgG4
isotype.
[0244] In some preferred embodiments, the expression vector encodes
for expression a human or humanized antibody, wherein the heavy
chain nucleic acid sequence comprises SEQ ID NO: 15 and the light
chain nucleic acid sequence comprises SEQ ID NO: 27, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% identical to a respective
SEQ ID NO, and is most preferably an IgG1, IgG2, or IgG4
isotype.
[0245] In some preferred embodiments, the expression vector encodes
for expression a human or humanized antibody, wherein the heavy
chain nucleic acid sequence comprises SEQ ID NO: 17 and the light
chain nucleic acid sequence comprises SEQ ID NO: 19, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% identical to a respective
SEQ ID NO or, in each case, at least 90%, 95%, 97%, 98%, or 99%
identical to a respective SEQ ID NO, and is most preferably an
IgG1, IgG2, or IgG4 isotype.
[0246] In some preferred embodiments, the expression vector encodes
for expression a human or humanized antibody, wherein the heavy
chain nucleic acid sequence comprises SEQ ID NO: 79 and the light
chain nucleic acid sequence comprises SEQ ID NO: 89, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% identical to a respective
SEQ ID NO, and is most preferably an IgG1, IgG2, or IgG4
isotype.
[0247] In some preferred embodiments, the expression vector encodes
for expression a human or humanized antibody, wherein the heavy
chain nucleic acid sequence comprises SEQ ID NO: 79 and the light
chain nucleic acid sequence comprises SEQ ID NO: 91, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% identical to a respective
SEQ ID NO, and is most preferably an IgG1, IgG2, or IgG4
isotype.
[0248] In some preferred embodiments, the expression vector encodes
for expression a human or humanized antibody, wherein the heavy
chain nucleic acid sequence comprises SEQ ID NO: 79 and the light
chain nucleic acid sequence comprises SEQ ID NO: 95, or, in each
case, at least 90%, 95%, 97%, 98%, or 99% identical to a respective
SEQ ID NO, and is most preferably an IgG1, IgG2, or IgG4
isotype.
[0249] In one embodiment, the host cell is Chinese hamster ovary
(CHO) cell. In one embodiment, the host cell is a mammalian cell
(e.g., a human cell such as an HEK293 cell, a hamster cell such as
a CHO cell, etc.), a bacterial cell (e.g., an E. coli cell) a yeast
cell (e.g., a Pichia pastoris cell, etc.), a plant cell (e.g., a
Nicotiana benthamiana cell), etc. . . . Mammalian cells are
preferred due to glycosylation patterns that are most
favorable.
[0250] The invention also provides pharmaceutical compositions
comprising an antibody or antigen binding fragment of the invention
and a pharmaceutically acceptable carrier or diluent.
[0251] In one embodiment, the composition comprises one or more
further therapeutic agents. In one embodiment, the further
therapeutic agent is selected from the group consisting of: an
anti-CD27 antibody or an antigen binding fragment thereof; an
anti-LAG3 antibody or an antigen binding fragment thereof; an
anti-APRIL antibody or an antigen binding fragment thereof; an
anti-TIGIT antibody or antigen biding fragment thereof; an
anti-VISTA antibody or an antigen binding fragment thereof; an
anti-BTLA antibody or an antigen binding fragment thereof; an
anti-TIM3 antibody or an antigen binding fragment thereof; an
anti-CTLA4 antibody or an antigen binding fragment thereof; an
anti-HVEM antibody or an antigen binding fragment thereof; an
anti-CD70 antibody or an antigen binding fragment thereof; an
anti-CD137 antibody or an antigen binding fragment thereof; an
anti-OX40 antibody or an antigen binding fragment thereof; an
anti-CD28 antibody or an antigen binding fragment thereof; thereof;
an anti-PD1 antibody or an antigen binding fragment thereof; an
anti-PDL1 antibody or an antigen binding fragment thereof; an
anti-PDL2 antibody or an antigen binding fragment thereof; an
anti-GITR antibody or an antigen binding fragment thereof; an
anti-ICOS antibody or an antigen binding fragment thereof; an
anti-ILT2 antibody or antigen binding fragment thereof; an
anti-ILT3 antibody or antigen binding fragment thereof; an
anti-ILT4 antibody or antigen binding fragment thereof; and an
anti-ILT5 antibody or an antigen binding fragment thereof; an anti
4-1BB antibody or an antigen binding fragment thereof; an
anti-NKG2A antibody or an antigen binding fragment thereof; an
anti-NKG2C antibody or an antigen binding fragment thereof; an
anti-NKG2E antibody or an antigen binding fragment thereof; an
anti-TSLP antibody or an antigen binding fragment thereof; an
anti-IL-10 antibody or an antigen binding fragment thereof; IL-10
or PEGylated IL-10; an agonist (e.g., an agonistic antibody or
antigen-binding fragment thereof, or a soluble fusion) of a TNF
receptor protein; an Immunoglobulin-like protein; a cytokine
receptor; an integrin; a signaling lymphocytic activation molecules
(SLAM proteins); an activating NK cell receptor; a Toll like
receptor; OX40; CD2; CD7; CD27; CD28; CD30; CD40; ICAM-1; LFA-1
(CD1 1a/CD18); 4-1BB (CD137); B7-H3; ICOS (CD278); GITR; BAFFR;
LIGHT; HVEM (LIGHTR); KIRDS2; SLAMF7; NKp80 (KLRF1); NKp44; NKp30;
NKp46; CD19; CD4; CD8alpha; CD8beta; IL2R beta; IL2R gamma; IL7R
alpha; ITGA4; VLA1; CD49a; ITGA4; IA4; CD49D; ITGA6; VLA-6; CD49f;
ITGAD; CD1 1d; ITGAE; CD103; ITGAL; ITGAM; CD1 1b; ITGAX; CD1 1c;
ITGB1; CD29; ITGB2; CD18; ITGB7; NKG2D; NKG2C; TNFR2; TRANCE/RANKL;
DNAM1 (CD226); SLAMF4 (CD244; 2B4); CD84; CD96 (Tactile); CEACAM1;
CRTAM; Ly9 (CD229); CD160 (BY55); PSGL1; CD100 (SEMA4D); CD69;
SLAMF6 (NTB-A; Ly108); SLAM (SLAMF1, CD150, IPO-3); SLAM7; BLAME
(SLAMF8); SELPLG (CD162); LTBR; LAT; GADS; PAG/Cbp; CD19a; a ligand
that specifically binds with CD83; an inhibitor of CD47, PD-1;
PD-L2; CTLA4; TIM3; LAG3; CEACAM (e.g.; CEACAM-1, -3 and/or -5);
VISTA; BTLA; TIGIT; LAIR1; IDO; TDO; CD160; TGFR beta; and a cyclic
dinculeotide or other STING pathway agonist.
[0252] The invention also comprises a combination comprising an
antibody or antigen binding fragment of the invention and a second
antibody that induces ADCC, wherein said antibody or antigen
binding fragment of the invention enhances the antibody-mediated
destruction of cells by the second antibody. Antibody-dependent
cell-mediated cytotoxicity (ADCC) is a mechanism of cell-mediated
immune defense whereby an effector cell of the immune system
actively lyses a target cell, whose membrane-surface antigens have
been bound by specific antibodies. ADCC is often thought of as
being mediated by natural killer (NK) cells, but dendritic cells,
macrophages, monocytes, and granulocytes can also mediate ADCC.
[0253] The invention also comprises a combination comprising an
antibody or antigen binding fragment of the invention and a second
antibody that induces ADCP, wherein said antibody or antigen
binding fragment of the invention enhances the antibody-mediated
phagocytosis of cells by the second antibody. Antibody-dependent
cell-mediated phagocytosis (ADCP) is a mechanism of cell-mediated
immune defense whereby target cells are killed via granulocyte,
monocyte, dendritic cell, or macrophage-mediated phagocytosis.
[0254] Natural killer (NK) cells play a major role in cancer
immunotherapies that involve tumor-antigen targeting by monoclonal
antibodies (mAbs). In the context of targeting cells, NK cells can
be "specifically activated" through certain Fc receptors that are
expressed on their cell surface. NK cells can express
Fc.gamma.RIIIA and/or Fc.gamma.RIIC, which can bind to the Fc
portion of immunoglobulins, transmitting activating signals within
NK cells. Once activated through Fc receptors by antibodies bound
to target cells, NK cells are able to lyse target cells without
priming, and secrete cytokines like interferon gamma to recruit
adaptive immune cells. Likewise, tumor-associated macrophages
(TAMs) express surface receptors that bind the Fc fragment of
antibodies and enable them to engage in Ab-dependent cellular
cytotoxicity/phagocytosis (ADCC/ADCP). Because SIRP.alpha./CD47
signalling induces a "don't eat me" response that reduces
ADCC/ADCP, blocking of this signaling by the anti-SIRP.alpha.
antibodies or antigen binding fragments of the invention can
enhance ADCC of tumor cells bearing the antigenic determinant to
which the therapeutic antibody is directed.
[0255] This ADCC/ADCP as a mode of action may be utilized in the
treatment of various cancers and infectious diseases. An exemplary
list of ADCC/ADCP-inducing antibodies and antibody conjugates that
can be combined with the antibodies or antigen binding fragments of
the present invention includes, but is not limited to, Rituximab,
ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab,
Obinutuzumab, ADCT-502, Hul4.18K322A, Hu3F8, Dinituximab,
Trastuzumab, Cetuximab, Rituximab-RLI, c.60C3-RLI, Hul4.18-IL2,
KM2812, AFM13, and (CD20).sub.2xCD16, erlotinib (Tarceva),
daratumumab, alemtuzumab, pertuzumab, brentuximab, elotuzumab,
ibritumomab, ifabotuzumab, farletuzumab, otlertuzumab, carotuximab,
epratuzumab, inebilizumab, lumretuzumab, 4G7SDIE, AFM21, AFM22,
LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20,
mogamulizumab, ChiLob-7/4, leukotuximab, isatuximab, DS-8895,
FPA144, GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04,
XOMA-213, PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005,
MDX-1097, MOR202, MOR-208, oportuzumab, ensituximab, vedotin
(Adcetris), ibritumomab tiuxetan, ABBV-838, HuMax-AXL-ADC, and
ado-trastuzumab emtansine (Kadcyla). An exemplary list of target
antigens for such ADCC/ADCP-inducing antibodies includes, but is
not limited to, AMHR2, AXL, BCMA, CA IX, CD4, CD16, CD19, CD20,
CD22, CD30, CD37, CD38, CD40, CD52, CD98, CSF1R, GD2, CCR4, CS1,
EpCam, EGFR, EGFRvIII, Endoglin, EPHA2, EphA3, FGFR2b, folate
receptor alpha, fucosyl-GM1, HER2, HER3, IL1RAP, kappa myeloma
antigen, MS4A1, prolactin receptor, TA-MUC1, and PSMA.
[0256] In certain embodiments, the second antibody or antigen
binding fragment thereof induces ADCP. By way of example only, such
antibodies may be selected from the group consisting of Rituximab,
ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab,
Obinutuzumab, Trastuzumab, Cetuximab, alemtuzumab, ibritumomab,
farletuzumab, inebilizumab, lumretuzumab, 4G7SDIE, BMS-986012,
BVX-20, mogamulizumab, ChiLob-7/4, GM102, GSK-2857916,
PankoMab-GEX, chKM-4927, MDX-1097, MOR202, and MOR-208.
[0257] In embodiments where the antibodies or antigen binding
fragments of the present invention are combined with one or more
ADCC/ADCP-inducing antibodies and antibody conjugates, such
combinations may also be used optionally in association with a
further therapeutic agent or therapeutic procedure. In one
embodiment, the further therapeutic agent is selected from the
group consisting of: an anti-LAG3 antibody or an antigen binding
fragment thereof; an anti-APRIL antibody or an antigen binding
fragment thereof; an anti-TIGIT antibody or an antigen binding
fragment thereof; an anti-VISTA antibody or an antigen binding
fragment thereof; an anti-BTLA antibody or an antigen binding
fragment thereof; an anti-TIM3 antibody or an antigen binding
fragment thereof; an anti-CTLA4 antibody or an antigen binding
fragment thereof; an anti-HVEM antibody or an antigen binding
fragment thereof; an anti-CD70 antibody or an antigen binding
fragment thereof; an anti-CD137 antibody or an antigen binding
fragment thereof; an anti-OX40 antibody or an antigen binding
fragment thereof; an anti-CD28 antibody or an antigen binding
fragment thereof; thereof; an anti-PD1 antibody or an antigen
binding fragment thereof; an anti-PDL1 antibody or an antigen
binding fragment thereof; an anti-PDL2 antibody or an antigen
binding fragment thereof; an anti-GITR antibody or an antigen
binding fragment thereof; an anti-ICOS antibody or an antigen
binding fragment thereof; an anti-ILT2 antibody or antigen binding
fragment thereof; an anti-ILT3 antibody or antigen binding fragment
thereof; an anti-ILT4 antibody or antigen binding fragment thereof;
an anti-ILT5 antibody or an antigen binding fragment thereof; and
an anti-4-1BB antibody or an antigen binding fragment thereof; an
anti-NKG2A antibody or an antigen binding fragment thereof; an
anti-NKG2C antibody or an antigen binding fragment thereof; an
anti-NKG2E antibody or an antigen binding fragment thereof; an
anti-TSLP antibody or an antigen binding fragment thereof; an
anti-IL-10 antibody or an antigen binding fragment thereof; and
IL-10 or PEGylated IL-10.
[0258] The invention also provides a vessel or injection device
comprising anyone of the anti-SIRP.alpha. antibodies or antigen
binding fragments of the invention.
[0259] The invention also provides a method of producing an
anti-SIRP.alpha. antibody or antigen binding fragment of the
invention comprising: culturing a host cell comprising a
polynucleotide encoding a heavy chain and/or light chain of an
antibody of the invention (or an antigen binding fragment thereof)
under conditions favorable to expression of the polynucleotide; and
optionally, recovering the antibody or antigen binding fragment
from the host cell and/or culture medium. In one embodiment, the
polynucleotide encoding the heavy chain and the polynucleotide
encoding the light chain are in a single vector. In another
embodiment, the polynucleotide encoding the heavy chain and the
polynucleotide encoding the light chain are in different
vectors.
[0260] The invention also provides a method of treating cancer in a
subject in need thereof, comprising administering to the subject an
effective amount of an anti-SIRP.alpha. antibody or antigen binding
fragment of the invention, optionally in association with a further
therapeutic agent or therapeutic procedure.
[0261] In one embodiment, the subject to be treated is a human
subject. In one embodiment, the further therapeutic agent is
selected from the group consisting of: an anti-LAG3 antibody or an
antigen binding fragment thereof; an anti-APRIL antibody or an
antigen binding fragment thereof; an an anti-TIGIT antibody or an
antigen binding fragment thereof; an anti-VISTA antibody or an
antigen binding fragment thereof; an anti-BTLA antibody or an
antigen binding fragment thereof; an anti-TIM3 antibody or an
antigen binding fragment thereof; an anti-CTLA4 antibody or an
antigen binding fragment thereof; an anti-HVEM antibody or an
antigen binding fragment thereof; an anti-CD70 antibody or an
antigen binding fragment thereof; an anti-CD137 antibody or an
antigen binding fragment thereof; an anti-OX40 antibody or an
antigen binding fragment thereof; an anti-CD28 antibody or an
antigen binding fragment thereof; thereof; an anti-PD1 antibody or
an antigen binding fragment thereof; an anti-PDL1 antibody or an
antigen binding fragment thereof; an anti-PDL2 antibody or an
antigen binding fragment thereof; an anti-GITR antibody or an
antigen binding fragment thereof; an anti-ICOS antibody or an
antigen binding fragment thereof; an anti-ILT2 antibody or antigen
binding fragment thereof; an anti-ILT3 antibody or antigen binding
fragment thereof; an anti-ILT4 antibody or antigen binding fragment
thereof; an anti-ILT5 antibody or an antigen binding fragment
thereof; and an anti-4-1BB antibody or an antigen binding fragment
thereof; an anti-NKG2A antibody or an antigen binding fragment
thereof; an anti-NKG2C antibody or an antigen binding fragment
thereof; an anti-NKG2E antibody or an antigen binding fragment
thereof; an anti-TSLP antibody or an antigen binding fragment
thereof; an anti-IL-10 antibody or an antigen binding fragment
thereof; and IL-10 or PEGylated IL-10.
[0262] The invention also provides a method of treating an
infection or infectious disease in a subject, comprising
administering to the subject an effective amount of an antibody or
antigen binding fragment of the invention, optionally in
association with a further therapeutic agent or therapeutic
procedure. In one embodiment, the subject to be treated is a human
subject.
[0263] In one embodiment, the further therapeutic agent is selected
from the group consisting of: an anti-LAG3 antibody or an antigen
binding fragment thereof; an anti-APRIL antibody or an antigen
binding fragment thereof; an an anti-TIGIT antibody or an antigen
binding fragment thereof; an anti-VISTA antibody or an antigen
binding fragment thereof; an anti-BTLA antibody or an antigen
binding fragment thereof; an anti-TIM3 antibody or an antigen
binding fragment thereof; an anti-CTLA4 antibody or an antigen
binding fragment thereof; an anti-HVEM antibody or an antigen
binding fragment thereof; an anti-CD70 antibody or an antigen
binding fragment thereof; an anti-CD137 antibody or an antigen
binding fragment thereof; an anti-OX40 antibody or an antigen
binding fragment thereof; an anti-CD28 antibody or an antigen
binding fragment thereof; thereof; an anti-PD1 antibody or an
antigen binding fragment thereof; an anti-PDL1 antibody or an
antigen binding fragment thereof; an anti-PDL2 antibody or an
antigen binding fragment thereof; an anti-GITR antibody or an
antigen binding fragment thereof; an anti-ICOS antibody or an
antigen binding fragment thereof; an anti-ILT2 antibody or antigen
binding fragment thereof; an anti-ILT3 antibody or antigen binding
fragment thereof; an anti-ILT4 antibody or antigen binding fragment
thereof; an anti-ILT5 antibody or an antigen binding fragment
thereof; and an anti-4-1BB antibody or an antigen binding fragment
thereof; an anti-NKG2A antibody or an antigen binding fragment
thereof; an anti-NKG2C antibody or an antigen binding fragment
thereof; an anti-NKG2E antibody or an antigen binding fragment
thereof; an anti-TSLP antibody or an antigen binding fragment
thereof; an anti-IL-10 antibody or an antigen binding fragment
thereof; and IL-10 or PEGylated IL-10.
[0264] The invention also provides a method for detecting the
presence of a SIRP.alpha. peptide or a fragment thereof in a sample
comprising contacting the sample with an antibody or antigen
binding fragment thereof of the invention and detecting the
presence of a complex between the antibody or fragment and the
peptide; wherein detection of the complex indicates the presence of
the SIRP.alpha. peptide.
BRIEF DESCRIPTION OF THE FIGURES
[0265] FIG. 1 depicts cross-reactivity of commercially available
anti-hSIRP.alpha. antibodies with hSIRP.beta.1 and allele-specific
binding to hSIRP.alpha.V1 and hSIRP.alpha.V2.
[0266] FIG. 2 depicts reactivity of KWAR23 antibody with
hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.1, and hSIRP.gamma..
[0267] FIG. 3 depicts reactivity of antibody clone hSIRP.alpha..50A
for various hSIRP.alpha. alleles.
[0268] FIG. 4 depicts the ability of hSIRP.alpha..50A antibody to
block recombinant hCD47/Fc-protein binding to cell surface
expressed hSIRP.alpha..
[0269] FIG. 5A depicts binding of hSIRP?0.50A antibody to primary
CD14+ enriched monocytes from a human donor.
[0270] FIG. 5B depicts binding of hSIRP?0.50A antibody to primary
CD14+ enriched monocytes from a second human donor.
[0271] FIG. 5C depicts the ability of hSIRP?0.50A antibody to block
hCD47 binding to to primary CD14+ enriched monocytes from a human
donor.
[0272] FIG. 5D depicts the ability of hSIRP?0.50A antibody to block
hCD47 binding to to primary CD14+ enriched monocytes from a second
human donor.
[0273] FIG. 6A depicts binding of hSIRP.alpha..50A antibody to
primary human granulocytes.
[0274] FIG. 6B depicts phagocytosis of tumor cells by primary human
granulocytes in the presence of rituximab plus or minus the
hSIRP.alpha..50A antibody.
[0275] FIG. 6C depicts phagocytosis of tumor cells by primary human
granulocytes in the presence of daratumumab plus or minus the
hSIRP.alpha..50A antibody.
[0276] FIG. 6D depicts phagocytosis of tumor cells by primary human
granulocytes in the presence of alemtuzumab plus or minus the
hSIRP.alpha..50A antibody.
[0277] FIG. 6E depicts phagocytosis of tumor cells by primary human
granulocytes in the presence of cetuximab plus or minus the
hSIRP.alpha..50A antibody.
[0278] FIG. 7 depicts phagocytosis of tumor cells by human
macrophages in the presence of the indicated antibody (rituximab or
daratumumab) plus or minus the hSIRP.alpha..50A antibody.
[0279] FIG. 8 depicts blocking of the hSIRP.alpha./hCD47
interaction by mouse hSIRP.alpha..50A and humanized
hSIRP.alpha..50A antibodies to hSIRP.alpha..
[0280] FIG. 9 depicts depicts hSIRP.alpha..50A antibody binding to
hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.1,
hSIRP.alpha.-V.beta.C1.alpha.C2.alpha.,
hSIRP.alpha.-V.alpha.C1.beta.C2.alpha., and
hSIRP.alpha.-V.alpha.C1.alpha.C2.beta..
[0281] FIG. 10A depicts an alignment of the hSIRP.alpha. and
hSIRP.beta.1 IgV domain amino acid sequences (SEQ ID NOS:
133-135).
[0282] FIG. 10B depicts loss of hSIRP.alpha..50A antibody binding
to hSIRP.alpha.V1(P74A).
[0283] FIG. 11 depicts binding of hSIRP.alpha..40A and
hSIRP.alpha..50A antibodies to hSIRP.alpha.V1, hSIRP.alpha.V2,
hSIRP.beta.1, hSIRP.beta.L, and hSIRP.gamma..
[0284] FIG. 12 depicts binding of hSIRP.alpha..40A and
hSIRP.alpha..50A antibodies to hSIRP.alpha.V1, hSIRP.alpha.V2,
hSIRP.alpha.V3, hSIRP.alpha.V4, hSIRP.alpha.V5, hSIRP.alpha.V6,
hSIRP.alpha.V8, and hSIRP.alpha.V9.
[0285] FIG. 13 depicts the ability of hSIRP.alpha..40A and
hSIRP.alpha..50A antibodies to block recombinant hCD47/Fc-protein
binding to cell surface expressed hSIRP.alpha..
[0286] FIG. 14A depicts binding of hSIRP?0.40A antibody to primary
CD14+ enriched monocytes from a human donor.
[0287] FIG. 14B depicts binding of hSIRP?0.40A antibody to primary
CD14+ enriched monocytes from a second human donor.
[0288] FIG. 14C depicts the ability of hSIRP?0.40A antibody to
block hCD47 binding to to primary CD14+ enriched monocytes from a
human donor.
[0289] FIG. 14D depicts the ability of hSIRP?0.40A antibody to
block hCD47 binding to to primary CD14+ enriched monocytes from a
second human donor.
[0290] FIG. 15A depicts binding of hSIRP.alpha..40A and
hSIRP.alpha..50A antibodies to primary human granulocytes.
[0291] FIG. 15B depicts phagocytosis of Ramos cells by primary
human granulocytes in the presence of rituximab plus or minus the
hSIRP.alpha..40A and hSIRP.alpha..50A antibodies.
[0292] FIG. 16 depicts enhancement of rituximab-induced Raji cell
phagocytosis by hSIRP.alpha..40A and hSIRP.alpha..50A
antibodies.
[0293] FIG. 17 depicts binding of mouse hSIRP.alpha..40A and
humanized hSIRP.alpha..40A antibodies to hSIRP.alpha..
[0294] FIG. 18 depicts the blockade of hCD47 binding to
hSIRP.alpha. in the presence of humanized hSIRP.alpha..40A antibody
variants.
[0295] FIG. 19 depicts binding of hSIRP.alpha..40A and
hSIRP.alpha..50A antibodies to hSIRP.alpha.V1, hSIRP.alpha.V2,
hSIRP.beta.1, hSIRP-V.gamma.C1.beta.C2.beta.,
hSIRP-V.beta.C1.gamma.C2.beta., and
hSIRP-V.beta.C1.beta.C2.gamma..
[0296] FIG. 20 depicts loss of hSIRP.alpha..40A and
hSIRP.alpha..50A antibody binding to hSIRP.alpha.V1(P74A).
[0297] FIG. 21 depicts the ability of chimeric hSIRP.alpha..40A
antibody variants to affect rituximab-mediated phagocytosis.
[0298] FIG. 22 depicts the ability of humanized hSIRP.alpha..40A
antibody variants to affect rituximab-mediated phagocytosis.
[0299] FIG. 23A depicts the ability of mouse hSIRP.alpha..50A and
chimeric hSIRP.alpha..50A hIgG2 and hIgG4 antibody variants to
affect rituximab-mediated phagocytosis.
[0300] FIG. 23B depicts the ability of chimeric hSIRP.alpha..50A
hIgG2 and hIgG4 antibody variants to affect rituximab-mediated
phagocytosis.
[0301] FIG. 23C depicts the ability of chimeric hSIRP.alpha..50A
hIgG2 and hIgG4 antibody variants to affect daratumumab-mediated
phagocytosis.
[0302] FIG. 23D depicts the ability of mouse hSIRP.alpha..50A and
chimeric hSIRP.alpha..50A hIgG2 antibody variants to affect
rituximab-mediated phagocytosis in granulocytes.
[0303] FIG. 24A depicts the ability of mouse hSIRP.alpha..50A and
chimeric hSIRP.alpha..50A.hIgG 1.N297Q,
hSIRP.alpha..50A.hIgG4.N297Q or hSIRP.alpha..50A.hIgG2 antibody
variants to affect rituximab-mediated phagocytosis.
[0304] FIG. 24B depicts the ability of mouse hSIRP.alpha..50A and
chimeric hSIRP.alpha..50A.hIgG 1.N297Q,
hSIRP.alpha..50A.hIgG4.N297Q or hSIRP.alpha..50A.hIgG2 antibody
variants to affect daratumumab-mediated phagocytosis.
[0305] FIG. 25 depicts the ability of chimeric
hSIRP.alpha..50A.hIgG1.N297Q, hSIRP.alpha..50A
hIgG1.L234A.L235A.P329G, and hSIRP.alpha..50A hIgG2 or hIgG4
antibody variants to affect rituximab-mediated phagocytosis.
DETAILED DESCRIPTION
Abbreviations
[0306] Throughout the detailed description and examples of the
invention the following abbreviations will be used: [0307] ADCC
Antibody-dependent cellular cytotoxicity [0308] ADCP
Antibody-dependent cellular phagocytosis [0309] CDC
Complement-dependent cytotoxicity [0310] CDR Complementarity
determining region in the immunoglobulin variable regions, defined
using the Kabat numbering system [0311] CHO Chinese hamster ovary
[0312] EC50 Concentration at which 50% of the total binding signal
is observed [0313] ELISA Enzyme-linked immunosorbant assay [0314]
FR Antibody framework region: the immunoglobulin variable regions
excluding the CDR regions. [0315] HRP Horseradish peroxidase [0316]
IFN interferon [0317] IC50 concentration resulting in 50%
inhibition [0318] IgG Immunoglobulin G [0319] Kabat An
immunoglobulin alignment and numbering system pioneered by Elvin A.
Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md.) [0320] mAb or Mab or MAb Monoclonal antibody [0321] SEB
Staphylococcus Enterotoxin B [0322] TT Tetanus toxoid [0323] V
region The segment of Ig chains which is variable in sequence
between different antibodies. It extends to Kabat residue 109 in
the light chain and 113 in the heavy chain. [0324] VH
Immunoglobulin heavy chain variable region [0325] VK Immunoglobulin
kappa light chain variable region [0326] VL Immunoglobulin light
chain variable region
Definitions
[0327] So that the invention may be more readily understood,
certain technical and scientific terms are specifically defined
below. Unless specifically defined elsewhere in this document, all
other technical and scientific terms used herein have the meaning
commonly understood by one of ordinary skill in the art to which
this invention belongs.
[0328] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the," include their
corresponding plural references unless the context clearly dictates
otherwise.
[0329] "Administration" and "treatment," as it applies to an
animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, or composition to the animal, human,
subject, cell, tissue, organ, or biological fluid. Treatment of a
cell encompasses contact of a reagent to the cell, as well as
contact of a reagent to a fluid, where the fluid is in contact with
the cell. "Administration" and "treatment" also means in vitro and
ex vivo treatments, e.g., of a cell, by a reagent, diagnostic,
binding compound, or by another cell.
[0330] "Treat" or "treating" means to administer a therapeutic
agent, such as a composition containing any of the antibodies or
antigen-binding fragments of the present invention, internally or
externally to a subject or patient having one or more disease
symptoms, or being suspected of having a disease, for which the
agent has therapeutic activity. Typically, the agent is
administered in an amount effective to alleviate one or more
disease symptoms in the treated subject or population, whether by
inducing the regression of or inhibiting the progression of such
symptom(s) by any clinically measurable degree. The amount of a
therapeutic agent that is effective to alleviate any particular
disease symptom may vary according to factors such as the disease
state, age, and weight of the patient, and the ability of the drug
to elicit a desired response in the subject. Whether a disease
symptom has been alleviated can be assessed by any clinical
measurement typically used by physicians or other skilled
healthcare providers to assess the severity or progression status
of that symptom.
[0331] "Recombinant expression" of a protein means the
transcription and translation of an exogenous gene in a host
organism to generate the protein, which is referred to herein as a
"recombinant protein."
SIRP.alpha. and Associated Proteins
[0332] SIRP.alpha. belongs to a class of membrane proteins known as
"paired receptors" that contain several genes coding for proteins
(e.g., SIRP.alpha., SIRP.beta.1, and SIRP.gamma.) with similar
extracellular regions but different transmembrane and/or
cytoplasmic regions having opposite (activating or inhibitory)
signaling abilities. Like SIRP.alpha., there are several examples
of paired receptors on NK cells and some on myeloid cells,
including the SIRP and CD200 receptor families (Hatherley et al.,
Mol Cell. 2008; 31: 266-277).
[0333] SIRP.alpha. contains an extracellular region that can be
subdivided into three separate domains: the Ig-like
(immunoglobulin-like) V-type (IgV), Ig-like C1-type (IgC1), and
Ig-like C2-type (IgC2) domain. The IgV domain is also known as the
ligand-binding N-terminal domain of SIRP.alpha.. Like SIRP.alpha.,
also the related proteins SIRP.beta.1 and SIRP.gamma. comprise an
extracellular region that can be subdivided into an IgV, IgC1, and
IgC2 domain. However, SIRP.alpha., SIRP.beta.1 and SIRP.gamma. have
different cytoplasmic regions. SIRP.beta.1 has a very short
cytoplasmic region of only 6 amino acids and lacks signalling
motifs for association with phosphatases. Instead, this protein
associates with DNAX activation protein 12 (DAP12), a dimeric
adaptor protein that binds an amino acid with a basic side chain in
the transmembrane region of SIRP.beta.1 and is able to transmit
activating signals through its immunoreceptor tyrosine-based
activation motif (ITAM). SIRP.gamma. also has a short cytoplasmic
region of 4 amino acids, but it lacks a charged amino-acid side
chain in the transmembrane region and therefore does not associate
with DAP12. Hence, SIRP.gamma. is annotated as a non-signalling
protein (Barclay, A. N. and Brown, M. H., Nat Rev Immunol. 2006; 6:
457-464).
[0334] The major ligand of SIRP.gamma. is CD47, which consists of
one extracellular IgV domain, a five times transmembrane-spanning
domain, and a short cytoplasmic tail. CD47 functions as a cellular
ligand with binding mediated through the NH2-terminal IgV domain of
SIRP.gamma.. Evidence that CD47 contributes to recognition of self
comes from the observation that splenic macrophages derived from
CD47-expressing mice clear infused blood cells from CD47.sup.-/-
mice (Oldenborg et al., Science. 2000; 288: 2051-2054).
[0335] In addition to CD47, two other SIRP.gamma. ligands have been
reported, known as surfactant proteins A and D (Sp-A and Sp-D),
both of which belong to the collectin family. Sp-D has been
reported to bind to the membrane-proximal IgC2 domain of
SIRP.gamma. in a calcium- and saccharide-dependent manner. It is
thought that Sp-A and Sp-D help maintain an anti-inflammatory
environment in the lung by stimulating SIRP.gamma. on alveolar
macrophages (Gardai et al., Cell. 2003; 115: 13-23).
[0336] The amino acid sequence of eight human SIRP.gamma. variants
are listed in SEQ ID NOs: 34, 36, 44, 46, 48, 50, 52, and 54;
exemplary nucleic acid sequences encoding these variants are listed
in SEQ ID NOs: 33, 35, 43, 45, 47, 49, 51, and 53,
respectively.
[0337] For comparison, the amino acid sequence of human SIRP.beta.1
and SIRP.gamma. are listed in SEQ ID NOs: 38 and 40, respectively,
and exemplary nucleic acid sequences in SEQ ID NOs: 37 and 39,
respectively.
[0338] The amino acid sequence of human CD47 is listed in SEQ ID
NO: 42, and an exemplary nucleic acid sequence in SEQ ID NO:
41.
[0339] Modified SIRP.gamma. polypeptides
hSIRP.alpha.-V.beta.C1.alpha.C2.alpha.,
hSIRP.alpha.-V.alpha.C1.beta.C2.alpha.,
hSIRP.alpha.-V.alpha.C1.alpha.C2.beta., and hSIRP.alpha.V1(P74A)
discussed hereinafter are listed in SEQ ID NOs: 56, 58, 60, and 62;
exemplary nucleic acid sequences encoding these variants are listed
in SEQ ID NOs: 55, 57, 59, and 61, respectively.
Anti-SIRP.alpha. Antibodies and Antigen-Binding Fragments
Thereof
[0340] The present invention provides antibodies or antigen-binding
fragments thereof that bind human SIRP.alpha. and uses of such
antibodies or fragments. In some embodiments, the anti-SIRP.alpha.
antibodies are isolated.
[0341] Whether an antibody specifically binds to a polypeptide
sequence (e.g., human SIRP.alpha., hSIRP.beta.1, etc.) can be
determined using any assay known in the art. Examples of assays
known in the art to determining binding affinity include surface
plasmon resonance (e.g., BIACORE) or a similar technique (e.g.
KinExa or OCTET).
[0342] As used herein, the term "antibody" refers to any form of
antibody that exhibits the desired biological activity. The term
antibody includes antigen-binding portions, i.e., "antigen binding
sites," (e.g., fragments, subsequences, complementarity determining
regions (CDRs)) that retain capacity to bind antigen, including (i)
a Fab fragment, a monovalent fragment consisting of the V.sub.L,
V.sub.H, C.sub.L and C.sub.H1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Single chain antibodies
are also included by reference in the term "antibody." Preferred
therapeutic antibodies are intact IgG antibodies. The term "intact
IgG" as used herein is meant as a polypeptide belonging to the
class of antibodies that are substantially encoded by a recognized
immunoglobulin gamma gene. In humans this class comprises IgG1,
IgG2, IgG3, and IgG4. In mice this class comprises IgG1, IgG2a,
IgG2b, IgG3. The known Ig domains in the IgG class of antibodies
are V.sub.H, C.gamma.1, C.gamma.2, C.gamma.3, V.sub.L, and
C.sub.L.
[0343] The present invention includes anti-SIRP.alpha.
antigen-binding fragments and methods of use thereof.
[0344] As used herein, a "full length antibody" is, in the case of
an IgG, a bivalent molecule comprising two heavy chains and two
light chains. Each heavy chain comprises a V.sub.H domain followed
by a constant domain (C.sub.H1), a hinge region, and two more
constant (C.sub.H2 and C.sub.H3) domains; while each light chain
comprises one V.sub.L domain and one constant (C.sub.L) domain. A
full length antibody in the case of an IgM is a decavalent or
dodecavalent molecule comprising 5 or 6 linked immunoglobulins in
which immunoglobulin each monomer has two antigen binding sites
formed of a heavy and light chain.
[0345] As used herein, unless otherwise indicated, "antibody
fragment" or "antigen-binding fragment" refers to antigen-binding
fragments of antibodies, i.e. antibody fragments that retain the
ability to bind specifically to the antigen bound by the
full-length antibody, e.g. fragments that retain one or more CDR
regions. Examples of antigen-binding fragments include, but are not
limited to, Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies;
linear antibodies; single-chain antibody molecules, e.g., sc-Fv;
nanobodies and multispecific antibodies formed from antibody
fragments.
[0346] The present invention includes anti-SIRP.alpha. Fab
fragments and methods of use thereof. A "Fab fragment" is comprised
of one light chain and the C.sub.H1 and variable regions of one
heavy chain. The heavy chain of a Fab molecule cannot form a
disulfide bond with another heavy chain molecule. A "Fab fragment"
can be the product of papain cleavage of an antibody.
[0347] The present invention includes anti-SIRP.alpha. antibodies
and antigen-binding fragments thereof which comprise an Fc region
and methods of use thereof. An "Fc" region contains two heavy chain
fragments comprising the C.sub.H3 and C.sub.H2 domains of an
antibody. The two heavy chain fragments are held together by two or
more disulfide bonds and by hydrophobic interactions of the
C.sub.H3 domains.
[0348] The present invention includes anti-SIRP.alpha. Fab'
fragments and methods of use thereof. A "Fab' fragment" contains
one light chain and a portion or fragment of one heavy chain that
contains the V.sub.H domain and the C.sub.H1 domain and also the
region between the C.sub.H1 and C H2 domains, such that an
interchain disulfide bond can be formed between the two heavy
chains of two Fab' fragments to form a F(ab') 2 molecule.
[0349] The present invention includes anti-SIRP.alpha. F(ab').sub.2
fragments and methods of use thereof. A "F(ab').sub.2 fragment"
contains two light chains and two heavy chains containing a portion
of the constant region between the C.sub.H1 and C.sub.H2 domains,
such that an interchain disulfide bond is formed between the two
heavy chains. A F(ab') 2 fragment thus is composed of two Fab'
fragments that are held together by a disulfide bond between the
two heavy chains. An "F(ab').sub.2 fragment" can be the product of
pepsin cleavage of an antibody.
[0350] The present invention includes anti-SIRP.alpha. Fv fragments
and methods of use thereof. The "Fv region" comprises the variable
regions from both the heavy and light chains, but lacks the
constant regions.
[0351] The present invention includes anti-SIRP.alpha. scFv
fragments and methods of use thereof. The term "single-chain Fv" or
"scFv" antibody refers to antibody fragments comprising the VH and
V.sub.L domains of an antibody, wherein these domains are present
in a single polypeptide chain. Generally, the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains which enables the scFv to form the desired
structure for antigen-binding. For a review of scFv, see Pluckthun
(1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.
See also, International Patent Application Publication No. WO
88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.
[0352] The present invention includes anti-SIRP.alpha. domain
antibodies and methods of use thereof. A "domain antibody" is an
immunologically functional immunoglobulin fragment containing only
the variable region of a heavy chain or the variable region of a
light chain. In some instances, two or more V.sub.H regions are
covalently joined with a peptide linker to create a bivalent domain
antibody. The two V.sub.H regions of a bivalent domain antibody may
target the same or different antigens.
[0353] The present invention includes anti-SIRP.alpha. bivalent
antibodies and methods of use thereof. A "bivalent antibody"
comprises two antigen-binding sites. In some instances, the two
binding sites have the same antigen specificities. However,
bivalent antibodies may be bispecific (see below).
[0354] The present invention includes anti-SIRP.alpha. diabodies
and methods of use thereof. As used herein, the term "diabodies"
refers to small antibody fragments with two antigen-binding sites,
which fragments comprise a heavy chain variable domain (V.sub.H)
connected to a light chain variable domain (V.sub.L) in the same
polypeptide chain (V.sub.H-V.sub.L or V.sub.L-V.sub.H). By using a
linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, e.g.,
EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 6444-6448. Duobodies are described in Labrijn et
al., 2013, Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150. For a
review of engineered antibody variants generally see Holliger and
Hudson (2005) Nat. Biotechnol. 23:1126-1136.
[0355] Typically, an antibody or antigen-binding fragment of the
invention which is modified in some way retains at least 10% of its
binding activity (when compared to the parental antibody) when that
activity is expressed on a molar basis. Preferably, an antibody or
antigen-binding fragment of the invention retains at least 20%,
50%, 70%, 80%, 90%, 95% or 100% or more of the SIRP.alpha. binding
affinity as the parental antibody. It is also intended that an
antibody or antigen-binding fragment of the invention can include
conservative or non-conservative amino acid substitutions (referred
to as "conservative variants" or "function conserved variants" of
the antibody) that do not substantially alter its biologic
activity.
[0356] The present invention includes isolated anti-SIRP.alpha.
antibodies and antigen-binding fragments thereof and methods of use
thereof. Herein, the term "isolated" is not intended to refer to a
complete absence of such biological molecules or to an absence of
water, buffers, or salts or to components of a pharmaceutical
formulation that includes the antibodies or fragments. An
"isolated" antibody, antigen-binding fragment, nucleic acid, etc.,
is one which has been identified and separated and/or recovered
from one or more components of its natural environment. In
preferred embodiments, the antibody, antigen-binding fragment,
nucleic acid, etc., is purified to 75% by weight or more, more
preferably to 90% by weight or more, still more preferably to 95%
by weight or more, an still more preferably to 98% by weight or
more. Thus, "isolated" biological molecules are at least partially
free of other biological molecules from the cells or cell cultures
in which they are produced. Such biological molecules include
nucleic acids, proteins, lipids, carbohydrates, or other material
such as cellular debris and growth medium. An isolated antibody or
antigen-binding fragment may further be at least partially free of
expression system components such as biological molecules from a
host cell or of the growth medium thereof.
[0357] The present invention includes anti-SIRP.alpha. chimeric
antibodies (e.g., human constant domain/mouse variable domain) and
methods of use thereof. As used herein, a "chimeric antibody" is an
antibody having the variable domain from a first antibody and the
constant domain from a second antibody, where the first and second
antibodies are from different species. (U.S. Pat. No. 4,816,567;
and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:
6851-6855). Typically, the variable domains are obtained from an
antibody from an experimental animal (the "parental antibody"),
such as a rodent, and the constant domain sequences are obtained
from human antibodies, so that the resulting chimeric antibody will
be less likely to elicit an adverse immune response in a human
subject than the parental (e.g., mouse) antibody.
[0358] The present invention includes anti-SIRP.alpha. humanized
antibodies and antigen-binding fragments thereof (e.g., rat or
mouse antibodies that have been humanized) and methods of use
thereof. As used herein, the term "humanized antibody" refers to
forms of antibodies that contain sequences from both human and
non-human (e.g., mouse or rat) antibodies. In general, the
humanized antibody will comprise substantially of at least one, and
typically two, variable domains, in which all or substantially all
of the hypervariable loops correspond to those of a non-human
immunoglobulin, and all or substantially all of the framework (FR)
regions are those of a human immunoglobulin sequence. The humanized
antibody may optionally comprise at least a portion of a human
immunoglobulin constant region (Fc). For more details about
humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525
(1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr.
Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21:
397-402 (2000).
[0359] In general, the basic antibody structural unit comprises a
tetramer. Each tetramer includes two identical pairs of polypeptide
chains, each pair having one "light" (about 25 kDa) and one "heavy"
chain (about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. The carboxy-terminal
portion of the heavy chain may define a constant region primarily
responsible for effector function. Typically, human light chains
are classified as kappa and lambda light chains. Furthermore, human
heavy chains are typically classified as mu, delta, gamma, alpha,
or epsilon, and define the antibody's isotype as IgM, IgD, IgG,
IgA, and IgE, respectively. Within light and heavy chains, the
variable and constant regions are joined by a "J" region of about
12 or more amino acids, with the heavy chain also including a "D"
region of about 10 more amino acids. See generally, Fundamental
Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.
(1989).
[0360] The variable regions of each light/heavy chain pair form the
antibody binding site. Thus, in general, an intact antibody has two
binding sites. Except in bifunctional or bispecific antibodies, the
two binding sites are, in general, the same.
[0361] Typically, the variable domains of both the heavy and light
chains comprise three hypervariable regions, also called
complementarity determining regions (CDRs), located within
relatively conserved framework regions (FR). The CDRs are usually
aligned by the framework regions, enabling binding to a specific
epitope. In general, from N-terminal to C-terminal, both light and
heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3,
CDR3 and FR4. The assignment of amino acids to each domain is,
generally, in accordance with the definitions of Sequences of
Proteins of Immunological Interest, Kabat, et al.; National
Institutes of Health, Bethesda, Md.; 5.sup.th ed.; NIH Publ. No.
91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et
al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J
Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature
342:878-883.
[0362] As used herein, the term "hypervariable region" refers to
the amino acid residues of an antibody or antigen-binding fragment
thereof that are responsible for antigen-binding. The hypervariable
region comprises amino acid residues from a "complementarity
determining region" or "CDR" (i.e. CDRL1, CDRL2 and CDRL3 in the
light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy
chain variable domain). See Kabat et al. (1991) Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (defining the CDR
regions of an antibody by sequence); see also Chothia and Lesk
(1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an
antibody by structure). As used herein, the term "framework" or
"FR" residues refers to those variable domain residues other than
the hypervariable region residues defined herein as CDR
residues.
[0363] "Isolated nucleic acid molecule" or "isolated
polynucleotide" means a DNA or RNA of genomic, mRNA, cDNA, or
synthetic origin or some combination thereof which is not
associated with all or a portion of a polynucleotide in which the
isolated polynucleotide is found in nature, or is linked to a
polynucleotide to which it is not linked in nature. For purposes of
this disclosure, it should be understood that "a nucleic acid
molecule comprising" a particular nucleotide sequence does not
encompass intact chromosomes. Isolated nucleic acid molecules
"comprising" specified nucleic acid sequences may include, in
addition to the specified sequences, coding sequences for up to ten
or even up to twenty or more other proteins or portions or
fragments thereof, or may include operably linked regulatory
sequences that control expression of the coding region of the
recited nucleic acid sequences, and/or may include vector
sequences.
[0364] The phrase "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to use promoters, polyadenylation
signals, and enhancers.
[0365] A nucleic acid or polynucleotide is "operably linked" when
it is placed into a functional relationship with another nucleic
acid sequence. For example, DNA for a presequence or secretory
leader is operably linked to DNA for a polypeptide if it is
expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence; or a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to facilitate translation. Generally, but not
always, "operably linked" means that the DNA sequences being linked
are contiguous, and, in the case of a secretory leader, contiguous
and in reading phase. However, enhancers do not have to be
contiguous. Linking is accomplished by ligation at convenient
restriction sites. If such sites do not exist, the synthetic
oligonucleotide adaptors or linkers are used in accordance with
conventional practice.
[0366] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that not all progeny will have precisely identical DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0367] As used herein, "germline sequence" refers to a sequence of
unrearranged immunoglobulin DNA sequences. Any suitable source of
unrearranged immunoglobulin sequences may be used. Human germline
sequences may be obtained, for example, from JOINSOLVER germline
databases on the website for the National Institute of Arthritis
and Musculoskeletal and Skin Diseases of the United States National
Institutes of Health. Mouse germline sequences may be obtained, for
example, as described in Giudicelli et al. (2005) Nucleic Acids
Res. 33: D256-D261.
[0368] Physical and Functional Properties of the Exemplary
Anti-SIRP.alpha. Antibodies
[0369] The present invention provides anti-SIRP.alpha. antibodies
and antigen-binding fragments thereof having specified structural
and functional features, and methods of use of the antibodies or
antigen-binding fragments thereof in the treatment or prevention of
disease (e.g., cancer or infectious disease).
[0370] As stated above, antibodies and fragments that bind to the
same epitope as any of the anti-SIRP.alpha. antibodies or
antigen-binding fragments thereof of the present invention also
form part of the present invention. In one embodiment, the
invention provides an antibody or antigen binding fragment thereof
that binds to the same epitope of human SIRP.alpha. as an antibody
comprising one of the following combinations of heavy chain
sequence/light chain sequence (or in each case an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto):
[0371] SEQ ID NO: 10/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H1L1)
[0372] SEQ ID NO: 10/SEQ ID NO: 22 (referred to herein as
hSIRP.alpha..50A.H1L2)
[0373] SEQ ID NO: 10/SEQ ID NO: 24 (referred to herein as
hSIRP.alpha..50A.H1L3)
[0374] SEQ ID NO: 10/SEQ ID NO: 26 (referred to herein as
hSIRP.alpha..50A.H1L4)
[0375] SEQ ID NO: 10/SEQ ID NO: 28 (referred to herein as
hSIRP.alpha..50A.H1L5)
[0376] SEQ ID NO: 12/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H2L1)
[0377] SEQ ID NO: 12/SEQ ID NO: 22 (referred to herein as
hSIRP.alpha..50A.H2L2)
[0378] SEQ ID NO: 12/SEQ ID NO: 24 (referred to herein as
hSIRP.alpha..50A.H2L3)
[0379] SEQ ID NO: 12/SEQ ID NO: 26 (referred to herein as
hSIRP.alpha..50A.H2L4)
[0380] SEQ ID NO: 12/SEQ ID NO: 28 (referred to herein as
hSIRP.alpha..50A.H2L5)
[0381] SEQ ID NO: 14/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H3L1)
[0382] SEQ ID NO: 14/SEQ ID NO: 22 (referred to herein as
hSIRP.alpha..50A.H3L2)
[0383] SEQ ID NO: 14/SEQ ID NO: 24 (referred to herein as
hSIRP.alpha..50A.H3L3)
[0384] SEQ ID NO: 14/SEQ ID NO: 26 (referred to herein as
hSIRP.alpha..50A.H3L4)
[0385] SEQ ID NO: 14/SEQ ID NO: 28 (referred to herein as
hSIRP.alpha..50A.H3L5)
[0386] SEQ ID NO: 16/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H4L1)
[0387] SEQ ID NO: 16/SEQ ID NO: 22 (referred to herein as
hSIRP.alpha..50A.H4L2)
[0388] SEQ ID NO: 16/SEQ ID NO: 24 (referred to herein as
hSIRP.alpha..50A.H4L3)
[0389] SEQ ID NO: 16/SEQ ID NO: 26 (referred to herein as
hSIRP.alpha..50A.H4L4)
[0390] SEQ ID NO: 16/SEQ ID NO: 28 (referred to herein as
hSIRP.alpha..50A.H4L5)
[0391] SEQ ID NO: 18/SEQ ID NO: 20 (referred to herein as
hSIRP.alpha..50A.H5L1)
[0392] SEQ ID NO: 18/SEQ ID NO: 22 (referred to herein as
hSIRP.alpha..50A.H5L2)
[0393] SEQ ID NO: 18/SEQ ID NO: 24 (referred to herein as
hSIRP.alpha..50A.H5L3)
[0394] SEQ ID NO: 18/SEQ ID NO: 26 (referred to herein as
hSIRP.alpha..50A.H5L4)
[0395] SEQ ID NO: 18/SEQ ID NO: 28 (referred to herein as
hSIRP.alpha..50A.H5L5)
[0396] SEQ ID NO: 78/SEQ ID NO: 90 (referred to herein as
hSIRP.alpha..40A.H1L1)
[0397] SEQ ID NO: 78/SEQ ID NO: 92 (referred to herein as
hSIRP.alpha..40A.H1L2)
[0398] SEQ ID NO: 78/SEQ ID NO: 94 (referred to herein as
hSIRP.alpha..40A.H1L3)
[0399] SEQ ID NO: 78/SEQ ID NO: 96 (referred to herein as
hSIRP.alpha..40A.H1L4)
[0400] SEQ ID NO: 78/SEQ ID NO: 98 (referred to herein as
hSIRP.alpha..40A.H1L5)
[0401] SEQ ID NO: 78/SEQ ID NO: 100 (referred to herein as
hSIRP.alpha..40A.H1L6)
[0402] SEQ ID NO: 80/SEQ ID NO: 90 (referred to herein as
hSIRP.alpha..40A.H2L1)
[0403] SEQ ID NO: 80/SEQ ID NO: 92 (referred to herein as
hSIRP.alpha..40A.H2L2)
[0404] SEQ ID NO: 80/SEQ ID NO: 94 (referred to herein as
hSIRP.alpha..40A.H2L3)
[0405] SEQ ID NO: 80/SEQ ID NO: 96 (referred to herein as
hSIRP.alpha..40A.H2L4)
[0406] SEQ ID NO: 80/SEQ ID NO: 98 (referred to herein as
hSIRP.alpha..40A.H2L5)
[0407] SEQ ID NO: 80/SEQ ID NO: 100 (referred to herein as
hSIRP.alpha..40A.H2L6)
[0408] SEQ ID NO: 82/SEQ ID NO: 90 (referred to herein as
hSIRP.alpha..40A.H3L1)
[0409] SEQ ID NO: 82/SEQ ID NO: 92 (referred to herein as
hSIRP.alpha..40A.H3L2)
[0410] SEQ ID NO: 82/SEQ ID NO: 94 (referred to herein as
hSIRP.alpha..40A.H3L3)
[0411] SEQ ID NO: 82/SEQ ID NO: 96 (referred to herein as
hSIRP.alpha..40A.H3L4)
[0412] SEQ ID NO: 82/SEQ ID NO: 98 (referred to herein as
hSIRP.alpha..40A.H3L5)
[0413] SEQ ID NO: 82/SEQ ID NO: 100 (referred to herein as
hSIRP.alpha..40A.H3L6)
[0414] SEQ ID NO: 84/SEQ ID NO: 90 (referred to herein as
hSIRP.alpha..40A.H4L1)
[0415] SEQ ID NO: 84/SEQ ID NO: 92 (referred to herein as
hSIRP.alpha..40A.H4L2)
[0416] SEQ ID NO: 84/SEQ ID NO: 94 (referred to herein as
hSIRP.alpha..40A.H4L3)
[0417] SEQ ID NO: 84/SEQ ID NO: 96 (referred to herein as
hSIRP.alpha..40A.H4L4)
[0418] SEQ ID NO: 84/SEQ ID NO: 98 (referred to herein as
hSIRP.alpha..40A.H4L5)
[0419] SEQ ID NO: 84/SEQ ID NO: 100 (referred to herein as
hSIRP.alpha..40A.H4L6)
[0420] SEQ ID NO: 86/SEQ ID NO: 90 (referred to herein as
hSIRP.alpha..40A.H5L1)
[0421] SEQ ID NO: 86/SEQ ID NO: 92 (referred to herein as
hSIRP.alpha..40A.H5L2)
[0422] SEQ ID NO: 86/SEQ ID NO: 94 (referred to herein as
hSIRP.alpha..40A.H5L3)
[0423] SEQ ID NO: 86/SEQ ID NO: 96 (referred to herein as
hSIRP.alpha..40A.H5L4)
[0424] SEQ ID NO: 86/SEQ ID NO: 98 (referred to herein as
hSIRP.alpha..40A.H5L5)
[0425] SEQ ID NO: 86/SEQ ID NO: 100 (referred to herein as
hSIRP.alpha..40A.H5L6)
[0426] SEQ ID NO: 88/SEQ ID NO: 90 (referred to herein as
hSIRP.alpha..40A.H6L1)
[0427] SEQ ID NO: 88/SEQ ID NO: 92 (referred to herein as
hSIRP.alpha..40A.H6L2)
[0428] SEQ ID NO: 88/SEQ ID NO: 94 (referred to herein as
hSIRP.alpha..40A.H6L3)
[0429] SEQ ID NO: 88/SEQ ID NO: 96 (referred to herein as
hSIRP.alpha..40A.H6L4)
[0430] SEQ ID NO: 88/SEQ ID NO: 98 (referred to herein as
hSIRP.alpha..40A.H6L5)
[0431] SEQ ID NO: 88/SEQ ID NO: 100 (referred to herein as
hSIRP.alpha..40A.H6L6).
[0432] There are several methods available for mapping antibody
epitopes on target antigens, including: H/D-Ex mass spectrometry,
crosslinking coupled mass spectrometry, X-ray crystallography,
pepscan analysis and site directed mutagenesis. For example, HDX
(Hydrogen Deuterium Exchange) coupled with proteolysis and mass
spectrometry can be used to determine the epitope of an antibody on
a specific antigen Y. HDX-MS relies on the accurate measurement and
comparison of the degree of deuterium incorporation by an antigen
when incubated in D.sub.2O on its own and in presence of its
antibody at various time intervals. Deuterium is exchanged with
hydrogen on the amide backbone of the proteins in exposed areas
whereas regions of the antigen bound to the antibody will be
protected and will show less or no exchange after analysis by
LC-MS/MS of proteolytic fragments. Crosslinking coupled mass
spectrometry begins by binding the antibody and the antigen with a
mass labeled chemical crosslinker. Next the presence of the complex
is confirmed using high mass MALDI detection. Because after
crosslinking chemistry the Ab/Ag complex is extremely stable, many
various enzymes and digestion conditions can be applied to the
complex to provide many different overlapping peptides.
Identification of these peptides is performed using high resolution
mass spectrometry and MS/MS techniques. Identification of the
crosslinked peptides is determined using mass tag linked to the
cross-linking reagents. After MS/MS fragmentation and data
analysis, both epitope and paratope are determined in the same
experiment.
[0433] The scope of the present invention also includes isolated
anti-SIRP.alpha. antibodies and antigen-binding fragments thereof
(e.g., humanized antibodies), comprising a variant of an
immunoglobulin chain set forth herein, wherein the variant exhibits
one or more of the following properties: [0434] binds human
SIRP.alpha.V1 protein having the sequence of SEQ ID NO: 34 with an
EC.sub.50<1 nM; and exhibits at least a 100-fold higher
EC.sub.50 for SIRP.alpha.V1(P74A) having the sequence of SEQ ID NO:
62; and optionally also at least a 100-fold higher EC.sub.50 for
human SIRP.beta.1 protein having the sequence of SEQ ID NO: 38 (in
each case wherein the reduced EC.sub.50 is relative to the
EC.sub.50 for human SIRP.alpha.V1 protein having the sequence of
[0435] SEQ ID NO: 34, and in each case preferably when measured by
cellular ELISA (CELISA) as described hereinafter; [0436] binds to a
cell expressing human SIRP.alpha.V1 protein with an EC.sub.50<10
nM, preferably <5 nM, more preferably <1.5 nM, still more
preferably <1.0 nM, even more preferably <0.5 nM, and most
preferably about 0.3 nM or less; [0437] binds to a cell expressing
human SIRP.alpha.V2 protein with an EC.sub.50<10 nM, preferably
<5 nM, more preferably <1.5 nM, still more preferably <1.0
nM, even more preferably <0.5 nM, and most preferably about 0.3
nM or less; [0438] does not appreciably bind to SIRP.beta.1 protein
at an antibody concentration of 50 nM, preferably 67 nM, and more
preferably 100 nM; or alternatively at a concentration that is
10-fold greater, preferably 50-fold greater, more preferably
100-fold greater, and still more preferably 200-fold greater than
the antibody's EC.sub.50 for SIRP.alpha.V1 or SIRP.alpha.V2; [0439]
inhibits binding between human SIRP.alpha. and CD47 with an
IC.sub.50<10.0 nM, more preferably <5.0 nM, still more
preferably <2.5 nM, and most preferably about 1.0 nM or less;
and [0440] exhibits a T20 "humanness" of at least 79, and more
preferably 85%.
[0441] In other embodiments, the invention provides antibodies or
antigen-binding fragment thereof that bind human SIRP.alpha. (e.g.,
humanized antibodies) and have V.sub.H domains and V.sub.L domains
with at least 90% sequence identity with SEQ ID NOs: 75, 78, 80,
82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92,
94, 96, 98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. In other
embodiments, the invention provides antibodies or antigen-binding
fragment thereof that bind human SIRP.alpha. (e.g., humanized
antibodies) and have V.sub.H domains and V.sub.L domains with at
least 95% sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84,
86, 88, 102, 7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96,
98, 100, 104, 8, 20, 22, 24, 26, 28, and 32. In other embodiments,
the invention provides antibodies or antigen-binding fragment
thereof that bind human SIRP.alpha. (e.g., humanized antibodies)
and have V.sub.H domains and V.sub.L domains with at least 97%
sequence identity with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102,
7, 10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100,
104, 8, 20, 22, 24, 26, 28, and 32. In other embodiments, the
invention provides antibodies or antigen-binding fragment thereof
that bind human SIRP.alpha. (e.g., humanized antibodies) and have
V.sub.H domains and V.sub.L domains with at least 98% sequence
identity with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10,
12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8,
20, 22, 24, 26, 28, and 32. In other embodiments, the invention
provides antibodies or antigen-binding fragment thereof that bind
human SIRP.alpha. (e.g., humanized antibodies) and have V.sub.H
domains and V.sub.L domains with at least 99% sequence identity
with SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14,
16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104, 8, 20, 22,
24, 26, 28, and 32. Preferably, in each case, the sequence
differences between SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7,
10, 12, 14, 16, 18, and 30; and 76, 90, 92, 94, 96, 98, 100, 104,
8, 20, 22, 24, 26, 28, and 32 and the variants consist of
conservative substitutions and are most preferably limited to
substitutions within the framework residues.
[0442] The following references relate to BLAST algorithms often
used for sequence analysis: BLAST ALGORITHMS: Camacho, C. et al.
(2009): BMC Bioinformatics 10:421; Altschul et al. (2005) FEBS J.
272(20): 5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol.
215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272;
Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul,
S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J.,
et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al.,
(1993) Comput. Chem. 17:149-163; Hancock, J. M. et al., (1994)
Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff,
M. O., et al., "A model of evolutionary change in proteins." in
Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3.
M. O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found.,
Washington, D.C.; Schwartz, R. M., et al., "Matrices for detecting
distant relationships." in Atlas of Protein Sequence and Structure,
(1978) vol. 5, suppl. 3." M. O. Dayhoff (ed.), pp. 353-358, Natl.
Biomed. Res. Found., Washington, D.C.; Altschul, S.F., (1991) J.
Mol. Biol. 219:555-565; States, D. J., et al., (1991) Methods
3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA
89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol.
36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc.
Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc.
Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann.
Prob. 22:2022-2039; and Altschul, S.F. "Evaluating the statistical
significance of multiple distinct local alignments." in Theoretical
and Computational Methods in Genome Research (S. Suhai, ed.),
(1997) pp. 1-14, Plenum, New York. In the present application,
percent identity comparisons are preferably performed by a BLAST
algorithm wherein the parameters of the algorithm are selected to
give the largest match between the respective sequences over the
entire length of the respective reference sequences (e.g. expect
threshold: 10; word size: 6; max matches in a query range: 0;
BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional
compositional score matrix adjustment).
[0443] "Conservatively modified variants" or "conservative
substitution" refers to substitutions of amino acids in a protein
with other amino acids having similar characteristics (e.g. charge,
side-chain size, hydrophobicity/hydrophilicity, backbone
conformation and rigidity, etc.), such that the changes can
frequently be made without altering the biological activity of the
protein. Those of skill in this art recognize that, in general,
single amino acid substitutions in non-essential regions of a
polypeptide do not substantially alter biological activity (see,
e.g., Watson et al. (1987) Molecular Biology of the Gene, The
Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,
substitutions of structurally or functionally similar amino acids
are less likely to disrupt biological activity. Exemplary
conservative substitutions are set forth the following Table 1.
TABLE-US-00001 TABLE 1 Exemplary Conservative Amino Acid
Substitutions Original residue Conservative substitution Ala (A)
Gly; Ser Arg (R) Lys; His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C)
Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln
Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu;
Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser
Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu
[0444] Function-conservative variants of the antibodies of the
invention are also contemplated by the present invention.
"Function-conservative variants," as used herein, refers to
antibodies or fragments in which one or more amino acid residues
have been changed without altering a desired property, such an
antigen affinity and/or specificity. Such variants include, but are
not limited to, replacement of an amino acid with one having
similar properties, such as the conservative amino acid
substitutions of Table 1. Also provided are isolated polypeptides
comprising the V.sub.L domains of the anti-SIRP.alpha. antibodies
of the invention (e.g., SEQ ID NOs: 76, 90, 92, 94, 96, 98, 100, 8,
20, 22, 24, 26, 28, and 32), and isolated polypeptides comprising
the V.sub.H domains of the anti-SIRP.alpha. antibodies of the
invention (e.g., SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 7, 10, 12,
14, 16, 18, and 30) having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more amino acid substitutions, and preferably conservative
substitutions.
[0445] The present invention further comprises the polynucleotides
encoding any of the polypeptides or immunoglobulin chains of
anti-SIRP.alpha. antibodies and antigen-binding fragments thereof
of the invention. For example, the present invention includes the
polynucleotides encoding the amino acids described in any one of
SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88, 102, 7, 10, 12, 14, 16, 18,
and 30; and SEQ ID NOs: 76, 90, 92, 94, 96, 98, 100, 104, 8, 20,
22, 24, 26, 28, and 32.
[0446] In one embodiment, an isolated polynucleotide, for example
DNA, encoding the polypeptide chains of the isolated antibodies or
antigen-binding fragments set forth herein is provided. In one
embodiment, the isolated polynucleotide encodes an antibody or
antigen-binding fragment thereof comprising at least one mature
immunoglobulin light chain variable (VL) domain according to the
invention and/or at least one mature immunoglobulin heavy chain
variable (VH) domain according to the invention. In some
embodiments, the isolated polynucleotide encodes both a light chain
and a heavy chain on a single polynucleotide molecule, and in other
embodiments the light and heavy chains are encoded on separate
polynucleotide molecules. In another embodiment, the
polynucleotides further encodes a signal sequence.
[0447] This present invention also provides vectors, e.g.,
expression vectors, such as plasmids, comprising the isolated
polynucleotides of the invention, wherein the polynucleotide is
operably linked to control sequences that are recognized by a host
cell when the host cell is transfected with the vector. Also
provided are host cells comprising a vector of the present
invention and methods for producing the antibody or antigen-binding
fragment thereof or polypeptide disclosed herein comprising
culturing a host cell harboring an expression vector or a nucleic
acid encoding the immunoglobulin chains of the antibody or
antigen-binding fragment thereof in culture medium, and isolating
the antigen or antigen-binding fragment thereof from the host cell
or culture medium.
Binding Affinity
[0448] By way of example, and not limitation, the antibodies and
antigen-binding fragments disclosed herein may bind human
SIRP.alpha. bivalently with a K.sub.D value of 10.times.10.sup.-9M
or lower) as determined by surface plasmon resonance (e.g.,
BIACORE) or a similar technique (e.g. KinExa or bio-layer
interferometry (OCTET)). In one embodiment, the antibodies and
antigen-binding fragments disclosed herein may bind human
SIRP.alpha. or bivalently with a K.sub.D value of about
5-10.times.10.sup.-9 M as determined by surface plasmon resonance
(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).
Affinity is calculated as K.sub.D=k.sub.off/k.sub.on (k.sub.off is
the dissociation rate constant, K. is the association rate constant
and K.sub.D is the equilibrium constant). Affinity can be
determined at equilibrium by measuring the fraction bound (r) of
labeled ligand at various concentrations (c). The data are graphed
using the Scatchard equation: r/c=K(n-r): where r=moles of bound
ligand/mole of receptor at equilibrium; c=free ligand concentration
at equilibrium; K=equilibrium association constant; and n=number of
ligand binding sites per receptor molecule. By graphical analysis,
r/c is plotted on the Y-axis versus r on the X-axis, thus producing
a Scatchard plot. Antibody affinity measurement by Scatchard
analysis is well known in the art. See, e.g., van Erp et al., J.
Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods
Programs Biomed. 27: 65-8, 1988.
Humanness
[0449] For purposes of this document, "humanness" is measured using
the T20 score analyzer to quantify the humanness of the variable
region of monoclonal antibodies as described in Gao S H, Huang K,
Tu H, Adler A S. Monoclonal antibody humanness score and its
applications. BMC Biotechnology. 2013: 13:55.
doi:10.1186/1472-6750-13-55).
[0450] A web-based tool is provided to calculate the T20 score of
antibody sequences using the T20 Cutoff Human Databases:
http://abAnalyzer.lakepharma.com. In computing a T20 score, an
input VH, VK, or VL variable region protein sequence is first
assigned Kabat numbering, and CDR residues are identified. The
full-length sequence or the framework only sequence (with CDR
residues removed) is compared to every sequence in a respective
antibody database using the blastp protein-protein BLAST algorithm.
The sequence identity between each pairwise comparison is isolated,
and after every sequence in the database has been analyzed, the
sequences are sorted from high to low based on the sequence
identity to the input sequence. The percent identity of the Top 20
matched sequences is averaged to obtain the T20 score.
[0451] For each chain type (VH, VK, VL) and sequence length
(full-length or framework only) in the "All Human Databases," each
antibody sequence was scored with its respective database using the
T20 score analyzer. The T20 score was obtained for the top 20
matched sequences after the input sequence itself was excluded (the
percent identity of sequences 2 through 21 were averaged since
sequence 1 was always the input antibody itself). The T20 scores
for each group were sorted from high to low. The decrease in score
was roughly linear for most of the sequences; however the T20
scores for the bottom .about.15% of antibodies started decreasing
sharply. Therefore, the bottom 15 percent of sequences were removed
and the remaining sequences formed the T20 Cutoff Human Databases,
where the T20 score cutoff indicates the lowest T20 score of a
sequence in the new database.
[0452] As used herein, a "Human" antibody is one that has a T20
humanness score of at least 79%, and more preferably at least
85%.
Ability of Anti-hSIRP.alpha. Antibodies to Block Binding to
CD47
[0453] In some embodiments, the anti-SIRP.alpha. antibodies or
antigen binding fragments of the invention are able to block
binding of human SIRP.alpha. to human CD47. The ability to block
binding of human SIRP.alpha. to human CD47 can be determined using
any method known in the art. In one embodiment, the ability of the
antibodies to block binding of human SIRP.alpha. to human CD47 is
determined using an ELISA assay.
Methods of Making Antibodies and Antigen-Binding Fragments
Thereof
[0454] Thus, the present invention includes methods for making an
anti-SIRP.alpha. antibody or antigen-binding fragment thereof of
the present invention comprising culturing a hybridoma cell that
expresses the antibody or fragment under condition favorable to
such expression and, optionally, isolating the antibody or fragment
from the hybridoma and/or the growth medium (e.g. cell culture
medium).
[0455] The anti-SIRP.alpha. antibodies disclosed herein may also be
produced recombinantly (e.g., in an E. coli/T7 expression system, a
mammalian cell expression system or a lower eukaryote expression
system). In this embodiment, nucleic acids encoding the antibody
immunoglobulin molecules of the invention (e.g., V.sub.H or
V.sub.L) may be inserted into a pET-based plasmid and expressed in
the E. coli/T7 system. For example, the present invention includes
methods for expressing an antibody or antigen-binding fragment
thereof or immunoglobulin chain thereof in a host cell (e.g.,
bacterial host cell such as E. coli such as BL21 or BL21DE3)
comprising expressing T7 RNA polymerase in the cell which also
includes a polynucleotide encoding an immunoglobulin chain that is
operably linked to a T7 promoter. For example, in an embodiment of
the invention, a bacterial host cell, such as a E. coli, includes a
polynucleotide encoding the T7 RNA polymerase gene operably linked
to a lac promoter and expression of the polymerase and the chain is
induced by incubation of the host cell with IPTG
(isopropyl-beta-D-thiogalactopyrano side).
[0456] There are several methods by which to produce recombinant
antibodies which are known in the art. One example of a method for
recombinant production of antibodies is disclosed in U.S. Pat. No.
4,816,567.
[0457] Transformation can be by any known method for introducing
polynucleotides into a host cell. Methods for introduction of
heterologous polynucleotides into mammalian cells are well known in
the art and include dextran-mediated transfection, calcium
phosphate precipitation, polybrene-mediated transfection,
protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, biolistic injection and direct
microinjection of the DNA into nuclei. In addition, nucleic acid
molecules may be introduced into mammalian cells by viral vectors.
Methods of transforming cells are well known in the art. See, for
example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461 and
4,959,455.
[0458] Thus, the present invention includes recombinant methods for
making an anti-SIRP.alpha. antibody or antigen-binding fragment
thereof of the present invention, or an immunoglobulin chain
thereof, comprising introducing a polynucleotide encoding one or
more immunoglobulin chains of the antibody or fragment (e.g., heavy
and/or light immunoglobulin chain); culturing the host cell (e.g.,
CHO or Pichia or Pichia pastoris) under condition favorable to such
expression and, optionally, isolating the antibody or fragment or
chain from the host cell and/or medium in which the host cell is
grown.
[0459] Anti-SIRP.alpha. antibodies can also be synthesized by any
of the methods set forth in U.S. Pat. No. 6,331,415.
[0460] Eukaryotic and prokaryotic host cells, including mammalian
cells as hosts for expression of the antibodies or fragments or
immunoglobulin chains disclosed herein are well known in the art
and include many immortalized cell lines available from the
American Type Culture Collection (ATCC). These include, inter alia,
Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3
cells, HEK-293 cells and a number of other cell lines. Mammalian
host cells include human, mouse, rat, dog, monkey, pig, goat,
bovine, horse and hamster cells. Cell lines of particular
preference are selected through determining which cell lines have
high expression levels. Other cell lines that may be used are
insect cell lines, such as Sf9 cells, amphibian cells, bacterial
cells, plant cells and fungal cells. Fungal cells include yeast and
filamentous fungus cells including, for example, Pichia pastoris,
Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia
membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri),
Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia
guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica,
Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula
polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida
albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus
oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium
sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens
and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula
polymorpha, any Kluyveromyces sp., Candida albicans, any
Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any
Fusarium sp., Yarrowia lipolytica, and Neurospora crassa. When
recombinant expression vectors encoding the heavy chain or
antigen-binding portion or fragment thereof, and/or the light chain
or antigen-binding fragment thereof are introduced into mammalian
host cells, the antibodies are produced by culturing the host cells
for a period of time sufficient to allow for expression of the
antibody or fragment or chain in the host cells or secretion into
the culture medium in which the host cells are grown.
[0461] Antibodies and antigen-binding fragments thereof and
immunoglobulin chains can be recovered from the culture medium
using standard protein purification methods. Further, expression of
antibodies and antigen-binding fragments thereof and immunoglobulin
chains of the invention (or other moieties therefrom) from
production cell lines can be enhanced using a number of known
techniques. For example, the glutamine synthetase gene expression
system (the GS system) is a common approach for enhancing
expression under certain conditions. The GS system is discussed in
whole or part in connection with European Patent Nos. 0216846,
0256055, and 0323997 and 0338841. Thus, in an embodiment of the
invention, the mammalian host cells (e.g., CHO) lack a glutamine
synthetase gene and are grown in the absence of glutamine in the
medium wherein, however, the polynucleotide encoding the
immunoglobulin chain comprises a glutamine synthetase gene which
complements the lack of the gene in the host cell.
[0462] The present invention includes methods for purifying an
anti-SIRP.alpha. antibody or antigen-binding fragment thereof of
the present invention comprising introducing a sample comprising
the antibody or fragment to a purification medium (e.g., cation
exchange medium, anion exchange medium, hydrophobic exchange
medium, affinity purification medium (e.g., protein-A, protein-G,
protein-A/G, protein-L)) and either collecting purified antibody or
fragment from the flow-through fraction of said sample that does
not bind to the medium; or, discarding the flow-through fraction
and eluting bound antibody or fragment from the medium and
collecting the eluate. In an embodiment of the invention, the
medium is in a column to which the sample is applied. In an
embodiment of the invention, the purification method is conducted
following recombinant expression of the antibody or fragment in a
host cell, e.g., wherein the host cell is first lysed and,
optionally, the lysate is purified of insoluble materials prior to
purification on a medium.
[0463] In general, glycoproteins produced in a particular cell line
or transgenic animal will have a glycosylation pattern that is
characteristic for glycoproteins produced in the cell line or
transgenic animal. Therefore, the particular glycosylation pattern
of an antibody will depend on the particular cell line or
transgenic animal used to produce the antibody. However, all
antibodies encoded by the nucleic acid molecules provided herein,
or comprising the amino acid sequences provided herein, comprise
the instant invention, independent of the glycosylation pattern the
antibodies may have. Similarly, in particular embodiments,
antibodies with a glycosylation pattern comprising only
non-fucosylated N-glycans may be advantageous, because these
antibodies have been shown to typically exhibit more potent
efficacy than their fucosylated counterparts both in vitro and in
vivo (See for example, Shinkawa et al., J. Biol. Chem. 278:
3466-3473 (2003); U.S. Pat. Nos. 6,946,292 and 7,214,775). These
antibodies with non-fucosylated N-glycans are not likely to be
immunogenic because their carbohydrate structures are a normal
component of the population that exists in human serum IgG.
[0464] The present invention includes bispecific and bifunctional
antibodies and antigen-binding fragments having a binding
specificity for SIRP.alpha. and another antigen such as, for
example, CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44,
CD52, CD56, CD70, CD96, CD97, CD99, CD117, CD123, c-Met, CEA, EGFR,
EpCAM, HER2, HER3, PSMA, PTHR2, mesothelin, PD-1, PD-L1, TIM3, and
methods of use thereof. A bispecific or bifunctional antibody is an
artificial hybrid antibody having two different heavy/light chain
pairs and two different binding sites. Bispecific antibodies can be
produced by a variety of methods including fusion of hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai, et al., (1990)
Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) J
Immunol. 148:1547-1553. In addition, bispecific antibodies may be
formed as "diabodies" (Holliger, et al., (1993) PNAS USA
90:6444-6448) or as "Janusins" (Traunecker, et al., (1991) EMBO J.
10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl.
7:51-52). Included are "Duobodies," which are bispecific antibodies
with normal IgG structures (Labrijn et al., 2013, Proc. Natl. Acad.
Sci. USA 110 (13): 5145-5150).
[0465] The present invention further includes anti-SIRP.alpha.
antigen-binding fragments of the anti-SIRP.alpha. antibodies
disclosed herein. The antibody fragments include F(ab).sub.2
fragments, which may be produced by enzymatic cleavage of an IgG
by, for example, pepsin. Fab fragments may be produced by, for
example, reduction of F(ab).sub.2 with dithiothreitol or
mercaptoethylamine.
[0466] Immunoglobulins may be assigned to different classes
depending on the amino acid sequences of the constant domain of
their heavy chains. In some embodiments, different constant domains
may be appended to humanized V.sub.L and V.sub.H regions derived
from the CDRs provided herein. There are at least five major
classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several
of these may be further divided into subclasses (isotypes), e.g.
IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2. The invention comprises
antibodies and antigen-binding fragments of any of these classes or
subclasses of antibodies.
[0467] In one embodiment, the antibody or antigen-binding fragment
comprises a heavy chain constant region, e.g. a human constant
region, such as .gamma.1, .gamma.2, .gamma.3, or .gamma.4 human
heavy chain constant region or a variant thereof. In another
embodiment, the antibody or antigen-binding fragment comprises a
light chain constant region, e.g. a human light chain constant
region, such as lambda or kappa human light chain region or variant
thereof. By way of example, and not limitation the human heavy
chain constant region can be .gamma.4 and the human light chain
constant region can be kappa. In an alternative embodiment, the Fc
region of the antibody is .gamma.4 with a Ser228Pro mutation
(Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).
[0468] In one embodiment, the antibody or antigen-binding fragment
comprises a heavy chain constant region of the IgG1 subtype. In one
embodiment, the antibody or antigen-binding fragment comprises a
heavy chain constant region of the IgG2 subtype. In one embodiment,
the antibody or antigen-binding fragment comprises a heavy chain
constant region of the IgG4 subtype.
[0469] Antibody Engineering
[0470] Further included are embodiments in which the
anti-SIRP.alpha. antibodies and antigen-binding fragments thereof
are engineered antibodies to include modifications to framework
residues within the variable domains the antibody, e.g. to improve
the properties of the antibody or fragment. Typically, such
framework modifications are made to decrease the immunogenicity of
the antibody or fragment. This is usually accomplished by replacing
non-CDR residues in the variable domains (i.e. framework residues)
in a parental (e.g. rodent) antibody or fragment with analogous
residues from the immune repertoire of the species in which the
antibody is to be used, e.g. human residues in the case of human
therapeutics. Such an antibody or fragment is referred to as a
"humanized" antibody or fragment. In some cases, it is desirable to
increase the affinity, or alter the specificity of an engineered
(e.g. humanized) antibody. One approach is to mutate one or more
framework residues to the corresponding germline sequence. More
specifically, an antibody or fragment that has undergone somatic
mutation can contain framework residues that differ from the
germline sequence from which the antibody is derived. Such residues
can be identified by comparing the antibody or fragment framework
sequences to the germline sequences from which the antibody or
fragment is derived. Another approach is to revert to the original
parental (e.g., rodent) residue at one or more positions of the
engineered (e.g. humanized) antibody, e.g. to restore binding
affinity that may have been lost in the process of replacing the
framework residues. (See, e.g., U.S. Pat. Nos. 5,693,762, 5,585,089
and 5,530,101).
[0471] In certain embodiments, the anti-SIRP.alpha. antibodies and
antigen-binding fragments thereof are engineered (e.g. humanized)
to include modifications in the framework and/or CDRs to improve
their properties. Such engineered changes can be based on molecular
modelling. A molecular model for the variable region for the
parental (non-human) antibody sequence can be constructed to
understand the structural features of the antibody and used to
identify potential regions on the antibody that can interact with
the antigen. Conventional CDRs are based on alignment of
immunoglobulin sequences and identifying variable regions. Kabat et
al., (1991) Sequences of Proteins of Immunological Interest, Kabat,
et al.; National Institutes of Health, Bethesda, Md.; 5.sup.th ed.;
NIH Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75;
Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and
coworkers carefully examined conformations of the loops in crystal
structures of antibodies and proposed hypervariable loops. Chothia,
et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989)
Nature 342:878-883. There are variations between regions classified
as "CDRs" and "hypervariable loops". Later studies (Raghunathan et
al, (2012) J. Mol Recog. 25, 3, 103-113) analyzed several
antibody-antigen crystal complexes and observed that the antigen
binding regions in antibodies do not necessarily conform strictly
to the "CDR" residues or "hypervariable" loops. The molecular model
for the variable region of the non-human antibody can be used to
guide the selection of regions that can potentially bind to the
antigen. In practice the potential antigen binding regions based on
the model differ from the conventional "CDR"s or "hypervariable"
loops. Commercial scientific software such as Discovery Studio
(BIOVIA, Dassault Systems)) can be used for molecular modeling.
Human frameworks can be selected based on best matches with the
non-human sequence both in the frameworks and in the CDRs. For FR4
(framework 4) in VH, VJ regions for the human germlines are
compared with the corresponding non-human region. In the case of
FR4 (framework 4) in VL, J-kappa and J-Lambda regions of human
germline sequences are compared with the corresponding non-human
region. Once suitable human frameworks are identified, the CDRs are
grafted into the selected human frameworks. In some cases, certain
residues in the VL-VH interface can be retained as in the non-human
(parental) sequence. Molecular models can also be used for
identifying residues that can potentially alter the CDR
conformations and hence binding to antigen. In some cases, these
residues are retained as in the non-human (parental) sequence.
Molecular models can also be used to identify solvent exposed amino
acids that can result in unwanted effects such as glycosylation,
deamidation and oxidation. Developability filters can be introduced
early on in the design stage to eliminate/minimize these potential
problems.
[0472] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Pat. No. 7,125,689.
[0473] In particular embodiments, it will be desirable to change
certain amino acids containing exposed side-chains to another amino
acid residue in order to provide for greater chemical stability of
the final antibody, so as to avoid deamidation or isomerization.
The deamidation of asparagine may occur on NG, DG, NG, NS, NA, NT,
QG or QS sequences and result in the creation of an isoaspartic
acid residue that introduces a kink into the polypeptide chain and
decreases its stability (isoaspartic acid effect). Isomerization
can occur at DG, DS, DA or DT sequences. In certain embodiments,
the antibodies of the present disclosure do not contain deamidation
or asparagine isomerism sites.
[0474] For example, an asparagine (Asn) residue may be changed to
Gln or Ala to reduce the potential for formation of isoaspartate at
any Asn-Gly sequences, particularly within a CDR. A similar problem
may occur at a Asp-Gly sequence. Reissner and Aswad (2003) Cell.
Mol. Life Sci. 60:1281. Isoaspartate formation may debilitate or
completely abrogate binding of an antibody to its target antigen.
See, Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. In one
embodiment, the asparagine is changed to glutamine (Gln). It may
also be desirable to alter an amino acid adjacent to an asparagine
(Asn) or glutamine (Gln) residue to reduce the likelihood of
deamidation, which occurs at greater rates when small amino acids
occur adjacent to asparagine or glutamine. See, Bischoff &
Kolbe (1994) J. Chromatog. 662:261. In addition, any methionine
residues (typically solvent exposed Met) in CDRs may be changed to
Lys, Leu, Ala, or Phe or other amino acids in order to reduce the
possibility that the methionine sulfur would oxidize, which could
reduce antigen-binding affinity and also contribute to molecular
heterogeneity in the final antibody preparation. Id. Additionally,
in order to prevent or minimize potential scissile Asn-Pro peptide
bonds, it may be desirable to alter any Asn-Pro combinations found
in a CDR to Gln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such
substitutions are subsequently screened to ensure that the
substitutions do not decrease the affinity or specificity of the
antibody for SIRP.alpha., or other desired biological activity to
unacceptable levels.
TABLE-US-00002 TABLE 2 Exemplary stabilizing CDR variants CDR
Residue Stabilizing Variant Sequence Asn-Gly Gln-Gly, Ala-Gly, or
Asn-Ala (N-G) (Q-G), (A-G), or (N-A) Asp-Gly Glu-Gly, Ala-Gly or
Asp-Ala (D-G) (E-G), (A-G), or (D-A) Met Lys, Leu, Ala, or Phe (M)
(K), (L), (A), or (F) Asn Gln or Ala (N) (Q) or (A) Asn-Pro
Gin-Pro, Ala-Pro, or Asn-Ala (N-P) (Q-P), (A-P), or (N-A)
[0475] Another type of framework modification involves mutating one
or more residues within the framework regions to prevent
aggregation. The risk of an antibody to aggregate can be assessed
using the spatial aggregation propensity--See, Chennamsetty, N et
al (2010) J. Phys. Chem. 114, 6614-6624. The method requires the
calculation of the Solvent Accessible Area (SAA) for each atom. The
molecular aggregation score is then calculated as the sum of all
atomic scores. For a given radius and size of molecule, this is an
approximate indication of its overall tendency to aggregate.
Residues with a high aggregation score are replaced by residues
with a lower score (e.g. more hydrophilic amino acids).
Antibody Engineering of the Fc Region
[0476] The antibodies (e.g., humanized antibodies) and
antigen-binding fragments thereof disclosed herein can also be
engineered to include modifications within the Fc region, typically
to alter one or more properties of the antibody, such as serum
half-life, complement fixation, Fc receptor binding, and/or
effector function (e.g., antigen-dependent cellular cytotoxicity).
Furthermore, the antibodies and antigen-binding fragments thereof
disclosed herein can be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more properties
of the antibody or fragment. Each of these embodiments is described
in further detail below. The numbering of residues in the Fc region
is that of the EU index of Kabat.
[0477] The antibodies and antigen-binding fragments thereof
disclosed herein also include antibodies and fragments with
modified (or blocked) Fc regions to provide altered effector
functions. See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310;
WO2005/120571; WO2006/0057702. Such modifications can be used to
enhance or suppress various reactions of the immune system, with
possible beneficial effects in diagnosis and therapy. Alterations
of the Fc region include amino acid changes (substitutions,
deletions and insertions), glycosylation or deglycosylation, and
adding multiple Fc regions. Changes to the Fc can also alter the
half-life of antibodies in therapeutic antibodies, enabling less
frequent dosing and thus increased convenience and decreased use of
material. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at
734-35.
[0478] In one embodiment, the antibody or antigen-binding fragment
of the invention is an IgG4 isotype antibody or fragment comprising
a Serine to Proline mutation at a position corresponding to
position 228 (S228P; EU index; SEQ ID NO: 66) in the hinge region
of the heavy chain constant region. This mutation has been reported
to abolish the heterogeneity of inter-heavy chain disulfide bridges
in the hinge region (Angal et al (1993). Mol. Immunol. 30:105-108;
position 241 is based on the Kabat numbering system).
[0479] In one embodiment of the invention, the hinge region of CH1
is modified such that the number of cysteine residues in the hinge
region is increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425. The number of cysteine residues
in the hinge region of CH1 is altered, for example, to facilitate
assembly of the light and heavy chains or to increase or decrease
the stability of the antibody.
[0480] In another embodiment, the Fc hinge region of an antibody or
antigen-binding fragment of the invention is mutated to decrease
the biological half-life of the antibody or fragment. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody or fragment has impaired Staphylococcyl protein A
(SpA) binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No.
6,165,745.
[0481] In another embodiment, the antibody or antigen-binding
fragment of the invention is modified to increase its biological
half-life. Various approaches are possible. For example, one or
more of the following mutations can be introduced: T252L, T254S,
T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, to
increase the biological half-life, the antibody can be altered
within the CH1 or CL region to contain a salvage receptor binding
epitope taken from two loops of a CH2 domain of an Fc region of an
IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022.
[0482] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector function(s) of the antibody or
antigen-binding fragment. For example, one or more amino acids
selected from amino acid residues 234, 235, 236, 237, 297, 318, 320
and 322 can be replaced with a different amino acid residue such
that the antibody has an altered affinity for an effector ligand
and retains the antigen-binding ability of the parent antibody. The
effector ligand to which affinity is altered can be, for example,
an Fc receptor or the C1 component of complement. This approach is
described in further detail in U.S. Pat. Nos. 5,624,821 and
5,648,260.
[0483] In another example, one or more amino acids selected from
amino acid residues 329, 331 and 322 can be replaced with a
different amino acid residue such that the antibody has altered C1q
binding and/or reduced or abolished complement dependent
cytotoxicity (CDC). This approach is described in further detail in
U.S. Pat. No. 6,194,551.
[0484] In another example, one or more amino acid residues within
amino acid positions 231 and 239 are altered to thereby alter the
ability of the antibody to fix complement. This approach is
described further in PCT Publication WO 94/29351.
[0485] The proteins of the invention, which are preferably
antibodies and most preferably IgG antibodies or fragments thereof,
may have altered (e.g., relative to an unmodified antibody)
Fc.gamma.R binding properties (examples of binding properties
include but are not limited to, binding specificity, equilibrium
dissociation constant (K.sub.D), dissociation and association rates
(k.sub.off and k.sub.on respectively), binding affinity and/or
avidity) and that certain alterations are more or less desirable.
It is known in the art that the equilibrium dissociation constant
(K.sub.D) is defined as k.sub.off/k.sub.on, and K.sub.a is the
reciprocal of K.sub.D.
[0486] The affinities and binding properties of an Fc region for
its ligand, may be determined by a variety of in vitro assay
methods (biochemical or immunological based assays) known in the
art for determining Fc-Fc.gamma.R interactions, i.e., specific
binding of an Fc region to an Fc.gamma.R including but not limited
to, equilibrium methods (e.g., enzyme-linked immuno absorbent assay
(ELISA) or radioimmunoassay (RIA)), or kinetics (e.g. BIACORE.RTM.,
Octet.RTM., or KinExa.RTM. analysis), and other methods such as
indirect binding assays, competitive inhibition assays,
fluorescence resonance energy transfer (FRET), gel electrophoresis
and chromatography (e.g., gel filtration). These and other methods
may utilize a label on one or more of the components being examined
and/or employ a variety of detection methods including but not
limited to chromogenic, fluorescent, luminescent, or isotopic
labels.
[0487] In certain embodiments, the proteins of the present
invention bind to one or more human Fc.gamma.Rs selected from the
group consisting of Fc.gamma.RI, Fc.gamma.RIIB, Fc.gamma.RIIC,
Fc.gamma.RIIIA-F158, and Fc.gamma.RIIIA-V158 with an affinity at
least 10-fold, preferably at least 30-fold, and more preferably at
least 100-fold, less than equivalent protein having a wild-type
human IgG1 heavy chain constant domain (SEQ ID NO: 119) Fc region
or a wild-type human IgG4 heavy chain constant domain (SEQ ID NO:
66) Fc region.
[0488] In various embodiments, the proteins of the invention
comprise an immunoglobulin Fc region comprising an immunoglobulin
C2 region and an immunoglobulin C3 region and an immunoglobulin
hinge region. By way of example, the immunoglobulin Fc region may
be an IgG Fc region, an IgE Fc region, or an IgA Fc region. In
certain preferred embodiments, the protein comprises two
immunoglobulin Fc regions, each immunoglobulin Fc region comprising
an immunoglobulin C2 region and an immunoglobulin C3 region and an
immunoglobulin hinge region, wherein the hinge region of one of the
immunoglobulin Fc regions is bound to the hinge region of the other
immunoglobulin Fc region to form a dimeric Fc structure. Most
preferably, such a protein is a human or humanized IgG protein.
[0489] In certain embodiments, the proteins of the invention
comprise a mutated IgG4 Fc region, and preferably the protein is an
IgG comprising two mutated IgG4 Fc regions to form a dimeric Fc
structure. By way of example, a mutated IgG4 Fc region may comprise
one of the mutations, or mutational combinations, recited in Table
3. The numbering system of the constant region referred to in this
table is that of the EU index as set forth in Kabat et al. (1991,
NIH Publication 91-3242, National Technical Information Service,
Springfield, Va.). In the table, the first letter and number
represent the unmodified amino acid and its position and the second
letter represents the substituted amino acid at said position. For
those entries that include combinations of more than one mutation,
each mutation in the combination is separated by a "/".
TABLE-US-00003 TABLE 3 N297Q L235E N297Q/L235E F234A Q268A
F234A/L235A/G237A/ P238A F234A/L235A/.DELTA.G236/
F234A/L235A/G237A/ F234A/L235A/.DELTA.G236/ G237A/P238A P238A/Q268A
G237A/P238A/Q268A F234A/L235A L235E/P329G L235A/G237A/E318A
F234A/L235A/G237A/ F234A/L235A/.DELTA.G236/ F234A/L235A/G237A/
P238S G237A/P238S P238S/Q268A F234A/L235A/.DELTA.G236/
G237A/P238S/Q268A
[0490] In certain embodiments, the proteins of the invention
comprise a mutated IgG1 Fc region, and preferably the protein is an
IgG comprising two mutated IgG1 Fc regions to form a dimeric Fc
structure. By way of example, a mutated IgG1 Fc region may comprise
one of the mutations recited in Table 4. The numbering system of
the constant region referred to in this table is that of the EU
index as set forth in Kabat et al. (1991, NIH Publication 91-3242,
National Technical Information Service, Springfield, Va.). In the
table, the first letter and number represent the unmodified amino
acid and its position and the second letter represents the
substituted amino acid at said position.
TABLE-US-00004 TABLE 4 K222Y P232K A231K E233N E233Q E233R E233S
E233T E233H E233A E233V E233L E233F E233M E233Y E233W E233G L234D
L234E L234N L234Q L234T L234H L234F L234K L234R L234S L234A L234M
L234V L235E L235T L235F L235K L235R L235A L235M L235W L235N L235Q
L235H L235V G236A G236N G236R G236H G236L G236F G236P G237A G237E
G237N G237Q G237K G237R G237S G237T G237H G237L G237I G237F G237M
G237Y G237P P238K P238N P238R P238S P238T P238Y P238G P238A S239A
S239N S239F S239K S239R S239V S239W S239P S239H S239Y D249H V240A
F241W F241L F243W F243L F243E P244H P245A P247V P247G V253I V263I
V263T V263M V264D V264E V264K V264F V264M V264H V264W V264G V264Q
V264A V264L D265A D265E D265Q D265S D265H D265V D265L D265F D265M
D265Y D265N D265G V266T V266M V266A S267G S267H S267N S267P S267R
S267T S267F S267W E269A E269K E269S E269V E269F E269I E269M E269W
E269H E269T E269L E269N E269Y E269R E269P E269G D270A D270N D270E
D270Q D270T D270H D270R D270S D270L D270I D270F D270W D270P D270G
P271H P271Q P271K P271R P271S P271V P271F P271W D280L D280W D280P
E293F E294A E293Y E294K E294R E294S E294V E294L E294F Q295A Q295W
Q295P Q295G Y296E Y296Q Y296D Y296N Y296S Y296T Y296L Y296I Y296A
Y296V Y296M N297S N297D N297Q N297A S298T S298N S298K S298R T299A
T299H T299D T299E T299N T299Q T299K T299R T299I T299F T299M T299Y
T299W T299S T299V T299P T299G Y300E Y300K Y300R Y300S Y300P Y300W
V303A V303D W313F E318A E318V E318Q E318H E318L E318Y K320A K322A
K322E N325A N325V N325H N325K N325Y N325W N325P N325G N325Q N325D
N325E N325L N325I A327Q A327E A327N A327L A327I A327F A327W L328N
L328F L328H L328R L328T L328V L328I L328P L328M L328E L328A P329A
P329F P329D P329N P329Q P329K P329S P329T P329H P329V P329L P329M
P329Y P329W P329G P329R A330L A330R A330P A330T A330V A330F A330H
P331A P331S P331N P331E I332K I332N I332Q I332T I332H I332Y I332A
I332R E333N E333R I336E I336Y S337H
[0491] In certain embodiments, a mutated IgG1 Fc region may
comprise one of the mutational combinations recited in Table 5. The
numbering system of the constant region referred to in this table
is that of the EU index as set forth in Kabat et al. (1991, NIH
Publication 91-3242, National Technical Information Service,
Springfield, Va.). In the table, the first letter and number
represent the unmodified amino acid and its position and the second
letter represents the substituted amino acid at said position. For
each of the combinations of more than one mutation, each mutation
in the combination is separated by a "I" and deletions are
indicated by a
TABLE-US-00005 TABLE 5 C220S/C226S/C229S/P238S
C226S/C229S/E233P/L234V/ E233P/L234V/L235A L235A
E233P/L234V/L235A/.DELTA.G236 E233P/L234V/L235A/.DELTA.G236/
L234A/L235A A327G/A330S/P331S L235A/G237A L235A/G237A/E318S/K320S/
L235A/G237A/P331A K322S L234F/L235E L234F/L235E/D265A
L234F/L235E/D265A/ N297Q/P331S L234F/L235E/N297Q L234F/L235E/P329G
L234F/L235A/K322Q/ M252Y/S254T/T256E L234F/L235Q/K322Q/M252Y/
L234F/L235Q/P331G/M252Y/ G236R/L328R S254T/T256E S254T/T256E
S239D/D265I/N297D/I332E S239D/D265L/N297D/I332E
S239D/D265F/N297D/I332E S239D/D265Y/N297D/I332E
S239D/D265T/N297D/I332E S239D/N297D/A330Y/I332E
S239D/F241S/F243H/V262T/ V264E/N297D/I332E D265A/P331S
V264T/N297D/K326E/I332E D265A/N297Q N297D/D265Y/T299L/I332E
N297D/D265Y/I332E N297D/I332E/Y296D N297D/I332E N297D/I332E/Y296E
N297D/I332E/Y296N N297D/I332E/Y296Q N297D/I332E/Y296H
N297D/I332E/Y296T N297D/I332E/T299V N297D/I332E/T299I
N297D/I332E/T299L N297D/I332E/T299F N297D/I332E/T299H
N297D/I332E/T299E N297D/I332E/A330Y N297D/I332E/S298A/A330Y
N297E/D265F/I332E N297E/I332E F241E/F243R/V262E/V264R
F241E/F243Q/V262T/V264E F241L/F243L/V262I/V264I F241W/F243W
F241W/F243W/V262A/V264A F241L/V262I F243L/V262I/V264W
F241Y/F243Y/V262T/V264T F241E/F243R/V262E/V264R
F241E/F243Q/V262T/V264E F241R/F243Q/V262T/V264R
F241E/F243Y/V262T/V264R P244H/P245A/P247V F241E/F243R/V262E/V264R/
F241E/F243Y/V262T/V264R F241E/F243Y/V262T/V264R/ I332E I332E
S239E/D265G S239E/D265N S239E/D265Q M252Y/S254T/T256E S267Q/A327S
S267L/A327S N297S/I332E S239N/I332N S239N/I332Q S239Q/I332N
S239Q/I332Q S298N/Y300S S298N/T299A/Y300S N297Q/S298N/Y300S
E318S/K320S/K322S E318S/K320S/K322S/P311A L328E/I332E L328N/I332E
L234A/L235A/G237A/P238A/ L234A/L235A/G237A/P238S/
L234A/L235A/G237A/P238A/ H268A/A330S/P331S H268A/A330S/P331S
H268A/A330S/P331S L328Q/I332E L328H/I332E
[0492] In certain embodiments, the proteins of the invention
comprise a wild type or mutated IgG2 Fc region, and preferably the
protein is an IgG comprising two wild type or mutated IgG2 Fc
regions to form a dimeric Fc structure. A mutated IgG2 Fc region
may comprise one of the mutations, or mutational combinations,
recited in Table 6. The numbering system of the constant region
referred to in this table is that of the EU index as set forth in
Kabat et al. (1991, NIH Publication 91-3242, National Technical
Information Service, Springfield, Va.). In the table, the first
letter and number represent the unmodified amino acid and its
position and the second letter represents the substituted amino
acid at said position. For those entries that include combinations
of more than one mutation, each mutation in the combination is
separated by a "I".
TABLE-US-00006 TABLE 6 V234A G237A A235E/G237A V234A/A235E/G237A
V234A/G237A V234A/G237A/P238S H268Q/V309L/A330S/P331S
V234A/G237A/H268A/V309L/ V234A/G237A/H268Q/V309L/ A330S/P331S
A330S/P331S V234A/G237A/P238S/H268A/ P233S/V234A/G237A/P238S
P233S/V234A/G237A/H268A/ V309L/A330S/P331S V309L/A330S/P331S
P233S/V234A/G237A/H268Q/ P233S/V234A/G237A/P238S/ V309L/A330S/P331S
H268A/V309L/A330S/P331S
Production of Antibodies with Modified Glycosylation
[0493] In still another embodiment, the antibodies or
antigen-binding fragments of the invention comprise a particular
glycosylation pattern. For example, an afucosylated or an
aglycosylated antibody or fragment can be made (i.e., the antibody
lacks fucose or glycosylation, respectively). The glycosylation
pattern of an antibody or fragment may be altered to, for example,
increase the affinity or avidity of the antibody or fragment for a
SIRP.alpha. antigen. Such modifications can be accomplished by, for
example, altering one or more of the glycosylation sites within the
antibody or fragment sequence. For example, one or more amino acid
substitutions can be made that result in removal of one or more of
the variable region framework glycosylation sites to thereby
eliminate glycosylation at that site. Such deglycosylation may
increase the affinity or avidity of the antibody or fragment for
antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.
[0494] Antibodies and antigen-binding fragments disclosed herein
may further include those produced in lower eukaryote host cells,
in particular fungal host cells such as yeast and filamentous fungi
have been genetically engineered to produce glycoproteins that have
mammalian- or human-like glycosylation patterns (See for example,
Choi et al, (2003) Proc. Natl. Acad. Sci. 100: 5022-5027; Hamilton
et al., (2003) Science 301: 1244-1246; Hamilton et al., (2006)
Science 313: 1441-1443; Nett et al., Yeast 28(3):237-52 (2011);
Hamilton et al., Curr Opin Biotechnol. 18(5): 387-92 (2007)). A
particular advantage of these genetically modified host cells over
currently used mammalian cell lines is the ability to control the
glycosylation profile of glycoproteins that are produced in the
cells such that compositions of glycoproteins can be produced
wherein a particular N-glycan structure predominates (see, e.g.,
U.S. Pat. Nos. 7,029,872 and 7,449,308). These genetically modified
host cells have been used to produce antibodies that have
predominantly particular N-glycan structures (See for example, Li
et al., (2006) Nat. Biotechnol. 24: 210-215).
[0495] In particular embodiments, the antibodies and
antigen-binding fragments thereof disclosed herein further include
those produced in lower eukaryotic host cells and which comprise
fucosylated and non-fucosylated hybrid and complex N-glycans,
including bisected and multiantennary species, including but not
limited to N-glycans such as GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2;
Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2;
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(1-4)Man.sub.3GlcNAc.sub.2.
[0496] In particular embodiments, the antibodies and
antigen-binding fragments thereof provided herein may comprise
antibodies or fragments having at least one hybrid N-glycan
selected from the group consisting of GlcNAcMan.sub.5GlcNAc.sub.2;
GalGlcNAcMan.sub.5GlcNAc.sub.2; and
NANAGalGlcNAcMan.sub.5GlcNAc.sub.2. In particular aspects, the
hybrid N-glycan is the predominant N-glycan species in the
composition.
[0497] In particular embodiments, the antibodies and
antigen-binding fragments thereof provided herein comprise
antibodies and fragments having at least one complex N-glycan
selected from the group consisting of GlcNAcMan.sub.3GlcNAc.sub.2;
GalGlcNAcMan.sub.3GlcNAc.sub.2; NANAGalGlcNAcMan.sub.3GlcNAc.sub.2;
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2;
GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2;
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2;
NANAGal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; and
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. In particular
aspects, the complex N-glycan are the predominant N-glycan species
in the composition. In further aspects, the complex N-glycan is a
particular N-glycan species that comprises about 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex
N-glycans in the composition. In one embodiment, the antibody and
antigen binding fragments thereof provided herein comprise complex
N-glycans, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%,
99%, or 100% of the complex N-glycans comprise the structure
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2, wherein such
structure is afucosylated. Such structures can be produced, e.g.,
in engineered Pichia pastoris host cells.
[0498] In particular embodiments, the N-glycan is fucosylated. In
general, the fucose is in an .alpha.1,3-linkage with the GlcNAc at
the reducing end of the N-glycan, an .alpha.1,6-linkage with the
GlcNAc at the reducing end of the N-glycan, an .alpha.1,2-linkage
with the Gal at the non-reducing end of the N-glycan, an
.alpha.1,3-linkage with the GlcNac at the non-reducing end of the
N-glycan, or an .alpha.1,4-linkage with a GlcNAc at the
non-reducing end of the N-glycan.
[0499] Therefore, in particular aspects of the above the
glycoprotein compositions, the glycoform is in an
.alpha.1,3-linkage or .alpha.1,6-linkage fucose to produce a
glycoform selected from the group consisting of
Man.sub.5GlcNAc.sub.2(Fuc), GlcNAcMan.sub.5GlcNAc.sub.2(Fuc),
Man.sub.3GlcNAc.sub.2(Fuc), GlcNAcMan.sub.3GlcNAc.sub.2(Fuc),
GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc),
GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc),
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc),
NANAGal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc), and
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2(Fuc); in an
.alpha..sub.1,3-linkage or .alpha..sub.1,4-linkage fucose to
produce a glycoform selected from the group consisting of
GlcNAc(Fuc)Man.sub.5GlcNAc.sub.2, GlcNAc(Fuc)Man.sub.3GlcNAc.sub.2,
GlcNAc.sub.2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2,
GalGlcNAc.sub.2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2,
Gal.sub.2GlcNAc.sub.2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2,
NANAGal.sub.2GlcNAc.sub.2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2, and
NANA.sub.2Gal.sub.2GlcNAc.sub.2(Fuc.sub.1-2)Man.sub.3GlcNAc.sub.2;
or in an .alpha..sub.1,2-linkage fucose to produce a glycoform
selected from the group consisting of
Gal(Fuc)GlcNAc.sub.2Man.sub.3GlcNAc.sub.2,
Gal.sub.2(Fuc.sub.1-2)GlcNAc.sub.2Man.sub.3GlcNAc.sub.2,
NANAGal.sub.2(Fuc.sub.1-2)GlcNAc.sub.2Man.sub.3GlcNAc.sub.2, and
NANA.sub.2Gal.sub.2(Fuc.sub.1-2)GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.
[0500] In further aspects, the antibodies (e.g., humanized
antibodies) or antigen-binding fragments thereof comprise high
mannose N-glycans, including but not limited to,
Man.sub.8GlcNAc.sub.2, Man.sub.7GlcNAc.sub.2,
Man.sub.6GlcNAc.sub.2, Man.sub.5GlcNAc.sub.2,
Man.sub.4GlcNAc.sub.2, or N-glycans that consist of the
Man.sub.3GlcNAc.sub.2 N-glycan structure.
[0501] In further aspects of the above, the complex N-glycans
further include fucosylated and non-fucosylated bisected and
multiantennary species.
[0502] As used herein, the terms "N-glycan" and "glycoform" are
used interchangeably and refer to an N-linked oligosaccharide, for
example, one that is attached by an asparagine-N-acetylglucosamine
linkage to an asparagine residue of a polypeptide. N-linked
glycoproteins contain an N-acetylglucosamine residue linked to the
amide nitrogen of an asparagine residue in the protein. The
predominant sugars found on glycoproteins are glucose, galactose,
mannose, fucose, N-acetylgalactosamine (GalNAc),
N-acetylglucosamine (GlcNAc) and sialic acid (e.g.,
N-acetyl-neuraminic acid (NANA)). The processing of the sugar
groups occurs co-translationally in the lumen of the ER and
continues post-translationally in the Golgi apparatus for N-linked
glycoproteins.
[0503] N-glycans have a common pentasaccharide core of
Man.sub.3GlcNAc.sub.2 ("Man" refers to mannose; "Glc" refers to
glucose; and "NAc" refers to N-acetyl; GlcNAc refers to
N-acetylglucosamine). Usually, N-glycan structures are presented
with the non-reducing end to the left and the reducing end to the
right. The reducing end of the N-glycan is the end that is attached
to the Asn residue comprising the glycosylation site on the
protein. N-glycans differ with respect to the number of branches
(antennae) comprising peripheral sugars (e.g., GlcNAc, galactose,
fucose and sialic acid) that are added to the Man.sub.3GlcNAc.sub.2
("Man3") core structure which is also referred to as the
"trimannose core", the "pentasaccharide core" or the "paucimannose
core". N-glycans are classified according to their branched
constituents (e.g., high mannose, complex or hybrid). A "high
mannose" type N-glycan has five or more mannose residues. A
"complex" type N-glycan typically has at least one GlcNAc attached
to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6
mannose arm of a "trimannose" core. Complex N-glycans may also have
galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues that
are optionally modified with sialic acid or derivatives (e.g.,
"NANA" or "NeuAc", where "Neu" refers to neuraminic acid and "Ac"
refers to acetyl). Complex N-glycans may also have intrachain
substitutions comprising "bisecting" GlcNAc and core fucose
("Fuc"). Complex N-glycans may also have multiple antennae on the
"trimannose core," often referred to as "multiple antennary
glycans." A "hybrid" N-glycan has at least one GlcNAc on the
terminal of the 1,3 mannose arm of the trimannose core and zero or
more mannoses on the 1,6 mannose arm of the trimannose core. The
various N-glycans are also referred to as "glycoforms."
[0504] With respect to complex N-glycans, the terms "G-2", "G-1",
"GO", "G1", "G2", "A1", and "A2" mean the following. "G-2" refers
to an N-glycan structure that can be characterized as
Man.sub.3GlcNAc.sub.2; the term "G-1" refers to an N-glycan
structure that can be characterized as GlcNAcMan.sub.3GlcNAc.sub.2;
the term "GO" refers to an N-glycan structure that can be
characterized as GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "G1"
refers to an N-glycan structure that can be characterized as
GalGlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "G2" refers to an
N-glycan structure that can be characterized as
Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; the term "A1" refers to
an N-glycan structure that can be characterized as
NANAGal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; and, the term "A2"
refers to an N-glycan structure that can be characterized as
NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. Unless
otherwise indicated, the terms G-2'', "G-1", "GO", "G1", "G2",
"A1", and "A2" refer to N-glycan species that lack fucose attached
to the GlcNAc residue at the reducing end of the N-glycan. When the
term includes an "F", the "F" indicates that the N-glycan species
contains a fucose residue on the GlcNAc residue at the reducing end
of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2F all
indicate that the N-glycan further includes a fucose residue
attached to the GlcNAc residue at the reducing end of the N-glycan.
Lower eukaryotes such as yeast and filamentous fungi do not
normally produce N-glycans that produce fucose.
[0505] With respect to multiantennary N-glycans, the term
"multiantennary N-glycan" refers to N-glycans that further comprise
a GlcNAc residue on the mannose residue comprising the non-reducing
end of the 1,6 arm or the 1,3 arm of the N-glycan or a GlcNAc
residue on each of the mannose residues comprising the non-reducing
end of the 1,6 arm and the 1,3 arm of the N-glycan. Thus,
multiantennary N-glycans can be characterized by the formulas
GlcNAc.sub.(2-4)Man.sub.3GlcNAc.sub.2,
Gal.sub.(1-4)GlcNAc.sub.(2-4)Man.sub.3GlcNAc.sub.2, or
NANA.sub.(1-4)Gal.sub.(1-4)GlcNAc.sub.(2-4)Man.sub.3GlcNAc.sub.2.
The term "1-4" refers to 1, 2, 3, or 4 residues.
[0506] With respect to bisected N-glycans, the term "bisected
N-glycan" refers to N-glycans in which a GlcNAc residue is linked
to the mannose residue at the reducing end of the N-glycan. A
bisected N-glycan can be characterized by the formula
GlcNAc.sub.3Man.sub.3GlcNAc.sub.2 wherein each mannose residue is
linked at its non-reducing end to a GlcNAc residue. In contrast,
when a multiantennary N-glycan is characterized as
GlcNAc.sub.3Man.sub.3GlcNAc.sub.2, the formula indicates that two
GlcNAc residues are linked to the mannose residue at the
non-reducing end of one of the two arms of the N-glycans and one
GlcNAc residue is linked to the mannose residue at the non-reducing
end of the other arm of the N-glycan.
[0507] In certain embodiments, the proteins of the invention
comprise an aglycosylated Fc region. By way of example, an IgG1 Fc
region may be aglycosylayed by deleting or substituting residue
N297.
Antibody Physical Properties
[0508] The antibodies and antigen-binding fragments thereof
disclosed herein may further contain one or more glycosylation
sites in either the light or heavy chain immunoglobulin variable
region. Such glycosylation sites may result in increased
immunogenicity of the antibody or fragment or an alteration of the
pK of the antibody due to altered antigen-binding (Marshall et al.
(1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J
Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109;
Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature
316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).
Glycosylation has been known to occur at motifs containing an
N-X-S/T sequence.
[0509] Each antibody or antigen-binding fragment will have a unique
isoelectric point (pI), which generally falls in the pH range
between 6 and 9.5. The pI for an IgG1 antibody typically falls
within the pH range of 7-9.5 and the pI for an IgG4 antibody
typically falls within the pH range of 6-8.
[0510] Each antibody or antigen-binding fragment will have a
characteristic melting temperature, with a higher melting
temperature indicating greater overall stability in vivo
(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol
3:361-71). In general, the T.sub.M1 (the temperature of initial
unfolding) may be greater than 60.degree. C., greater than
65.degree. C., or greater than 70.degree. C. The melting point of
an antibody or fragment can be measured using differential scanning
calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et
al (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et
al. (2002) J. Chromatogr Sci 40:343-9).
[0511] In a further embodiment, antibodies and antigen-binding
fragments thereof are selected that do not degrade rapidly.
Degradation of an antibody or fragment can be measured using
capillary electrophoresis (CE) and MALDI-MS (Alexander A J and
Hughes D E (1995) Anal Chem 67:3626-32).
[0512] In a further embodiment, antibodies and antigen-binding
fragments thereof are selected that have minimal aggregation
effects, which can lead to the triggering of an unwanted immune
response and/or altered or unfavorable pharmacokinetic properties.
Generally, antibodies and fragments are acceptable with aggregation
of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or
less. Aggregation can be measured by several techniques, including
size-exclusion column (SEC), high performance liquid chromatography
(HPLC), and light scattering.
Antibody Conjugates
[0513] The anti-SIRP.alpha. antibodies and antigen-binding
fragments thereof disclosed herein may also be conjugated to a
chemical moiety. The chemical moiety may be, inter alia, a polymer,
a radionucleotide or a cytotoxic factor. In particular embodiments,
the chemical moiety is a polymer which increases the half-life of
the antibody or fragment in the body of a subject. Suitable
polymers include, but are not limited to, hydrophilic polymers
which include but are not limited to polyethylene glycol (PEG)
(e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa,
20 kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene
glycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981)
discloses PEG conjugated single-chain antibodies. Wen, et al.,
(2001) (Bioconj. Chem. 12:545-553) disclose conjugating antibodies
with PEG which is attached to a radiometal chelator
(diethylenetriaminpentaacetic acid (DTPA)).
[0514] The antibodies and antigen-binding fragments thereof
disclosed herein may also be conjugated with labels such as
.sup.99Tc, .sup.90Y, .sup.111In, .sup.32P, .sup.14C, .sup.125I,
.sup.3H, .sup.131I, .sup.11C, .sup.15O, .sup.13N, .sup.18F,
.sup.35S, .sup.51Cr, .sup.57To, .sup.226Ra, .sup.60Co, .sup.59Fe,
.sup.57Se, .sup.152Eu, .sup.67CU, .sup.217Ci, .sup.211At,
.sup.212Pb, .sup.47Sc, .sup.109Pd, .sup.234Th, and .sup.40K,
.sup.157Gd, .sup.55Mn, .sup.52Tr, and .sup.56Fe.
[0515] The antibodies and antigen-binding fragments disclosed
herein may also be PEGylated, for example to increase its
biological (e.g., serum) half-life. To PEGylate an antibody or
fragment, the antibody or fragment, typically is reacted with a
reactive form of polyethylene glycol (PEG), such as a reactive
ester or aldehyde derivative of PEG, under conditions in which one
or more PEG groups become attached to the antibody or antibody
fragment. In particular embodiments, the PEGylation is carried out
via an acylation reaction or an alkylation reaction with a reactive
PEG molecule (or an analogous reactive water-soluble polymer). As
used herein, the term "polyethylene glycol" is intended to
encompass any of the forms of PEG that have been used to derivatize
other proteins, such as mono (C1-C10) alkoxy- or
aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In
certain embodiments, the antibody or fragment to be PEGylated is an
aglycosylated antibody or fragment. Methods for PEGylating proteins
are known in the art and can be applied to the antibodies of the
invention. See, e.g., EP 0 154 316 and EP 0 401 384.
[0516] The antibodies and antigen-binding fragments disclosed
herein may also be conjugated with fluorescent or chemilluminescent
labels, including fluorophores such as rare earth chelates,
fluorescein and its derivatives, rhodamine and its derivatives,
isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin,
o-phthaladehyde, fluorescamine, .sup.152Eu, dansyl, umbelliferone,
luciferin, luminal label, isoluminal label, an aromatic acridinium
ester label, an imidazole label, an acridimium salt label, an
oxalate ester label, an aequorin label,
2,3-dihydrophthalazinediones, biotin/avidin, spin labels and stable
free radicals.
[0517] The antibodies and antigen-binding fragments thereof of the
invention may also be conjugated to a cytotoxic factor such as
diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins and compounds (e.g., fatty acids), dianthin
proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S,
Momordica charantia inhibitor, curcin, crotin, Saponaria
officinalis inhibitor, mitogellin, restrictocin, phenomycin, and
enomycin.
[0518] Any method known in the art for conjugating the antibodies
and antigen-binding fragments thereof of the invention to the
various moieties may be employed, including those methods described
by Hunter, et al., (1962) Nature 144:945; David, et al., (1974)
Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth.
40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407.
Methods for conjugating antibodies and fragments are conventional
and very well known in the art.
Therapeutic Uses of Anti-SIRP.alpha. Antibodies
[0519] Further provided are methods for treating subjects,
including human subjects, in need of treatment with the isolated
antibodies or antigen-binding fragments thereof disclosed herein.
In one embodiment of the invention, such subject suffers from an
infection or an infectious disease.
[0520] In another embodiment of the invention, such subject suffers
from cancer. In one embodiment the cancer is, e.g., osteosarcoma,
rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal
transitional cell cancer, bladder cancer, Wilm's cancer, ovarian
cancer, pancreatic cancer, breast cancer, prostate cancer, bone
cancer, lung cancer (e.g., non-small cell lung cancer), gastric
cancer, colorectal cancer, cervical cancer, synovial sarcoma, head
and neck cancer, squamous cell carcinoma, multiple myeloma, renal
cell cancer, retinoblastoma, hepatoblastoma, hepatocellular
carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma,
chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary
adenoma, vestibular schwannoma, a primitive neuroectodermal tumor,
medulloblastoma, astrocytoma, anaplastic astrocytoma,
oligodendroglioma, ependymoma, choroid plexus papilloma,
polycythemia vera, thrombocythemia, idiopathic myelfibrosis, soft
tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid
cancer or liver cancer, breast cancer or gastric cancer. In an
embodiment of the invention, the cancer is metastatic cancer, e.g.,
of the varieties described above.
[0521] Cancers that may be treated by the antibodies or
antigen-binding fragments, compositions and methods of the
invention include, but are not limited to: Cardiac: sarcoma
(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),
myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung:
bronchogenic carcinoma (squamous cell, undifferentiated small cell,
undifferentiated large cell, adenocarcinoma), alveolar
(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,
chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus
(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma,
lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas
(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma,
carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma,
carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma,
neurofibroma, fibroma), large bowel (adenocarcinoma, tubular
adenoma, villous adenoma, hamartoma, leiomyoma) colorectal;
Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor
[nephroblastoma], lymphoma, leukemia), bladder and urethra
(squamous cell carcinoma, transitional cell carcinoma,
adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis
(seminoma, teratoma, embryonal carcinoma, teratocarcinoma,
choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,
fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma
(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,
angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic
sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous
histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma
(reticulum cell sarcoma), multiple myeloma, malignant giant cell
tumor chordoma, osteochronfroma (osteocartilaginous exostoses),
benign chondroma, chondroblastoma, chondromyxofibroma, osteoid
osteoma and giant cell tumors; Nervous system: skull (osteoma,
hemangioma, granuloma, xanthoma, osteitis deformans), meninges
(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,
medulloblastoma, glioma, ependymoma, germinoma [pinealoma],
glioblastoma multiform, oligodendroglioma, schwannoma,
retinoblastoma, congenital tumors), spinal cord neurofibroma,
meningioma, glioma, sarcoma); Gynecological: uterus (endometrial
carcinoma), cervix (cervical carcinoma, pre tumor cervical
dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,
mucinous cystadenocarcinoma, unclassified carcinoma], granulosa
thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma,
malignant teratoma), vulva (squamous cell carcinoma,
intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),
vagina (clear cell carcinoma, squamous cell carcinoma, botryoid
sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma),
breast; Hematologic: blood (myeloid leukemia [acute and chronic],
acute lymphoblastic leukemia, chronic lymphocytic leukemia,
myeloproliferative diseases, multiple myeloma, myelodysplastic
syndrome), Hodgkin's disease, non Hodgkin's lymphoma [malignant
lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous
cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma,
angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands:
neuroblastoma. Thus, the term "cancerous cell" as provided herein,
includes a cell afflicted by any one of the above-identified
conditions.
[0522] In one embodiment, cancers that may be treated by the
antibodies or antigen-binding fragments thereof disclosed herein,
compositions and methods of the invention include, but are not
limited to: breast cancer, gastric cancer, esophageal cancer,
gastroesophageal junction carcinoma, colorectal cancer, head and
neck cancer, non-small cell lung cancer, osteosarcoma,
neuroblastoma, bladder cancer, cervical cancer, endometrial cancer,
ovarian cancer, lung cancer, squamous cell carcinoma, melanoma,
pancreatic cancer, prostate cancer, small cell lung cancer, kidney
cancer, renal cell carcinoma, thyroid cancer, glioblastoma
multiforme, fallopian tube cancer, peritoneal cancer, angiosarcoma,
hepatocellular carcinoma, choriocarcinoma, soft tissue sarcoma,
chronic lymphocytic leukemia, chronic myelocytic leukemia,
non-Hodgkin's lymphoma, B-cell non-hodgkin's lymphoma, diffuse
large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma,
myelodysplastic syndrome, acute myelocytic leukemia, T-cell
lymphoma, natural killer cell lymphoma, extranodal marginal zone
B-cell lymphoma, acute lymphocytic leukemia, multiple myeloma.
[0523] In one embodiment, the antibodies or antigen-binding
fragments thereof disclosed herein may be used for the treatment of
infections and infectious diseases. As used herein, the term
"infection" refers to any state in at least one cell of an organism
(i.e., a subject) is infected by an infectious agent (e.g., a
subject has an intracellular pathogen infection, e.g., a chronic
intracellular pathogen infection). As used herein, the term
"infectious agent" refers to a foreign biological entity (i.e. a
pathogen) that induces CD47 expression (e.g., increased CD47
expression) in at least one cell of the infected organism. For
example, infectious agents include, but are not limited to
bacteria, viruses, protozoans, and fungi.
[0524] Intracellular pathogens are of particular interest.
Infectious diseases are disorders caused by infectious agents. Some
infectious agents cause no recognizable symptoms or disease under
certain conditions, but have the potential to cause symptoms or
disease under changed conditions. The subject methods can be used
in the treatment of chronic pathogen infections, for example
including but not limited to viral infections, e.g. retrovirus,
lentivirus, hepadna virus, herpes viruses, pox viruses, human
papilloma viruses, etc.; intracellular bacterial infections, e.g.
Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella,
Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella,
Yersinia sp, Helicobacter pylori etc.; and intracellular protozoan
pathogens, e.g. Plasmodium sp, Trypanosoma sp., Giardia sp.,
Toxoplasma sp., Leishmania sp., etc.
[0525] In an embodiment, the invention provides methods for
treating subjects using an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention, wherein the
subject suffers from a viral infection. In one embodiment, the
viral infection is an infection with a virus selected from the
group consisting of human immunodeficiency virus (HIV), hepatitis
virus (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II,
and CMV, Epstein Barr virus), adenovirus, influenza virus,
flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus,
respiratory syncytial virus, mumps virus, rotavirus, measles virus,
rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue
virus, papillomavirus, molluscum virus, poliovirus, rabies virus,
JC virus or arboviral encephalitis virus.
[0526] In an embodiment, the invention provides methods for
treating subjects using an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention, wherein the
subject suffers from a bacterial infection. In one embodiment, the
bacterial infection is infection with a bacteria selected from the
group consisting of Chlamydia, rickettsial bacteria, mycobacteria,
staphylococci, streptococci, pneumonococci, meningococci and
gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella,
Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae,
Clostridium tetan, Clostridium botulinum, Bacillus anthricis,
Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis,
and Borriella.
[0527] In an embodiment, the invention provides methods for
treating subjects using an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention, wherein the
subject suffers from a fungal infection. In one embodiment, the
fungal infection is an infection with a fungus selected from the
group consisting of Candida (albicans, krusei, glabrata,
tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,
niger, etc.), Genus Mucorales (mucor, absidia, rhizopus),
Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides
brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
[0528] In an embodiment, the invention provides methods for
treating subjects using an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention, wherein the
subject suffers from a parasitic infection. In one embodiment, the
parasitic infection is infection with a parasite selected from the
group consisting of Entamoeba histolytica, Balantidium coli,
Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium,
Pneumocystis carinii, Plasmodium vivax, Babesia microti,
Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,
Toxoplasma gondii and Nippostrongylus brasiliensis.
[0529] A "subject" may be a mammal such as a human, dog, cat,
horse, cow, mouse, rat, monkey (e.g., cynomolgous monkey, e.g.,
Macaca fascicularis) or rabbit. In preferred embodiments of the
invention, the subject is a human subject.
[0530] The term "in association with" indicates that the components
administered in a method of the present invention (e.g., an
anti-SIRP.alpha. antibody (e.g., humanized antibody) or
antigen-binding fragment thereof along with an anti-cancer agent
can be formulated into a single composition for simultaneous
delivery or formulated separately into two or more compositions
(e.g., a kit). Each component can be administered to a subject at a
different time than when the other component is administered; for
example, each administration may be given non-simultaneously (e.g.,
separately or sequentially) at several intervals over a given
period of time. Moreover, the separate components may be
administered to a subject by the same or by a different route.
[0531] In particular embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein may be used alone, or in
association with other, further therapeutic agents and/or
therapeutic procedures, for treating or preventing any disease such
as cancer, e.g., as discussed herein, in a subject in need of such
treatment or prevention. Compositions, e.g., pharmaceutical
compositions comprising a pharmaceutically acceptable carrier,
comprising such antibodies and fragments in association with
further therapeutic agents are also part of the present
invention.
[0532] Therefore, the present invention provides a method of
treating cancer in a human subject, comprising administering to the
subject an effective amount of the antibody or antigen binding
fragment disclosed herein, optionally in association with a further
therapeutic agent or therapeutic procedure. The present invention
also provides a method of treating an infection or infectious
disease in a human subject, comprising administering to the subject
an effective amount of the antibody or antigen binding fragment
disclosed herein, optionally in association with a further
therapeutic agent or therapeutic procedure. The present invention
also provides a method of increasing the activity of an immune
cell, comprising administering to a subject in need thereof an
effective amount of an antibody or antigen binding fragment
disclosed herein. In one embodiment, the method is used for: the
treatment of cancer; the treatment of an infection or infectious
disease; or as a vaccine adjuvant.
[0533] In particular embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein may be used alone, or in
association with tumor vaccines. Examples of tumor vaccines include
but are not limited to vaccines for Human Papillomavirus (HPV)
infection caused cancer such as Gardasil.RTM., Gardisil9.RTM. and
Cervarix.RTM.; vaccines that prevent hepatitis B virus caused liver
cancer such as Engerix-B.RTM. and Recombivax HB.RTM.; oncolytic
virus therapy that triggers immune response such as Imlygic.RTM.;
DNA vaccines such as Synchotrope MA2M plasmid DNA vaccine and
ZYC101; mammaglobin-a DNA vaccine (see Clinical Cancer Res. 2014
20(23):5964-75); vector based vaccines such as PSA-TRICOM
(prostvac), PANVAC-VF, Listeria monocytogenes-based vaccines (see,
e.g., Therapeutic Advances in Vaccines, 2014, 2(5) 137-148),
Listeria-based vaccines (Listeria expressing one or more cancer
vaccines such as Listeria-mesothelin (e.g., CRS-207), ADXS-HPV,
Axalimogene Filolisbac, Listeria-HER2/Neu, Listeria-EGFRvIII),
Adeno-CEA; allogeneic vaccines such as GVAX, BLP-25
(anti-Ankara-mucin 1), Belagenpumatucel-L, TG4010, CIMAvax
epidermal growth factor vaccine, NY-ESO, GM.CD40L-CCL21; autologous
vaccines such as:Adeno-CD40L, BCG, INGN-225, Dendritic cell
vaccines such as Provenge(Sipuleucel-T), rF-CEA-MUC1-TRICOM
(panvac-DC); antigen vaccines such as MUC-1 (stimuvax), NY-ESO-1,
GP-100, MAGE-A3 (melanoma antigen encoding gene A3), INGN-225 (see
Pharmacology & Therapeutics 153 (2015) 1-9).
[0534] Eat-me signals could be elevated by cytotoxic therapies like
radiotherapy or chemotherapeutic agents including, but not limited
to anthracyclines (doxorubicin, epirubicin, daunorubicin,
idarubicin, mitoxantrone), oxaliplatin, bortezomib,
cyclophosphamide, bleomycin, vorinostat, paclitaxel,
5-fluorouracil, cytarabine, prednisolone, docetaxel, mitomycin C,
topotecan/camptothecin, etoposide, zoledronic acid, methotrexate,
ibrutinib, aflibercept, bevacizumab, toremifene, vinblastine,
vincristine, idelalisib, mercaptopurine, thalidomide, sorafenib.
Thus, in certain embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein may be used in association with
chemotherapeutic agents, in association with radiation therapy,
etc. In particular embodiments, the antibodies or antigen-binding
fragments thereof disclosed herein may be used alone, or in
association with targeted therapies. Examples of targeted therapies
include: hormone therapies, signal transduction inhibitors (e.g.,
EGFR inhibitors, such as cetuximab (Erbitux) and erlotinib
(Tarceva)); CD20 inhibitors (e.g., rituximab (Rituxan) and
ofatumumab (Arzerra)); CD38 inhibitors (e.g., daratumumab
(DARZALEX)); CD52 inhibitors (e.g., alemtuzumab (Campath)); HER2
inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab
(Perjeta)); BCR-ABL inhibitors (such as imatinib (Gleevec) and
dasatinib (Sprycel)); ALK inhibitors (such as crizotinib (Xalkori)
and ceritinib (Zykadia)); BRAF inhibitors (such as vemurafenib
(Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators
(e.g., decitabine (Dacogen) and Vorinostat (Zolinza)), apoptosis
inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)),
angiogenesis inhibitors (e.g., bevacizumab (Avastin) and
ramucirumab (Cyramza)), immunomodulatory imide drugs (e.g.,
thalidomide, lenalidomide, pomalidomide, and apremilast),
monoclonal antibodies attached to toxins (e.g., brentuximab vedotin
(Adcetris) and ado-trastuzumab emtansine (Kadcyla)).
[0535] The antibodies or antigen-binding fragments thereof
disclosed herein may preferably find use in association with
targeted therapies in which antibodies are employed to mediate
ADCC/ADCP. Functional bioassays are available to analyze the mode
of action of an antibody drug and to distinguish ADCP as a mode of
action from ADCC. By way of example, an antibody-dependent
cell-mediated cytotoxicity (ADCC) assay typically utilizes normal
human peripheral blood mononuclear cells (PBMCs) or effector cells
isolated thereof. Assay variation can be reduced by using selective
donor pools with defined Fc.gamma. receptor Ha
(Fc.gamma.RIIa/CD32a), IIIa (Fc.gamma.RIIIa/CD16a) or IIIb
(Fc.gamma.RIIIb/CD16b) gene copy number variation (CNV) or
genotypes such as Fc.gamma.RIIIa-158 V/V versus V/F or F/F,
Fc.gamma.RIIIa-131 H/H versus H/R or R/R, and the
Fc.gamma.RIIIb-NA1 and -NA2 polymorphic variants. Alternatively,
effector cells such as PBMCs, PBMC-derived natural killer (NK)
cells, granulocytes, monocytes, monocyte-derived macrophages, or
dendritic cells (DCs) can be replaced with a
Fc.gamma.RIIIa-expressing cell line (for example, engineered NK92).
Killing of the target cells can be assessed by measuring the
release of specific probes from pre-labelled target cells, using
.sup.51chromium (Cr.sup.51) or fluorescent dyes such as
calcein-acetoxymethyl (calcein-AM), carboxyfluorescein succinimidyl
ester (CFSE),
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF),
europium (Eu) or propidium iodide (PI), or by measuring the release
of cytosolic enzymes such as lactate dehydrogenase (LDH) or the
release of nucleoside triphosphate (ATP).
[0536] In contrast, antibody-dependent cellular phagocytosis (ADCP)
may be assessed by measuring the destruction of target cells via
granulocyte, monocyte, dendritic cell, or macrophage-mediated
phagocytosis. ADCP assays use PBMC-derived cells or myeloid cell
lines such as HL-60, THP-1, and U937 cells differentiated into
macrophages or granulocytes. Stimuli that are commonly used to
induce macrophage differentiation in monocytic cell lines include
phorbol-12-myristate-13-acetate (PMA), 1,25-dihydroxyvitamin D3
(VD3), and retinoic acid (RA). RA is also known to induce terminal
granulocytic differentiation of for example HL-60 cells.
Phagocytosis of the target cells can be assessed by monitoring
effector cells for the internalization of specific probes from
target cells pre-labelled with fluorescent dyes such as cell
proliferation dye eFluor450, CFSE, and pH-sensitive dyes including
pHrodo and CypHer5E. Phagocytosis is measured by an increase in
fluorescently labelled effector cells using flow cytometry or
fluorescence microscopy. "Reporter gene" assays are also available
to assess ADCP. In order to measure ADCP function in a reporter
gene assay, target cells are first incubated with a titration of an
antibody of interest. Once the antibody is bound to its cognate
target on the target cell surface, engineered Jurkat effector cells
are added. If ADCP pathway activation ensues, the Jurkat cells
produce a luciferase product by expression of the reporter gene
NFAT-RE-luc2. Luciferase activity is then measured following a 4-24
hour induction period, after addition of the luciferase assay
reagent. The dose-dependent response in the microtiter plate-based
assay can be used to quantify the relative biological activity of
the therapeutic antibody compared to the dose-response curve of a
suitable reference item.
[0537] In particular embodiments, the anti-SIRP.alpha. antibodies
or antigen-binding fragments thereof of the invention may be used
in combination with an anti-cancer therapeutic agent or
immunomodulatory drug such as an immunomodulatory receptor
inhibitor, e.g., an antibody or antigen-binding fragment thereof
that specifically binds to the receptor.
[0538] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with one or more of: [0539] an agonist (e.g., an
agonistic antibody or antigen-binding fragment thereof, or a
soluble fusion) of a TNF receptor protein, an Immunoglobulin-like
protein, a cytokine receptor, an integrin, a signaling lymphocytic
activation molecules (SLAM proteins), an activating NK cell
receptor, a Toll like receptor, OX40, CD2, CD7, CD27, CD28, CD30,
CD40, ICAM-1, LFA-1 (CD1 1a/CD18), 4-1BB (CD137), B7-H3, ICOS
(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80
(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R
beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4,
CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL,
ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, ITGB7,
NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244,
2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160
(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM
(SLAMF1, CD150, IPO-3), SLAM7, BLAME (SLAMF8), SELPLG (CD162),
LTBR, LAT, GADS, PAG/Cbp, CD19a, and a ligand that specifically
binds with CD83; or [0540] an inhibitor of CD47, PD-1, PD-L1,
PD-L2, CTLA4, TIM3, LAGS, CEACAM (e.g., CEACAM-1, -3 and/or -5),
VISTA, BTLA, TIGIT, LAIR1, IDO, TDO, CD160 and/or TGFR beta.
[0541] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with one or more cyclic dinculeotides or other STING
pathway agonists. STING (stimulator of interferon genes, also known
as TMEM173, MITA, ERIS, and MPYS) is a transmembrane protein
localized to the ER that undergoes a conformational change in
response to direct binding of cyclic dinucleotides (CDNs),
resulting in a downstream signaling cascade involving TBK1
activation, IRF-3 phosphorylation, and production of IFN-.beta. and
other cytokines. The STING pathway in tumor-resident host antigen
presenting c3ellss is involved in the induction of a spontaneous
CD8+ T cell response against tumor-derived antigens. Activation of
this pathway and the subsequent production of IFN-.beta. also
reportedly contributes to the anti-tumor effect of radiation. STING
agonists and their uses are described in, for example,
US20060040887, US20080286296, US20120041057, US20140205653,
WO2014179335, WO 2014179760, US20150056224, WO 2015185565, WO
2016096174, WO 2016145102, WO 2017011444, WO 2017027645, WO
2017027646, WO 2017123657, WO 2017123669, WO 2017175147, WO
2017175156, WO 2018045204, WO 2018009648, WO 2018006652, WO
2018013887, WO 2018013908, US20180002369, US20180092937, and
US20180093964.
[0542] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with one or more of: anti-CD47 antibody, anti-PD-1
antibody (e.g., nivolumab, pembrolizumab, anti-PDL1 antibody,
anti-TIGIT antibody, anti-APRIL antibody, anti-CTLA4 antibody,
anti-CS1 antibody (e.g., elotuzumab), anti-KIR2DL1/2/3 antibody
(e.g., lirilumab), anti-CD137 antibody (e.g., urelumab), anti-GITR
antibody (e.g., TRX518), anti-PD-L1 antibody (e.g., BMS-936559,
MSB0010718C or MPDL3280A), anti-PD-L2 antibody, anti-ILT1 antibody,
anti-ILT2 antibody, anti-ILT3 antibody, anti-ILT4 antibody,
anti-ILT5 antibody, anti-ILT6 antibody, anti-ILT7 antibody,
anti-ILT8 antibody, anti-CD40 antibody, anti-OX40 antibody,
anti-ICOS, anti-KIR2DL1 antibody, anti-KIR2DL2/3 antibody,
anti-KIR2DL4 antibody, anti-KIR2DL5A antibody, anti-KIR2DL5B
antibody, anti-KIR3DL1 antibody, anti-KIR3DL2 antibody,
anti-KIR3DL3 antibody, anti-NKG2A antibody, anti-NKG2C antibody,
anti-NKG2E antibody, anti-4-1BB antibody (e.g., PF-05082566),
anti-TSLP antibody, anti-IL-10 antibody, IL-10 or PEGylated IL-10,
or any small organic molecule inhibitor of such targets.
[0543] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CD20 antibody (e.g., rituximab,
ofatumumab, ocrelizumab, obinutuzumab, ocaratuzumab, ublituximab,
veltuzumab, ibritumomab tiuxetan, tositumomab, BVX-20, SCT-400 or
PRO131921).
[0544] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CD38 antibody (e.g., daratumumab,
isatuximab or MOR202).
[0545] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-EGFR antibody (e.g., cetuximab, CetuGEX,
panitumumab, nimotuzumab, depatuxizumab or AFM-21).
[0546] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-HER2 antibody (e.g., trastuzumab, TrasGEX,
pertuzumab, margetuximab or ADCT-502).
[0547] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-HER3 antibody (e.g., lumretuzumab,
patritumab or LJM716).
[0548] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CD19 antibody (e.g., inebilizumab,
blinatumomab, DI-B4, MDX-1342, MEDI-551, MOR208 or 4-G7SDIE).
[0549] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CD52 antibody (e.g., alemtuzumab).
[0550] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-EpCAM antibody (e.g., adecatumumab,
catumaxomab, edrecolomab or ING-1).
[0551] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-SLAMF7 antibody (e.g., elotuzumab or
ABBV-838).
[0552] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-PD-1 antibody (e.g., nivolumab or
pembrolizumab).
[0553] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-PD-L1 antibody (e.g., BMS-936559,
MSB0010718C or MPDL3280A).
[0554] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CTLA4 antibody (e.g., ipilimumab or
tremelimumab).
[0555] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CD137 antibody (e.g., urelumab).
[0556] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-GITR antibody (e.g., TRX518 or
FPA154).
[0557] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-OX40 antibody (e.g., MEDI6469, MOXR0916 or
INCAGN1949).
[0558] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CD40 antibody (e.g., lucatumumab,
dacetuzmumab, APX005M, ChiLob7/4, CP-870,893 or JNJ-64457107) In an
embodiment of the invention, an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention is in association
with an anti-CS1 antibody.
[0559] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR2DL1/2/3 antibody.
[0560] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CD137 (e.g., urelumab) antibody.
[0561] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-GITR (e.g., TRX518) antibody.
[0562] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-PD-L2 antibody.
[0563] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ITL1 antibody.
[0564] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ITL2 antibody.
[0565] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ITL3 antibody.
[0566] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ITL4 antibody.
[0567] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ITL5 antibody.
[0568] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ITL6 antibody.
[0569] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ITL7 antibody.
[0570] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ITL8 antibody.
[0571] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-CD40 antibody.
[0572] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-OX40 antibody.
[0573] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR2DL1 antibody.
[0574] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR2DL2/3 antibody.
[0575] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR2DL4 antibody.
[0576] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR2DL5A antibody.
[0577] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR2DL5B antibody.
[0578] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR3DL1 antibody.
[0579] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR3DL2 antibody.
[0580] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-KIR3DL3 antibody.
[0581] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-NKG2A antibody.
[0582] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-NKG2C antibody.
[0583] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-ICOS antibody.
[0584] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-4-1BB antibody.
[0585] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-IL-10 antibody.
[0586] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with an anti-TSLP antibody.
[0587] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with IL-10 or PEGylated IL-10.
[0588] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with one or more of an inhibitor (e.g., a small organic
molecule or an antibody or antigen-binding fragment thereof) such
as: an MTOR (mammalian target of rapamycin) inhibitor, a cytotoxic
agent, a platinum agent, an EGFR inhibitor, a VEGF inhibitor, a
microtubule stabilizer, a taxane, a CD20 inhibitor, a CD52
inhibitor, a CD30 inhibitor, a RANK (Receptor activator of nuclear
factor kappa-B) inhibitor, a RANKL (Receptor activator of nuclear
factor kappa-B ligand) inhibitor, an ERK inhibitor, a MAP Kinase
inhibitor, an AKT inhibitor, a MEK inhibitor, a PI3K inhibitor, a
HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4
inhibitor, a Bcl2 inhibitor, a CD22 inhibitor, a CD79b inhibitor,
an ErbB2 inhibitor, or a farnesyl protein transferase
inhibitor.
[0589] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with any one or more of: 13-cis-retinoic acid,
3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone,
4-hydroxytamoxifen, 5-deooxyuridine, 5'-deoxy-5-fluorouridine,
5-fluorouracil, 6-mecaptopurine, 7-hydroxystaurosporine, A-443654,
abirateroneacetate, abraxane, ABT-578, acolbifene, ADS-100380,
ALT-110, altretamine, amifostine, aminoglutethimide, amrubicin,
Amsacrine, anagrelide, anastrozole, angiostatin, AP-23573, ARQ-197,
arzoxifene, AS-252424, AS-605240, asparaginase, AT-9263,
atrasentan, axitinib, AZD1152, Bacillus Calmette-Guerin (BCG)
vaccine, batabulin, BC-210, besodutox, bevacizumab, bicalutamide,
Bio111, BIO140, bleomycin, BMS-214662, BMS-247550, BMS-275291,
BMS-310705, bortezomib, buserelin, busulfan, calcitriol,
camptothecin, canertinib, capecitabine, carboplatin, carmustine,
CC8490, Cediranib, CG-1521, CG-781, chlamydocin, chlorambucil,
chlorotoxin, cilengitide, cimitidine, cisplatin, cladribine,
clodronate, COL-3, CP-724714, cyclophosphamide, cyproterone,
cyproteroneacetate, cytarabine, cytosinearabinoside, dacarbazine,
dacinostat, dactinomycin, dalotuzumab, danusertib, dasatanib,
daunorubicin, decatanib, deguelin, denileukin, deoxycoformycin,
depsipeptide, diarylpropionitrile, diethylstilbestrol, diftitox,
docetaxel, dovitinib, doxorubicin, droloxifene, edotecarin,
yttrium-90 labeled-edotreotide, edotreotide, EKB-569, EMD121974,
endostatin, enzalutamide, enzastaurin, epirubicin, epithilone B,
ERA-923, Erbitux, erlotinib, estradiol, estramustine, etoposide,
everolimus, exemestane, ficlatuzumab, finasteride, flavopiridol,
floxuridine, fludarabine, fludrocortisone, fluoxymesterone,
flutamide, FOLFOX regimen, Fulvestrant, galeterone, gefitinib,
gemcitabine, gimatecan, goserelin, goserelin acetate, gossypol,
GSK461364, GSK690693, HMR-3339, hydroxyprogesteronecaproate,
hydroxyurea, IC87114, idarubicin, idoxyfene, ifosfamide, IM862,
imatinib, IMC-1C11, INCB24360, INO1001, interferon, interleukin-12,
ipilimumab, irinotecan, JNJ-16241199, ketoconazole, KRX-0402,
thalidomide, lenalidomide, pomalidomide, apremilast,lapatinib,
lasofoxifene, letrozole, leucovorin, leuprolide, leuprolide
acetate, levamisole, liposome entrapped paclitaxel, lomustine,
lonafarnib, lucanthone, LY292223, LY292696, LY293646, LY293684,
LY294002, LY317615, marimastat, mechlorethamine,
medroxyprogesteroneacetate, megestrolacetate, melphalan,
mercaptopurine, mesna, methotrexate, mithramycin, mitomycin,
mitotane, mitoxantrone, tozasertib, MLN8054, neovastat, Neratinib,
neuradiab, nilotinib, nilutimide, nolatrexed, NVP-BEZ235,
oblimersen, octreotide, ofatumumab, oregovomab, orteronel,
oxaliplatin, paclitaxel, palbociclib, pamidronate, panitumumab,
pazopanib, PD0325901, PD184352, PEG-interferon, pemetrexed,
pentostatin, perifosine, phenylalaninemustard, PI-103, pictilisib,
PIK-75, pipendoxifene, PKI-166, plicamycin, porfimer, prednisone,
procarbazine, progestins, PX-866, R-763, raloxifene, raltitrexed,
razoxin, ridaforolimus, rituximab, romidepsin, RTA744, rubitecan,
scriptaid, Sdx102, seliciclib, selumetinib, semaxanib, SF1126,
sirolimus, SN36093, sorafenib, spironolactone, squalamine, SR13668,
streptozocin, SU6668, suberoylanalide hydroxamic acid, sunitinib,
synthetic estrogen, talampanel, talimogene laherparepvec,
tamoxifen, temozolomide, temsirolimus, teniposide, tesmilifene,
testosterone, tetrandrine, TGX-221, thalidomide, thioguanine,
thiotepa, tremelimumab, tipifarnib, tivozanib, TKI-258, TLK286,
topotecan, toremifene citrate, trabectedin, trastuzumab, tretinoin,
trichostatin A, triciribinephosphate monohydrate, triptorelin
pamoate, TSE-424, uracil mustard, valproic acid, valrubicin,
vandetanib, vatalanib, VEGF trap, vinblastine, vincristine,
vindesine, vinorelbine, vitaxin, vitespan, vorinostat, VX-745,
wortmannin, Xr311, zanolimumab, ZK186619, ZK-304709, ZM336372,
ZSTK474.
[0590] Non-limiting examples of suitable anti-cancer agents to be
used in combination with an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention include
cytostatic agents, immune modulating imide drugs, cytotoxic agents,
targeted therapeutic agents (small molecules, biologics, siRNA and
microRNA) against cancer and neoplastic diseases, [0591] 1)
anti-metabolites (such as methotrexate, 5-fluorouracil,
gemcitabine, fludarabine, capecitabine); [0592] 2) alkylating
agents, such as temozolomide, cyclophosphamide, [0593] 3) DNA
interactive and DNA damaging agents, such as cisplatin,
oxaliplatin, doxorubicin, [0594] 4) Ionizing irradiation, such as
radiation therapy, [0595] 5) topoisomerase II inhibitors, such as
etoposide, doxorubicin, [0596] 6) topoisomerase I inhibitors, such
as irinotecan, topotecan, [0597] 7) tubulin interacting agents,
such as paclitaxel, docetaxel, Abraxane, epothilones, [0598] 8)
kinesin spindle protein inhibitors, [0599] 9) spindle checkpoint
inhibitors, [0600] 10) Poly(ADP-ribose) polymerase (PARP)
inhibitors, such as olaparib, MK-4827 and veliparib [0601] 11)
Matrix metalloprotease (MMP) inhibitors [0602] 12) Protease
inhibitors, such as cathepsin D and cathepsin K inhibitors [0603]
13) Proteosome or ubiquitination inhibitors, such as bortezomib,
[0604] 14) Activator of mutant p53 to restore its wild-type p53
activity [0605] 15) Adenoviral-p53 [0606] 16) Bcl-2 inhibitors,
such as ABT-263 [0607] 17) Heat shock protein (HSP) modulators,
such as geldanamycin and 17-AAG [0608] 18) Histone deacetylase
(HDAC) inhibitors, such as vorinostat (SAHA), [0609] 19) sex
hormone modulating agents, [0610] a. anti-estrogens, such as
tamoxifen, fulvestrant, [0611] b. selective estrogen receptor
modulators (SERM), such as raloxifene, [0612] c. anti-androgens,
such as bicalutamide, flutamide [0613] d. LHRH agonists, such as
leuprolide, [0614] e. 5.alpha.-reductase inhibitors, such as
finasteride, [0615] f. Cytochrome P450 C17 lyase (CYP450c17, also
called 17aC); [0616] g. aromatase inhibitors, such as letrozole,
anastrozole, exemestane, [0617] 20) EGFR kinase inhibitors, such as
geftinib, erlotinib, laptinib [0618] 21) dual erbB1 and erbB2
inhibitors, such as Lapatinib [0619] 22) multi-targeted kinases
(serine/threonine and/or tyrosine kinase) inhibitors, [0620] a. ABL
kinase inhibitors, imatinib and nilotinib, dasatinib [0621] b.
VEGFR-1, VEGFR-2, PDGFR, KDR, FLT, c-Kit, Tie2, Raf, MEK and ERK
inhibitors, such as sunitinib, sorafenib, Vandetanib, pazopanib,
PLX-4032, Axitinib, PTK787, GSK-1120212 [0622] c. Polo-like kinase
inhibitors [0623] d. Aurora kinase inhibitors [0624] e. JAK
inhibitor [0625] f. c-MET kinase inhibitors [0626] g.
Cyclin-dependent kinase inhibitors, such as CDK1 and CDK2 inhibitor
Dinaciclib SCH 727965 (see Parry et al, Molecular Cancer
Therapeutics 9 (8): 2344-53 (2010)) and CDK4/6 inhibitors, such as
Ribociclib, Palbociclib, Abemaciclib, and Trilaciclib. [0627] h.
PI3K and mTOR inhibitors, such as GDC-0941, BEZ-235, BKM-120 and
AZD-8055 [0628] i. Rapamycin and its analogs, such as Temsirolimus,
everolimus, and deforolimus [0629] 23) and other anti-cancer (also
know as anti-neoplastic) agents include but are not limited to
ara-C, adriamycin, cytoxan, Carboplatin, Uracil mustard,
Clormethine, Ifosfsmide, Melphalan, Chlorambucil, Pipobroman,
Triethylenemelamine, Triethylenethiophosphoramine, Busulfan,
Carmustine, Lomustine, Streptozocin, Dacarbazine, Floxuridine,
Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,
Pentostatine, Vinblastine, Vincristine, Vindesine, Vinorelbine,
Navelbine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin,
Epirubicin, teniposide, cytarabine, pemetrexed, Idarubicin,
Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase,
Teniposide, Ethinylestradiol, Diethylstilbestrol, Testosterone,
Prednisone, Fluoxymesterone, Dromostanolone propionate,
Testolactone, Megestrolacetate, Methylprednisolone,
Methyltestosterone, Prednisolone, Triamcinolone, Chlorotrianisene,
Hydroxyprogesterone, Aminoglutethimide, Estramustine, Flutamide
Medroxyprogesteroneacetate, Toremifene, goserelin, Carboplatin,
Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone,
Levamisole, Drolloxafine, Hexamethylmelamine, Bexxar, Zevalin,
Trisenox, Profimer, Thiotepa, Altretamine, Doxil, Ontak, Depocyt,
Aranesp, Neupogen, Neulasta, Kepivance. [0630] 24) Farnesyl protein
transferase inhibitors, such as, SARASAR.TM.
(4-[2-[4-[(11R)-3,10-dibromo-8-chloro-6,
11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-
-oxoethyl]-piperidinecarboxamide, tipifarnib [0631] 25)
interferons, such as Intron A, Peg-Intron, [0632] 26) anti-erbB1
antibodies, such as cetuximab, panitumumab, [0633] 27) anti-erbB2
antibodies, such as trastuzumab, [0634] 28) anti-CD52 antibodies,
such as Alemtuzumab, [0635] 29) anti-CD20 antibodies, such as
Rituximab [0636] 30) anti-CD33 antibodies, such as Gemtuzumab
ozogamicin [0637] 31) anti-VEGF antibodies, such as Avastin, [0638]
32) TRIAL ligands, such as Lexatumumab, mapatumumab, and AMG-655
[0639] 33) anti-CTLA-4 antibodies, such as ipilimumab [0640] 34)
antibodies against CTA1, CEA, CD5, CD19, CD22, CD30, CD44, CD44V6,
CD55, CD56, EpCAM, FAP, MHCII, HGF, IL-6, MUC1, PSMA, TAL6, TAG-72,
TRAILR, VEGFR, IGF-2, FGF, [0641] 35) anti-IGF-1R antibodies, such
as dalotuzumab (MK-0646) and robatumumab (SCH 717454).
[0642] "Estrogen receptor modulators" refers to compounds that
interfere with or inhibit the binding of estrogen to the receptor,
regardless of mechanism. Examples of estrogen receptor modulators
include, but are not limited to, tamoxifen, raloxifene, idoxifene,
LY353381, LY117081, toremifene, fulvestrant,
4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]ph-
enyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,
4,4'-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and
SH646.
[0643] "Androgen receptor modulators" refers to compounds which
interfere or inhibit the binding of androgens to the receptor,
regardless of mechanism. Examples of androgen receptor modulators
include finasteride and other 5.alpha.-reductase inhibitors,
nilutamide, flutamide, bicalutamide, liarozole, and abiraterone
acetate.
[0644] "Retinoid receptor modulators" refers to compounds which
interfere or inhibit the binding of retinoids to the receptor,
regardless of mechanism. Examples of such retinoid receptor
modulators include bexarotene, tretinoin, 13-cis-retinoic acid,
9-cis-retinoic acid, .alpha.-difluoromethylornithine, ILX23-7553,
trans-N-(4'-hydroxyphenyl) retinamide, and N-4-carboxyphenyl
retinamide.
[0645] "Cytotoxic/cytostatic agents" refer to compounds which cause
cell death or inhibit cell proliferation primarily by interfering
directly with the cell's functioning or inhibit or interfere with
cell myosis, including alkylating agents, tumor necrosis factors,
intercalators, hypoxia activatable compounds, microtubule
inhibitors/microtubule-stabilizing agents, inhibitors of mitotic
kinesins, histone deacetylase inhibitors, inhibitors of kinases
involved in mitotic progression, inhibitors of kinases involved in
growth factor and cytokine signal transduction pathways,
antimetabolites, biological response modifiers,
hormonal/anti-hormonal therapeutic agents, haematopoietic growth
factors, monoclonal antibody targeted therapeutic agents,
topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase
inhibitors, and aurora kinase inhibitors.
[0646] Examples of cytotoxic/cytostatic agents include, but are not
limited to, platinum coordinator compounds, sertenef, cachectin,
ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,
prednimustine, dibromodulcitol, ranimustine, fotemustine,
nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine,
improsulfan tosilate, trofosfamide, nimustine, dibrospidium
chloride, pumitepa, lobaplatin, satraplatin, profiromycin,
cisplatin, irofulven, dexifosfamide,
cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine,
glufosfamide, GPX100, (trans, trans,
trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(c-
hloro)platinum (Th]tetrachloride, diarizidinylspermine, arsenic
trioxide,
1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine,
zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone,
pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston,
3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin,
annamycin, galarubicin, elinafide, MEN10755,
4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin
(see WO 00/50032).
[0647] An example of a hypoxia activatable compound is
tirapazamine.
[0648] Examples of proteosome inhibitors include but are not
limited to lactacystin and MLN-341 (Velcade).
[0649] Examples of microtubule inhibitors/microtubule-stabilising
agents include taxanes in general. Specific compounds include
paclitaxel (Taxol.RTM.), vindesine sulfate,
3',4'-didehydro-4'-deoxy-8'-norvincaleukoblastine, docetaxol
(Taxotere.RTM.), rhizoxin, dolastatin, mivobulin isethionate,
auristatin, cemadotin, RPR109881, BMS184476, vinflunine,
cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)
benzene sulfonamide, anhydrovinblastine,
N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butyla-
mide, TDX258, the epothilones (see for example U.S. Pat. Nos.
6,284,781 and 6,288,237) and BMS188797.
[0650] Some examples of topoisomerase inhibitors are topotecan,
hycaptamine, irinotecan, rubitecan,
6-ethoxypropionyl-3',4'-O-exo-benzylidene-chartreusin,
9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)
propanamine,
1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]p-
yrano[3',4':b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione,
lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin,
BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate,
teniposide, sobuzoxane, 2'-dimethylamino-2'-deoxy-etoposide, GL331,
N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazo-
le-1-carboxamide, asulacrine, (5a, 5aB, 8aa,
9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3-
,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3',4':6,7)naphtho(2,3-d)-1-
,3-dioxol-6-one,
2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridiniu-
m, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,
5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-py-
razolo[4,5,1-de]acridin-6-one,
N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethy-
l]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,
6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]
quinolin-7-one, and dimesna.
[0651] Examples of inhibitors of mitotic kinesins, and in
particular the human mitotic kinesin KSP, are described in
Publications WO03/039460, WO03/050064, WO03/050122, WO03/049527,
WO03/049679, WO03/049678, WO04/039774, WO03/079973, WO03/099211,
WO03/105855, WO03/106417, WO04/037171, WO04/058148, WO04/058700,
WO04/126699, WO05/018638, WO05/019206, WO05/019205, WO05/018547,
WO05/017190, US2005/0176776. In an embodiment inhibitors of mitotic
kinesins include, but are not limited to inhibitors of KSP,
inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK and
inhibitors of Rab6-KIFL.
[0652] Examples of "histone deacetylase inhibitors" include, but
are not limited to, SAHA, TSA, oxamflatin, PXD101, MG98 and
scriptaid. Further reference to other histone deacetylase
inhibitors may be found in the following manuscript; Miller, T. A.
et al. J. Med. Chem. 46(24):5097-5116 (2003).
[0653] "Inhibitors of kinases involved in mitotic progression"
include, but are not limited to, inhibitors of aurora kinase,
inhibitors of Polo-like kinases (PLK; in particular inhibitors of
PLK-1), inhibitors of bub-1 and inhibitors of bub-R1. An example of
an "aurora kinase inhibitor" is VX-680.
[0654] "Antiproliferative agents" includes antisense RNA and DNA
oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and
INX3001, and antimetabolites such as enocitabine, carmofur,
tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine,
capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium
hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin,
decitabine, nolatrexed, pemetrexed, nelzarabine,
2'-deoxy-2'-methylidenecytidine,
2'-fluoromethylene-2'-deoxycytidine,
N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-(3,4-dichlorophenyl)urea,
N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L--
manno-heptopyranosyl]adenine, aplidine, ecteinascidin,
troxacitabine,
4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-
-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin,
5-flurouracil, alanosine,
11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetr-
acyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester,
swainsonine, lometrexol, dexrazoxane, methioninase,
2'-cyano-2'-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine,
3-aminopyridine-2-carboxaldehyde thiosemicarbazone and
trastuzumab.
[0655] Examples of monoclonal antibody targeted therapeutic agents
include those therapeutic agents which have cytotoxic agents or
radioisotopes attached to a cancer cell specific or target cell
specific monoclonal antibody. Examples include Bexxar.
[0656] "Prenyl-protein transferase inhibitor" refers to a compound
which inhibits any one or any combination of the prenyl-protein
transferase enzymes, including farnesyl-protein transferase
(FPTase), geranylgeranyl-protein transferase type I (GGPTase-I),
and geranylgeranyl-protein transferase type-II (GGPTase-II, also
called Rab GGPTase).
[0657] Examples of prenyl-protein transferase inhibitors can be
found in the following publications and patents: WO 96/30343, WO
97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO
98/29119, WO 95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430,
5,532,359, 5,510,510, 5,589,485, 5,602,098, European Patent Publ. 0
618 221, European Patent Publ. 0 675 112, European Patent Publ. 0
604 181, European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542,
WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No.
5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO
95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO
96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO
96/05168, WO 96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO
96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO
96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO
96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO
97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO
97/30053, WO 97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359.
For an example of the role of a prenyl-protein transferase
inhibitor on angiogenesis see European J. of Cancer, Vol. 35, No.
9, pp. 1394-1401 (1999).
[0658] "Angiogenesis inhibitors" refers to compounds that inhibit
the formation of new blood vessels, regardless of mechanism.
Examples of angiogenesis inhibitors include, but are not limited
to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine
kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors
of epidermal-derived, fibroblast-derived, or platelet derived
growth factors, MMP (matrix metalloprotease) inhibitors, integrin
blockers, interferon-a, interleukin-12, pentosan polysulfate,
cyclooxygenase inhibitors, including nonsteroidal
anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as
selective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib
(PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch.
Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68
(1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol.
313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996);
Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57,
p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med.,
Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),
steroidal anti-inflammatories (such as corticosteroids,
mineralocorticoids, dexamethasone, prednisone, prednisolone,
methylpred, betamethasone), carboxyamidotriazole, combretastatin
A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,
thalidomide, angiostatin, troponin-1, angiotensin II antagonists
(see Fernandez et al., J. Lab. Clin. Med. 105: 141-145 (1985)), and
antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968
(October 1999); Kim et al., Nature, 362, 841-844 (1993); WO
00/44777; and WO 00/61186).
[0659] Other examples of angiogenesis inhibitors include, but are
not limited to, endostatin, ukrain, ranpirnase, IM862,
5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct--
6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-1-[
[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carbo-
xamide,CM101, squalamine, combretastatin, RPI4610, NX31838,
sulfated mannopentaose phosphate,
7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-py-
rrole]-carbonylimino]-bis-(1,3-naphthalene disulfonate), and
3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).
[0660] Other therapeutic agents that modulate or inhibit
angiogenesis and may also be used in combination with the compounds
of the instant invention include agents that modulate or inhibit
the coagulation and fibrinolysis systems (see review in Clin. Chem.
La. Med. 38:679-692 (2000)). Examples of such agents that modulate
or inhibit the coagulation and fibrinolysis pathways include, but
are not limited to, heparin (see Thromb. Haemost. 80:10-23 (1998)),
low molecular weight heparins and carboxypeptidase U inhibitors
(also known as inhibitors of active thrombin activatable
fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354
(2001)). TAFIa inhibitors have been described in U.S. Ser. Nos.
60/310,927 (filed Aug. 8, 2001) and 60/349,925 (filed Jan. 18,
2002).
[0661] "Agents that interfere with cell cycle checkpoints" refer to
compounds that inhibit protein kinases that transduce cell cycle
checkpoint signals, thereby sensitizing the cancer cell to DNA
damaging agents. Such agents include inhibitors of ATR, ATM, the
CHK11 and CHK12 kinases and cdk and cdc kinase inhibitors and are
specifically exemplified by 7-hydroxystaurosporin, flavopiridol,
CYC202 (Cyclacel) and BMS-387032.
[0662] "Agents that interfere with receptor tyrosine kinases
(RTKs)" refer to compounds that inhibit RTKs and therefore
mechanisms involved in oncogenesis and tumor progression. Such
agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met.
Further agents include inhibitors of RTKs as described by
Bume-Jensen and Hunter, Nature, 411:355-365, 2001.
[0663] "Inhibitors of cell proliferation and survival signalling
pathway" refer to compounds that inhibit signal transduction
cascades downstream of cell surface receptors. Such agents include
inhibitors of serine/threonine kinases (including but not limited
to inhibitors of Akt such as described in WO 02/083064, WO
02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US
2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO
03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO
2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO
2005/100344, US 2005/029941, US 2005/44294, US 2005/43361,
60/734188, 60/652737, 60/670469), inhibitors of Raf kinase (for
example PLX-4032), inhibitors of MEK (for example Arry-162,
RO-4987655 and GSK-1120212), inhibitors of mTOR (for example
AZD-8055, BEZ-235 and everolimus), and inhibitors of PI3K (for
example GDC-0941, BKM-120).
[0664] As used above, "integrin blockers" refers to compounds which
selectively antagonize, inhibit or counteract binding of a
physiological ligand to the .alpha..sub.v.beta..sub.3integrin, to
compounds which selectively antagonize, inhibit or counteract
binding of a physiological ligand to the .alpha.v.beta.5 integrin,
to compounds which antagonize, inhibit or counteract binding of a
physiological ligand to both the .alpha..sub.v.beta..sub.3 integrin
and the .alpha..sub.v.beta..sub.5 integrin, and to compounds which
antagonize, inhibit or counteract the activity of the particular
integrin(s) expressed on capillary endothelial cells. The term also
refers to antagonists of the .alpha..sub.v.beta..sub.6,
.alpha..sub.v.beta..sub.8, .alpha..sub.1.beta..sub.1,
.alpha..sub.2.beta..sub.1, .alpha..sub.5.beta..sub.1,
.alpha..sub.6.beta..sub.1, and .alpha..sub.6.beta..sub.4 integrins.
The term also refers to antagonists of any combination of
.alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5,
.alpha..sub.v.beta..sub.8, .alpha..sub.1.beta..sub.1,
.alpha..sub.2.beta..sub.1, .alpha..sub.5.beta..sub.1,
.alpha..sub.6.beta..sub.1, and .alpha..sub.6.beta..sub.4
integrins.
[0665] Some specific examples of tyrosine kinase inhibitors include
N-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,
3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,
17-(allylamino)-17-demethoxygeldanamycin,
4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]q-
uinazoline,
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,
BIBX1382,
2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epox-
y-1H-diindolo[1,2,3-fg:3`',2`,1'-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one-
, SH268, genistein, STI571, CEP2563,
4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane
sulfonate,
4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,
4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668,
STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine,
and EMD121974.
[0666] Combinations of the instantly claimed antibodies or antigen
binding fragments with PPAR-.gamma. (i.e., PPAR-gamma) agonists and
PPAR-.delta. (i.e., PPAR-delta) agonists may be useful in the
treatment of certain malignancies. PPAR-.gamma. and PPAR-.delta.
are the nuclear peroxisome proliferator-activated receptors .gamma.
and .delta.. The expression of PPAR-.gamma. on endothelial cells
and its involvement in angiogenesis has been reported in the
literature (see J. Cardiovasc. Pharmacol. 1998; 31: 909-913; J.
Biol. Chem. 1999; 274: 9116-9121; Invest. Ophthalmol Vis. Sci.
2000; 41: 2309-2317). More recently, PPAR-.gamma. agonists have
been shown to inhibit the angiogenic response to VEGF in vitro;
both troglitazone and rosiglitazone maleate inhibit the development
of retinal neovascularization in mice. (Arch. Ophthamol. 2001; 119:
709-717). Examples of PPAR-.gamma. agonists and
PPAR-.gamma./.alpha. agonists include, but are not limited to,
Lynparza.RTM., Rucaparib.RTM., Talazoparib.RTM., niraparib,
Veliparib.RTM., thiazolidinediones (such as DRF2725, CS-011,
troglitazone, rosiglitazone, and pioglitazone), fenofibrate,
gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501,
MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622,
GI262570, PNU182716, DRF552926,
2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpro-
pionic acid, and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)
phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid.
[0667] The antibody or antigen binding fragment of the instant
invention may also be useful for treating or preventing breast
cancer in combination with aromatase inhibitors. Examples of
aromatase inhibitors include but are not limited to: anastrozole,
letrozole and exemestane.
[0668] The antibody or antigen binding fragment of the instant
invention may also be useful for treating cancer in combination
with the following chemotherapeutic agents: abarelix (Plenaxis
depot.RTM.); aldesleukin (Prokine.RTM.); Aldesleukin
(Proleukin.RTM.); Alemtuzumab (Campath.RTM.); alitretinoin
(Panretin.RTM.); allopurinol (Zyloprim.RTM.); altretamine
(Hexalen.RTM.); amifostine (Ethyol.RTM.); anastrozole
(Arimidex.RTM.); arsenic trioxide (Trisenox.RTM.); asparaginase
(Elspar.RTM.); azacitidine (Vidaza.RTM.); bendamustine
hydrochloride (Treanda.RTM.); bevacuzimab (Avastin.RTM.);
bexarotene capsules (Targretin.RTM.); bexarotene gel
(Targretin.RTM.); bleomycin (Blenoxane.RTM.); bortezomib
(Velcade.RTM.); brefeldin A; busulfan intravenous (Busulfex.RTM.);
busulfan oral (Myleran.RTM.); calusterone (Methosarb.RTM.);
capecitabine (Xeloda.RTM.); carboplatin (Paraplatin.RTM.);
carmustine (BCNU.RTM., BiCNU.RTM.); carmustine (Gliadel.RTM.);
carmustine with Polifeprosan 20 Implant (Gliadel Wafer.RTM.);
celecoxib (Celebrex.RTM.); cetuximab (Erbitux.RTM.); chlorambucil
(Leukeran.RTM.); cisplatin (Platinol.RTM.); cladribine
(Leustatin.RTM., 2-CdA.RTM.); clofarabine (Clolar.RTM.);
cyclophosphamide (Cytoxan.RTM., Neosar.RTM.); cyclophosphamide
(Cytoxan Injection.RTM.); cyclophosphamide (Cytoxan Tablet.RTM.);
cytarabine (Cytosar-U.RTM.); cytarabine liposomal (DepoCyt.RTM.);
dacarbazine (DTIC-Dome.RTM.); dactinomycin, actinomycin D
(Cosmegen.RTM.); dalteparin sodium injection (Fragmin.RTM.);
daratumumab (DARZALEX.RTM.); Darbepoetin alfa (Aranesp.RTM.);
dasatinib (Sprycel.RTM.); daunorubicin liposomal (DanuoXome.RTM.);
daunorubicin, daunomycin (Daunorubicin.RTM.); daunorubicin,
daunomycin (Cerubidine.RTM.); degarelix (Firmagon.RTM.); Denileukin
diftitox (Ontak.RTM.); dexrazoxane (Zinecard.RTM.); dexrazoxane
hydrochloride (Totect.RTM.); didemnin B; 17-DMAG; docetaxel
(Taxotere.RTM.); doxorubicin (Adriamycin PFS.RTM.); doxorubicin
(Adriamycin.RTM., Rubex.RTM.); doxorubicin (Adriamycin PFS
Injection.RTM.); doxorubicin liposomal (Doxil.RTM.); dromostanolone
propionate (Dromostanolone.RTM.); dromostanolone propionate
(Masterone Injection.RTM.); eculizumab injection (Soliris.RTM.);
Elliott's B Solution (Elliott's B Solution.RTM.); eltrombopag
(Promacta.RTM.); epirubicin (Ellence.RTM.); Epoetin alfa
(Epogen.RTM.); erlotinib (Tarceva.RTM.); estramustine (Emcyt.RTM.);
ethinyl estradiol; etoposide phosphate (Etopophos.RTM.); etoposide,
VP-16 (Vepesid.RTM.); everolimus tablets (Afinitor.RTM.);
exemestane (Aromasin.RTM.); ferumoxytol (Feraheme Injection.RTM.);
Filgrastim (Neupogen.RTM.); floxuridine (intraarterial)
(FUDR.RTM.); fludarabine (Fludara.RTM.); fluorouracil, 5-FU
(Adrucil.RTM.); fulvestrant (Faslodex.RTM.); gefitinib
(Iressa.RTM.); geldanamycin; gemcitabine (Gemzar.RTM.); gemtuzumab
ozogamicin (Mylotarg.RTM.); goserelin acetate (Zoladex
Implant.RTM.); goserelin acetate (Zoladex.RTM.); histrelin acetate
(Histrelin implant.RTM.); hydroxyurea (Hydrea.RTM.); Ibritumomab
Tiuxetan (Zevalin.RTM.); idarubicin (Idamycin.RTM.); ifosfamide
(IFEX.RTM.); imatinib mesylate (Gleevec.RTM.); interferon alfa 2a
(Roferon A.RTM.); Interferon alfa-2b (Intron A.RTM.); iobenguane I
123 injection (AdreView.RTM.); irinotecan (Camptosar.RTM.);
ixabepilone (Ixempra.RTM.); lapatinib tablets (Tykerb.RTM.);
lenalidomide (Revlimid.RTM.); letrozole (Femara.RTM.); leucovorin
(Wellcovorin.RTM., Leucovorin.RTM.); Leuprolide Acetate
(Eligard.RTM.); levamisole (Ergamisol.RTM.); lomustine, CCNU
(CeeBU.RTM.); meclorethamine, nitrogen mustard (Mustargen.RTM.);
megestrol acetate (Megace.RTM.); melphalan, L-PAM (Alkeran.RTM.);
mercaptopurine, 6-MP (Purinethol.RTM.); mesna (Mesnex.RTM.); mesna
(Mesnex tabs.RTM.); methotrexate (Methotrexate.RTM.); methoxsalen
(Uvadex.RTM.); 8-methoxypsoralen; mitomycin C (Mutamycin.RTM.);
mitotane (Lysodren.RTM.); mitoxantrone (Novantrone.RTM.);
mitramycin; nandrolone phenpropionate (Durabolin-50.RTM.);
nelarabine (Arranon.RTM.); nilotinib (Tasigna.RTM.); Nofetumomab
(Verluma.RTM.); ofatumumab (Arzerra.RTM.); Oprelvekin
(Neumega.RTM.); oxaliplatin (Eloxatin.RTM.); paclitaxel
(Paxene.RTM.); paclitaxel (Taxol.RTM.); paclitaxel protein-bound
particles (Abraxane.RTM.); palifermin (Kepivance.RTM.); pamidronate
(Aredia.RTM.); panitumumab (Vectibix.RTM.); pazopanib tablets
(Votrienttm.RTM.); pegademase (Adagen (Pegademase Bovine).RTM.);
pegaspargase (Oncaspar.RTM.); Pegfilgrastim (Neulasta.RTM.);
pemetrexed disodium (Alimta.RTM.); pentostatin (Nipent.RTM.);
pipobroman (Vercyte.RTM.); plerixafor (Mozobil.RTM.); plicamycin,
mithramycin (Mithracin.RTM.); porfimer sodium (Photofrin.RTM.);
pralatrexate injection (Folotyn.RTM.); procarbazine
(Matulane.RTM.); quinacrine (Atabrine.RTM.); rapamycin; Rasburicase
(Elitek.RTM.); raloxifene hydrochloride (Evista.RTM.); Rituximab
(Rituxan.RTM.); romidepsin (Istodax.RTM.); romiplostim
(Nplate.RTM.); sargramostim (Leukine.RTM.); Sargramostim
(Prokine.RTM.); sorafenib (Nexavar.RTM.); streptozocin
(Zanosar.RTM.); sunitinib maleate (Sutent.RTM.); talc
(Sclerosol.RTM.); tamoxifen (Nolvadex.RTM.); temozolomide
(Temodar.RTM.); temsirolimus (Torisel.RTM.); teniposide, VM-26
(Vumon.RTM.); testolactone (Teslac.RTM.); thioguanine, 6-TG
(Thioguanine.RTM.); thiopurine; thiotepa (Thioplex.RTM.); topotecan
(Hycamtin.RTM.); toremifene (Fareston.RTM.); Tositumomab
(Bexxar.RTM.); Tositumomab/I-131 tositumomab (Bexxar.RTM.);
trans-retinoic acid; Trastuzumab (Herceptin.RTM.); tretinoin, ATRA
(Vesanoid.RTM.); triethylenemelamine; Uracil Mustard (Uracil
Mustard Capsules.RTM.); valrubicin (Valstar.RTM.); vinblastine
(Velban.RTM.); vincristine (Oncovin.RTM.); vinorelbine
(Navelbine.RTM.); vorinostat (Zolinza.RTM.); wortmannin; and
zoledronate (Zometa.RTM.).
[0669] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is in
association with one or more antiemetics including, but not limited
to: casopitant (GlaxoSmithKline), Netupitant (MGI-Helsinn) and
other NK-1 receptor antagonists, palonosetron (sold as Aloxi by MGI
Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, N.J.),
diphenhydramine (sold as Benadryl.RTM. by Pfizer; New York, N.Y.),
hydroxyzine (sold as Atarax.RTM. by Pfizer; New York, N.Y.),
metoclopramide (sold as Reglan.RTM. by AH Robins Co,; Richmond,
Va.), lorazepam (sold as Ativan.RTM. by Wyeth; Madison, N.J.),
alprazolam (sold as Xanax.RTM. by Pfizer; New York, N.Y.),
haloperidol (sold as Haldol.RTM. by Ortho-McNeil; Raritan, N.J.),
droperidol (Inapsine.RTM.), dronabinol (sold as Marinol.RTM. by
Solvay Pharmaceuticals, Inc.; Marietta, Ga.), dexamethasone (sold
as Decadron.RTM. by Merck and Co.; Rahway, N.J.),
methylprednisolone (sold as Medrol.RTM. by Pfizer; New York, N.Y.),
prochlorperazine (sold as Compazine.RTM. by Glaxosmithkline;
Research Triangle Park, NC), granisetron (sold as Kytril.RTM. by
Hoffmann-La Roche Inc.; Nutley, N.J.), ondansetron (sold as
Zofran.RTM. by Glaxosmithkline; Research Triangle Park, NC),
dolasetron (sold as Anzemet.RTM. by Sanofi-Aventis; New York,
N.Y.), tropisetron (sold as Navoban.RTM. by Novartis; East Hanover,
N.J.).
[0670] Other side effects of cancer treatment include red and white
blood cell deficiency. Accordingly, in an embodiment of the
invention, an anti-SIRP.alpha. antibody or antigen-binding fragment
thereof is in association with an agent which treats or prevents
such a deficiency, such as, e.g., filgrastim, PEG-filgrastim,
erythropoietin, epoetin alfa or darbepoetin alfa.
[0671] In an embodiment of the invention, an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention is
administered in association with anti-cancer radiation therapy. For
example, in an embodiment of the invention, the radiation therapy
is external beam therapy (EBT): a method for delivering a beam of
high-energy X-rays to the location of the tumor. The beam is
generated outside the patient (e.g., by a linear accelerator) and
is targeted at the tumor site. These X-rays can destroy the cancer
cells and careful treatment planning allows the surrounding normal
tissues to be spared. No radioactive sources are placed inside the
patient's body. In an embodiment of the invention, the radiation
therapy is proton beam therapy: a type of conformal therapy that
bombards the diseased tissue with protons instead of X-rays. In an
embodiment of the invention, the radiation therapy is conformal
external beam radiation therapy: a procedure that uses advanced
technology to tailor the radiation therapy to an individual's body
structures. In an embodiment of the invention, the radiation
therapy is brachytherapy: the temporary placement of radioactive
materials within the body, usually employed to give an extra
dose--or boost--of radiation to an area.
[0672] In an embodiment of the invention, a surgical procedure is
administered in association with an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof is surgical tumorectomy.
Experimental and Diagnostic Uses
[0673] The anti-SIRP.alpha. antibodies and antigen-binding
fragments thereof disclosed herein may be used as affinity
purification agents. In this process, the anti-SIRP.alpha.
antibodies and antigen-binding fragments thereof are immobilized on
a solid phase such a Sephadex, glass or agarose resin or filter
paper, using methods well known in the art. The immobilized
antibody or fragment is contacted with a sample containing the
SIRP.alpha. protein (or a fragment thereof) to be purified, and
thereafter the support is washed with a suitable solvent that will
remove substantially all the material in the sample except the
SIRP.alpha. protein, which is bound to the immobilized antibody or
fragment. Finally, the support is washed with a solvent which
elutes the bound SIRP.alpha. (e.g., protein A). Such immobilized
antibodies and fragments form part of the present invention.
[0674] Further provided are antigens for generating secondary
antibodies which are useful for example for performing Western
blots and other immunoassays discussed herein.
[0675] Anti-SIRP.alpha. antibodies (e.g., humanized antibodies) and
antigen-binding fragments thereof may also be useful in diagnostic
assays for SIRP.alpha. protein, e.g., detecting its expression in
specific cells, tissues, or serum, e.g., myeloid cells such as
monocytes, macrophages, neutrophils, basophils, eosinophils, and
dendritic cells. Such diagnostic methods may be useful in various
disease diagnoses.
[0676] The present invention includes ELISA assays (enzyme-linked
immunosorbent assay) incorporating the use of an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof disclosed herein.
[0677] For example, such a method comprises the following
steps:
(a) coat a substrate (e.g., surface of a microtiter plate well,
e.g., a plastic plate) with anti-SIRP.alpha. antibody or
antigen-binding fragment thereof; (b) apply a sample to be tested
for the presence of SIRP.alpha. to the substrate; (c) wash the
plate, so that unbound material in the sample is removed; (d) apply
detectably labeled antibodies (e.g., enzyme-linked antibodies)
which are also specific to the SIRP.alpha. antigen; (e) wash the
substrate, so that the unbound, labeled antibodies are removed; (f)
if the labeled antibodies are enzyme linked, apply a chemical which
is converted by the enzyme into a fluorescent signal; and (g)
detect the presence of the labeled antibody.
[0678] Detection of the label associated with the substrate
indicates the presence of the SIRP.alpha. protein.
[0679] In a further embodiment, the labeled antibody or
antigen-binding fragment thereof is labeled with peroxidase which
react with ABTS (e.g.,
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) or
3,3',5,5'-Tetramethylbenzidine to produce a color change which is
detectable. Alternatively, the labeled antibody or fragment is
labeled with a detectable radioisotope (e.g., .sup.3H) which can be
detected by scintillation counter in the presence of a
scintillant.
[0680] An anti-SIRP.alpha. antibody or antigen-binding fragment
thereof of the invention may be used in a Western blot or
immune-protein blot procedure. Such a procedure forms part of the
present invention and includes e.g.: [0681] (1) optionally
transferring proteins from a sample to be tested for the presence
of SIRP.alpha. (e.g., from a PAGE or SDS-PAGE electrophoretic
separation of the proteins in the sample) onto a membrane or other
solid substrate using a method known in the art (e.g., semi-dry
blotting or tank blotting); contacting the membrane or other solid
substrate to be tested for the presence of bound SIRP.alpha. or a
fragment thereof with an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention. [0682] (2)
washing the membrane one or more times to remove unbound
anti-SIRP.alpha. antibody or fragment and other unbound substances;
and [0683] (3) detecting the bound anti-SIRP.alpha. antibody or
fragment.
[0684] Such a membrane may take the form of a nitrocellulose or
vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membrane to
which the proteins to be tested for the presence of SIRP.alpha. in
a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis) gel have been transferred (e.g., following
electrophoretic separation in the gel). Before contacting the
membrane with the anti-SIRP.alpha. antibody or fragment, the
membrane is optionally blocked, e.g., with non-fat dry milk or the
like so as to bind non-specific protein binding sites on the
membrane.
[0685] Detection of the bound antibody or fragment indicates that
the SIRP.alpha. protein is present on the membrane or substrate and
in the sample. Detection of the bound antibody or fragment may be
by binding the antibody or fragment with a secondary antibody (an
anti-immunoglobulin antibody) which is detectably labeled and,
then, detecting the presence of the secondary antibody.
[0686] The anti-SIRP.alpha. antibodies and antigen-binding
fragments thereof disclosed herein may also be used for
immunohistochemistry. Such a method forms part of the present
invention and comprises, e.g., [0687] (1) contacting a cell (e.g.,
a sample containing myeloid cells such as monocytes, macrophages,
neutrophils, basophils, eosinophils, and dendritic cells) to be
tested for the presence of SIRP.alpha. protein with an
anti-SIRP.alpha. antibody or antigen-binding fragment thereof of
the invention; and [0688] (2) detecting the antibody or fragment on
or in the cell.
[0689] If the antibody or fragment itself is detectably labeled, it
can be detected directly. Alternatively, the antibody or fragment
may be bound by a detectably labeled secondary antibody which is
detected.
[0690] Certain anti-SIRP.alpha. antibodies and antigen-binding
fragments thereof disclosed herein may also be used for in vivo
tumor imaging. Such a method may include injection of a
radiolabeled anti-SIRP.alpha. antibody or antigen-binding fragment
thereof into the body of a patient to be tested for the presence of
a tumor associated with SIRP.alpha. expression (e.g., which
expresses SIRP.alpha., for example, on the tumor cell surface)
followed by nuclear imaging of the body of the patient to detect
the presence of the labeled antibody or fragment e.g., at loci
comprising a high concentration of the antibody or fragment which
are bound to the tumor. The detection of the loci indicates the
presence of the SIRP.alpha..sup.+ tumor and tumor cells.
[0691] Imaging techniques include SPECT imaging (single photon
emission computed tomography) or PET imaging (positron emission
tomography). Labels include e.g., iodine-123 (.sup.123I) and
technetium-99m (.sup.99mTc), e.g., in conjunction with SPECT
imaging or .sup.11C, .sup.13N, .sup.15O or .sup.18F, e.g., in
conjunction with PET imaging or Indium-111 (See e.g., Gordon et
al., (2005) International Rev. Neurobiol. 67:385-440).
Pharmaceutical Compositions and Administration
[0692] To prepare pharmaceutical or sterile compositions of the
anti-SIRP.alpha. antibodies and antigen-binding fragments of the
invention, the antibody or antigen-binding fragment thereof is
admixed with a pharmaceutically acceptable carrier or excipient.
See, e.g., Remington's Pharmaceutical Sciences and U.S.
Pharmacopeia: National Formulary, Mack Publishing Company, Easton,
Pa. (1984).
[0693] Formulations of therapeutic and diagnostic agents may be
prepared by mixing with acceptable carriers, excipients, or
stabilizers in the form of, e.g., lyophilized powders, slurries,
aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001)
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science
and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New
York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, N.Y.; Lieberman, et al.
(eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker,
N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Disperse Systems, Marcel Dekker, N.Y.; Weiner and Kotkoskie (2000)
Excipient Toxicity and Safety, Marcel Dekker, Inc., New York,
N.Y.).
[0694] Toxicity and therapeutic efficacy of the antibodies of the
invention, administered alone or in combination with another
therapeutic agent, can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index (LD.sub.50/ED.sub.50).
The data obtained from these cell culture assays and animal studies
can be used in formulating a range of dosage for use in human. The
dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration.
[0695] In a further embodiment, a further therapeutic agent that is
administered to a subject in association with an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention in
accordance with the Physicians' Desk Reference 2003 (Thomson
Healthcare; 57th edition (Nov. 1, 2002)).
[0696] The mode of administration can vary. Routes of
administration include oral, rectal, transmucosal, intestinal,
parenteral; intramuscular, subcutaneous, intradermal,
intramedullary, intrathecal, direct intraventricular, intravenous,
intraperitoneal, intranasal, intraocular, inhalation, insufflation,
topical, cutaneous, transdermal, or intra-arterial.
[0697] In particular embodiments, the anti-SIRP.alpha. antibodies
or antigen-binding fragments thereof of the invention can be
administered by an invasive route such as by injection. In further
embodiments of the invention, an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof, or pharmaceutical composition
thereof, is administered intravenously, subcutaneously,
intramuscularly, intraarterially, intratumorally, or by inhalation,
aerosol delivery. Administration by non-invasive routes (e.g.,
orally; for example, in a pill, capsule or tablet) is also within
the scope of the present invention.
[0698] The present invention provides a vessel (e.g., a plastic or
glass vial, e.g., with a cap or a chromatography column, hollow
bore needle or a syringe cylinder) comprising any of the antibodies
or antigen-binding fragments of the invention or a pharmaceutical
composition thereof. The present invention also provides an
injection device comprising any of the antibodies or
antigen-binding fragments of the invention or a pharmaceutical
composition thereof. An injection device is a device that
introduces a substance into the body of a patient via a parenteral
route, e.g., intramuscular, subcutaneous or intravenous. For
example, an injection device may be a syringe (e.g., pre-filled
with the pharmaceutical composition, such as an auto-injector)
which, for example, includes a cylinder or barrel for holding fluid
to be injected (e.g., antibody or fragment or a pharmaceutical
composition thereof), a needle for piecing skin and/or blood
vessels for injection of the fluid; and a plunger for pushing the
fluid out of the cylinder and through the needle bore. In an
embodiment of the invention, an injection device that comprises an
antibody or antigen-binding fragment thereof of the present
invention or a pharmaceutical composition thereof is an intravenous
(IV) injection device. Such a device includes the antibody or
fragment or a pharmaceutical composition thereof in a cannula or
trocar/needle which may be attached to a tube which may be attached
to a bag or reservoir for holding fluid (e.g., saline; or lactated
ringer solution comprising NaCl, sodium lactate, KCl, CaCl.sub.2)
and optionally including glucose) introduced into the body of the
patient through the cannula or trocar/needle. The antibody or
fragment or a pharmaceutical composition thereof may, in an
embodiment of the invention, be introduced into the device once the
trocar and cannula are inserted into the vein of a subject and the
trocar is removed from the inserted cannula. The IV device may, for
example, be inserted into a peripheral vein (e.g., in the hand or
arm); the superior vena cava or inferior vena cava, or within the
right atrium of the heart (e.g., a central IV); or into a
subclavian, internal jugular, or a femoral vein and, for example,
advanced toward the heart until it reaches the superior vena cava
or right atrium (e.g., a central venous line). In an embodiment of
the invention, an injection device is an autoinjector; a jet
injector or an external infusion pump. A jet injector uses a
high-pressure narrow jet of liquid which penetrate the epidermis to
introduce the antibody or fragment or a pharmaceutical composition
thereof to a patient's body. External infusion pumps are medical
devices that deliver the antibody or fragment or a pharmaceutical
composition thereof into a patient's body in controlled amounts.
External infusion pumps may be powered electrically or
mechanically. Different pumps operate in different ways, for
example, a syringe pump holds fluid in the reservoir of a syringe,
and a moveable piston controls fluid delivery, an elastomeric pump
holds fluid in a stretchable balloon reservoir, and pressure from
the elastic walls of the balloon drives fluid delivery. In a
peristaltic pump, a set of rollers pinches down on a length of
flexible tubing, pushing fluid forward. In a multi-channel pump,
fluids can be delivered from multiple reservoirs at multiple
rates.
[0699] The pharmaceutical compositions disclosed herein may also be
administered with a needleless hypodermic injection device; such as
the devices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002;
5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or
4,596,556. Such needleless devices comprising the pharmaceutical
composition are also part of the present invention. The
pharmaceutical compositions disclosed herein may also be
administered by infusion. Examples of well-known implants and
modules for administering the pharmaceutical compositions include
those disclosed in: U.S. Pat. No. 4,487,603, which discloses an
implantable micro-infusion pump for dispensing medication at a
controlled rate; U.S. Pat. No. 4,447,233, which discloses a
medication infusion pump for delivering medication at a precise
infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable
flow implantable infusion apparatus for continuous drug delivery;
U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery
system having multi-chamber compartments. Many other such implants,
delivery systems, and modules are well known to those skilled in
the art and those comprising the pharmaceutical compositions of the
present invention are within the scope of the present
invention.
[0700] Alternately, one may administer the anti-SIRP.alpha.
antibody or antigen-binding fragment of the invention in a local
rather than systemic manner, for example, via injection of the
antibody or fragment directly into a tumor. Furthermore, one may
administer the antibody or fragment in a targeted drug delivery
system, for example, in a liposome coated with a tissue-specific
antibody, targeting, for example, a tumor. The liposomes will be
targeted to and taken up selectively by the afflicted tissue. Such
methods and liposomes are part of the present invention.
[0701] The administration regimen depends on several factors,
including the serum or tissue turnover rate of the therapeutic
antibody or antigen-binding fragment, the level of symptoms, the
immunogenicity of the therapeutic antibody, and the accessibility
of the target cells in the biological matrix. Preferably, the
administration regimen delivers sufficient therapeutic antibody or
fragment to effect improvement in the target disease state, while
simultaneously minimizing undesired side effects. Accordingly, the
amount of biologic delivered depends in part on the particular
therapeutic antibody and the severity of the condition being
treated. Guidance in selecting appropriate doses of therapeutic
antibodies or fragments is available (see, e.g., Wawrzynczak (1996)
Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;
Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and
Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993)
Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases,
Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New Engl. J.
Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.
341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;
Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et
al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New
Engl. J. Med. 343:1594-1602).
[0702] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment. Generally, the dose begins with an
amount somewhat less than the optimum dose and it is increased by
small increments thereafter until the desired or optimum effect is
achieved relative to any negative side effects. Important
diagnostic measures include those of symptoms of, e.g., the
inflammation or level of inflammatory cytokines produced. In
general, it is desirable that a biologic that will be used is
derived from the same species as the animal targeted for treatment,
thereby minimizing any immune response to the reagent. In the case
of human subjects, for example, humanized and fully human
antibodies may be desirable.
[0703] Antibodies or antigen-binding fragments thereof disclosed
herein may be provided by continuous infusion, or by doses
administered, e.g., daily, 1-7 times per week, weekly, bi-weekly,
monthly, bimonthly, quarterly, semiannually, annually etc. Doses
may be provided, e.g., intravenously, subcutaneously, topically,
orally, nasally, rectally, intramuscular, intracerebrally,
intraspinally, or by inhalation. A total weekly dose is generally
at least 0.05 .mu.g/kg body weight, more generally at least 0.2
.mu.g/kg, 0.5 .mu.g/kg, 1 .mu.g/kg, 10 .mu.g/kg, 100 .mu.g/kg, 0.25
mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/mL, 10 mg/kg, 25 mg/kg, 50
mg/kg or more (see, e.g., Yang, et al. (2003) New Engl. J. Med.
349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;
Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67: 451-456;
Portielji, et al. (20003) Cancer Immunol. Immunother. 52: 151-144).
Doses may also be provided to achieve a pre-determined target
concentration of anti-SIRP.alpha. antibody in the subject's serum,
such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 .mu.g/mL or more. In other
embodiments, An anti-SIRP.alpha. antibody of the present invention
is administered, e.g., subcutaneously or intravenously, on a
weekly, biweekly, "every 4 weeks," monthly, bimonthly, or quarterly
basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500
mg/subject.
[0704] As used herein, the term "effective amount" refer to an
amount of an anti-SIRP.alpha. or antigen-binding fragment thereof
of the invention that, when administered alone or in combination
with an additional therapeutic agent to a cell, tissue, or subject,
is effective to cause a measurable improvement in one or more
symptoms of disease, for example cancer or the progression of
cancer. An effective dose further refers to that amount of the
antibody or fragment sufficient to result in at least partial
amelioration of symptoms, e.g., tumor shrinkage or elimination,
lack of tumor growth, increased survival time. When applied to an
individual active ingredient administered alone, an effective dose
refers to that ingredient alone. When applied to a combination, an
effective dose refers to combined amounts of the active ingredients
that result in the therapeutic effect, whether administered in
combination, serially or simultaneously. An effective amount of a
therapeutic will result in an improvement of a diagnostic measure
or parameter by at least 10%; usually by at least 20%; preferably
at least about 30%; more preferably at least 40%, and most
preferably by at least 50%. An effective amount can also result in
an improvement in a subjective measure in cases where subjective
measures are used to assess disease severity.
Kits
[0705] Further provided are kits comprising one or more components
that include, but are not limited to, an anti-SIRP.alpha. antibody
or antigen-binding fragment, as discussed herein in association
with one or more additional components including, but not limited
to a pharmaceutically acceptable carrier and/or a therapeutic
agent, as discussed herein. The antibody or fragment and/or the
therapeutic agent can be formulated as a pure composition or in
combination with a pharmaceutically acceptable carrier, in a
pharmaceutical composition.
[0706] In one embodiment, the kit includes an anti-SIRP.alpha.
antibody or antigen-binding fragment thereof of the invention or a
pharmaceutical composition thereof in one container (e.g., in a
sterile glass or plastic vial) and/or a therapeutic agent and a
pharmaceutical composition thereof in another container (e.g., in a
sterile glass or plastic vial).
[0707] In another embodiment, the kit comprises a combination of
the invention, including an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention along with a
pharmaceutically acceptable carrier, optionally in combination with
one or more therapeutic agents formulated together, optionally, in
a pharmaceutical composition, in a single, common container.
[0708] If the kit includes a pharmaceutical composition for
parenteral administration to a subject, the kit can include a
device for performing such administration. For example, the kit can
include one or more hypodermic needles or other injection devices
as discussed above.
[0709] The kit can include a package insert including information
concerning the pharmaceutical compositions and dosage forms in the
kit. Generally, such information aids patients and physicians in
using the enclosed pharmaceutical compositions and dosage forms
effectively and safely. For example, the following information
regarding a combination of the invention may be supplied in the
insert: pharmacokinetics, pharmacodynamics, clinical studies,
efficacy parameters, indications and usage, contraindications,
warnings, precautions, adverse reactions, overdosage, proper dosage
and administration, how supplied, proper storage conditions,
references, manufacturer/distributor information and patent
information.
[0710] The kit can also comprise a second therapeutic, for example
one or more of: anti-CD47 antibody, anti-APRIL antibody, anti-PD-1
antibody (e.g., nivolumab, pembrolizumab, anti-PDL1 antibody,
anti-TIGIT antibody, anti-CTLA4 antibody, anti-CS1 antibody (e.g.,
elotuzumab), anti-KIR2DL1/2/3 antibody (e.g., lirilumab),
anti-CD137 antibody (e.g., urelumab), anti-GITR antibody (e.g.,
TRX518), anti-PD-L1 antibody (e.g., BMS-936559, MSB0010718C or
MPDL3280A), anti-PD-L2 antibody, anti-ILT1 antibody, anti-ILT2
antibody, anti-ILT3 antibody, anti-ILT4 antibody, anti-ILT5
antibody, anti-ILT6 antibody, anti-ILT7 antibody, anti-ILT8
antibody, anti-CD40 antibody, anti-OX40 antibody, anti-ICOS,
anti-KIR2DL1 antibody, anti-KIR2DL2/3 antibody, anti-KIR2DL4
antibody, anti-KIR2DL5A antibody, anti-KIR2DL5B antibody,
anti-KIR3DL1 antibody, anti-KIR3DL2 antibody, anti-KIR3DL3
antibody, anti-NKG2A antibody, anti-NKG2C antibody, anti-NKG2E
antibody, anti-4-1BB antibody (e.g., PF-05082566), anti-TSLP
antibody, anti-IL-10 antibody, IL-10 or PEGylated IL-10, or any
small organic molecule inhibitor of such targets; an antibody or
antigen binding fragment thereof binds to an antigen selected from
the group consisting of AMHR2, AXL, BCMA, CA IX, CD4, CD16, CD19,
CD20, CD22, CD30, CD37, CD38, CD40, CD52, CD98, CSF1R, GD2, CCR4,
CS1, EpCam, EGFR, EGFRvIII, Endoglin, EPHA2, EphA3, FGFR2b, folate
receptor alpha, fucosyl-GM1, HER2, HER3, IL1RAP, kappa myeloma
antigen, MS4A1, prolactin receptor, TA-MUC1, and PSMA; Rituximab,
ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab,
Obinutuzumab, ADCT-502, Hul4.18K322A, Hu3F8, Dinituximab,
Trastuzumab, Cetuximab, Rituximab-RLI, c.60C3-RLI, Hul4.18-IL2,
KM2812, AFM13, and (CD20).sub.2.times.CD16, erlotinib (Tarceva),
daratumumab, alemtuzumab, pertuzumab, brentuximab, elotuzumab,
ibritumomab, ifabotuzumab, farletuzumab, otlertuzumab, carotuximab,
epratuzumab, inebilizumab, lumretuzumab, 4G7SDIE, AFM21, AFM22,
LY-3022855, SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20,
mogamulizumab, ChiLob-7/4, leukotuximab, isatuximab, DS-8895,
FPA144, GM102, GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04,
XOMA-213, PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005,
MDX-1097, MOR202, MOR-208, oportuzumab, ensituximab, vedotin
(Adcetris), ibritumomab tiuxetan, ABBV-838, HuMax-AXL-ADC, and
ado-trastuzumab emtansine (Kadcyla); radiotherapy or
chemotherapeutic agents including, but not limited to
Anthracyclines (Doxorubicin, Epirubicin, Daunorubicin, Idarubicin,
Mitoxantrone), Oxaliplatin, Bortezomib, Cyclophosphamide,
Bleomycin, Vorinostat, Paclitaxel, 5-Fluorouracil, Cytarabine,
Prednisolone, Docetaxel, Mitomycin C, Topotecan/Camptothecin,
Etoposide, Zoledronic acid, Methotrexate, Ibrutinib, Aflibercept,
Bevacizumab, Toremifene, Vinblastine, Vincristine, Idelalisib,
Mercaptopurine, Thalidomide, Sorafenib; a cyclic dinculeotide or
other STING pathway agonist; etc.
Detection Kits and Therapeutic Kits
[0711] As a matter of convenience, an anti-SIRP.alpha. antibody or
antigen-binding fragment thereof of the invention can be provided
in a kit, i.e., a packaged combination of reagents in predetermined
amounts with instructions for performing the diagnostic or
detection assay. Where the antibody or fragment is labeled with an
enzyme, the kit will include substrates and cofactors required by
the enzyme (e.g., a substrate precursor which provides the
detectable chromophore or fluorophore). In addition, other
additives may be included such as stabilizers, buffers (e.g., a
block buffer or lysis buffer) and the like. The relative amounts of
the various reagents may be varied widely to provide for
concentrations in solution of the reagents which substantially
optimize the sensitivity of the assay. Particularly, the reagents
may be provided as dry powders, usually lyophilized, including
excipients which on dissolution will provide a reagent solution
having the appropriate concentration.
[0712] Also provided are diagnostic or detection reagents and kits
comprising one or more such reagents for use in a variety of
detection assays, including for example, immunoassays such as ELISA
(sandwich-type or competitive format). The kit's components may be
pre-attached to a solid support, or may be applied to the surface
of a solid support when the kit is used. In some embodiments of the
invention, the signal generating means may come pre-associated with
an antibody or fragment of the invention or may require combination
with one or more components, e.g., buffers, antibody-enzyme
conjugates, enzyme substrates, or the like, prior to use. Kits may
also include additional reagents, e.g., blocking reagents for
reducing nonspecific binding to the solid phase surface, washing
reagents, enzyme substrates, and the like. The solid phase surface
may be in the form of a tube, a bead, a microtiter plate, a
microsphere, or other materials suitable for immobilizing proteins,
peptides, or polypeptides. In particular aspects, an enzyme that
catalyzes the formation of a chemilluminescent or chromogenic
product or the reduction of a chemilluminescent or chromogenic
substrate is a component of the signal generating means. Such
enzymes are well known in the art. Kits may comprise any of the
capture agents and detection reagents described herein. Optionally
the kit may also comprise instructions for carrying out the methods
of the invention.
[0713] Also provided is a kit comprising an anti-SIRP.alpha.
antibody (e.g., humanized antibody) or antigen-binding fragment
thereof packaged in a container, such as a vial or bottle, and
further comprising a label attached to or packaged with the
container, the label describing the contents of the container and
providing indications and/or instructions regarding use of the
contents of the container to treat one or more disease states as
described herein.
[0714] In one aspect, the kit is for treating cancer and comprises
an anti-SIRP.alpha. antibody (e.g., humanized antibody) or
antigen-binding fragment thereof and a further therapeutic agent or
a vaccine. The kit may optionally further include a syringe for
parenteral, e.g., intravenous, administration. In another aspect,
the kit comprises an anti-SIRP.alpha. antibody (e.g., humanized
antibody) or antigen-binding fragment thereof and a label attached
to or packaged with the container describing use of the antibody or
fragment with the vaccine or further therapeutic agent. In yet
another aspect, the kit comprises the vaccine or further
therapeutic agent and a label attached to or packaged with the
container describing use of the vaccine or further therapeutic
agent with the anti-SIRP.alpha. antibody or fragment. In certain
embodiments, an anti-SIRP.alpha. antibody and vaccine or further
therapeutic agent are in separate vials or are combined together in
the same pharmaceutical composition.
[0715] As discussed above in the combination therapy section,
concurrent administration of two therapeutic agents does not
require that the agents be administered at the same time or by the
same route, as long as there is an overlap in the time period
during which the agents are exerting their therapeutic effect.
Simultaneous or sequential administration is contemplated, as is
administration on different days or weeks.
[0716] The therapeutic and detection kits disclosed herein may also
be prepared that comprise at least one of the antibody, peptide,
antigen-binding fragment, or polynucleotide disclosed herein and
instructions for using the composition as a detection reagent or
therapeutic agent. Containers for use in such kits may typically
comprise at least one vial, test tube, flask, bottle, syringe or
other suitable container, into which one or more of the detection
and/or therapeutic composition(s) may be placed, and preferably
suitably aliquoted. Where a second therapeutic agent is also
provided, the kit may also contain a second distinct container into
which this second detection and/or therapeutic composition may be
placed. Alternatively, a plurality of compounds may be prepared in
a single pharmaceutical composition, and may be packaged in a
single container means, such as a vial, flask, syringe, bottle, or
other suitable single container. The kits disclosed herein will
also typically include a means for containing the vial(s) in close
confinement for commercial sale, such as, e.g., injection or
blow-molded plastic containers into which the desired vial(s) are
retained. Where a radiolabel, chromogenic, fluorigenic, or other
type of detectable label or detecting means is included within the
kit, the labeling agent may be provided either in the same
container as the detection or therapeutic composition itself, or
may alternatively be placed in a second distinct container means
into which this second composition may be placed and suitably
aliquoted. Alternatively, the detection reagent and the label may
be prepared in a single container means, and in most cases, the kit
will also typically include a means for containing the vial(s) in
close confinement for commercial sale and/or convenient packaging
and delivery.
[0717] A device or apparatus for carrying out the detection or
monitoring methods described herein is also provided. Such an
apparatus may include a chamber or tube into which sample can be
input, a fluid handling system optionally including valves or pumps
to direct flow of the sample through the device, optionally filters
to separate plasma or serum from blood, mixing chambers for the
addition of capture agents or detection reagents, and optionally a
detection device for detecting the amount of detectable label bound
to the capture agent immunocomplex. The flow of sample may be
passive (e.g., by capillary, hydrostatic, or other forces that do
not require further manipulation of the device once sample is
applied) or active (e.g., by application of force generated via
mechanical pumps, electroosmotic pumps, centrifugal force, or
increased air pressure), or by a combination of active and passive
forces.
[0718] In further embodiments, also provided is a processor, a
computer readable memory, and a routine stored on the computer
readable memory and adapted to be executed on the processor to
perform any of the methods described herein. Examples of suitable
computing systems, environments, and/or configurations include
personal computers, server computers, hand-held or laptop devices,
multiprocessor systems, microprocessor-based systems, set top
boxes, programmable consumer electronics, network PCs,
minicomputers, mainframe computers, distributed computing
environments that include any of the above systems or devices, or
any other systems known in the art.
Preferred Embodiments
[0719] Embodiment 1. An antibody or antigen binding fragment
thereof that binds to human SIRP.alpha., wherein the antibody or
antigen binding fragment comprises one or more, and optionally
each, of: [0720] a. a heavy chain variable region CDR1 comprising
the amino acid sequence of SEQ ID NO:69 or an amino acid sequence
differing from SEQ ID NO: 1 by 1, 2, or 3 conservative
substitutions, [0721] b. a heavy chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO:70 or an amino acid
sequence differing from SEQ ID NO: 2 by 1, 2, or 3 conservative
substitutions, [0722] c. a heavy chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO:71 or an amino acid
sequence differing from SEQ ID NO: 3 by 1, 2, or 3 conservative
substitutions, [0723] d. a light chain variable region CDR1
comprising the amino acid sequence of SEQ ID NO:72 or an amino acid
sequence differing from SEQ ID NO: 4 by 1, 2, or 3 conservative
substitutions, [0724] e. a light chain variable region CDR2
comprising the amino acid sequence of SEQ ID NO:73 or an amino acid
sequence differing from SEQ ID NO: 5 by 1, 2, or 3 conservative
substitutions, and [0725] f. a light chain variable region CDR3
comprising the amino acid sequence of SEQ ID NO:74 or an amino acid
sequence differing from SEQ ID NO: 6 by 1, 2, or 3 conservative
substitutions. or wherein the antibody or antigen binding fragment
comprises one or more, and optionally each, of: [0726] g. a heavy
chain variable region CDR1 comprising the amino acid sequence of
SEQ ID NO:1 or an amino acid sequence differing from SEQ ID NO: 1
by 1, 2, or 3 conservative substitutions, [0727] h. a heavy chain
variable region CDR2 comprising the amino acid sequence of SEQ ID
NO:2 or an amino acid sequence differing from SEQ ID NO: 2 by 1, 2,
or 3 conservative substitutions, [0728] i. a heavy chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:3 or an
amino acid sequence differing from SEQ ID NO: 3 by 1, 2, or 3
conservative substitutions, [0729] j. a light chain variable region
CDR1 comprising the amino acid sequence of SEQ ID NO:4 or an amino
acid sequence differing from SEQ ID NO: 4 by 1, 2, or 3
conservative substitutions, [0730] k. a light chain variable region
CDR2 comprising the amino acid sequence of SEQ ID NO:5 or an amino
acid sequence differing from SEQ ID NO: 5 by 1, 2, or 3
conservative substitutions, and [0731] l. a light chain variable
region CDR3 comprising the amino acid sequence of SEQ ID NO:6 or an
amino acid sequence differing from SEQ ID NO: 6 by 1, 2, or 3
conservative substitutions.
[0732] Embodiment 2. The antibody or antigen binding fragment of
embodiment 1, wherein the antibody or antigen binding fragment
comprises [0733] each of a heavy chain sequence comprising the
amino acid sequence of SEQ ID NO:69 or an amino acid sequence
differing from SEQ ID NO: 69 by 1, 2, or 3 conservative
substitutions; the amino acid sequence of SEQ ID NO:70 or an amino
acid sequence differing from SEQ ID NO: 70 by 1, 2, or 3
conservative substitutions; and the amino acid sequence of SEQ ID
NO: 71 or an amino acid sequence differing from SEQ ID NO: 71 by 1,
2, or 3 conservative substitutions; and/or [0734] each of a light
chain sequence comprising the amino acid sequence of SEQ ID NO: 72
or an amino acid sequence differing from SEQ ID NO: 72 by 1, 2, or
3 conservative substitutions; the amino acid sequence of SEQ ID NO:
73 or an amino acid sequence differing from SEQ ID NO: 73 by 1, 2,
or 3 conservative substitutions; and the amino acid sequence of SEQ
ID NO: 74 or an amino acid sequence differing from SEQ ID NO: 74 by
1, 2, or 3 conservative substitutions; or wherein the antibody or
antigen binding fragment comprises [0735] each of a heavy chain
sequence comprising the amino acid sequence of SEQ ID NO:1 or an
amino acid sequence differing from SEQ ID NO: 1 by 1, 2, or 3
conservative substitutions; the amino acid sequence of SEQ ID NO:2
or an amino acid sequence differing from SEQ ID NO: 2 by 1, 2, or 3
conservative substitutions; and the amino acid sequence of SEQ ID
NO:3 or an amino acid sequence differing from SEQ ID NO: 3 by 1, 2,
or 3 conservative substitutions; and/or [0736] each of a light
chain sequence comprising the amino acid sequence of SEQ ID NO:4 or
an amino acid sequence differing from SEQ ID NO: 4 by 1, 2, or 3
conservative substitutions; the amino acid sequence of SEQ ID NO:5
or an amino acid sequence differing from SEQ ID NO: 5 by 1, 2, or 3
conservative substitutions; and the amino acid sequence of SEQ ID
NO:6 or an amino acid sequence differing from SEQ ID NO: 6 by 1, 2,
or 3 conservative substitutions.
[0737] Embodiment 3. The antibody or antigen binding fragment of
embodiment 2, wherein the antibody or antigen binding fragment
comprises one or both of: [0738] a heavy chain variable region
comprising an amino acid sequence selected from the group
consisting of: [0739] SEQ ID NO: 75 or an amino acid sequence at
least 90%, 95%, 97%, 98%, or 99% identical thereto, [0740] SEQ ID
NO: 78 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% identical thereto, [0741] SEQ ID NO: 80 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,
[0742] SEQ ID NO: 82 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% identical thereto, [0743] SEQ ID NO: 84 or an
amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0744] SEQ ID NO: 86 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% identical thereto, [0745] SEQ ID NO: 88
or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, and [0746] SEQ ID NO: 102 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto; and
[0747] a light chain variable region comprising an amino acid
sequence selected from the group consisting of: [0748] SEQ ID NO:
76 or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto, [0749] SEQ ID NO: 90 or an amino acid sequence
at least 90%, 95%, 97%, 98%, or 99% identical thereto, [0750] SEQ
ID NO: 92 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% identical thereto, [0751] SEQ ID NO: 94 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,
[0752] SEQ ID NO: 96 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% identical thereto, [0753] SEQ ID NO: 98 or an
amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0754] SEQ ID NO: 100 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% identical thereto, and [0755] SEQ ID NO:
104 or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto; or wherein the antibody or antigen binding
fragment comprises one or both of: [0756] a heavy chain variable
region comprising an amino acid sequence selected from the group
consisting of: [0757] SEQ ID NO: 7 or an amino acid sequence at
least 90%, 95%, 97%, 98%, or 99% identical thereto, [0758] SEQ ID
NO: 10 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% identical thereto, [0759] SEQ ID NO: 12 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,
[0760] SEQ ID NO: 14 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% identical thereto, [0761] SEQ ID NO: 16 or an
amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0762] SEQ ID NO: 18 or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% identical thereto, and [0763] SEQ ID NO:
30 or an amino acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto; and [0764] a light chain variable region
comprising an amino acid sequence selected from the group
consisting of: [0765] SEQ ID NO: 8 or an amino acid sequence at
least 90%, 95%, 97%, 98%, or 99% identical thereto, [0766] SEQ ID
NO: 20 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% identical thereto, [0767] SEQ ID NO: 22 or an amino acid
sequence at least 90%, 95%, 97%, 98%, or 99% identical thereto,
[0768] SEQ ID NO: 24 or an amino acid sequence at least 90%, 95%,
97%, 98%, or 99% identical thereto, [0769] SEQ ID NO: 26 or an
amino acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, and [0770] SEQ ID NO: 28 or an amino acid sequence at
least 90%, 95%, 97%, 98%, or 99% identical thereto, and [0771] SEQ
ID NO: 32 or an amino acid sequence at least 90%, 95%, 97%, 98%, or
99% identical thereto.
[0772] Embodiment 4. The antibody or antigen binding fragment of
embodiment 3, wherein the antibody or fragment thereof has the
following characteristics: [0773] binds to a cell expressing human
SIRP.alpha.V1 protein with an EC.sub.50<10 nM, preferably <5
nM, more preferably <1.5 nM, still more preferably <1.0 nM,
even more preferably <0.5 nM, and most preferably about 0.3 nM
or less; [0774] binds to a cell expressing human SIRP.alpha.V2
protein with an EC.sub.50<10 nM, preferably <5 nM, more
preferably <1.5 nM, still more preferably <1.0 nM, even more
preferably <0.5 nM, and most preferably about 0.3 nM or less;
[0775] does not appreciably bind to SIRP.beta.1 protein at an
antibody concentration of 50 nM, preferably 67 nM, and more
preferably 100 nM; or alternatively at a concentration that is
10-fold greater, preferably 50-fold greater, more preferably
100-fold greater, and still more preferably 200-fold greater than
the antibody's EC.sub.50 for SIRP.alpha.V1 or SIRP.alpha.V2; [0776]
inhibits binding between human SIRP.alpha. and CD47 with an
IC.sub.50<10.0 nM, more preferably <5.0 nM, still more
preferably <2.5 nM, and most preferably about 1.0 nM or less;
and [0777] exhibits a T20 "humanness" score of at least 79, and
more preferably 85.
[0778] Embodiment 5. The antibody or antigen binding fragment of
embodiment 1, wherein the antibody or antigen binding fragment
thereof comprises one of the following combinations of heavy chain
sequence/light chain sequence:
SEQ ID NO: 78/SEQ ID NO: 90,
SEQ ID NO: 78/SEQ ID NO: 92,
SEQ ID NO: 78/SEQ ID NO: 94,
SEQ ID NO: 78/SEQ ID NO: 96,
SEQ ID NO: 78/SEQ ID NO: 98,
SEQ ID NO: 78/SEQ ID NO: 100,
SEQ ID NO: 80/SEQ ID NO: 90,
SEQ ID NO: 80/SEQ ID NO: 92,
SEQ ID NO: 80/SEQ ID NO: 94,
SEQ ID NO: 80/SEQ ID NO: 96,
SEQ ID NO: 80/SEQ ID NO: 98,
SEQ ID NO: 80/SEQ ID NO: 100,
SEQ ID NO: 82/SEQ ID NO: 90,
SEQ ID NO: 82/SEQ ID NO: 92,
SEQ ID NO: 82/SEQ ID NO: 94,
SEQ ID NO: 82/SEQ ID NO: 96,
SEQ ID NO: 82/SEQ ID NO: 98,
SEQ ID NO: 82/SEQ ID NO: 100,
SEQ ID NO: 84/SEQ ID NO: 90,
SEQ ID NO: 84/SEQ ID NO: 92,
SEQ ID NO: 84/SEQ ID NO: 94,
SEQ ID NO: 84/SEQ ID NO: 96,
SEQ ID NO: 84/SEQ ID NO: 98,
SEQ ID NO: 84/SEQ ID NO: 100,
SEQ ID NO: 86/SEQ ID NO: 90,
SEQ ID NO: 86/SEQ ID NO: 92,
SEQ ID NO: 86/SEQ ID NO: 94,
SEQ ID NO: 86/SEQ ID NO: 96,
SEQ ID NO: 86/SEQ ID NO: 98,
SEQ ID NO: 86/SEQ ID NO: 100,
SEQ ID NO: 88/SEQ ID NO: 90,
SEQ ID NO: 88/SEQ ID NO: 92,
SEQ ID NO: 88/SEQ ID NO: 94,
SEQ ID NO: 88/SEQ ID NO: 96,
SEQ ID NO: 88/SEQ ID NO: 98,
SEQ ID NO: 88/SEQ ID NO: 100,
SEQ ID NO: 10/SEQ ID NO: 20,
SEQ ID NO: 10/SEQ ID NO: 22,
SEQ ID NO: 10/SEQ ID NO: 24,
SEQ ID NO: 10/SEQ ID NO: 26,
SEQ ID NO: 10/SEQ ID NO: 28,
SEQ ID NO: 12/SEQ ID NO: 20,
SEQ ID NO: 12/SEQ ID NO: 22,
SEQ ID NO: 12/SEQ ID NO: 24,
SEQ ID NO: 12/SEQ ID NO: 26,
SEQ ID NO: 12/SEQ ID NO: 28,
SEQ ID NO: 14/SEQ ID NO: 20,
SEQ ID NO: 14/SEQ ID NO: 22,
SEQ ID NO: 14/SEQ ID NO: 24,
SEQ ID NO: 14/SEQ ID NO: 26,
SEQ ID NO: 14/SEQ ID NO: 28,
SEQ ID NO: 16/SEQ ID NO: 20,
SEQ ID NO: 16/SEQ ID NO: 22,
SEQ ID NO: 16/SEQ ID NO: 24,
SEQ ID NO: 16/SEQ ID NO: 26,
SEQ ID NO: 16/SEQ ID NO: 28,
SEQ ID NO: 18/SEQ ID NO: 20,
SEQ ID NO: 18/SEQ ID NO: 22,
SEQ ID NO: 18/SEQ ID NO: 24,
SEQ ID NO: 18/SEQ ID NO: 26,
SEQ ID NO: 18/SEQ ID NO: 28,
[0779] or, in each case, at least 90%, 95%, 97%, 98%, or 99%
identical to a respective SEQ ID.
[0780] Embodiment 6. The antibody or antigen binding fragment of
one of embodiments 1-5, wherein the antibody is an intact IgG.
[0781] Embodiment 7. The antibody or antigen binding fragment of
one of embodiments 1-6, wherein the antibody comprises a wild-type
or mutated IgG2 Fc region.
[0782] Embodiment 8. The antibody or antigen binding fragment of
one of embodiments 1-6, wherein the antibody comprises a mutated
IgG1 Fc region.
[0783] Embodiment 9. The antibody or antigen binding fragment of
one of embodiments 1-6, wherein the antibody comprises a mutated
IgG4 Fc region.
[0784] Embodiment 10. An antibody or antigen binding fragment
thereof that binds to the same epitope of human SIRP.alpha. as an
antibody as an antibody according to embodiment 5.
[0785] Embodiment 11. The antibody or antigen binding fragment of
any of embodiments 1-10, wherein the antibody or antigen binding
fragment is humanized.
[0786] Embodiment 12. The antibody or antigen binding fragment of
any of embodiments 1-11 that is a humanized antibody that comprises
two heavy chains and two light chains, wherein each heavy chain
comprises SEQ ID NO: 10 and each light chain comprises SEQ ID NO:
20.
[0787] Embodiment 13. The antibody or antigen binding fragment of
any of embodiments 1-11 that is a humanized antibody that comprises
two heavy chains and two light chains, wherein each heavy chain
comprises SEQ ID NO: 16 and each light chain comprises SEQ ID NO:
28.
[0788] Embodiment 14. The antibody or antigen binding fragment of
any of embodiments 1-11 that is a humanized antibody that comprises
two heavy chains and two light chains, wherein each heavy chain
comprises SEQ ID NO: 18 and each light chain comprises SEQ ID NO:
20.
[0789] Embodiment 15. The antibody or antigen binding fragment of
any of embodiments 1-11 that is a humanized antibody that comprises
two heavy chains and two light chains, wherein each heavy chain
comprises SEQ ID NO: 80 and each light chain comprises SEQ ID NO:
90.
[0790] Embodiment 16. The antibody or antigen binding fragment of
any of embodiments 1-11 that is a humanized antibody that comprises
two heavy chains and two light chains, wherein each heavy chain
comprises SEQ ID NO: 80 and each light chain comprises SEQ ID NO:
92.
[0791] Embodiment 17. The antibody or antigen binding fragment of
any of embodiments 1-11 that is a humanized antibody that comprises
two heavy chains and two light chains, wherein each heavy chain
comprises SEQ ID NO: 80 and each light chain comprises SEQ ID NO:
95.
[0792] Embodiment 18. The antibody or antigen binding fragment of
any one of embodiments 1-17 that comprises a glycosylation pattern
characteristic of expression by a mammalian cell, and optionally is
glycosylated by expression from a CHO cell.
[0793] Embodiment 19. An isolated polypeptide comprising the amino
acid sequence of any one of SEQ ID NOs: 75, 78, 80, 82, 84, 86, 88,
76, 90, 92, 94, 96, 98, 100, 102, 104, 7, 10, 12, 14, 16, 18, 30,
8, 20, 22, 24, 26, 28, and 32, or an amino acid sequence at least
90%, 95%, 97%, 98%, or 99% identical thereto.
[0794] Embodiment 20. An isolated nucleic acid encoding any one of
the antibodies or antigen binding fragments of embodiments 1-18, or
any one of the polypeptides of embodiment 19.
[0795] Embodiment 21. An isolated nucleic acid of embodiment 20
comprising: [0796] a nucleic acid sequence of SEQ ID NO: 77 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0797] a nucleic acid sequence of SEQ ID NO: 79 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0798] a nucleic acid sequence of SEQ ID NO: 81 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0799] a nucleic acid sequence of SEQ ID NO: 83 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0800] a nucleic acid sequence of SEQ ID NO: 85 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0801] a nucleic acid sequence of SEQ ID NO: 87 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0802] a nucleic acid sequence of SEQ ID NO: 101 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0803] a nucleic acid sequence of SEQ ID NO: 89 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0804] a nucleic acid sequence of SEQ ID NO: 91 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0805] a nucleic acid sequence of SEQ ID NO: 93 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0806] a nucleic acid sequence of SEQ ID NO: 95 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0807] a nucleic acid sequence of SEQ ID NO: 97 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0808] a nucleic acid sequence of SEQ ID NO: 99 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0809] a nucleic acid sequence of SEQ ID NO: 103 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0810] a nucleic acid sequence of SEQ ID NO: 9 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0811] a nucleic acid sequence of SEQ ID NO: 11 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0812] a nucleic acid sequence of SEQ ID NO: 13 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0813] a nucleic acid sequence of SEQ ID NO: 15 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0814] a nucleic acid sequence of SEQ ID NO: 17 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0815] a nucleic acid sequence of SEQ ID NO: 29 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0816] a nucleic acid sequence of SEQ ID NO: 19 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0817] a nucleic acid sequence of SEQ ID NO: 21 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0818] a nucleic acid sequence of SEQ ID NO: 23 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0819] a nucleic acid sequence of SEQ ID NO: 25 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, [0820] a nucleic acid sequence of SEQ ID NO: 27 or a
nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99% identical
thereto, and/or [0821] a nucleic acid sequence of SEQ ID NO: 31 or
a nucleic acid sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto.
[0822] Embodiment 22. An expression vector comprising the isolated
nucleic acid of embodiment 20 or 21.
[0823] Embodiment 23. An expression vector of embodiment 22,
encoding both a heavy chain sequence and a light chain sequence of
an anti-SIRP.alpha. antibody, the expression vectors comprising the
following a first nucleic acid sequence/second nucleic acid
sequence selected from the group consisting of:
SEQ ID NO: 77/SEQ ID NO: 89,
SEQ ID NO: 77/SEQ ID NO: 91,
SEQ ID NO: 77/SEQ ID NO: 93,
SEQ ID NO: 77/SEQ ID NO: 95,
SEQ ID NO: 77/SEQ ID NO: 97,
SEQ ID NO: 77/SEQ ID NO: 99,
SEQ ID NO: 79/SEQ ID NO: 89,
SEQ ID NO: 79/SEQ ID NO: 91,
SEQ ID NO: 79/SEQ ID NO: 93,
SEQ ID NO: 79/SEQ ID NO: 95,
SEQ ID NO: 79/SEQ ID NO: 97,
SEQ ID NO: 79/SEQ ID NO: 99,
SEQ ID NO: 81/SEQ ID NO: 89,
SEQ ID NO: 81/SEQ ID NO: 91,
SEQ ID NO: 81/SEQ ID NO: 93,
SEQ ID NO: 81/SEQ ID NO: 95,
SEQ ID NO: 81/SEQ ID NO: 97,
SEQ ID NO: 81/SEQ ID NO: 99,
SEQ ID NO: 83/SEQ ID NO: 89,
SEQ ID NO: 83/SEQ ID NO: 91,
SEQ ID NO: 83/SEQ ID NO: 93,
SEQ ID NO: 83/SEQ ID NO: 95,
SEQ ID NO: 83/SEQ ID NO: 97,
SEQ ID NO: 83/SEQ ID NO: 99,
SEQ ID NO: 85/SEQ ID NO: 89,
SEQ ID NO: 85/SEQ ID NO: 91,
SEQ ID NO: 85/SEQ ID NO: 93,
SEQ ID NO: 85/SEQ ID NO: 95,
SEQ ID NO: 85/SEQ ID NO: 97,
SEQ ID NO: 85/SEQ ID NO: 99,
SEQ ID NO: 87/SEQ ID NO: 89,
SEQ ID NO: 87/SEQ ID NO: 91,
SEQ ID NO: 87/SEQ ID NO: 93,
SEQ ID NO: 87/SEQ ID NO: 95,
SEQ ID NO: 87/SEQ ID NO: 97,
SEQ ID NO: 87/SEQ ID NO: 99,
SEQ ID NO: 9/SEQ ID NO: 19,
SEQ ID NO: 9/SEQ ID NO: 21,
SEQ ID NO: 9/SEQ ID NO: 23,
SEQ ID NO: 9/SEQ ID NO: 25,
SEQ ID NO: 9/SEQ ID NO: 27,
SEQ ID NO: 11/SEQ ID NO: 19,
SEQ ID NO: 11/SEQ ID NO: 21,
SEQ ID NO: 11/SEQ ID NO: 23,
SEQ ID NO: 11/SEQ ID NO: 25,
SEQ ID NO: 11/SEQ ID NO: 27,
SEQ ID NO: 13/SEQ ID NO: 19,
SEQ ID NO: 13/SEQ ID NO: 21,
SEQ ID NO: 13/SEQ ID NO: 23,
SEQ ID NO: 13/SEQ ID NO: 25,
SEQ ID NO: 13/SEQ ID NO: 27,
SEQ ID NO: 15/SEQ ID NO: 19,
SEQ ID NO: 15/SEQ ID NO: 21,
SEQ ID NO: 15/SEQ ID NO: 23,
SEQ ID NO: 15/SEQ ID NO: 25,
SEQ ID NO: 15/SEQ ID NO: 27,
SEQ ID NO: 17/SEQ ID NO: 19,
SEQ ID NO: 17/SEQ ID NO: 21,
SEQ ID NO: 17/SEQ ID NO: 23,
SEQ ID NO: 17/SEQ ID NO: 25, and
SEQ ID NO: 17/SEQ ID NO: 27,
[0824] or, in each case, at least 90%, 95%, 97%, 98%, or 99%
identical to a respective SEQ ID NO.
[0825] Embodiment 24. A host cell comprising expression vector of
embodiment 22 or 23.
[0826] Embodiment 25. A host cell of embodiment 24 which produces a
full length anti-SIRP.alpha. antibody.
[0827] Embodiment 26. The host cell of one of embodiments 24 or 25,
which is a bacterial cell, a human cell, a mammalian cell, a Pichia
cell, a plant cell, an HEK293 cell, or a Chinese hamster ovary
cell.
[0828] Embodiment 27. A composition comprising the antibody or
antigen binding fragment of any one of embodiments 1-18 and a
pharmaceutically acceptable carrier or diluent.
[0829] Embodiment 28. The composition of embodiment 27, further
comprising a second antibody or antigen binding fragment thereof
that induces ADCC and/or ADCP, wherein said antibody or antigen
binding fragment of the invention enhances the antibody-mediated
destruction of cells by the second antibody.
[0830] Embodiment 29. The composition according to embodiment 28,
wherein the second antibody or antigen binding fragment thereof
binds to an antigen selected from the group consisting of AMHR2,
AXL, BCMA, CA IX, CD4, CD16, CD19, CD20, CD22, CD30, CD37, CD38,
CD40, CD52, CD98, CSF1R, GD2, CCR4, CS1, EpCam, EGFR, EGFRvIII,
Endoglin, EPHA2, EphA3, FGFR2b, folate receptor alpha, fucosyl-GM1,
HER2, HER3, IL1RAP, kappa myeloma antigen, MS4A1, prolactin
receptor, TA-MUC1, and PSMA.
[0831] Embodiment 30. The composition according to embodiment 29,
wherein the second antibody or antigen binding fragment thereof is
selected from the group consisting of Rituximab, ublituximab,
margetuximab, IMGN-529, SCT400, veltuzumab, Obinutuzumab, ADCT-502,
Hul4.18K322A, Hu3F8, Dinituximab, Trastuzumab, Cetuximab,
Rituximab-RLI, c.60C3-RLI, Hul4.18-IL2, KM2812, AFM13,
(CD20)2xCD16, erlotinib (Tarceva), daratumumab, alemtuzumab,
pertuzumab, brentuximab, elotuzumab, ibritumomab, ifabotuzumab,
farletuzumab, otlertuzumab, carotuximab, epratuzumab, inebilizumab,
lumretuzumab, 4G7SDIE, AFM21, AFM22, LY-3022855, SNDX-6352, AFM-13,
BI-836826, BMS-986012, BVX-20, mogamulizumab, ChiLob-7/4,
leukotuximab, isatuximab, DS-8895, FPA144, GM102, GSK-2857916,
IGN523, IT1208, ADC-1013, CAN-04, XOMA-213, PankoMab-GEX,
chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202, MOR-208,
oportuzumab, ensituximab, vedotin (Adcetris), ibritumomab tiuxetan,
ABBV-838, HuMax-AXL-ADC, and ado-trastuzumab emtansine
(Kadcyla).
[0832] Embodiment 31. The composition according to embodiment 28,
wherein the second antibody or antigen binding fragment thereof
induces ADCP.
[0833] Embodiment 32. The composition according to embodiment 31,
wherein the second antibody or antigen binding fragment thereof is
selected from the group consisting of Rituximab, ublituximab,
margetuximab, IMGN-529, SCT400, veltuzumab, Obinutuzumab,
Trastuzumab, Cetuximab, alemtuzumab, ibritumomab, farletuzumab,
inebilizumab, lumretuzumab, 4G7SDIE, BMS-986012, BVX-20,
mogamulizumab, ChiLob-7/4, GM102, GSK-2857916, PankoMab-GEX,
chKM-4927, MDX-1097, MOR202, and MOR-208.
[0834] Embodiment 33. The composition of embodiment 27, further
comprising one or more agents selected from the group consisting of
anti-CD27 antibody, anti-CD47 antibody, anti-APRIL antibody,
anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody,
anti-CTLA4 antibody, anti-CS1 antibody, anti-KIR2DL1/2/3 antibody,
anti-CD137 antibody, anti-GITR antibody, anti-PD-L2 antibody,
anti-ILT1 antibody, anti-ILT2 antibody, anti-ILT3 antibody,
anti-ILT4 antibody, anti-ILT5 antibody, anti-ILT6 antibody,
anti-ILT7 antibody, anti-ILT8 antibody, anti-CD40 antibody,
anti-OX40 antibody, anti-ICOS, anti-KIR2DL1 antibody,
anti-KIR2DL2/3 antibody, anti-KIR2DL4 antibody, anti-KIR2DL5A
antibody, anti-KIR2DL5B antibody, anti-KIR3DL1 antibody,
anti-KIR3DL2 antibody, anti-KIR3DL3 antibody, anti-NKG2A antibody,
anti-NKG2C antibody, anti-NKG2E antibody, anti-4-1BB antibody,
anti-TSLP antibody, anti-IL-10 antibody, IL-10 PEGylated IL-10, an
agonist (e.g., an agonistic antibody or antigen-binding fragment
thereof, or a soluble fusion) of a TNF receptor protein, an
Immunoglobulin-like protein, a cytokine receptor, an integrin, a
signaling lymphocytic activation molecules (SLAM proteins), an
activating NK cell receptor, a Toll like receptor, OX40, CD2, CD7,
CD27, CD28, CD30, CD40, ICAM-1, LFA-1 (CD1 1a/CD18), 4-1BB (CD137),
B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,
SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,
CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a,
ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE,
CD103, ITGAL, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2,
CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226),
SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9
(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,
Ly108), SLAM (SLAMF1, CD150, IPO-3), SLAM7, BLAME (SLAMF8), SELPLG
(CD162), LTBR, LAT, GADS, PAG/Cbp, CD19a, a ligand that
specifically binds with CD83, inhibitor of CD47, an inhibitor of
PD-1, an an inhibitor of PD-L1, an inhibitor of PD-L2, an inhibitor
of CTLA4, an inhibitor of TIM3, an inhibitor of LAGS, an inhibitor
of CEACAM (e.g., CEACAM-1, -3 and/or -5), an inhibitor of VISTA, an
inhibitor of BTLA, an inhibitor of TIGIT, an inhibitor of LAIR1, an
inhibitor of IDO, an inhibitor of TDO, an inhibitor of CD160 an
inhibitor of TGFR beta, and a cyclic dinculeotide or other STING
pathway agonist.
[0835] Embodiment 34. A method of producing an antibody or antigen
binding fragment comprising: [0836] culturing a host cell
comprising a polynucleotide encoding the heavy chain and/or the
light chain of any one of the antibodies or antigen binding
fragments of embodiments 1-18 under conditions favorable to
expression of the polynucleotide; and optionally, recovering the
antibody or antigen binding fragment from the host cell and/or
culture medium.
[0837] Embodiment 35. A method for detecting the presence of a
SIRP.alpha. peptide or a fragment thereof in a sample comprising
contacting the sample with an antibody or fragment of any of
embodiments 1-18 and detecting the presence of a complex between
the antibody or fragment and the peptide; wherein detection of the
complex indicates the presence of the SIRP.alpha. peptide.
[0838] Embodiment 36. An antibody or antigen binding fragment
according to any one of embodiments 1-18 or a composition according
to any one of embodiments 21-25, for the treatment of cancer or an
infectious disease.
[0839] Embodiment 37. An antibody or antigen binding fragment of
embodiments 1-18 or a composition according to any one of
embodiments 27-33 for decreasing SIRP.alpha./CD47 signalling in a
human subject.
[0840] Embodiment 38. A method of treating cancer in a human
subject, comprising administering to the subject an effective
amount of an antibody or antigen binding fragment of any one of
embodiments 1-18, or an expression vector according to one of
embodiments 22 or 23, or a host cell according to one of
embodiments 24-26, or a composition according one of embodiments
27-33, optionally in association with a further therapeutic agent
or therapeutic procedure.
[0841] Embodiment 39. A method of treating cancer in a human
subject, comprising:
administering to the subject an effective amount of (i) an antibody
or antigen binding fragment thereof that induces ADCC and/or ADCP;
and (ii) an antibody or antigen binding fragment of any one of
embodiments 1-18, or an expression vector according to one of
embodiments 22 or 23, or a host cell according to one of
embodiments 24-26, or a composition according one of embodiments
27-33, optionally in association with a further therapeutic agent
or therapeutic procedure, wherein the administration of (ii)
enhances the antibody-mediated destruction of cells by the antibody
or antigen binding fragment thereof that induces ADCC and/or
ADCP.
[0842] Embodiment 40. The method according to embodiment 39,
wherein the antibody or antigen binding fragment thereof that
induces ADCC and/or ADCP binds to an antigen selected from the
group consisting of AMHR2, AXL, BCMA, CA IX, CD4, CD16, CD19, CD20,
CD22, CD30, CD37, CD38, CD40, CD52, CD98, CSF1R, GD2, CCR4, CS1,
EpCam, EGFR, EGFRvIII, Endoglin, EPHA2, EphA3, FGFR2b, folate
receptor alpha, fucosyl-GM1, HER2, HER3, IL1RAP, kappa myeloma
antigen, MS4A1, prolactin receptor, TA-MUC1, and PSMA.
[0843] Embodiment 41. The method according to embodiment 40,
wherein the antibody or antigen binding fragment thereof that
induces ADCC and/or ADCP is selected from the group consisting of
Rituximab, ublituximab, margetuximab, IMGN-529, SCT400, veltuzumab,
Obinutuzumab, ADCT-502, Hul4.18K322A, Hu3F8, Dinituximab,
Trastuzumab, Cetuximab, Rituximab-RLI, c.60C3-RLI, Hul4.18-IL2,
KM2812, AFM13, (CD20)2xCD16, erlotinib (Tarceva), daratumumab,
alemtuzumab, pertuzumab, brentuximab, elotuzumab, ibritumomab,
ifabotuzumab, farletuzumab, otlertuzumab, carotuximab, epratuzumab,
inebilizumab, lumretuzumab, 4G7SDIE, AFM21, AFM22, LY-3022855,
SNDX-6352, AFM-13, BI-836826, BMS-986012, BVX-20, mogamulizumab,
ChiLob-7/4, leukotuximab, isatuximab, DS-8895, FPA144, GM102,
GSK-2857916, IGN523, IT1208, ADC-1013, CAN-04, XOMA-213,
PankoMab-GEX, chKM-4927, IGN003, IGN004, IGN005, MDX-1097, MOR202,
MOR-208, oportuzumab, ensituximab, vedotin (Adcetris), ibritumomab
tiuxetan, ABBV-838, HuMax-AXL-ADC, and ado-trastuzumab emtansine
(Kadcyla).
[0844] Embodiment 42. The method according to embodiment 39 or 40,
wherein the second antibody or antigen binding fragment thereof
induces ADCP.
[0845] Embodiment 43. The method according to embodiment 42,
wherein the second antibody or antigen binding fragment thereof is
selected from the group consisting of Rituximab, ublituximab,
margetuximab, IMGN-529, SCT400, veltuzumab, Obinutuzumab,
Trastuzumab, Cetuximab, alemtuzumab, ibritumomab, farletuzumab,
inebilizumab, lumretuzumab, 4G7SDIE, BMS-986012, BVX-20,
mogamulizumab, ChiLob-7/4, GM102, GSK-2857916, PankoMab-GEX,
chKM-4927, MDX-1097, MOR202, and MOR-208.
[0846] Embodiment 44. A method of treating an infection or
infectious disease in a human subject, comprising administering to
the subject an effective amount of an antibody or antigen binding
fragment of any one of embodiments 1-18, or an expression vector
according to one of embodiments 22 or 23, or a host cell according
to one of embodiments 24-26, or a composition according one of
embodiments 27-33, optionally in association with a further
therapeutic agent or therapeutic procedure.
[0847] Embodiment 45. An antibody having one or more of the
following characteristics: [0848] binds human SIRP.alpha.V1 protein
having the sequence of SEQ ID NO: 34 with an EC.sub.50<1 nM;
exhibits at least a 100-fold higher EC.sub.50 for
SIRP.alpha.V1(P74A) having the sequence of SEQ ID NO: 62; and
exhibits at least a 100-fold higher EC.sub.50 for human SIRP.beta.1
protein having the sequence of SEQ ID NO: 38, preferably when
measured by cellular ELISA; [0849] binds to a cell expressing human
SIRP.alpha.V1 protein with an EC.sub.50<10 nM, preferably <5
nM, more preferably <1.5 nM, still more preferably <1.0 nM,
even more preferably <0.5 nM, and most preferably about 0.3 nM
or less; [0850] binds to a cell expressing human SIRP.alpha.V2
protein with an EC.sub.50<10 nM, preferably <5 nM, more
preferably <1.5 nM, still more preferably <1.0 nM, even more
preferably <0.5 nM, and most preferably about 0.3 nM or less;
[0851] does not appreciably bind to SIRP.beta.1 protein at an
antibody concentration of 50 nM, preferably 67 nM, and more
preferably 100 nM; or alternatively at a concentration that is
10-fold greater, preferably 50-fold greater, more preferably
100-fold greater, and still more preferably 200-fold greater than
the antibody's EC.sub.50 for SIRP.alpha.V1 or SIRP.alpha.V2; [0852]
inhibits binding between human SIRP.alpha. and CD47 with an
IC.sub.50<10.0 nM, more preferably <5.0 nM, still more
preferably <2.5 nM, and most preferably about 1.0 nM or less;
and [0853] exhibits a T20 "humanness" score of at least 79, and
more preferably 85.
[0854] Embodiment 46. The antibody or antigen binding fragment of
embodiment 45 that binds human SIRP.alpha.V1 protein having the
sequence of SEQ ID NO: 34 with an EC.sub.50<1 nM; exhibits at
least a 100-fold higher EC.sub.50 for SIRP.alpha.V1(P74A) having
the sequence of SEQ ID NO: 62; and exhibits at least a 100-fold
higher EC.sub.50 for human SIRP.beta.1 protein having the sequence
of SEQ ID NO: 38.
[0855] Embodiment 47. The antibody or antigen binding fragment of
embodiment 45 or 46 that comprises one or two light chains
comprising SEQ ID NO: 20 or a sequence at least 90%, 95%, 97%, 98%,
or 99% identical thereto and one or two heavy chains comprising SEQ
ID NO: 10 or a sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto.
[0856] Embodiment 48. The antibody or antigen binding fragment of
embodiment 45 or 46 that comprises one or two light chains
comprising SEQ ID NO: 28 or a sequence at least 90%, 95%, 97%, 98%,
or 99% identical thereto and one or two heavy chains comprising SEQ
ID NO: 16 or a sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto.
[0857] Embodiment 49. The antibody or antigen binding fragment of
embodiment 45 or 46 that comprises one or two light chains
comprising SEQ ID NO: 20 or a sequence at least 90%, 95%, 97%, 98%,
or 99% identical thereto and and one or two heavy chains comprising
SEQ ID NO: 18 or a sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto.
[0858] Embodiment 50. The antibody or antigen binding fragment of
embodiment 45 or 46 that comprises one or two light chains
comprising SEQ ID NO: 90 or a sequence at least 90%, 95%, 97%, 98%,
or 99% identical thereto and one or two heavy chains comprising SEQ
ID NO: 80 or a sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto.
[0859] Embodiment 51. The antibody or antigen binding fragment of
embodiment 45 or 46 that comprises one or two light chains
comprising SEQ ID NO: 92 or a sequence at least 90%, 95%, 97%, 98%,
or 99% identical thereto and one or two heavy chains comprising SEQ
ID NO: 80 or a sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto.
[0860] Embodiment 52. The antibody or antigen binding fragment of
embodiment 45 or 46 that comprises one or two light chains
comprising SEQ ID NO: 96 or a sequence at least 90%, 95%, 97%, 98%,
or 99% identical thereto and and one or two heavy chains comprising
SEQ ID NO: 80 or a sequence at least 90%, 95%, 97%, 98%, or 99%
identical thereto.
[0861] Embodiment 53. The antibody or antigen binding fragment of
one of embodiments 45-52, wherein the antibody is an intact
IgG.
[0862] Embodiment 54. The antibody or antigen binding fragment of
one of embodiments 45-52, wherein the antibody comprises a
wild-type or mutated IgG2 Fc region.
[0863] Embodiment 55. The antibody or antigen binding fragment of
one of embodiments 45-52, wherein the antibody comprises a mutated
IgG1 Fc region.
[0864] Embodiment 56. The antibody or antigen binding fragment of
one of embodiments 45-52, wherein the antibody comprises a mutated
IgG4 Fc region.
[0865] Embodiment 57. An antibody or antigen binding fragment
thereof that binds to the same epitope of human SIRP.alpha. as an
antibody as an antibody according to one of embodiments 45-52.
[0866] Embodiment 58. The antibody or antigen binding fragment of
any of embodiments 45-52, wherein the antibody or antigen binding
fragment is humanized.
[0867] Embodiment 59. A composition comprising the antibody or
antigen binding fragment of any one of embodiments 45-52 and a
pharmaceutically acceptable carrier or diluent.
[0868] Embodiment 60. An antibody or antigen binding fragment
according to any one of embodiments 45-52 or a composition
according to embodiment 59, for the treatment of cancer or an
infectious disease.
[0869] Embodiment 61. An antibody or antigen binding fragment
according to any one of embodiments 45-52 or a composition
according to embodiment 59 for decreasing SIRP.alpha./CD47
signalling in a human subject.
[0870] Embodiment 62. A method of treating cancer in a human
subject, comprising administering to the subject an effective
amount of an antibody or antigen binding fragment according to any
one of embodiments 45-52 or a composition according to embodiment
59, optionally in association with a further therapeutic agent or
therapeutic procedure.
[0871] Embodiment 63. A method of treating an infection or
infectious disease in a human subject, comprising administering to
the subject an effective amount of an antibody or antigen binding
fragment according to any one of embodiments 45-52 or a composition
according to embodiment 59, optionally in association with a
further therapeutic agent or therapeutic procedure.
General Methods
[0872] Standard methods in molecular biology are described
Sambrook, Fritsch and Maniatis (1982 & 1989 2.sup.nd Edition,
2001 3.sup.rd Edition) Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook
and Russell (2001) Molecular Cloning, 3.sup.rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993)
Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.).
Standard methods also appear in Ausbel, et al. (2001) Current
Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,
Inc. New York, N.Y., which describes cloning in bacterial cells and
DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast
(Vol. 2), glycoconjugates and protein expression (Vol. 3), and
bioinformatics (Vol. 4).
[0873] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described (Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York). Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described (see, e.g., Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies are described (Coligan, et al. (2001) Current
Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New
York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane,
supra). Standard techniques for characterizing ligand/receptor
interactions are available (see, e.g., Coligan, et al. (2001)
Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New
York).
[0874] Monoclonal, polyclonal, and humanized antibodies can be
prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal
Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and
Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New
York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.
139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al.
(1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem.
274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684;
Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992)
J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).
[0875] An alternative to humanization is to use human antibody
libraries displayed on phage or human antibody libraries in
transgenic mice (Vaughan et al. (1996) Nature Biotechnol.
14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al.
(1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000)
Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Kay et al. (1996) Phage Display of Peptides and
Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.;
de Bruin et al. (1999) Nature Biotechnol. 17:397-399).
[0876] Single chain antibodies and diabodies are described (see,
e.g., Malecki et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218;
Conrath et al. (2001) J. Biol. Chem. 276:7346-7350; Desmyter et al.
(2001) J. Biol. Chem. 276:26285-26290; Hudson and Kortt (1999) J.
Immunol. Methods 231:177-189; and U.S. Pat. No. 4,946,778).
Bifunctional antibodies are provided (see, e.g., Mack, et al.
(1995) Proc. Natl. Acad. Sci. USA 92:7021-7025; Carter (2001) J.
Immunol. Methods 248:7-15; Volkel, et al. (2001) Protein
Engineering 14:815-823; Segal, et al. (2001) J. Immunol. Methods
248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al.
(1997) J. Biol. Chem. 272:27623; Morrison (1985) Science
229:1202-1207; Traunecker, et al. (1991) EMBO J. 10:3655-3659; and
U.S. Pat. Nos. 5,932,448, 5,532,210, and 6,129,914).
[0877] Bispecific antibodies are also provided (see, e.g., Azzoni
et al. (1998) J. Immunol. 161:3493; Kita et al. (1999) J. Immunol.
162:6901; Merchant et al. (2000) J. Biol. Chem. 74:9115; Pandey et
al. (2000) J. Biol. Chem. 275:38633; Zheng et al. (2001) J. Biol
Chem. 276:12999; Propst et al. (2000) J. Immunol. 165:2214; Long
(1999) Ann. Rev. Immunol. 17:875). Purification of antigen is not
necessary for the generation of antibodies. Animals can be
immunized with cells bearing the antigen of interest. Splenocytes
can then be isolated from the immunized animals, and the
splenocytes can fused with a myeloma cell line to produce a
hybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290;
Wright et al. (2000) Immunity 13:233-242; Preston et al., supra;
Kaithamana et al. (1999) J. Immunol. 163:5157-5164).
[0878] Antibodies can be conjugated, e.g., to small drug molecules,
enzymes, liposomes, polyethylene glycol (PEG). Antibodies are
useful for therapeutic, diagnostic, kit or other purposes, and
include antibodies coupled, e.g., to dyes, radioisotopes, enzymes,
or metals, e.g., colloidal gold (see, e.g., Le Doussal et al.
(1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol.
160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811;
Everts et al. (2002) J. Immunol. 168:883-889).
[0879] Methods for flow cytometry, including fluorescence activated
cell sorting (FACS), are available (see, e.g., Owens, et al. (1994)
Flow Cytometry Principles for Clinical Laboratory Practice, John
Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry,
2.sup.nd ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical
Flow Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent
reagents suitable for modifying nucleic acids, including nucleic
acid primers and probes, polypeptides, and antibodies, for use,
e.g., as diagnostic reagents, are available (Molecular Probes
(2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0880] Standard methods of histology of the immune system are
described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus:
Histopathology and Pathology, Springer Verlag, New York, N.Y.;
Hiatt, et al. (2000) Color Atlas of Histology, Lippincott,
Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic
Histology: Text and Atlas, McGraw-Hill, New York, N.Y.).
[0881] Software packages and databases for determining, e.g.,
antigenic fragments, leader sequences, protein folding, functional
domains, glycosylation sites, and sequence alignments, are
available (see, e.g., GenBank, Vector NTI.RTM. Suite (Informax,
Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San
Diego, Calif.); DeCypher.RTM. (TimeLogic Corp., Crystal Bay, Nev.);
Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al.
(2000) Bioinformatics Applications Note 16:741-742; Wren, et al.
(2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne
(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids
Res. 14:4683-4690).
EXAMPLES
[0882] The following examples serve to illustrate the present
invention. These examples are in no way intended to limit the scope
of the invention.
Example 1: Specificity of Commercial hSIRP.alpha. Antibodies
[0883] The specificity of various commercially available monoclonal
anti-hSIRP.alpha. antibodies (Table 7) for binding to hSIRP.alpha.
variant 1 (hSIRP.alpha.V1; GenBank accession: NM_001040022.1) (SEQ
ID NO: 34), hSIRP.alpha. variant 2 (hSIRP.alpha.V2; GenBank
accession: D86043.1) (SEQ ID NO: 36), hSIRP.beta.1 (GenBank
accession: NM_006065.4) (SEQ ID NO: 38), hSIRP.beta.1 transcript
variant 3/hSIRP.beta.L (NCBI accession: NM_001135844.3) (SEQ ID NO:
117), and hSIRP.gamma. (NCBI accession: NM_018556.3) (SEQ ID NO:
40) was evaluated by cellular ELISA (CELISA). Reactivity was
confirmed using CHO-K1 cells (ATCC CCL-61) that had been
transiently transfected, using Lipofectamine 2000, with cDNA
encoding the full length open reading frame of hSIRP.alpha.V1,
hSIRP.alpha.V2, hSIRP.beta.1, hSIRP.beta.L, and hSIRP.gamma.
subcloned into the pCI-neo vector (Promega, Madison, Wis.).
CHO-K1.hSIRP.alpha.V1, CHO-K1.hSIRP.alpha.V2, CHO-K1.hSIRP.beta.1,
CHO-K1.hSIRP.beta.L, and CHO-K1.hSIRP.gamma. cells were seeded in
culture medium (DMEM-F12 (Gibco) supplemented with 5% New Born Calf
Serum (BioWest) and Pen/Strep (Gibco)) in 96-well flat-bottom
tissue culture plates and incubated at 37.degree. C., 5% CO.sub.2
and 95% humidity for 24 hours. Subsequently, culture medium was
removed and cells were incubated for 1 hour at 37.degree. C., 5%
CO.sub.2 and 95% humidity with purified hSIRP.gamma. antibodies
(used at 10 .mu.g/mL and dilutions thereof). Next, cells were
washed with PBS-T and incubated for 1 hour at 37.degree. C., 5%
CO.sub.2 and 95% humidity with goat-anti-mouse IgG-HRP (Southern
Biotech). Subsequently, cells were washed three times with PBS-T
and immunoreactivity against hSIRP.alpha.V1, hSIRP.alpha.V2,
hSIRP.beta.1, hSIRP.beta.L, and hSIRP.gamma. was visualized with
TMB Stabilized Chromogen (Invitrogen). Reactions were stopped with
0.5 M H.sub.2SO.sub.4 and absorbances were read at 450 and 610 nm.
EC.sub.50 values, the concentration at which 50% of the total
binding signal is observed, were calculated using GraphPad Prism 6
(GraphPad Software, Inc.).
TABLE-US-00007 TABLE 7 Commercially available hSIRP.alpha.
antibodies used for comparison with antibodies generated herein.
Target Clone Cat. # Company Species Reactivity Isotype hSIRP.alpha.
SE5A5 323802 Biolegend mouse human IgG1 hSIRP.alpha. 7B3 LS-C340387
LifeSpan Biosciences mouse human IgG1 hSIRP.alpha. 1B5 LS-C338479
LifeSpan Biosciences mouse human IgG1 hSIRP.alpha. 1C6 LS-C338477
LifeSpan Biosciences mouse human IgG1 hSIRP.alpha. 27 sc-136067
Santa Cruz mouse human, mouse, rat IgG1 Biotechnology hSIRP.alpha.
SE7C2 sc-23863 Santa Cruz mouse human IgG1 Biotechnology
hSIRP.alpha. P3C4 LS-C179629-100 CliniSciences mouse human IgG2a
hSIRP.alpha. 2A4A5 W172-3 MBL International mouse human IgG2a
hSIRP.alpha. 15-414 LS-C58098 LifeSpan Biosciences mouse human
IgG2a hSIRP.alpha. 1H1 LS-C338476 LifeSpan Biosciences mouse human
IgG2a hSIRP.alpha. C-7 sc-376884 Santa Cruz mouse human IgG2a
Biotechnology hSIRP.alpha. 03 11612-MM03-100 Sino Biological Inc.
mouse human IgG2b hSIRP.alpha. 5E10 LS C83566 LifeSpan Biosciences
mouse human IgG2b hSIRP.alpha. 602411 MAB4546 R&D mouse human
IgG2b hSIRP.alpha. EPR16264 ab191419 Abcam rabbit human, mouse, rat
IgG hSIRP.alpha. D6I3M 13379S Cell Signaling rabbit human, mouse,
rat, IgG Technology monkey hSIRP.alpha. 001 50956- Sino Biological
Inc. rabbit mouse, human IgG R001_100ug hSIRP.alpha. REA144
130-099-768 Miltenyi Biotec human human IgG1 hSIRP.alpha. KWAR23
TAB-453CT Creative Biolabs human human IgG4
[0884] As depicted in FIG. 1 and the following Table 8,
commercially available hSIRP.alpha. antibodies cross-react with at
least hSIRP.beta.1, hSIRP.beta.L, or hSIRP.gamma. or demonstrate
allele-specific binding to hSIRP.alpha.V2. The KWAR23 antibody
cross-reacts with all members of the SIRP receptor family tested:
it binds to hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.1,
hSIRP.beta.L, and hSIRP.gamma..
TABLE-US-00008 TABLE 8 hSIRP.alpha.V1 hSIRP.alpha.V2 hSIRP.beta.1
hSIRP.gamma. hSIRP.beta.L binding binding binding binding binding
EC50 EC50 EC50 EC50 EC50 Antibody (nM) (nM) (nM) (nM) (nM)
hSIRP.alpha..50A 1.626 1.627 nd 1.475 0.639 anti-hSIRP.alpha.
(clone SE5A5) 0.372 0.186 0.185 0.200 0.122 anti-hSIRP.alpha.
(clone 7B3) 0.187 0.300 0.255 nd 0.206 anti-hSIRP.alpha. (clone
1B5) nd 0.122 nd nd nd anti-hSIRP.alpha. (clone 1C6) 0.739 0.167
2.965 15.589 2.008 anti-hSIRP.alpha. (clone 27) nd nd nd nd nd
anti-hSIRP.alpha. (clone SE7C2) 1.269* 0.300 nd 1.525 26.818*
anti-hSIRP.alpha. (clone P3C4) 0.288 2.154 0.383 0.365 0.136
anti-hSIRP.alpha. (clone 2A4A5) nd 1.005 8.633 nd 12.156*
anti-hSIRP.alpha. (clone 15-414) nd nd nd nd nd anti-hSIRP.alpha.
(clone 1H1) nd 0.204 nd nd nd anti-hSIRP.alpha. (clone C-7) nd nd
nd nd nd anti-hSIRP.alpha. (clone 03) 96.016* 15.059* 16.043*
17.303* 9.109* anti-hSIRP.alpha. (clone 5E10) nd nd nd nd nd
anti-hSIRP.alpha. (clone 602411) 0.068 nd 0.081 3.622 0.060
anti-hSIRP.alpha. (clone EPR16264) nd 2.450* nd nd nd
anti-hSIRP.alpha. (clone D6I3M) 18.690* 8.762* nd nd nd
anti-hSIRP.alpha. (clone 001) 18.081* nd nd 0.494 6.253*
anti-hSIRP.alpha. (clone REA144) 5.243* 3.274* 4.534* 3.212* 2.147*
KWAR23 0.067 0.062 0.140 0.043 0.097 Values indicated with * were
extrapolated; nd, not detected
Example 2: Immunization and Selection of Anti-hSIRP.alpha.
Antibodies
[0885] To generate SIRP.alpha. antibodies that bind to all known
SIRP.alpha. alleles and are not binding SIRP.beta.1 mice were
immunized with a pCI-neo expression construct encoding
hSIRP.alpha.V1 and hSIRP.alpha.V2. Mice were immunized by gene gun
immunization using a Helios Gene gun (BioRad, Hercules, Calif.) and
DNA coated gold bullets (BioRad) following manufacturer's
instructions. Briefly, 1 .mu.m gold particles were coated with
pCI-neo-hSIRP.alpha.V1 or pCI-neo-hSIRP.alpha.V2 cDNA and
commercial expression vectors for mouse Flt3L and mouse GM-CSF in a
2:1:1 ratio (both from Aldevron, Fargo, N. Dak.). A total of 1
.mu.g of plasmid DNA was used to coat 500 .mu.g of gold particles.
Specifically, 7-8 weeks old female BALB/C mice (Harlan) were
immunized in the ears with a gene gun, receiving 3 administration
cycles in both ears.
[0886] For positive and negative B-cell selection and CELISA
purposes, CHO-K1.hSIRP.alpha.V1, CHO-K1.hSIRP.alpha.V2,
CHO-K1.hSIRP.beta.1, and CHO-K1.hCD47 stable cell lines were
generated by transfecting CHO-K1 cells with pCI-neo vector encoding
the full length open reading frame of hSIRP.alpha.V1,
hSIRP.alpha.V2, hSIRP.beta.1, and hCD47 (NCBI accession:
NM_001777.3) (SEQ ID NO: 42), respectively. Stable clones were
obtained by limiting dilution.
[0887] Antibody titer was assessed by CELISA, using the
CHO-K1.hSIRP.alpha.V1 and CHO-K1.hSIRP.alpha.V2 stable cell lines.
These hSIRP.alpha.-expressing CHO-K1 cell lines were maintained in
DMEM-F12 (Gibco) supplemented with 10% Fetal Bovine Serum (Hyclone)
and 80U Pen/Strep (Gibco). Cells were seeded into 96-well
flat-bottom tissue culture plates at 8.times.10.sup.4 cells/well
and cultured at 37.degree. C., 5% CO.sub.2 and 95% humidity until
cell layers were confluent. Cells were incubated with each sample
of the diluted mouse sera for 1 hour at 37.degree. C., 5% CO.sub.2
and 95% humidity. Next, cells were washed with Phosphate buffered
Saline (PBS)/0.05% Tween-20 (PBS-T) and incubated with
goat-anti-mouse IgG-HRP conjugate (Southern Biotech) for 1 hour at
37.degree. C., 5% CO.sub.2 and 95% humidity. Subsequently, cells
were washed three times with PBS-T and anti-hSIRP.alpha.
immunoreactivity was visualized with TMB Stabilized Chromogen
(Invitrogen). Reactions were stopped with 0.5 M H.sub.2SO.sub.4 and
absorbances were read at 450 and 610 nm. The anti-hSIRP.alpha.
titer was higher than 1:2,500 in each individual mouse serum sample
as detected after two DNA immunizations. All mice that demonstrated
reactivity against hSIRP.alpha.V1 and hSIRP.alpha.V2 were immunized
for a final, third time and sacrificed 14 days later.
Erythrocyte-depleted spleen and lymph-node cell populations were
prepared as described previously (Steenbakkers et al., 1992, J.
Immunol. Meth. 152: 69-77; Steenbakkers et al., 1994, Mol. Biol.
Rep. 19: 125-134) and frozen at -180.degree. C.
[0888] To select anti-hSIRP.alpha. antibody producing B-cells, a
selection strategy was designed and developed that preferentially
bound B-cells expressing antibodies that bind to hSIRP.alpha.V1 and
hSIRP.alpha.V2. Splenocytes and lymph nodes were harvested from the
hSIRP.alpha.V1/V2 immunized mice and isolated cells were incubated
with CHO-K1.hSIRP.beta.1 that were seeded into T25 culture flasks
and irradiated at 30 Gray. After 1 hour unbound cells were gently
removed by moving the flask back and forth. Medium containing
unbound cells was then transferred to a new T25 flask containing
irradiated CHO-K1.hSIRP.beta.1 cells. This procedure was followed
for in total three times on ice in order to negatively select
hSIRP.beta.1-reactive B-cells. Next, medium containing unbound
B-cells was incubated with CHO-K1.hSIRP.alpha.V1 and
CHO-K1.hSIRP.alpha.V2 cells that were irradiated at 3,000 Gray.
After 1.5 hours incubation on ice unbound cells were removed with
multiple wash steps using culture medium. Subsequently, T25 flasks
containing CHO-K1.hSIRP.alpha.V1 and CHO-K1.hSIRP.alpha.V2 cells
with bound lymphocytes were harvested with Trypsin-EDTA (Sigma).
Bound B-cells were cultured, as described by Steenbakkers et al.,
1994, Mol. Biol. Rep. 19: 125-134. Briefly, selected B-cells were
mixed with 10% (v/v) T-cell supernatant and 50,000 irradiated (25
Gray) EL-4 B5 feeder cells in a final volume of 200 .mu.l medium in
96-well flat-bottom tissue culture plates. On day eight,
supernatants were screened for hSIRP.alpha.V1 and hSIRP.alpha.V2
reactivity by CELISA as described below.
[0889] CHO-K1.hSIRP.alpha.V1, CHO-K1.hSIRP.alpha.V2, and
CHO-K1.hSIRP.beta.1 were seeded in culture medium (DMEM-F12 (Gibco)
supplemented with 10% Fetal Bovine Serum (Hyclone) and 80U
Pen/Strep (Gibco)) in 96-well flat-bottom tissue culture plates and
cultured at 37.degree. C., 5% CO.sub.2 and 95% humidity until they
were confluent. Subsequently, culture medium was removed and cells
were incubated for 1 hour at 37.degree. C., 5% CO.sub.2 and 95%
humidity with supernatants from the B-cell cultures. Next, cells
were washed with PBS-T and incubated for 1 hour at 37.degree. C.,
5% CO.sub.2 and 95% humidity with goat-anti-mouse IgG-HRP conjugate
(Southern Biotech). Subsequently, cells were washed three times
with PBS-T and anti-hSIRP.alpha.V1, anti-hSIRP.alpha.V2, and
anti-hSIRP.beta.1 immunoreactivity was visualized with TMB
Stabilized Chromogen (Invitrogen). Reactions were stopped with 0.5
M H.sub.2SO.sub.4 and absorbances were read at 450 and 610 nm.
[0890] Immunoreactivity to human SIRP.gamma. was assessed by ELISA
using recombinant hSIRP.gamma./Fc-protein (R&D Systems,
Cat.#4486-SB-050; SEQ ID NO: 108) coated 96-well MaxiSorp
flat-bottom plates. Protein coated 96-well plates were blocked in
PBS/1% bovine serum albumin (BSA) for 1 hour at room temperature
(RT). PBS/1% BSA was removed and plates were incubated for 1 hour
at RT with supernatants from the B-cell cultures. Next, plates were
washed with PBS-T and incubated for 1 hour at RT with
goat-anti-mouse IgG-HRP conjugate (Southern Biotech). Subsequently,
wells were washed three times with PBS-T and anti-hSIRP.gamma.
immunoreactivity was visualized with TMB Stabilized Chromogen
(Invitrogen). Reactions were stopped with 0.5 M H.sub.2SO.sub.4 and
absorbances were read at 450 and 610 nm.
[0891] B-cell clones from the hSIRP.alpha. reactive supernatants,
which were not or which were minimally reactive to hSIRP.beta.1
were immortalized by mini-electrofusion following published
procedures (Steenbakkers et al., 1992, J. Immunol. Meth. 152:
69-77; Steenbakkers et al., 1994, Mol. Biol. Rep. 19:125-34) with
some minor deviations (e.g. pronase reaction was omitted). Briefly,
B-cells were mixed with 10.sup.6 Sp2/0-Ag14 murine myeloma cells
(ATCC CRL-1581) in Electrofusion Isomolar Buffer (Eppendorf).
Electrofusions were performed in a 50 .mu.L fusion chamber by an
alternating electric field of 15 s, 1 MHz, 23 Vrms AC followed by a
square, high field DC pulse of 10 .mu.s, 180 Volt DC and again by
an alternating electric field of 15 s, 1 MHz, 23 Vrms AC. Content
of the chamber was transferred to hybridoma selective medium and
plated in a 96-well plate under limiting dilution conditions. On
day 10 following the electrofusion, hybridoma supernatants were
screened for hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.1, and
hSIRP.gamma. binding activity by CELISA and ELISA, as described
above. Hybridomas that secreted antibodies in the supernatant that
specifically bound hSIRP.alpha.V1 and hSIRP.alpha.V2 were both
frozen at -180.degree. C. (-1 batch) and subcloned by limited
dilution to safeguard their integrity and stability. Stable
hybridomas were frozen at -180.degree. C. (-LD1 batch) until cell
layers were confluent.
[0892] Further selection of the hybridomas was performed by
assessing the blocking abilities of the hSIRP.alpha.V1/hCD47
interaction in CELISA format. For the assessment of hCD47 blockade
CHO-K1.hCD47 cells were seeded in 384-well flat-bottom tissue
culture plates and incubated at 37.degree. C., 5% CO.sub.2 and 95%
humidity in culture medium. Recombinant hSIRP.alpha./Fc-protein
(R&D Systems, Cat.#4546-SA-050; SEQ ID NO: 107) was
pre-incubated with a dilution series of the hybridoma supernatants
containing hSIRP.alpha. reactive antibodies and control antibodies
(at 10 .mu.g/mL and dilutions thereof) for 30 minutes at 37.degree.
C., 5% CO.sub.2 and 95% humidity. Confluent CHO-K1.hCD47 cells were
washed with PBS-T and incubated for 1 hour with the mixtures
containing hSIRP.alpha. reactive antibodies and recombinant
hSIRP.alpha./Fc-protein at 37.degree. C., 5% CO.sub.2 and 95%
humidity. Next, cells were washed with PBS-T followed by addition
of goat-anti-human IgG-HRP conjugate (Jackson Immuno Research) to
the cells, which was incubated for 1 hour at 37.degree. C., 5%
CO.sub.2 and 95% humidity. Subsequently cells were washed three
times with PBS-T and binding of hSIRP.alpha./Fc-protein was
visualized with TMB Stabilized Chromogen (Invitrogen). Reactions
were stopped with 0.5 M H.sub.2SO.sub.4 and absorbances were read
at 450 and 610 nm.
[0893] Selected stable hybridomas were cultured in serum-free media
for 7 days; supernatants were harvested and antibodies were
purified using MabSelect Sure Protein A resin according to the
manufacturer's instructions (GE Healthcare). Antibody
concentrations were quantified using spectrophotometry.
Supernatants of the hybridoma cultures were used to isotype the
hybridomas. In short, isotyping was done using a mouse monoclonal
antibody isotyping kit (Biorad) based on a dipstick with
immobilized goat-anti-mouse antibody bands to each of the common
mouse isotypes and light chains. Recovered antibodies were all
identified as mouse IgG1. Antibody sequences were elucidated by
sequencing of variable regions of the mouse IgG1 hybridoma material
performed at LakePharma, using the following method: the total RNA
of the hybridoma cells was extracted, which allowed cDNA synthesis.
Rapid Amplification of cDNA Ends (RACE) was performed that allowed
cloning of positive fragments in a TOPO (Thermo Fisher Scientific)
vector. TOPO clones were sequenced and sequences were annotated
using VBASE2 (Retter et al., VBASE2, an integrative V gene
database. Nucleic Acids Res. 2005 Jan. 1; 33(Database
issue):D671-4).
Example 3: Characterization of hSIRP.alpha. Antibodies
[0894] The binding specificity of antibody hSIRP.alpha..50A to
hSIRP.alpha. was compared antibody KWAR23 (Canadian Patent 2939293
A1), in a CELISA format. CHO-K1 cells were transiently transfected
with hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.1, and hSIRP.gamma.
(GenBank accession: NM_018556.3) (SEQ ID NO: 39) cDNAs.
Subsequently, hSIRP.alpha. binding was assessed by CELISA using
CHO-K1.hSIRP.alpha.V1, CHO-K1.hSIRP.alpha.V2, CHO-K1.hSIRP.beta.1,
and CHO-K1.hSIRP.gamma. cells. Detection of bound antibody was
performed with goat-anti-mouse IgG-HRP (Southern Biotech) for mouse
antibodies including hSIRP.alpha..50A and control antibodies or,
alternatively, with goat-anti-human IgG-HRP conjugate (Jackson
Immuno Research) for the KWAR23 antibody. KWAR23 (SEQ ID NO: 130;
SEQ ID NO: 131) was expressed as a chimeric human IgG4 kappa
antibody in CHO cells. As shown in FIG. 2 and the following Table
9, KWAR23 antibody cross-reacts with all members of the SIRP
receptor family tested: it binds to hSIRP.alpha.V1, hSIRP.alpha.V2,
hSIRP.beta.1, and hSIRP.gamma.. EC.sub.50 values represent the
concentration at which 50% of the total binding signal is observed
(average and SD were calculated from values of two independent
experiments).
TABLE-US-00009 TABLE 9 hSIRP.alpha.V1 binding EC50 (nM)
hSIRP.alpha.V2 binding EC50 (nM) Antibody Average SD Average SD
KWAR23 0.081 0.001 0.051 0.004 hSIRP.alpha..50A 1.365 0.164 1.296
0.186 anti-hSIRP.alpha. (clone SE5A5) 0.304 0.200 anti-hSIRP.gamma.
(clone LSB2.20) nd nd hSIRP.beta.1 binding EC50 (nM) hSIRP.gamma.
binding EC50 (nM) Antibody Average SD Average SD KWAR23 0.161 0.007
0.040 0.002 hSIRP.alpha..50A nd nd 1.249 0.179 anti-hSIRP.alpha.
(clone SE5A5) 0.192 0.168 anti-hSIRP.gamma. (clone LSB2.20) nd
0.265 Empty squares indicate n = 1 measurements. nd, not
detected
[0895] In addition, the specificity of hSIRP.alpha..50A for all
known of hSIRP.alpha. alleles (allelic variants as described by
Takenaka et al., 2007, Nat Immunol. 8:1313-1323) was further
investigated by CELISA using the same strategy as above. To this
end, hSIRP.alpha..50A binding was assessed using CHO-K1 cells that
were transiently transfected with cDNAs encoding full length
hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.alpha.V3 (NA07056_V3) (SEQ ID
NO: 43), hSIRP.alpha.V4 (NA11832_V4) (SEQ ID NO: 45),
hSIRP.alpha.V5 (NA18502_V5) (SEQ ID NO: 47), hSIRP.alpha.V6
(NA18507_V6) (SEQ ID NO: 49), hSIRP.alpha.V8 (NA18570_V8) (SEQ ID
NO: 51), and hSIRP.alpha.V9 (NA18943_V9) (SEQ ID NO: 53). FIG. 3
and the following Table 10 demonstrate the reactivity of antibody
clone hSIRP.alpha..50A for each of these hSIRP.alpha. alleles. EC50
values represent the concentration at which 50% of the total
binding signal is observed (average and SD were calculated from
values of two independent experiments).
TABLE-US-00010 TABLE 10 Antibody anti-hSIRP.alpha. hSIRP.alpha..50A
(clone SE5A5) hSIRP.alpha.V1 EC50 (nM) 0.936 0.327 SD 0.285 0.107
hSIRP.alpha.V2 EC50 (nM) 0.665 0.200 SD 0.106 0.046 hSIRP.alpha.V3
EC50 (nM) 0.688 0.226 SD 0.097 0.052 hSIRP.alpha.V4 EC50 (nM) 0.824
0.256 SD 0.280 0.085 hSIRP.alpha.V5 EC50 (nM) 0.765 0.276 SD 0.210
0.086 hSIRP.alpha.V6 EC50 (nM) 0.954 0.098 SD 0.437 0.050
hSIRP.alpha.V8 EC50 (nM) 0.644 0.300 SD 0.066 0.061 hSIRP.alpha.V9
EC50 (nM) 0.733 0.260 SD 0.205 0.079
Example 4: hCD47 Blocking Ability of hSIRP.alpha..50A
[0896] The hSIRP.alpha..50A antibody was analyzed by flow cytometry
for its ability to block recombinant hCD47/Fc-protein (R&D
Systems, Cat.#4670-CD-050; SEQ ID NO: 109) binding to cell surface
expressed hSIRP.alpha.. For this purpose, THP-1 (ATCC TIB-202) and
U-937 (ATCC CRL-1593.2) monocyte cell lines were used as the source
of hSIRP.alpha. in the assay. THP-1 and U-937 cells were seeded in
96-well round bottomed tissue culture plates and incubated for 45
minutes with FcR Blocking Reagent (Miltenyi Biotec) and
hSIRP.alpha..50A antibody (200 .mu.g/mL and dilutions thereof) in
PBS/1% BSA at 4.degree. C. Next, cells were washed three times with
PBS/1% BSA and incubated with DyLight 488-labeled recombinant
hCD47/Fc-protein for 30 minutes at 4.degree. C. After this labeling
procedure, cells were washed two times, resuspended in PBS/1% BSA
containing 0.1 .mu.g/mL DAPI (BioLegend), and analysed by flow
cytometry on the FACSCanto II (BD Biosciences). Data were processed
and analysed with FlowJo V10 software (FlowJo, LLC).
[0897] As depicted in FIG. 4 and the following Table 11, binding of
recombinant hCD47 fused to an Fc domain of human IgG1 was monitored
in the presence of increasing amounts of the hSIRP.alpha..50A
antibody. Antibody hSIRP.alpha..50A blocked the hSIRP.alpha./hCD47
interaction, using the flow cytometry-based method described above.
IC50 values for the blockade of hCD47 were calculated from this
data. IC50 values represent the concentration at which half of the
inhibition is observed.
TABLE-US-00011 TABLE 11 THP-1 U-937 Antibody IC50 (nM) IC50 (nM)
hSIRP.alpha..50A 4.605 7.164
[0898] Next, the binding of hSIRP.alpha..50A to hSIRP.alpha.
expressed on primary human CD14.sup.+ monocytes was investigated.
In addition, the ability of hSIRP.alpha..50A to block the
interaction between hSIRP.alpha. and recombinant hCD47/Fc-protein
was assessed. For this purpose, CD14+ monocytes were isolated from
Ficoll-purified human peripheral blood mononuclear cells (PBMCs)
using RosetteSep human monocyte enrichment cocktail (Stemcell). The
percentage of monocytes present after the enrichment was determined
by flow cytometry on the FACSVerse (BD Biosciences) based on CD14
staining using an APC-Cy7-conjugated mouse-anti-human CD14
detection antibody (BD Biosciences). Subsequently, CD14+ enriched
PBMCs were seeded in 96-well round bottomed tissue culture plates
and incubated for 40 minutes with FcR Blocking Reagent (Miltenyi
Biotec) containing hSIRP.alpha..50A antibody (25 .mu.g/mL and
dilutions thereof) in PBS/1% BSA at 4.degree. C. Next, cells were
washed three times with PBS/1% BSA and incubated with a
FITC-labeled goat-anti-mouse Ig (BD Biosciences) detection antibody
in PBS/1% BSA for 40 minutes at 4.degree. C. After this labeling
procedure, cells were washed two times, resuspended in PBS/1% BSA
containing 0.1m/mL DAPI (BioLegend), and analysed by flow cytometry
on the FACSVerse (BD Biosciences). Data were processed and analysed
with FlowJo V10 software (FlowJo, LLC).
[0899] FIGS. 5A and B and the following Table 12 indicates that
hSIRP.alpha..50A binds to primary human CD14+ enriched monocytes.
EC50 values represent the concentration at which 50% of the total
binding signal is observed. To assess the blocking ability of
hSIRP.alpha..50A, CD14+ enriched monocytes cells were seeded in
96-well round bottomed tissue culture plates and incubated for 45
minutes with FcR Blocking Reagent (Miltenyi Biotec) and
hSIRP.alpha..50A antibody (200m/mL and dilutions thereof) in PBS/1%
BSA at 4.degree. C. Thereafter, cells were washed three times with
PBS/1% BSA and incubated with 10m/mL DyLight 488-labeled
recombinant hCD47/Fc-protein for 45 minutes at 4.degree. C. After
this labeling procedure, cells were washed two times, resuspended
in PBS/1% BSA containing 0.1m/mL DAPI (BioLegend), and analysed by
flow cytometry on the FACSVerse (BD Biosciences). Data were
processed and analysed with FlowJo V10 software (FlowJo, LLC).
FIGS. 5 C and D and the following Table 12 demonstrates the ability
of antibody hSIRP.alpha..50A to block the hSIRP.alpha./hCD47
interaction. IC50 values for the blockade of hCD47 were calculated
from this data. IC50 values represent the concentration at which
half of the inhibition is observed.
TABLE-US-00012 TABLE 12 Donor 1 Donor 2 Antibody EC50 (nM) IC50
(nM) EC50 (nM) IC50 (nM) hSIRP.alpha..50A 7.381 4.618 3.081
1.035
Example 5: Functionality of hSIRP.alpha..50A mAb in the Human
Granulocyte Phagocytosis Assay
[0900] To confirm the functionality of hSIRP.alpha..50A in primary
immune cells, granulocytes (e.g. effector cells) were isolated from
healthy human donor EDTA blood. First, the EDTA blood of each donor
was pooled and centrifuged at 300 g for 6 minutes at 20.degree. C.
Next, plasma was removed by aspiration, and the remaining blood
cells were gently resuspended. Cells were recovered in red blood
cell (RBC) lysis buffer (155 mM NH4C1; 10 mM KHCO3) and incubated
for 10 minutes on ice. Next, cells were centrifuged at 300 g for 7
minutes. Supernatants containing lysed RBCs were removed by
aspiration, and the remaining blood cells were gently resuspended
in RBC lysis buffer and kept on ice for 1 minute. RBC lysis was
neutralized by adding assay medium (IMDM (Gibco) supplemented with
10% Fetal Bovine Serum (Gibco) and Pen/Strep (Gibco)). Blood cells
were centrifuged at 300 g for 6 minutes and supernatants were
removed by aspiration to remove remaining RBCs as much as possible.
Subsequently, erythrocyte-lysed blood cells were resuspended in
assay medium containing 10 ng/mL IFN.gamma. and cells were
incubated for 1 hour at 37.degree. C., 5% CO.sub.2 and 95%
humidity. Non-adherent blood cells containing human granulocytes
were collected by mild washing of the tissue culture plate with
assay medium (monocytes are depleted due to adherence to the
plastic surface). The percentage of granulocytes present in the
cell suspension was determined by flow cytometry on the FACSCanto
II (BD Biosciences) based on high forward scatter (FSC) and side
scatter (SSC). Binding of hSIRP.alpha..50A to human granulocytes
was assessed by incubating the cells for 30 minutes at 4.degree. C.
with hSIRP.alpha..50A antibody (25 .mu.g/mL and dilutions thereof)
in PBS/1% BSA containing 10% autologous serum (PBS/1% BSA/10%
serum). Next, cells were washed three times with PBS/1% BSA/10%
serum and incubated for 30 minutes at 4.degree. C. with a
FITC-labeled goat-anti-mouse Ig (BD Biosciences) detection
antibody. After this labeling procedure, cells were washed two
times, resuspended in PBS/1% BSA/10% serum and analysed by flow
cytometry on the FACSCanto II (BD Biosciences). Data were processed
and analysed with FlowJo V10 software (FlowJo, LLC). FIG. 6A shows
that hSIRP.alpha..50A binds to primary human granulocytes. EC50
values represent the concentration at which 50% of the total
binding signal is observed.
[0901] Next, target cells were fluorescently labeled with either
cell proliferation dye eFluor450 (eBioscience) in the case of Raji
(ECACC 85011429), Daudi (ECACC 85011437), Ramos (ECACC 85030802),
and BJAB (DSMZ ACC-757) lymphoma cells or, alternatively, with
Vybrant DiD cell-labeling solution (Thermo Fisher Scientific) for
FaDu cells. Labeling was performed according to manufacturer's
instructions. Labeled target cells were co-cultured for 2-3 hours
at 37.degree. C., 5% CO.sub.2 and 95% humidity with isolated
primary human granulocytes in a 1:1 ratio (7.5*10.sup.4 cells of
each target and effector per well of a 96-well round bottomed
tissue culture plate) in the presence of 0.1 .mu.g/mL rituximab
(anti-hCD20). In addition, cells were co-cultured with 0.1 .mu.g/mL
rituximab in presence of 10 .mu.g/mL hSIRP.alpha..50A. Phagocytosis
was assayed by determining the percentage of granulocytes positive
for eFluor450 (or DID) using flow cytometry on the FACSCanto II (BD
Biosciences). Data were processed and analysed with FlowJo V10
software (FlowJo, LLC).
[0902] Compared to the mouse IgG1 isotype control, hSIRP.alpha..50A
potently enhances tumor cell phagocytosis induced by rituximab
(FIG. 6B). The same procedure was followed with other existing
therapeutic antibodies such as 0.05 .mu.g/mL daratumumab
(anti-hCD38), 0.1 .mu.g/mL alemtuzumab (anti-hCD52), and 0.1
.mu.g/mL cetuximab (anti-hEGFR) (FIG. 6C-E). These data demonstrate
that hSIRP.alpha..50A enhances antibody-mediated tumor cell
phagocytosis by human granulocytes.
Example 6: Functionality of hSIRP.alpha..50A mAb in the Human
Macrophage Phagocytosis Assay
[0903] Blockade of CD47 by hSIRP.alpha..50A enhances the
phagocytosis of human lymphoma cells tumor cells by human
macrophages. Human macrophages were generated by first enriching
CD14+ monocytes from Ficoll-purified human peripheral blood
mononuclear cells (PBMCs) using RosetteSep human monocyte
enrichment cocktail (Stemcell). Monocytes were seeded into
CellCarrier 96-well flat-bottom microplates (Perkin Elmer) and
cultured in macrophage medium (IMDM (Gibco) supplemented with 8.5%
Fetal Bovine Serum (Gibco) and Pen/Strep (Gibco)) containing 50
ng/mL human monocyte colony stimulating factor (M-CSF) for 7 days
at 37.degree. C., 5% CO.sub.2 and 95% humidity to promote
differentiation into macrophages. These monocyte-derived
macrophages (MDMs) become adherent allowing other cells to be
washed away. Human Raji, Daudi, Ramos, and BJAB lymphoma cells were
counted and labeled with cell proliferation dye eFluor450
(eBioscience) following manufacturer's instructions. After
labeling, the lymphoma cells were mixed with assay medium (RPMI
(Gibco) supplemented with 10% Fetal Bovine Serum (Gibco) and
Pen/Strep (Gibco)) containing 10 .mu.g/mL anti-hSIRP.alpha.
antibodies, respective isotype controls and either 0.1 .mu.g/mL
rituximab (anti-hCD20) or 0.05 .mu.g/mL daratumumab (anti-hCD38).
The lymphoma cells were then added to the individual wells
containing MDMs at a ratio of 2.5:1 tumor cells per phagocyte,
mixed and incubated at 37.degree. C., 5% CO.sub.2 and 95% humidity
for 2 hours. After the incubation, the wells were washed with PBS
to remove most of the non-phagocytosed tumor cells, and cells were
fixed with 2% formaldehyde for 10 min at RT. The wells were then
washed and maintained in PBS/3% BSA in dark at 4.degree. C.
overnight. Lymphoma cells present in the wells were stained with
biotin-conjugated anti-human CD19 clone HIB19 (eBioscience) for 1
hour at RT, and subsequently were counterstained with Alexa Fluor
488-conjugated streptavidin (Thermo Fisher Scientific) for 1 hour
at RT. Next, nuclei were stained with DRAQ5 (Thermo Fisher
Scientific) for 10 minutes at RT, mixture was removed, and PBS was
added to each well. Cells were analysed with the Operetta automated
fluorescence microscope (Perkin Elmer). Data were processed and
analysed with Columbus V2.6 software.
[0904] As shown in FIG. 7, hSIRP.alpha..50A enhances rituximab and
daratumumab-mediated phagocytosis activity. The phagocytosis of
human lymphoma cells was quantified using a phagocytosis index, as
follows: (number of tumor cells inside macrophages/number of
macrophages)*100; counting at least 200 macrophages per sample.
Example 7: Humanized Antibody Design and CDR Grafting
[0905] The mouse hSIRP.alpha..50A antibody was humanized using
CDR-grafting technology (see e.g. U.S. Pat. No. 5,225,539 and
Williams, D. G. et al., 2010, Antibody Engineering, volume 1,
Chapter 21).
[0906] First, human germline sequences were identified using
IgBLAST (Ye J. et al., 2013, Nucleic Acids Res. 41:W34-40). For the
hSIRP.alpha..50A VH human germline sequence, V-gene IGHV1/OR15-2*02
was identified (75.2% identity) and for the VL human germline
sequence IGKV1-27*01 was identified (74.0% identity). These two
germline sequences were used to directly graft the mouse CDRs,
resulting in the following two cDNA constructs: SEQ ID NO: 17 (VH)
and SEQ ID NO: 25 (VL).
[0907] Next, a database was constructed containing all human
sequences available in the IMGT database (Lefranc, M.-P. et al.,
1999, Nucleic Acid Res. 27:209-212) identifying 85,848 individual
sequences. These sequences were queried using TBLASTN (2.2.31+) to
identify template sequences that demonstrated the highest identify
to the framework of hSIRP.alpha..50A VH and VL sequences. Three VH
and three VL sequences were identified that demonstrated a
similarity score of 75% or higher and that displayed similar CDR
lengths, preferably identical to those in hSIRP.alpha..50A VH CDR1,
CDR2, CDR3 and VL CDR1, CDR2 and CDR3, respectively.
[0908] For the heavy chain, the frameworks encoded by GenBank
(Benson, D. A. et al., 2013, Nucleic Acids Res. 41(D1): D36-42)
accession # AB066948, AB067235, and U84168 were selected as
templates for straight grafting of the hSIRP.alpha..50A VH CDRs,
resulting in the following cDNA constructs: SEQ ID NO: 9, 11 and
13, respectively. For the light chain, the frameworks encoded by
GenBank accession # JF894288, AB363321, and L12101 were selected as
templates for straight grafting of the hSIRP.alpha..50A VL CDRs,
resulting in the following cDNA constructs: SEQ ID NO: 19, 21 and
23. Framework and CDR definition were those as described by Kabat
et al. ("Sequences of Proteins of Immunological Interest", Kabat,
E., et al., US Department of Health and Human Services,
(1983)).
[0909] To understand the effect of humanized framework residues on
the structure of the Fv, a homology model of the mouse
hSIRP.alpha..50A Fv was made using the `Antibody Modeling Cascade`
(default parameters) within Discovery Studio 4.5. The homology
model was built on basis of PDB ID 1CIC, for the light chain and
Fv, and PDB ID 4QOX for the heavy chain. The CDRs were grafted in
silico to study residues that are close to any of the CDRs and
which might affect the loop conformation, referred as Vernier
residues. Residues that might affect the loop conformation, and
which are within <5 .ANG. to the CDR surface were identified and
substituted with the mouse amino acid at this position. The
resulting templates were checked for the presence of post
translational modification (PTM) motifs using Discovery Studio 4.5
and where possible (i.e. non-CDR, non-Vernier residues) changed to
prevent a PTM. For the heavy chain, removal of the predicted
sequence PTM motifs and structural considerations (i.e. rigidity of
the backbone) in the hSIRP.alpha..50A VH resulted in the design of
one additional construct: SEQ ID NO: 15. For the light chain the
PTM removal resulted in the following construct: SEQ ID NO: 27.
[0910] CDRs were grafted on each of the identified templates,
expressed as a human IgG4 (SEQ ID NO: 65), kappa (SEQ ID NO: 63)
antibody cloned in the pcDNA3.1(+) vector (Thermo Fisher
Scientific) and for transient transfection in FreeStyle 293-F human
embryonic kidney cells (HEK293T/17, ATCC CRL-11268). In each case,
an IgG4 version carrying the stabilizing Adair mutation (Angal S.
et al., 1993, Mol Immunol. 30: 105-108), where Serine 228 is
converted to Proline, was used.
Example 8: Synthesis, Expression and Purification of Humanized
Constructs
[0911] Plasmids encoding the heavy chain and light chain constructs
were mixed in a 1:1 ratio (30m in total) and transiently expressed
by transfection into FreeStyle 293-F cells using 293fectin
transfection reagent (Invitrogen) following the manufacturer's
instructions. Supernatants (30 ml) were harvested after 7 days and
antibodies were purified using MabSelect Sure Protein A resin
according to the manufacturer's instructions (GE Healthcare).
Buffer was exchanged for 10 mM Histidine, 100 mM NaCl pH 5.5 buffer
using Zeba desalting columns (Thermo Fisher Scientific). The
concentration of purified antibodies was determined based on OD280
(Nanodrop ND-1000). Endotoxin level was determined by LAL-test
according to the manufacturer's instructions (Lonza).
Example 9: Binding of Humanized SIRP.alpha. Antibodies
[0912] Binding of the humanized antibodies to hSIRP.gamma. was
studied in CELISA format.
[0913] Binding of the hSIRP.gamma. antibodies to human
SIRP.alpha.V1, SIRP.alpha.V2, hSIRP.beta.1, and hSIRP.gamma. was
confirmed using CHO-K1 cells that had been transiently transfected
with cDNA encoding the full length open reading frame of each of
these respective targets subcloned into the pCI-neo vector.
CHO-K1.hSIRP.alpha.V1, CHO-K1.hSIRP.alpha.V2, CHO-K1.hSIRP.beta.1,
and CHO-K1.hSIRP.gamma. cells were seeded in culture medium
(DMEM-F12 (Gibco) supplemented with 5% New Born Calf Serum
(BioWest) and Pen/Strep (Gibco)) in 96-well flat-bottom tissue
culture plates and incubated at 37.degree. C., 5% CO.sub.2 and 95%
humidity until cell layers were confluent. Subsequently, culture
medium was removed and cells were incubated for 1 hour at
37.degree. C., 5% CO.sub.2 and 95% humidity with purified
hSIRP.gamma. antibodies (10m/mL and dilutions thereof). Next, cells
were washed with PBS-T and incubated for 1 hour at 37.degree. C.,
5% CO.sub.2 and 95% humidity with goat-anti-human IgG-HRP conjugate
(Jackson Immuno Research) or goat-anti-mouse IgG-HRP (Southern
Biotech). Subsequently, cells were washed three times with PBS-T
and anti-hSIRP.gamma. immunoreactivity was visualized with TMB
Stabilized Chromogen (Invitrogen). Reactions were stopped with 0.5
M H.sub.2SO.sub.4 and absorbances were read at 450 and 610 nm. EC50
values, the concentration at which 50% of the total binding signal
is observed, were calculated using GraphPad Prism 6 (GraphPad
Software, Inc.). In Table 13 the EC50 values of the humanized
hSIRP.gamma. antibodies are depicted.
[0914] Table 13: Binding of humanized and parental hSIRP.alpha..50A
antibodies to CHO-K1.hSIRP.alpha.V1, CHO-K1.hSIRP.alpha.V2,
CHO-K1.hSIRP.beta.1, and CHO-K1.hSIRP.gamma. cells. EC50 values
represent the concentration at which 50% of the total binding
signal is observed (average and SD were calculated from values of
two independent experiments).
TABLE-US-00013 TABLE 13 hSIRP.alpha.V1 binding hSIRP.alpha.V2
binding hSIRP.beta.1 binding hSIRP.gamma. binding EC50 (nM) EC50
(nM) EC50 (nM) EC50 (nM) Antibody Average SD Average SD Average SD
Average SD hSIRP.alpha..50H 0.883 0.212 0.864 0.109 nd nd 1.485*
0.120 1L1 hSIRP.alpha..50H 0.781 0.104 0.816 0.161 nd nd 1.259*
0.155 1L2 hSIRP.alpha..50H 1.094 0.112 1.107 0.238 nd nd 2.579*
0.672 1L3 hSIRP.alpha..50H 1.488 0.259 1.621 0.320 nd nd 7.435*
0.208 1L4 hSIRP.alpha..50H 0.962 0.235 0.848 0.239 nd nd 1.013*
0.115 1L5 hSIRP.alpha..50H 1.097 0.286 1.056 0.303 nd nd 1.424*
0.080 3L1 hSIRP.alpha..50H 1.055 0.347 0.999 0.450 nd nd 1.502*
0.305 3L2 hSIRP.alpha..50H 1.159 0.417 1.160 0.429 nd nd 2.471*
0.530 3L3 hSIRP.alpha..50H 1.261 0.317 1.520 0.333 nd nd 5.175*
0.210 3L4 hSIRP.alpha..50H 0.878 0.097 0.868 0.190 nd nd 1.199*
0.120 3L5 hSIRP.alpha..50H 0.683 0.027 0.681 0.156 nd nd 0.950*
0.171 4L1 hSIRP.alpha..50H 0.737 0.110 0.651 0.147 nd nd 0.871*
0.062 4L2 hSIRP.alpha..50H 0.933 0.078 0.898 0.133 nd nd 1.596*
0.144 4L3 hSIRP.alpha..50H 1.197 0.175 1.240 0.238 nd nd 1.980*
0.681 4L4 hSIRP.alpha..50H 0.701 0.136 0.661 0.161 nd nd 0.808*
0.038 4L5 hSIRP.alpha..50H 0.731 0.039 0.709 0.063 nd nd 1.028*
0.087 5L1 hSIRP.alpha..50H 0.675 0.086 0.572 0.023 nd nd 0.822*
0.046 5L2 hSIRP.alpha..50H 1.029 0.084 0.796 0.004 nd nd 1.612*
0.247 5L3 hSIRP.alpha..50H 1.169 0.197 1.115 0.060 nd nd 4.028*
0.342 5L4 hSIRP.alpha..50H 0.681 0.066 0.611 0.030 nd nd 0.868*
0.028 5L5 hSIRP.alpha..50A 1.365 0.164 1.296 0.186 nd nd 1.249*
0.179 Note that variants with the H2 heavy chain could not be
expressed in FreeStyle 293-F cells; values indicated with * were
extrapolated; nd, not detected
[0915] Binding of the parental and humanized hSIRP.gamma.
antibodies to hSIRP.gamma. was further assessed using NK-92MI cells
(ATCC CRL-2408), an interleukin-2 (IL-2) independent natural killer
cell line derived from the NK-92 cell line. NK-92MI cells were
seeded in 96-well round bottomed tissue culture plates and
incubated for 30 minutes with the humanized hSIRP.alpha..50A
antibody variants (100m/mL and dilutions thereof) in PBS/1% BSA at
4.degree. C. Next, cells were washed three times with PBS/1% BSA
and incubated for 30 minutes at 4.degree. C. with a FITC-labeled
mouse-anti-human IgG4 (Abcam) or donkey-anti-mouse IgG (Jackson
Immuno Research) detection antibody in PBS/1% BSA. After this
labeling procedure, cells were washed two times, resuspended in
PBS/1% BSA and analysed by flow cytometry on the FACSCanto II (BD
Biosciences). Data were processed and analysed with FlowJo V10
software (FlowJo, LLC).
Example 10: Blockade of hCD47 Binding to hSIRP.gamma. by Humanized
hSIRP.alpha..50A Antibodies
[0916] hCD47 blockade was assessed by flow cytometry for the full
panel of humanized hSIRP.alpha..50A antibodies. To this end, HEK293
cells (ATCC CRL-1573) were transiently transfected using
Lipofectamine 2000 (Invitrogen) with the pCI-neo vector encoding
the full length open reading frame of human SIRP.alpha.V1. The
transfected cells were cultured at 37.degree. C., 5% CO.sub.2 and
95% humidity in medium (DMEM-F12 (Gibco) with 10% Fetal Bovine
Serum (Gibco) and Pen/Strep (Gibco)) until confluent. Subsequently,
cells were dissociated and seeded in 96-well round bottomed tissue
culture plates and incubated for 30 minutes with the humanized
hSIRP.alpha..50A antibody variants (100m/mL and dilutions thereof)
in PBS/1% BSA at 4.degree. C. Next, cells were washed three times
with PBS/1% BSA and incubated with recombinant hCD47/Fc-protein
(ModiQuest; SEQ ID NO: 42) for 30 minutes at 4.degree. C.
Afterwards, cells were washed three times with PBS/1% BSA and
incubated for 30 minutes at 4.degree. C. with a mouse-anti-human
IgG1 Hinge-FITC (Southern Biotech) detection antibody. After this
labeling procedure, cells were washed two times, resuspended in
PBS/1% BSA and analysed by flow cytometry on the FACSCanto II (BD
Biosciences). Data were processed and analysed with FlowJo V10
software (FlowJo, LLC) and plotted using GraphPad Prism 6 (GraphPad
Software, Inc.) (FIG. 8).
[0917] As depicted in FIG. 8, binding of recombinant hCD47 fused to
an Fc domain of human IgG1 was monitored in the presence of
increasing amounts of the humanized hSIRP.alpha..50A antibody
variants. All antibody variants blocked the hSIRP.alpha./hCD47
interaction.
Example 11: Binding Domain of hSIRP.alpha..50A
[0918] To identify the binding region of hSIRP.alpha..50A, several
SIRP.alpha. exchange-mutants were designed based on the human
SIRP.alpha.V1 and hSIRP.beta.1 amino acid sequence. Based on the
fold of SIRP.alpha., the extracellular region can be subdivided
into three separate domains: the Ig-like (immunoglobulin-like)
V-type (IgV), Ig-like C1-type (IgC1), and Ig-like C2-type (IgC2)
domain. The IgV domain is also known as the ligand-binding
N-terminal domain of SIRP.alpha. (which binds to CD47). The human
SIRP.alpha.V1/31 mutants were designed on the basis of the full
length hSIRP.alpha.V1 sequence (SEQ ID NO: 33) and each individual
Ig-like domain was substituted for the equivalent domain of human
SIRP.beta.1 (SEQ ID NO: 37). The cDNAs encoding the constructs,
hSIRP.alpha.-V.beta.C1.alpha.C2.alpha.(SEQ ID NO: 55),
hSIRP.alpha.-V.alpha.C1.beta.C2.alpha.(SEQ ID NO: 57), and
hSIRP.alpha.-V.alpha.C1.alpha.C2.beta. (SEQ ID NO: 59) were
synthesized (GeneArt) and subcloned into the pCI-neo vector.
Binding of hSIRP.alpha..50A to the exchange mutants was tested
using CELISA. To this end, CHO-K1 cells were transiently
transfected, using Lipofectamine 2000, with the pCI-neo vectors
encoding hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.1,
hSIRP.alpha.-V.beta.C1.alpha.C2.alpha.,
hSIRP.alpha.-V.alpha.C1.beta.C2.alpha., and
hSIRP.alpha.-VaC1.alpha.C2.beta., respectively. The transfected
cells were cultured at 37.degree. C., 5% CO.sub.2 and 95% humidity
in medium (DMEM-F12 (Gibco) with 5% New Born Calf serum (Biowest)
and Pen/Strep (Gibco)) until confluent. Subsequently, cells were
trypsinized and seeded in 96-well flat-bottom tissue culture plates
and cultured at 37.degree. C., 5% CO.sub.2 and 95% humidity in
culture medium until confluent. Then, culture medium was removed
and cells were incubated for 1 hour at 37.degree. C., 5% CO.sub.2
and 95% humidity with hSIRP.alpha..50A and anti-hSIRP.alpha. clone
SE5A5 antibodies. Next, cells were washed with PBS-T and incubated
for 1 hour at 37.degree. C., 5% CO.sub.2 and 95% humidity with
goat-anti-mouse IgG-HRP conjugate (Southern Biotech). After that,
cells were washed three times with PBS-T and anti-hSIRP.alpha.
immunoreactivity was visualized with TMB Stabilized Chromogen
(Invitrogen). Reactions were stopped with 0.5 M H.sub.2SO.sub.4 and
absorbances were read at 450 and 610 nm.
[0919] The antibody of the present invention demonstrated loss of
binding to the hSIRP.alpha.-V.beta.C1.alpha.C2.alpha.mutant,
indicating that hSIRP.alpha..50A binds to the IgV domain of
hSIRP.alpha. (FIG. 9; Table 14). EC50 values represent the
concentration at which 50% of the total binding signal is observed
(average and SD were calculated from values of two independent
experiments).
TABLE-US-00014 TABLE 14 Antibody anti-hSIRP.alpha. hSIRP.alpha..50A
(clone SE5A5) hSIRP.alpha.V1 EC50 (nM) 0.321 0.117 SD 0.018 0.001
hSIRP.alpha.V2 EC50 (nM) 0.215 0.084 SD 0.012 0.012 hSIRP.beta.1
EC50 (nM) nd 0.180 SD nd 0.025
hSIRP.alpha.-V.beta.C1.alpha.C2.alpha. EC50 (nM) nd 0.121 SD nd
0.003 hSIRP.alpha.-V.alpha.C1.beta.C2.alpha. EC50 (nM) 0.345 0.135
SD 0.008 0.013 hSIRP.alpha.-V.alpha.C1.alpha.C2.beta. EC50 (nM)
0.408 0.127 SD 0.039 0.028
[0920] To pinpoint the amino acids for interaction of
hSIRP.alpha..50A with the IgV domain, several point mutants of
hSIRP.alpha.V1 were generated based on single amino acid
differences between hSIRP.alpha.V1/V2 and hSIRP.beta.1. FIG. 10A
shows an alignment of the hSIRP.alpha. and hSIRP.beta.1 IgV domain.
Amino acids in the hSIRP.alpha. IgV domain that are altered in
hSIRP.beta.1 were mutated by using the QuikChange II Site-Directed
Mutagenesis Kit (Stratagene) and the full length hSIRP.alpha.V1
sequence (SEQ ID NO: 33) as donor cDNA. Binding of hSIRP.alpha..50A
to hSIRP.alpha.V1 point mutants was tested using CELISA. To this
end, CHO-K1 cells were transiently transfected, using Lipofectamine
2000, with cDNA encoding the full length open reading frame of
hSIRP.alpha.V1 and mutants thereof, and hSIRP.beta.1 subcloned into
the pCI-neo vector. Transfected cells were seeded in culture medium
(DMEM-F12 (Gibco) supplemented with 5% New Born Calf Serum
(BioWest) and Pen/Strep (Gibco)) in 96-well flat-bottom tissue
culture plates and incubated at 37.degree. C., 5% CO.sub.2 and 95%
humidity for 24 hours. Subsequently, culture medium was removed and
cells were incubated for 1 hour at 37.degree. C., 5% CO.sub.2 and
95% humidity with purified hSIRP.alpha. antibodies (used at 10m/mL
and dilutions thereof). Next, cells were washed with PBS-T and
incubated for 1 hour at 37.degree. C., 5% CO.sub.2 and 95% humidity
with goat-anti-mouse IgG-HRP (Southern Biotech). Subsequently,
cells were washed three times with PBS-T and immunoreactivity
against hSIRP.alpha.V1, hSIRP.alpha.V1 mutants, and hSIRP.beta.1
was visualized with TMB Stabilized Chromogen (Invitrogen).
Reactions were stopped with 0.5 M H.sub.2SO.sub.4 and absorbances
were read at 450 and 610 nm. EC50 values, the concentration at
which 50% of the total binding signal is observed, were calculated
using GraphPad Prism 6 (GraphPad Software, Inc.) (average and SD
were calculated from values of two independent experiments). As
shown in FIG. 10B and the following Table 15, the Proline at
position 74 (P74) constitutes a crucial amino acid for the specific
binding of hSIRP.alpha..50A to hSIRP.alpha.V1. Expression of
hSIRP.alpha.V1(P74A) (SEQ ID NO: 61), where P74 is converted to
Alanine, on CHO-K1 cells results in loss of hSIRP.alpha..50A
antibody binding. This proline is absent in the IgV domain sequence
of hSIRP.beta.1.
TABLE-US-00015 TABLE 15 hSIRP.alpha.V1 hSIRP.beta.1
hSIRP.alpha.V1(P74A) binding binding binding EC50 (nM) EC50 (nM)
EC50 (nM) Antibody Average SD Average SD Average SD
hSIRP.alpha..50A 0.535 0.152 nd nd nd nd anti-hSIRP.alpha. 0.164
0.008 0.156 0.009 0.150 0.013 (clone SE5A5)
Example 12: Characterization of hSIRP.alpha..40A and
hSIRP.alpha..50A Antibodies
[0921] The binding specificity of antibodies hSIRP.alpha..40A and
hSIRP.alpha..50A to hSIRP.alpha. were compared in a CELISA format.
In short, CHO-K1 cells were transiently transfected with
hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.1, hSIRP.beta.L, and
hSIRP.gamma. cDNAs. Subsequently, hSIRP.alpha. binding was assessed
by CELISA using CHO-K1.hSIRP.alpha.V1, CHO-K1.hSIRP.alpha.V2,
CHO-K1.hSIRP.beta.1, CHO-K1.hSIRP.beta.3L, and CHO-K1.hSIRP.gamma.
cells. Detection of bound antibody was done with goat-anti-mouse
IgG-HRP (Southern Biotech). As shown in FIG. 11 and the following
Table 16, hSIRP.alpha..40A and hSIRP.alpha..50A antibodies bind to
hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.L, and hSIRP.gamma., but
do not display detectable hSIRP.beta.1 binding. EC.sub.50 values
represent the concentration at which 50% of the total binding
signal is observed (average and SD were calculated from values of
two independent experiments).
TABLE-US-00016 TABLE 16 hSIRP.alpha.V1 binding hSIRP.alpha.V2
binding hSIRP.beta.1 binding hSIRP.gamma. binding hSIRP.beta.L
binding EC50 (nM) EC50 (nM) EC50 (nM) EC50 (nM) EC50 (nM) Antibody
Average SD Average SD Average SD Average SD Average SD
hSIRP.alpha..40A 0.109 0.036 0.088 0.002 nd nd 0.099 0.055 0.141
0.078 hSIRP.alpha..50A 1.428 0.371 1.156 0.127 nd nd 1.990 0.827
0.632 0.277 nd, not detected
[0922] In addition, the specificity of hSIRP.alpha..40A for all
known of hSIRP.alpha. alleles (allelic variants as described by
Takenaka et al., Nat Immunol. 8:1313-1323 (2007) was further
investigated by CELISA using the same strategy as above. To this
end, hSIRP.alpha..40A binding was assessed using CHO-K1 cells that
were transiently transfected with cDNAs encoding full length
hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.alpha.V3 (NA07056_V3) (SEQ ID
NO: 44), hSIRP.alpha.V4 (NA11832_V4) (SEQ ID NO: 46),
hSIRP.alpha.V5 (NA18502_V5) (SEQ ID NO: 48), hSIRP.alpha.V6
(NA18507_V6) (SEQ ID NO: 50), hSIRP.alpha.V8 (NA18570_V8) (SEQ ID
NO: 52), and hSIRP.alpha.V9 (NA18943_V9) (SEQ ID NO: 54). FIG. 12
and the following Table 17 demonstrates the reactivity of antibody
clone hSIRP.alpha..40A for each of these hSIRP.alpha. alleles. EC50
values represent the concentration at which 50% of the total
binding signal is observed (average and SD were calculated from
values of two independent experiments).
TABLE-US-00017 TABLE 17 Antibody hSIRP.alpha..40A hSIRP.alpha..50A
hSIRP.alpha.V1 EC50 (nM) 0.134 1.690 hSIRP.alpha.V2 EC50 (nM) 0.089
1.066 hSIRP.alpha.V3 EC50 (nM) 0.107 1.767 hSIRP.alpha.V4 EC50 (nM)
0.100 1.297 hSIRP.alpha.V5 EC50 (nM) 0.115 1.260 hSIRP.alpha.V6
EC50 (nM) 0.136 2.219 hSIRP.alpha.V8 EC50 (nM) 0.089 1.508
hSIRP.alpha.V9 EC50 (nM) 0.115 1.367
Example 13: hCD47 Blocking Ability of hSIRP.alpha..40A
[0923] The hSIRP.alpha..40A antibody was analyzed by flow cytometry
for its ability to block recombinant hCD47/Fc-protein (R&D
Systems, Cat.#4670-CD-050; SEQ ID NO: 109) binding to cell surface
expressed hSIRP.alpha.. For this purpose, THP-1 (ATCC TIB-202) and
U-937 (ATCC CRL-1593.2) monocyte cell lines were used as the source
of hSIRP.alpha. in the assay. THP-1 and U-937 cells were seeded in
96-well round bottomed tissue culture plates and incubated for 45
minutes with FcR Blocking Reagent (Miltenyi Biotec) and
hSIRP.alpha..40A antibody (100 .mu.g/mL and dilutions thereof) in
PBS/1% BSA at 4.degree. C. Next, cells were washed three times with
PBS/1% BSA and incubated with DyLight 488-labeled recombinant
hCD47/Fc-protein for 30 minutes at 4.degree. C. After this labeling
procedure, cells were washed two times, resuspended in PBS/1% BSA
containing 0.1 .mu.g/mL DAPI (BioLegend), and analysed by flow
cytometry on the FACSCanto II (BD Biosciences). Data were processed
and analysed with FlowJo V10 software (FlowJo, LLC).
[0924] As depicted in FIG. 13 and the following Table 18, binding
of recombinant hCD47 fused to an Fc domain of human IgG1 was
monitored in the presence of increasing amounts of the
hSIRP.alpha..40A antibody. Antibody hSIRP.alpha..40A blocked the
hSIRP.alpha./hCD47 interaction, using the flow cytometry-based
method described above. IC50 values for the blockade of hCD47 were
calculated from this data. IC50 values represent the concentration
at which half of the inhibition is observed.
TABLE-US-00018 TABLE 18 THP-1 U-937 Antibody IC50 (nM) IC50 (nM)
hSIRP.alpha..40A 0.646 1.344 hSIRP.alpha..50A 7.833 19.501
[0925] Next, the binding of hSIRP.alpha..40A to hSIRP.alpha.
expressed on primary human CD14.sup.+ monocytes was investigated.
In addition, the ability of hSIRP.alpha..40A to block the
interaction between hSIRP.alpha. and recombinant hCD47/Fc-protein
was assessed. For this purpose, CD14+ monocytes were isolated from
Ficoll-purified human peripheral blood mononuclear cells (PBMCs)
using RosetteSep human monocyte enrichment cocktail (Stemcell). The
percentage of monocytes present after the enrichment was determined
by flow cytometry on the FACSVerse (BD Biosciences) based on CD14
staining using an APC-Cy7-conjugated mouse-anti-human CD14
detection antibody (BD Biosciences). Subsequently, CD14+ enriched
PBMCs were seeded in 96-well round bottomed tissue culture plates
and incubated for 40 minutes with FcR Blocking Reagent (Miltenyi
Biotec) containing hSIRP.alpha..40A antibody (20m/mL and dilutions
thereof) in PBS/1% BSA at 4.degree. C. Next, cells were washed
three times with PBS/1% BSA and incubated with an Alexa Fluor
647-labeled goat-anti-mouse IgG (Invitrogen) detection antibody in
PBS/1% BSA for 40 minutes at 4.degree. C. After this labeling
procedure, cells were washed two times, resuspended in PBS/1% BSA
containing 0.1m/mL DAPI (BioLegend), and analysed by flow cytometry
on the FACSVerse (BD Biosciences). Data were processed and analysed
with FlowJo V10 software (FlowJo, LLC).
[0926] FIGS. 14A and B shows that hSIRP.alpha..40A binds to primary
human CD14+ enriched monocytes. EC50 values represent the
concentration at which 50% of the total binding signal is observed.
To assess the blocking ability of hSIRP.alpha..40A, CD14+ enriched
monocytes cells were seeded in 96-well round bottomed tissue
culture plates and incubated for 45 minutes with FcR Blocking
Reagent (Miltenyi Biotec) and hSIRP.alpha..40A antibody (20m/mL and
dilutions thereof) in PBS/1% BSA at 4.degree. C. Thereafter, cells
were washed three times with PBS/1% BSA and incubated with 10
.mu.g/mL DyLight 488-labeled recombinant hCD47/Fc-protein for 45
minutes at 4.degree. C. After this labeling procedure, cells were
washed two times, resuspended in PBS/1% BSA containing 0.1 .mu.g/mL
DAPI (BioLegend), and analysed by flow cytometry on the FACSVerse
(BD Biosciences). Data were processed and analysed with FlowJo V10
software (FlowJo, LLC). FIGS. 14 C and D demonstrates the ability
of antibody hSIRP.alpha..40A to block the hSIRP.alpha./hCD47
interaction. IC50 values for the blockade of hCD47 were calculated
from this data. IC50 values represent the concentration at which
half of the inhibition is observed.
Example 14: Functionality of hSIRP.alpha..40A mAb in the Human
Granulocyte Phagocytosis Assay
[0927] To confirm the functionality of hSIRP.alpha..40A in primary
immune cells, granulocytes (e.g. effector cells) were isolated from
healthy human donor EDTA blood. First, the EDTA blood of each donor
was pooled and centrifuged at 300 g for 6 minutes at 20.degree. C.
Next, plasma was removed by aspiration, and the remaining blood
cells were gently resuspended. Cells were recovered in red blood
cell (RBC) lysis buffer (155 mM NH4C1; 10 mM KHCO3) and incubated
for 10 minutes on ice. Next, cells were centrifuged at 300 g for 7
minutes. Supernatants containing lysed RBCs were removed by
aspiration, and the remaining blood cells were gently resuspended
in RBC lysis buffer and kept on ice for 1 minute. RBC lysis was
neutralized by adding assay medium (IMDM (Gibco) supplemented with
10% Fetal Bovine Serum (Gibco) and Pen/Strep (Gibco)). Blood cells
were centrifuged at 300 g for 6 minutes and supernatants were
removed by aspiration to remove remaining RBCs as much as possible.
Subsequently, erythrocyte-lysed blood cells were resuspended in
assay medium containing 10 ng/mL IFN.gamma. and cells were
incubated for 1 hour at 37.degree. C., 5% CO.sub.2 and 95%
humidity. Non-adherent blood cells containing human granulocytes
were collected by mild washing of the tissue culture plate with
assay medium (monocytes are depleted due to adherence to the
plastic surface). The percentage of granulocytes present in the
cell suspension was determined by flow cytometry on the FACSCanto
II (BD Biosciences) based on high forward scatter (FSC) and side
scatter (SSC). Binding of hSIRP.alpha..40A to human granulocytes
was assessed by incubating the cells for 30 minutes at 4.degree. C.
with hSIRP.alpha..40A antibody (25 .mu.g/mL and dilutions thereof)
in PBS/1% BSA containing 10% autologous serum (PBS/1% BSA/10%
serum). Next, cells were washed three times with PBS/1% BSA/10%
serum and incubated for 30 minutes at 4.degree. C. with a
FITC-labeled goat-anti-mouse Ig (BD Biosciences) detection
antibody. After this labeling procedure, cells were washed two
times, resuspended in PBS/1% BSA/10% serum and analysed by flow
cytometry on the FACSCanto II (BD Biosciences). Data were processed
and analysed with FlowJo V10 software (FlowJo, LLC). FIG. 15A and
the following Table 19 shows that hSIRP.alpha..40A binds to primary
human granulocytes. EC50 values represent the concentration at
which 50% of the total binding signal is observed.
TABLE-US-00019 TABLE 19 Donor 1 Antibody EC50 (nM) hSIRP.alpha..40A
1.227 hSIRP.alpha..50A 4.298
[0928] Next, Ramos (ECACC 85030802) target cells were fluorescently
labeled with cell proliferation dye eFluor450 (eBioscience).
Labeling was performed according to manufacturer's instructions.
Labeled target cells were co-cultured for 2-3 hours at 37.degree.
C., 5% CO.sub.2 and 95% humidity with isolated primary human
granulocytes in a 1:1 ratio (7.5*10.sup.4 cells of each target and
effector per well of a 96-well round bottomed tissue culture plate)
in the presence of 0.1 .mu.g/mL rituximab (anti-hCD20). In
addition, cells were co-cultured with 0.1 .mu.g/mL rituximab in
presence of 10 .mu.g/mL hSIRP.alpha..40A. Phagocytosis was assayed
by determining the percentage of granulocytes positive for
eFluor450 using flow cytometry on the FACSCanto II (BD
Biosciences). Data were processed and analysed with FlowJo V10
software (FlowJo, LLC).
[0929] Compared to the mouse IgG1 isotype control, hSIRP.alpha..40A
potently enhances tumor cell phagocytosis induced by rituximab
(FIG. 15B).
Example 15: Functionality of hSIRP.alpha..40A mAb in the Human
Macrophage Phagocytosis Assay
[0930] Blockade of CD47 by hSIRP.alpha..40A enhances the
phagocytosis of human lymphoma cells tumor cells by human
macrophages. Human macrophages were generated by first enriching
CD14+ monocytes from Ficoll-purified human peripheral blood
mononuclear cells (PBMCs) using RosetteSep human monocyte
enrichment cocktail (Stemcell). Monocytes were seeded into
CellCarrier 96-well flat-bottom microplates (Perkin Elmer) and
cultured in macrophage medium (IMDM (Gibco) supplemented with 8.5%
Fetal Bovine Serum (Gibco) and Pen/Strep (Gibco)) containing 50
ng/mL human monocyte colony stimulating factor (M-CSF) for 7 days
at 37.degree. C., 5% CO.sub.2 and 95% humidity to promote
differentiation into macrophages. These monocyte-derived
macrophages (MDMs) become adherent allowing other cells to be
washed away. Human Raji lymphoma cells were counted and labeled
with cell proliferation dye eFluor450 (eBioscience) following
manufacturer's instructions. After labeling, the lymphoma cells
were mixed with assay medium (RPMI (Gibco) supplemented with 10%
Fetal Bovine Serum (Gibco) and Pen/Strep (Gibco)) containing 100
.mu.g/mL anti-hSIRP.alpha. antibodies and dilutions thereof, the
respective isotype control antibody, and 1 .mu.g/mL rituximab
(anti-hCD20). The lymphoma cells were then added to the individual
wells containing MDMs at a ratio of 2.5:1 tumor cells per
phagocyte, mixed and incubated at 37.degree. C., 5% CO.sub.2 and
95% humidity for 2 hours. After the incubation, the wells were
washed with PBS to remove most of the non-phagocytosed tumor cells,
and cells were fixed with 2% formaldehyde for 10 min at RT. The
wells were then washed and maintained in PBS/3% BSA in dark at
4.degree. C. overnight. Lymphoma cells present in the wells were
stained with biotin-conjugated anti-human CD19 clone HIB19
(eBioscience) for 1 hour at RT, and subsequently were
counterstained with Alexa Fluor 488-conjugated streptavidin (Thermo
Fisher Scientific) for 1 hour at RT. Next, nuclei were stained with
DRAQ5 (Thermo Fisher Scientific) for 10 minutes at RT, mixture was
removed, and PBS was added to each well. Cells were analysed with
the Operetta automated fluorescence microscope (Perkin Elmer). Data
were processed and analysed with Columbus V2.6 software.
[0931] As shown in FIG. 16, hSIRP.alpha..40A enhances
rituximab-mediated phagocytosis activity. The phagocytosis of human
lymphoma cells was quantified using a phagocytosis index, as
follows: (number of tumor cells inside macrophages/number of
macrophages)*100; counting at least 200 macrophages per sample.
Example 16: Humanized Antibody Design and CDR Grafting
[0932] The mouse hSIRP.alpha..40A antibody was humanized using
CDR-grafting technology (see e.g. U.S. Pat. No. 5,225,539 and
Williams, D. G. et al., 2010, Antibody Engineering, volume 1,
Chapter 21). First, human germline sequences were identified using
IgBLAST (Ye J. et al., Nucleic Acids Res. 41:W34-40 (2013). For the
hSIRP.alpha..40A VH human germline sequence, V-gene IGHV1-46*01 was
identified (62.2% identity) and for the VL human germline sequence
IGKV1-39*01 was identified (68.4% identity). These two germline
sequences were used as template to graft the mouse CDRs, resulting
in the following two cDNA constructs: SEQ ID NO: 87 (VH) and SEQ ID
NO: 99 (VL).
[0933] Next, a database was constructed containing all human
sequences available in the IMGT database (Lefranc, M.-P. et al.,
Nucleic Acid Res. 27:209-212 (1999)) identifying 85,848 individual
sequences. These sequences were queried using TBLASTN (2.2.31+) to
identify template sequences that demonstrated the highest identify
to the framework of hSIRP.alpha..40A VH and VL sequences. Four VH
and four VL sequences were identified that demonstrated a
similarity score of 80% or higher and that displayed similar CDR
lengths, preferably identical to those in hSIRP.alpha..40A VH CDR1,
CDR2, CDR3 and VL CDR1, CDR2 and CDR3, respectively.
[0934] For the heavy chain, the frameworks encoded by GenBank
(Benson, D. A. et al., Nucleic Acids Res. 41(D1):D36-42 (2013))
accession # L39130, DJ031925, DJ326840, and EF177968 were selected
as templates for grafting of the hSIRP.alpha..40A VH CDRs,
resulting in the following cDNA constructs: SEQ ID NO: 77, 79, 81
and 83, respectively. For the light chain, the frameworks encoded
by GenBank accession # AY731031, DQ840993, AY942002 and DQ535171
were selected as templates for straight grafting of the
hSIRP.alpha..40A VL CDRs, resulting in the following cDNA
constructs: SEQ ID NO: 89, 91, 93 and 95. Additionally, a database
was constructed containing all humanized antibody sequences
available in the public domain, identifying 300 sequences. These
sequences were queried using BLASTP (2.2.31+) to identify template
sequences that demonstrated the highest identify to the framework
of hSIRP.alpha..40A VH and VL sequences. For the heavy chain, the
framework of Gemtuzumab was selected as template, for grafting of
the hSIRP.alpha..40A VH CDRs, resulting in the following cDNA
construct: SEQ ID NO: 85. For the light chain, the framework of
Alacizumab was selected as template, for grafting of the
hSIRP.alpha..40A VL CDRs, resulting in the following cDNA
construct: SEQ ID NO: 97
[0935] Framework and CDR definition were those as described by
Kabat et al. ("Sequences of Proteins of Immunological Interest",
Kabat, E., et al., US Department of Health and Human Services,
(1983)).
[0936] To study the effect of humanized framework residues on the
structure of the Fv, a homology model of the mouse hSIRP.alpha..40A
Fv was made using the `Antibody Modeling Cascade` (default
parameters) within Discovery Studio 4.5. The homology model was
built on basis of PDB ID 3UMT, for the light chain, PDB ID 1EHL for
the heavy chain, and PDB ID 3BGF for the Fv. The CDRs were grafted
in silico to study residues that are close to any of the CDRs and
which might affect the loop conformation, referred to as Vernier
residues. Residues that might affect the loop conformation, and
which are within <5 .ANG. to the CDR surface were identified and
substituted with the mouse amino acid at this position. The
resulting templates were checked for the presence of post
translational modification (PTM) motifs using Discovery Studio 4.5
and where possible (i.e. non-CDR, non-Vernier residues) changed to
prevent a PTM. The VH CDR2 contained a glycosylation site that was
removed by an aspargine to serine mutation.
[0937] CDRs were grafted on each of the identified templates,
expressed as a human IgG2 (SEQ ID NO: 68), kappa (SEQ ID NO: 64)
antibody cloned in the pcDNA3.1(+) vector (Thermo Fisher
Scientific) and for transient transfection in FreeStyle 293-F human
embryonic kidney cells (HEK293T/17, ATCC CRL-11268).
Example 17: Synthesis, Expression and Purification of Chimeric and
Humanized Constructs
[0938] Plasmids encoding the heavy chain and light chain humanized
constructs were mixed in a 1:1 ratio (30m in total) and transiently
expressed by transfection into FreeStyle 293-F cells using
293fectin transfection reagent (Invitrogen) following the
manufacturer's instructions. Supernatants (30 ml) were harvested
after 7 days, filtered over a 0.22 .mu.m filter, and antibodies
were purified using MabSelect Sure Protein A resin according to the
manufacturer's instructions (GE Healthcare). Buffer was exchanged
for 10 mM Histidine, 100 mM NaCl pH 5.5 buffer using Zeba desalting
columns (Thermo Fisher Scientific). The concentration of purified
antibodies was determined based on OD280 (Nanodrop ND-1000).
Endotoxin level was determined by LAL-test according to the
manufacturer's instructions (Lonza).
Example 18: Binding of Humanized SIRP.alpha. Antibodies
[0939] Binding of the parental and humanized antibodies to
hSIRP.alpha. was assessed by flow cytometry using the
CHO-K1.hSIRP.alpha.V1 stable cell line. CHO-K1.hSIRP.alpha.V1 cells
were seeded in 96-well round bottomed tissue culture plates and
incubated for 40 minutes with the humanized hSIRP.alpha..40A
antibody variants (20 .mu.g/mL and dilutions thereof) in PBS/1% BSA
at 4.degree. C. Next, cells were washed three times with PBS/1% BSA
and incubated for 40 minutes at 4.degree. C. with either an Alexa
Fluor 647-labeled goat-anti-mouse IgG (Invitrogen), or an Alexa
Fluor 647-labeled donkey-anti-human IgG (Jackson Immuno Research)
detection antibody in PBS/1% BSA. After this labeling procedure,
cells were washed two times, resuspended in PBS/1% BSA, containing
0.1 .mu.g/mL DAPI (BioLegend), and analysed by flow cytometry on
the FACSVerse (BD Biosciences). Data were processed and analysed
with FlowJo V10 software (FlowJo, LLC). EC50 values, the
concentration at which 50% of the total binding signal is observed,
were calculated using GraphPad Prism 6 (GraphPad Software, Inc.)
(FIG. 17 and Table 20).
TABLE-US-00020 TABLE 20 hSIRP.alpha.V1 Antibody EC50 (nM)
hSIRP.alpha..40A 0.022 hSIRP.alpha..40H1L1 nd hSIRP.alpha..40H1L2
nd hSIRP.alpha..40H1L3 nd hSIRP.alpha..40H1L4 nd
hSIRP.alpha..40H1L5 nd hSIRP.alpha..40H1L6 nd hSIRP.alpha..40H2L1
0.264 hSIRP.alpha..40H2L2 0.298 hSIRP.alpha..40H2L3 0.300
hSIRP.alpha..40H2L4 0.315 hSIRP.alpha..40H2L5 0.284
hSIRP.alpha..40H2L6 0.251 hSIRP.alpha..40H3L1 1.644
hSIRP.alpha..40H3L2 1.404 hSIRP.alpha..40H3L3 1.501
hSIRP.alpha..40H3L4 0.693 hSIRP.alpha..40H3L5 2.302
hSIRP.alpha..40H3L6 0.833 hSIRP.alpha..40H4L1 3.308
hSIRP.alpha..40H4L2 3.360 hSIRP.alpha..40H4L3 3.072
hSIRP.alpha..40H4L4 3.471 hSIRP.alpha..40H4L5 4.828
hSIRP.alpha..40H4L6 3.028 hSIRP.alpha..40H5L1 2.011
hSIRP.alpha..40H5L2 1.919 hSIRP.alpha..40H5L3 2.268
hSIRP.alpha..40H5L4 0.869 hSIRP.alpha..40H5L5 2.954
hSIRP.alpha..40H5L6 2.197 hSIRP.alpha..40H6L1 2.349
hSIRP.alpha..40H6L2 3.002 hSIRP.alpha..40H6L3 3.014
hSIRP.alpha..40H6L4 1.279 hSIRP.alpha..40H6L5 3.785
hSIRP.alpha..40H6L6 2.677 nd, not detected
Example 19: Blockade of hCD47 Binding to hSIRP.alpha. by Humanized
hSIRP.alpha..40A Antibodies
[0940] hCD47 blockade was assessed by flow cytometry for the full
panel of humanized hSIRP.alpha..40A antibodies. To this end, the
U-937 (ATCC CRL-1593.2) monocyte cell line was used as the source
of hSIRP.alpha. in the assay. U-937 cells were seeded in 96-well
round bottomed tissue culture plates and incubated for 45 minutes
with FcR Blocking Reagent (Miltenyi Biotec) and the parental or
humanized hSIRP.alpha..40A antibody variants (20 .mu.g/mL and
dilutions thereof) in PBS/1% BSA at 4.degree. C. Next, cells were
washed three times with PBS/1% BSA and incubated with 10 .mu.g/mL
DyLight 488-labeled recombinant hCD47/Fc-protein for 30 minutes at
4.degree. C. After this labeling procedure, cells were washed two
times, resuspended in PBS/1% BSA containing 0.1 .mu.g/mL DAPI
(BioLegend), and analysed by flow cytometry on the FACSVerse (BD
Biosciences). Data were processed and analysed with FlowJo V10
software (FlowJo, LLC) and plotted using GraphPad Prism 6 (GraphPad
Software, Inc.).
[0941] As depicted in FIG. 18 and the following Table 21, binding
of recombinant hCD47 fused to an Fc domain of human IgG1 was
monitored in the presence of increasing amounts of the humanized
hSIRP.alpha..40A antibody variants. Humanized hSIRP.alpha..40A
blocked the hSIRP.alpha./hCD47 interaction, using the flow
cytometry-based method described above. IC50 values for the
blockade of hCD47 were calculated from this data. IC50 values
represent the concentration at which half of the inhibition is
observed.
TABLE-US-00021 TABLE 21 U-937 Antibody IC50 (nM) hSIRP.alpha..40A
1.122 hSIRP.alpha..40H1L1 nd hSIRP.alpha..40H1L2 nd
hSIRP.alpha..40H1L3 nd hSIRP.alpha..40H1L4 nd hSIRP.alpha..40H1L5
nd hSIRP.alpha..40H1L6 nd hSIRP.alpha..40H2L1 0.638
hSIRP.alpha..40H2L2 0.773 hSIRP.alpha..40H2L3 0.685
hSIRP.alpha..40H2L4 0.718 hSIRP.alpha..40H2L5 0.745
hSIRP.alpha..40H2L6 0.901 hSIRP.alpha..40H3L1 0.980*
hSIRP.alpha..40H3L2 nd h5IRP.alpha..40H3L3 2.625*
hSIRP.alpha..40H3L4 1.784* hSIRP.alpha..40H3L5 2.435*
hSIRP.alpha..40H3L6 97.762* hSIRP.alpha..40H4L1 10.002*
hSIRP.alpha..40H4L2 7.579* hSIRP.alpha..40H4L3 75.422*
hSIRP.alpha..40H4L4 3.153* hSIRP.alpha..40H4L5 5.171*
hSIRP.alpha..40H4L6 3.512* hSIRP.alpha..40H5L1 34.977*
hSIRP.alpha..40H5L2 nd hSIRP.alpha..40H5L3 nd hSIRP.alpha..40H5L4
10.772* hSIRP.alpha..40H5L5 nd hSIRP.alpha..40H5L6 0.247*
hSIRP.alpha..40H6L1 2.391* hSIRP.alpha..40H6L2 20.427*
hSIRP.alpha..40H6L3 9.208* hSIRP.alpha..40H6L4 3.797*
hSIRP.alpha..40H6L5 20.421* hSIRP.alpha..40H6L6 9.750* Values
indicated with * were extrapolated; nd, not detected
Example 20: Binding domain of hSIRP.alpha..40A
[0942] To identify the binding region of hSIRP.alpha..40A, several
SIRP.beta.1 exchange-mutants were designed based on the human
SIRP.beta.1 and SIRP.gamma. amino acid sequences. Based on the fold
of SIRP.alpha./.beta.1/.gamma., the extracellular region can be
subdivided into three separate domains: the Ig-like
(immunoglobulin-like) V-type (IgV), Ig-like C1-type (IgC1), and
Ig-like C2-type (IgC2) domain. The IgV domain is also known as the
ligand-binding N-terminal domain of SIRP.alpha. and SIRP.gamma.
(which binds to CD47). The human SIRP.beta.1/.gamma. mutants were
designed based on the full length hSIRP.beta.1 sequence (SEQ ID NO:
38) and each individual Ig-like domain was substituted for the
equivalent domain of human SIRP.gamma. (SEQ ID NO: 40). The cDNAs
encoding the constructs, hSIRP-V.gamma.C1.beta.C2.beta. (SEQ ID NO:
110), hSIRP-V.beta.C1.gamma.C2.beta. (SEQ ID NO: 112), and
hSIRP-V.beta.C1.beta.C2.gamma. (SEQ ID NO: 114) were synthesized
(GeneArt) and subcloned into the pCI-neo vector. Binding of
hSIRP.alpha..40A to the exchange mutants was tested using CELIS A.
To this end, CHO-K1 cells were transiently transfected, using
Lipofectamine 2000, with the pCI-neo vectors encoding
hSIRP.alpha.V1, hSIRP.alpha.V2, hSIRP.beta.1,
hSIRP-V.gamma.C1.beta.C2.beta., hSIRP-V.beta.C1.gamma.C2.beta., and
hSIRP-V.beta.C1.beta.C2.gamma., respectively. The transfected cells
were cultured at 37.degree. C., 5% CO.sub.2 and 95% humidity in
medium (DMEM-F12 (Gibco) with 5% New Born Calf serum (Biowest) and
Pen/Strep (Gibco)) until confluent. Subsequently, cells were
trypsinized and seeded in 96-well flat-bottom tissue culture plates
and cultured at 37.degree. C., 5% CO.sub.2 and 95% humidity in
culture medium until confluent. Then, culture medium was removed
and cells were incubated for 1 hour at 37.degree. C., 5% CO.sub.2
and 95% humidity with hSIRP.alpha..40A, hSIRP.alpha..50A, and
anti-hSIRP.gamma. clone SE5A5 antibodies. Next, cells were washed
with PBS-T and incubated for 1 hour at 37.degree. C., 5% CO.sub.2
and 95% humidity with goat-anti-mouse IgG-HRP conjugate (Southern
Biotech). After that, cells were washed three times with PBS-T and
anti-hSIRP.gamma. immunoreactivity was visualized with TMB
Stabilized Chromogen (Invitrogen). Reactions were stopped with 0.5
M H.sub.2SO.sub.4 and absorbances were read at 450 and 610 nm.
[0943] The antibody of the present invention demonstrated gain of
binding to the hSIRP-V.gamma.C1.beta.C2.beta. mutant, indicating
that hSIRP.alpha..40A binds to the IgV domain of hSIRP.gamma. and
hSIRP.gamma. (FIG. 19 and Table 22). EC50 values represent the
concentration at which 50% of the total binding signal is observed
(average and SD were calculated from values of two independent
experiments).
TABLE-US-00022 TABLE 22 Antibody anti-hSIRP.alpha. hSIRP.alpha..40A
hSIRP.alpha..50A (clone SE5A5) hSIRP.alpha.V1 EC50 (nM) 0.133 0.968
0.350 SD 0.065 0.432 0.136 hSIRP.alpha.V2 EC50 (nM) 0.101 0.821
0.224 SD 0.051 0.183 0.076 hSIRP.beta.1 EC50 (nM) nd nd 0.249 SD nd
nd 0.091 hSIRP- EC50 (nM) 0.123 2.524 0.287
V.gamma.C1.beta.C2.beta. SD 0.040 0.609 0.026 hSIRP- EC50 (nM) nd
nd 0.309 V.beta.C1.gamma.C2.beta. SD nd nd 0.140 hSIRP- EC50 (nM)
nd nd 0.231 V.beta.C1.beta.C2.gamma. SD nd nd 0.079 nd, not
detected
[0944] To pinpoint the amino acids for interaction of
hSIRP.alpha..40A with the IgV domain, several point mutants of
hSIRP.alpha.V1 were generated based on single amino acid
differences between hSIRP.alpha.V1/V2 and hSIRP.beta.1. The
following sequence alignment shows an alignment of the hSIRP.alpha.
and hSIRP.beta.1 IgV domain.
Sequence Alignment of the IgV Domain:
TABLE-US-00023 ##STR00001##
[0946] Amino acids in the hSIRP.alpha. IgV domain that are altered
in hSIRP.beta.1 were mutated by using the QuikChange II
Site-Directed Mutagenesis Kit (Stratagene) and the full length
hSIRP.alpha.V1 sequence (SEQ ID NO: 33) as donor cDNA. Binding of
hSIRP.alpha..40A to hSIRP.alpha.V1 point mutants was tested using
CELISA. To this end, CHO-K1 cells were transiently transfected,
using Lipofectamine 2000, with cDNA encoding the full length open
reading frame of hSIRP.alpha.V1 and mutants thereof, and
hSIRP.beta.1 subcloned into the pCI-neo vector. Transfected cells
were seeded in culture medium (DMEM-F12 (Gibco) supplemented with
5% New Born Calf Serum (BioWest) and Pen/Strep (Gibco)) in 96-well
flat-bottom tissue culture plates and incubated at 37.degree. C.,
5% CO.sub.2 and 95% humidity for 24 hours. Subsequently, culture
medium was removed and cells were incubated for 1 hour at
37.degree. C., 5% CO.sub.2 and 95% humidity with purified
hSIRP.alpha. antibodies (used at 10m/mL and dilutions thereof).
Next, cells were washed with PBS-T and incubated for 1 hour at
37.degree. C., 5% CO.sub.2 and 95% humidity with goat-anti-mouse
IgG-HRP (Southern Biotech). Subsequently, cells were washed three
times with PBS-T and immunoreactivity against hSIRP.alpha.V1,
hSIRP.alpha.V1 mutants, and hSIRP.beta.1 was visualized with TMB
Stabilized Chromogen (Invitrogen). Reactions were stopped with 0.5
M H.sub.2SO.sub.4 and absorbances were read at 450 and 610 nm. EC50
values, the concentration at which 50% of the total binding signal
is observed, were calculated using GraphPad Prism 6 (GraphPad
Software, Inc.) (average and SD were calculated from values of two
independent experiments).
[0947] As shown in FIG. 20 and the following Table 23, the Proline
at position 74 (P74) constitutes a crucial amino acid for the
specific binding of hSIRP.alpha..40A to hSIRP.alpha.V1. Expression
of hSIRP.alpha.V1(P74A) (SEQ ID NO: 61), where P74 is converted to
Alanine, on CHO-K1 cells results in loss of hSIRP.alpha..40A
antibody binding. This proline is not present in the IgV domain
sequence of hSIRP.beta.1, and could play a role in the correct
conformation of the IgV domain.
TABLE-US-00024 TABLE 23 hSIRP.alpha.V1 hSIRP.beta.1
hSIRP.alpha.V1(P74A) binding binding binding EC50 (nM) EC50 (nM)
EC50 (nM) Antibody Average SD Average SD Average SD
hSIRP.alpha..40A 0.065 0.006 nd nd nd nd hSIRP.alpha..50A 0.534
0.152 nd nd nd nd anti-hSIRP.alpha. 0.163 0.008 0.156 0.009 0.149
0.013 (clone SE5A5) nd, not detected
Example 21: Functionality of Chimeric hSIRP.alpha..40A mAb Variants
in the Human Macrophage Phagocytosis Assay
[0948] The functionality of hSIRP.alpha..40A variable domains,
grafted on different Fc constant domains, was assessed by an in
vitro phagocytosis assay using human macrophages. Experimental
conditions for the human macrophage phagocytosis assay were similar
as explained in Example 15 above. Labelled Raji lymphoma cells were
mixed with assay medium containing either 10 .mu.g/mL or 1 .mu.g/mL
chimeric hSIRP.alpha..40A antibody variants and 1 .mu.g/mL
rituximab and then added to MDMs at a ratio of 2.5:1 tumor cells
per phagocyte. Cells were incubated at 37.degree. C., 5% CO.sub.2
and 95% humidity for 2 hours.
[0949] Analysis was performed with the Operetta automated
fluorescence microscope (Perkin Elmer) and data were processed and
analysed with Columbus V2.6 software. The phagocytosis of human
lymphoma cells was quantified using a phagocytosis index, as
follows: (number of tumor cells inside macrophages/number of
macrophages)*100; counting at least 200 macrophages per sample.
[0950] As shown in FIG. 21, the wild-type (WT) chimeric
hSIRP.alpha..40A.hIgG4 antibody does not enhance rituximab-mediated
phagocytosis, whereas inert chimeric hSIRP.alpha..40A.hIgG1 (SEQ ID
NO: 119) antibody variants containing N297Q (SEQ ID NO: 126),
L234A.L235A (LALA) (SEQ ID NO: 123), or L234A.L235A.P329G (LALAPG)
(SEQ ID NO: 125) mutations enhance rituximab-mediated phagocytosis
activity in a concentration-dependent manner. Likewise,
hSIRP.alpha..40A.hIgG2 and the inert chimeric
hSIRP.alpha..40A.hIgG2 antibody variant containing
V234A.G237A.P238S.H268A.V309L.A330S.P331S (Sigma) (SEQ ID NO: 122)
mutations enhance rituximab-mediated phagocytosis activity in a
concentration-dependent manner.
Example 22: Functionality of Humanized hSIRP.alpha..40A mAb
Variants in the Human Macrophage Phagocytosis Assay
[0951] The functionality of a selected set of the humanized
hSIRP.alpha..40A antibody variants was assessed by an in vitro
phagocytosis assay using human macrophages. Experimental conditions
for the human macrophage phagocytosis assay were similar as
explained in Example 6.
[0952] As shown in FIG. 22, the humanized hSIRP.alpha..40A antibody
variants enhance rituximab-mediated phagocytosis activity in a
concentration-dependent manner similar to antibody KWAR23 grafted
on a hIgG2 Fc.
Example 23: Functionality of Chimeric hSIRP.alpha..50A mAb Variants
in the Human Macrophage Phagocytosis Assay
[0953] The functionality of hSIRP.alpha..50A variable domains,
grafted on different Fc constant domains, was assessed by in vitro
phagocytosis assays using human macrophages. As shown in FIG. 23A,
the chimeric hSIRP.alpha..50A.hIgG4 antibody marginally enhances
rituximab-mediated phagocytosis, whereas the chimeric
hSIRP.alpha..50A.hIgG2 antibody enhances rituximab-mediated
phagocytosis activity similar to the murine hSIRP.alpha..50A.mIgG1
(SEQ ID NO: 120) antibody. FIG. 23B demonstrates that the chimeric
hSIRP.alpha..50A.hIgG2 antibody potently enhances tumor cell
phagocytosis induced by rituximab in a concentration-dependent
manner as compared to the human IgG2 isotype control. Similarly,
hSIRP.alpha..50A.hIgG2 enhanced daratumumab-mediated phagocytosis
(anti-hCD38, used at 0.05 .mu.g/mL) (FIG. 23C).
[0954] In addition, hSIRP.alpha..50A.hIgG2 also enhanced
rituximab-mediated phagocytosis in human granulocytes. As shown in
FIG. 23D, the chimeric hSIRP.alpha..50A.hIgG2 antibody enhances
phagocytosis activity induced by rituximab to a similar extend as
the murine hSIRP.alpha..50A.mIgG1 antibody. Likewise, as shown in
FIG. 24A, the chimeric hSIRP.alpha..50A.hIgG1.N297Q,
hSIRP.alpha..50A.hIgG4.N297Q (SEQ ID NO: 127) or
hSIRP.alpha..50A.hIgG2 antibodies enhance rituximab-mediated
phagocytosis activity by human MDMs to a similar extent as the
murine hSIRP.alpha..50A.mIgG1 antibody (rituximab used at 1
.mu.g/mL). Similar observations were made in FIG. 24B when
phagocytosis was induced by daratumumab (0.05 .mu.g/mL). As shown
in FIG. 25, the chimeric hSIRP.alpha..50A.hIgG1.N297Q and
hSIRP.alpha..50A hIgG1.L234A.L235A.P329G antibodies also enhance
rituximab-mediated phagocytosis activity by human MDMs to a similar
extent as the or hSIRP.alpha..50A.hIgG2 antibody (rituximab used at
1 .mu.g/mL). Chimeric variants of hSIRP.alpha..50A mAb containing a
wild-type hIgG1 or hIgG4 Fc region did not enhance tumor cell
phagocytosis.
Example 24: Comparison of KWAR23, Clone 18D5, hSIRP.alpha..50A, and
hSIRP.alpha..40A Antibodies
[0955] A direct comparison of the specificity of monoclonal
anti-hSIRP.alpha. antibodies KWAR23, clone 18D5 (SEQ ID NO: 128;
SEQ ID NO: 129) from WO2017/178653, hSIRP.alpha..50A, and
hSIRP.alpha..40A for binding to hSIRP.alpha.V1,
hSIRP.alpha.V1(P74A), hSIRP.alpha.V2, and hSIRP.beta.1 was
evaluated by CELISA. Reactivity was confirmed using CHO-K1 cells
(ATCC CCL-61) expressing a cDNA encoding the full length open
reading frame of hSIRP.alpha.V1, hSIRP.alpha.V1(P74A),
hSIRP.alpha.V2, and hSIRP.beta.1 subcloned into the pCI-neo vector
(Promega, Madison, Wis.). CHO-K1.hSIRP.alpha.V1,
CHO-K1.hSIRP.alpha.V1(P74A), CHO-K1.hSIRP.alpha.V2, and
CHO-K1.hSIRP.beta.1 cells were seeded in culture medium (DMEM-F12
(Gibco) supplemented with 5% New Born Calf Serum (BioWest) and
Pen/Strep (Gibco)) in 96-well flat-bottom tissue culture plates and
incubated at 37.degree. C., 5% CO.sub.2 and 95% humidity for 24
hours. Subsequently, culture medium was removed and cells were
incubated for 1 hour at 37.degree. C., 5% CO.sub.2 and 95% humidity
with purified hSIRP.alpha. antibodies (used at 10m/mL and dilutions
thereof). Next, cells were washed with PBS-T and incubated for 1
hour at 37.degree. C., 5% CO.sub.2 and 95% humidity with
goat-anti-mouse IgG-HRP (Southern Biotech). Subsequently, cells
were washed three times with PBS-T and immunoreactivity against
hSIRP.alpha.V1, hSIRP.alpha.V1(P74A), hSIRP.alpha.V2, and
hSIRP.beta.1 was visualized with TMB Stabilized Chromogen
(Invitrogen). Reactions were stopped with 0.5 M H.sub.2SO.sub.4 and
absorbances were read at 450 and 610 nm. EC50 values, the
concentration at which 50% of the total binding signal is observed,
were calculated using GraphPad Prism 6 (GraphPad Software,
Inc.).
[0956] Binding to hSIRP.gamma. was assessed by flow cytometry using
the Jurkat E6.1 T cell leukemia cell line (ECACC 88042803). Jurkat
cells were seeded in 96-well round bottomed tissue culture plates
and incubated for 40 minutes with the anti-hSIRP.alpha. antibodies
(20m/mL and dilutions thereof) in PBS/1% BSA at 4.degree. C. Next,
cells were washed three times with PBS/1% BSA and incubated for 40
minutes at 4.degree. C. with an Alexa Fluor 647-labeled
goat-anti-mouse IgG (Invitrogen) detection antibody in PBS/1% BSA.
After this labeling procedure, cells were washed two times,
resuspended in PBS/1% BSA, containing 0.1m/mL DAPI (BioLegend), and
analysed by flow cytometry on the FACSVerse (BD Biosciences). Data
were processed and analysed with FlowJo V10 software (FlowJo, LLC).
EC.sub.50 values, the concentration at which 50% of the total
binding signal is observed, were calculated using GraphPad Prism 6
(GraphPad Software, Inc.).
[0957] As depicted in Table 24, KWAR23 and clone 18D5 antibodies
cross-react with at least hSIRP.beta.1 and the P74A variant of
hSIRP.alpha.V1. The hSIRP.alpha..50A, and hSIRP.alpha..40A
antibodies of the present invention do not bind to either
hSIRP.beta.1 or the P74A variant of hSIRP.alpha.V1 under the tested
conditions. In this regard, the hSIRP.alpha..50A, and
hSIRP.alpha..40A antibodies of the present invention similarly
distinguish from antibody clone SIRP29 from WO2013/056352. FIGS. 7A
and B of WO2017/178653 compares clone SIRP29 and KWAR23 binding to
SIRP.beta.1 (referred to as "sirp-b", Product No. ABIN3077231 from
antibodies-online.com), demonstrating that each of clone SIRP29 and
KWAR23 has nanomolar affinity for SIRP.beta.1.
TABLE-US-00025 TABLE 24 hSIRP.alpha.V1 hSIRP.alpha.V1
hSIRP.alpha.V2 hSIRP.beta.1 hSIRP.gamma. binding (P74A) binding
binding binding EC50 binding EC50 EC50 EC50 Antibody (nM) EC50 (nM)
(nM) (nM) (nM) hSIRP.alpha..40A 0.114 nd 0.093 nd 0.369
hSIRP.alpha..50A 0.773 nd 0.645 nd -- KWAR23 0.070 0.049 0.049
0.033 0.003 18D5 0.134 0.055 nd 0.055 nd nd, not detected; -- not
tested
[0958] hCD47 blockade for the KWAR23, clone 18D5, and
hSIRP.alpha..40A antibodies was assessed by flow cytometry. For
this purpose, THP-1 (ATCC TIB-202) and U-937 (ATCC CRL-1593.2)
monocyte cell lines were used as the source of hSIRP.alpha. in the
assay. THP-1 and U-937 cells were seeded in 96-well round bottomed
tissue culture plates and incubated for 45 minutes with FcR
Blocking Reagent (Miltenyi Biotec) and indicated anti-hSIRP.alpha.
antibodies (20 .mu.g/mL and dilutions thereof) in PBS/1% BSA at
4.degree. C. Next, cells were washed three times with PBS/1% BSA
and incubated with 10 .mu.g/mL DyLight 488-labeled recombinant
hCD47/Fc-protein for 30 minutes at 4.degree. C. After this labeling
procedure, cells were washed two times, resuspended in PBS/1% BSA
containing 0.1 .mu.g/mL DAPI (BioLegend), and analysed by flow
cytometry on the FACSVerse (BD Biosciences). Data were processed
and analysed with FlowJo V10 software (FlowJo, LLC) and plotted
using GraphPad Prism 6 (GraphPad Software, Inc.). Binding of
recombinant hCD47 fused to an Fc domain of human IgG1 was monitored
in the presence of increasing amounts of the anti-hSIRP.alpha.
antibodies. IC50 values for the blockade of hCD47 were calculated
from this data. IC50 values represent the concentration at which
half of the inhibition is observed.
[0959] As depicted in Table 18 and Table 25, hSIRP.alpha..40A,
hSIRP.alpha..50A, and KWAR23 antibodies block rhCD47/Fc binding to
both the THP-1 and U-937 monocyte cell lines which express the
hSIRP.alpha.V2 and hSIRP.alpha.V1 allele, respectively. Antibody
clone 18D5 blocks rhCD47/Fc binding to the U-937 monocyte cell line
but does not block rhCD47/Fc binding to the THP-1 monocyte cell
line, in line with the observation that 18D5 does not bind to
hSIRP.alpha.V2 (Table 24). In this regard, the hSIRP.alpha..50A,
and hSIRP.alpha..40A antibodies of the present invention similarly
distinguish from antibody clone 18D5.
TABLE-US-00026 TABLE 25 THP-1 U-937 Antibody IC50 (nM) IC50 (nM)
hSIRP.alpha..40A 0.548 1.417 KWAR23 0.132 0.284 18D5 nd 1.522 nd,
not detected
Example 25: Mapping the Interaction Interface Between
hSIRP.alpha.-hSIRP.alpha..40A and hSIRP.alpha.-hSIRP.alpha..50A
[0960] The amino acids on hSIRP.alpha. that are bound by
hSIRP.alpha..40A or hSIRP.alpha..50A were elucidated by a procedure
that involves deuterated chemical cross-linking followed by
enzymatic digestion and detection using mass spectrometry. First,
antibody hSIRP.alpha..40A and antigen rhSIRP.alpha.-HIS
(SinoBiological 11612-H08H-100, SEQ ID NO: 132), or antibody
hSIRP.alpha..50A and antigen rhSIRP.alpha.-HIS were incubated to
promote binding and integrity and aggregation level were verified
by Ultraflex III MALDI TOF mass spectrometer (Bruker) equipped with
a HM4 interaction module (CovalX). For these control experiments a
dilution series of 10 .mu.L samples of antibody or antigen (1- to
128-fold dilution, starting at 1 mg/mL) were prepared. Of each
sample 9 .mu.L was submitted to cross-linking using K200 MALDI MS
analysis kit, according to the manufacturer's instructions (CovalX)
and incubated for 180 minutes, while 1 .mu.L was directly used for
mass spectrometry analysis (High-Mass MALDI). The mass spectrometry
analysis showed the antibody and antigen had the expected molecular
weight: hSIRP.alpha..40A=151.68 kDa (152.78 kDa with cross-linker),
hSIRP.alpha..50A=151.80 kD (153.17 kDa with cross-linker), and
rhSIRP.alpha.-HIS=46.05 kDa (48.67 kDa with cross-linker). For
characterization of the antigen-antibody complex, a mixture was
made with an excess of antigen (antigen:antibody ratio for
rhSIRP.alpha.-HIS:hSIRP.alpha..40A 10.8 .mu.M:8.5 .mu.M, and
antigen:antibody ratio for rhSIRP.alpha.-HIS:hSIRP.alpha..50A 5.4
.mu.M:2.13 .mu.M). A 9 .mu.L sample of the antigen-antibody mixture
was submitted to cross-linking using K200 MALDI MS analysis kit,
according to the manufacturer's instructions, while 1 .mu.L was
directly used for mass spectrometry analysis. The detected mass of
the antibody and antigen (hSIRP.alpha..40A: 151.18 kDa,
rhSIRP.alpha.-HIS 45.93 kDa, hSIRP.alpha..50A: 151.69 kDa,
rhSIRP.alpha.-HIS 46.18 kDa) corresponds to the molecular weight as
detected previously. The antigen-antibody complexes, after
cross-linking, were detected as two non-covalent complexes with a
1:1 (195.24 kDa) and 2:1 (240.48 kDa) stoichiometry for
rhSIRP.alpha.-HIS:hSIRP.alpha..40A, and as one non-covalent complex
with a 1:1 (198.24 kDa) stoichiometry for
rhSIRP.alpha.-HIS:hSIRP.alpha..50A. Antibody and antigen bound
non-covalent; non-covalent aggregates or non-specific multimers
were not detected.
[0961] Next, peptide mass fingerprinting of rhSIRP.alpha.-HIS was
performed. Samples were submitted to ASP-N, trypsin, chymotrypsin,
elastase and thermolysin (Roche Diagnostic) proteolysis, following
manufacturer's instructions followed by analysis by nLC-LTQ
Orbitrap MS/MS using an Ultimate 3000 (Dionex) system in line with
a LTQ Orbitrap XL mass spectrometer (Thermo Scientific). This
proteolysis array resulted in 98% of the sequence being covered by
the identified peptides.
[0962] To determine the interacting amino acids of antibody
hSIRP.alpha..40A and hSIRP.alpha..50A on rhSIRP.alpha.-HIS antigen
with high resolution, the antigen-antibody complex
(rhSIRP.alpha.-HIS:hSIRP.alpha..40A ratio 10.8 .mu.M:8.5 .mu.M,
rhSIRP.alpha.-HIS:hSIRP.alpha..50A ratio 5.4 .mu.M:2.13 .mu.M) was
incubated with deuterated cross-linkers d0/d12 (K200 MALDI Kit) for
180 minutes and subjected to multi-enzymatic cleavage with the
enzymes ASP-N, trypsin, chymotrypsin, elastase and thermolysin.
After enrichment of the cross-linked peptides, the samples were
analyzed by high-resolution mass spectrometry (nLC-Orbitrap MS) and
the data generated were analyzed using XQuest (Jin Lee, Mol.
Biosyst. 4:816-823 (2008)) and Stavrox (Gotze et al., J. Am. Soc.
Mass Spectrom. 23:76-87 (2012)). The interacting amino acids of
hSIRP.alpha..40A and hSIRP.alpha..50A to rhSIRP.alpha.-HIS were
mapped onto human SIRP.alpha.V1 (SEQ ID NO: 34). Cross-linked
residues of hSIRP.alpha..40A are depicted as bold, boxed, and
hSIRP.alpha..50A as bold, underlined:
TABLE-US-00027
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVG
##STR00002## ##STR00003##
DFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTL
EVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNV
SAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLV
ALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPN
NHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK
[0963] The C-alpha distance between residue P74 and the identified
cross-linked residues was measured in Discovery Studio using the
crystal structure of SIRP.alpha. (PDB ID 4CMM). The cross-linked
residues identified for hSIRP.alpha..50A are within 14.0 to 21.4
angstrom C-alpha atom distance from residue P74; the cross-linked
residues identified for hSIRP.alpha..40A are within 16.2 to 33.5
angstrom C-alpha atom distance from residue P74. The C-alpha
distances fit within the expected range for an epitope-paratope
surface area of 700 .ANG..sup.2 (Rowley et al., Biotech. Ann. Rev.
10:151-188 (2004)). The identified residues and surface area are
distinctly different from the binding epitope of anti-hSIRP.alpha.
antibody KWAR23 (Ring et al., Proc. Natl Acad. Sci. USA
114:E10578-E10585 (2017)).
Example 26: Comparison of hSIRP.alpha. Antibodies for Binding to
hSIRP.alpha.V1, hSIRP.alpha.V1(P74A), and hSIRP.beta.1
[0964] The specificity of monoclonal anti-hSIRP.alpha. antibodies
(e.g., including the hSIRP.alpha. antibodies known in the art,
KWAR23 (U.S. Patent CA2939293 A1), 18D5 (Patent WO2017/178653 A2),
and various commercially available hSIRP.alpha. antibodies) for
binding to hSIRP.alpha.V1, hSIRP.alpha.V1(P74A), and hSIRP.beta.1
was evaluated by CELISA. Reactivity was confirmed using CHO-K1
cells (ATCC CCL-61) expressing a cDNA encoding the full length open
reading frame of hSIRP.alpha.V1, hSIRP.alpha.V1(P74A), and
hSIRP.beta.1 subcloned into the pCI-neo vector (Promega, Madison,
Wis.). CHO-K1.hSIRP.alpha.V1, CHO-K1.hSIRP.alpha.V1(P74A), and
CHO-K1.hSIRP.beta.1 cells were seeded in culture medium (DMEM-F12
(Gibco) supplemented with 5% New Born Calf Serum (BioWest) and
Pen/Strep (Gibco)) in 96-well flat-bottom tissue culture plates and
incubated at 37.degree. C., 5% CO.sub.2 and 95% humidity for 24
hours. Subsequently, culture medium was removed and cells were
incubated for 1 hour at 37.degree. C., 5% CO.sub.2 and 95% humidity
with purified hSIRP.alpha. antibodies (used at 10 .mu.g/ml and
dilutions thereof). Next, cells were washed with PBS-T and
incubated for 1 hour at 37.degree. C., 5% CO.sub.2 and 95% humidity
with either goat-anti-mouse IgG-HRP (Southern Biotech),
goat-anti-human IgG-HRP (Jackson Immuno Research), or
goat-anti-rabbit IgG-HRP (Southern Biotech). Subsequently, cells
were washed three times with PBS-T and immunoreactivity against
hSIRP.alpha.V1, hSIRP.alpha.V1(P74A), and hSIRP.beta.1 was
visualized with TMB Stabilized Chromogen (Invitrogen). Reactions
were stopped with 0.5 M H.sub.2SO.sub.4 and absorbances were read
at 450 and 610 nm. EC.sub.50 values, the concentration at which 50%
of the total binding signal is observed, were calculated using
GraphPad Prism 6 (GraphPad Software, Inc.)
[0965] As depicted in Table 26, KWAR23, clone 18D5, and all
commercially available monoclonal anti-hSIRP.alpha. antibodies are
able to bind to the P74A variant of hSIRP.alpha.V1 whereas the
hSIRP.alpha..40A and hSIRP.alpha..50A antibodies of the present
invention do not bind to the P74A variant of hSIRP.alpha.V1 under
the tested conditions.
TABLE-US-00028 TABLE 26 hSIRP.alpha.V1 hSIRP.alpha.V1 hSIRP.beta.1
binding (P74A) binding EC50 binding EC50 Antibody (nM) EC50 (nM)
(nM) hSIRP.alpha..40A 0.053 nd nd hSIRP.alpha..50A 0.307 nd nd
KWAR23 0.135 0.077 0.065 18D5 0.128 0.073 0.064 anti-hSIRP.alpha.
(clone SE5A5) 0.156 0.207 0.105 anti-hSIRP.alpha. (clone 7B3) 0.122
0.141 0.115 anti-hSIRP.alpha. (clone 1C6) 0.329 0.440 >2.817
anti-hSIRP.alpha. (clone 27) nd nd nd anti-hSIRP.alpha. (clone
SE7C2) >7.010 >6.139 nd anti-hSIRP.alpha. (clone P3C4) 0.179
0.197 0.160 anti-hSIRP.alpha. (clone 2A4A5) nd nd >6.456
anti-hSIRP.alpha. (clone 15-414) nd nd nd anti-hSIRP.alpha. (clone
1H1) nd nd nd anti-hSIRP.alpha. (clone C-7) nd nd nd
anti-hSIRP.alpha. (clone 03) >8.247 >8.992 >6.092
anti-hSIRP.alpha. (clone 5E10) nd nd nd anti-hSIRP.alpha. (clone
602411) 0.047 0.076 0.051 anti-hSIRP.alpha. (clone EPR16264)
>1.166 >1.999 nd anti-hSIRP.alpha. (clone D6I3M) >6.413
>121.509 nd anti-hSIRP.alpha. (clone 001) >0.868 >1.192 nd
anti-hSIRP.alpha. (clone REA144) >3.661 >4.793 >3.075 nd,
not detected
Example 27: Sequences Referred to in the Specification
TABLE-US-00029 [0966] SEQ ID Description NO: SEQUENCE 50A heavy
chain 1 NYYIH CDR1 (amino acid sequence) 50A heavy chain 2
WIYPGNVNTKYNEKFKA CDR2 (amino acid sequence) 50A heavy chain 3
PTIIATDFDV CDR3 (amino acid sequence) 50A light chain CDR1 4
KASQGVGTAVG (amino acid sequence) 50A light chain CDR2 5 WASTRHT
(amino acid sequence) 50A light chain CDR3 6 QQYSTYPFT (amino acid
sequence) humanized 50 heavy 7
EVQLX.sub.1X.sub.2SGX.sub.3EX.sub.4VKPGASVX.sub.5X.sub.6SCKASGFTFTNYYIHWV-
RQX.sub.7P chain variable region
X.sub.8QGLEWX.sub.9GWIYPGNVNTKYNEKFKAX.sub.10X.sub.11X.sub.12X.sub.13TADK-
STSTX.sub.14 (consensus sequence)
YMX.sub.15LSSLX.sub.16SX.sub.17DX.sub.18AVYYCARPTIIATDFDVWGQGTX.sub.19VTV-
S S wherein: X.sub.1 = Q, V X.sub.2 = Q, E X.sub.3 = A, S X.sub.4 =
V, L X.sub.5 = K, M X.sub.6 = V, I X.sub.7 = A, R X.sub.8 = G, E
X.sub.9 = I, M X.sub.10 = R, K X.sub.11 = V, A X.sub.12 = T, I
X.sub.13 = I, M X.sub.14 = A, V X.sub.15 = D, E, Q X.sub.16 = R, T
X.sub.17 = E, D X.sub.18 = T, M X.sub.19 = T, L humanized 50 light
8
X.sub.1X.sub.2X.sub.3X.sub.4TQSPSX.sub.5LSASVGDRVTITCKASQGVGTAVGWYQX.sub.-
6KPGK chain variable region
X.sub.7PKLLIYWASTRHTGVPDRFSGSGSGTX.sub.8FTLX.sub.9IX.sub.10X.sub.11LQPEDX
(consensus sequence) .sub.12AX.sub.13YYCQQYSTYPFTFGGGTKX.sub.14EIK
wherein: X.sub.1 = D, E X.sub.2 = I, L X.sub.3 = V, Q X.sub.4 = L,
M X.sub.5 = F, S X.sub.6 = Q, K X.sub.7 = A, S, V X.sub.8 = E, D
X.sub.9 = T, A X.sub.10 = S, N X.sub.11 = S, N, G X.sub.12 = F, I,
V X.sub.13 = A, D, T X.sub.14 = L, V hSIRP.alpha..50AVH1 9
GAAGTGCAGCTGCAGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCG (nucleotide
sequence) CCTCTGTGAAGGTGTCCTGCAAGGCCTCCGGCTTCACCTTCACCAA
CTACTACATCCACTGGGTGCGACAGGCCCCAGGCCAGGGACTGGAA
TGGATCGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACG
AGAAGTTCAAGGCCCGCGTGACCATCACCGCCGACAAGTCTACCTC
CACCGCCTACATGGACCTGTCCTCCCTGAGATCCGAGGACACCGCC
GTGTACTACTGCGCCAGACCCACCATCATTGCCACCGACTTCGACG
TGTGGGGCCAGGGCACAACCGTGACCGTGTCCTCT hSIRP.alpha..50AVH1 10
EVQLQQSGAEVVKPGASVKVSCKASGFTFTNYYIHWVRQAPGQGLE (amino acid
sequence) WIGWIYPGNVNTKYNEKFKARVTITADKSTSTAYMDLSSLRSEDTA
VYYCARPTIIATDFDVWGQGTTVTVSS hSIRP.alpha..50AVH2 11
GAAGTGCAGCTGGTGGAATCCGGCTCCGAGCTCGTGAAGCCTGGCG (nucleotide
sequence) CCTCCGTGAAGGTGTCCTGCAAGGCCTCTGGCTTCACCTTCACCAA
CTACTACATCCACTGGGTGCGACAGGCCCCAGGCCAGGGACTGGAA
TGGATGGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACG
AGAAGTTCAAGGCCAAGGCCACCATCACCGCCGACAAGTCCACCTC
CACCGCCTACATGGAACTGTCCTCCCTGCGGAGCGAGGACACCGCC
GTGTACTACTGTGCCCGGCCTACCATCATTGCCACCGACTTCGATG
TGTGGGGCCAGGGCACACTCGTGACCGTGTCCTCT hSIRP.alpha..50AVH2 12
EVQLVESGSELVKPGASVKVSCKASGFTFTNYYIHWVRQAPGQGLE (amino acid
sequence) WMGWIYPGNVNTKYNEKFKAKATITADKSTSTAYMELSSLRSEDTA
VYYCARPTIIATDFDVWGQGTLVTVSS hSIRP.alpha..50AVH3 13
GAAGTGCAGCTGGTGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCG (nucleotide
sequence) CCTCCGTGATGATCTCCTGCAAGGCCTCCGGCTTCACCTTCACCAA
CTACTACATCCACTGGGTGCGACAGCGGCCAGGCCAGGGACTGGAA
TGGATCGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACG
AGAAGTTCAAGGCCCGCGTGATCATGACCGCCGACAAGTCCACCTC
CACCGTGTACATGCAGCTGTCCTCCCTGACCTCCGAGGACACCGCC
GTGTACTACTGCGCCAGACCCACCATCATTGCCACCGACTTCGACG
TGTGGGGCCAGGGCACACTCGTGACCGTGTCCTCT hSIRP.alpha..50AVH3 14
EVQLVQSGAEVVKPGASVMISCKASGFTFTNYYIHWVRQRPGQGLE (amino acid
sequence) WIGWIYPGNVNTKYNEKFKARVIMTADKSTSTVYMQLSSLTSEDTA
VYYCARPTIIATDFDVWGQGTLVTVSS hSIRP.alpha..50AVH4 15
GAAGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCG (nucleotide
sequence) CCTCTGTGAAGGTGTCCTGCAAGGCCTCCGGCTTCACCTTCACCAA
CTACTACATCCACTGGGTGCGACAGCGGCCAGGCCAGGGACTGGAA
TGGATGGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACG
AGAAGTTCAAGGCCAAGGCCACCATCACCGCCGACAAGTCCACCTC
CACCGCCTACATGGAACTGTCCTCCCTGACCTCCGAGGACACCGCC
GTGTACTACTGCGCCAGACCCACCATCATTGCCACCGACTTCGACG
TGTGGGGCCAGGGCACAACCGTGACCGTGTCCTCT hSIRP.alpha..50AVH4 16
EVQLQQSGAELVKPGASVKVSCKASGFTFTNYYIHWVRQRPGQGLE (amino acid
sequence) WMGWIYPGNVNTKYNEKFKAKATITADKSTSTAYMELSSLTSEDTA
VYYCARPTIIATDFDVWGQGTTVTVSS hSIRP.alpha..50AVH5 17
GAAGTGCAGCTGGTGCAGTCTGGCGCCGAGGTCGTGAAACCTGGCG (nucleotide
sequence) CCTCTGTGAAGGTGTCCTGCAAGGCCTCCGGCTTCACCTTCACCAA
CTACTACATCCACTGGGTGCGACAGGCCCCCGAGCAGGGACTGGAA
TGGATCGGCTGGATCTACCCCGGCAACGTGAACACCAAGTACAACG
AGAAGTTCAAGGCCCGCGTGACCATGACCGCCGACAAGTCTACCTC
CACCGCCTACATGGAACTGTCCTCCCTGCGGAGCGACGACATGGCC
GTGTACTACTGCGCCAGACCCACCATCATTGCCACCGACTTCGACG
TGTGGGGCCAGGGCACAACCGTGACCGTGTCCTCT hSIRP.alpha..50AVH5 18
EVQLVQSGAEVVKPGASVKVSCKASGFTFTNYYIHWVRQAPEQGLE (amino acid
sequence) WIGWIYPGNVNTKYNEKFKARVTMTADKSTSTAYMELSSLRSDDMA
VYYCARPTIIATDFDVWGQGTTVTVSS hSIRP.alpha..50AVL1 19
GACATCGTGCTGACCCAGTCCCCCAGCTTCCTGTCTGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCAC
CGCTGTGGGATGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTG
CTGATCTACTGGGCCTCTACCAGACACACCGGCGTGCCCGACAGAT
TCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCAG
CCTGCAGCCCGAGGATTTCGCCGCCTACTACTGCCAGCAGTACTCC
ACCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG hSIRP.alpha..50AVL1
20 DIVLTQSPSFLSASVGDRVTITCKASQGVGTAVGWYQQKPGKAPKL (amino acid
sequence) LIYWASTRHTGVPDRFSGSGSGTEFTLTISSLQPEDFAAYYCQQYS
TYPFTFGGGTKLEIK hSIRP.alpha..50AVL2 21
GACATCGTGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCAC
CGCTGTGGGATGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTG
CTGATCTACTGGGCCTCTACCAGACACACCGGCGTGCCCGACAGAT
TCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCTCCAA
CCTGCAGCCCGAGGACTTCGCCGACTACTACTGCCAGCAGTACTCC
ACCTACCCCTTCACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG hSIRP.alpha..50AVL2
22 DIVMTQSPSSLSASVGDRVTITCKASQGVGTAVGWYQQKPGKAPKL (amino acid
sequence) LIYWASTRHTGVPDRFSGSGSGTDFTLTISNLQPEDFADYYCQQYS
TYPFTFGGGTKVEIK hSIRP.alpha..50AVL3 23
GAGCTCGTGATGACCCAGTCCCCTTCCAGCCTGTCTGCCTCCGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCAC
CGCTGTGGGATGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTG
CTGATCTACTGGGCCTCTACCAGACACACCGGCGTGCCCGACAGAT
TCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGGCCATCTCCAG
CCTGCAGCCCGAGGATATCGCCGACTACTACTGCCAGCAGTACTCC
ACCTACCCCTTCACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG hSIRP.alpha..50AVL3
24 ELVMTQSPSSLSASVGDRVTITCKASQGVGTAVGWYQQKPGKAPKL (amino acid
sequence) LIYWASTRHTGVPDRFSGSGSGTDFTLAISSLQPEDIADYYCQQYS
TYPFTFGGGTKVEIK hSIRP.alpha..50AVL4 25
GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCAC
CGCTGTGGGCTGGTATCAGAAAAAGCCCGGCAAGGTGCCCAAGCTG
CTGATCTACTGGGCCTCCACCAGACACACCGGCGTGCCCGATAGAT
TCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCAACGG
CCTGCAGCCTGAGGACGTGGCCACCTACTACTGCCAGCAGTACTCC
ACCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG hSIRP.alpha..50AVL4
26 DIQMTQSPSSLSASVGDRVTITCKASQGVGTAVGWYQKKPGKVPKL (amino acid
sequence) LIYWASTRHTGVPDRFSGSGSGTDFTLTINGLQPEDVATYYCQQYS
TYPFTFGGGTKLEIK hSIRP.alpha..50AVL5 27
GACATCGTGCTGACCCAGTCCCCCAGCTTCCTGTCTGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGGCGTGGGCAC
CGCTGTGGGATGGTATCAGCAGAAGCCCGGCAAGTCCCCCAAGCTG
CTGATCTACTGGGCCTCCACCAGACACACCGGCGTGCCCGATAGAT
TCTCCGGCTCTGGCTCTGGCACCGAGTTCACCCTGACCATCTCCAA
CCTGCAGCCCGAGGACTTCGCCGCCTACTACTGCCAGCAGTACTCC
ACCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG hSIRP.alpha..50AVL5
28 DIVLTQSPSFLSASVGDRVTITCKASQGVGTAVGWYQQKPGKSPKL (amino acid
sequence) LIYWASTRHTGVPDRFSGSGSGTEFTLTISNLQPEDFAAYYCQQYS
TYPFTFGGGTKLEIK hSIRP.alpha..50A mouse 29
CAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGGGG VH (nucleotide
CTTCAGTTAGGATATCCTGCAAGGCTTCTGGCTTCACCTTCACAAA sequence)
CTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAG
TGGATTGGATGGATTTATCCTGGAAATGTTAATACTAAGTACAATG
AGAAGTTCAAGGCCAAGGCCACACTGACTGCAGACAAATCCTCCAC
CACAGCCTACATGCAGCTCAGCAGCCTGGCCTCTGAGGACTCTGCG
GTCTATTTCTGTGCAAGACCTACGATAATAGCTACGGACTTCGATG
TCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA hSIRP.alpha..50A mouse 30
QVQLQQSGPELVKPGASVRISCKASGFTFTNYYIHWVKQRPGQGLE VH (amino acid
WIGWIYPGNVNTKYNEKFKAKATLTADKSSTTAYMQLSSLASEDSA sequence)
VYFCARPTIIATDFDVWGAGTTVTVSS hSIRP.alpha..50A mouse 31
GACATTGTCATGACCCAGTCTCACAAATTCATGTCCACATCAGTAG VL (nucleotide
GAGACAGGGTCAACATCACCTGCAAGGCCAGTCAGGGTGTGGGTAC sequence)
TGCTGTAGGCTGGTATCAACAGAAACCAGGGCAATCTCCTAGACTA
CTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCT
TCACAGGCAGTGGATCTGGGACAGATTTCAGTCTCGCCATTAGCAA
TGTGCAGTCTGAAGACCTGGCAGATTATTTCTGTCAGCAATATAGC
ACCTATCCGTTCACGTTCGGAGGGGGGACCAATCTAGAAATAAAA hSIRP.alpha..50A
mouse 32 DIVMTQSHKFMSTSVGDRVNITCKASQGVGTAVGWYQQKPGQSPRL VL (amino
acid LIYWASTRHTGVPDRFTGSGSGTDFSLAISNVQSEDLADYFCQQYS sequence)
TYPFTFGGGTNLEIK human SIRP.alpha.V1 33
ATGGAGCCCGCCGGCCCGGCCCCCGGCCGCCTCGGGCCGCTGCTCT (nucleotide
sequence) GCCTGCTGCTCGCCGCGTCCTGCGCCTGGTCAGGAGTGGCGGGTGA
GGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCA
GCTGGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCTGATCC
CTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGGACCAGGCCGGGA
ATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACT
GTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCA
TCGGTAACATCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAA
GTTCCGGAAAGGGAGCCCCGATGACGTGGAGTTTAAGTCTGGAGCA
GGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGGTAT
CGGGCCCTGCGGCGAGGGCCACACCTCAGCACACAGTGAGCTTCAC
CTGCGAGTCCCACGGCTTCTCACCCAGAGACATCACCCTGAAATGG
TTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACC
CCGTAGGAGAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGT
GGTGCTGACCCGCGAGGACGTTCACTCTCAAGTCATCTGCGAGGTG
GCCCACGTCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACT
TGTCTGAGACCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACA
GCCCGTGAGGGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGG
AAGTTCTACCCCCAGAGACTACAGCTGACCTGGTTGGAGAATGGAA
ACGTGTCCCGGACAGAAACGGCCTCAACCGTTACAGAGAACAAGGA
TGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGTATCTGCC
CACAGGGATGATGTGAAGCTCACCTGCCAGGTGGAGCATGACGGGC
AGCCAGCGGTCAGCAAAAGCCATGACCTGAAGGTCTCAGCCCACCC
GAAGGAGCAGGGCTCAAATACCGCCGCTGAGAACACTGGATCTAAT
GAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCTGG
TGGCCCTACTGATGGCGGCCCTCTACCTCGTCCGAATCAGACAGAA
GAAAGCCCAGGGCTCCACTTCTTCTACAAGGTTGCATGAGCCCGAG
AAGAATGCCAGAGAAATAACACAGGACACAAATGATATCACATATG
CAGACCTGAACCTGCCCAAGGGGAAGAAGCCTGCTCCCCAGGCTGC
GGAGCCCAACAACCACACGGAGTATGCCAGCATTCAGACCAGCCCG
CAGCCCGCGTCGGAGGACACCCTCACCTATGCTGACCTGGACATGG
TCCACCTCAACCGGACCCCCAAGCAGCCGGCCCCCAAGCCTGAGCC
GTCCTTCTCAGAGTACGCCAGCGTCCAGGTCCCGAGGAAG human SIRP.alpha.V1 34
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid
sequence) AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTT
VSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRP.alpha.V2 35
ATGGAACCTGCCGGACCTGCCCCTGGCAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGAACTGCAAGTGATCCAGCCCGACAAGAGCGTGTCCGTGGCT
GCTGGCGAGTCTGCCATCCTGCACTGTACCGTGACCAGCCTGATCC
CCGTGGGCCCCATCCAGTGGTTTAGAGGCGCTGGCCCTGCCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGAGAGCACCAAGCGCGAGAACATGGACTTCAGCATCAGCA
TCTCCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAA
GTTCAGAAAGGGCAGCCCCGACACCGAGTTCAAGAGCGGCGCTGGA
ACCGAGCTGTCTGTGCGGGCTAAGCCTTCTGCCCCTGTGGTGTCTG
GACCTGCCGCCAGAGCTACACCTCAGCACACCGTGTCTTTCACATG
CGAGAGCCACGGCTTCAGCCCCAGAGACATCACCCTGAAGTGGTTC
AAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTG
TGGGCGAGTCCGTGTCCTACAGCATCCACAGCACCGCCAAGGTGGT
GCTGACCCGCGAGGATGTGCACAGCCAAGTGATCTGCGAGGTGGCC
CACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCTAACCTGA
GCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCC
CGTGCGGGCTGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAG
TTCTACCCTCAGAGACTGCAGCTGACCTGGCTGGAAAACGGAAACG
TGTCCAGAACCGAGACAGCCAGCACCGTGACAGAGAACAAGGACGG
CACATACAACTGGATGAGCTGGCTGCTCGTGAACGTGTCCGCCCAC
AGAGATGACGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGC
CTGCCGTGTCTAAGAGCCACGACCTGAAGGTGTCCGCTCACCCCAA
AGAGCAGGGCAGCAACACCGCCGCTGAGAACACAGGCAGCAACGAG
AGAAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGG
CTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCAGACAGAAGAA
GGCCCAGGGCTCCACCTCCAGCACCAGACTGCACGAGCCTGAGAAG
AACGCCCGCGAGATCACCCAGGACACCAACGACATCACCTACGCCG
ACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGA
GCCTAACAACCACACAGAGTACGCCAGCATCCAGACCAGCCCTCAG
CCTGCCAGCGAGGACACACTGACATACGCCGATCTGGACATGGTGC
ACCTGAACAGAACCCCCAAGCAGCCCGCTCCCAAGCCCGAGCCTAG
CTTCTCTGAGTACGCCTCCGTGCAGGTGCCCAGAAAA human SIRP.alpha.V2 36
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVSVA (amino acid
sequence) AGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTT
VSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG
TELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWF
KNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVA
HVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRK
FYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAH
RDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNE
RNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEK
NAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQ
PASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRP.beta.1 37
ATGCCCGTGCCAGCCTCCTGGCCCCACCTTCCTAGTCCTTTCCTGC (nucleotide
sequence) TGATGACGCTACTGCTGGGGAGACTCACAGGAGTGGCAGGTGAGGA
CGAGCTACAGGTGATTCAGCCTGAAAAGTCCGTATCAGTTGCAGCT
GGAGAGTCGGCCACTCTGCGCTGTGCTATGACGTCCCTGATCCCTG
TGGGGCCCATCATGTGGTTTAGAGGAGCTGGAGCAGGCCGGGAATT
AATCTACAATCAGAAAGAAGGCCACTTCCCACGGGTAACAACTGTT
TCAGAACTCACAAAGAGAAACAACCTGGACTTTTCCATCAGCATCA
GTAACATCACCCCAGCAGACGCCGGCACCTACTACTGTGTGAAGTT
CCGGAAAGGGAGCCCTGACGACGTGGAGTTTAAGTCTGGAGCAGGC
ACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGGTATCGG
GCCCTGCGGTGAGGGCCACACCTGAGCACACAGTGAGCTTCACCTG
CGAGTCCCATGGCTTCTCTCCCAGAGACATCACCCTGAAATGGTTC
AAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCG
CAGGAGACAGTGTGTCCTACAGCATCCACAGCACAGCCAGGGTGGT
GCTGACCCGTGGGGACGTTCACTCTCAAGTCATCTGCGAGATAGCC
CACATCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACTTGT
CTGAGGCCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACAGCC
CATGAGGGCAGAGAACCAGGCAAACGTCACCTGCCAGGTGAGCAAT
TTCTACCCCCGGGGACTACAGCTGACCTGGTTGGAGAATGGAAATG
TGTCCCGGACAGAAACAGCTTCGACCCTCATAGAGAACAAGGATGG
CACCTACAACTGGATGAGCTGGCTCCTGGTGAACACCTGTGCCCAC
AGGGACGATGTGGTGCTCACCTGTCAGGTGGAGCATGATGGGCAGC
AAGCAGTCAGCAAAAGCTATGCCCTGGAGATCTCAGCGCACCAGAA
GGAGCACGGCTCAGATATCACCCATGAAGCAGCGCTGGCTCCTACT
GCTCCACTCCTCGTAGCTCTCCTCCTGGGCCCCAAGCTGCTACTGG
TGGTTGGTGTCTCTGCCATCTACATCTGCTGGAAACAGAAGGCC human SIRP.beta.1
(amino 38 MPVPASWPHLPSPFLLMTLLLGRLTGVAGEDELQVIQPEKSVSVAA acid
sequence) GESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTV
SELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAG
TELSVRAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWF
KNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIA
HITLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAENQANVTCQVSN
FYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAH
RDDVVLTCQVEHDGQQAVSKSYALEISAHQKEHGSDITHEAALAPT
APLLVALLLGPKLLLVVGVSAIYICWKQKA human SIRP.gamma. 39
ATGCCTGTCCCAGCCTCCTGGCCCCATCCTCCTGGTCCTTTCCTGC (nucleotide
sequence) TTCTGACTCTACTGCTGGGACTTACAGAAGTGGCAGGTGAGGAGGA
GCTACAGATGATTCAGCCTGAGAAGCTCCTGTTGGTCACAGTTGGA
AAGACAGCCACTCTGCACTGCACTGTGACCTCCCTGCTTCCCGTGG
GACCCGTCCTGTGGTTCAGAGGAGTTGGACCAGGCCGGGAATTAAT
CTACAATCAAAAAGAAGGCCACTTCCCCAGGGTAACAACAGTTTCA
GACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCAGTA
GCATCACCCCAGCAGATGTCGGCACATACTACTGTGTGAAGTTTCG
AAAAGGGAGCCCTGAGAACGTGGAGTTTAAGTCTGGACCAGGCACT
GAGATGGCTTTGGGTGCCAAACCCTCTGCCCCCGTGGTATTGGGCC
CTGCGGCGAGGACCACACCTGAGCATACAGTGAGTTTCACCTGTGA
GTCCCATGGCTTCTCTCCCAGAGACATCACCCTGAAATGGTTCAAA
AATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCACAG
GACAGAGTGTGGCCTACAGCATCCGCAGCACAGCCAGGGTGGTACT
GGACCCCTGGGACGTTCGCTCTCAGGTCATCTGCGAGGTGGCCCAT
GTCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACTTGTCTG
AGGCCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACAGCCCAT
GAGGGIGGGGAACCAGGTAAACGTCACCTGCCAGGTGAGGAAGTIC
TACCCCCAGAGCCTACAGCTGACCTGGTCGGAGAATGGAAACGTGT
GCCAGAGAGAAACAGCCICGACCCTTACAGAGAACAAGGATGGTAC
CTACAACTGGACAAGCTGGTTCCTGGTGAACATATCTGACCAAAGG
GATGATGTGGTCCTCACCTGCCAGGTGAAGCATGATGGGCAGCTGG
CGGTCAGCAAACGCCTTGCCCTAGAGGTCACAGTCCACCAGAAGGA
CCAGAGCTCAGATGCTACCCCTGGCCCGGCATCATCCCTTACTGCG
CTGCTCCTCATAGCTGTCCTCCTGGGCCCCATCTACGTCCCCTGGA AGCAGAAGACC human
SIRP.gamma. (amino 40
MPVPASWPHPPGPFLLLTLLLGLTEVAGEEELQMIQPEKLLLVTVG acid sequence)
KTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVS
DLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGT
EMALGAKPSAPVVLGPAARTTPEHTVSFTCESHGFSPRDITLKWFK
NGNELSDFQTNVDPTGQSVAYSIRSTARVVLDPWDVRSQVICEVAH
VTLQGDPLRGTANLSEAIRVPPTLEVTQQPMRVGNQVNVTCQVRKF
YPQSLQLTWSENGNVCQRETASTLTENKDGTYNWTSWFLVNISDQR
DDVVLTCQVKHDGQLAVSKRLALEVTVHQKDQSSDATPGPASSLTA LLLIAVLLGPIYVPWKQKT
human CD47 41 ATGTGGCCTCTGGTGGCCGCTCTGCTGCTGGGCTCTGCTTGTTGTG
(nucleotide sequence)
GATCCGCCCAGCTGCTGTTCAACAAGACCAAGTCCGTGGAGTTCAC
CTTCTGCAACGATACCGTCGTGATCCCCTGCTTCGTGACCAACATG
GAAGCCCAGAACACCACCGAGGTGTACGTGAAGTGGAAGTTCAAGG
GCCGGGACATCTACACCTTCGACGGCGCCCTGAACAAGTCCACCGT
GCCCACCGATTTCTCCAGCGCCAAGATCGAGGTGTCACAGCTGCTG
AAGGGCGACGCCTCCCTGAAGATGGACAAGTCCGACGCCGTGTCCC
ACACCGGCAACTACACCTGTGAAGTGACCGAGCTGACCAGAGAGGG
CGAGACAATCATCGAGCTGAAGTACCGGGTGGTGTCCTGGTTCAGC
CCCAACGAGAACATCCTGATCGTGATCTTCCCCATCTTCGCCATCC
TGCTGTTCTGGGGCCAGTTCGGCATCAAGACCCTGAAGTACAGATC
CGGCGGCATGGACGAAAAGACAATCGCCCTGCTGGTGGCTGGCCTC
GTGATCACCGTGATTGTGATCGTGGGCGCTATCCTGTTCGTGCCCG
GCGAGTACAGCCTGAAGAATGCTACCGGCCTGGGCCTGATTGTGAC
CTCCACCGGAATCCTGATCCTGCTGCACTACTACGTGTTCTCCACC
GCTATCGGCCTGACCTCCTTCGTGATCGCCATTCTCGTGATCCAAG
TGATCGCCTACATCCTGGCCGTCGTGGGCCTGTCCCTGTGTATCGC
CGCCTGCATCCCTATGCACGGCCCCCTGCTGATCTCCGGCCTGTCT
ATTCTGGCCCTGGCTCAGCTGCTGGGACTGGTGTACATGAAGTTCG
TGGCCTCCAACCAGAAAACCATCCAGCCCCCTCGGAAGGCCGTGGA
AGAACCCCTGAACGCCTTCAAAGAATCCAAGGGCATGATGAACGAC GAA human CD47
(amino 42 MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNM acid
sequence) EAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLL
KGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFS
PNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGL
VITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFST
AIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLS
ILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMND E human
SIRP.alpha.V3 43 ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT
(nucleotide sequence)
GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGTCTGTGGCC
GCTGGCGAGTCTGCCATCCTGCTGTGTACCGTGACCTCCCTGATCC
CCGTGGGCCCCATCCAGTGGTTTAGAGGCGCTGGCCCTGCCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGAGTCCACCAAGCGCGAGAACATGGACTTCTCCATCTCCA
TCAGCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACACCGAGTTCAAGTCTGGCGCTGGC
ACCGAGCTGTCTGTGCGGGCTAAACCTICTGCCCCTGTGGTGTCTG
GACCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTG
CGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTC
AAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTG
TGGGCGAGAGCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGT
GCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCC
CACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGT
CCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCC
AGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAG
TTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATG
TGTCCCGGACCGAGACAGCCAGCACCGTGACCGAGAACAAGGATGG
CACCTACAATTGGATGTCTTGGCTGCTCGTGAACGTGTCCGCCCAC
CGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGC
CTGCCGTGTCCAAGAGCCACGATCTGAAGGTGTCCGCTCATCCCAA
AGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAG
CGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGG
CTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCCGGCAGAAGAA
GGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAG
AACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCG
ACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGA
GCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAG
CCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGC
ACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAG
CTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRP.alpha.V3 44
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVSVA (amino acid
sequence) AGESAILLCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTT
VSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG
TELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWF
KNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVA
HVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRK
FYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAH
RDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNE
RNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEK
NAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQ
PASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRP.alpha.V4 45
ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGGCCTGCAAGTGATCCAGCCCGACAAGTCCGTGTCTGTGGCC
GCTGGCGAGTCTGCCATCCTGCACTGTACCGCCACCTCCCTGATCC
CCGTGGGACCCATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGA
TCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCT
GGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGT
CTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTAC
CTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGG
TTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACC
CTGTGGGCGAGAGCGTGTCCTACTCCATCCACTCCACCGCCAAGGT
GGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTG
GCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACC
TGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCA
GCCAGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGG
AAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCA
ATGTGTCCCGGACCGAGACAGCCTCCACCGTGACCGAGAACAAGGA
TGGCACCTACAATTGGATGTCTTGGCTGCTCGTGAACGTGTCCGCC
CACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCC
AGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCC
CAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAAC
GAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGG
TGGCTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCCGGCAGAA
GAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAG
AAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACG
CCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGC
CGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCT
CAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGG
TGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCC
TAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRP.alpha.V4 46
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEGLQVIQPDKSVSVA (amino acid
sequence) AGESAILHCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTT
VSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRP.alpha.V5 47
ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGAACTGCAAGTGATCCAGCCCGACAAGTTCGTGCTGGTGGCC
GCTGGCGAGACAGCCACCCTGAGATGTACCGCCACCTCCCTGATCC
CCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGA
TCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCT
GGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGT
CTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTAC
CTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGG
TTCAAGAACGGCAACGAGCTGAGCGACTICCAGACCAACGTGGACC
CTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGT
GGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTG
GCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACC
TGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCA
GCCAGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGG
AAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCA
ATGTGTCCCGGACCGAGACTGCCTCCACCGTGACCGAGAACAAGGA
TGGCACCTACAATTGGATGTCTTGGCTGCTCGTGAACGTGTCCGCC
CACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCC
AGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCC
CAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAAC
GAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGG
TGGCTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCCGGCAGAA
GAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAG
AAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACG
CCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGC
CGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCT
CAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGG
TGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCC
TAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRP.alpha.V5 48
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKFVLVA (amino acid
sequence) AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTT
VSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRP.alpha.V6 49
ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCT
GCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCC
CCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGACCTGACCAAGCGGAACAACATGGACTTCCCCATCCGGA
TCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCT
GGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGT
CTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTAC
CTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGG
TTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACC
CTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGT
GGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTG
GCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACC
TGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCA
GCCCGTGCGGGCTGAGAACCAAGTGAACGTGACCTGCCAAGTGCGG
AAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCA
ATGTGTCCCGGACCGAGACAGCCTCCACCGTGACCGAGAACAAGGA
TGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACGTGTCCGCC
CACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCC
AGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCC
CAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAAC
GAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGG
TGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAA
GAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAG
AAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACG
CCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGC
CGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCT
CAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGG
TGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCC
TAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRP.alpha.V6 50
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid
sequence) AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTT
VSDLTKRNNMDFPIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KEYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRP.alpha.V8 51
ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCT
GCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCC
CCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGCCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGAGTCCACCAAGCGCGAGAACATGGACTTCTCCATCTCCA
TCAGCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACACCGAGTTCAAGTCTGGCGCTGGC
ACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTG
GACCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTACCTG
CGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTC
AAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTG
TGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGTGGT
GCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTGGCC
CACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACCTGT
CCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCC
CGTGCGGGCTGAGAACCAAGTGAACGTGACCTGCCAAGTGCGGAAG
TTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCAATG
TGTCCCGGACCGAGACAGCCAGCACCGTGACCGAGAACAAGGATGG
CACCTACAATTGGATGTCCTGGCTGCTCGTGAACGTGTCCGCCCAC
CGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCCAGC
CTGCCGTGTCCAAGAGCCACGATCTGAAGGTGTCCGCTCATCCCAA
AGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAACGAG
CGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGGTGG
CACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAAGAA
GGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAGAAG
AACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACGCCG
ACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGCCGA
GCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCTCAG
CCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGGTGC
ACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCCTAG
CTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRP.alpha.V8 52
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid
sequence) AGETATLRCTATSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTT
VSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG
TELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWF
KNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVA
HVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRK
FYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAH
RDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNE
RNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEK
NAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQ
PASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human SIRP.alpha.V9 53
ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCT
GCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCC
CCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGA
TCTCCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCT
GGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGT
CTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTAC
CTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGG
TTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACC
CTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGT
GGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTG
GCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACC
TGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCA
GCCCGTGCGGGCTGAGAACCAAGTGAACGTGACCTGCCAAGTGCGG
AAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCA
ATGTGTCCCGGACCGAGACAGCCTCCACCGTGACCGAGAACAAGGA
TGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACGTGTCCGCC
CACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCC
AGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCC
CAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAAC
GAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGG
TGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAA
GAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAG
AAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACG
CCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGC
CGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCT
CAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGG
TGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCC
TAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA human SIRP.alpha.V9 54
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid
sequence) AGETATLRCTATSLIPVGPIQWFRGAGPORELIYNQKEGHFPRVTT
VSDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK
hSIRP.alpha.-V.beta.C1.alpha.C2.alpha. 55
ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
GGACGAGCTGCAAGTGATCCAGCCCGAGAAGTCCGTGTCTGTGGCC
GCTGGCGAGTCTGCCACCCTGAGATGCGCTATGACCTCCCTGATCC
CCGTGGGCCCCATCATGTGGTTTAGAGGCGCTGGCGCTGGCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGAGCTGACCAAGCGGAACAACCTGGACTTCTCCATCTCCA
TCAGCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCT
GGAACCGAGCTGTCCGTGCGGGCTAAACCTTCTGCCCCTGTGGTGT
CTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTAC
CTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGG
TTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACC
CTGTGGGCGAGAGCGTGTCCTACTCCATCCACTCCACCGCCAAGGT
GGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTG
GCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACC
TGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCA
GCCTGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGG
AAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCA
ATGTGTCCCGGACCGAGACAGCCAGCACCGTGACCGAGAACAAGGA
TGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACGTGTCCGCC
CACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCC
AGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCC
CAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAAC
GAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGG
TGGCTCTGCTGATGGCTGCCCTGTACCTCGTGCGGATCCGGCAGAA
GAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCTGAG
AAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACG
CCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCCGC
CGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCT
CAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGG
TGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCC
TAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA
hSIRP.alpha.-V.beta.C1.alpha.C2.alpha. 56
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEDELQVIQPEKSVSVA (amino acid
sequence) AGESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTT
VSELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK
hSIRP.alpha.-V.alpha.C1.beta.C2.alpha. 57
ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCT
GCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCC
CCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGA
TCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCT
GGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCCGTGGTGT
CTGGACCTGCCGTGCGAGCTACCCCTGAGCACACCGTGTCTTTTAC
CTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGG
TTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACC
CAGCCGGCGACTCCGTGTCCTACTCCATCCACTCTACCGCCAGAGT
GGTGCTGACCAGAGGCGACGTGCACTCCCAAGTGATCTGCGAGATC
GCCCATATCACACTGCAGGGCGACCCCCTGAGAGGCACCGCTAACC
TGTCTGAGACAATCCGGGTGCCCCCCACCCTGGAAGTGACTCAGCA
GCCAGTGCGGGCCGAGAACCAAGTGAACGTGACCTGCCAAGTGCGG
AAGTTCTACCCCCAGCGGCTGCAGCTGACCTGGCTGGAAAACGGCA
ATGTGTCCCGGACCGAGACAGCCTCCACCGTGACCGAGAACAAGGA
TGGCACCTACAATTGGATGTCTTGGCTGCTCGTGAACGTGTCCGCC
CACCGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGACGGCC
AGCCTGCCGTGTCCAAGTCCCACGATCTGAAGGTGTCCGCTCATCC
CAAAGAGCAGGGCTCCAACACCGCCGCTGAGAACACCGGCTCTAAC
GAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGG
TGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAA
GAAGGCCCAGGGCTCTACCTCCTCCACCAGACTGCACGAGCCCGAG
AAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACG
CCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCCGC
CGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCT
CAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGG
TGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCC
TAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA
hSIRP.alpha.-V.alpha.C1.beta.C2.alpha. 58
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid
sequence) AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTT
VSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEI
AHITLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK
hSIRP.alpha.-V.alpha.C1.alpha.C2.beta. 59
ATGGAACCTGCCGGCCCTGCTCCTGGTAGACTGGGACCTCTGCTGT (nucleotide
sequence) GTCTGCTGCTGGCCGCCTCTTGTGCTTGGAGCGGAGTGGCTGGCGA
AGAGGAACTGCAAGTGATCCAGCCCGACAAGTCCGTGCTGGTGGCT
GCTGGCGAGACTGCCACCCTGAGATGTACCGCCACCTCCCTGATCC
CCGTGGGCCCTATCCAGTGGTTTAGAGGCGCTGGCCCTGGCAGAGA
GCTGATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACC
GTGTCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGA
TCGGCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAA
GTTCCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCT
GGCACCGAGCTGTCTGTGCGGGCTAAACCTTCTGCCCCTGTGGTGT
CTGGCCCTGCCGCTAGAGCTACCCCTCAGCACACCGTGTCTTTTAC
CTGCGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGG
TTCAAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACC
CTGTGGGCGAGTCCGTGTCCTACTCCATCCACTCCACCGCCAAGGT
GGTGCTGACACGCGAGGACGTGCACTCCCAAGTGATCTGCGAGGTG
GCCCACGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAACC
TGTCCGAGACAATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCA
GCCTATGAGAGCCGAGAACCAGGCCAACGTGACCTGCCAGGTGTCC
AACTTCTACCCTCGGGGCCTGCAGCTGACCTGGCTGGAAAACGGCA
ATGTGTCCCGGACCGAGACAGCCTCCACCCTGATCGAGAACAAGGA
TGGCACCTACAATTGGATGTCCTGGCTGCTCGTGAACACCTGTGCC
CACCGGGACGATGTGGTGCTGACCTGTCAGGTGGAACACGATGGCC
AGCAGGCCGTGTCCAAGTCCTACGCTCTGGAAGTGTCCGCCCACCC
CAAAGAGCAGGGCTCTAATACCGCCGCTGAGAACACCGGCTCCAAC
GAGCGGAACATCTACATCGTCGTGGGCGTCGTGTGCACCCTGCTGG
TGGCACTGCTGATGGCCGCTCTGTACCTCGTGCGGATCCGGCAGAA
GAAGGCTCAGGGCTCCACCTCCTCCACCAGACTGCACGAGCCTGAG
AAGAACGCCAGAGAGATCACCCAGGACACCAACGACATCACCTACG
CCGACCTGAACCTGCCCAAGGGCAAGAAGCCTGCCCCTCAGGCTGC
CGAGCCTAACAACCACACCGAGTACGCCTCCATCCAGACCAGCCCT
CAGCCTGCCTCTGAGGACACCCTGACCTACGCTGATCTGGACATGG
TGCACCTGAACCGGACCCCCAAGCAGCCAGCTCCTAAGCCCGAGCC
TAGCTTCTCTGAGTACGCCAGCGTGCAGGTGCCCCGGAAA
hSIRP.alpha.-V.alpha.C1.alpha.C2.beta. 60
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA (amino acid
sequence) AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTT
VSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPMRAENQANVTCQVS
NFYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCA
HRDDVVLTCQVEHDGQQAVSKSYALEVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human 61
ATGGAGCCCGCCGGCCCGGCCCCCGGCCGCCTCGGGCCGCTGCTCT SIRP.alpha.V1(P74A)
GCCTGCTGCTCGCCGCGTCCTGCGCCTGGTCAGGAGTGGCGGGTGA (nucleotide
sequence) GGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCA
GCTGGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCTGATCC
CTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGGAGCAGGCCGGGA
ATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACT
GTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCA
TCGGTAACATCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAA
GTTCCGGAAAGGGAGCCCCGATGACGTGGAGTTTAAGTCTGGAGCA
GGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGGTAT
CGGGCCCTGCGGCGAGGGCCACACCTCAGCACACAGTGAGCTTCAC
CTGCGAGTCCCACGGCTTCTCACCCAGAGACATCACCCTGAAATGG
TTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACC
CCGTAGGAGAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGT
GGTGCTGACCCGCGAGGACGTTCACTCTCAAGTCATCTGCGAGGTG
GCCCACGTCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACT
TGTCTGAGACCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACA
GCCCGTGAGGGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGG
AAGTTCTACCCCCAGAGACTACAGCTGACCTGGTTGGAGAATGGAA
ACGTGTCCCGGACAGAAACGGCCTCAACCGTTACAGAGAACAAGGA
TGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGTATCTGCC
CACAGGGATGATGTGAAGCTCACCTGCCAGGTGGAGCATGACGGGC
AGCCAGCGGTCAGCAAAAGCCATGACCTGAAGGTCTCAGCCCACCC
GAAGGAGCAGGGCTCAAATACCGCCGCTGAGAACACTGGATCTAAT
GAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCTGG
TGGCCCTACTGATGGCGGCCCTCTACCTCGTCCGAATCAGACAGAA
GAAAGCCCAGGGCTCCACTTCTTCTACAAGGTTGCATGAGCCCGAG
AAGAATGCCAGAGAAATAACACAGGACACAAATGATATCACATATG
CAGACCTGAACCTGCCCAAGGGGAAGAAGCCTGCTCCCCAGGCTGC
GGAGCCCAACAACCACACGGAGTATGCCAGCATTCAGACCAGCCCG
CAGCCCGCGTCGGAGGACACCCTCACCTATGCTGACCTGGACATGG
TCCACCTCAACCGGACCCCCAAGCAGCCGGCCCCCAAGCCTGAGCC
GTCCTTCTCAGAGTACGCCAGCGTCCAGGTCCCGAGGAAG human 62
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA SIRP.alpha.V1(P74A)
AGETATLRCTATSLIPVGPIQWFRGAGAGRELIYNQKEGHFPRVTT (amino acid
sequence) VSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN
ERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPE
KNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK human kappa constant
63 CGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACG domain
(nucleotide AGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAA
sequence) CTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCC
CTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCA
AGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGC
CGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAG
GGCCTGTCTAGCCCTGTGACCAAGTCCTTCAACCGGGGCGAGTGC human kappa constant
64 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA domain (protein
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ sequence)
GLSSPVTKSFNRGEC human IgG4 constant 65
GCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCA domains (including
GATCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTCGTGAAGGA S228P)
CTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTG (nucleotide
sequence) ACCTCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCC
TGTACTCCCTGTCCAGCGTCGTGACAGTGCCCTCCAGCTCTCTGGG
CACCAAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACC
AAGGTGGACAAGCGGGTGGAATCTAAGTACGGCCCTCCCTGCCCTC
CTTGCCCAGCCCCTGAATTTCTGGGCGGACCTTCTGTGTTTCTGTT
CCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAA
GTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGC
AGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGAC
CAAGCCTAGAGAGGAACAGTTCAACTCCACCTACCGGGTGGTGTCC
GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACA
AGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAAAAGAC
CATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACA
CTGCCTCCAAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGA
CCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATG
GGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCT
GTGCTGGACTCCGACGGCTCCTTCTTTCTGTACTCTCGCCTGACCG
TGGACAAGTCCCGGTGGCAGGAAGGCAACGTGTTCTCCTGCAGCGT
GATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCC CTGTCTCTGGGAAAA
human IgG4 constant 66
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL domains (including
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT S228P)
KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE (protein sequence)
VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK human IgG2
constant 67 GCTTCTACAAAGGGCCCCAGCGTGTTCCCTCTGGCTCCTTGTAGCA domains
(nucleotide GAAGCACCAGCGAGTCTACAGCCGCTCTGGGCTGTCTGGTCAAGGA
sequence) CTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCACTG
ACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCTCCGGCC
TGTACTCTCTGTCCAGCGTGGTCACAGTGCCCAGCAGCAATTTTGG
CACCCAGACCTACACCTGTAATGTGGACCACAAGCCTAGCAACACC
AAGGTGGACAAGACCGTGGAACGGAAGTGCTGCGTGGAATGCCCTC
CTTGTCCTGCTCCTCCAGTGGCTGGCCCTTCCGTGTTTCTGTTCCC
TCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTG
ACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCTGAGGTGCAGT
TCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAA
GCCTAGAGAGGAACAGTTCAACAGCACCTTCAGAGTGGTGTCCGTG
CTGACCGTGGTGCATCAGGATTGGCTGAACGGCAAAGAGTACAAGT
GCAAGGTGTCCAACAAGGGCCTGCCTGCTCCTATCGAGAAAACCAT
CAGCAAGACCAAAGGCCAGCCTCGCGAGCCCCAGGTTTACACACTT
CCTCCAAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCT
GCCTCGTGAAGGGCTTCTACCCCAGCGACATCX.sub.1CCGTGGAATGGG
AGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTAT
GCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTG
GACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCCTGCAGCGTGA
TGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCT GAGCCCCGGCAAA
wherein: X.sub.1 = G, T human IgG2 constant 68
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL domains (protein
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT sequence)
KVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV
LTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIX.sub.1VEWESNGQPENNYKTTPP
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK wherein:
X.sub.1 = A, S 40A heavy chain 69 SYWMH CDR1 (amino acid sequence)
40A heavy chain 70 AIYPVNNDTTYNQKFKG CDR2 (amino acid sequence) 40A
heavy chain 71 SFYYSLDAAWFVY CDR3 (amino acid sequence) 40A light
chain CDR1 72 RASQDIGSRLN (amino acid sequence) 40A light chain
CDR2 73 ATSSLDS (amino acid sequence) 40A light chain CDR3 74
LQYASSPFT (amino acid sequence) humanized 40 heavy 75
EVQX.sub.1X.sub.2QSGAX.sub.3X.sub.4X.sub.5KPGASVKX.sub.6SCKASGSTFTSYWMHWV-
X.sub.7QX.sub.8 chain variable region
PGQGLEWX.sub.9GAIYPVNSDTTYNQKFKGX.sub.10X.sub.11TX.sub.12TVX.sub.13X.sub.-
14SX.sub.15S
(consensus sequence)
TX.sub.16YMX.sub.17LSSLX.sub.18X.sub.19EDX.sub.20AVYYCX.sub.21RSFYYSLDAAW-
FVYWGQG TX.sub.22X.sub.23TVSS wherein: X.sub.1 = F, L X.sub.2 = Q,
R, V X.sub.3 = E, V X.sub.4 = L, V X.sub.5 = A, K, V X.sub.6 = L,
M, V X.sub.7 = K, R X.sub.8 = A, R, T X.sub.9 = I, M X.sub.10 = K,
R X.sub.11 = A, V X.sub.12 = L, M X.sub.13 = D, V X.sub.14 = K, T
X.sub.15 = A, S, T X.sub.16 = A, V X.sub.17 = E, Q X.sub.18 = R, T
X.sub.19 = F, S X.sub.20 = S, T X.sub.21 = A, T X.sub.22 = L, T
X.sub.23 = L, V humanized 40 light 76
DIQMTQSPSSLSASX.sub.1GX.sub.2RVX.sub.3ITCRASQDIGSRLNWLQQX.sub.4PGKA
chain variable region
X.sub.5KRLIYATSSLDSGVPX.sub.6RFSGSX.sub.7SGX.sub.8X.sub.9X.sub.10X.sub.11-
LTISX.sub.12LQPE (consensus sequence)
DFATYYCLQYASSPFTFGX.sub.13GTKX.sub.14EIX.sub.15 wherein: X.sub.1 =
L, V X.sub.2 = D, E X.sub.3 = S, T X.sub.4 = K, T X.sub.5 = I, P
X.sub.6 = K, S X.sub.7 = G, R X.sub.8 = S, T X.sub.9 = D, E
X.sub.10 = F, Y X.sub.11 = S, T X.sub.12 = G, S X.sub.13 = G, Q
X.sub.14 = L, V X.sub.15 = H, K hSIRP.alpha..40AVH1 77
GAGGTGCAGTTCTTGCAGTCTGGTGCCGTGCTGGCTAGACCTGGAA (nucleotide
sequence) CCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTCCACCTTCACCTC
TTACTGGATGCACTGGGTCAAGCAGAGGCCTGGACAGGGACTCGAA
TGGATCGGCGCTCTGTACCCTGTGAACTCCGACACCACCTACAACC
AGAAGTTCAAGGGCAGAGCCAAGCTGACCGTGGCCACCTCTGCTTC
TATCGCCTACCTGGAATTTTCCAGCCTGACCAACGAGGACTCCGCC
GTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTT
GGTTTGTGTACTGGGGCCAGGGAACTCTGGTGACCGTGTCCTCT hSIRP.alpha..40AVH1 78
EVQFLQSGAVLARPGTSVKISCKASGSTFTSYWMHWVKQRPGQGLE (amino acid
sequence) WIGALYPVNSDTTYNQKFKGRAKLTVATSASIAYLEFSSLTNEDSA
VYYCARSFYYSLDAAWFVYWGQGTLVTVSS hSIRP.alpha..40AVH2 79
GAGGTGCAGCTGGTTCAGTCTGGCGCTGAGGTTGTGAAGCCTGGCG (nucleotide
sequence) CTTCCGTGAAGCTGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAG
CTACTGGATGCACTGGGTCAAGCAGGCCCCTGGACAAGGCCTGGAA
TGGATCGGCGCTATCTACCCCGTGAACTCCGACACCACCTACAACC
AGAAGTTCAAGGGCAAAGCTACCCTGACCGTGGACAAGTCTGCCTC
CACCGCCTACATGGAACTGTCCAGCCTGAGATCTGAGGACACCGCC
GTGTACTACTGCACCCGGTCCTTCTACTACTCCCTGGACGCCGCTT
GGTTTGTGTATTGGGGCCAGGGAACACTGGTGACCGTGTCCTCT hSIRP.alpha..40AVH2 80
EVQLVQSGAEVVKPGASVKLSCKASGSTFTSYWMHWVKQAPGQGLE (amino acid
sequence) WIGAIYPVNSDTTYNQKFKGKATLTVDKSASTAYMELSSLRSEDTA
VYYCTRSFYYSLDAAWFVYWGQGTLVTVSS hSIRP.alpha..40AVH3 81
GAGGTGCAGCTGAGACAGTCTGGCGCTGTGCTTGTGAAGCCTGGCG (nucleotide
sequence) CCTCCGTGAAGATGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAG
CTACTGGATGCACTGGGTCAAGCAGACCCCTGGACAGGGACTCGAG
TGGATCGGCGCTATCTACCCTGTGAACTCCGACACCACCTACAACC
AGAAGTTCAAGGGCAAAGCTACCCTGACCGTGGACAAGTCCTCCTC
CACCGCTTACATGCAGCTGTCCAGCCTGACCTCTGAGGACTCCGCC
GTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTT
GGTTTGTGTACTGGGGCCAGGGCACAACCCTGACAGTGTCCTCT hSIRP.alpha..40AVH3 82
EVQLRQSGAVLVKPGASVKMSCKASGSTFTSYWMHWVKQTPGQGLE (amino acid
sequence) WIGAIYPVNSDTTYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSA
VYYCARSFYYSLDAAWFVYWGQGTTLTVSS hSIRP.alpha..40AVH4 83
GAGGTGCAGTTCGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCG (nucleotide
sequence) CCTCTGTGAAGGTGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAG
CTACTGGATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTGGAA
TGGATGGGCGCTATCTACCCCGTGAACTCCGACACCACCTACAACC
AGAAATTCAAGGGCAGAGTGACCATGACCGTCGTGACCTCCACCTC
CACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCC
GTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTT
GGTTTGTGTACTGGGGCCAGGGAACTCTGGTGACCGTGTCCTCT hSIRP.alpha..40AVH4 84
EVQFVQSGAEVKKPGASVKVSCKASGSTFTSYWMHWVRQAPGQGLE (amino acid
sequence) WMGAIYPVNSDTTYNQKFKGRVTMTVVTSTSTVYMELSSLRSEDTA
VYYCARSFYYSLDAAWFVYWGQGTLVTVSS hSIRP.alpha..40AVH5 85
GAGGTCCAGCTGCAACAGTCTGGTGCCGTGTTGGCTAAGCCTGGCG (nucleotide
sequence) CCTCCGTGAAGATGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAG
CTACTGGATGCACTGGGTCAAGCAGAGGCCTGGACAGGGACTCGAG
TGGATCGGCGCTATCTACCCTGTGAACTCCGACACCACCTACAACC
AGAAGTTCAAGGGCAAAGCTACCCTGACCGTGGACAAGTCCTCCTC
CACCGCTTACATGCAGCTGTCCAGCCTGACCTTCGAGGACTCCGCC
GTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTT
GGTTTGTGTACTGGGGCCAGGGCACAACCCTGACAGTGTCCTCT hSIRP.alpha..40AVH5 86
EVQLQQSGAVLAKPGASVKMSCKASGSTFTSYWMHWVKQRPGQGLE (amino acid
sequence) WIGAIYPVNSDTTYNQKFKGKATLTVDKSSSTAYMQLSSLTFEDSA
VYYCARSFYYSLDAAWFVYWGQGTTLTVSS hSIRP.alpha..40AVH6 87
GAGGTGCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCG (nucleotide
sequence) CCTCTGTGAAGGTGTCCTGCAAGGCTTCTGGCTCCACCTTCACCAG
CTACTGGATGCACTGGGTCCGACAGGCTCCAGGACAAGGCTTGGAA
TGGATGGGCGCTATCTACCCCGTGAACTCCGACACCACCTACAACC
AGAAATTCAAGGGCAGAGTGACCATGACCGTGGACACCTCCACCAG
CACCGTGTACATGGAACTGTCCAGCCTGAGATCCGAGGACACCGCC
GTGTACTACTGCGCCCGGTCCTTCTACTACTCTCTGGACGCCGCTT
GGTTTGTGTACTGGGGCCAGGGAACTCTGGTGACCGTGTCCTCT hSIRP.alpha..40AVH6 88
EVQLVQSGAEVKKPGASVKVSCKASGSTFTSYWMHWVRQAPGQGLE (amino acid
sequence) WMGAIYPVNSDTTYNQKFKGRVTMTVDTSTSTVYMELSSLRSEDTA
VYYCARSFYYSLDAAWFVYWGQGTLVTVSS hSIRP.alpha..40AVL1 89
GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTC
CAGACTGAACTGGCTGCAGCAGACCCCTGGCAAGGCCATCAAGAGA
CTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGAT
TCTCCGGCTCTAGATCTGGCACCGACTTCTCCCTGACCATCTCTGG
ACTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCC
AGCTCTCCATTCACCTTTGGCGGAGGCACCAAGGTGGAAATCCAC hSIRP.alpha..40AVL1
90 DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQTPGKAIKR (amino acid
sequence) LIYATSSLDSGVPSRFSGSRSGTDFSLTISGLQPEDFATYYCLQYA
SSPFTFGGGTKVEIH hSIRP.alpha..40AVL2 91
GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTC
CAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCATCAAGAGA
CTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGAT
TCTCCGGCTCTAGATCTGGCACCGACTTTACCCTGACAATCAGCTC
CCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCC
TCCTCTCCATTCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG hSIRP.alpha..40AVL2
92 DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAIKR (amino acid
sequence) LIYATSSLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQYA
SSPFTFGQGTKVEIK hSIRP.alpha..40AVL3 93
GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTC
CAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCATCAAGAGA
CTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGAT
TCTCCGGCTCTAGATCTGGCACCGACTTTACCCTGACAATCAGCTC
CCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCC
AGCTCTCCATTCACCTTTGGCGGAGGCACCAAGCTGGAAATCAAG hSIRP.alpha..40AVL3
94 DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAIKR (amino acid
sequence) LIYATSSLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQYA
SSPFTFGGGTKLEIK hSIRP.alpha..40AVL4 95
GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTC
CAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCCCTAAGAGA
CTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGAT
TCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACAATCAGCTC
CCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCC
AGCTCTCCATTCACCTTTGGCGGAGGCACCAAGGTGGAAATCAAG hSIRP.alpha..40AVL4
96 DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAPKR (amino acid
sequence) LIYATSSLDSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYA
SSPFTFGGGTKVEIK hSIRP.alpha..40AVL5 97
GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGG (nucleotide
sequence) GCGACAGAGTGACCATCACCTGTAGAGCCTCTCAGGACATCGGCTC
CAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCATCAAGAGA
CTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCAAGAGAT
TCTCCGGCTCTAGATCCGGCTCCGACTATACCCTGACAATCAGCTC
CCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCC
TCCTCTCCATTCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG hSIRP.alpha..40AVL5
98 DIQMTQSPSSLSASVGDRVTITCRASQDIGSRLNWLQQKPGKAIKR (amino acid
sequence) LIYATSSLDSGVPKRFSGSRSGSDYTLTISSLQPEDFATYYCLQYA
SSPFTFGQGTKVEIK hSIRP.alpha..40AVL6 99
GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCTGCTTCCCTGG (nucleotide
sequence) GCGAGAGAGTGTCCATCACCTGTAGAGCCTCTCAGGACATCGGCTC
CAGACTGAACTGGCTGCAGCAGAAGCCTGGCAAGGCCATCAAGAGA
CTGATCTACGCCACCTCCAGCCTGGATTCTGGCGTGCCCTCTAGAT
TCTCCGGCTCTAGATCTGGCACCGACTTTACCCTGACAATCAGCTC
CCTGCAGCCTGAGGACTTCGCCACCTACTACTGTCTGCAGTACGCC
AGCTCTCCATTCACCTTTGGCGGAGGCACCAAGGTGGAAATCAAG hSIRP.alpha..40AVL6
100 DIQMTQSPSSLSASLGERVSITCRASQDIGSRLNWLQQKPGKAIKR (amino acid
sequence) LIYATSSLDSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQYA
SSPFTFGGGTKVEIK hSIRP.alpha..40A mouse 101
GAGGTTCAGTTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCAGGGA VH (nucleotide
CTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTCCACCTTTACCAG sequence)
CTACTGGATGCACTGGGTAAAACAGGGGCCTGGACAGGGTCTGCAA
TGGATTGGCGCTATTTATCCTGTAAATAATGATACTACCTATAATC
AGAAGTTCAAGGGCAAGGCCGAACTCACTGTAGTCACTTCCACCAG
CACTGCCTACATGGAGGTCAGTAGTCTGACAAATGAGGACTCTGCG
GTCTATTACTGTACAAGATCGTTCTACTATAGTCTCGACGCGGCCT
GGTTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA hSIRP.alpha..40A mouse
102 EVQFQQSGTVLARPGTSVKMSCKASGSTFTSYWMHWVKQGPGQGLQ VH (amino acid
WIGAIYPVNNDTTYNQKFKGKAELTVVTSTSTAYMEVSSLTNEDSA sequence)
VYYCTRSFYYSLDAAWFVYWGQGTLVTVSA hSIRP.alpha..40A mouse 103
GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGG VL (nucleotide
GAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGACATTGGTAG sequence)
TAGGTTAAACTGGCTTCAGCAGGAACCAGATGGAACTATTAAACGC
CTGATCTACGCCACATCCAGTTTAGATTCTGGTGTCCCCAAAAGGT
TCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCGG
CCTTGAGTCTGAAGACTTTGTAGACTATTACTGTCTACAATATGCT
AGTTCTCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAC hSIRP.alpha..40A
mouse 104 DIQMTQSPSSLSASLGERVSLTCRASQDIGSRLNWLQQEPDGTIKR VL (amino
acid LIYATSSLDSGVPKRFSGSRSGSDYSLTISGLESEDFVDYYCLQYA sequence)
SSPFTFGGGTKLEIN hSIRP.alpha..40A mouse 105
EVQFQQSGTVLARPGTSVKMSCKASGSTFTSYWMHWVKQGPGQGLQ heavy chain (amino
WIGAIYPVNNDTTYNQKFKGKAELTVVTSTSTAYMEVSSLTNEDSA acid sequence;
VYYCTRSFYYSLDAAWFVYWGQGTLVTVSAAKTTPPSVYPLAPGSA constant domain
AQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY underlined, signal
TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI peptide not shown)
CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSW
FVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRV
NSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMI
TDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKS
NWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK hSIRP.alpha..40A mouse 106
DIQMTQSPSSLSASLGERVSLTCRASQDIGSRLNWLQQEPDGTIKR light chain (amino
acid LIYATSSLDSGVPKRFSGSRSGSDYSLTISGLESEDFVDYYCLQYA sequence;
constant SSPFTFGGGTKLEINRADAAPTVSIFPPSSEQLTSGGASVVCFLNN domain
underlined, FYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKD signal
peptide not EYERHNSYTCEATHKTSTSPIVKSFNRNEC shown) rhSIRP.alpha./Fc
(amino 107 (GVAG)EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFR acid
sequence) GAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADA
GTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATP
QHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYS
IHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPP
TLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETAS
TVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHD
LKVSAHPKEQGSNTAAENTGSNERIEGRMDPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK rhSIRP.gamma./Fc (amino 108
VLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSI acid sequence)
TPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKPSAPVVLGPA
ARTTPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPTGQ
SVAYSIRSTARVVLDPWDVRSQVICEVAHVTLQGDPLRGTANLSEA
IRVPPTLEVTQQPMRAGNQVNVTCQVRKFYPQSLQLTWLENGNVCQ
RETASTLTENKDGTYNWTSWFLVNISDQRDDVVLTCQVKHDGQLAV
SKRLALEVTVHQKDQSSDATPGPASIEGRMDPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK rhCD47/Fc (amino 109
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRD acid sequence)
IYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTG
NYTCEVTELTREGETIIELKYRVVSWFSPIEGRMDPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hSIRP-V.gamma.C1.beta.C2.beta.
110 ATGCCCGTGCCTGCCTCTTGGCCTCATCTGCCCAGCCCCTTTCTGC (nucleotide
sequence) TGATGACCCTGCTGCTGGGCAGGCTGACAGGCGTGGCAGGCGAAGA
GGAACTGCAGATGATCCAGCCCGAGAAGCTGCTGCTCGTGACCGTG
GGCAAGACCGCCACCCTGCACTGCACCGTGACATCCCTGCTGCCTG
TGGGACCCGTGCTGTGGTTTAGAGGCGTGGGCCCTGGCAGAGAGCT
GATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTG
TCCGACCTGACCAAGCGGAACAACATGGACTTCTCCATCCGGATCT
CCAGCATCACCCCTGCCGACGTGGGCACCTACTACTGCGTGAAGTT
CCGGAAGGGCTCCCCCGAGAACGTGGAGTTCAAGTCTGGCCCAGGC
ACCGAGATGGCCCTGGGCGCTAAACCTTCTGCCCCTGTGGTGTCTG
GACCTGCCGTGCGGGCTACCCCTGAGCACACCGTGTCTTTTACCTG
CGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTC
AAGAACGGCAACGAGCTGTCCGACTTCCAGACCAACGTGGACCCTG
CCGGCGACTCCGTGTCCTACTCCATCCACTCTACCGCCAGAGTGGT
GCTGACCAGAGGCGACGTGCACTCCCAAGTGATCTGCGAGATCGCC
CATATCACACTGCAGGGCGACCCCCTGAGAGGCACCGCCAATCTGT
CTGAGGCCATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCC
TATGAGAGCCGAGAACCAGGCCAACGTGACCTGTCAGGTGTCCAAC
TTCTACCCTCGGGGCCTGCAGCTGACCTGGCTGGAAAACGGCAATG
TGTCCCGGACCGAGACAGCCTCCACCCTGATCGAGAACAAGGACGG
CACCTACAATTGGATGTCCTGGCTGCTCGTGAACACCTGTGCCCAC
AGGGACGACGTGGTGCTGACATGCCAGGTGGAACACGATGGCCAGC
AGGCCGTGTCCAAGTCCTACGCCCTGGAAATCTCCGCCCATCAGAA
AGAGCACGGCTCCGATATCACCCACGAGGCCGCTCTGGCTCCTACC
GCTCCTCTGCTGGTGGCTCTGCTGCTGGGACCTAAGCTGCTGCTGG
TCGTGGGCGTGTCCGCCATCTACATCTGCTGGAAGCAGAAGGCCTG A
hSIRP-V.gamma.C1.beta.C2.beta. 111
MPVPASWPHLPSPFLLMTLLLGRLTGVAGEEELQMIQPEKLLLVTV (amino acid
sequence) GKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTV
SDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPG
TEMALGAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWF
KNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIA
HITLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAENQANVTCQVSN
FYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAH
RDDVVLTCQVEHDGQQAVSKSYALEISAHQKEHGSDITHEAALAPT
APLLVALLLGPKLLLVVGVSAIYICWKQKA hSIRP-V.beta.C1.gamma.C2.beta. 112
ATGCCCGTGCCTGCCTCTTGGCCTCATCTGCCCAGCCCCTTTCTGC (nucleotide
sequence) TGATGACCCTGCTGCTGGGCAGGCTGACAGGCGTGGCAGGCGAAGA
TGAGCTGCAAGTGATCCAGCCCGAGAAGTCCGTGTCTGTGGCCGCT
GGCGAGTCTGCCACCCTGAGATGCGCTATGACCTCCCTGATCCCCG
TGGGCCCCATCATGTGGTTTAGAGGCGCTGGCGCTGGCAGAGAGCT
GATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTG
TCCGAGCTGACCAAGCGGAACAACCTGGACTTCTCCATCTCCATCA
GCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTT
CCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGA
ACCGAGCTGTCCGTGCGGGCTAAACCTTCTGCCCCTGTGGTGCTGG
GACCTGCCGCTAGAACCACCCCTGAGCACACCGTGTCTTTTACCTG
CGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTC
AAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTA
CCGGCCAGTCCGTGGCCTACTCCATCAGATCCACCGCCAGAGTGGT
GCTGGACCCTTGGGATGTGCGGTCCCAAGTGATCTGCGAGGTGGCC
CATGTGACACTGCAGGGCGATCCTCTGAGAGGCACCGCCAATCTGT
CTGAGGCCATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCC
TATGAGAGCCGAGAACCAGGCCAACGTGACCTGCCAGGTGTCCAAC
TTCTACCCTCGGGGCCTGCAGCTGACCTGGCTGGAAAACGGCAATG
TGTCCCGGACCGAGACAGCCTCCACCCTGATCGAGAACAAGGATGG
CACCTACAATTGGATGTCCTGGCTGCTCGTGAACACCTGTGCCCAC
CGGGATGACGTGGTGCTGACTTGTCAGGTGGAACACGACGGCCAGC
AGGCCGTGTCCAAGTCCTACGCCCTGGAAATCTCCGCCCATCAGAA
AGAGCACGGCTCCGATATCACCCACGAGGCCGCTCTGGCTCCTACC
GCTCCTCTGCTGGTGGCTCTGCTGCTGGGACCTAAGCTGCTGCTGG
TCGTGGGCGTGTCCGCCATCTACATCTGCTGGAAGCAGAAGGCCTG A
hSIRP-V.beta.C1.gamma.C2.beta. 113
MPVPASWPHLPSPFLLMTLLLGRLTGVAGEDELQVIQPEKSVSVAA (amino acid
sequence) GESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTV
SELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAG
TELSVRAKPSAPVVLGPAARTTPEHTVSFTCESHGFSPRDITLKWF
KNGNELSDFQTNVDPTGQSVAYSIRSTARVVLDPWDVRSQVICEVA
HVTLQGDPLRGTANLSEAIRVPPTLEVTQQPMRAENQANVTCQVSN
FYPRGLQLTWLENGNVSRTETASTLIENKDGTYNWMSWLLVNTCAH
RDDVVLTCQVEHDGQQAVSKSYALEISAHQKEHGSDITHEAALAPT
APLLVALLLGPKLLLVVGVSAIYICWKQKA hSIRP-V.beta.C1.beta.C2.gamma. 114
ATGCCCGTGCCTGCCTCTTGGCCTCATCTGCCCAGCCCCTTTCTGC (nucleotide
sequence) TGATGACCCTGCTGCTGGGCAGGCTGACAGGCGTGGCAGGCGAAGA
TGAGCTGCAAGTGATCCAGCCCGAGAAGTCCGTGTCTGTGGCCGCT
GGCGAGTCTGCCACCCTGAGATGCGCTATGACCTCCCTGATCCCCG
TGGGCCCCATCATGTGGTTTAGAGGCGCTGGCGCTGGCAGAGAGCT
GATCTACAACCAGAAAGAGGGCCACTTCCCCAGAGTGACCACCGTG
TCCGAGCTGACCAAGCGGAACAACCTGGACTTCTCCATCTCCATCA
GCAACATCACCCCTGCCGACGCCGGCACCTACTACTGCGTGAAGTT
CCGGAAGGGCTCCCCCGACGACGTGGAGTTCAAATCCGGCGCTGGA
ACCGAGCTGTCCGTGCGGGCTAAACCTTCTGCCCCTGTGGTGTCTG
GACCTGCTGTGCGCGCTACCCCTGAGCACACCGTGTCTTTTACCTG
CGAGTCCCACGGCTTCAGCCCTCGGGACATCACCCTGAAGTGGTTC
AAGAACGGCAACGAGCTGAGCGACTTCCAGACCAACGTGGACCCTG
CCGGCGACTCCGTGTCCTACTCCATCCACTCTACCGCCAGAGTGGT
GCTGACCAGAGGCGACGTGCACTCCCAAGTGATCTGCGAGATCGCC
CATATCACACTGCAGGGCGACCCCCTGAGAGGCACCGCCAATCTGT
CTGAGGCCATCAGAGTGCCCCCCACCCTGGAAGTGACCCAGCAGCC
TATGAGAGTGGGCAACCAAGTGAACGTGACCTGCCAAGTGCGGAAG
TTCTACCCCCAGTCCCTGCAGCTGACTTGGAGCGAGAATGGCAACG
TGTGCCAGAGAGAGACAGCCTCCACCCTGACCGAGAACAAGGACGG
AACCTACAACTGGACCTCCTGGTTCCTCGTGAACATCTCCGACCAG
CGGGACGACGTGGTGCTGACATGCCAAGTGAAGCACGATGGACAGC
TGGCCGTGTCCAAGCGGCTGGCTCTGGAAGTGACAGTGCACCAGAA
AGAGCACGGCTCCGACATCACCCACGAGGCCGCTCTGGCTCCTACA
GCTCCTCTGCTGGTGGCTCTGCTGCTGGGACCTAAGCTGCTGCTGG
TCGTGGGCGTGTCCGCCATCTACATCTGCTGGAAGCAGAAGGCCTG A
hSIRP-V.beta.C1.beta.C2.gamma. 115
MPVPASWPHLPSPFLLMTLLLGRLTGVAGEDELQVIQPEKSVSVAA (amino acid
sequence) GESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTV
SELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAG
TELSVRAKPSAPVVSGPAVRATPEHTVSFTCESHGFSPRDITLKWF
KNGNELSDFQTNVDPAGDSVSYSIHSTARVVLTRGDVHSQVICEIA
HITLQGDPLRGTANLSEAIRVPPTLEVTQQPMRVGNQVNVTCQVRK
FYPQSLQLTWSENGNVCQRETASTLTENKDGTYNWTSWFLVNISDQ
RDDVVLTCQVKHDGQLAVSKRLALEVTVHQKEHGSDITHEAALAPT
APLLVALLLGPKLLLVVGVSAIYICWKQKA human SIRP.beta.L 116
ATGCCTGTGCCTGCCTCTTGGCCTCATCTGCCCTCTCCATTTCTGC (nucleotide
sequence) TGATGACCCTGCTGCTGGGCAGACTGACAGGTGTTGCTGGCGAAGA
GGAACTGCAAGTGATCCAGCCTGACAAGAGCATCTCTGTGGCCGCT
GGCGAATCTGCCACACTGCACTGTACCGTGACATCTCTGATCCCTG
TGGGCCCCATCCAGTGGTTTAGAGGTGCTGGACCTGGCAGAGAGCT
GATCTACAACCAGAAAGAGGGACACTTCCCCAGAGTGACCACCGTG
TCCGACCTGACCAAGCGGAACAACATGGACTTCAGCATCCGGATCA
GCAACATCACCCCTGCCGATGCCGGCACCTACTACTGCGTGAAGTT
CAGAAAGGGCAGCCCCGACCACGTCGAGTTTAAAAGCGGAGCCGGC
ACAGAGCTGAGCGTGCGGGCTAAACCTTCTGCTCCTGTGGTGTCTG
GACCAGCCGCTAGAGCTACACCTCAGCACACCGTGTCTTTTACCTG
CGAGAGCCACGGCTTCAGCCCCAGAGATATCACCCTGAAGTGGTTC
AAGAACGGCAACGAGCTGTCCGACTTCCAGACCAATGTGGACCCAG
CCGGCGATAGCGTGTCCTACAGCATTCACAGCACCGCCAAGGTGGT
GCTGACCCGGGAAGATGTGCACAGCCAAGTGATTTGCGAGGTGGCC
CACGTTACCCTGCAAGGCGATCCTCTGAGAGGAACCGCCAACCTGA
GCGAGACAATCCGGGTGCCACCTACACTGGAAGTGACCCAGCAGCC
TGTGCGGGCCGAGAATCAAGTGAACGTGACCTGCCAAGTGCGGAAG
TTCTACCCTCAGAGACTGCAGCTGACCTGGCTGGAAAACGGCAATG
TGTCCCGGACCGAGACAGCCAGCACACTGACCGAGAACAAGGATGG
CACCTACAATTGGATGAGCTGGCTGCTGGTCAATGTGTCTGCCCAC
CGGGACGATGTGAAGCTGACATGCCAGGTGGAACACGATGGCCAGC
CTGCCGTGTCTAAGAGCCACGACCTGAAGGTGTCCGCTCATCCCAA
AGAGCAGGGCAGCAATACTGCCCCTGGACCTGCTCTTGCTTCTGCC
GCTCCTCTGCTGATCGCCTTTCTGCTGGGACCTAAGGTGCTGCTGG
TTGTGGGAGTGTCCGTGATCTACGTGTACTGGAAGCAGAAGGCC human SIRP.beta.L
(amino 117 MPVPASWPHLPSPFLLMTLLLGRLTGVAGEEELQVIQPDKSISVAA acid
sequence) GESATLHCTVTSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTV
SDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDHVEFKSGAG
TELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWF
KNGNELSDFQTNVDPAGDSVSYSIHSTAKVVLTREDVHSQVICEVA
HVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRK
FYPQRLQLTWLENGNVSRTETASTLTENKDGTYNWMSWLLVNVSAH
RDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAPGPALASA
APLLIAFLLGPKVLLVVGVSVIYVYWKQKA human IgG1 constant 118
GCCAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA domains (nucleotide
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA sequence)
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGAC
TCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG
CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACA
CATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGG
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC
CGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC
ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCA
AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC
ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG
AGCCTCTCCCTGTCTCCGGGTAAA human IgG1 constant 119
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL domains (amino acid
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT sequence)
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK mouse IgG1
constant 120 AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSL domains
(amino acid SSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTT
sequence) VDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKD
VLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRED
YNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKG
LVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHT
EENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLK NYYLKKTISRSPGK mouse
kappa constant 121 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGS
domain (amino acid ERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK
sequence) TSTSPIVKSFNRNEC human IgG2 constant 122
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL domains, V234A-
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT G237A-P238S-
KVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEV H268A-V309L-
TCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV A330S-P331S (Sigma)
LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTL mutant (amino acid
PPSREEMTKNQVSLTCLVKGFYPSDIX.sub.1VEWESNGQPENNYKTTPP sequence)
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK wherein:
X.sub.1 = A, S human IgG1 constant 123
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL domains, L234A-
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT L235A mutant (amino
KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR acid sequence)
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK human IgG1
constant 124 GCTAGCACAAAGGGCCCTAGTGTGTTTCCTCTGGCTCCCTCTTCCA
domains, L234A- AATCCACTTCTGGTGGCACTGCTGCTCTGGGATGCCTGGTGAAGGA
L235A-P329G mutant TTACTTTCCTGAACCTGTGACTGTCTCATGGAACTCTGGTGCTCTG
(nucleotide sequence)
ACTTCTGGTGTCCACACTTTCCCTGCTGTGCTGCAGTCTAGTGGAC
TGTACTCTCTGTCATCTGTGGTCACTGTGCCCTCTTCATCTCTGGG
AACCCAGACCTACATTTGTAATGTGAACCACAAACCATCCAACACT
AAAGTGGACAAAAAAGTGGAACCCAAATCCTGTGACAAAACCCACA
CCTGCCCACCTTGTCCGGCGCCTGAAGCGGCGGGAGGACCTTCTGT
GTTTCTGTTCCCCCCCAAACCAAAGGATACCCTGATGATCTCGCGA
ACCCCTGAGGTGACATGTGTGGTGGTGGATGTGTCTCATGAGGACC
CCGAAGTCAAATTTAATTGGTATGTCGACGGCGTCGAGGTGCATAA
TGCCAAAACCAAGCCTAGAGAGGAACAGTACAATTCAACCTACAGA
GTCGTCAGTGTGCTGACTGTGCTGCATCAGGATTGGCTGAATGGCA
AGGAATACAAGTGTAAAGTCTCAAACAAGGCCCTGGGAGCTCCAAT
TGAGAAAACAATCTCAAAGGCCAAAGGACAGCCTAGGGAACCCCAG
GTCTACACCCTGCCACCTTCGAGAGACGAACTGACCAAAAACCAGG
TGTCCCTGACATGCCTGGTCAAAGGCTTCTACCCTTCTGACATTGC
TGTGGAGTGGGAGTCAAATGGACAGCCTGAGAACAACTACAAAACA
ACCCCCCCTGTGCTGGATTCTGATGGCTCTTTCTTTCTGTACTCCA
AACTGACTGTGGACAAGTCTAGATGGCAGCAGGGGAATGTCTTTTC
TTGCTCTGTCATGCATGAGGCTCTGCATAACCACTACACTCAGAAA
TCCCTGTCTCTGTCTCCCGGGAAA human IgG1 constant 125
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL domains, L234A-
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT L235A-P329G mutant
KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR (amino acid
sequence) TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK human IgG1
constant 126 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
domains, N297Q TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
mutant (amino acid KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
sequence) TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK human IgG4
constant 127 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
domains, S228P- TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT
N297Q mutant (amino KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE
acid sequence) VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 18D5 VH (amino
acid 128 QVQLQQPGAELVRPGSSVKLSCKASGYTFTSYWVHWVKQRPIQGLE sequence)
WIGNIDPSDSDTHYNQKFKDKASLTVDKSSSTAYMQLSSLTFEDSA
VYYCVRGGTGTMAWFAYWGQGTLVTVSA 18D5 VL (amino acid 129
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSYGNTYLYWYLQKPG sequence)
QSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYF CFQGTHVPYTFGSGTKLEIK
KWAR23 VH (amino 130 EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLE
acid sequence) WIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTA
VYYCARWGAYWGQGTLVTVSS KWAR23 VL (amino 131
QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPK acid sequence)
LWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQW SSYPRTFGAGTKLELK
rhSIRP.alpha.-HIS (amino 132
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVA acid sequence)
AGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTT
VSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA
GTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKW
FKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEV
AHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVR
KFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSN ERHHHHHH
[0967] All references cited herein are incorporated by reference to
the same extent as if each individual publication, database entry
(e.g. Genbank sequences or GeneID entries), patent application, or
patent, was specifically and individually indicated to be
incorporated by reference. This statement of incorporation by
reference is intended by Applicants, pursuant to 37 C.F.R. .sctn.
1.57(b)(1), to relate to each and every individual publication,
database entry (e.g. Genbank sequences or GeneID entries), patent
application, or patent, each of which is clearly identified in
compliance with 37 C.F.R. .sctn. 1.57(b)(2), even if such citation
is not immediately adjacent to a dedicated statement of
incorporation by reference. The inclusion of dedicated statements
of incorporation by reference, if any, within the specification
does not in any way weaken this general statement of incorporation
by reference. Citation of the references herein is not intended as
an admission that the reference is pertinent prior art, nor does it
constitute any admission as to the contents or date of these
publications or documents. To the extent that the references
provide a definition for a claimed term that conflicts with the
definitions provided in the instant specification, the definitions
provided in the instant specification shall be used to interpret
the claimed invention.
[0968] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0969] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. Various modifications of the invention in addition to
those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims.
Sequence CWU 1
1
13715PRTArtificial SequenceSynthetic 50A heavy chain CDR1 1Asn Tyr
Tyr Ile His1 5217PRTArtificial SequenceSynthetic 50A heavy chain
CDR2 2Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe
Lys1 5 10 15Ala310PRTArtificial SequenceSynthetic 50A heavy chain
CDR3 3Pro Thr Ile Ile Ala Thr Asp Phe Asp Val1 5 10411PRTArtificial
SequenceSynthetic 50A light chain CDR1 4Lys Ala Ser Gln Gly Val Gly
Thr Ala Val Gly1 5 1057PRTArtificial SequenceSynthetic 50A light
chain CDR2 5Trp Ala Ser Thr Arg His Thr1 569PRTArtificial
SequenceSynthetic 50A light chain CDR3 6Gln Gln Tyr Ser Thr Tyr Pro
Phe Thr1 57119PRTArtificial SequenceSynthetic humanized 50 heavy
chain variable region (consensus sequence)misc_feature(5)..(5)X = Q
or Vmisc_feature(6)..(6)X = Q or Emisc_feature(9)..(9)X = A or
Smisc_feature(11)..(11)X = V or Lmisc_feature(19)..(19)X = K or
Mmisc_feature(20)..(20)X = V or Imisc_feature(40)..(40)X = A or
Rmisc_feature(42)..(42)X = G or Emisc_feature(48)..(48)X = I or
Mmisc_feature(67)..(67)X = R or Kmisc_feature(68)..(68)X = V or
Amisc_feature(69)..(69)X = T or Imisc_feature(70)..(70)X = I or
Mmisc_feature(79)..(79)X = A or Vmisc_feature(82)..(82)X = D or E
or Qmisc_feature(87)..(87)X = R or Tmisc_feature(89)..(89)X = E or
Dmisc_feature(91)..(91)X = T or Mmisc_feature(114)..(114)X = T or L
7Glu Val Gln Leu Xaa Xaa Ser Gly Xaa Glu Xaa Val Lys Pro Gly Ala1 5
10 15Ser Val Xaa Xaa Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn
Tyr 20 25 30Tyr Ile His Trp Val Arg Gln Xaa Pro Xaa Gln Gly Leu Glu
Trp Xaa 35 40 45Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn
Glu Lys Phe 50 55 60Lys Ala Xaa Xaa Xaa Xaa Thr Ala Asp Lys Ser Thr
Ser Thr Xaa Tyr65 70 75 80Met Xaa Leu Ser Ser Leu Xaa Ser Xaa Asp
Xaa Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Thr Ile Ile Ala Thr Asp
Phe Asp Val Trp Gly Gln Gly 100 105 110Thr Xaa Val Thr Val Ser Ser
1158107PRTArtificial SequenceSynthetic humanized 50 light chain
variable region (consensus sequence)misc_feature(1)..(4)X = D or
Emisc_feature(2)..(2)X = I or Lmisc_feature(3)..(3)X = V or
Qmisc_feature(4)..(4)X = L or Mmisc_feature(10)..(10)X = F or
Smisc_feature(38)..(38)X = Q or Kmisc_feature(43)..(43)X = A or S
or Vmisc_feature(70)..(70)X = E or Dmisc_feature(74)..(74)X = T or
Amisc_feature(76)..(76)X = S or Nmisc_feature(77)..(77)X = S or N
or Gmisc_feature(83)..(83)X = F or I or Vmisc_feature(85)..(85)X =
A or D or Tmisc_feature(104)..(104)X = L or V 8Xaa Xaa Xaa Xaa Thr
Gln Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Gly Val Gly Thr Ala 20 25 30Val Gly Trp
Tyr Gln Xaa Lys Pro Gly Lys Xaa Pro Lys Leu Leu Ile 35 40 45Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Xaa Phe Thr Leu Xaa Ile Xaa Xaa Leu Gln Pro65 70 75
80Glu Asp Xaa Ala Xaa Tyr Tyr Cys Gln Gln Tyr Ser Thr Tyr Pro Phe
85 90 95Thr Phe Gly Gly Gly Thr Lys Xaa Glu Ile Lys 100
1059357DNAArtificial SequenceSynthetic hSIRP alpha.50AVH1
9gaagtgcagc tgcagcagtc tggcgccgag gtcgtgaaac ctggcgcctc tgtgaaggtg
60tcctgcaagg cctccggctt caccttcacc aactactaca tccactgggt gcgacaggcc
120ccaggccagg gactggaatg gatcggctgg atctaccccg gcaacgtgaa
caccaagtac 180aacgagaagt tcaaggcccg cgtgaccatc accgccgaca
agtctacctc caccgcctac 240atggacctgt cctccctgag atccgaggac
accgccgtgt actactgcgc cagacccacc 300atcattgcca ccgacttcga
cgtgtggggc cagggcacaa ccgtgaccgt gtcctct 35710119PRTArtificial
SequenceSynthetic hSIRP alpha.50AVH1 10Glu Val Gln Leu Gln Gln Ser
Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr
Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ala Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Pro Thr Ile Ile Ala Thr Asp Phe Asp Val Trp Gly Gln Gly
100 105 110Thr Thr Val Thr Val Ser Ser 11511357DNAArtificial
SequenceSynthetic hSIRP alpha.50AVH2 11gaagtgcagc tggtggaatc
cggctccgag ctcgtgaagc ctggcgcctc cgtgaaggtg 60tcctgcaagg cctctggctt
caccttcacc aactactaca tccactgggt gcgacaggcc 120ccaggccagg
gactggaatg gatgggctgg atctaccccg gcaacgtgaa caccaagtac
180aacgagaagt tcaaggccaa ggccaccatc accgccgaca agtccacctc
caccgcctac 240atggaactgt cctccctgcg gagcgaggac accgccgtgt
actactgtgc ccggcctacc 300atcattgcca ccgacttcga tgtgtggggc
cagggcacac tcgtgaccgt gtcctct 35712119PRTArtificial
SequenceSynthetic hSIRP alpha.50AVH2 12Glu Val Gln Leu Val Glu Ser
Gly Ser Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Tyr
Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ala Lys
Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Pro Thr Ile Ile Ala Thr Asp Phe Asp Val Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser Ser 11513357DNAArtificial
SequenceSynthetic hSIRP alpha.50AVH3 13gaagtgcagc tggtgcagtc
tggcgccgag gtcgtgaaac ctggcgcctc cgtgatgatc 60tcctgcaagg cctccggctt
caccttcacc aactactaca tccactgggt gcgacagcgg 120ccaggccagg
gactggaatg gatcggctgg atctaccccg gcaacgtgaa caccaagtac
180aacgagaagt tcaaggcccg cgtgatcatg accgccgaca agtccacctc
caccgtgtac 240atgcagctgt cctccctgac ctccgaggac accgccgtgt
actactgcgc cagacccacc 300atcattgcca ccgacttcga cgtgtggggc
cagggcacac tcgtgaccgt gtcctct 35714119PRTArtificial
SequenceSynthetic hSIRP alpha.50AVH3 14Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Met Ile Ser Cys
Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30Tyr Ile His Trp Val
Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr
Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ala Arg
Val Ile Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr65 70 75 80Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Pro Thr Ile Ile Ala Thr Asp Phe Asp Val Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser Ser 11515357DNAArtificial
SequenceSynthetic hSIRP alpha.50AVH4 15gaagtgcagc tgcagcagtc
tggcgccgag ctcgtgaaac ctggcgcctc tgtgaaggtg 60tcctgcaagg cctccggctt
caccttcacc aactactaca tccactgggt gcgacagcgg 120ccaggccagg
gactggaatg gatgggctgg atctaccccg gcaacgtgaa caccaagtac
180aacgagaagt tcaaggccaa ggccaccatc accgccgaca agtccacctc
caccgcctac 240atggaactgt cctccctgac ctccgaggac accgccgtgt
actactgcgc cagacccacc 300atcattgcca ccgacttcga cgtgtggggc
cagggcacaa ccgtgaccgt gtcctct 35716119PRTArtificial
SequenceSynthetic hSIRP alpha.50AVH4 16Glu Val Gln Leu Gln Gln Ser
Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30Tyr Ile His Trp Val
Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Tyr
Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ala Lys
Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Pro Thr Ile Ile Ala Thr Asp Phe Asp Val Trp Gly Gln Gly
100 105 110Thr Thr Val Thr Val Ser Ser 11517357DNAArtificial
SequenceSynthetic hSIRP alpha.50AVH5 17gaagtgcagc tggtgcagtc
tggcgccgag gtcgtgaaac ctggcgcctc tgtgaaggtg 60tcctgcaagg cctccggctt
caccttcacc aactactaca tccactgggt gcgacaggcc 120cccgagcagg
gactggaatg gatcggctgg atctaccccg gcaacgtgaa caccaagtac
180aacgagaagt tcaaggcccg cgtgaccatg accgccgaca agtctacctc
caccgcctac 240atggaactgt cctccctgcg gagcgacgac atggccgtgt
actactgcgc cagacccacc 300atcattgcca ccgacttcga cgtgtggggc
cagggcacaa ccgtgaccgt gtcctct 35718119PRTArtificial
SequenceSynthetic hSIRP alpha.50AVH5 18Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30Tyr Ile His Trp Val
Arg Gln Ala Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr
Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Ala Arg
Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Asp Asp Met Ala Val Tyr Tyr Cys 85 90
95Ala Arg Pro Thr Ile Ile Ala Thr Asp Phe Asp Val Trp Gly Gln Gly
100 105 110Thr Thr Val Thr Val Ser Ser 11519321DNAArtificial
SequenceSynthetic hSIRP alpha.50AVL1 19gacatcgtgc tgacccagtc
ccccagcttc ctgtctgcct ctgtgggcga cagagtgacc 60atcacatgca aggcctctca
gggcgtgggc accgctgtgg gatggtatca gcagaagcct 120ggcaaggccc
ccaagctgct gatctactgg gcctctacca gacacaccgg cgtgcccgac
180agattctccg gctctggctc tggcaccgag tttaccctga ccatctccag
cctgcagccc 240gaggatttcg ccgcctacta ctgccagcag tactccacct
accccttcac cttcggcgga 300ggcaccaagc tggaaatcaa g
32120107PRTArtificial SequenceSynthetic hSIRP alpha.50AVL1 20Asp
Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Val Gly Thr Ala
20 25 30Val Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Ala Tyr Tyr Cys Gln Gln Tyr
Ser Thr Tyr Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 10521321DNAArtificial SequenceSynthetic hSIRP alpha.50AVL2
21gacatcgtga tgacccagtc cccctccagc ctgtctgcct ctgtgggcga cagagtgacc
60atcacatgca aggcctctca gggcgtgggc accgctgtgg gatggtatca gcagaagcct
120ggcaaggccc ccaagctgct gatctactgg gcctctacca gacacaccgg
cgtgcccgac 180agattctccg gctctggctc tggcaccgac ttcaccctga
ccatctccaa cctgcagccc 240gaggacttcg ccgactacta ctgccagcag
tactccacct accccttcac cttcggcgga 300ggcaccaagg tggaaatcaa g
32122107PRTArtificial SequenceSynthetic hSIRP alpha.50AVL2 22Asp
Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Val Gly Thr Ala
20 25 30Val Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Asp Tyr Tyr Cys Gln Gln Tyr
Ser Thr Tyr Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 10523321DNAArtificial SequenceSynthetic hSIRP alpha.50AVL3
23gagctcgtga tgacccagtc cccttccagc ctgtctgcct ccgtgggcga cagagtgacc
60atcacatgca aggcctctca gggcgtgggc accgctgtgg gatggtatca gcagaagcct
120ggcaaggccc ccaagctgct gatctactgg gcctctacca gacacaccgg
cgtgcccgac 180agattctccg gctctggctc tggcaccgac tttaccctgg
ccatctccag cctgcagccc 240gaggatatcg ccgactacta ctgccagcag
tactccacct accccttcac cttcggcgga 300ggcaccaagg tggaaatcaa g
32124107PRTArtificial SequenceSynthetic hSIRP alpha.50AVL3 24Glu
Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Val Gly Thr Ala
20 25 30Val Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Tyr
Ser Thr Tyr Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 10525321DNAArtificial SequenceSynthetic hSIRP alpha.50AVL4
25gacatccaga tgacccagtc cccctccagc ctgtctgcct ctgtgggcga cagagtgacc
60atcacatgca aggcctctca gggcgtgggc accgctgtgg gctggtatca gaaaaagccc
120ggcaaggtgc ccaagctgct gatctactgg gcctccacca gacacaccgg
cgtgcccgat 180agattctccg gctctggctc tggcaccgac ttcaccctga
ccatcaacgg cctgcagcct 240gaggacgtgg ccacctacta ctgccagcag
tactccacct accccttcac cttcggcgga 300ggcaccaagc tggaaatcaa g
32126107PRTArtificial SequenceSynthetic hSIRP alpha.50AVL4 26Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Val Gly Thr Ala
20 25 30Val Gly Trp Tyr Gln Lys Lys Pro Gly Lys Val Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Gly
Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ser Thr Tyr Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 10527321DNAArtificial SequenceSynthetic hSIRP alpha.50AVL5
27gacatcgtgc tgacccagtc ccccagcttc ctgtctgcct ctgtgggcga cagagtgacc
60atcacatgca aggcctctca gggcgtgggc accgctgtgg gatggtatca gcagaagccc
120ggcaagtccc ccaagctgct gatctactgg gcctccacca gacacaccgg
cgtgcccgat 180agattctccg gctctggctc tggcaccgag ttcaccctga
ccatctccaa cctgcagccc 240gaggacttcg ccgcctacta ctgccagcag
tactccacct accccttcac cttcggcgga 300ggcaccaagc tggaaatcaa g
32128107PRTArtificial SequenceSynthetic hSIRP alpha.50AVL5 28Asp
Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Val Gly Thr Ala
20 25 30Val Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asn
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Ala Tyr Tyr Cys Gln Gln Tyr
Ser Thr Tyr Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 10529357DNAArtificial SequenceSynthetic hSIRP alpha.50A
mouse VH 29caggtccagc tgcagcagtc tggacctgaa ctggtgaagc ctggggcttc
agttaggata 60tcctgcaagg cttctggctt caccttcaca aactactata tacactgggt
gaagcagagg 120cctggacagg gacttgagtg gattggatgg atttatcctg
gaaatgttaa tactaagtac 180aatgagaagt tcaaggccaa ggccacactg
actgcagaca aatcctccac cacagcctac 240atgcagctca gcagcctggc
ctctgaggac tctgcggtct atttctgtgc aagacctacg 300ataatagcta
cggacttcga tgtctggggc gcagggacca cggtcaccgt ctcctca
35730119PRTArtificial
SequenceSynthetic hSIRP alpha.50A mouse VH 30Gln Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Arg Ile
Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30Tyr Ile His
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp
Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys
Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95Ala Arg Pro Thr Ile Ile Ala Thr Asp Phe Asp Val Trp Gly Ala
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 11531321DNAArtificial
SequenceSynthetic hSIRP alpha.50A mouse VL 31gacattgtca tgacccagtc
tcacaaattc atgtccacat cagtaggaga cagggtcaac 60atcacctgca aggccagtca
gggtgtgggt actgctgtag gctggtatca acagaaacca 120gggcaatctc
ctagactact gatttactgg gcatccaccc ggcacactgg agtccctgat
180cgcttcacag gcagtggatc tgggacagat ttcagtctcg ccattagcaa
tgtgcagtct 240gaagacctgg cagattattt ctgtcagcaa tatagcacct
atccgttcac gttcggaggg 300gggaccaatc tagaaataaa a
32132107PRTArtificial SequenceSynthetic hSIRP alpha.50A mouse VL
32Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1
5 10 15Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Gly Val Gly Thr
Ala 20 25 30Val Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Ser Leu Ala Ile Ser
Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
Tyr Ser Thr Tyr Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Asn Leu Glu
Ile Lys 100 105331512DNAHomo sapiensmisc_feature(1)..(1512)human
SIRP alpha V1 33atggagcccg ccggcccggc ccccggccgc ctcgggccgc
tgctctgcct gctgctcgcc 60gcgtcctgcg cctggtcagg agtggcgggt gaggaggagc
tgcaggtgat tcagcctgac 120aagtccgtgt tggttgcagc tggagagaca
gccactctgc gctgcactgc gacctctctg 180atccctgtgg ggcccatcca
gtggttcaga ggagctggac caggccggga attaatctac 240aatcaaaaag
aaggccactt cccccgggta acaactgttt cagacctcac aaagagaaac
300aacatggact tttccatccg catcggtaac atcaccccag cagatgccgg
cacctactac 360tgtgtgaagt tccggaaagg gagccccgat gacgtggagt
ttaagtctgg agcaggcact 420gagctgtctg tgcgcgccaa accctctgcc
cccgtggtat cgggccctgc ggcgagggcc 480acacctcagc acacagtgag
cttcacctgc gagtcccacg gcttctcacc cagagacatc 540accctgaaat
ggttcaaaaa tgggaatgag ctctcagact tccagaccaa cgtggacccc
600gtaggagaga gcgtgtccta cagcatccac agcacagcca aggtggtgct
gacccgcgag 660gacgttcact ctcaagtcat ctgcgaggtg gcccacgtca
ccttgcaggg ggaccctctt 720cgtgggactg ccaacttgtc tgagaccatc
cgagttccac ccaccttgga ggttactcaa 780cagcccgtga gggcagagaa
ccaggtgaat gtcacctgcc aggtgaggaa gttctacccc 840cagagactac
agctgacctg gttggagaat ggaaacgtgt cccggacaga aacggcctca
900accgttacag agaacaagga tggtacctac aactggatga gctggctcct
ggtgaatgta 960tctgcccaca gggatgatgt gaagctcacc tgccaggtgg
agcatgacgg gcagccagcg 1020gtcagcaaaa gccatgacct gaaggtctca
gcccacccga aggagcaggg ctcaaatacc 1080gccgctgaga acactggatc
taatgaacgg aacatctata ttgtggtggg tgtggtgtgc 1140accttgctgg
tggccctact gatggcggcc ctctacctcg tccgaatcag acagaagaaa
1200gcccagggct ccacttcttc tacaaggttg catgagcccg agaagaatgc
cagagaaata 1260acacaggaca caaatgatat cacatatgca gacctgaacc
tgcccaaggg gaagaagcct 1320gctccccagg ctgcggagcc caacaaccac
acggagtatg ccagcattca gaccagcccg 1380cagcccgcgt cggaggacac
cctcacctat gctgacctgg acatggtcca cctcaaccgg 1440acccccaagc
agccggcccc caagcctgag ccgtccttct cagagtacgc cagcgtccag
1500gtcccgagga ag 151234504PRTHomo
sapiensmisc_feature(1)..(504)human SIRP alpha V1 34Met 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 Ile Arg Gln Lys Lys385 390 395 400Ala Gln Gly Ser Thr
Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn 405 410 415Ala Arg Glu
Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu 420 425 430Asn
Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn 435 440
445Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser
450 455 460Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu
Asn Arg465 470 475 480Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro
Ser Phe Ser Glu Tyr 485 490 495Ala Ser Val Gln Val Pro Arg Lys
500351509DNAHomo sapiensmisc_feature(1)..(1509)human SIRP alpha V2
35atggaacctg ccggacctgc ccctggcaga ctgggacctc tgctgtgtct gctgctggcc
60gcctcttgtg cttggagcgg agtggctggc gaagaggaac tgcaagtgat ccagcccgac
120aagagcgtgt ccgtggctgc tggcgagtct gccatcctgc actgtaccgt
gaccagcctg 180atccccgtgg gccccatcca gtggtttaga ggcgctggcc
ctgccagaga gctgatctac 240aaccagaaag agggccactt ccccagagtg
accaccgtgt ccgagagcac caagcgcgag 300aacatggact tcagcatcag
catctccaac atcacccctg ccgacgccgg cacctactac 360tgcgtgaagt
tcagaaaggg cagccccgac accgagttca agagcggcgc tggaaccgag
420ctgtctgtgc gggctaagcc ttctgcccct gtggtgtctg gacctgccgc
cagagctaca 480cctcagcaca ccgtgtcttt cacatgcgag agccacggct
tcagccccag agacatcacc 540ctgaagtggt tcaagaacgg caacgagctg
agcgacttcc agaccaacgt ggaccctgtg 600ggcgagtccg tgtcctacag
catccacagc accgccaagg tggtgctgac ccgcgaggat 660gtgcacagcc
aagtgatctg cgaggtggcc cacgtgacac tgcagggcga tcctctgaga
720ggcaccgcta acctgagcga gacaatcaga gtgcccccca ccctggaagt
gacccagcag 780cccgtgcggg ctgagaacca agtgaacgtg acctgccaag
tgcggaagtt ctaccctcag 840agactgcagc tgacctggct ggaaaacgga
aacgtgtcca gaaccgagac agccagcacc 900gtgacagaga acaaggacgg
cacatacaac tggatgagct ggctgctcgt gaacgtgtcc 960gcccacagag
atgacgtgaa gctgacatgc caggtggaac acgacggcca gcctgccgtg
1020tctaagagcc acgacctgaa ggtgtccgct caccccaaag agcagggcag
caacaccgcc 1080gctgagaaca caggcagcaa cgagagaaac atctacatcg
tcgtgggcgt cgtgtgcacc 1140ctgctggtgg ctctgctgat ggctgccctg
tacctcgtgc ggatcagaca gaagaaggcc 1200cagggctcca cctccagcac
cagactgcac gagcctgaga agaacgcccg cgagatcacc 1260caggacacca
acgacatcac ctacgccgac ctgaacctgc ccaagggcaa gaagcctgcc
1320cctcaggctg ccgagcctaa caaccacaca gagtacgcca gcatccagac
cagccctcag 1380cctgccagcg aggacacact gacatacgcc gatctggaca
tggtgcacct gaacagaacc 1440cccaagcagc ccgctcccaa gcccgagcct
agcttctctg agtacgcctc cgtgcaggtg 1500cccagaaaa 150936503PRTHomo
sapiensmisc_feature(1)..(503)human SIRP alpha V2 36Met 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 Ser Val Ala Ala Gly 35 40 45Glu
Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly 50 55
60Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr65
70 75 80Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
Ser 85 90 95Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser 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 Thr Glu Phe Lys Ser Gly Ala Gly
Thr Glu Leu Ser Val Arg 130 135 140Ala Lys Pro Ser Ala Pro Val Val
Ser Gly Pro Ala Ala Arg Ala Thr145 150 155 160Pro Gln His Thr Val
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro 165 170 175Arg Asp Ile
Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp 180 185 190Phe
Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile 195 200
205His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln
210 215 220Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
Leu Arg225 230 235 240Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val
Pro Pro Thr Leu Glu 245 250 255Val Thr Gln Gln Pro Val Arg Ala Glu
Asn Gln Val Asn Val Thr Cys 260 265 270Gln Val Arg Lys Phe Tyr Pro
Gln Arg Leu Gln Leu Thr Trp Leu Glu 275 280 285Asn Gly Asn Val Ser
Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn 290 295 300Lys Asp Gly
Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser305 310 315
320Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly
325 330 335Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala
His Pro 340 345 350Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr
Gly Ser Asn Glu 355 360 365Arg Asn Ile Tyr Ile Val Val Gly Val Val
Cys Thr Leu Leu Val Ala 370 375 380Leu Leu Met Ala Ala Leu Tyr Leu
Val Arg Ile Arg Gln Lys Lys Ala385 390 395 400Gln Gly Ser Thr Ser
Ser Thr Arg Leu His Glu Pro Glu Lys Asn Ala 405 410 415Arg Glu Ile
Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu Asn 420 425 430Leu
Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn Asn 435 440
445His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser Glu
450 455 460Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn
Arg Thr465 470 475 480Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser
Phe Ser Glu Tyr Ala 485 490 495Ser Val Gln Val Pro Arg Lys
500371194DNAHomo sapiensmisc_feature(1)..(1194)human SIRP beta 1
37atgcccgtgc cagcctcctg gccccacctt cctagtcctt tcctgctgat gacgctactg
60ctggggagac tcacaggagt ggcaggtgag gacgagctac aggtgattca gcctgaaaag
120tccgtatcag ttgcagctgg agagtcggcc actctgcgct gtgctatgac
gtccctgatc 180cctgtggggc ccatcatgtg gtttagagga gctggagcag
gccgggaatt aatctacaat 240cagaaagaag gccacttccc acgggtaaca
actgtttcag aactcacaaa gagaaacaac 300ctggactttt ccatcagcat
cagtaacatc accccagcag acgccggcac ctactactgt 360gtgaagttcc
ggaaagggag ccctgacgac gtggagttta agtctggagc aggcactgag
420ctgtctgtgc gcgccaaacc ctctgccccc gtggtatcgg gccctgcggt
gagggccaca 480cctgagcaca cagtgagctt cacctgcgag tcccatggct
tctctcccag agacatcacc 540ctgaaatggt tcaaaaatgg gaatgagctc
tcagacttcc agaccaacgt ggaccccgca 600ggagacagtg tgtcctacag
catccacagc acagccaggg tggtgctgac ccgtggggac 660gttcactctc
aagtcatctg cgagatagcc cacatcacct tgcaggggga ccctcttcgt
720gggactgcca acttgtctga ggccatccga gttccaccca ccttggaggt
tactcaacag 780cccatgaggg cagagaacca ggcaaacgtc acctgccagg
tgagcaattt ctacccccgg 840ggactacagc tgacctggtt ggagaatgga
aatgtgtccc ggacagaaac agcttcgacc 900ctcatagaga acaaggatgg
cacctacaac tggatgagct ggctcctggt gaacacctgt 960gcccacaggg
acgatgtggt gctcacctgt caggtggagc atgatgggca gcaagcagtc
1020agcaaaagct atgccctgga gatctcagcg caccagaagg agcacggctc
agatatcacc 1080catgaagcag cgctggctcc tactgctcca ctcctcgtag
ctctcctcct gggccccaag 1140ctgctactgg tggttggtgt ctctgccatc
tacatctgct ggaaacagaa ggcc 119438398PRTHomo
sapiensmisc_feature(1)..(398)human SIRP beta 1 38Met Pro Val Pro
Ala Ser Trp Pro His Leu Pro Ser Pro Phe Leu Leu1 5 10 15Met Thr Leu
Leu Leu Gly Arg Leu Thr Gly Val Ala Gly Glu Asp Glu 20 25 30Leu Gln
Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala Gly Glu 35 40 45Ser
Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val Gly Pro 50 55
60Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile Tyr Asn65
70 75 80Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Leu
Thr 85 90 95Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile
Thr Pro 100 105 110Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg
Lys Gly Ser Pro 115 120 125Asp Asp Val Glu Phe Lys Ser Gly Ala Gly
Thr Glu Leu Ser Val Arg 130 135 140Ala Lys Pro Ser Ala Pro Val Val
Ser Gly Pro Ala Val Arg Ala Thr145 150 155 160Pro Glu His Thr Val
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro 165 170 175Arg Asp Ile
Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp 180 185 190Phe
Gln Thr Asn Val Asp Pro Ala Gly Asp Ser Val Ser Tyr Ser Ile 195 200
205His Ser Thr Ala Arg Val Val Leu Thr Arg Gly Asp Val His Ser Gln
210 215 220Val Ile Cys Glu Ile Ala His Ile Thr Leu Gln Gly Asp Pro
Leu Arg225 230 235 240Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val
Pro Pro Thr Leu Glu 245 250 255Val Thr Gln Gln Pro Met Arg Ala Glu
Asn Gln Ala Asn Val Thr Cys 260 265 270Gln Val Ser Asn Phe Tyr Pro
Arg Gly Leu Gln Leu Thr Trp Leu Glu 275 280 285Asn Gly Asn Val Ser
Arg Thr Glu Thr Ala Ser Thr Leu Ile Glu Asn 290 295 300Lys Asp Gly
Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Thr Cys305 310 315
320Ala His Arg Asp Asp Val Val Leu Thr Cys Gln Val Glu His Asp Gly
325 330 335Gln Gln Ala Val Ser Lys Ser Tyr Ala Leu Glu Ile Ser Ala
His Gln 340 345 350Lys Glu His Gly Ser Asp Ile Thr His Glu Ala Ala
Leu Ala Pro Thr 355 360 365Ala Pro Leu Leu Val Ala Leu Leu Leu Gly
Pro Lys Leu Leu Leu Val 370 375 380Val Gly Val Ser Ala Ile Tyr Ile
Cys Trp Lys Gln Lys Ala385 390
395391161DNAHomo sapiensmisc_feature(1)..(1161)human SIRP?
39atgcctgtcc cagcctcctg gccccatcct cctggtcctt tcctgcttct gactctactg
60ctgggactta cagaagtggc aggtgaggag gagctacaga tgattcagcc tgagaagctc
120ctgttggtca cagttggaaa gacagccact ctgcactgca ctgtgacctc
cctgcttccc 180gtgggacccg tcctgtggtt cagaggagtt ggaccaggcc
gggaattaat ctacaatcaa 240aaagaaggcc acttccccag ggtaacaaca
gtttcagacc tcacaaagag aaacaacatg 300gacttttcca tccgcatcag
tagcatcacc ccagcagatg tcggcacata ctactgtgtg 360aagtttcgaa
aagggagccc tgagaacgtg gagtttaagt ctggaccagg cactgagatg
420gctttgggtg ccaaaccctc tgcccccgtg gtattgggcc ctgcggcgag
gaccacacct 480gagcatacag tgagtttcac ctgtgagtcc catggcttct
ctcccagaga catcaccctg 540aaatggttca aaaatgggaa tgagctctca
gacttccaga ccaacgtgga ccccacagga 600cagagtgtgg cctacagcat
ccgcagcaca gccagggtgg tactggaccc ctgggacgtt 660cgctctcagg
tcatctgcga ggtggcccat gtcaccttgc agggggaccc tcttcgtggg
720actgccaact tgtctgaggc catccgagtt ccacccacct tggaggttac
tcaacagccc 780atgagggtgg ggaaccaggt aaacgtcacc tgccaggtga
ggaagttcta cccccagagc 840ctacagctga cctggtcgga gaatggaaac
gtgtgccaga gagaaacagc ctcgaccctt 900acagagaaca aggatggtac
ctacaactgg acaagctggt tcctggtgaa catatctgac 960caaagggatg
atgtggtcct cacctgccag gtgaagcatg atgggcagct ggcggtcagc
1020aaacgccttg ccctagaggt cacagtccac cagaaggacc agagctcaga
tgctacccct 1080ggcccggcat catcccttac tgcgctgctc ctcatagctg
tcctcctggg ccccatctac 1140gtcccctgga agcagaagac c 116140387PRTHomo
sapiensmisc_feature(1)..(387)human SIRP? 40Met Pro Val Pro Ala Ser
Trp Pro His Pro Pro Gly Pro Phe Leu Leu1 5 10 15Leu Thr Leu Leu Leu
Gly Leu Thr Glu Val Ala Gly Glu Glu Glu Leu 20 25 30Gln Met Ile Gln
Pro Glu Lys Leu Leu Leu Val Thr Val Gly Lys Thr 35 40 45Ala Thr Leu
His Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Val 50 55 60Leu Trp
Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln65 70 75
80Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys
85 90 95Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro
Ala 100 105 110Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
Ser Pro Glu 115 120 125Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu
Met Ala Leu Gly Ala 130 135 140Lys Pro Ser Ala Pro Val Val Leu Gly
Pro Ala Ala Arg Thr Thr Pro145 150 155 160Glu His Thr Val Ser Phe
Thr Cys Glu Ser His Gly Phe Ser Pro Arg 165 170 175Asp Ile Thr Leu
Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe 180 185 190Gln Thr
Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile Arg 195 200
205Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln Val
210 215 220Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
Arg Gly225 230 235 240Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro
Pro Thr Leu Glu Val 245 250 255Thr Gln Gln Pro Met Arg Val Gly Asn
Gln Val Asn Val Thr Cys Gln 260 265 270Val Arg Lys Phe Tyr Pro Gln
Ser Leu Gln Leu Thr Trp Ser Glu Asn 275 280 285Gly Asn Val Cys Gln
Arg Glu Thr Ala Ser Thr Leu Thr Glu Asn Lys 290 295 300Asp Gly Thr
Tyr Asn Trp Thr Ser Trp Phe Leu Val Asn Ile Ser Asp305 310 315
320Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys His Asp Gly Gln
325 330 335Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr Val His
Gln Lys 340 345 350Asp Gln Ser Ser Asp Ala Thr Pro Gly Pro Ala Ser
Ser Leu Thr Ala 355 360 365Leu Leu Leu Ile Ala Val Leu Leu Gly Pro
Ile Tyr Val Pro Trp Lys 370 375 380Gln Lys Thr38541969DNAHomo
sapiensmisc_feature(1)..(969)human CD47 41atgtggcctc tggtggccgc
tctgctgctg ggctctgctt gttgtggatc cgcccagctg 60ctgttcaaca agaccaagtc
cgtggagttc accttctgca acgataccgt cgtgatcccc 120tgcttcgtga
ccaacatgga agcccagaac accaccgagg tgtacgtgaa gtggaagttc
180aagggccggg acatctacac cttcgacggc gccctgaaca agtccaccgt
gcccaccgat 240ttctccagcg ccaagatcga ggtgtcacag ctgctgaagg
gcgacgcctc cctgaagatg 300gacaagtccg acgccgtgtc ccacaccggc
aactacacct gtgaagtgac cgagctgacc 360agagagggcg agacaatcat
cgagctgaag taccgggtgg tgtcctggtt cagccccaac 420gagaacatcc
tgatcgtgat cttccccatc ttcgccatcc tgctgttctg gggccagttc
480ggcatcaaga ccctgaagta cagatccggc ggcatggacg aaaagacaat
cgccctgctg 540gtggctggcc tcgtgatcac cgtgattgtg atcgtgggcg
ctatcctgtt cgtgcccggc 600gagtacagcc tgaagaatgc taccggcctg
ggcctgattg tgacctccac cggaatcctg 660atcctgctgc actactacgt
gttctccacc gctatcggcc tgacctcctt cgtgatcgcc 720attctcgtga
tccaagtgat cgcctacatc ctggccgtcg tgggcctgtc cctgtgtatc
780gccgcctgca tccctatgca cggccccctg ctgatctccg gcctgtctat
tctggccctg 840gctcagctgc tgggactggt gtacatgaag ttcgtggcct
ccaaccagaa aaccatccag 900ccccctcgga aggccgtgga agaacccctg
aacgccttca aagaatccaa gggcatgatg 960aacgacgaa 96942323PRTHomo
sapiensmisc_feature(1)..(323)human CD47 42Met Trp Pro Leu Val Ala
Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly1 5 10 15Ser Ala Gln Leu Leu
Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30Cys Asn Asp Thr
Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45Gln Asn Thr
Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60Ile Tyr
Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp65 70 75
80Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala
85 90 95Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn
Tyr 100 105 110Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr
Ile Ile Glu 115 120 125Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro
Asn Glu Asn Ile Leu 130 135 140Ile Val Ile Phe Pro Ile Phe Ala Ile
Leu Leu Phe Trp Gly Gln Phe145 150 155 160Gly Ile Lys Thr Leu Lys
Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175Ile Ala Leu Leu
Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190Gly Ala
Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200
205Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His
210 215 220Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val
Ile Ala225 230 235 240Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu
Ala Val Val Gly Leu 245 250 255Ser Leu Cys Ile Ala Ala Cys Ile Pro
Met His Gly Pro Leu Leu Ile 260 265 270Ser Gly Leu Ser Ile Leu Ala
Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285Met Lys Phe Val Ala
Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys 290 295 300Ala Val Glu
Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met305 310 315
320Asn Asp Glu431509DNAHomo sapiensmisc_feature(1)..(1509)human
SIRP alpha V3 43atggaacctg ccggccctgc tcctggtaga ctgggacctc
tgctgtgtct gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc gaagaggaac
tgcaagtgat ccagcccgac 120aagtccgtgt ctgtggccgc tggcgagtct
gccatcctgc tgtgtaccgt gacctccctg 180atccccgtgg gccccatcca
gtggtttaga ggcgctggcc ctgccagaga gctgatctac 240aaccagaaag
agggccactt ccccagagtg accaccgtgt ccgagtccac caagcgcgag
300aacatggact tctccatctc catcagcaac atcacccctg ccgacgccgg
cacctactac 360tgcgtgaagt tccggaaggg ctcccccgac accgagttca
agtctggcgc tggcaccgag 420ctgtctgtgc gggctaaacc ttctgcccct
gtggtgtctg gacctgccgc tagagctacc 480cctcagcaca ccgtgtcttt
tacctgcgag tcccacggct tcagccctcg ggacatcacc 540ctgaagtggt
tcaagaacgg caacgagctg agcgacttcc agaccaacgt ggaccctgtg
600ggcgagagcg tgtcctactc catccactcc accgccaagg tggtgctgac
acgcgaggac 660gtgcactccc aagtgatctg cgaggtggcc cacgtgacac
tgcagggcga tcctctgaga 720ggcaccgcca acctgtccga gacaatcaga
gtgcccccca ccctggaagt gacccagcag 780ccagtgcggg ccgagaacca
agtgaacgtg acctgccaag tgcggaagtt ctacccccag 840cggctgcagc
tgacctggct ggaaaacggc aatgtgtccc ggaccgagac agccagcacc
900gtgaccgaga acaaggatgg cacctacaat tggatgtctt ggctgctcgt
gaacgtgtcc 960gcccaccggg acgatgtgaa gctgacatgc caggtggaac
acgacggcca gcctgccgtg 1020tccaagagcc acgatctgaa ggtgtccgct
catcccaaag agcagggctc caacaccgcc 1080gctgagaaca ccggctctaa
cgagcggaac atctacatcg tcgtgggcgt cgtgtgcacc 1140ctgctggtgg
ctctgctgat ggctgccctg tacctcgtgc ggatccggca gaagaaggcc
1200cagggctcta cctcctccac cagactgcac gagcccgaga agaacgccag
agagatcacc 1260caggacacca acgacatcac ctacgccgac ctgaacctgc
ccaagggcaa gaagcctgcc 1320cctcaggctg ccgagcctaa caaccacacc
gagtacgcct ccatccagac cagccctcag 1380cctgcctctg aggacaccct
gacctacgct gatctggaca tggtgcacct gaaccggacc 1440cccaagcagc
cagctcctaa gcccgagcct agcttctctg agtacgccag cgtgcaggtg
1500ccccggaaa 150944503PRTHomo sapiensmisc_feature(1)..(503)human
SIRP alpha V3 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
Ser Val Ala Ala Gly 35 40 45Glu Ser Ala Ile Leu Leu Cys Thr Val Thr
Ser Leu Ile Pro Val Gly 50 55 60Pro Ile Gln Trp Phe Arg Gly Ala Gly
Pro Ala Arg Glu Leu Ile Tyr65 70 75 80Asn Gln Lys Glu Gly His Phe
Pro Arg Val Thr Thr Val Ser Glu Ser 85 90 95Thr Lys Arg Glu Asn Met
Asp Phe Ser Ile Ser Ile Ser 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
Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg 130 135
140Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala
Thr145 150 155 160Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His
Gly Phe Ser Pro 165 170 175Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn
Gly Asn Glu Leu Ser Asp 180 185 190Phe Gln Thr Asn Val Asp Pro Val
Gly Glu Ser Val Ser Tyr Ser Ile 195 200 205His Ser Thr Ala Lys Val
Val Leu Thr Arg Glu Asp Val His Ser Gln 210 215 220Val Ile Cys Glu
Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg225 230 235 240Gly
Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu 245 250
255Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys
260 265 270Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp
Leu Glu 275 280 285Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr
Val Thr Glu Asn 290 295 300Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp
Leu Leu Val Asn Val Ser305 310 315 320Ala His Arg Asp Asp Val Lys
Leu Thr Cys Gln Val Glu His Asp Gly 325 330 335Gln Pro Ala Val Ser
Lys Ser His Asp Leu Lys Val Ser Ala His Pro 340 345 350Lys Glu Gln
Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn Glu 355 360 365Arg
Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val Ala 370 375
380Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys Lys
Ala385 390 395 400Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro
Glu Lys Asn Ala 405 410 415Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile
Thr Tyr Ala Asp Leu Asn 420 425 430Leu Pro Lys Gly Lys Lys Pro Ala
Pro Gln Ala Ala Glu Pro Asn Asn 435 440 445His Thr Glu Tyr Ala Ser
Ile Gln Thr Ser Pro Gln Pro Ala Ser Glu 450 455 460Asp Thr Leu Thr
Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg Thr465 470 475 480Pro
Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr Ala 485 490
495Ser Val Gln Val Pro Arg Lys 500451512DNAHomo
sapiensmisc_feature(1)..(1512)human SIRP alpha V4 45atggaacctg
ccggccctgc tcctggtaga ctgggacctc tgctgtgtct gctgctggcc 60gcctcttgtg
cttggagcgg agtggctggc gaagagggcc tgcaagtgat ccagcccgac
120aagtccgtgt ctgtggccgc tggcgagtct gccatcctgc actgtaccgc
cacctccctg 180atccccgtgg gacccatcca gtggtttaga ggcgctggcc
ctggcagaga gctgatctac 240aaccagaaag agggccactt ccccagagtg
accaccgtgt ccgacctgac caagcggaac 300aacatggact tctccatccg
gatcggcaac atcacccctg ccgatgccgg cacctactac 360tgcgtgaagt
tccggaaggg ctcccccgac gacgtggagt tcaaatccgg cgctggcacc
420gagctgtctg tgcgggctaa accttctgcc cctgtggtgt ctggccctgc
cgctagagct 480acccctcagc acaccgtgtc ttttacctgc gagtcccacg
gcttcagccc tcgggacatc 540accctgaagt ggttcaagaa cggcaacgag
ctgagcgact tccagaccaa cgtggaccct 600gtgggcgaga gcgtgtccta
ctccatccac tccaccgcca aggtggtgct gacacgcgag 660gacgtgcact
cccaagtgat ctgcgaggtg gcccacgtga cactgcaggg cgatcctctg
720agaggcaccg ccaacctgtc cgagacaatc agagtgcccc ccaccctgga
agtgacccag 780cagccagtgc gggccgagaa ccaagtgaac gtgacctgcc
aagtgcggaa gttctacccc 840cagcggctgc agctgacctg gctggaaaac
ggcaatgtgt cccggaccga gacagcctcc 900accgtgaccg agaacaagga
tggcacctac aattggatgt cttggctgct cgtgaacgtg 960tccgcccacc
gggacgatgt gaagctgaca tgccaggtgg aacacgacgg ccagcctgcc
1020gtgtccaagt cccacgatct gaaggtgtcc gctcatccca aagagcaggg
ctccaacacc 1080gccgctgaga acaccggctc taacgagcgg aacatctaca
tcgtcgtggg cgtcgtgtgc 1140accctgctgg tggctctgct gatggctgcc
ctgtacctcg tgcggatccg gcagaagaag 1200gcccagggct ctacctcctc
caccagactg cacgagcccg agaagaacgc cagagagatc 1260acccaggaca
ccaacgacat cacctacgcc gacctgaacc tgcccaaggg caagaagcct
1320gcccctcagg ctgccgagcc taacaaccac accgagtacg cctccatcca
gaccagccct 1380cagcctgcct ctgaggacac cctgacctac gctgatctgg
acatggtgca cctgaaccgg 1440acccccaagc agccagctcc taagcccgag
cctagcttct ctgagtacgc cagcgtgcag 1500gtgccccgga aa 151246504PRTHomo
sapiensmisc_feature(1)..(504)human SIRP alpha V4 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 30Gly Leu
Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly 35 40 45Glu
Ser Ala Ile Leu His 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 Ile Arg Gln Lys Lys385 390 395 400Ala Gln Gly Ser
Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn 405 410 415Ala Arg
Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu 420 425
430Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn
435 440 445Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro
Ala Ser 450 455 460Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val
His Leu Asn Arg465 470 475 480Thr Pro Lys Gln Pro Ala Pro Lys Pro
Glu Pro Ser Phe Ser Glu Tyr 485 490 495Ala Ser Val Gln Val Pro Arg
Lys 500471512DNAHomo sapiensmisc_feature(1)..(1512)human SIRP alpha
V5 47atggaacctg ccggccctgc tcctggtaga ctgggacctc tgctgtgtct
gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc gaagaggaac tgcaagtgat
ccagcccgac 120aagttcgtgc tggtggccgc tggcgagaca gccaccctga
gatgtaccgc cacctccctg 180atccccgtgg gccctatcca gtggtttaga
ggcgctggcc ctggcagaga gctgatctac 240aaccagaaag agggccactt
ccccagagtg accaccgtgt ccgacctgac caagcggaac 300aacatggact
tctccatccg gatcggcaac atcacccctg ccgatgccgg cacctactac
360tgcgtgaagt tccggaaggg ctcccccgac gacgtggagt tcaaatccgg
cgctggcacc 420gagctgtctg tgcgggctaa accttctgcc cctgtggtgt
ctggccctgc cgctagagct 480acccctcagc acaccgtgtc ttttacctgc
gagtcccacg gcttcagccc tcgggacatc 540accctgaagt ggttcaagaa
cggcaacgag ctgagcgact tccagaccaa cgtggaccct 600gtgggcgagt
ccgtgtccta ctccatccac tccaccgcca aggtggtgct gacacgcgag
660gacgtgcact cccaagtgat ctgcgaggtg gcccacgtga cactgcaggg
cgatcctctg 720agaggcaccg ccaacctgtc cgagacaatc agagtgcccc
ccaccctgga agtgacccag 780cagccagtgc gggccgagaa ccaagtgaac
gtgacctgcc aagtgcggaa gttctacccc 840cagcggctgc agctgacctg
gctggaaaac ggcaatgtgt cccggaccga gactgcctcc 900accgtgaccg
agaacaagga tggcacctac aattggatgt cttggctgct cgtgaacgtg
960tccgcccacc gggacgatgt gaagctgaca tgccaggtgg aacacgacgg
ccagcctgcc 1020gtgtccaagt cccacgatct gaaggtgtcc gctcatccca
aagagcaggg ctccaacacc 1080gccgctgaga acaccggctc taacgagcgg
aacatctaca tcgtcgtggg cgtcgtgtgc 1140accctgctgg tggctctgct
gatggctgcc ctgtacctcg tgcggatccg gcagaagaag 1200gcccagggct
ctacctcctc caccagactg cacgagcccg agaagaacgc cagagagatc
1260acccaggaca ccaacgacat cacctacgcc gacctgaacc tgcccaaggg
caagaagcct 1320gcccctcagg ctgccgagcc taacaaccac accgagtacg
cctccatcca gaccagccct 1380cagcctgcct ctgaggacac cctgacctac
gctgatctgg acatggtgca cctgaaccgg 1440acccccaagc agccagctcc
taagcccgag cctagcttct ctgagtacgc cagcgtgcag 1500gtgccccgga aa
151248504PRTHomo sapiensmisc_feature(1)..(504)human SIRP alpha V5
48Met 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 Phe 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 Ile Arg Gln Lys Lys385 390 395
400Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn
405 410 415Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala
Asp Leu 420 425 430Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala
Ala Glu Pro Asn 435 440 445Asn His Thr Glu Tyr Ala Ser Ile Gln Thr
Ser Pro Gln Pro Ala Ser 450 455 460Glu Asp Thr Leu Thr Tyr Ala Asp
Leu Asp Met Val His Leu Asn Arg465 470 475 480Thr Pro Lys Gln Pro
Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr 485 490 495Ala Ser Val
Gln Val Pro Arg Lys 500491512DNAHomo
sapiensmisc_feature(1)..(1512)human SIRP alpha V6 49atggaacctg
ccggccctgc tcctggtaga ctgggacctc tgctgtgtct gctgctggcc 60gcctcttgtg
cttggagcgg agtggctggc gaagaggaac tgcaagtgat ccagcccgac
120aagtccgtgc tggtggctgc tggcgagact gccaccctga gatgtaccgc
cacctccctg 180atccccgtgg gccctatcca gtggtttaga ggcgctggcc
ctggcagaga gctgatctac 240aaccagaaag agggccactt ccccagagtg
accaccgtgt ccgacctgac caagcggaac 300aacatggact tccccatccg
gatcggcaac atcacccctg ccgatgccgg cacctactac 360tgcgtgaagt
tccggaaggg ctcccccgac gacgtggagt tcaaatccgg cgctggcacc
420gagctgtctg tgcgggctaa accttctgcc cctgtggtgt ctggccctgc
cgctagagct 480acccctcagc acaccgtgtc ttttacctgc gagtcccacg
gcttcagccc tcgggacatc 540accctgaagt ggttcaagaa cggcaacgag
ctgagcgact tccagaccaa cgtggaccct 600gtgggcgagt ccgtgtccta
ctccatccac tccaccgcca aggtggtgct gacacgcgag 660gacgtgcact
cccaagtgat ctgcgaggtg gcccacgtga cactgcaggg cgatcctctg
720agaggcaccg ccaacctgtc cgagacaatc agagtgcccc ccaccctgga
agtgacccag 780cagcccgtgc gggctgagaa ccaagtgaac gtgacctgcc
aagtgcggaa gttctacccc 840cagcggctgc agctgacctg gctggaaaac
ggcaatgtgt cccggaccga gacagcctcc 900accgtgaccg agaacaagga
tggcacctac aattggatgt cctggctgct cgtgaacgtg 960tccgcccacc
gggacgatgt gaagctgaca tgccaggtgg aacacgacgg ccagcctgcc
1020gtgtccaagt cccacgatct gaaggtgtcc gctcatccca aagagcaggg
ctccaacacc 1080gccgctgaga acaccggctc taacgagcgg aacatctaca
tcgtcgtggg cgtcgtgtgc 1140accctgctgg tggcactgct gatggccgct
ctgtacctcg tgcggatccg gcagaagaag 1200gcccagggct ctacctcctc
caccagactg cacgagcccg agaagaacgc cagagagatc 1260acccaggaca
ccaacgacat cacctacgcc gacctgaacc tgcccaaggg caagaagcct
1320gcccctcagg ctgccgagcc taacaaccac accgagtacg cctccatcca
gaccagccct 1380cagcctgcct ctgaggacac cctgacctac gctgatctgg
acatggtgca cctgaaccgg 1440acccccaagc agccagctcc taagcccgag
cctagcttct ctgagtacgc cagcgtgcag 1500gtgccccgga aa 151250504PRTHomo
sapiensmisc_feature(1)..(504)human SIRP alpha V6 50Met 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 Pro 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 Ile Arg Gln Lys Lys385 390 395 400Ala Gln Gly Ser Thr
Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn 405 410 415Ala Arg Glu
Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu 420 425 430Asn
Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn 435 440
445Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser
450 455 460Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu
Asn Arg465 470 475 480Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro
Ser Phe Ser Glu Tyr 485 490 495Ala Ser Val Gln Val Pro Arg Lys
500511509DNAHomo sapiensmisc_feature(1)..(1509)human SIRP alpha V8
51atggaacctg ccggccctgc tcctggtaga ctgggacctc tgctgtgtct gctgctggcc
60gcctcttgtg cttggagcgg agtggctggc gaagaggaac tgcaagtgat ccagcccgac
120aagtccgtgc tggtggctgc tggcgagact gccaccctga gatgtaccgc
cacctccctg 180atccccgtgg gccctatcca gtggtttaga ggcgctggcc
ctgccagaga gctgatctac 240aaccagaaag agggccactt ccccagagtg
accaccgtgt ccgagtccac caagcgcgag 300aacatggact tctccatctc
catcagcaac atcacccctg ccgacgccgg cacctactac 360tgcgtgaagt
tccggaaggg ctcccccgac accgagttca agtctggcgc tggcaccgag
420ctgtctgtgc gggctaaacc ttctgcccct gtggtgtctg gacctgccgc
tagagctacc 480cctcagcaca ccgtgtcttt tacctgcgag tcccacggct
tcagccctcg ggacatcacc 540ctgaagtggt tcaagaacgg caacgagctg
agcgacttcc agaccaacgt ggaccctgtg 600ggcgagtccg tgtcctactc
catccactcc accgccaagg tggtgctgac acgcgaggac 660gtgcactccc
aagtgatctg cgaggtggcc cacgtgacac tgcagggcga tcctctgaga
720ggcaccgcca acctgtccga gacaatcaga gtgcccccca ccctggaagt
gacccagcag 780cccgtgcggg ctgagaacca agtgaacgtg acctgccaag
tgcggaagtt ctacccccag 840cggctgcagc tgacctggct ggaaaacggc
aatgtgtccc ggaccgagac agccagcacc 900gtgaccgaga acaaggatgg
cacctacaat tggatgtcct ggctgctcgt gaacgtgtcc 960gcccaccggg
acgatgtgaa gctgacatgc caggtggaac acgacggcca gcctgccgtg
1020tccaagagcc acgatctgaa ggtgtccgct catcccaaag agcagggctc
caacaccgcc 1080gctgagaaca ccggctctaa cgagcggaac atctacatcg
tcgtgggcgt cgtgtgcacc 1140ctgctggtgg cactgctgat ggccgctctg
tacctcgtgc ggatccggca gaagaaggcc 1200cagggctcta cctcctccac
cagactgcac gagcccgaga agaacgccag agagatcacc 1260caggacacca
acgacatcac ctacgccgac ctgaacctgc ccaagggcaa gaagcctgcc
1320cctcaggctg ccgagcctaa caaccacacc gagtacgcct ccatccagac
cagccctcag 1380cctgcctctg aggacaccct gacctacgct gatctggaca
tggtgcacct gaaccggacc 1440cccaagcagc cagctcctaa gcccgagcct
agcttctctg agtacgccag cgtgcaggtg 1500ccccggaaa 150952503PRTHomo
sapiensmisc_feature(1)..(503)human SIRP alpha V8 52Met 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 Ala Arg Glu Leu Ile Tyr65
70 75 80Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
Ser 85 90 95Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser 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 Thr Glu Phe Lys Ser Gly Ala Gly
Thr Glu Leu Ser Val Arg 130 135 140Ala Lys Pro Ser Ala Pro Val Val
Ser Gly Pro Ala Ala Arg Ala Thr145 150 155 160Pro Gln His Thr Val
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro 165 170 175Arg Asp Ile
Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp 180 185 190Phe
Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile 195 200
205His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln
210 215 220Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
Leu Arg225 230 235 240Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val
Pro Pro Thr Leu Glu 245 250 255Val Thr Gln Gln Pro Val Arg Ala Glu
Asn Gln Val Asn Val Thr Cys 260 265 270Gln Val Arg Lys Phe Tyr Pro
Gln Arg Leu Gln Leu Thr Trp Leu Glu 275 280 285Asn Gly Asn Val Ser
Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn 290 295 300Lys Asp Gly
Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser305 310 315
320Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly
325 330 335Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala
His Pro 340 345 350Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr
Gly Ser Asn Glu 355 360 365Arg Asn Ile Tyr Ile Val Val Gly Val Val
Cys Thr Leu Leu Val Ala 370 375 380Leu Leu Met Ala Ala Leu Tyr Leu
Val Arg Ile Arg Gln Lys Lys Ala385 390 395 400Gln Gly Ser Thr Ser
Ser Thr Arg Leu His Glu Pro Glu Lys Asn Ala 405 410 415Arg Glu Ile
Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu Asn 420 425 430Leu
Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn Asn 435 440
445His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln
Pro Ala Ser Glu 450 455 460Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met
Val His Leu Asn Arg Thr465 470 475 480Pro Lys Gln Pro Ala Pro Lys
Pro Glu Pro Ser Phe Ser Glu Tyr Ala 485 490 495Ser Val Gln Val Pro
Arg Lys 500531512DNAHomo sapiensmisc_feature(1)..(1512)human SIRP
alpha V9 53atggaacctg ccggccctgc tcctggtaga ctgggacctc tgctgtgtct
gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc gaagaggaac tgcaagtgat
ccagcccgac 120aagtccgtgc tggtggctgc tggcgagact gccaccctga
gatgtaccgc cacctccctg 180atccccgtgg gccctatcca gtggtttaga
ggcgctggcc ctggcagaga gctgatctac 240aaccagaaag agggccactt
ccccagagtg accaccgtgt ccgacctgac caagcggaac 300aacatggact
tctccatccg gatctccaac atcacccctg ccgacgccgg cacctactac
360tgcgtgaagt tccggaaggg ctcccccgac gacgtggagt tcaaatccgg
cgctggcacc 420gagctgtctg tgcgggctaa accttctgcc cctgtggtgt
ctggccctgc cgctagagct 480acccctcagc acaccgtgtc ttttacctgc
gagtcccacg gcttcagccc tcgggacatc 540accctgaagt ggttcaagaa
cggcaacgag ctgagcgact tccagaccaa cgtggaccct 600gtgggcgagt
ccgtgtccta ctccatccac tccaccgcca aggtggtgct gacacgcgag
660gacgtgcact cccaagtgat ctgcgaggtg gcccacgtga cactgcaggg
cgatcctctg 720agaggcaccg ccaacctgtc cgagacaatc agagtgcccc
ccaccctgga agtgacccag 780cagcccgtgc gggctgagaa ccaagtgaac
gtgacctgcc aagtgcggaa gttctacccc 840cagcggctgc agctgacctg
gctggaaaac ggcaatgtgt cccggaccga gacagcctcc 900accgtgaccg
agaacaagga tggcacctac aattggatgt cctggctgct cgtgaacgtg
960tccgcccacc gggacgatgt gaagctgaca tgccaggtgg aacacgacgg
ccagcctgcc 1020gtgtccaagt cccacgatct gaaggtgtcc gctcatccca
aagagcaggg ctccaacacc 1080gccgctgaga acaccggctc taacgagcgg
aacatctaca tcgtcgtggg cgtcgtgtgc 1140accctgctgg tggcactgct
gatggccgct ctgtacctcg tgcggatccg gcagaagaag 1200gcccagggct
ctacctcctc caccagactg cacgagcccg agaagaacgc cagagagatc
1260acccaggaca ccaacgacat cacctacgcc gacctgaacc tgcccaaggg
caagaagcct 1320gcccctcagg ctgccgagcc taacaaccac accgagtacg
cctccatcca gaccagccct 1380cagcctgcct ctgaggacac cctgacctac
gctgatctgg acatggtgca cctgaaccgg 1440acccccaagc agccagctcc
taagcccgag cctagcttct ctgagtacgc cagcgtgcag 1500gtgccccgga aa
151254504PRTHomo sapiensmisc_feature(1)..(504)human SIRP alpha V9
54Met 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 Ser 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 Ile Arg Gln Lys Lys385 390 395
400Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn
405 410 415Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala
Asp Leu 420 425 430Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala
Ala Glu Pro Asn 435 440 445Asn His Thr Glu Tyr Ala Ser Ile Gln Thr
Ser Pro Gln Pro Ala Ser 450 455 460Glu Asp Thr Leu Thr Tyr Ala Asp
Leu Asp Met Val His Leu Asn Arg465 470 475 480Thr Pro Lys Gln Pro
Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr 485 490 495Ala Ser Val
Gln Val Pro Arg Lys 500551512DNAArtificial SequenceSynthetic hSIRP
alpha-V beta C1 alphaC2 alpha 55atggaacctg ccggccctgc tcctggtaga
ctgggacctc tgctgtgtct gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc
gaggacgagc tgcaagtgat ccagcccgag 120aagtccgtgt ctgtggccgc
tggcgagtct gccaccctga gatgcgctat gacctccctg 180atccccgtgg
gccccatcat gtggtttaga ggcgctggcg ctggcagaga gctgatctac
240aaccagaaag agggccactt ccccagagtg accaccgtgt ccgagctgac
caagcggaac 300aacctggact tctccatctc catcagcaac atcacccctg
ccgacgccgg cacctactac 360tgcgtgaagt tccggaaggg ctcccccgac
gacgtggagt tcaaatccgg cgctggaacc 420gagctgtccg tgcgggctaa
accttctgcc cctgtggtgt ctggccctgc cgctagagct 480acccctcagc
acaccgtgtc ttttacctgc gagtcccacg gcttcagccc tcgggacatc
540accctgaagt ggttcaagaa cggcaacgag ctgagcgact tccagaccaa
cgtggaccct 600gtgggcgaga gcgtgtccta ctccatccac tccaccgcca
aggtggtgct gacacgcgag 660gacgtgcact cccaagtgat ctgcgaggtg
gcccacgtga cactgcaggg cgatcctctg 720agaggcaccg ccaacctgtc
cgagacaatc agagtgcccc ccaccctgga agtgacccag 780cagcctgtgc
gggccgagaa ccaagtgaac gtgacctgcc aagtgcggaa gttctacccc
840cagcggctgc agctgacctg gctggaaaac ggcaatgtgt cccggaccga
gacagccagc 900accgtgaccg agaacaagga tggcacctac aattggatgt
cctggctgct cgtgaacgtg 960tccgcccacc gggacgatgt gaagctgaca
tgccaggtgg aacacgacgg ccagcctgcc 1020gtgtccaagt cccacgatct
gaaggtgtcc gctcatccca aagagcaggg ctccaacacc 1080gccgctgaga
acaccggctc taacgagcgg aacatctaca tcgtcgtggg cgtcgtgtgc
1140accctgctgg tggctctgct gatggctgcc ctgtacctcg tgcggatccg
gcagaagaag 1200gcccagggct ctacctcctc caccagactg cacgagcctg
agaagaacgc cagagagatc 1260acccaggaca ccaacgacat cacctacgcc
gacctgaacc tgcccaaggg caagaagcct 1320gcccctcagg ccgccgagcc
taacaaccac accgagtacg cctccatcca gaccagccct 1380cagcctgcct
ctgaggacac cctgacctac gctgatctgg acatggtgca cctgaaccgg
1440acccccaagc agccagctcc taagcccgag cctagcttct ctgagtacgc
cagcgtgcag 1500gtgccccgga aa 151256504PRTArtificial
SequenceSynthetic hSIRP alpha-V beta C1 alphaC2 alpha 56Met 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 Asp 20 25 30Glu
Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala Gly 35 40
45Glu Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val Gly
50 55 60Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile
Tyr65 70 75 80Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val
Ser Glu Leu 85 90 95Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile
Ser 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 Ile Arg Gln Lys Lys385 390 395 400Ala Gln Gly Ser
Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn 405 410 415Ala Arg
Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu 420 425
430Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn
435 440 445Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro
Ala Ser 450 455 460Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val
His Leu Asn Arg465 470 475 480Thr Pro Lys Gln Pro Ala Pro Lys Pro
Glu Pro Ser Phe Ser Glu Tyr 485 490 495Ala Ser Val Gln Val Pro Arg
Lys 500571512DNAArtificial SequenceSynthetic hSIRP alpha-V alphaC1
beta C2 alpha 57atggaacctg ccggccctgc tcctggtaga ctgggacctc
tgctgtgtct gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc gaagaggaac
tgcaagtgat ccagcccgac 120aagtccgtgc tggtggctgc tggcgagact
gccaccctga gatgtaccgc cacctccctg 180atccccgtgg gccctatcca
gtggtttaga ggcgctggcc ctggcagaga gctgatctac 240aaccagaaag
agggccactt ccccagagtg accaccgtgt ccgacctgac caagcggaac
300aacatggact tctccatccg gatcggcaac atcacccctg ccgatgccgg
cacctactac 360tgcgtgaagt tccggaaggg ctcccccgac gacgtggagt
tcaaatccgg cgctggcacc 420gagctgtctg tgcgggctaa accttctgcc
cccgtggtgt ctggacctgc cgtgcgagct 480acccctgagc acaccgtgtc
ttttacctgc gagtcccacg gcttcagccc tcgggacatc 540accctgaagt
ggttcaagaa cggcaacgag ctgagcgact tccagaccaa cgtggaccca
600gccggcgact ccgtgtccta ctccatccac tctaccgcca gagtggtgct
gaccagaggc 660gacgtgcact cccaagtgat ctgcgagatc gcccatatca
cactgcaggg cgaccccctg 720agaggcaccg ctaacctgtc tgagacaatc
cgggtgcccc ccaccctgga agtgactcag 780cagccagtgc gggccgagaa
ccaagtgaac gtgacctgcc aagtgcggaa gttctacccc 840cagcggctgc
agctgacctg gctggaaaac ggcaatgtgt cccggaccga gacagcctcc
900accgtgaccg agaacaagga tggcacctac aattggatgt cttggctgct
cgtgaacgtg 960tccgcccacc gggacgatgt gaagctgaca tgccaggtgg
aacacgacgg ccagcctgcc 1020gtgtccaagt cccacgatct gaaggtgtcc
gctcatccca aagagcaggg ctccaacacc 1080gccgctgaga acaccggctc
taacgagcgg aacatctaca tcgtcgtggg cgtcgtgtgc 1140accctgctgg
tggcactgct gatggccgct ctgtacctcg tgcggatccg gcagaagaag
1200gcccagggct ctacctcctc caccagactg cacgagcccg agaagaacgc
cagagagatc 1260acccaggaca ccaacgacat cacctacgcc gacctgaacc
tgcccaaggg caagaagcct 1320gcccctcagg ccgccgagcc taacaaccac
accgagtacg cctccatcca gaccagccct 1380cagcctgcct ctgaggacac
cctgacctac gctgatctgg acatggtgca cctgaaccgg 1440acccccaagc
agccagctcc taagcccgag cctagcttct ctgagtacgc cagcgtgcag
1500gtgccccgga aa 151258504PRTArtificial SequenceSynthetic hSIRP
alpha-V alphaC1 beta C2 alpha 58Met 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 Val Arg Ala145 150 155 160Thr Pro Glu 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 Ala Gly Asp Ser Val Ser Tyr Ser 195 200 205Ile His Ser
Thr Ala Arg Val Val Leu Thr Arg Gly Asp Val His Ser 210 215 220Gln
Val Ile Cys Glu Ile Ala His Ile 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 Ile
Arg Gln Lys Lys385 390 395 400Ala Gln Gly Ser Thr Ser Ser Thr Arg
Leu His Glu Pro Glu Lys Asn 405 410 415Ala Arg Glu Ile Thr Gln Asp
Thr Asn Asp Ile Thr Tyr Ala Asp Leu 420 425 430Asn Leu Pro Lys Gly
Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn 435 440 445Asn His Thr
Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser 450 455 460Glu
Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg465 470
475 480Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu
Tyr 485 490 495Ala Ser Val Gln Val Pro Arg Lys
500591512DNAArtificial SequenceSynthetic hSIRP alpha-V alphaC1
alphaC2 beta 59atggaacctg ccggccctgc tcctggtaga ctgggacctc
tgctgtgtct gctgctggcc 60gcctcttgtg cttggagcgg agtggctggc gaagaggaac
tgcaagtgat ccagcccgac 120aagtccgtgc tggtggctgc tggcgagact
gccaccctga gatgtaccgc cacctccctg 180atccccgtgg gccctatcca
gtggtttaga ggcgctggcc ctggcagaga gctgatctac 240aaccagaaag
agggccactt ccccagagtg accaccgtgt ccgacctgac caagcggaac
300aacatggact tctccatccg gatcggcaac atcacccctg ccgatgccgg
cacctactac 360tgcgtgaagt tccggaaggg ctcccccgac gacgtggagt
tcaaatccgg cgctggcacc 420gagctgtctg tgcgggctaa accttctgcc
cctgtggtgt ctggccctgc cgctagagct 480acccctcagc acaccgtgtc
ttttacctgc gagtcccacg gcttcagccc tcgggacatc 540accctgaagt
ggttcaagaa cggcaacgag ctgagcgact tccagaccaa cgtggaccct
600gtgggcgagt ccgtgtccta ctccatccac tccaccgcca aggtggtgct
gacacgcgag 660gacgtgcact cccaagtgat ctgcgaggtg gcccacgtga
cactgcaggg cgatcctctg 720agaggcaccg ccaacctgtc cgagacaatc
agagtgcccc ccaccctgga agtgacccag 780cagcctatga gagccgagaa
ccaggccaac gtgacctgcc aggtgtccaa cttctaccct 840cggggcctgc
agctgacctg gctggaaaac ggcaatgtgt cccggaccga gacagcctcc
900accctgatcg agaacaagga tggcacctac aattggatgt cctggctgct
cgtgaacacc 960tgtgcccacc gggacgatgt ggtgctgacc tgtcaggtgg
aacacgatgg ccagcaggcc 1020gtgtccaagt cctacgctct ggaagtgtcc
gcccacccca aagagcaggg ctctaatacc 1080gccgctgaga acaccggctc
caacgagcgg aacatctaca tcgtcgtggg cgtcgtgtgc 1140accctgctgg
tggcactgct gatggccgct ctgtacctcg tgcggatccg gcagaagaag
1200gctcagggct ccacctcctc caccagactg cacgagcctg agaagaacgc
cagagagatc 1260acccaggaca ccaacgacat cacctacgcc gacctgaacc
tgcccaaggg caagaagcct 1320gcccctcagg ctgccgagcc taacaaccac
accgagtacg cctccatcca gaccagccct 1380cagcctgcct ctgaggacac
cctgacctac gctgatctgg acatggtgca cctgaaccgg 1440acccccaagc
agccagctcc taagcccgag cctagcttct ctgagtacgc cagcgtgcag
1500gtgccccgga aa 151260504PRTArtificial SequenceSynthetic hSIRP
alpha-V alphaC1 alphaC2 beta 60Met 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 Met Arg Ala Glu Asn Gln Ala
Asn Val Thr 260 265 270Cys Gln Val Ser Asn Phe Tyr Pro Arg Gly Leu
Gln Leu Thr Trp Leu 275 280 285Glu Asn Gly Asn Val Ser Arg Thr Glu
Thr Ala Ser Thr Leu Ile Glu 290 295 300Asn Lys Asp Gly Thr Tyr Asn
Trp Met Ser Trp Leu Leu Val Asn Thr305 310 315 320Cys Ala His Arg
Asp Asp Val Val Leu Thr Cys Gln Val Glu His Asp 325 330 335Gly Gln
Gln Ala Val Ser Lys Ser Tyr Ala Leu Glu 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 Ile
Arg Gln Lys Lys385 390 395 400Ala Gln Gly Ser Thr Ser Ser Thr Arg
Leu His Glu Pro Glu Lys Asn 405 410 415Ala Arg Glu Ile Thr Gln Asp
Thr Asn Asp Ile Thr Tyr Ala Asp Leu 420 425 430Asn Leu Pro Lys Gly
Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn 435 440 445Asn His Thr
Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser 450 455 460Glu
Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg465 470
475 480Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu
Tyr 485 490 495Ala Ser Val Gln Val Pro Arg Lys 500611512DNAHomo
sapiensmisc_feature(1)..(1512)human SIRP alpha V1(P74A)
61atggagcccg ccggcccggc ccccggccgc ctcgggccgc tgctctgcct gctgctcgcc
60gcgtcctgcg cctggtcagg agtggcgggt gaggaggagc tgcaggtgat tcagcctgac
120aagtccgtgt tggttgcagc tggagagaca gccactctgc gctgcactgc
gacctctctg 180atccctgtgg ggcccatcca gtggttcaga ggagctggag
caggccggga attaatctac 240aatcaaaaag aaggccactt cccccgggta
acaactgttt cagacctcac aaagagaaac 300aacatggact tttccatccg
catcggtaac atcaccccag cagatgccgg cacctactac 360tgtgtgaagt
tccggaaagg gagccccgat gacgtggagt ttaagtctgg agcaggcact
420gagctgtctg tgcgcgccaa accctctgcc cccgtggtat cgggccctgc
ggcgagggcc 480acacctcagc acacagtgag cttcacctgc gagtcccacg
gcttctcacc cagagacatc 540accctgaaat ggttcaaaaa tgggaatgag
ctctcagact tccagaccaa cgtggacccc 600gtaggagaga gcgtgtccta
cagcatccac agcacagcca aggtggtgct gacccgcgag 660gacgttcact
ctcaagtcat ctgcgaggtg gcccacgtca ccttgcaggg ggaccctctt
720cgtgggactg ccaacttgtc tgagaccatc cgagttccac ccaccttgga
ggttactcaa 780cagcccgtga gggcagagaa ccaggtgaat gtcacctgcc
aggtgaggaa gttctacccc 840cagagactac agctgacctg gttggagaat
ggaaacgtgt cccggacaga aacggcctca 900accgttacag agaacaagga
tggtacctac aactggatga gctggctcct ggtgaatgta 960tctgcccaca
gggatgatgt gaagctcacc tgccaggtgg agcatgacgg gcagccagcg
1020gtcagcaaaa gccatgacct gaaggtctca gcccacccga aggagcaggg
ctcaaatacc 1080gccgctgaga acactggatc taatgaacgg aacatctata
ttgtggtggg tgtggtgtgc 1140accttgctgg tggccctact gatggcggcc
ctctacctcg tccgaatcag acagaagaaa 1200gcccagggct ccacttcttc
tacaaggttg catgagcccg agaagaatgc cagagaaata 1260acacaggaca
caaatgatat cacatatgca gacctgaacc tgcccaaggg gaagaagcct
1320gctccccagg ctgcggagcc caacaaccac acggagtatg ccagcattca
gaccagcccg 1380cagcccgcgt cggaggacac cctcacctat gctgacctgg
acatggtcca cctcaaccgg 1440acccccaagc agccggcccc caagcctgag
ccgtccttct cagagtacgc cagcgtccag 1500gtcccgagga ag 151262504PRTHomo
sapiensmisc_feature(1)..(504)human SIRP alpha V1(P74A) 62Met 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 Ala 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 Ile Arg Gln Lys Lys385 390 395 400Ala
Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn 405 410
415Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu
420 425 430Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu
Pro Asn 435 440 445Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro
Gln Pro Ala Ser 450 455 460Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp
Met Val His Leu Asn Arg465 470 475 480Thr Pro Lys Gln Pro Ala Pro
Lys Pro Glu Pro Ser Phe Ser Glu Tyr 485 490 495Ala Ser Val Gln Val
Pro Arg Lys 50063321DNAHomo sapiensmisc_feature(1)..(321)human
kappa constant domain 63cggaccgtgg ccgctccctc cgtgttcatc ttcccacctt
ccgacgagca gctgaagtcc 60ggcaccgctt ctgtcgtgtg cctgctgaac aacttctacc
cccgcgaggc caaggtgcag 120tggaaggtgg acaacgccct gcagtccggc
aactcccagg aatccgtgac cgagcaggac 180tccaaggaca gcacctactc
cctgtcctcc accctgaccc tgtccaaggc cgactacgag 240aagcacaagg
tgtacgcctg cgaagtgacc caccagggcc tgtctagccc tgtgaccaag
300tccttcaacc ggggcgagtg c 32164107PRTHomo
sapiensmisc_feature(1)..(107)human kappa constant domain 64Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25
30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 10565981DNAHomo sapiensmisc_feature(1)..(981)human IgG4
constant domains (including S228P) 65gcttccacca agggcccctc
cgtgtttcct ctggcccctt gctccagatc cacctccgag 60tctaccgccg ctctgggctg
cctcgtgaag gactacttcc ccgagcctgt gacagtgtcc 120tggaactctg
gcgccctgac ctctggcgtg cacacctttc cagctgtgct gcagtcctcc
180ggcctgtact ccctgtccag cgtcgtgaca gtgccctcca gctctctggg
caccaagacc 240tacacctgta acgtggacca caagccctcc aacaccaagg
tggacaagcg ggtggaatct 300aagtacggcc ctccctgccc tccttgccca
gcccctgaat ttctgggcgg accttctgtg 360tttctgttcc ccccaaagcc
caaggacacc ctgatgatct cccggacccc cgaagtgacc 420tgcgtggtgg
tggatgtgtc ccaggaagat cccgaggtgc agttcaattg gtacgtggac
480ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagttcaa
ctccacctac 540cgggtggtgt ccgtgctgac cgtgctgcac caggattggc
tgaacggcaa agagtacaag 600tgcaaggtgt ccaacaaggg cctgcccagc
tccatcgaaa agaccatctc caaggccaag 660ggccagcccc gggaacccca
ggtgtacaca ctgcctccaa gccaggaaga gatgaccaag 720aaccaggtgt
ccctgacctg tctcgtgaaa ggcttctacc cctccgatat cgccgtggaa
780tgggagtcca acggccagcc tgagaacaac tacaagacca ccccccctgt
gctggactcc 840gacggctcct tctttctgta ctctcgcctg accgtggaca
agtcccggtg gcaggaaggc 900aacgtgttct cctgcagcgt gatgcacgag
gccctgcaca accactacac ccagaagtcc 960ctgtccctgt ctctgggaaa a
98166327PRTHomo sapiensmisc_feature(1)..(327)human IgG4 constant
domains (including S228P) 66Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
Gly Lys 32567978DNAHomo sapiensmisc_feature(1)..(978)human IgG2
constant domainsmisc_feature(769)..(769)n is g or t 67gcttctacaa
agggccccag cgtgttccct ctggctcctt gtagcagaag caccagcgag 60tctacagccg
ctctgggctg tctggtcaag gactactttc ccgagcctgt gaccgtgtcc
120tggaatagcg gagcactgac aagcggcgtg cacacctttc cagctgtgct
gcaaagctcc 180ggcctgtact ctctgtccag cgtggtcaca gtgcccagca
gcaattttgg cacccagacc 240tacacctgta atgtggacca caagcctagc
aacaccaagg tggacaagac cgtggaacgg 300aagtgctgcg tggaatgccc
tccttgtcct gctcctccag tggctggccc ttccgtgttt 360ctgttccctc
caaagcctaa ggacaccctg atgatcagca gaacccctga agtgacctgc
420gtggtggtgg atgtgtccca cgaggatcct gaggtgcagt tcaattggta
cgtggacggc 480gtggaagtgc acaacgccaa gaccaagcct agagaggaac
agttcaacag caccttcaga 540gtggtgtccg tgctgaccgt ggtgcatcag
gattggctga acggcaaaga gtacaagtgc 600aaggtgtcca acaagggcct
gcctgctcct atcgagaaaa ccatcagcaa gaccaaaggc 660cagcctcgcg
agccccaggt ttacacactt cctccaagcc gggaagagat gaccaagaac
720caggtgtccc tgacctgcct cgtgaagggc ttctacccca gcgacatcnc
cgtggaatgg 780gagagcaatg gccagcctga gaacaactac aagaccacac
ctcctatgct ggactccgac 840ggctcattct tcctgtacag caagctgaca
gtggacaagt ccagatggca gcagggcaac 900gtgttctcct gcagcgtgat
gcacgaggcc ctgcacaacc actacaccca gaagtccctg 960tctctgagcc ccggcaaa
97868326PRTHomo sapiensmisc_feature(1)..(326)human IgG2 constant
domainsmisc_feature(257)..(257)X is A or S 68Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75
80Tyr Thr Cys Asn Val Asp
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135
140Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg Val Val Ser Val Leu Thr
Val Val His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235 240Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250
255Xaa Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro Gly Lys
325695PRTArtificial SequenceSynthetic 40A heavy chain CDR1 69Ser
Tyr Trp Met His1 57017PRTArtificial SequenceSynthetic 40A heavy
chain CDR2 70Ala Ile Tyr Pro Val Asn Asn Asp Thr Thr Tyr Asn Gln
Lys Phe Lys1 5 10 15Gly7113PRTArtificial SequenceSynthetic 40A
heavy chain CDR3 71Ser Phe Tyr Tyr Ser Leu Asp Ala Ala Trp Phe Val
Tyr1 5 107211PRTArtificial SequenceSynthetic 40A light chain CDR1
72Arg Ala Ser Gln Asp Ile Gly Ser Arg Leu Asn1 5 10737PRTArtificial
SequenceSynthetic 40A light chain CDR2 73Ala Thr Ser Ser Leu Asp
Ser1 5749PRTArtificial SequenceSynthetic 40A light chain CDR3 74Leu
Gln Tyr Ala Ser Ser Pro Phe Thr1 575122PRTArtificial
SequenceSynthetic humanized 40 heavy chain variable region
(consensus sequence)misc_feature(4)..(4)X = F or
Lmisc_feature(5)..(5)X = Q or R or Vmisc_feature(10)..(10)X = E or
Vmisc_feature(11)..(11)X = L or Vmisc_feature(12)..(12)X = A or K
or Vmisc_feature(20)..(20)X = L or M or Vmisc_feature(38)..(38)X =
K or Rmisc_feature(40)..(40)X = A or R or Tmisc_feature(48)..(48)X
= I or Mmisc_feature(67)..(67)X = K or Rmisc_feature(68)..(68)X = A
or Vmisc_feature(70)..(70)X = L or Mmisc_feature(73)..(73)X = D or
Vmisc_feature(74)..(74)X = K or Tmisc_feature(76)..(76)X = A or S
or Tmisc_feature(79)..(79)X = A or Vmisc_feature(82)..(82)X = E or
Qmisc_feature(87)..(87)X = R or Tmisc_feature(88)..(88)X = F or
Smisc_feature(91)..(91)X = S or Tmisc_feature(97)..(97)X = A or
Tmisc_feature(117)..(117)X = L or Tmisc_feature(118)..(118)X = L or
V 75Glu Val Gln Xaa Xaa Gln Ser Gly Ala Xaa Xaa Xaa Lys Pro Gly
Ala1 5 10 15Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Ser Thr Phe Thr
Ser Tyr 20 25 30Trp Met His Trp Val Xaa Gln Xaa Pro Gly Gln Gly Leu
Glu Trp Xaa 35 40 45Gly Ala Ile Tyr Pro Val Asn Ser Asp Thr Thr Tyr
Asn Gln Lys Phe 50 55 60Lys Gly Xaa Xaa Thr Xaa Thr Val Xaa Xaa Ser
Xaa Ser Thr Xaa Tyr65 70 75 80Met Xaa Leu Ser Ser Leu Xaa Xaa Glu
Asp Xaa Ala Val Tyr Tyr Cys 85 90 95Xaa Arg Ser Phe Tyr Tyr Ser Leu
Asp Ala Ala Trp Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr Xaa Xaa
Thr Val Ser Ser 115 12076107PRTArtificial SequenceSynthetic
humanized 40 light chain variable region (consensus
sequence)misc_feature(15)..(15)X = L or Vmisc_feature(17)..(17)X =
D or Emisc_feature(20)..(20)X = S or Tmisc_feature(39)..(39)X = K
or Tmisc_feature(44)..(44)X = I or Pmisc_feature(60)..(60)X = K or
Smisc_feature(66)..(66)X = G or Rmisc_feature(69)..(69)X = S or
Tmisc_feature(70)..(70)X = D or Emisc_feature(71)..(71)X = F or
Ymisc_feature(72)..(72)X = S or Tmisc_feature(77)..(77)X = G or
Smisc_feature(100)..(100)X = G or Qmisc_feature(104)..(104)X = L or
Vmisc_feature(107)..(107)X = H or K 76Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Xaa Gly1 5 10 15Xaa Arg Val Xaa Ile Thr
Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg 20 25 30Leu Asn Trp Leu Gln
Gln Xaa Pro Gly Lys Ala Xaa Lys Arg Leu Ile 35 40 45Tyr Ala Thr Ser
Ser Leu Asp Ser Gly Val Pro Xaa Arg Phe Ser Gly 50 55 60Ser Xaa Ser
Gly Xaa Xaa Xaa Xaa Leu Thr Ile Ser Xaa Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Phe 85 90
95Thr Phe Gly Xaa Gly Thr Lys Xaa Glu Ile Xaa 100
10577366DNAArtificial SequenceSynthetic hSIRP alpha.40AVH1
77gaggtgcagt tcttgcagtc tggtgccgtg ctggctagac ctggaacctc cgtgaagatc
60tcctgcaagg cctccggctc caccttcacc tcttactgga tgcactgggt caagcagagg
120cctggacagg gactcgaatg gatcggcgct ctgtaccctg tgaactccga
caccacctac 180aaccagaagt tcaagggcag agccaagctg accgtggcca
cctctgcttc tatcgcctac 240ctggaatttt ccagcctgac caacgaggac
tccgccgtgt actactgcgc ccggtccttc 300tactactctc tggacgccgc
ttggtttgtg tactggggcc agggaactct ggtgaccgtg 360tcctct
36678122PRTArtificial SequenceSynthetic hSIRP alpha.40AVH1 78Glu
Val Gln Phe Leu Gln Ser Gly Ala Val Leu Ala Arg Pro Gly Thr1 5 10
15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Ser Thr Phe Thr Ser Tyr
20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45Gly Ala Leu Tyr Pro Val Asn Ser Asp Thr Thr Tyr Asn Gln
Lys Phe 50 55 60Lys Gly Arg Ala Lys Leu Thr Val Ala Thr Ser Ala Ser
Ile Ala Tyr65 70 75 80Leu Glu Phe Ser Ser Leu Thr Asn Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe Tyr Tyr Ser Leu Asp Ala
Ala Trp Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12079366DNAArtificial SequenceSynthetic hSIRP
alpha.40AVH2 79gaggtgcagc tggttcagtc tggcgctgag gttgtgaagc
ctggcgcttc cgtgaagctg 60tcctgcaagg cttctggctc caccttcacc agctactgga
tgcactgggt caagcaggcc 120cctggacaag gcctggaatg gatcggcgct
atctaccccg tgaactccga caccacctac 180aaccagaagt tcaagggcaa
agctaccctg accgtggaca agtctgcctc caccgcctac 240atggaactgt
ccagcctgag atctgaggac accgccgtgt actactgcac ccggtccttc
300tactactccc tggacgccgc ttggtttgtg tattggggcc agggaacact
ggtgaccgtg 360tcctct 36680122PRTArtificial SequenceSynthetic hSIRP
alpha.40AVH2 80Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys
Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Ser Thr
Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Ala Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Val Asn Ser Asp Thr
Thr Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Ser Phe Tyr Tyr
Ser Leu Asp Ala Ala Trp Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 12081366DNAArtificial SequenceSynthetic
hSIRP alpha.40AVH3 81gaggtgcagc tgagacagtc tggcgctgtg cttgtgaagc
ctggcgcctc cgtgaagatg 60tcctgcaagg cttctggctc caccttcacc agctactgga
tgcactgggt caagcagacc 120cctggacagg gactcgagtg gatcggcgct
atctaccctg tgaactccga caccacctac 180aaccagaagt tcaagggcaa
agctaccctg accgtggaca agtcctcctc caccgcttac 240atgcagctgt
ccagcctgac ctctgaggac tccgccgtgt actactgcgc ccggtccttc
300tactactctc tggacgccgc ttggtttgtg tactggggcc agggcacaac
cctgacagtg 360tcctct 36682122PRTArtificial SequenceSynthetic hSIRP
alpha.40AVH3 82Glu Val Gln Leu Arg Gln Ser Gly Ala Val Leu Val Lys
Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ser Thr
Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Thr Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Val Asn Ser Asp Thr
Thr Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe Tyr Tyr
Ser Leu Asp Ala Ala Trp Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser 115 12083366DNAArtificial SequenceSynthetic
hSIRP alpha.40AVH4 83gaggtgcagt tcgttcagtc tggcgccgaa gtgaagaaac
ctggcgcctc tgtgaaggtg 60tcctgcaagg cttctggctc caccttcacc agctactgga
tgcactgggt ccgacaggct 120ccaggacaag gcttggaatg gatgggcgct
atctaccccg tgaactccga caccacctac 180aaccagaaat tcaagggcag
agtgaccatg accgtcgtga cctccacctc caccgtgtac 240atggaactgt
ccagcctgag atccgaggac accgccgtgt actactgcgc ccggtccttc
300tactactctc tggacgccgc ttggtttgtg tactggggcc agggaactct
ggtgaccgtg 360tcctct 36684122PRTArtificial SequenceSynthetic hSIRP
alpha.40AVH4 84Glu Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ser Thr
Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Tyr Pro Val Asn Ser Asp Thr
Thr Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Val Val
Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe Tyr Tyr
Ser Leu Asp Ala Ala Trp Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 12085366DNAArtificial SequenceSynthetic
hSIRP alpha.40AVH5 85gaggtccagc tgcaacagtc tggtgccgtg ttggctaagc
ctggcgcctc cgtgaagatg 60tcctgcaagg cttctggctc caccttcacc agctactgga
tgcactgggt caagcagagg 120cctggacagg gactcgagtg gatcggcgct
atctaccctg tgaactccga caccacctac 180aaccagaagt tcaagggcaa
agctaccctg accgtggaca agtcctcctc caccgcttac 240atgcagctgt
ccagcctgac cttcgaggac tccgccgtgt actactgcgc ccggtccttc
300tactactctc tggacgccgc ttggtttgtg tactggggcc agggcacaac
cctgacagtg 360tcctct 36686122PRTArtificial SequenceSynthetic hSIRP
alpha.40AVH5 86Glu Val Gln Leu Gln Gln Ser Gly Ala Val Leu Ala Lys
Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ser Thr
Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Val Asn Ser Asp Thr
Thr Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr
Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe Tyr Tyr
Ser Leu Asp Ala Ala Trp Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser 115 12087366DNAArtificial SequenceSynthetic
hSIRP alpha.40AVH6 87gaggtgcagc tggttcagtc tggcgccgaa gtgaagaaac
ctggcgcctc tgtgaaggtg 60tcctgcaagg cttctggctc caccttcacc agctactgga
tgcactgggt ccgacaggct 120ccaggacaag gcttggaatg gatgggcgct
atctaccccg tgaactccga caccacctac 180aaccagaaat tcaagggcag
agtgaccatg accgtggaca cctccaccag caccgtgtac 240atggaactgt
ccagcctgag atccgaggac accgccgtgt actactgcgc ccggtccttc
300tactactctc tggacgccgc ttggtttgtg tactggggcc agggaactct
ggtgaccgtg 360tcctct 36688122PRTArtificial SequenceSynthetic hSIRP
alpha.40AVH6 88Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ser Thr
Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Tyr Pro Val Asn Ser Asp Thr
Thr Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Val Asp
Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe Tyr Tyr
Ser Leu Asp Ala Ala Trp Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 12089321DNAArtificial SequenceSynthetic
hSIRP alpha.40AVL1 89gacatccaga tgacccagtc tccatcctct ctgtccgcct
ctgtgggcga cagagtgacc 60atcacctgta gagcctctca ggacatcggc tccagactga
actggctgca gcagacccct 120ggcaaggcca tcaagagact gatctacgcc
acctccagcc tggattctgg cgtgccctct 180agattctccg gctctagatc
tggcaccgac ttctccctga ccatctctgg actgcagcct 240gaggacttcg
ccacctacta ctgtctgcag tacgccagct ctccattcac ctttggcgga
300ggcaccaagg tggaaatcca c 32190107PRTArtificial SequenceSynthetic
hSIRP alpha.40AVL1 90Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Gly Ser Arg 20 25 30Leu Asn Trp Leu Gln Gln Thr Pro Gly
Lys Ala Ile Lys Arg Leu Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe
Ser Leu Thr Ile Ser Gly Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Phe 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile His 100 10591321DNAArtificial
SequenceSynthetic hSIRP alpha.40AVL2 91gacatccaga tgacccagtc
tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60atcacctgta gagcctctca
ggacatcggc tccagactga actggctgca gcagaagcct 120ggcaaggcca
tcaagagact gatctacgcc acctccagcc tggattctgg cgtgccctct
180agattctccg gctctagatc tggcaccgac tttaccctga caatcagctc
cctgcagcct 240gaggacttcg ccacctacta ctgtctgcag tacgcctcct
ctccattcac ctttggccag 300ggcaccaagg tggaaatcaa g
32192107PRTArtificial SequenceSynthetic hSIRP alpha.40AVL2 92Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg
20 25 30Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu
Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr
Ala Ser Ser Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10593321DNAArtificial SequenceSynthetic hSIRP alpha.40AVL3
93gacatccaga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc
60atcacctgta gagcctctca ggacatcggc tccagactga actggctgca gcagaagcct
120ggcaaggcca tcaagagact gatctacgcc acctccagcc tggattctgg
cgtgccctct 180agattctccg gctctagatc tggcaccgac tttaccctga
caatcagctc cctgcagcct 240gaggacttcg ccacctacta ctgtctgcag
tacgccagct ctccattcac ctttggcgga 300ggcaccaagc tggaaatcaa g
32194107PRTArtificial SequenceSynthetic
hSIRP alpha.40AVL3 94Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Gly Ser Arg 20 25 30Leu Asn Trp Leu Gln Gln Lys Pro Gly
Lys Ala Ile Lys Arg Leu Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Phe 85 90 95Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 10595321DNAArtificial
SequenceSynthetic hSIRP alpha.40AVL4 95gacatccaga tgacccagtc
tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60atcacctgta gagcctctca
ggacatcggc tccagactga actggctgca gcagaagcct 120ggcaaggccc
ctaagagact gatctacgcc acctccagcc tggattctgg cgtgccctct
180agattctccg gctctggctc tggcaccgag tttaccctga caatcagctc
cctgcagcct 240gaggacttcg ccacctacta ctgtctgcag tacgccagct
ctccattcac ctttggcgga 300ggcaccaagg tggaaatcaa g
32196107PRTArtificial SequenceSynthetic hSIRP alpha.40AVL4 96Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg
20 25 30Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu
Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr
Ala Ser Ser Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 10597321DNAArtificial SequenceSynthetic hSIRP alpha.40AVL5
97gacatccaga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc
60atcacctgta gagcctctca ggacatcggc tccagactga actggctgca gcagaagcct
120ggcaaggcca tcaagagact gatctacgcc acctccagcc tggattctgg
cgtgcccaag 180agattctccg gctctagatc cggctccgac tataccctga
caatcagctc cctgcagcct 240gaggacttcg ccacctacta ctgtctgcag
tacgcctcct ctccattcac ctttggccag 300ggcaccaagg tggaaatcaa g
32198107PRTArtificial SequenceSynthetic hSIRP alpha.40AVL5 98Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg
20 25 30Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu
Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe
Ser Gly 50 55 60Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr
Ala Ser Ser Pro Phe 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10599321DNAArtificial SequenceSynthetic hSIRP alpha.40AVL6
99gacatccaga tgacccagtc tccatcctct ctgtctgctt ccctgggcga gagagtgtcc
60atcacctgta gagcctctca ggacatcggc tccagactga actggctgca gcagaagcct
120ggcaaggcca tcaagagact gatctacgcc acctccagcc tggattctgg
cgtgccctct 180agattctccg gctctagatc tggcaccgac tttaccctga
caatcagctc cctgcagcct 240gaggacttcg ccacctacta ctgtctgcag
tacgccagct ctccattcac ctttggcgga 300ggcaccaagg tggaaatcaa g
321100107PRTArtificial SequenceSynthetic hSIRP alpha.40AVL6 100Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10
15Glu Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg
20 25 30Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu
Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr
Ala Ser Ser Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105101366DNAArtificial SequenceSynthetic hSIRP alpha.40A
mouse VH 101gaggttcagt tccagcagtc tgggactgtg ctggcaaggc cagggacttc
agtgaagatg 60tcctgcaagg cttctggctc cacctttacc agctactgga tgcactgggt
aaaacagggg 120cctggacagg gtctgcaatg gattggcgct atttatcctg
taaataatga tactacctat 180aatcagaagt tcaagggcaa ggccgaactc
actgtagtca cttccaccag cactgcctac 240atggaggtca gtagtctgac
aaatgaggac tctgcggtct attactgtac aagatcgttc 300tactatagtc
tcgacgcggc ctggtttgtt tactggggcc aagggactct ggtcactgtc 360tctgca
366102122PRTArtificial SequenceSynthetic hSIRP alpha.40A mouse VH
102Glu Val Gln Phe Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Thr1
5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Ser Thr Phe Thr Ser
Tyr 20 25 30Trp Met His Trp Val Lys Gln Gly Pro Gly Gln Gly Leu Gln
Trp Ile 35 40 45Gly Ala Ile Tyr Pro Val Asn Asn Asp Thr Thr Tyr Asn
Gln Lys Phe 50 55 60Lys Gly Lys Ala Glu Leu Thr Val Val Thr Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Val Ser Ser Leu Thr Asn Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Thr Arg Ser Phe Tyr Tyr Ser Leu Asp
Ala Ala Trp Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ala 115 120103321DNAArtificial SequenceSynthetic hSIRP
alpha.40A mouse VL 103gacatccaga tgacccagtc tccatcctcc ttatctgcct
ctctgggaga aagagtcagt 60ctcacttgtc gggcaagtca ggacattggt agtaggttaa
actggcttca gcaggaacca 120gatggaacta ttaaacgcct gatctacgcc
acatccagtt tagattctgg tgtccccaaa 180aggttcagtg gcagtaggtc
tgggtcagat tattctctca ccatcagcgg ccttgagtct 240gaagactttg
tagactatta ctgtctacaa tatgctagtt ctccgttcac gttcggaggg
300gggaccaagc tggaaataaa c 321104107PRTArtificial SequenceSynthetic
hSIRP alpha.40A mouse VL 104Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Leu Gly1 5 10 15Glu Arg Val Ser Leu Thr Cys Arg Ala
Ser Gln Asp Ile Gly Ser Arg 20 25 30Leu Asn Trp Leu Gln Gln Glu Pro
Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp
Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Ser Asp
Tyr Ser Leu Thr Ile Ser Gly Leu Glu Ser65 70 75 80Glu Asp Phe Val
Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Phe 85 90 95Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Asn 100 105105446PRTArtificial
SequenceSynthetic hSIRP alpha.40A mouse heavy chain 105Glu Val Gln
Phe Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Thr1 5 10 15Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Ser Thr Phe Thr Ser Tyr 20 25 30Trp
Met His Trp Val Lys Gln Gly Pro Gly Gln Gly Leu Gln Trp Ile 35 40
45Gly Ala Ile Tyr Pro Val Asn Asn Asp Thr Thr Tyr Asn Gln Lys Phe
50 55 60Lys Gly Lys Ala Glu Leu Thr Val Val Thr Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Val Ser Ser Leu Thr Asn Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Thr Arg Ser Phe Tyr Tyr Ser Leu Asp Ala Ala Trp
Phe Val Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ala
Ala Lys Thr Thr Pro Pro 115 120 125Ser Val Tyr Pro Leu Ala Pro Gly
Ser Ala Ala Gln Thr Asn Ser Met 130 135 140Val Thr Leu Gly Cys Leu
Val Lys Gly Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Thr Trp
Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175Ala
Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185
190Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His
195 200 205Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg
Asp Cys 210 215 220Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val
Ser Ser Val Phe225 230 235 240Ile Phe Pro Pro Lys Pro Lys Asp Val
Leu Thr Ile Thr Leu Thr Pro 245 250 255Lys Val Thr Cys Val Val Val
Asp Ile Ser Lys Asp Asp Pro Glu Val 260 265 270Gln Phe Ser Trp Phe
Val Asp Asp Val Glu Val His Thr Ala Gln Thr 275 280 285Gln Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300Leu
Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys305 310
315 320Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
Ser 325 330 335Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr
Ile Pro Pro 340 345 350Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser
Leu Thr Cys Met Ile 355 360 365Thr Asp Phe Phe Pro Glu Asp Ile Thr
Val Glu Trp Gln Trp Asn Gly 370 375 380Gln Pro Ala Glu Asn Tyr Lys
Asn Thr Gln Pro Ile Met Asp Thr Asp385 390 395 400Gly Ser Tyr Phe
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 405 410 415Glu Ala
Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 420 425
430Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440
445106214PRTArtificial SequenceSynthetic hSIRP alpha.40A mouse
light chain 106Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Leu Gly1 5 10 15Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp
Ile Gly Ser Arg 20 25 30Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr
Ile Lys Arg Leu Ile 35 40 45Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val
Pro Lys Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Ser Asp Tyr Ser Leu
Thr Ile Ser Gly Leu Glu Ser65 70 75 80Glu Asp Phe Val Asp Tyr Tyr
Cys Leu Gln Tyr Ala Ser Ser Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Asn Arg Ala Asp Ala Ala 100 105 110Pro Thr Val Ser
Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125Gly Ala
Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135
140Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val
Leu145 150 155 160Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Met Ser 165 170 175Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr
Glu Arg His Asn Ser Tyr 180 185 190Thr Cys Glu Ala Thr His Lys Thr
Ser Thr Ser Pro Ile Val Lys Ser 195 200 205Phe Asn Arg Asn Glu Cys
210107581PRTArtificial SequenceSynthetic rhSIRP alpha/Fc 107Gly Val
Ala Gly Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser1 5 10 15Val
Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr 20 25
30Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
35 40 45Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg
Val 50 55 60Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe
Ser Ile65 70 75 80Arg Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr
Tyr Tyr Cys Val 85 90 95Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu
Phe Lys Ser Gly Ala 100 105 110Gly Thr Glu Leu Ser Val Arg Ala Lys
Pro Ser Ala Pro Val Val Ser 115 120 125Gly Pro Ala Ala Arg Ala Thr
Pro Gln His Thr Val Ser Phe Thr Cys 130 135 140Glu Ser His Gly Phe
Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys145 150 155 160Asn Gly
Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly 165 170
175Glu Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr
180 185 190Arg Glu Asp Val His Ser Gln Val Ile Cys Glu Val Ala His
Val Thr 195 200 205Leu Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu
Ser Glu Thr Ile 210 215 220Arg Val Pro Pro Thr Leu Glu Val Thr Gln
Gln Pro Val Arg Ala Glu225 230 235 240Asn Gln Val Asn Val Thr Cys
Gln Val Arg Lys Phe Tyr Pro Gln Arg 245 250 255Leu Gln Leu Thr Trp
Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr 260 265 270Ala Ser Thr
Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser 275 280 285Trp
Leu Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr 290 295
300Cys Gln Val Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His
Asp305 310 315 320Leu Lys Val Ser Ala His Pro Lys Glu Gln Gly Ser
Asn Thr Ala Ala 325 330 335Glu Asn Thr Gly Ser Asn Glu Arg Ile Glu
Gly Arg Met Asp Pro Lys 340 345 350Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu 355 360 365Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 370 375 380Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val385 390 395 400Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 405 410
415Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
420 425 430Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 435 440 445Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 450 455 460Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro465 470 475 480Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln 485 490 495Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 500 505 510Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 515 520 525Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 530 535
540Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser545 550 555 560Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 565 570 575Leu Ser Pro Gly Lys
580108538PRTArtificial SequenceSynthetic rhSIRPg/Fc 108Val Leu Trp
Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile Tyr Asn1 5 10 15Gln Lys
Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr 20 25 30Lys
Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile Thr Pro 35 40
45Ala Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro
50 55 60Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met Ala Leu
Gly65 70 75 80Ala Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala
Arg Thr Thr 85 90 95Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser His
Gly Phe Ser Pro 100 105 110Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn
Gly Asn Glu Leu Ser Asp 115 120 125Phe Gln Thr Asn Val Asp Pro Thr
Gly Gln Ser Val Ala Tyr Ser Ile 130 135 140Arg Ser Thr Ala Arg Val
Val Leu Asp Pro Trp
Asp Val Arg Ser Gln145 150 155 160Val Ile Cys Glu Val Ala His Val
Thr Leu Gln Gly Asp Pro Leu Arg 165 170 175Gly Thr Ala Asn Leu Ser
Glu Ala Ile Arg Val Pro Pro Thr Leu Glu 180 185 190Val Thr Gln Gln
Pro Met Arg Ala Gly Asn Gln Val Asn Val Thr Cys 195 200 205Gln Val
Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr Trp Leu Glu 210 215
220Asn Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu
Asn225 230 235 240Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu
Val Asn Ile Ser 245 250 255Asp Gln Arg Asp Asp Val Val Leu Thr Cys
Gln Val Lys His Asp Gly 260 265 270Gln Leu Ala Val Ser Lys Arg Leu
Ala Leu Glu Val Thr Val His Gln 275 280 285Lys Asp Gln Ser Ser Asp
Ala Thr Pro Gly Pro Ala Ser Ile Glu Gly 290 295 300Arg Met Asp Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys305 310 315 320Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 325 330
335Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
340 345 350Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp 355 360 365Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 370 375 380Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu385 390 395 400His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 405 410 415Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 420 425 430Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 435 440 445Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 450 455
460Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn465 470 475 480Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 485 490 495Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 500 505 510Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr 515 520 525Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 530 535109358PRTArtificial SequenceSynthetic
rhCD47/Fc 109Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr
Phe Cys Asn1 5 10 15Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met
Glu Ala Gln Asn 20 25 30Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys
Gly Arg Asp Ile Tyr 35 40 45Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr
Val Pro Thr Asp Phe Ser 50 55 60Ser Ala Lys Ile Glu Val Ser Gln Leu
Leu Lys Gly Asp Ala Ser Leu65 70 75 80Lys Met Asp Lys Ser Asp Ala
Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95Glu Val Thr Glu Leu Thr
Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110Tyr Arg Val Val
Ser Trp Phe Ser Pro Ile Glu Gly Arg Met Asp Pro 115 120 125Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 130 135
140Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp145 150 155 160Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp 165 170 175Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly 180 185 190Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn 195 200 205Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp 210 215 220Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro225 230 235 240Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 245 250
255Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
260 265 270Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 275 280 285Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr 290 295 300Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys305 310 315 320Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 325 330 335Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 340 345 350Ser Leu Ser
Pro Gly Lys 3551101197DNAArtificial SequenceSynthetic hSIRP-V?C1
beta C2 beta 110atgcccgtgc ctgcctcttg gcctcatctg cccagcccct
ttctgctgat gaccctgctg 60ctgggcaggc tgacaggcgt ggcaggcgaa gaggaactgc
agatgatcca gcccgagaag 120ctgctgctcg tgaccgtggg caagaccgcc
accctgcact gcaccgtgac atccctgctg 180cctgtgggac ccgtgctgtg
gtttagaggc gtgggccctg gcagagagct gatctacaac 240cagaaagagg
gccacttccc cagagtgacc accgtgtccg acctgaccaa gcggaacaac
300atggacttct ccatccggat ctccagcatc acccctgccg acgtgggcac
ctactactgc 360gtgaagttcc ggaagggctc ccccgagaac gtggagttca
agtctggccc aggcaccgag 420atggccctgg gcgctaaacc ttctgcccct
gtggtgtctg gacctgccgt gcgggctacc 480cctgagcaca ccgtgtcttt
tacctgcgag tcccacggct tcagccctcg ggacatcacc 540ctgaagtggt
tcaagaacgg caacgagctg tccgacttcc agaccaacgt ggaccctgcc
600ggcgactccg tgtcctactc catccactct accgccagag tggtgctgac
cagaggcgac 660gtgcactccc aagtgatctg cgagatcgcc catatcacac
tgcagggcga ccccctgaga 720ggcaccgcca atctgtctga ggccatcaga
gtgcccccca ccctggaagt gacccagcag 780cctatgagag ccgagaacca
ggccaacgtg acctgtcagg tgtccaactt ctaccctcgg 840ggcctgcagc
tgacctggct ggaaaacggc aatgtgtccc ggaccgagac agcctccacc
900ctgatcgaga acaaggacgg cacctacaat tggatgtcct ggctgctcgt
gaacacctgt 960gcccacaggg acgacgtggt gctgacatgc caggtggaac
acgatggcca gcaggccgtg 1020tccaagtcct acgccctgga aatctccgcc
catcagaaag agcacggctc cgatatcacc 1080cacgaggccg ctctggctcc
taccgctcct ctgctggtgg ctctgctgct gggacctaag 1140ctgctgctgg
tcgtgggcgt gtccgccatc tacatctgct ggaagcagaa ggcctga
1197111398PRTArtificial SequenceSynthetic hSIRP-V?C1 beta C2 beta
111Met Pro Val Pro Ala Ser Trp Pro His Leu Pro Ser Pro Phe Leu Leu1
5 10 15Met Thr Leu Leu Leu Gly Arg Leu Thr Gly Val Ala Gly Glu Glu
Glu 20 25 30Leu Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val
Gly Lys 35 40 45Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro
Val Gly Pro 50 55 60Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu
Leu Ile Tyr Asn65 70 75 80Gln Lys Glu Gly His Phe Pro Arg Val Thr
Thr Val Ser Asp Leu Thr 85 90 95Lys Arg Asn Asn Met Asp Phe Ser Ile
Arg Ile Ser Ser Ile Thr Pro 100 105 110Ala Asp Val Gly Thr Tyr Tyr
Cys Val Lys Phe Arg Lys Gly Ser Pro 115 120 125Glu Asn Val Glu Phe
Lys Ser Gly Pro Gly Thr Glu Met Ala Leu Gly 130 135 140Ala Lys Pro
Ser Ala Pro Val Val Ser Gly Pro Ala Val Arg Ala Thr145 150 155
160Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro
165 170 175Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu
Ser Asp 180 185 190Phe Gln Thr Asn Val Asp Pro Ala Gly Asp Ser Val
Ser Tyr Ser Ile 195 200 205His Ser Thr Ala Arg Val Val Leu Thr Arg
Gly Asp Val His Ser Gln 210 215 220Val Ile Cys Glu Ile Ala His Ile
Thr Leu Gln Gly Asp Pro Leu Arg225 230 235 240Gly Thr Ala Asn Leu
Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu 245 250 255Val Thr Gln
Gln Pro Met Arg Ala Glu Asn Gln Ala Asn Val Thr Cys 260 265 270Gln
Val Ser Asn Phe Tyr Pro Arg Gly Leu Gln Leu Thr Trp Leu Glu 275 280
285Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Leu Ile Glu Asn
290 295 300Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn
Thr Cys305 310 315 320Ala His Arg Asp Asp Val Val Leu Thr Cys Gln
Val Glu His Asp Gly 325 330 335Gln Gln Ala Val Ser Lys Ser Tyr Ala
Leu Glu Ile Ser Ala His Gln 340 345 350Lys Glu His Gly Ser Asp Ile
Thr His Glu Ala Ala Leu Ala Pro Thr 355 360 365Ala Pro Leu Leu Val
Ala Leu Leu Leu Gly Pro Lys Leu Leu Leu Val 370 375 380Val Gly Val
Ser Ala Ile Tyr Ile Cys Trp Lys Gln Lys Ala385 390
3951121197DNAArtificial SequenceSynthetic hSIRP-V beta C1?C2 beta
112atgcccgtgc ctgcctcttg gcctcatctg cccagcccct ttctgctgat
gaccctgctg 60ctgggcaggc tgacaggcgt ggcaggcgaa gatgagctgc aagtgatcca
gcccgagaag 120tccgtgtctg tggccgctgg cgagtctgcc accctgagat
gcgctatgac ctccctgatc 180cccgtgggcc ccatcatgtg gtttagaggc
gctggcgctg gcagagagct gatctacaac 240cagaaagagg gccacttccc
cagagtgacc accgtgtccg agctgaccaa gcggaacaac 300ctggacttct
ccatctccat cagcaacatc acccctgccg acgccggcac ctactactgc
360gtgaagttcc ggaagggctc ccccgacgac gtggagttca aatccggcgc
tggaaccgag 420ctgtccgtgc gggctaaacc ttctgcccct gtggtgctgg
gacctgccgc tagaaccacc 480cctgagcaca ccgtgtcttt tacctgcgag
tcccacggct tcagccctcg ggacatcacc 540ctgaagtggt tcaagaacgg
caacgagctg agcgacttcc agaccaacgt ggaccctacc 600ggccagtccg
tggcctactc catcagatcc accgccagag tggtgctgga cccttgggat
660gtgcggtccc aagtgatctg cgaggtggcc catgtgacac tgcagggcga
tcctctgaga 720ggcaccgcca atctgtctga ggccatcaga gtgcccccca
ccctggaagt gacccagcag 780cctatgagag ccgagaacca ggccaacgtg
acctgccagg tgtccaactt ctaccctcgg 840ggcctgcagc tgacctggct
ggaaaacggc aatgtgtccc ggaccgagac agcctccacc 900ctgatcgaga
acaaggatgg cacctacaat tggatgtcct ggctgctcgt gaacacctgt
960gcccaccggg atgacgtggt gctgacttgt caggtggaac acgacggcca
gcaggccgtg 1020tccaagtcct acgccctgga aatctccgcc catcagaaag
agcacggctc cgatatcacc 1080cacgaggccg ctctggctcc taccgctcct
ctgctggtgg ctctgctgct gggacctaag 1140ctgctgctgg tcgtgggcgt
gtccgccatc tacatctgct ggaagcagaa ggcctga 1197113398PRTArtificial
SequenceSynthetic hSIRP-V beta C1?C2 beta 113Met Pro Val Pro Ala
Ser Trp Pro His Leu Pro Ser Pro Phe Leu Leu1 5 10 15Met Thr Leu Leu
Leu Gly Arg Leu Thr Gly Val Ala Gly Glu Asp Glu 20 25 30Leu Gln Val
Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala Gly Glu 35 40 45Ser Ala
Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val Gly Pro 50 55 60Ile
Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile Tyr Asn65 70 75
80Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Leu Thr
85 90 95Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr
Pro 100 105 110Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
Gly Ser Pro 115 120 125Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr
Glu Leu Ser Val Arg 130 135 140Ala Lys Pro Ser Ala Pro Val Val Leu
Gly Pro Ala Ala Arg Thr Thr145 150 155 160Pro Glu His Thr Val Ser
Phe Thr Cys Glu Ser His Gly Phe Ser Pro 165 170 175Arg Asp Ile Thr
Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp 180 185 190Phe Gln
Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala Tyr Ser Ile 195 200
205Arg Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val Arg Ser Gln
210 215 220Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
Leu Arg225 230 235 240Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val
Pro Pro Thr Leu Glu 245 250 255Val Thr Gln Gln Pro Met Arg Ala Glu
Asn Gln Ala Asn Val Thr Cys 260 265 270Gln Val Ser Asn Phe Tyr Pro
Arg Gly Leu Gln Leu Thr Trp Leu Glu 275 280 285Asn Gly Asn Val Ser
Arg Thr Glu Thr Ala Ser Thr Leu Ile Glu Asn 290 295 300Lys Asp Gly
Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Thr Cys305 310 315
320Ala His Arg Asp Asp Val Val Leu Thr Cys Gln Val Glu His Asp Gly
325 330 335Gln Gln Ala Val Ser Lys Ser Tyr Ala Leu Glu Ile Ser Ala
His Gln 340 345 350Lys Glu His Gly Ser Asp Ile Thr His Glu Ala Ala
Leu Ala Pro Thr 355 360 365Ala Pro Leu Leu Val Ala Leu Leu Leu Gly
Pro Lys Leu Leu Leu Val 370 375 380Val Gly Val Ser Ala Ile Tyr Ile
Cys Trp Lys Gln Lys Ala385 390 3951141197DNAArtificial
SequenceSynthetic hSIRP-V beta C1 beta C2? 114atgcccgtgc ctgcctcttg
gcctcatctg cccagcccct ttctgctgat gaccctgctg 60ctgggcaggc tgacaggcgt
ggcaggcgaa gatgagctgc aagtgatcca gcccgagaag 120tccgtgtctg
tggccgctgg cgagtctgcc accctgagat gcgctatgac ctccctgatc
180cccgtgggcc ccatcatgtg gtttagaggc gctggcgctg gcagagagct
gatctacaac 240cagaaagagg gccacttccc cagagtgacc accgtgtccg
agctgaccaa gcggaacaac 300ctggacttct ccatctccat cagcaacatc
acccctgccg acgccggcac ctactactgc 360gtgaagttcc ggaagggctc
ccccgacgac gtggagttca aatccggcgc tggaaccgag 420ctgtccgtgc
gggctaaacc ttctgcccct gtggtgtctg gacctgctgt gcgcgctacc
480cctgagcaca ccgtgtcttt tacctgcgag tcccacggct tcagccctcg
ggacatcacc 540ctgaagtggt tcaagaacgg caacgagctg agcgacttcc
agaccaacgt ggaccctgcc 600ggcgactccg tgtcctactc catccactct
accgccagag tggtgctgac cagaggcgac 660gtgcactccc aagtgatctg
cgagatcgcc catatcacac tgcagggcga ccccctgaga 720ggcaccgcca
atctgtctga ggccatcaga gtgcccccca ccctggaagt gacccagcag
780cctatgagag tgggcaacca agtgaacgtg acctgccaag tgcggaagtt
ctacccccag 840tccctgcagc tgacttggag cgagaatggc aacgtgtgcc
agagagagac agcctccacc 900ctgaccgaga acaaggacgg aacctacaac
tggacctcct ggttcctcgt gaacatctcc 960gaccagcggg acgacgtggt
gctgacatgc caagtgaagc acgatggaca gctggccgtg 1020tccaagcggc
tggctctgga agtgacagtg caccagaaag agcacggctc cgacatcacc
1080cacgaggccg ctctggctcc tacagctcct ctgctggtgg ctctgctgct
gggacctaag 1140ctgctgctgg tcgtgggcgt gtccgccatc tacatctgct
ggaagcagaa ggcctga 1197115398PRTArtificial SequenceSynthetic
hSIRP-V beta C1 beta C2? 115Met Pro Val Pro Ala Ser Trp Pro His Leu
Pro Ser Pro Phe Leu Leu1 5 10 15Met Thr Leu Leu Leu Gly Arg Leu Thr
Gly Val Ala Gly Glu Asp Glu 20 25 30Leu Gln Val Ile Gln Pro Glu Lys
Ser Val Ser Val Ala Ala Gly Glu 35 40 45Ser Ala Thr Leu Arg Cys Ala
Met Thr Ser Leu Ile Pro Val Gly Pro 50 55 60Ile Met Trp Phe Arg Gly
Ala Gly Ala Gly Arg Glu Leu Ile Tyr Asn65 70 75 80Gln Lys Glu Gly
His Phe Pro Arg Val Thr Thr Val Ser Glu Leu Thr 85 90 95Lys Arg Asn
Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr Pro 100 105 110Ala
Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro 115 120
125Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg
130 135 140Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Val Arg
Ala Thr145 150 155 160Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser
His Gly Phe Ser Pro 165 170 175Arg Asp Ile Thr Leu Lys Trp Phe Lys
Asn Gly Asn Glu Leu Ser Asp 180 185 190Phe Gln Thr Asn Val Asp Pro
Ala Gly Asp Ser Val Ser Tyr Ser Ile 195 200 205His Ser Thr Ala Arg
Val Val Leu Thr Arg Gly Asp Val His Ser Gln 210 215 220Val Ile Cys
Glu Ile Ala His Ile Thr Leu Gln Gly Asp Pro Leu Arg225 230 235
240Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu
245 250 255Val Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn Val
Thr Cys 260 265 270Gln Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu
Thr Trp Ser Glu 275
280 285Asn Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu Thr Glu
Asn 290 295 300Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val
Asn Ile Ser305 310 315 320Asp Gln Arg Asp Asp Val Val Leu Thr Cys
Gln Val Lys His Asp Gly 325 330 335Gln Leu Ala Val Ser Lys Arg Leu
Ala Leu Glu Val Thr Val His Gln 340 345 350Lys Glu His Gly Ser Asp
Ile Thr His Glu Ala Ala Leu Ala Pro Thr 355 360 365Ala Pro Leu Leu
Val Ala Leu Leu Leu Gly Pro Lys Leu Leu Leu Val 370 375 380Val Gly
Val Ser Ala Ile Tyr Ile Cys Trp Lys Gln Lys Ala385 390
3951161194DNAHomo sapiensmisc_feature(1)..(1194)human SIRP beta L
116atgcctgtgc ctgcctcttg gcctcatctg ccctctccat ttctgctgat
gaccctgctg 60ctgggcagac tgacaggtgt tgctggcgaa gaggaactgc aagtgatcca
gcctgacaag 120agcatctctg tggccgctgg cgaatctgcc acactgcact
gtaccgtgac atctctgatc 180cctgtgggcc ccatccagtg gtttagaggt
gctggacctg gcagagagct gatctacaac 240cagaaagagg gacacttccc
cagagtgacc accgtgtccg acctgaccaa gcggaacaac 300atggacttca
gcatccggat cagcaacatc acccctgccg atgccggcac ctactactgc
360gtgaagttca gaaagggcag ccccgaccac gtcgagttta aaagcggagc
cggcacagag 420ctgagcgtgc gggctaaacc ttctgctcct gtggtgtctg
gaccagccgc tagagctaca 480cctcagcaca ccgtgtcttt tacctgcgag
agccacggct tcagccccag agatatcacc 540ctgaagtggt tcaagaacgg
caacgagctg tccgacttcc agaccaatgt ggacccagcc 600ggcgatagcg
tgtcctacag cattcacagc accgccaagg tggtgctgac ccgggaagat
660gtgcacagcc aagtgatttg cgaggtggcc cacgttaccc tgcaaggcga
tcctctgaga 720ggaaccgcca acctgagcga gacaatccgg gtgccaccta
cactggaagt gacccagcag 780cctgtgcggg ccgagaatca agtgaacgtg
acctgccaag tgcggaagtt ctaccctcag 840agactgcagc tgacctggct
ggaaaacggc aatgtgtccc ggaccgagac agccagcaca 900ctgaccgaga
acaaggatgg cacctacaat tggatgagct ggctgctggt caatgtgtct
960gcccaccggg acgatgtgaa gctgacatgc caggtggaac acgatggcca
gcctgccgtg 1020tctaagagcc acgacctgaa ggtgtccgct catcccaaag
agcagggcag caatactgcc 1080cctggacctg ctcttgcttc tgccgctcct
ctgctgatcg cctttctgct gggacctaag 1140gtgctgctgg ttgtgggagt
gtccgtgatc tacgtgtact ggaagcagaa ggcc 1194117398PRTHomo
sapiensmisc_feature(1)..(398)human SIRP beta L 117Met Pro Val Pro
Ala Ser Trp Pro His Leu Pro Ser Pro Phe Leu Leu1 5 10 15Met Thr Leu
Leu Leu Gly Arg Leu Thr Gly Val Ala Gly Glu Glu Glu 20 25 30Leu Gln
Val Ile Gln Pro Asp Lys Ser Ile Ser Val Ala Ala Gly Glu 35 40 45Ser
Ala Thr Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly Pro 50 55
60Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr Asn65
70 75 80Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu
Thr 85 90 95Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn Ile
Thr Pro 100 105 110Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg
Lys Gly Ser Pro 115 120 125Asp His Val Glu Phe Lys Ser Gly Ala Gly
Thr Glu Leu Ser Val Arg 130 135 140Ala Lys Pro Ser Ala Pro Val Val
Ser Gly Pro Ala Ala Arg Ala Thr145 150 155 160Pro Gln His Thr Val
Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro 165 170 175Arg Asp Ile
Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp 180 185 190Phe
Gln Thr Asn Val Asp Pro Ala Gly Asp Ser Val Ser Tyr Ser Ile 195 200
205His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln
210 215 220Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
Leu Arg225 230 235 240Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val
Pro Pro Thr Leu Glu 245 250 255Val Thr Gln Gln Pro Val Arg Ala Glu
Asn Gln Val Asn Val Thr Cys 260 265 270Gln Val Arg Lys Phe Tyr Pro
Gln Arg Leu Gln Leu Thr Trp Leu Glu 275 280 285Asn Gly Asn Val Ser
Arg Thr Glu Thr Ala Ser Thr Leu Thr Glu Asn 290 295 300Lys Asp Gly
Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser305 310 315
320Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly
325 330 335Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala
His Pro 340 345 350Lys Glu Gln Gly Ser Asn Thr Ala Pro Gly Pro Ala
Leu Ala Ser Ala 355 360 365Ala Pro Leu Leu Ile Ala Phe Leu Leu Gly
Pro Lys Val Leu Leu Val 370 375 380Val Gly Val Ser Val Ile Tyr Val
Tyr Trp Lys Gln Lys Ala385 390 395118990DNAHomo
sapiensmisc_feature(1)..(990)human IgG1 constant domains
118gccagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag
cacctctggg 60ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 120tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 180ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 240tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagaa agttgagccc 300aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
360ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 420gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
ctgaggtcaa gttcaactgg 480tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc cgcgggagga gcagtacaac 540agcacgtacc gggtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
660aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggatgag 720ctgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 780gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 840ctggactccg acggctcctt
cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
960cagaagagcc tctccctgtc tccgggtaaa 990119330PRTHomo
sapiensmisc_feature(1)..(330)human IgG1 constant domains 119Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
330120336PRTMus musculusmisc_feature(1)..(336)mouse IgG1 constant
domains 120Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly
Cys Gly1 5 10 15Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val
Lys Gly Tyr 20 25 30Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly
Ser Leu Ser Ser 35 40 45Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser
Gly Leu Tyr Thr Met 50 55 60Ser Ser Ser Val Thr Val Pro Ser Ser Thr
Trp Pro Ser Gln Thr Val65 70 75 80Thr Cys Ser Val Ala His Pro Ala
Ser Ser Thr Thr Val Asp Lys Lys 85 90 95Leu Glu Pro Ser Gly Pro Ile
Ser Thr Ile Asn Pro Cys Pro Pro Cys 100 105 110Lys Glu Cys His Lys
Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser 115 120 125Val Phe Ile
Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu 130 135 140Thr
Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro145 150
155 160Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr
Ala 165 170 175Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile
Arg Val Val 180 185 190Ser Thr Leu Pro Ile Gln His Gln Asp Trp Met
Ser Gly Lys Glu Phe 195 200 205Lys Cys Lys Val Asn Asn Lys Asp Leu
Pro Ser Pro Ile Glu Arg Thr 210 215 220Ile Ser Lys Ile Lys Gly Leu
Val Arg Ala Pro Gln Val Tyr Ile Leu225 230 235 240Pro Pro Pro Ala
Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys 245 250 255Leu Val
Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser 260 265
270Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp
275 280 285Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys
Thr Ser 290 295 300Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val
Arg His Glu Gly305 310 315 320Leu Lys Asn Tyr Tyr Leu Lys Lys Thr
Ile Ser Arg Ser Pro Gly Lys 325 330 335121107PRTMus
musculusmisc_feature(1)..(107)mouse kappa constant domain 121Arg
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu1 5 10
15Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu
Arg 35 40 45Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys
Asp Ser 50 55 60Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp
Glu Tyr Glu65 70 75 80Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His
Lys Thr Ser Thr Ser 85 90 95Pro Ile Val Lys Ser Phe Asn Arg Asn Glu
Cys 100 105122326PRTHomo sapiensmisc_feature(1)..(326)human IgG2
constant domains, V234A-G237A-P238S-H268A-V309L-A330S-P331S (Sigma)
mutantmisc_feature(257)..(257)X is A or S 122Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75
80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro 100 105 110Pro Ala Ala Ala Ser Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 130 135 140Val Ser Ala Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200
205Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile 245 250 255Xaa Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro Met Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305 310 315
320Ser Leu Ser Pro Gly Lys 325123330PRTHomo
sapiensmisc_feature(1)..(330)human IgG1 constant domains,
L234A-L235A mutant 123Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 330124990DNAHomo
sapiensmisc_feature(1)..(990)human IgG1 constant domains,
L234A-L235A-P329G mutant 124gctagcacaa agggccctag tgtgtttcct
ctggctccct cttccaaatc cacttctggt 60ggcactgctg ctctgggatg cctggtgaag
gattactttc ctgaacctgt gactgtctca 120tggaactctg gtgctctgac
ttctggtgtc cacactttcc ctgctgtgct gcagtctagt 180ggactgtact
ctctgtcatc tgtggtcact gtgccctctt catctctggg aacccagacc
240tacatttgta atgtgaacca caaaccatcc
aacactaaag tggacaaaaa agtggaaccc 300aaatcctgtg acaaaaccca
cacctgccca ccttgtccgg cgcctgaagc ggcgggagga 360ccttctgtgt
ttctgttccc ccccaaacca aaggataccc tgatgatctc gcgaacccct
420gaggtgacat gtgtggtggt ggatgtgtct catgaggacc ccgaagtcaa
atttaattgg 480tatgtcgacg gcgtcgaggt gcataatgcc aaaaccaagc
ctagagagga acagtacaat 540tcaacctaca gagtcgtcag tgtgctgact
gtgctgcatc aggattggct gaatggcaag 600gaatacaagt gtaaagtctc
aaacaaggcc ctgggagctc caattgagaa aacaatctca 660aaggccaaag
gacagcctag ggaaccccag gtctacaccc tgccaccttc gagagacgaa
720ctgaccaaaa accaggtgtc cctgacatgc ctggtcaaag gcttctaccc
ttctgacatt 780gctgtggagt gggagtcaaa tggacagcct gagaacaact
acaaaacaac cccccctgtg 840ctggattctg atggctcttt ctttctgtac
tccaaactga ctgtggacaa gtctagatgg 900cagcagggga atgtcttttc
ttgctctgtc atgcatgagg ctctgcataa ccactacact 960cagaaatccc
tgtctctgtc tcccgggaaa 990125330PRTHomo
sapiensmisc_feature(1)..(330)human IgG1 constant domains,
L234A-L235A-P329G mutant 125Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Gly Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 325 330126330PRTHomo
sapiensmisc_feature(1)..(330)human IgG1 constant domains, N297Q
mutant 126Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330127327PRTHomo sapiensmisc_feature(1)..(327)human IgG4
constant domains, S228P-N297Q mutant 127Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Gln Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215
220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu
Ser Leu Ser Leu Gly Lys 325128120PRTArtificial SequenceSynthetic
18D5 VH 128Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro
Gly Ser1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Trp Val His Trp Val Lys Gln Arg Pro Ile Gln Gly
Leu Glu Trp Ile 35 40 45Gly Asn Ile Asp Pro Ser Asp Ser Asp Thr His
Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Ser Leu Thr Val Asp Lys
Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Phe
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Val Arg Gly Gly Thr Gly Thr
Met Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ala 115 120129112PRTArtificial SequenceSynthetic 18D5 VL
129Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1
5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
Ser 20 25 30Tyr Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Phe Cys Phe Gln Gly 85 90 95Thr His Val Pro Tyr Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile Lys 100 105 110130113PRTArtificial
SequenceSynthetic KWAR23 VH 130Glu Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala
Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30Tyr Ile His Trp Val Gln Gln
Arg Thr Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Asp Pro Glu
Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe 50 55 60Gln Asp Lys Ala Thr
Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu His Leu
Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Trp Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105
110Ser131108PRTArtificial SequenceSynthetic KWAR23 VL 131Gln Ile
Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu
Lys Val Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser 20 25
30Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp
35 40 45Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser
Met Glu65 70 75 80Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp
Ser Ser Tyr Pro 85 90 95Arg Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
Lys 100 105132376PRTArtificial SequenceSynthetic rhSIRP alpha-HIS
132Met 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 His His His
His His His 370 375133107PRTArtificial SequenceSynthetic hSIRP
alphaV1 133Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu
Val Ala1 5 10 15Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser
Leu Ile Pro 20 25 30Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
Gly Arg Glu Leu 35 40 45Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg
Val Thr Thr Val Ser 50 55 60Asp Leu Thr Lys Arg Asn Asn Met Asp Phe
Ser Ile Arg Ile Gly Asn65 70 75 80Ile Thr Pro Ala Asp Ala Gly Thr
Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95Gly Ser Pro Asp Asp Val Glu
Phe Lys Ser Gly 100 105134106PRTArtificial SequenceSynthetic hSIRP
alphaV2 134Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser
Val Ala1 5 10 15Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser
Leu Ile Pro 20 25 30Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro
Ala Arg Glu Leu 35 40 45Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg
Val Thr Thr Val Ser 50 55 60Glu Ser Thr Lys Arg Glu Asn Met Asp Phe
Ser Ile Ser Ile Ser Asn65 70 75 80Ile Thr Pro Ala Asp Ala Gly Thr
Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95Gly Ser Pro Asp Thr Glu Phe
Lys Ser Gly 100 105135107PRTArtificial SequenceSynthetic hSIRP beta
1 135Glu Asp Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val
Ala1 5 10 15Ala Gly Glu Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu
Ile Pro 20 25 30Val Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly
Arg Glu Leu 35 40 45Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
Thr Thr Val Ser 50 55 60Glu Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser
Ile Ser Ile Ser Asn65 70 75 80Ile Thr Pro Ala Asp Ala Gly Thr Tyr
Tyr Cys Val Lys Phe Arg Lys 85 90 95Gly Ser Pro Asp Asp Val Glu Phe
Lys Ser Gly 100 10513617PRTArtificial SequenceSynthetic subsequence
from SEQ ID NO 78 136Ala Ile Tyr Pro Val Asn Ser Asp Thr Thr Tyr
Asn Gln Lys Phe Lys1 5 10 15Gly13717PRTArtificial SequenceSynthetic
subsequence from SEQ ID NO 80 137Ala Leu Tyr Pro Val Asn Ser Asp
Thr Thr Tyr Asn Gln Lys Phe Lys1 5 10 15Gly
* * * * *
References