U.S. patent application number 16/760899 was filed with the patent office on 2022-09-22 for cd47 antigen-binding molecules.
This patent application is currently assigned to Hummingbird Bioscience Holdings Pte. Ltd.. The applicant listed for this patent is Hummingbird Bioscience Holdings Pte. Ltd.. Invention is credited to Jerome Douglas Boyd-Kirkup, Peter Brauer, Siyu Guan, Piers Ingram, Konrad Paszkiewicz, Dipti Thakkar, Zhihao Wu.
Application Number | 20220298254 16/760899 |
Document ID | / |
Family ID | 1000006447135 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220298254 |
Kind Code |
A1 |
Boyd-Kirkup; Jerome Douglas ;
et al. |
September 22, 2022 |
CD47 ANTIGEN-BINDING MOLECULES
Abstract
CD47 antigen-binding molecules are disclosed. Also disclosed are
nucleic acids and expression vectors encoding, compositions
comprising, and methods using, the CD47 antigen-binding
molecules.
Inventors: |
Boyd-Kirkup; Jerome Douglas;
(Singapore, SG) ; Thakkar; Dipti; (Singapore,
SG) ; Ingram; Piers; (Singapore, SG) ; Wu;
Zhihao; (Singapore, SG) ; Paszkiewicz; Konrad;
(Singapore, SG) ; Brauer; Peter; (Singapore,
SG) ; Guan; Siyu; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hummingbird Bioscience Holdings Pte. Ltd. |
Singapore |
|
SG |
|
|
Assignee: |
Hummingbird Bioscience Holdings
Pte. Ltd.
Singapore
SG
|
Family ID: |
1000006447135 |
Appl. No.: |
16/760899 |
Filed: |
November 1, 2018 |
PCT Filed: |
November 1, 2018 |
PCT NO: |
PCT/EP2018/079931 |
371 Date: |
April 30, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/55 20130101;
C12N 15/62 20130101; C07K 2317/622 20130101; A61K 2039/505
20130101; C07K 2317/76 20130101; C07K 2317/24 20130101; C07K
16/2896 20130101; C12N 15/09 20130101; C07K 2317/565 20130101; A61P
35/00 20180101; C07K 2317/92 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 1, 2017 |
GB |
1718101.7 |
Dec 7, 2017 |
GB |
1720425.6 |
Dec 7, 2017 |
GB |
1720426.4 |
Claims
1.-39. (canceled)
40. An antigen-binding molecule which binds to CD47, comprising:
(i) a heavy chain variable (VH) region incorporating the following
CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:169
HC-CDR2 having the amino acid sequence of SEQ ID NO:170 HC-CDR3
having the amino acid sequence of SEQ ID NO:26; and (ii) a light
chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:171 LC-CDR2
having the amino acid sequence of SEQ ID NO:172 LC-CDR3 having the
amino acid sequence of SEQ ID NO:173.
41. The antigen-binding molecule according to claim 40, wherein the
antigen-binding molecule comprises: (a) (i) a heavy chain variable
(VH) region incorporating the following CDRs: HC-CDR1 having the
amino acid sequence of SEQ ID NO:137 HC-CDR2 having the amino acid
sequence of SEQ ID NO:138 HC-CDR3 having the amino acid sequence of
SEQ ID NO:26; and (ii) a light chain variable (VL) region
incorporating the following CDRs: LC-CDR1 having the amino acid
sequence of SEQ ID NO:32 LC-CDR2 having the amino acid sequence of
SEQ ID NO:33 LC-CDR3 having the amino acid sequence of SEQ ID
NO:34; or (b) (i) a heavy chain variable (VH) region incorporating
the following CDRs: HC-CDR1 having the amino acid sequence of SEQ
ID NO:24 HC-CDR2 having the amino acid sequence of SEQ ID NO:25
HC-CDR3 having the amino acid sequence of SEQ ID NO:26; and (ii) a
light chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:32 LC-CDR2
having the amino acid sequence of SEQ ID NO:33 LC-CDR3 having the
amino acid sequence of SEQ ID NO:34; or (c) (i) a heavy chain
variable (VH) region incorporating the following CDRs: HC-CDR1
having the amino acid sequence of SEQ ID NO:24 HC-CDR2 having the
amino acid sequence of SEQ ID NO:25 HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and (ii) a light chain variable (VL)
region incorporating the following CDRs: LC-CDR1 having the amino
acid sequence of SEQ ID NO:139 LC-CDR2 having the amino acid
sequence of SEQ ID NO:141 LC-CDR3 having the amino acid sequence of
SEQ ID NO:34; or (d) (i) a heavy chain variable (VH) region
incorporating the following CDRs: HC-CDR1 having the amino acid
sequence of SEQ ID NO:137 HC-CDR2 having the amino acid sequence of
SEQ ID NO:138 HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and (ii) a light chain variable (VL) region incorporating
the following CDRs: LC-CDR1 having the amino acid sequence of SEQ
ID NO:139 LC-CDR2 having the amino acid sequence of SEQ ID NO:141
LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or (e) (i)
a heavy chain variable (VH) region incorporating the following
CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:24
HC-CDR2 having the amino acid sequence of SEQ ID NO:25 HC-CDR3
having the amino acid sequence of SEQ ID NO:26; and (ii) a light
chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:140 LC-CDR2
having the amino acid sequence of SEQ ID NO:141 LC-CDR3 having the
amino acid sequence of SEQ ID NO:34; or (f) (i) a heavy chain
variable (VH) region incorporating the following CDRs: HC-CDR1
having the amino acid sequence of SEQ ID NO:137 HC-CDR2 having the
amino acid sequence of SEQ ID NO:138 HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and (ii) a light chain variable (VL)
region incorporating the following CDRs: LC-CDR1 having the amino
acid sequence of SEQ ID NO:140 LC-CDR2 having the amino acid
sequence of SEQ ID NO:141 LC-CDR3 having the amino acid sequence of
SEQ ID NO:34; or (g) (i) a heavy chain variable (VH) region
incorporating the following CDRs: HC-CDR1 having the amino acid
sequence of SEQ ID NO:137 HC-CDR2 having the amino acid sequence of
SEQ ID NO:138 HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and (ii) a light chain variable (VL) region incorporating
the following CDRs: LC-CDR1 having the amino acid sequence of SEQ
ID NO:32 LC-CDR2 having the amino acid sequence of SEQ ID NO:141
LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or (h) (i)
a heavy chain variable (VH) region incorporating the following
CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:137
HC-CDR2 having the amino acid sequence of SEQ ID NO:138 HC-CDR3
having the amino acid sequence of SEQ ID NO:26; and (ii) a light
chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:32 LC-CDR2
having the amino acid sequence of SEQ ID NO:33 LC-CDR3 having the
amino acid sequence of SEQ ID NO:142.
42. The antigen-binding molecule according to claim 40, wherein the
antigen-binding molecule comprises: a VH region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:132, 23, 39, 178, 127, 129, 130 or 131;
and a VL region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:128,
31, 44, 179, 133, 134, 135 or 136.
43. The antigen-binding molecule according to claim 40, wherein the
antigen-binding molecule comprises: (i) a VH region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:132; and a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:128; or (ii) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:23; and a VL
region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:31; or
(iii) a VH region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:39;
and a VL region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:44;
or (iv) a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:178; and a VL region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:179; or (v) a VH region comprising an amino acid sequence having
at least 70% sequence identity to the amino acid sequence of SEQ ID
NO:127; and a VL region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:128; or (vi) a VH region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:129; and a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:128; or (vii) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:130; and a VL region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:128; or (viii) a VH region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:131; and a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:128; or (ix) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:131; and a VL
region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:133; or
(x) a VH region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:132;
and a VL region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:133;
or (xi) a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:131; and a VL region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:134; or (xii) a VH region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:132; and a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:134; or (xiii) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:132; and a VL region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:135; or (xiv) a VH region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:132; and a VL region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:136.
44. The antigen-binding molecule according to claim 40, wherein the
antigen-binding molecule inhibits interaction between CD47 and
SIRP.alpha..
45. The antigen-binding molecule according to claim 40, wherein the
antigen-binding molecule is a multispecific antigen-binding
molecule, and further comprises an antigen-binding domain specific
for a target antigen other than CD47.
46. A nucleic acid, or a plurality of nucleic acids, encoding an
antigen-binding molecule which binds to CD47, comprising: (i) a
heavy chain variable (VH) region incorporating the following CDRs:
HC-CDR1 having the amino acid sequence of SEQ ID NO:169 HC-CDR2
having the amino acid sequence of SEQ ID NO:170 HC-CDR3 having the
amino acid sequence of SEQ ID NO:26; and (ii) a light chain
variable (VL) region incorporating the following CDRs: LC-CDR1
having the amino acid sequence of SEQ ID NO:171 LC-CDR2 having the
amino acid sequence of SEQ ID NO:172 LC-CDR3 having the amino acid
sequence of SEQ ID NO:173.
47. The nucleic acid or plurality of nucleic acids according to
claim 46, wherein the antigen-binding molecule comprises: (a) (i) a
heavy chain variable (VH) region incorporating the following CDRs:
HC-CDR1 having the amino acid sequence of SEQ ID NO:137 HC-CDR2
having the amino acid sequence of SEQ ID NO:138 HC-CDR3 having the
amino acid sequence of SEQ ID NO:26; and (ii) a light chain
variable (VL) region incorporating the following CDRs: LC-CDR1
having the amino acid sequence of SEQ ID NO:32 LC-CDR2 having the
amino acid sequence of SEQ ID NO:33 LC-CDR3 having the amino acid
sequence of SEQ ID NO:34; or (b) (i) a heavy chain variable (VH)
region incorporating the following CDRs: HC-CDR1 having the amino
acid sequence of SEQ ID NO:24 HC-CDR2 having the amino acid
sequence of SEQ ID NO:25 HC-CDR3 having the amino acid sequence of
SEQ ID NO:26; and (ii) a light chain variable (VL) region
incorporating the following CDRs: LC-CDR1 having the amino acid
sequence of SEQ ID NO:32 LC-CDR2 having the amino acid sequence of
SEQ ID NO:33 LC-CDR3 having the amino acid sequence of SEQ ID
NO:34; or (c) (i) a heavy chain variable (VH) region incorporating
the following CDRs: HC-CDR1 having the amino acid sequence of SEQ
ID NO:24 HC-CDR2 having the amino acid sequence of SEQ ID NO:25
HC-CDR3 having the amino acid sequence of SEQ ID NO:26; and (ii) a
light chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:139 LC-CDR2
having the amino acid sequence of SEQ ID NO:141 LC-CDR3 having the
amino acid sequence of SEQ ID NO:34; or (d) (i) a heavy chain
variable (VH) region incorporating the following CDRs: HC-CDR1
having the amino acid sequence of SEQ ID NO:137 HC-CDR2 having the
amino acid sequence of SEQ ID NO:138 HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and (ii) a light chain variable (VL)
region incorporating the following CDRs: LC-CDR1 having the amino
acid sequence of SEQ ID NO:139 LC-CDR2 having the amino acid
sequence of SEQ ID NO:141 LC-CDR3 having the amino acid sequence of
SEQ ID NO:34; or (e) (i) a heavy chain variable (VH) region
incorporating the following CDRs: HC-CDR1 having the amino acid
sequence of SEQ ID NO:24 HC-CDR2 having the amino acid sequence of
SEQ ID NO:25 HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and (ii) a light chain variable (VL) region incorporating
the following CDRs: LC-CDR1 having the amino acid sequence of SEQ
ID NO:140 LC-CDR2 having the amino acid sequence of SEQ ID NO:141
LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or (f) (i)
a heavy chain variable (VH) region incorporating the following
CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:137
HC-CDR2 having the amino acid sequence of SEQ ID NO:138 HC-CDR3
having the amino acid sequence of SEQ ID NO:26; and (ii) a light
chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:140 LC-CDR2
having the amino acid sequence of SEQ ID NO:141 LC-CDR3 having the
amino acid sequence of SEQ ID NO:34; or (g) (i) a heavy chain
variable (VH) region incorporating the following CDRs: HC-CDR1
having the amino acid sequence of SEQ ID NO:137 HC-CDR2 having the
amino acid sequence of SEQ ID NO:138 HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and (ii) a light chain variable (VL)
region incorporating the following CDRs: LC-CDR1 having the amino
acid sequence of SEQ ID NO:32 LC-CDR2 having the amino acid
sequence of SEQ ID NO:141 LC-CDR3 having the amino acid sequence of
SEQ ID NO:34; or (h) (i) a heavy chain variable (VH) region
incorporating the following CDRs: HC-CDR1 having the amino acid
sequence of SEQ ID NO:137 HC-CDR2 having the amino acid sequence of
SEQ ID NO:138 HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and (ii) a light chain variable (VL) region incorporating
the following CDRs: LC-CDR1 having the amino acid sequence of SEQ
ID NO:32 LC-CDR2 having the amino acid sequence of SEQ ID NO:33
LC-CDR3 having the amino acid sequence of SEQ ID NO:142.
48. The nucleic acid or plurality of nucleic acids according to
claim 46, wherein the antigen-binding molecule comprises: a VH
region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:132, 23,
39, 178, 127, 129, 130 or 131; and a VL region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:128, 31, 44, 179, 133, 134, 135 or
136
49. The nucleic acid or plurality of nucleic acids according to
claim 46, wherein the antigen-binding molecule comprises: (i) a VH
region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:132; and
a VL region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:128; or
(ii) a VH region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:23;
and a VL region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:31;
or (iii) a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:39; and a VL region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:44; or (iv) a VH region comprising an amino acid sequence having
at least 70% sequence identity to the amino acid sequence of SEQ ID
NO:178; and a VL region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:179; or (v) a VH region comprising an amino acid sequence having
at least 70% sequence identity to the amino acid sequence of SEQ ID
NO:127; and a VL region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:128; or (vi) a VH region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:129; and a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:128; or (vii) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:130; and a VL region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:128; or (viii) a VH region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:131; and a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:128; or (ix) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:131; and a VL
region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:133; or
(x) a VH region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:132;
and a VL region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:133;
or (xi) a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:131; and a VL region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:134; or (xii) a VH region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:132; and a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:134; or (xiii) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:132; and a VL region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:135; or (xiv) a VH region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:132; and a VL region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:136.
50. The nucleic acid or plurality of nucleic acids according to
claim 46, wherein the antigen-binding molecule inhibits interaction
between CD47 and SIRP.alpha..
51. The nucleic acid or plurality of nucleic acids according to
claim 46, wherein the antigen-binding molecule is a multispecific
antigen-binding molecule, and further comprises an antigen-binding
domain specific for a target antigen other than CD47.
52. A method of treating or preventing a cancer, comprising
administering to a subject a therapeutically or prophylactically
effective amount of an antigen-binding molecule which binds to
CD47, comprising: (i) a heavy chain variable (VH) region
incorporating the following CDRs: HC-CDR1 having the amino acid
sequence of SEQ ID NO:169 HC-CDR2 having the amino acid sequence of
SEQ ID NO:170 HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and (ii) a light chain variable (VL) region incorporating
the following CDRs: LC-CDR1 having the amino acid sequence of SEQ
ID NO:171 LC-CDR2 having the amino acid sequence of SEQ ID NO:172
LC-CDR3 having the amino acid sequence of SEQ ID NO:173.
53. The method according to claim 52, wherein the antigen-binding
molecule comprises: (a) (i) a heavy chain variable (VH) region
incorporating the following CDRs: HC-CDR1 having the amino acid
sequence of SEQ ID NO:137 HC-CDR2 having the amino acid sequence of
SEQ ID NO:138 HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and (ii) a light chain variable (VL) region incorporating
the following CDRs: LC-CDR1 having the amino acid sequence of SEQ
ID NO:32 LC-CDR2 having the amino acid sequence of SEQ ID NO:33
LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or (b) (i)
a heavy chain variable (VH) region incorporating the following
CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:24
HC-CDR2 having the amino acid sequence of SEQ ID NO:25 HC-CDR3
having the amino acid sequence of SEQ ID NO:26; and (ii) a light
chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:32 LC-CDR2
having the amino acid sequence of SEQ ID NO:33 LC-CDR3 having the
amino acid sequence of SEQ ID NO:34; or (c) (i) a heavy chain
variable (VH) region incorporating the following CDRs: HC-CDR1
having the amino acid sequence of SEQ ID NO:24 HC-CDR2 having the
amino acid sequence of SEQ ID NO:25 HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and (ii) a light chain variable (VL)
region incorporating the following CDRs: LC-CDR1 having the amino
acid sequence of SEQ ID NO:139 LC-CDR2 having the amino acid
sequence of SEQ ID NO:141 LC-CDR3 having the amino acid sequence of
SEQ ID NO:34; or (d) (i) a heavy chain variable (VH) region
incorporating the following CDRs: HC-CDR1 having the amino acid
sequence of SEQ ID NO:137 HC-CDR2 having the amino acid sequence of
SEQ ID NO:138 HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and (ii) a light chain variable (VL) region incorporating
the following CDRs: LC-CDR1 having the amino acid sequence of SEQ
ID NO:139 LC-CDR2 having the amino acid sequence of SEQ ID NO:141
LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or (e) (i)
a heavy chain variable (VH) region incorporating the following
CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:24
HC-CDR2 having the amino acid sequence of SEQ ID NO:25 HC-CDR3
having the amino acid sequence of SEQ ID NO:26; and (ii) a light
chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:140 LC-CDR2
having the amino acid sequence of SEQ ID NO:141 LC-CDR3 having the
amino acid sequence of SEQ ID NO:34; or (f) (i) a heavy chain
variable (VH) region incorporating the following CDRs: HC-CDR1
having the amino acid sequence of SEQ ID NO:137 HC-CDR2 having the
amino acid sequence of SEQ ID NO:138 HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and (ii) a light chain variable (VL)
region incorporating the following CDRs: LC-CDR1 having the amino
acid sequence of SEQ ID NO:140 LC-CDR2 having the amino acid
sequence of SEQ ID NO:141 LC-CDR3 having the amino acid sequence of
SEQ ID NO:34; or (g) (i) a heavy chain variable (VH) region
incorporating the following CDRs: HC-CDR1 having the amino acid
sequence of SEQ ID NO:137 HC-CDR2 having the amino acid sequence of
SEQ ID NO:138 HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and (ii) a light chain variable (VL) region incorporating
the following CDRs: LC-CDR1 having the amino acid sequence of SEQ
ID NO:32 LC-CDR2 having the amino acid sequence of SEQ ID NO:141
LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or (h) (i)
a heavy chain variable (VH) region incorporating the following
CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:137
HC-CDR2 having the amino acid sequence of SEQ ID NO:138 HC-CDR3
having the amino acid sequence of SEQ ID NO:26; and (ii) a light
chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NO:32 LC-CDR2
having the amino acid sequence of SEQ ID NO:33 LC-CDR3 having the
amino acid sequence of SEQ ID NO:142.
54. The method according to claim 52, wherein the antigen-binding
molecule comprises: a VH region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:132, 23, 39, 178, 127, 129, 130 or 131; and a VL region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:128, 31, 44, 179,
133, 134, 135 or 136.
55. The method according to claim 52, wherein the antigen-binding
molecule comprises: (i) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:132; and a VL region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:128; or (ii) a VH region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:23; and a VL region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:31; or (iii) a VH region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:39; and a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:44; or (iv) a VH region comprising
an amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:178; and a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:179; or (v) a VH region comprising
an amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:127; and a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:128; or (vi) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:129; and a VL
region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:128; or
(vii) a VH region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:130;
and a VL region comprising an amino acid sequence having at least
70% sequence identity to the amino acid sequence of SEQ ID NO:128;
or (viii) a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:131; and a VL region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:128; or (ix) a VH region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:131; and a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:133; or (x) a VH region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:132; and a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:133; or (xi) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:131; and a VL region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:134; or (xii) a VH region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:132; and a VL region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:134; or (xiii) a VH region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:132; and a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:135; or (xiv) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:132; and a VL
region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:136.
56. The method according to claim 52, wherein the antigen-binding
molecule inhibits interaction between CD47 and SIRP.alpha..
57. The method according to claim 52, wherein the antigen-binding
molecule is a multispecific antigen-binding molecule, and further
comprises an antigen-binding domain specific for a target antigen
other than CD47.
58. The method according to claim 52, wherein the cancer is
selected from: a hematologic malignancy, a myeloid hematologic
malignancy, a lymphoblastic hematologic malignancy, myelodysplastic
syndrome (MDS), acute myeloid leukemia (AML), chronic myeloid
leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's
lymphoma (NHL), multiple myeloma (MM), bladder cancer, brain
cancer, glioblastoma, ovarian cancer, breast cancer, colon cancer,
liver cancer, hepatocellular carcinoma, prostate cancer, lung
cancer, Non-small Cell Lung Cancer (NSCLC), skin cancer and
melanoma.
Description
RELATED APPLICATIONS
[0001] This application is a national stage filing under 35 U.S.C.
371 of International Patent Application Serial No.
PCT/EP2018/079931, filed Nov. 1, 2018, which claims priority to
British Application No. GB 1718101.7, filed Nov. 1, 2017, British
Application No. GB 1720425.6, filed Dec. 7, 2017 and British
Application No. GB 1720426.4, filed Dec. 7, 2017, the contents and
elements of each of which are herein incorporated by reference for
all purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 6, 2022, is named H096970002US00-SUBSEQ-AZW and is 193,533
bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to the fields of molecular
biology, more specifically antibody technology. The present
invention also relates to methods of medical treatment and
prophylaxis.
BACKGROUND TO THE INVENTION
[0004] CD47 is the "don't-eat-me" signal and is ubiquitously
expressed on normal cells where binding to SIRP.alpha. on
macrophages inhibits phagocytosis. CD47 is commonly over-expressed
in tumors where it correlates with immune evasion and poor
prognosis. Blocking CD47-SIRPalpha interaction restores macrophage
phagocytosis of tumor cells and anti-CD47 mAbs have shown
anti-tumor efficacy in mouse models of solid tumors and
hematological malignancies.
[0005] WO 2014/087248 A2 discloses monospecific anti-CD47
antibodies having an affinity for human CD47 as high as .about.23.6
nM. The high-affinity CD47 antibodies disclosed therein induce
substantial hemagglutination (see e.g. Example 8 of WO 2014/087248
A2).
SUMMARY OF THE INVENTION
[0006] In a first aspect, the present invention provides an
antigen-binding molecule, optionally isolated, which is capable of
binding to CD47.
[0007] Also provided is an antigen-binding molecule, optionally
isolated, which is capable of binding to CD47 in extracellular
region 1.
[0008] In some embodiments the antigen-binding molecule is capable
of binding to the V-type Ig-like domain of CD47. In some
embodiments the antigen-binding molecule is capable of binding to a
polypeptide comprising or consisting of the amino acid sequence
shown in SEQ ID NO:9. In some embodiments the antigen-binding
molecule is capable of inhibiting interaction between CD47 and
SIRP.alpha.. In some embodiments the antigen-binding molecule is
capable of increasing phagocytosis of CD47-expressing cells.
[0009] In some embodiments the antigen-binding molecule is capable
of binding to a peptide or polypeptide comprising or consisting of
the amino acid sequence of SEQ ID NO:21. In some embodiments the
antigen-binding molecule comprises: [0010] (i) a heavy chain
variable (VH) region incorporating the following CDRs: [0011]
HC-CDR1 having the amino acid sequence of SEQ ID NO:169 [0012]
HC-CDR2 having the amino acid sequence of SEQ ID NO:170 [0013]
HC-CDR3 having the amino acid sequence of SEQ ID NO:26, [0014] or a
variant thereof in which one or two or three amino acids in one or
more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another
amino acid; and [0015] (ii) a light chain variable (VL) region
incorporating the following CDRs: [0016] LC-CDR1 having the amino
acid sequence of SEQ ID NO:171 [0017] LC-CDR2 having the amino acid
sequence of SEQ ID NO:172 [0018] LC-CDR3 having the amino acid
sequence of SEQ ID NO:173; or a variant thereof in which one or two
or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3
are substituted with another amino acid.
[0019] In some embodiments the antigen-binding molecule
comprises:
(a) [0020] (i) a heavy chain variable (VH) region incorporating the
following CDRs: [0021] HC-CDR1 having the amino acid sequence of
SEQ ID NO:24 [0022] HC-CDR2 having the amino acid sequence of SEQ
ID NO:25 [0023] HC-CDR3 having the amino acid sequence of SEQ ID
NO:26, [0024] or a variant thereof in which one or two or three
amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are
substituted with another amino acid; and [0025] (ii) a light chain
variable (VL) region incorporating the following CDRs: [0026]
LC-CDR1 having the amino acid sequence of SEQ ID NO:32 [0027]
LC-CDR2 having the amino acid sequence of SEQ ID NO:33 [0028]
LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or a
variant thereof in which one or two or three amino acids in one or
more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another
amino acid; or (b) [0029] (i) a heavy chain variable (VH) region
incorporating the following CDRs: [0030] HC-CDR1 having the amino
acid sequence of SEQ ID NO:137 [0031] HC-CDR2 having the amino acid
sequence of SEQ ID NO:138 [0032] HC-CDR3 having the amino acid
sequence of SEQ ID NO:26, [0033] or a variant thereof in which one
or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or
HC-CDR3 are substituted with another amino acid; and [0034] (ii) a
light chain variable (VL) region incorporating the following CDRs:
[0035] LC-CDR1 having the amino acid sequence of SEQ ID NO:32
[0036] LC-CDR2 having the amino acid sequence of SEQ ID NO:33
[0037] LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or a
variant thereof in which one or two or three amino acids in one or
more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another
amino acid; or (c) [0038] (i) a heavy chain variable (VH) region
incorporating the following CDRs: [0039] HC-CDR1 having the amino
acid sequence of SEQ ID NO:24 [0040] HC-CDR2 having the amino acid
sequence of SEQ ID NO:25 [0041] HC-CDR3 having the amino acid
sequence of SEQ ID NO:26, [0042] or a variant thereof in which one
or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or
HC-CDR3 are substituted with another amino acid; and [0043] (ii) a
light chain variable (VL) region incorporating the following CDRs:
[0044] LC-CDR1 having the amino acid sequence of SEQ ID NO:139
[0045] LC-CDR2 having the amino acid sequence of SEQ ID NO:141
[0046] LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
[0047] or a variant thereof in which one or two or three amino
acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted
with another amino acid; or (d) [0048] (i) a heavy chain variable
(VH) region incorporating the following CDRs: [0049] HC-CDR1 having
the amino acid sequence of SEQ ID NO:137 [0050] HC-CDR2 having the
amino acid sequence of SEQ ID NO:138 [0051] HC-CDR3 having the
amino acid sequence of SEQ ID NO:26, [0052] or a variant thereof in
which one or two or three amino acids in one or more of HC-CDR1,
HC-CDR2, or HC-CDR3 are substituted with another amino acid; and
[0053] (ii) a light chain variable (VL) region incorporating the
following CDRs: [0054] LC-CDR1 having the amino acid sequence of
SEQ ID NO:139 [0055] LC-CDR2 having the amino acid sequence of SEQ
ID NO:141 [0056] LC-CDR3 having the amino acid sequence of SEQ ID
NO:34;
[0057] or a variant thereof in which one or two or three amino
acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted
with another amino acid;
or (e) [0058] (i) a heavy chain variable (VH) region incorporating
the following CDRs: [0059] HC-CDR1 having the amino acid sequence
of SEQ ID NO:24 [0060] HC-CDR2 having the amino acid sequence of
SEQ ID NO:25 [0061] HC-CDR3 having the amino acid sequence of SEQ
ID NO:26, [0062] or a variant thereof in which one or two or three
amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are
substituted with another amino acid; and [0063] (ii) a light chain
variable (VL) region incorporating the following CDRs: [0064]
LC-CDR1 having the amino acid sequence of SEQ ID NO:140 [0065]
LC-CDR2 having the amino acid sequence of SEQ ID NO:141 [0066]
LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
[0067] or a variant thereof in which one or two or three amino
acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted
with another amino acid;
or (f) [0068] (i) a heavy chain variable (VH) region incorporating
the following CDRs: [0069] HC-CDR1 having the amino acid sequence
of SEQ ID NO:137 [0070] HC-CDR2 having the amino acid sequence of
SEQ ID NO:138 [0071] HC-CDR3 having the amino acid sequence of SEQ
ID NO:26, [0072] or a variant thereof in which one or two or three
amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are
substituted with another amino acid; and [0073] (ii) a light chain
variable (VL) region incorporating the following CDRs: [0074]
LC-CDR1 having the amino acid sequence of SEQ ID NO:140 [0075]
LC-CDR2 having the amino acid sequence of SEQ ID NO:141 [0076]
LC-CDR3 having the amino acid sequence of SEQ ID NO:34; [0077] or a
variant thereof in which one or two or three amino acids in one or
more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another
amino acid; or (g) [0078] (i) a heavy chain variable (VH) region
incorporating the following CDRs: [0079] HC-CDR1 having the amino
acid sequence of SEQ ID NO:137 [0080] HC-CDR2 having the amino acid
sequence of SEQ ID NO:138 [0081] HC-CDR3 having the amino acid
sequence of SEQ ID NO:26, [0082] or a variant thereof in which one
or two or three amino acids in one or more of HC-CDR1, HC-CDR2, or
HC-CDR3 are substituted with another amino acid; and [0083] (ii) a
light chain variable (VL) region incorporating the following CDRs:
[0084] LC-CDR1 having the amino acid sequence of SEQ ID NO:32
[0085] LC-CDR2 having the amino acid sequence of SEQ ID NO:141
[0086] LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
[0087] or a variant thereof in which one or two or three amino
acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted
with another amino acid; or (h) [0088] (i) a heavy chain variable
(VH) region incorporating the following CDRs: [0089] HC-CDR1 having
the amino acid sequence of SEQ ID NO:137 [0090] HC-CDR2 having the
amino acid sequence of SEQ ID NO:138 [0091] HC-CDR3 having the
amino acid sequence of SEQ ID NO:26, [0092] or a variant thereof in
which one or two or three amino acids in one or more of HC-CDR1,
HC-CDR2, or HC-CDR3 are substituted with another amino acid; and
[0093] (ii) a light chain variable (VL) region incorporating the
following CDRs: [0094] LC-CDR1 having the amino acid sequence of
SEQ ID NO:32 [0095] LC-CDR2 having the amino acid sequence of SEQ
ID NO:33 [0096] LC-CDR3 having the amino acid sequence of SEQ ID
NO:142; [0097] or a variant thereof in which one or two or three
amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are
substituted with another amino acid.
[0098] In some embodiments the antigen-binding molecule
comprises:
(a) [0099] (i) a heavy chain variable (VH) region incorporating the
following CDRs: [0100] HC-CDR1 having the amino acid sequence of
SEQ ID NO:24 [0101] HC-CDR2 having the amino acid sequence of SEQ
ID NO:25 [0102] HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and [0103] (ii) a light chain variable (VL) region
incorporating the following CDRs: [0104] LC-CDR1 having the amino
acid sequence of SEQ ID NO:32 [0105] LC-CDR2 having the amino acid
sequence of SEQ ID NO:33 LC-CDR3 having the amino acid sequence of
SEQ ID NO:34; or (b) [0106] (i) a heavy chain variable (VH) region
incorporating the following CDRs: [0107] HC-CDR1 having the amino
acid sequence of SEQ ID NO:137 [0108] HC-CDR2 having the amino acid
sequence of SEQ ID NO:138 [0109] HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and [0110] (ii) a light chain variable
(VL) region incorporating the following CDRs: [0111] LC-CDR1 having
the amino acid sequence of SEQ ID NO:32 [0112] LC-CDR2 having the
amino acid sequence of SEQ ID NO:33 [0113] LC-CDR3 having the amino
acid sequence of SEQ ID NO:34; or (c) [0114] (i) a heavy chain
variable (VH) region incorporating the following CDRs: [0115]
HC-CDR1 having the amino acid sequence of SEQ ID NO:24 [0116]
HC-CDR2 having the amino acid sequence of SEQ ID NO:25 [0117]
HC-CDR3 having the amino acid sequence of SEQ ID NO:26; and [0118]
(ii) a light chain variable (VL) region incorporating the following
CDRs: [0119] LC-CDR1 having the amino acid sequence of SEQ ID
NO:139 [0120] LC-CDR2 having the amino acid sequence of SEQ ID
NO:141 [0121] LC-CDR3 having the amino acid sequence of SEQ ID
NO:34; or (d) [0122] (i) a heavy chain variable (VH) region
incorporating the following CDRs: [0123] HC-CDR1 having the amino
acid sequence of SEQ ID NO:137 [0124] HC-CDR2 having the amino acid
sequence of SEQ ID NO:138 [0125] HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and [0126] (ii) a light chain variable
(VL) region incorporating the following CDRs: [0127] LC-CDR1 having
the amino acid sequence of SEQ ID NO:139 [0128] LC-CDR2 having the
amino acid sequence of SEQ ID NO:141 [0129] LC-CDR3 having the
amino acid sequence of SEQ ID NO:34; or (e) [0130] (i) a heavy
chain variable (VH) region incorporating the following CDRs: [0131]
HC-CDR1 having the amino acid sequence of SEQ ID NO:24 [0132]
HC-CDR2 having the amino acid sequence of SEQ ID NO:25 [0133]
HC-CDR3 having the amino acid sequence of SEQ ID NO:26; and [0134]
(ii) a light chain variable (VL) region incorporating the following
CDRs: [0135] LC-CDR1 having the amino acid sequence of SEQ ID
NO:140 [0136] LC-CDR2 having the amino acid sequence of SEQ ID
NO:141 [0137] LC-CDR3 having the amino acid sequence of SEQ ID
NO:34; or (f) [0138] (i) a heavy chain variable (VH) region
incorporating the following CDRs: [0139] HC-CDR1 having the amino
acid sequence of SEQ ID NO:137 [0140] HC-CDR2 having the amino acid
sequence of SEQ ID NO:138 [0141] HC-CDR3 having the amino acid
sequence of SEQ ID NO:26; and [0142] (ii) a light chain variable
(VL) region incorporating the following CDRs: [0143] LC-CDR1 having
the amino acid sequence of SEQ ID NO:140 [0144] LC-CDR2 having the
amino acid sequence of SEQ ID NO:141 [0145] LC-CDR3 having the
amino acid sequence of SEQ ID NO:34; or (g) [0146] (i) a heavy
chain variable (VH) region incorporating the following CDRs: [0147]
HC-CDR1 having the amino acid sequence of SEQ ID NO:137 [0148]
HC-CDR2 having the amino acid sequence of SEQ ID NO:138 [0149]
HC-CDR3 having the amino acid sequence of SEQ ID NO:26; and [0150]
(ii) a light chain variable (VL) region incorporating the following
CDRs: [0151] LC-CDR1 having the amino acid sequence of SEQ ID NO:32
[0152] LC-CDR2 having the amino acid sequence of SEQ ID NO:141
[0153] LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or
(h) [0154] (i) a heavy chain variable (VH) region incorporating the
following CDRs: [0155] HC-CDR1 having the amino acid sequence of
SEQ ID NO:137 [0156] HC-CDR2 having the amino acid sequence of SEQ
ID NO:138 [0157] HC-CDR3 having the amino acid sequence of SEQ ID
NO:26; and [0158] (ii) a light chain variable (VL) region
incorporating the following CDRs: [0159] LC-CDR1 having the amino
acid sequence of SEQ ID NO:32 [0160] LC-CDR2 having the amino acid
sequence of SEQ ID NO:33 [0161] LC-CDR3 having the amino acid
sequence of SEQ ID NO:142.
[0162] In some embodiments the antigen-binding molecule comprises:
[0163] a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:23, 39, 178, 127, 129, 130, 131 or 132; and [0164] a VL region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:31, 44, 179, 128,
133, 134, 135 or 136.
[0165] In some embodiments the antigen-binding molecule comprises:
[0166] (i) a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:23; and [0167] a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:31; or [0168] (ii) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:39; and [0169] a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:44; or [0170] (iii) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:178; and [0171] a
VL region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:179; or
[0172] (iv) a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:127; and [0173] a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:128; or [0174] (v) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:129; and [0175] a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:128; or [0176] (vi) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:130; and [0177] a
VL region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:128; or
[0178] (vii) a VH region comprising an amino acid sequence having
at least 70% sequence identity to the amino acid sequence of SEQ ID
NO:131; and [0179] a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:128; or [0180] (viii) a VH region comprising an amino
acid sequence having at least 70% sequence identity to the amino
acid sequence of SEQ ID NO:132; and [0181] a VL region comprising
an amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:128; or [0182] (ix) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:131; and [0183] a
VL region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:133; or
[0184] (x) a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:132; and [0185] a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:133; or [0186] (xi) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:131; and [0187] a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:134; or [0188] (xii) a VH region
comprising an amino acid sequence having at least 70% sequence
identity to the amino acid sequence of SEQ ID NO:132; and [0189] a
VL region comprising an amino acid sequence having at least 70%
sequence identity to the amino acid sequence of SEQ ID NO:134; or
[0190] (xiii) a VH region comprising an amino acid sequence having
at least 70% sequence identity to the amino acid sequence of SEQ ID
NO:132; and [0191] a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:135; or [0192] (xiv) a VH region comprising an amino acid
sequence having at least 70% sequence identity to the amino acid
sequence of SEQ ID NO:132; and [0193] a VL region comprising an
amino acid sequence having at least 70% sequence identity to the
amino acid sequence of SEQ ID NO:136.
[0194] In some embodiments the antigen-binding molecule is capable
of binding to a peptide or polypeptide comprising or consisting of
the amino acid sequence of SEQ ID NO:22.
[0195] In some embodiments the antigen-binding molecule comprises:
[0196] (i) a heavy chain variable (VH) region incorporating the
following CDRs: [0197] HC-CDR1 having the amino acid sequence of
SEQ ID NO:50 [0198] HC-CDR2 having the amino acid sequence of SEQ
ID NO:51 [0199] HC-CDR3 having the amino acid sequence of SEQ ID
NO:52, [0200] or a variant thereof in which one or two or three
amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are
substituted with another amino acid; and [0201] (ii) a light chain
variable (VL) region incorporating the following CDRs: [0202]
LC-CDR1 having the amino acid sequence of SEQ ID NO:58 [0203]
LC-CDR2 having the amino acid sequence of SEQ ID NO:59 [0204]
LC-CDR3 having the amino acid sequence of SEQ ID NO:60; [0205] or a
variant thereof in which one or two or three amino acids in one or
more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another
amino acid.
[0206] In some embodiments the antigen-binding molecule comprises:
[0207] (i) a heavy chain variable (VH) region incorporating the
following CDRs: [0208] HC-CDR1 having the amino acid sequence of
SEQ ID NO:50 [0209] HC-CDR2 having the amino acid sequence of SEQ
ID NO:51 [0210] HC-CDR3 having the amino acid sequence of SEQ ID
NO:52; and [0211] (ii) a light chain variable (VL) region
incorporating the following CDRs: [0212] LC-CDR1 having the amino
acid sequence of SEQ ID NO:58 [0213] LC-CDR2 having the amino acid
sequence of SEQ ID NO:59 [0214] LC-CDR3 having the amino acid
sequence of SEQ ID NO:60.
[0215] In some embodiments the antigen-binding molecule comprises:
[0216] a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:49; and [0217] a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:57.
[0218] In some embodiments the antigen-binding molecule comprises:
[0219] (i) a heavy chain variable (VH) region incorporating the
following CDRs: [0220] HC-CDR1 having the amino acid sequence of
SEQ ID NO:66 [0221] HC-CDR2 having the amino acid sequence of SEQ
ID NO:67 [0222] HC-CDR3 having the amino acid sequence of SEQ ID
NO:68, [0223] or a variant thereof in which one or two or three
amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are
substituted with another amino acid; and [0224] (ii) a light chain
variable (VL) region incorporating the following CDRs: [0225]
LC-CDR1 having the amino acid sequence of SEQ ID NO:74 [0226]
LC-CDR2 having the amino acid sequence of SEQ ID NO:75 [0227]
LC-CDR3 having the amino acid sequence of SEQ ID NO:76; [0228] or a
variant thereof in which one or two or three amino acids in one or
more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another
amino acid.
[0229] In some embodiments the antigen-binding molecule comprises:
[0230] (i) a heavy chain variable (VH) region incorporating the
following CDRs: [0231] HC-CDR1 having the amino acid sequence of
SEQ ID NO:66 [0232] HC-CDR2 having the amino acid sequence of SEQ
ID NO:67 [0233] HC-CDR3 having the amino acid sequence of SEQ ID
NO:68; and [0234] (ii) a light chain variable (VL) region
incorporating the following CDRs: [0235] LC-CDR1 having the amino
acid sequence of SEQ ID NO:74 [0236] LC-CDR2 having the amino acid
sequence of SEQ ID NO:75 [0237] LC-CDR3 having the amino acid
sequence of SEQ ID NO:76.
[0238] In some embodiments the antigen-binding molecule comprises:
[0239] a VH region comprising an amino acid sequence having at
least 70% sequence identity to the amino acid sequence of SEQ ID
NO:65; and [0240] a VL region comprising an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of
SEQ ID NO:73.
[0241] In another aspect the present invention provides an
antigen-binding molecule, optionally isolated, comprising (i) an
antigen-binding molecule according to the invention, and (ii) an
antigen-binding molecule capable of binding to an antigen other
than CD47.
[0242] In some embodiments the antigen-binding molecule is capable
of binding to cells expressing CD47 at the cell surface.
[0243] In some embodiments the antigen-binding molecule is capable
of inhibiting interaction between CD47 and SIRP.alpha..
[0244] In some embodiments the antigen-binding molecule is capable
of increasing phagocytosis of CD47-expressing cells.
[0245] In another aspect the present invention provides a chimeric
antigen receptor (CAR) comprising an antigen-binding molecule
according to the invention.
[0246] In another aspect the present invention provides a nucleic
acid, or a plurality of nucleic acids, optionally isolated,
encoding an antigen-binding molecule or a CAR according to the
invention.
[0247] In another aspect the present invention provides an
expression vector, or a plurality of expression vectors, comprising
a nucleic acid or a plurality of nucleic acids according to the
invention.
[0248] In another aspect the present invention provides a cell
comprising an antigen-binding molecule, a CAR, a nucleic acid or a
plurality of nucleic acids, or an expression vector or a plurality
of expression vectors according to the invention.
[0249] In another aspect the present invention provides a method
comprising culturing a cell comprising a nucleic acid or a
plurality of nucleic acids, or an expression vector or a plurality
of expression vectors according to the invention, under conditions
suitable for expression of the antigen-binding molecule or CAR from
the nucleic acid(s) or expression vector(s).
[0250] In another aspect the present invention provides a
composition comprising an antigen-binding molecule, a CAR, a
nucleic acid or a plurality of nucleic acids, an expression vector
or a plurality of expression vectors, or a cell according to the
invention.
[0251] In another aspect the present invention provides an
antigen-binding molecule, a CAR, a nucleic acid or a plurality of
nucleic acids, an expression vector or a plurality of expression
vectors, a cell, or a composition according to the invention for
use in a method of medical treatment or prophylaxis.
[0252] In another aspect the present invention provides an
antigen-binding molecule, a CAR, a nucleic acid or a plurality of
nucleic acids, an expression vector or a plurality of expression
vectors, a cell, or a composition according to the invention for
use in a method of treatment or prevention of a cancer.
[0253] In another aspect the present invention provides the use of
an antigen-binding molecule, a CAR, a nucleic acid or a plurality
of nucleic acids, an expression vector or a plurality of expression
vectors, a cell, or a composition according to the invention in the
manufacture of a medicament for use in a method of treatment or
prevention of a cancer.
[0254] In another aspect the present invention provides a method of
treating or preventing a cancer, comprising administering to a
subject a therapeutically or prophylactically effective amount of
an antigen-binding molecule, a CAR, a nucleic acid or a plurality
of nucleic acids, an expression vector or a plurality of expression
vectors, a cell, or a composition according to the invention.
[0255] In another aspect the present invention provides a method
for increasing phagocytosis of CD47-expressing cells, comprising
contacting CD47-expressing cells with an antigen-binding molecule
according to the invention.
[0256] In another aspect the present invention provides an in vitro
complex, optionally isolated, comprising an antigen-binding
molecule according to the invention bound to CD47.
[0257] In another aspect the present invention provides a method
comprising contacting a sample containing, or suspected to contain,
CD47 with an antigen-binding molecule according to the invention,
and detecting the formation of a complex of the antigen-binding
molecule with CD47.
[0258] In another aspect the present invention provides a subject
for treatment with a CD47-targeted agent, the method comprising
contacting, in vitro, a sample from the subject with an
antigen-binding molecule according to the invention and detecting
the formation of a complex of the antigen-binding molecule with
CD47.
[0259] In another aspect the present invention provides the use of
an antigen-binding molecule according to the invention as an in
vitro or in vivo diagnostic or prognostic agent.
[0260] In some embodiments in connection with various aspects of
the present invention the cancer is selected from: a hematologic
malignancy, a myeloid hematologic malignancy, a lymphoblastic
hematologic malignancy, myelodysplastic syndrome (MDS), acute
myeloid leukemia (AML), chronic myeloid leukemia (CML), acute
lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL),
multiple myeloma (MM), bladder cancer, brain cancer, glioblastoma,
ovarian cancer, breast cancer, colon cancer, liver cancer,
hepatocellular carcinoma, prostate cancer, lung cancer, Non-small
Cell Lung Cancer (NSCLC), skin cancer and melanoma.
DESCRIPTION
[0261] The present invention provides antigen-binding molecules
having combinations of desirable biophysical and/or functional
properties as compared to antigen-binding molecules disclosed in
the prior art.
[0262] Aspects of the present invention relate to antigen-binding
molecules capable of binding to CD47.
[0263] In aspects described herein antigen-binding molecules are
provided which bind to human CD47 with high affinity, which are
cross-reactive with non-human primate CD47, and which display
potent inhibition of interaction between CD47 and SIRP.alpha..
[0264] In particular, the antigen-binding molecules described
herein bind to CD47 with greater affinity than prior art anti-CD47
antibodies, and are more potent as a CD47-targeted therapeutic
agents.
[0265] Also, the antigen-binding molecules described herein bind to
a particular epitope of CD47 that provides for more effective
inhibition of the interaction between CD47 and SIPR.alpha. as
compared to prior art anti-CD47 antibodies. The antigen-binding
molecules described herein are thus more effective at enhancing
phagocytosis of cells expressing CD47 than prior art anti-CD47
antibodies.
[0266] CD47
[0267] Human CD47 (also known as IAP, MERG and OA3) is the protein
identified by UniProt Q08722. Alternative splicing of mRNA encoded
by the human CD47 gene yields four isoforms which differ in the
sequence of the C-terminal cytoplasmic tail region: isoform OA3-323
(UniProt: Q08722-1, v1; SEQ ID NO:1); isoform OA3-293 (UniProt:
Q08722-2; SEQ ID NO:2), which lacks the amino acid sequence
corresponding to positions 293 to 323 of SEQ ID NO:1; isoform
OA3-305 (UniProt: Q08722-3; SEQ ID NO:3), which comprises the
substitutions K304N and A305N relative to SEQ ID NO:1, and which
lacks the amino acid sequence corresponding to positions 306 to 323
of SEQ ID NO:1; and isoform OA3-312 (UniProt: Q08722-4; SEQ ID
NO:4), which lacks the amino acid sequence corresponding to
positions 312 to 323 of SEQ ID NO:1.
[0268] The N-terminal 18 amino acids of SEQ ID NOs:1 to 4
constitute a signal peptide, and so the mature form of isoforms
OA3-323, OA3-293, OA3-305 and OA3-312 (i.e. after processing to
remove the signal peptide) have the amino acid sequences shown in
SEQ ID NOs:5 to 8, respectively.
[0269] The structure and function of CD47 is reviewed e.g. in Sick
et al., Br J Pharmacol. (2012) 167(7): 1415-1430 and Willingham et
al. Proc Natl Acad Sci USA. (2012) 109(17): 6662-6667, both of
which are hereby incorporated by reference in its entirety. CD47 is
a ubiquitously-expressed .about.50 kDa multi-pass membrane receptor
that belongs to the immunoglobulin superfamily, comprising an
N-terminal extracellular region (SEQ ID NO:10) having a V-type
Ig-like domain (SEQ ID NO:9), five transmembrane domains (SEQ ID
NOs:11, 13, 15, 17 and 19), and a short C-terminal intracellular
tail (SEQ ID NO:20).
[0270] CD47 is involved in cell-to-cell communication through
ligating to the transmembrane signal-regulatory proteins (SIRPs)
SIRP.alpha. and SIRP.gamma. and integrins (e.g. .alpha.v.beta.3
integrin), and also mediates cell-extracellular matrix interactions
through binding to thrombospondin-1 (TSP-1). CD47 is involved in a
wide range of cellular processes including adhesion, migration,
proliferation and apoptosis, and plays a key role in immune
processes and angiogenesis.
[0271] CD47 is the ligand for SIRP.alpha., which expressed on
macrophages and dendritic cells. Binding of CD47 to SIRP.alpha. on
the surface of phagocytic cells, triggers SIRP.alpha. ITIM
signalling, inhibiting phagocytosis of the CD47 expressing cell.
CD47 is a multi-pass transmembrane protein, whereas SIRP.alpha.
consists of 4 extracellular domains and an intracellular
ITIM-domain. The terminal V-set domain of SIRP.alpha. interacts
with the Ig V-like domain of CD47.
[0272] Upon binding CD47, SIRP.alpha. initiates a signalling
cascade that results in the inhibition of phagocytosis of the
CD47-expressing cell. This "don't eat me" signal is transmitted by
phosphorylation by Src kinases of immunoreceptor tyrosine-based
inhibitor motifs (ITIMs) in the cytoplasmic domain of SIRP.alpha..
Subsequent binding and activation of Src homology-2 (SH2)
domain-containing tyrosine phosphatases SHP-1 and SHP-2 blocks
phagocytosis, potentially through preventing the accumulation of
myosin-IIA at the phagocytic synapse. Disrupting the interaction
along the antiparallel beta sheets of CD47 prevents downstream
ITIM-mediated signalling, enabling phagocytes to `eat` and destroy
cancer cells.
[0273] Aberrant CD47 expression/activity is implicated in the
development and progression of many cancers, and accumulating
evidence suggests that cell-surface expression of CD47 is a common
mechanism by which cancer cells protect themselves from
phagocytosis.
[0274] In this specification "CD47" refers to CD47 from any species
and includes CD47 isoforms, fragments, variants or homologues from
any species.
[0275] As used herein, a "fragment", "variant" or "homologue" of a
protein may optionally be characterised as having at least 60%,
preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% amino acid sequence identity to the
amino acid sequence of the reference protein (e.g. a reference
isoform). In some embodiments fragments, variants, isoforms and
homologues of a reference protein may be characterised by ability
to perform a function performed by the reference protein.
[0276] A "fragment" generally refers to a fraction of the reference
protein. A "variant" generally refers to a protein having an amino
acid sequence comprising one or more amino acid substitutions,
insertions, deletions or other modifications relative to the amino
acid sequence of the reference protein, but retaining a
considerable degree of sequence identity (e.g. at least 60%) to the
amino acid sequence of the reference protein. An "isoform"
generally refers to a variant of the reference protein expressed by
the same species as the species of the reference protein (e.g.
OA3-323, OA3-293, OA3-305 and OA3-312 are all isoforms of one
another). A "homologue" generally refers to a variant of the
reference protein produced by a different species as compared to
the species of the reference protein. For example, human CD47
isoform OA3-323 (Q08722-1, v1; SEQ ID NO:1) and Rhesus macaque CD47
(UniProt: F7F5Y9-1, v2; SEQ ID NO:117) are homologues of one
another. Homologues include orthologues.
[0277] A "fragment" of a reference protein may be of any length (by
number of amino acids), although may optionally be at least 25% of
the length of the reference protein (that is, the protein from
which the fragment is derived) and may have a maximum length of one
of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or 99% of the length of the reference protein.
[0278] A fragment of CD47 may have a minimum length of one of 10,
20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids, and may have
a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250 or
300 amino acids.
[0279] In some embodiments, the CD47 is CD47 from a mammal (e.g. a
primate (rhesus, cynomolgous, non-human primate or human) and/or a
rodent (e.g. rat or murine) CD47). Isoforms, fragments, variants or
homologues of CD47 may optionally be characterised as having at
least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the
amino acid sequence of an immature or mature CD47 isoform from a
given species, e.g. human.
[0280] Isoforms, fragments, variants or homologues may optionally
be functional isoforms, fragments, variants or homologues, e.g.
having a functional property/activity of the reference CD47 (e.g.
human CD47 isoform OA3-323), as determined by analysis by a
suitable assay for the functional property/activity. For example,
an isoform, fragment, variant or homologue of CD47 may display
association with one or more of: SIRP.alpha., SIRP.gamma., TSP-1
and .alpha.v.beta.3 integrin.
[0281] In some embodiments, the CD47 comprises, or consists of, an
amino acid sequence having at least 70%, preferably one of 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino
acid sequence identity to one of SEQ ID NOs:1 to 8.
[0282] In some embodiments, a fragment of CD47 comprises, or
consists of, an amino acid sequence having at least 70%, preferably
one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or 100% amino acid sequence identity to one of SEQ ID NOs:9 or
10.
[0283] CD47 is an attractive therapeutic target. CD47 is usually
expressed on the surface of normal healthy cells and migrating
hematopoietic stem cells to prevent phagocytosis, and is
upregulated in nearly all hematological and solid tumors, to evade
immune surveillance and escape phagocytosis. Disrupting the
interaction between CD47 and SIRP.alpha. enables phagocytes to
"eat" and destroy cancer cells. CD47 blockade repolarises
tumor-associated macrophages into a pro-inflammatory, anti-tumor
state, and clearance of malignant cells by phagocytic cells offers
an additional route for neo-antigen presentation to adaptive immune
system.
[0284] Antigen-Binding Molecules
[0285] The present invention provides antigen-binding molecules
capable of binding to CD47.
[0286] An "antigen-binding molecule" refers to a molecule which is
capable of binding to a target antigen, and encompasses monoclonal
antibodies, polyclonal antibodies, monospecific and multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments
(e.g. Fv, scFv, Fab, scFab, F(ab').sub.2, Fab.sub.2, diabodies,
triabodies, scFv-Fc, minibodies, single domain antibodies (e.g.
VhH), etc.), as long as they display binding to the relevant target
molecule(s).
[0287] The antigen-binding molecule of the present invention
comprises a moiety or moieties capable of binding to a target
antigen(s). In some embodiments, the moiety capable of binding to a
target antigen comprises an antibody heavy chain variable region
(VH) and an antibody light chain variable region (VL) of an
antibody capable of specific binding to the target antigen. In some
embodiments, the moiety capable of binding to a target antigen
comprises or consists of an aptamer capable of binding to the
target antigen, e.g. a nucleic acid aptamer (reviewed, for example,
in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). In some
embodiments, the moiety capable of binding to a target antigen
comprises or consists of a antigen-binding peptide/polypeptide,
e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz
domain, avimer, knottin, fynomer, atrimer, DARPin, affibody,
nanobody (i.e. a single-domain antibody (sdAb)) affilin, armadillo
repeat protein (ArmRP), OBody or fibronectin--reviewed e.g. in
Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101,
which is hereby incorporated by reference in its entirety (see also
e.g. Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et
al., Mabs (2011) 3:38-48).
[0288] The antigen-binding molecules of the present invention
generally comprise an antigen-binding domain comprising a VH and a
VL of an antibody capable of specific binding to the target
antigen. The antigen-binding domain formed by a VH and a VL may
also be referred to herein as an Fv region.
[0289] An antigen-binding molecule may be, or may comprise, an
antigen-binding polypeptide, or an antigen-binding polypeptide
complex. An antigen-binding molecule may comprise more than one
polypeptide which together form an antigen-binding domain. The
polypeptides may associate covalently or non-covalently. In some
embodiments the polypeptides form part of a larger polypeptide
comprising the polypeptides (e.g. in the case of scFv comprising VH
and VL, or in the case of scFab comprising VH-CH1 and VL-CL).
[0290] An antigen-binding molecule may refer to a non-covalent or
covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or
8 polypeptides), e.g. an IgG-like antigen-binding molecule
comprising two heavy chain polypeptides and two light chain
polypeptides.
[0291] The antigen-binding molecules of the present invention may
be designed and prepared using the sequences of monoclonal
antibodies (mAbs) capable of binding to CD47. Antigen-binding
regions of antibodies, such as single chain variable fragment
(scFv), Fab and F(ab')2 fragments may also be used/provided. An
"antigen-binding region" is any fragment of an antibody which is
capable of binding to the target for which the given antibody is
specific.
[0292] Antibodies generally comprise six
complementarity-determining regions CDRs; three in the heavy chain
variable (VH) region: HC-CDR1, HC-CDR2 and HC-CDR3, and three in
the light chain variable (VL) region: LC-CDR1, LC-CDR2, and
LC-CDR3. The six CDRs together define the paratope of the antibody,
which is the part of the antibody which binds to the target
antigen.
[0293] The VH region and VL region comprise framework regions (FRs)
either side of each CDR, which provide a scaffold for the CDRs.
From N-terminus to C-terminus, VH regions comprise the following
structure: N
term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C
term; and VL regions comprise the following structure: N
term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C
term.
[0294] There are several different conventions for defining
antibody CDRs and FRs, such as those described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and
VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33
(suppl 1): D671-D674. The CDRs and FRs of the VH regions and VL
regions of the antibody clones described herein were defined
according to the international IMGT (ImMunoGeneTics) information
system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database
issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as
described in Lefranc et al., Dev. Comp. Immunol. (2003)
27:55-77.
[0295] In some embodiments, the antigen-binding molecule comprises
the CDRs of an antigen-binding molecule which is capable of binding
to CD47. In some embodiments, the antigen-binding molecule
comprises the FRs of an antigen-binding molecule which is capable
of binding to CD47. In some embodiments, the antigen-binding
molecule comprises the CDRs and the FRs of an antigen-binding
molecule which is capable of binding to CD47. That is, in some
embodiments the antigen-binding molecule comprises the VH region
and the VL region of an antigen-binding molecule which is capable
of binding to CD47.
[0296] In some embodiments the antigen-binding molecule comprises a
VH region and a VL region which is, or which is derived from, the
VH/VL region of a CD47-binding antibody clone described herein
(i.e. anti-CD47 antibody clones 1-1-A1_BM, 1-1-A1, 5-48-A6,
5-48-D2, 11A1H1, 11A1H2, 11A1H3, 11A1H4, 11A1H5, 11A1H6, 11A1H7,
11A1H8, 11A1H9, 11A1H10 or 11A1H11).
[0297] In some embodiments the antigen-binding molecule comprises a
VH region according to one of (1) to (4) below:
(1) a VH region incorporating the following CDRs: [0298] HC-CDR1
having the amino acid sequence of SEQ ID NO:24 [0299] HC-CDR2
having the amino acid sequence of SEQ ID NO:25 [0300] HC-CDR3
having the amino acid sequence of SEQ ID NO:26, [0301] or a variant
thereof in which one or two or three amino acids in one or more of
HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another amino
acid. (2) a VH region incorporating the following CDRs: [0302]
HC-CDR1 having the amino acid sequence of SEQ ID NO:50 [0303]
HC-CDR2 having the amino acid sequence of SEQ ID NO:51 [0304]
HC-CDR3 having the amino acid sequence of SEQ ID NO:52, [0305] or a
variant thereof in which one or two or three amino acids in one or
more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with another
amino acid. (3) a VH region incorporating the following CDRs:
[0306] HC-CDR1 having the amino acid sequence of SEQ ID NO:66
[0307] HC-CDR2 having the amino acid sequence of SEQ ID NO:67
[0308] HC-CDR3 having the amino acid sequence of SEQ ID NO:68,
[0309] or a variant thereof in which one or two or three amino
acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are
substituted with another amino acid. (4) a VH region incorporating
the following CDRs: [0310] HC-CDR1 having the amino acid sequence
of SEQ ID NO:169 [0311] HC-CDR2 having the amino acid sequence of
SEQ ID NO:170 [0312] HC-CDR3 having the amino acid sequence of SEQ
ID NO:26, [0313] or a variant thereof in which one or two or three
amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are
substituted with another amino acid. (5) a VH region incorporating
the following CDRs: [0314] HC-CDR1 having the amino acid sequence
of SEQ ID NO:137 [0315] HC-CDR2 having the amino acid sequence of
SEQ ID NO:138 [0316] HC-CDR3 having the amino acid sequence of SEQ
ID NO:26, [0317] or a variant thereof in which one or two or three
amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are
substituted with another amino acid.
[0318] In some embodiments the antigen-binding molecule comprises a
VH region according to one of (6) to (15) below:
(6) a VH region incorporating the following FRs: [0319] HC-FR1
having the amino acid sequence of SEQ ID NO:27 [0320] HC-FR2 having
the amino acid sequence of SEQ ID NO:28 [0321] HC-FR3 having the
amino acid sequence of SEQ ID NO:29 [0322] HC-FR4 having the amino
acid sequence of SEQ ID NO:30, [0323] or a variant thereof in which
one or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (7) a VH
region incorporating the following FRs: [0324] HC-FR1 having the
amino acid sequence of SEQ ID NO:40 [0325] HC-FR2 having the amino
acid sequence of SEQ ID NO:41 [0326] HC-FR3 having the amino acid
sequence of SEQ ID NO:42 [0327] HC-FR4 having the amino acid
sequence of SEQ ID NO:43, [0328] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (8) a VH
region incorporating the following FRs: [0329] HC-FR1 having the
amino acid sequence of SEQ ID NO:53 [0330] HC-FR2 having the amino
acid sequence of SEQ ID NO:54 [0331] HC-FR3 having the amino acid
sequence of SEQ ID NO:55 [0332] HC-FR4 having the amino acid
sequence of SEQ ID NO:56, [0333] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (9) a VH
region incorporating the following FRs: [0334] HC-FR1 having the
amino acid sequence of SEQ ID NO:69 [0335] HC-FR2 having the amino
acid sequence of SEQ ID NO:70 [0336] HC-FR3 having the amino acid
sequence of SEQ ID NO:71 [0337] HC-FR4 having the amino acid
sequence of SEQ ID NO:72, [0338] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (10) a
VH region incorporating the following FRs: [0339] HC-FR1 having the
amino acid sequence of SEQ ID NO:143 [0340] HC-FR2 having the amino
acid sequence of SEQ ID NO:174 [0341] HC-FR3 having the amino acid
sequence of SEQ ID NO:175 [0342] HC-FR4 having the amino acid
sequence of SEQ ID NO:176, [0343] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (11) a
VH region incorporating the following FRs: [0344] HC-FR1 having the
amino acid sequence of SEQ ID NO:143 [0345] HC-FR2 having the amino
acid sequence of SEQ ID NO:144 [0346] HC-FR3 having the amino acid
sequence of SEQ ID NO:147 [0347] HC-FR4 having the amino acid
sequence of SEQ ID NO:152, [0348] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (12) a
VH region incorporating the following FRs: [0349] HC-FR1 having the
amino acid sequence of SEQ ID NO:143 [0350] HC-FR2 having the amino
acid sequence of SEQ ID NO:144 [0351] HC-FR3 having the amino acid
sequence of SEQ ID NO:148 [0352] HC-FR4 having the amino acid
sequence of SEQ ID NO:152, [0353] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (13) a
VH region incorporating the following FRs: [0354] HC-FR1 having the
amino acid sequence of SEQ ID NO:143 [0355] HC-FR2 having the amino
acid sequence of SEQ ID NO:145 [0356] HC-FR3 having the amino acid
sequence of SEQ ID NO:149 [0357] HC-FR4 having the amino acid
sequence of SEQ ID NO:153, [0358] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (14) a
VH region incorporating the following FRs: [0359] HC-FR1 having the
amino acid sequence of SEQ ID NO:143 [0360] HC-FR2 having the amino
acid sequence of SEQ ID NO:146 [0361] HC-FR3 having the amino acid
sequence of SEQ ID NO:150 [0362] HC-FR4 having the amino acid
sequence of SEQ ID NO:153, [0363] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid. (15) a
VH region incorporating the following FRs: [0364] HC-FR1 having the
amino acid sequence of SEQ ID NO:143 [0365] HC-FR2 having the amino
acid sequence of SEQ ID NO:146 [0366] HC-FR3 having the amino acid
sequence of SEQ ID NO:151 [0367] HC-FR4 having the amino acid
sequence of SEQ ID NO:152, [0368] or a variant thereof in which one
or two or three amino acids in one or more of HC-FR1, HC-FR2,
HC-FR3, or HC-FR4 are substituted with another amino acid.
[0369] In some embodiments the antigen-binding molecule comprises a
VH region comprising the CDRs according to one of (1), (2), (3),
(4) or (5) above, and the FRs according to one of (5), (6), (7),
(8), (9), (10), (11), (12), (13), (14) or (15) above.
[0370] In some embodiments the antigen-binding molecule comprises a
VH region according to one of (16) to (25) below:
(16) a VH region comprising the CDRs according to (1) and the FRs
according to (6). (17) a VH region comprising the CDRs according to
(1) and the FRs according to (7). (18) a VH region comprising the
CDRs according to (2) and the FRs according to (8). (19) a VH
region comprising the CDRs according to (3) and the FRs according
to (9). (20) a VH region comprising the CDRs according to (4) and
the FRs according to (10). (21) a VH region comprising the CDRs
according to (1) and the FRs according to (11). (22) a VH region
comprising the CDRs according to (1) and the FRs according to (12).
(23) a VH region comprising the CDRs according to (1) and the FRs
according to (13). (24) a VH region comprising the CDRs according
to (1) and the FRs according to (14). (25) a VH region comprising
the CDRs according to (5) and the FRs according to (15).
[0371] In some embodiments the antigen-binding molecule comprises a
VH region according to one of (26) to (34) below:
(26) a VH region comprising an amino acid sequence having at least
70% sequence identity more preferably one of at least 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100%, sequence identity to the amino acid sequence of
SEQ ID NO:23. (27) a VH region comprising an amino acid sequence
having at least 70% sequence identity more preferably one of at
least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino
acid sequence of SEQ ID NO:39. (28) a VH region comprising an amino
acid sequence having at least 70% sequence identity more preferably
one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to
the amino acid sequence of SEQ ID NO:49. (29) a VH region
comprising an amino acid sequence having at least 70% sequence
identity more preferably one of at least 75%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100%, sequence identity to the amino acid sequence of SEQ ID NO:65.
(31) a VH region comprising an amino acid sequence having at least
70% sequence identity more preferably one of at least 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100%, sequence identity to the amino acid sequence of
SEQ ID NO:178. (31) a VH region comprising an amino acid sequence
having at least 70% sequence identity more preferably one of at
least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino
acid sequence of SEQ ID NO:127. (32) a VH region comprising an
amino acid sequence having at least 70% sequence identity more
preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence
identity to the amino acid sequence of SEQ ID NO:129. (33) a VH
region comprising an amino acid sequence having at least 70%
sequence identity more preferably one of at least 75%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%, sequence identity to the amino acid sequence of SEQ
ID NO:130. (34) a VH region comprising an amino acid sequence
having at least 70% sequence identity more preferably one of at
least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino
acid sequence of SEQ ID NO:131. (35) a VH region comprising an
amino acid sequence having at least 70% sequence identity more
preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence
identity to the amino acid sequence of SEQ ID NO:132.
[0372] In some embodiments the antigen-binding molecule comprises a
VL region according to one of (36) to (43) below:
(36) a VL region incorporating the following CDRs: [0373] LC-CDR1
having the amino acid sequence of SEQ ID NO:32 [0374] LC-CDR2
having the amino acid sequence of SEQ ID NO:33 [0375] LC-CDR3
having the amino acid sequence of SEQ ID NO:34; [0376] or a variant
thereof in which one or two or three amino acids in one or more of
LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino
acid. (37) a VL region incorporating the following CDRs: [0377]
LC-CDR1 having the amino acid sequence of SEQ ID NO:58 [0378]
LC-CDR2 having the amino acid sequence of SEQ ID NO:59 [0379]
LC-CDR3 having the amino acid sequence of SEQ ID NO:60; [0380] or a
variant thereof in which one or two or three amino acids in one or
more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another
amino acid. (38) a VL region incorporating the following CDRs:
[0381] LC-CDR1 having the amino acid sequence of SEQ ID NO:74
[0382] LC-CDR2 having the amino acid sequence of SEQ ID NO:75
[0383] LC-CDR3 having the amino acid sequence of SEQ ID NO:76;
[0384] or a variant thereof in which one or two or three amino
acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted
with another amino acid. (39) a VL region incorporating the
following CDRs: [0385] LC-CDR1 having the amino acid sequence of
SEQ ID NO:171 [0386] LC-CDR2 having the amino acid sequence of SEQ
ID NO:172 [0387] LC-CDR3 having the amino acid sequence of SEQ ID
NO:173; [0388] or a variant thereof in which one or two or three
amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are
substituted with another amino acid. (40) a VL region incorporating
the following CDRs: [0389] LC-CDR1 having the amino acid sequence
of SEQ ID NO:139 [0390] LC-CDR2 having the amino acid sequence of
SEQ ID NO:141 [0391] LC-CDR3 having the amino acid sequence of SEQ
ID NO:34; [0392] or a variant thereof in which one or two or three
amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are
substituted with another amino acid. (41) a VL region incorporating
the following CDRs: [0393] LC-CDR1 having the amino acid sequence
of SEQ ID NO:140 [0394] LC-CDR2 having the amino acid sequence of
SEQ ID NO:141 [0395] LC-CDR3 having the amino acid sequence of SEQ
ID NO:34; [0396] or a variant thereof in which one or two or three
amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are
substituted with another amino acid. (42) a VL region incorporating
the following CDRs: [0397] LC-CDR1 having the amino acid sequence
of SEQ ID NO:32 [0398] LC-CDR2 having the amino acid sequence of
SEQ ID NO:141 [0399] LC-CDR3 having the amino acid sequence of SEQ
ID NO:34; [0400] or a variant thereof in which one or two or three
amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are
substituted with another amino acid. (43) a VL region incorporating
the following CDRs: [0401] LC-CDR1 having the amino acid sequence
of SEQ ID NO:32 [0402] LC-CDR2 having the amino acid sequence of
SEQ ID NO:33 [0403] LC-CDR3 having the amino acid sequence of SEQ
ID NO:142; [0404] or a variant thereof in which one or two or three
amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are
substituted with another amino acid.
[0405] In some embodiments the antigen-binding molecule comprises a
VL region according to one of (44) to (50) below:
(44) a VL region incorporating the following FRs: [0406] LC-FR1
having the amino acid sequence of SEQ ID NO:35 [0407] LC-FR2 having
the amino acid sequence of SEQ ID NO:36 [0408] LC-FR3 having the
amino acid sequence of SEQ ID NO:37 [0409] LC-FR4 having the amino
acid sequence of SEQ ID NO:38, [0410] or a variant thereof in which
one or two or three amino acids in one or more of LC-FR1, LC-FR2,
LC-FR3, or LC-FR4 are substituted with another amino acid. (45) a
VL region incorporating the following FRs: [0411] LC-FR1 having the
amino acid sequence of SEQ ID NO:45 [0412] LC-FR2 having the amino
acid sequence of SEQ ID NO:46 [0413] LC-FR3 having the amino acid
sequence of SEQ ID NO:47 [0414] LC-FR4 having the amino acid
sequence of SEQ ID NO:48, [0415] or a variant thereof in which one
or two or three amino acids in one or more of LC-FR1, LC-FR2,
LC-FR3, or LC-FR4 are substituted with another amino acid. (46) a
VL region incorporating the following FRs: [0416] LC-FR1 having the
amino acid sequence of SEQ ID NO:61 [0417] LC-FR2 having the amino
acid sequence of SEQ ID NO:62 [0418] LC-FR3 having the amino acid
sequence of SEQ ID NO:63 [0419] LC-FR4 having the amino acid
sequence of SEQ ID NO:64, [0420] or a variant thereof in which one
or two or three amino acids in one or more of LC-FR1, LC-FR2,
LC-FR3, or LC-FR4 are substituted with another amino acid. (47) a
VL region incorporating the following FRs: [0421] LC-FR1 having the
amino acid sequence of SEQ ID NO:77 [0422] LC-FR2 having the amino
acid sequence of SEQ ID NO:78 [0423] LC-FR3 having the amino acid
sequence of SEQ ID NO:79 [0424] LC-FR4 having the amino acid
sequence of SEQ ID NO:80, [0425] or a variant thereof in which one
or two or three amino acids in one or more of LC-FR1, LC-FR2,
LC-FR3, or LC-FR4 are substituted with another amino acid. (48) a
VL region incorporating the following FRs: [0426] LC-FR1 having the
amino acid sequence of SEQ ID NO:154 [0427] LC-FR2 having the amino
acid sequence of SEQ ID NO:155 [0428] LC-FR3 having the amino acid
sequence of SEQ ID NO:177 [0429] LC-FR4 having the amino acid
sequence of SEQ ID NO:158, [0430] or a variant thereof in which one
or two or three amino acids in one or more of LC-FR1, LC-FR2,
LC-FR3, or LC-FR4 are substituted with another amino acid. (49) a
VL region incorporating the following FRs: [0431] LC-FR1 having the
amino acid sequence of SEQ ID NO:154 [0432] LC-FR2 having the amino
acid sequence of SEQ ID NO:155 [0433] LC-FR3 having the amino acid
sequence of SEQ ID NO:156 [0434] LC-FR4 having the amino acid
sequence of SEQ ID NO:158, [0435] or a variant thereof in which one
or two or three amino acids in one or more of LC-FR1, LC-FR2,
LC-FR3, or LC-FR4 are substituted with another amino acid. (50) a
VL region incorporating the following FRs: [0436] LC-FR1 having the
amino acid sequence of SEQ ID NO:154 [0437] LC-FR2 having the amino
acid sequence of SEQ ID NO:155 [0438] LC-FR3 having the amino acid
sequence of SEQ ID NO:157 [0439] LC-FR4 having the amino acid
sequence of SEQ ID NO:158, [0440] or a variant thereof in which one
or two or three amino acids in one or more of LC-FR1, LC-FR2,
LC-FR3, or LC-FR4 are substituted with another amino acid.
[0441] In some embodiments the antigen-binding molecule comprises a
VL region comprising the CDRs according to one of (36), (37), (38),
(39), (40), (41), (42) or (43) above, and the FRs according to one
of (44), (45), (46), (47), (48), (49) or (50) above.
[0442] In some embodiments the antigen-binding molecule comprises a
VL region according to one of (51) to (60) below:
(51) a VL region comprising the CDRs according to (36) and the FRs
according to (43). 52) a VL region comprising the CDRs according to
(36) and the FRs according to (44). (53) a VL region comprising the
CDRs according to (37) and the FRs according to (45). (54) a VL
region comprising the CDRs according to (38) and the FRs according
to (46). (55) a VL region comprising the CDRs according to (39) and
the FRs according to (48). (56) a VL region comprising the CDRs
according to (36) and the FRs according to (49). (57) a VL region
comprising the CDRs according to (40) and the FRs according to
(50). (58) a VL region comprising the CDRs according to (41) and
the FRs according to (50). (59) a VL region comprising the CDRs
according to (42) and the FRs according to (50). (60) a VL region
comprising the CDRs according to (43) and the FRs according to
(49).
[0443] In some embodiments the antigen-binding molecule comprises a
VL region according to one of (61) to (70) below:
(61) a VL region comprising an amino acid sequence having at least
70% sequence identity more preferably one of at least 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100%, sequence identity to the amino acid sequence of
SEQ ID NO:31. (62) a VL region comprising an amino acid sequence
having at least 70% sequence identity more preferably one of at
least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino
acid sequence of SEQ ID NO:44. (63) a VL region comprising an amino
acid sequence having at least 70% sequence identity more preferably
one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to
the amino acid sequence of SEQ ID NO:57. (64) a VL region
comprising an amino acid sequence having at least 70% sequence
identity more preferably one of at least 75%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100%, sequence identity to the amino acid sequence of SEQ ID NO:73.
(65) a VL region comprising an amino acid sequence having at least
70% sequence identity more preferably one of at least 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100%, sequence identity to the amino acid sequence of
SEQ ID NO:179. (66) a VL region comprising an amino acid sequence
having at least 70% sequence identity more preferably one of at
least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino
acid sequence of SEQ ID NO:128. (67) a VL region comprising an
amino acid sequence having at least 70% sequence identity more
preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence
identity to the amino acid sequence of SEQ ID NO:133. (68) a VL
region comprising an amino acid sequence having at least 70%
sequence identity more preferably one of at least 75%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%, sequence identity to the amino acid sequence of SEQ
ID NO:134. (69) a VL region comprising an amino acid sequence
having at least 70% sequence identity more preferably one of at
least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino
acid sequence of SEQ ID NO:135. (70) a VL region comprising an
amino acid sequence having at least 70% sequence identity more
preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence
identity to the amino acid sequence of SEQ ID NO:136.
[0444] In some embodiments the antigen-binding molecule comprises a
VH region according to any one of (1) to (35) above, and a VL
region according to any one of (36) to (70) above.
[0445] In embodiments in accordance with the present invention in
which one or more amino acids are substituted with another amino
acid, the substitutions may conservative substitutions, for example
according to the following Table. In some embodiments, amino acids
in the same block in the middle column are substituted. In some
embodiments, amino acids in the same line in the rightmost column
are substituted:
TABLE-US-00001 ALIPHATIC Non-polar G A P I L V Polar - uncharged C
S T M N Q Polar - charged D E K R AROMATIC H F W Y
[0446] In some embodiments, substitution(s) may be functionally
conservative. That is, in some embodiments the substitution may not
affect (or may not substantially affect) one or more functional
properties (e.g. target binding) of the antigen-binding molecule
comprising the substitution as compared to the equivalent
unsubstituted molecule.
[0447] The VH and VL region of an antigen-binding region of an
antibody together constitute the Fv region. In some embodiments,
the antigen-binding molecule according to the present invention
comprises, or consists of, an Fv region which binds to CD47. In
some embodiments the VH and VL regions of the Fv are provided as
single polypeptide joined by a linker region, i.e. a single chain
Fv (scFv).
[0448] In some embodiments the antigen-binding molecule of the
present invention comprises one or more regions of an
immunoglobulin heavy chain constant sequence. In some embodiments
the immunoglobulin heavy chain constant sequence is, or is derived
from, the heavy chain constant sequence of an IgG (e.g. IgG1, IgG2,
IgG3, IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE or IgM.
[0449] In some embodiments the immunoglobulin heavy chain constant
sequence is human immunoglobulin G 1 constant (IGHG1; UniProt:
P01857-1, v1; SEQ ID NO:118). Positions 1 to 98 of SEQ ID NO:118
form the CH1 region (SEQ ID NO:119). Positions 99 to 110 of SEQ ID
NO:118 form a hinge region between CH1 and CH2 regions (SEQ ID
NO:120). Positions 111 to 223 of SEQ ID NO:118 form the CH2 region
(SEQ ID NO:121). Positions 224 to 330 of SEQ ID NO:118 form the CH3
region (SEQ ID NO:122).
[0450] The exemplified antigen-binding molecules were prepared
using pFUSE-CHIg-hG1, which comprises the substitutions D356E,
L358M (positions numbered according to EU numbering) in the CH3
region relative to SEQ ID NO:118. The amino acid sequence of the
CH3 region encoded by pFUSE-CHIg-hG1 is shown in SEQ ID NO:123. It
will be appreciated that CH3 regions may be provided with further
substitutions in accordance with modification to an Fc region of
the antigen-binding molecule as described herein.
[0451] In some embodiments a CH1 region comprises or consists of
the sequence of SEQ ID NO:119, or a sequence having at least 60%,
preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% amino acid sequence identity to the
amino acid sequence of SEQ ID NO:119. In some embodiments a CH1-CH2
hinge region comprises or consists of the sequence of SEQ ID
NO:120, or a sequence having at least 60%, preferably one of 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% amino acid sequence identity to the amino acid sequence of SEQ
ID NO:120. In some embodiments a CH2 region comprises or consists
of the sequence of SEQ ID NO:121, or a sequence having at least
60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the
amino acid sequence of SEQ ID NO:121. In some embodiments a CH3
region comprises or consists of the sequence of SEQ ID NO:122 or
123, or a sequence having at least 60%, preferably one of 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
amino acid sequence identity to the amino acid sequence of SEQ ID
NO:122 or 123.
[0452] In some embodiments the antigen-binding molecule of the
present invention comprises one or more regions of an
immunoglobulin light chain constant sequence. In some embodiments
the immunoglobulin light chain constant sequence is human
immunoglobulin kappa constant (IGKC; C.kappa.; UniProt: P01834-1,
v2; SEQ ID NO:124). In some embodiments the immunoglobulin light
chain constant sequence is a human immunoglobulin lambda constant
(IGLC; C.lamda.), e.g. IGLC1, IGLC2, IGLC3, IGLC6 or IGLC7. In some
embodiments a CL region comprises or consists of the sequence of
SEQ ID NO:124, or a sequence having at least 60%, preferably one of
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or 100% amino acid sequence identity to the amino acid sequence
of SEQ ID NO:124.
[0453] The VL and light chain constant (CL) region, and the VH
region and heavy chain constant 1 (CH1) region of an
antigen-binding region of an antibody together constitute the Fab
region. In some embodiments the antigen-binding molecule comprises
a Fab region comprising a VH, a CH1, a VL and a CL (e.g. C.kappa.
or C.lamda.). In some embodiments the Fab region comprises a
polypeptide comprising a VH and a CH1 (e.g. a VH-CH1 fusion
polypeptide), and a polypeptide comprising a VL and a CL (e.g. a
VL-CL fusion polypeptide). In some embodiments the Fab region
comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL
fusion polypeptide) and a polypeptide comprising a VL and a CH
(e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments
the Fab region is a CrossFab region. In some embodiments the VH,
CH1, VL and CL regions of the Fab or CrossFab are provided as
single polypeptide joined by linker regions, i.e. as a single chain
Fab (scFab) or a single chain CrossFab (scCrossFab).
[0454] In some embodiments, the antigen-binding molecule of the
present invention comprises, or consists of, a Fab region which
binds to CD47.
[0455] In some embodiments, the antigen-binding molecule described
herein comprises, or consists of, a whole antibody which binds to
CD47. As used herein, "whole antibody" refers to an antibody having
a structure which is substantially similar to the structure of an
immunoglobulin (Ig). Different kinds of immunoglobulins and their
structures are described e.g. in Schroeder and Cavacini J Allergy
Clin Immunol. (2010) 125(202): S41-S52, which is hereby
incorporated by reference in its entirety.
[0456] Immunoglobulins of type G (i.e. IgG) are .about.150 kDa
glycoproteins comprising two heavy chains and two light chains.
From N- to C-terminus, the heavy chains comprise a VH followed by a
heavy chain constant region comprising three constant domains (CH1,
CH2, and CH3), and similarly the light chain comprise a VL followed
by a CL. Depending on the heavy chain, immunoglobulins may be
classed as IgG (e.g. IgG1, IgG2, IgG3, IgG4), IgA (e.g. IgA1,
IgA2), IgD, IgE, or IgM. The light chain may be kappa (.kappa.) or
lambda (.lamda.).
[0457] In some embodiments, the antigen-binding molecule described
herein comprises, or consists of, an IgG (e.g. IgG1, IgG2, IgG3,
IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE, or IgM which binds to CD47.
Aspects of the present invention relate to multispecific
antigen-binding molecules. By "multispecific" it is meant that the
antigen-binding molecule displays specific binding to more than one
target. In some embodiments the antigen-binding molecule is a
bispecific antigen-binding molecule. In some embodiments the
antigen-binding molecule comprises at least two different
antigen-binding domains (i.e. at least two antigen-binding domains,
e.g. comprising non-identical VHs and VLs).
[0458] In some embodiments the antigen-binding molecule binds to
CD47 and an antigen other than CD47, and so is at least bispecific.
The term "bispecific" means that the antigen-binding molecule is
able to bind specifically to at least two distinct antigenic
determinants.
[0459] It will be appreciated that an antigen-binding molecule
according to the present invention (e.g. a multispecific
antigen-binding molecule) may comprise antigen-binding molecules
capable of binding to the targets for which the antigen-binding
molecule is specific. For example, an antigen-binding molecule
which is capable of binding to CD47 and an antigen other than CD47
may comprise: (i) an antigen-binding molecule which is capable of
binding to CD47, and (ii) an antigen-binding molecule which is
capable of binding to an antigen other than CD47. By way of
illustration, an antigen-binding molecule which is capable of
binding to CD47 and an antigen other than CD47 may comprise (i) an
antigen-binding molecule which is capable of binding to CD47, (e.g.
a CD47-binding Fab or scFv), and (ii) an antigen-binding molecule
which is capable of binding to an antigen other than CD47 (e.g. a
Fab or scFv specific for the antigen other than CD47).
[0460] It will also be appreciated that an antigen-binding molecule
according to the present invention (e.g. a multispecific
antigen-binding molecule) may comprise antigen-binding polypeptides
or antigen-binding polypeptide complexes capable of binding to the
targets for which the antigen-binding molecule is specific.
[0461] In some embodiments, a component antigen-binding molecule of
a larger antigen-binding molecule (e.g. a multispecific
antigen-binding molecule) may be referred to e.g. as an
"antigen-binding domain" or "antigen-binding region" of the larger
antigen-binding molecule.
[0462] In some embodiments the antigen-binding molecule comprises
an antigen-binding molecule capable of binding to CD47, and an
antigen-binding molecule capable of binding to an antigen other
than CD47. In some embodiments, the antigen other than CD47 is an
immune cell surface molecule. In some embodiments, the antigen
other than CD47 is a cancer cell antigen. In some embodiments the
antigen other than CD47 is a receptor molecule, e.g. a cell surface
receptor. In some embodiments the antigen other than CD47 is a cell
signalling molecule, e.g. a cytokine, chemokine, interferon,
interleukin or lymphokine. In some embodiments the antigen other
than CD47 is a growth factor or a hormone.
[0463] A cancer cell antigen is an antigen which is expressed or
over-expressed by a cancer cell. A cancer cell antigen may be any
peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid,
lipid, or fragment thereof. A cancer cell antigen's expression may
be associated with a cancer. A cancer cell antigen may be
abnormally expressed by a cancer cell (e.g. the cancer cell antigen
may be expressed with abnormal localisation), or may be expressed
with an abnormal structure by a cancer cell. A cancer cell antigen
may be capable of eliciting an immune response. In some
embodiments, the antigen is expressed at the cell surface of the
cancer cell (i.e. the cancer cell antigen is a cancer cell surface
antigen). In some embodiments, the part of the antigen which is
bound by the antigen-binding molecule described herein is displayed
on the external surface of the cancer cell (i.e. is extracellular).
The cancer cell antigen may be a cancer-associated antigen. In some
embodiments the cancer cell antigen is an antigen whose expression
is associated with the development, progression or severity of
symptoms of a cancer. The cancer-associated antigen may be
associated with the cause or pathology of the cancer, or may be
expressed abnormally as a consequence of the cancer. In some
embodiments, the cancer cell antigen is an antigen whose expression
is upregulated (e.g. at the RNA and/or protein level) by cells of a
cancer, e.g. as compared to the level of expression of by
comparable non-cancerous cells (e.g. non-cancerous cells derived
from the same tissue/cell type). In some embodiments, the
cancer-associated antigen may be preferentially expressed by
cancerous cells, and not expressed by comparable non-cancerous
cells (e.g. non-cancerous cells derived from the same tissue/cell
type). In some embodiments, the cancer-associated antigen may be
the product of a mutated oncogene or mutated tumor suppressor gene.
In some embodiments, the cancer-associated antigen may be the
product of an overexpressed cellular protein, a cancer antigen
produced by an oncogenic virus, an oncofetal antigen, or a cell
surface glycolipid or glycoprotein.
[0464] An immune cell surface molecule may be any
peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid,
lipid, or fragment thereof expressed at or on the cell surface of
an immune cell. In some embodiments, the part of the immune cell
surface molecule which is bound by the antigen-binding molecule of
the present invention is on the external surface of the immune cell
(i.e. is extracellular). The immune cell surface molecule may be
expressed at the cell surface of any immune cell. In some
embodiments, the immune cell may be a cell of hematopoietic origin,
e.g. a neutrophil, eosinophil, basophil, dendritic cell,
lymphocyte, or monocyte. The lymphocyte may be e.g. a T cell, B
cell, natural killer (NK) cell, NKT cell or innate lymphoid cell
(ILC), or a precursor thereof (e.g. a thymocyte or pre-B cell).
[0465] In some embodiments the antigen other than CD47 is an
antigen expressed by cells of a hematologic malignancy, a myeloid
hematologic malignancy, a lymphoblastic hematologic malignancy,
myelodysplastic syndrome (MDS), acute myeloid leukemia (AML),
chronic myeloid leukemia, acute lymphoblastic leukemia (ALL),
non-Hodgkin's lymphoma, multiple myeloma, bladder cancer or brain
cancer.
[0466] In some embodiments the antigen other than CD47 is an
antigen expressed by cells of AML, e.g. as described in Hoseini and
Cheung Blood Cancer J. (2017) 7(2):e522, which is hereby
incorporated by reference in its entirety. In some embodiments the
antigen other than CD47 is selected from: CD33, CD123, Wilms' tumor
protein (WT1), CD13, CD15, CD30, CD45, C-type lectin-like molecule
1 (CLL1), Fms-like tyrosine kinase 3 (FLT-3), VEGF and
angiopoietin-2 (Ang-2). In some embodiments the antigen other than
CD47 is CD33.
[0467] Multispecific antigen-binding molecules described herein
display at least monovalent binding with respect to CD47, and also
display at least monovalent binding with respect to the antigen
other than CD47. In some embodiments the antigen-binding molecule
comprises an antigen-binding region (e.g. an Fv, Fab or antibody)
capable of binding to CD47, and an antigen-binding region (e.g. an
Fv, Fab or antibody) capable of binding to an antigen other than
CD47. In some embodiments the antigen-binding molecule comprises
the VH and VL of an antibody capable of binding to CD47, and the VH
and VL of an antibody capable of binding to an antigen other than
CD47.
[0468] Binding valency refers to the number of binding sites in an
antigen-binding molecule for a given antigenic determinant. For
example, in the IgG1 format described herein the anti-CD47 antibody
is bivalent with respect to binding to CD47.
[0469] Multispecific antigen-binding molecules according to the
invention may be provided in any suitable format, such as those
formats described in described in Brinkmann and Kontermann MAbs
(2017) 9(2): 182-212, which is hereby incorporated by reference in
its entirety. Suitable formats include those shown in FIG. 2 of
Brinkmann and Kontermann MAbs (2017) 9(2): 182-212: antibody
conjugates, e.g. IgG2, F(ab').sub.2 or CovX-Body; IgG or IgG-like
molecules, e.g. IgG, chimeric IgG, .kappa..lamda.-body common HC;
CH1/CL fusion proteins, e.g. scFv2-CH1/CL, VHH2-CH1/CL; `variable
domain only` bispecific antigen-binding molecules, e.g. tandem scFv
(taFV), triplebodies, diabodies (db), dsDb, db(kih), DART, scDB,
dsFv-dsFv, tandAbs, triple heads, tandem dAbNHH, tertravalent
dAb.VHH; Non-Ig fusion proteins, e.g. scFv2-albumin, scDb-albumin,
taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv,
DNL-Fab2-IgG-cytokine.sub.2, ImmTAC (TCR-scFv); modified Fc and CH3
fusion proteins, e.g. scFv-Fc(kih), scFv-Fc(CH3 charge pairs),
scFv-Fc (EW-RVT), scFv-fc (HA-TF), scFv-Fc (SEEDbody),
taFv-Fc(kih), scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih),
Fab-scFv-Fc(BEAT), Fab-scFv-Fc (SEEDbody), DART-Fc, scFv-CH3(kih),
TriFabs; Fc fusions, e.g. Di-diabody, scDb-Fc, taFv-Fc,
scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv.sub.4-Ig,
scFv.sub.2-Fcab; CH3 fusions, e.g. Dia-diabody, scDb-CH3; IgE/IgM
CH2 fusions, e.g. scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion
proteins, e.g. Fab-scFv (bibody), Fab-scFv.sub.2 (tribody), Fab-Fv,
Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, e.g.
DNL-Fab.sub.3, DNL-Fab.sub.2-scFv,
DNL-Fab.sub.2-IgG-cytokine.sub.2; asymmetric IgG or IgG-like
molecules, e.g. IgG(kih), IgG(kih) common LC, ZW1 IgG common LC,
Biclonics common LC, CrossMab, CrossMab(kih), scFab-IgG(kih),
Fab-scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge
pairs+CH1/CL charge pairs, hinge/CH3 charge pairs, SEED-body,
Duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y
scFab-IgG, FcFc*; appended and Fc-modified IgGs, e.g. IgG(kih)-Fv,
IgG HA-TF-Fv, IgG(kih)scFab, scFab-Fc(kih)-scFv.sub.2,
scFab-Fc(kih)-scFv, half DVD-Ig, DVI-Ig (four-in-one),
CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g.
Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT),
Fab-scFv-Fc-SEEDbody, Tri Fab; appended IgGs-HC fusions, e.g.
IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab,
IgG-(C.alpha.C.beta.) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal
Fab) Fab-IgG(C.alpha.C.beta. Fab), Fab-IgG(CR3),
Fab-hinge-IgG(CR3); appended IgGs-LC fusions, e.g. IgG-scFv(LC),
scFv(LC)-IgG, dAb-IgG; appended IgGs-HC and LC fusions, e.g.
DVD-Ig, TVD-Ig, CODV-Ig, scFv.sub.4-IgG, Zybody; Fc fusions, e.g.
Fab-scFv-Fc, scFv.sub.4-Ig; F(ab')2 fusions, e.g.
F(ab')2-scFv.sub.2; CH1/CL fusion proteins e.g.
scFv.sub.2-CH1-hinge/CL; modified IgGs, e.g. DAF (two-in one-IgG),
DutaMab, Mabe; and non-Ig fusions, e.g. DNL-Fab.sub.4-IgG.
[0470] The skilled person is able to design and prepare bispecific
antigen-binding molecules. Methods for producing bispecific
antigen-binding molecules include chemically crosslinking of
antigen-binding molecules or antibody fragments, e.g. with
reducible disulphide or non-reducible thioether bonds, for example
as described in Segal and Bast, 2001. Production of Bispecific
Antigen-binding molecules. Current Protocols in Immunology.
14:IV:2.13:2.13.1-2.13.16, which is hereby incorporated by
reference in its entirety. For example,
N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used
to chemically crosslink e.g. Fab fragments via hinge region SH--
groups, to create disulfide-linked bispecific F(ab).sub.2
heterodimers.
[0471] Other methods for producing bispecific antigen-binding
molecules include fusing antibody-producing hybridomas e.g. with
polyethylene glycol, to produce a quadroma cell capable of
secreting bispecific antibody, for example as described in D. M.
and Bast, B. J. 2001. Production of Bispecific Antigen-binding
molecules. Current Protocols in Immunology.
14:IV:2.13:2.13.1-2.13.16.
[0472] Bispecific antigen-binding molecules according to the
present invention can also be produced recombinantly, by expression
from e.g. a nucleic acid construct encoding polypeptides for the
antigen-binding molecules, for example as described in Antibody
Engineering: Methods and Protocols, Second Edition (Humana Press,
2012), at Chapter 40: Production of Bispecific Antigen-binding
molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz),
or French, How to make bispecific antigen-binding molecules,
Methods Mol. Med. 2000; 40:333-339, the entire contents of both of
which are hereby incorporated by reference.
[0473] For example, a DNA construct encoding the light and heavy
chain variable domains for the two antigen-binding fragments (i.e.
the light and heavy chain variable domains for the antigen-binding
fragment capable of binding CD47, and the light and heavy chain
variable domains for the antigen-binding fragment capable of
binding to another target protein), and including sequences
encoding a suitable linker or dimerization domain between the
antigen-binding fragments can be prepared by molecular cloning
techniques. Recombinant bispecific antibody can thereafter be
produced by expression (e.g. in vitro) of the construct in a
suitable host cell (e.g. a mammalian host cell), and expressed
recombinant bispecific antibody can then optionally be
purified.
[0474] Fc Regions
[0475] In some embodiments the antigen-binding molecules of the
present invention comprise an Fc region.
[0476] An Fc region is composed of CH2 and CH3 regions from one
polypeptide, and CH2 and CH3 regions from another polypeptide. The
CH2 and CH3 regions from the two polypeptides together form the Fc
region.
[0477] In some embodiments, the antigen-binding molecule of the
present invention comprises an Fc region comprising modification in
one or more of the CH2 and CH3 regions promoting association of the
Fc region. Recombinant co-expression of constituent polypeptides of
an antigen-binding molecule and subsequent association leads to
several possible combinations. To improve the yield of the desired
combinations of polypeptides in antigen-binding molecules in
recombinant production, it is advantageous to introduce in the Fc
regions modification(s) promoting association of the desired
combination of heavy chain polypeptides. Modifications may promote
e.g. hydrophobic and/or electrostatic interaction between CH2
and/or CH3 regions of different polypeptide chains. Suitable
modifications are described e.g. in Ha et al., Front. Immnol (2016)
7:394, which is hereby incorporated by reference in its
entirety.
[0478] In some embodiments the antigen antigen-binding molecule of
the present invention comprises an Fc region comprising paired
substitutions in the CH3 regions of the Fc region according to one
of the following formats, as shown in Table 1 of Ha et al., Front.
Immnol (2016) 7:394: KiH, KiH.sub.s-s, HA-TF, ZW1, 7.8.60, DD-KK,
EW-RVT, EW-RVT.sub.s-s, SEED or A107.
[0479] In some embodiments, the Fc region comprises the
"knob-into-hole" or "KiH" modification, e.g. as described e.g. in
U.S. Pat. No. 7,695,936 and Carter, J Immunol Meth 248, 7-15
(2001). In such embodiments, one of the CH3 regions of the Fc
region comprises a "knob" modification, and the other CH3 region
comprises a "hole" modification. The "knob" and "hole"
modifications are positioned within the respective CH3 regions so
that the "knob" can be positioned in the "hole" in order to promote
heterodimerisation (and inhibit homodimerisation) of the
polypeptides and/or stabilise heterodimers. Knobs are constructed
by substituting amino acids having small chains with those having
larger side chains (e.g. tyrosine or tryptophan). Holes are created
by substituting amino acids having large side chains with those
having smaller side chains (e.g. alanine or threonine).
[0480] In some embodiments, one of the CH3 regions of the Fc region
of the antigen-binding molecule of the present invention comprises
the substitution (numbering of positions/substitutions in the Fc,
CH2 and CH3 regions herein is according to the EU numbering system
as described in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md., 1991) T366W, and the other CH3
region of the Fc region comprises the substitution Y407V. In some
embodiments, one of the CH3 regions of the Fc region of the
antigen-binding molecule comprises the substitution T366W, and the
other CH3 region of the Fc region comprises the substitutions T3665
and L368A. In some embodiments, one of the CH3 regions of the Fc
region of the antigen-binding molecule comprises the substitution
T366W, and the other CH3 region of the Fc region comprises the
substitutions Y407V, T3665 and L368A.
[0481] In some embodiments, the Fc region comprises the "DD-KK"
modification as described e.g. in WO 2014/131694 A1. In some
embodiments, one of the CH3 regions comprises the substitutions
K392D and K409D, and the other CH3 region of the Fc region
comprises the substitutions E356K and D399K. The modifications
promote electrostatic interaction between the CH3 regions.
[0482] In some embodiments, the antigen-binding molecule of the
present invention comprises an Fc region modified as described in
Labrijn et al., Proc Natl Acad Sci USA. (2013) 110(13):5145-50,
referred to as `Duobody` format. In some embodiments one of the CH3
regions comprises the substitution K409R, and the other CH3 region
of the Fc region comprises the substitution K405L.
[0483] In some embodiments, the antigen-binding molecule of the
present invention comprises an Fc region comprising the "EEE-RRR"
modification as described in Strop et al., J Mol Biol. (2012)
420(3):204-19. In some embodiments one of the CH3 regions comprises
the substitutions D221E, P228E and L368E, and the other CH3 region
of the Fc region comprises the substitutions D221R, P228R and
K409R.
[0484] In some embodiments, the antigen-binding molecule comprises
an Fc region comprising the "EW-RVT" modification described in Choi
et al., Mol Cancer Ther (2013) 12(12):2748-59. In some embodiments
one of the CH3 regions comprises the substitutions K360E and K409W,
and the other CH3 region of the Fc region comprises the
substitutions Q347R, D399V and F405T.
[0485] In some embodiments, one of the CH3 regions comprises the
substitution S354C, and the other CH3 region of the Fc region
comprises the substitution Y349C. Introduction of these cysteine
residues results in formation of a disulphide bridge between the
two CH3 regions of the Fc region, further stabilizing the
heterodimer (Carter (2001), J Immunol Methods 248, 7-15).
[0486] In some embodiments, the Fc region comprises the
"KiH.sub.S-S" modification. In some embodiments one of the CH3
regions comprises the substitutions T366W and S354C, and the other
CH3 region of the Fc region comprises the substitutions T3665,
L368A, Y407V and Y349C.
[0487] In some embodiments, the antigen-binding molecule of the
present invention comprises an Fc region comprising the "SEED"
modification as described in Davis et al., Protein Eng Des Sel
(2010) 23(4):195-202, in which .beta.-strand segments of human IgG1
CH3 and IgA CH3 are exchanged.
[0488] In some embodiments, one of the CH3 regions comprises the
substitutions S364H and F405A, and the other CH3 region of the Fc
region comprises the substitutions Y349T and T394F (see e.g. Moore
et al., MAbs (2011) 3(6):546-57).
[0489] In some embodiments, one of the CH3 regions comprises the
substitutions T350V, L351Y, F405A and Y407V, and the other CH3
region of the Fc region comprises the substitutions T350V, T366L,
K392L and T394W (see e.g. Von Kreudenstein et al., MAbs (2013)
5(5):646-54).
[0490] In some embodiments, one of the CH3 regions comprises the
substitutions K360D, D399M and Y407A, and the other CH3 region of
the Fc region comprises the substitutions E345R, Q347R, T366V and
K409V (see e.g. Leaver-Fay et al., Structure (2016)
24(4):641-51).
[0491] In some embodiments, one of the CH3 regions comprises the
substitutions K370E and K409W, and the other CH3 region of the Fc
region comprises the substitutions E357N, D399V and F405T (see e.g.
Choi et al., PLoS One (2015) 10(12):e0145349).
[0492] Polypeptides
[0493] The present invention also provides polypeptide constituents
of antigen-binding molecules. The polypeptides may be provided in
isolated or substantially purified form.
[0494] The antigen-binding molecule of the present invention may
be, or may comprise, a complex of polypeptides.
[0495] In the present specification where a polypeptide comprises
more than one domain or region, it will be appreciated that the
plural domains/regions are preferably present in the same
polypeptide chain. That is, the polypeptide comprises more than one
domain or region is a fusion polypeptide comprising the
domains/regions.
[0496] In some embodiments a polypeptide according to the present
invention comprises, or consists of, a VH as described herein. In
some embodiments a polypeptide according to the present invention
comprises, or consists of, a VL as described herein.
[0497] In some embodiments, the polypeptide additionally comprises
one or more antibody heavy chain constant regions (CH). In some
embodiments, the polypeptide additionally comprises one or more
antibody light chain constant regions (CL). In some embodiments,
the polypeptide comprises a CH1, CH2 region and/or a CH3 region of
an immunoglobulin (Ig).
[0498] In some embodiments the polypeptide comprises one or more
regions of an immunoglobulin heavy chain constant sequence. In some
embodiments the polypeptide comprises a CH1 region as described
herein. In some embodiments the polypeptide comprises a CH1-CH2
hinge region as described herein. In some embodiments the
polypeptide comprises a CH2 region as described herein. In some
embodiments the polypeptide comprises a CH3 region as described
herein.
[0499] In some embodiments the polypeptide comprises a CH3 region
comprising any one of the following amino acid
substitutions/combinations of amino acid substitutions (shown e.g.
in Table 1 of Ha et al., Front. Immnol (2016) 7:394, incorporated
by reference hereinabove): T366W; T3665, L368A and Y407V; T366W and
S354C; T3665, L368A, Y407V and Y349C; S364H and F405A; Y349T and
T394F; T350V, L351Y, F405A and Y407V; T350V, T366L, K392L and
T394W; K360D, D399M and Y407A; E345R, Q347R, T366V and K409V; K409D
and K392D; D399K and E356K; K360E and K409W; Q347R, D399V and
F405T; K360E, K409W and Y349C; Q347R, D399V, F405T and S354C; K370E
and K409W; and E357N, D399V and F405T.
[0500] In some embodiments the CH2 and/or CH3 regions of the
polypeptide comprise one or more amino acid substitutions for
promoting association of the polypeptide with another polypeptide
comprising a CH2 and/or CH3 region.
[0501] In some embodiments the polypeptide comprises one or more
regions of an immunoglobulin light chain constant sequence. In some
embodiments the polypeptide comprises a CL region as described
herein.
[0502] In some embodiments, the polypeptide according to the
present invention comprises a structure from N- to C-terminus
according to one of the following: [0503] (i) VH [0504] (ii) VL
[0505] (iii) VH-CH1 [0506] (iv) VL-CL [0507] (v) VL-CH1 [0508] (vi)
VH-CL [0509] (vii) VH-CH1-CH2-CH3 [0510] (viii) VL-CL-CH2-CH3
[0511] (ix) VL-CH1-CH2-CH3 [0512] (x) VH-CL-CH2-CH3
[0513] Also provided by the present invention are antigen-binding
molecules composed of the polypeptides of the present invention. In
some embodiments, the antigen-binding molecule of the present
invention comprises one of the following combinations of
polypeptides: [0514] (A) VH+VL [0515] (B) VH-CH1+VL-CL [0516] (C)
VL-CH1+VH-CL [0517] (D) VH-CH1-CH2-CH3+VL-CL [0518] (E)
VH-CL-CH2-CH3+VL-CH1 [0519] (F) VL-CH1-CH2-CH3+VH-CL [0520] (G)
VL-CL-CH2-CH3+VH-CH1 [0521] (H) VH-CH1-CH2-CH3+VL-CL-CH2-CH3 [0522]
(I) VH-CL-CH2-CH3+VL-CH1-CH2-CH3
[0523] In some embodiments the antigen-binding molecule comprises
more than one of a polypeptide of the combinations shown in (A) to
(I) above. By way of example, with reference to (D) above, in some
embodiments the antigen-binding molecule comprises two polypeptides
comprising the structure VH-CH1-CH2-CH3, and two polypeptides
comprising the structure VL-CL.
[0524] In some embodiments, the antigen-binding molecule of the
present invention comprises one of the following combinations of
polypeptides: [0525] (J) VH (anti-CD47)+VL (anti-CD47) [0526] (K)
VH (anti-CD47)-CH1+VL (anti-CD47)-CL [0527] (L) VL
(anti-CD47)-CH1+VH (anti-CD47)-CL [0528] (M) VH
(anti-CD47)-CH1-CH2-CH3+VL (anti-CD47)-CL [0529] (N) VH
(anti-CD47)-CL-CH2-CH3+VL (anti-CD47)-CH1 [0530] (O) VL
(anti-CD47)-CH1-CH2-CH3+VH (anti-CD47)-CL [0531] (P) VL
(anti-CD47)-CL-CH2-CH3+VH (anti-CD47)-CH1 [0532] (Q) VH
(anti-CD47)-CH1-CH2-CH3+VL (anti-CD47)-CL-CH2-CH3 [0533] (R) VH
(anti-CD47)-CL-CH2-CH3+VL (anti-CD47)-CH1-CH2-CH3
[0534] Wherein: "VH(anti-CD47)" refers to the VH of an
antigen-binding molecule capable of binding to CD47 as described
herein, e.g. as defined in one of (1) to (35); and "VL(anti-CD47)"
refers to the VL of an antigen-binding molecule capable of binding
to CD47 as described herein, e.g. as defined in one of (36) to
(70); In some embodiments the polypeptide comprises or consists of
an amino acid sequence having at least 70%, preferably one of 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
amino acid sequence identity to the amino acid sequence of one of
SEQ ID NOs:23, 31, 39, 44, 49, 57, 65, 73, 178, 179, 127, 128, 129,
130, 131, 132, 133, 134, 135 or 136.
[0535] In some embodiments the polypeptide comprises or consists of
an amino acid sequence having at least 70%, preferably one of 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
amino acid sequence identity to the amino acid sequence of one of
SEQ ID NOs:107, 108, 109, 110, 111, 112, 113, 114, 159, 160, 161,
162, 163, 164, 165, 166, 167 or 168.
[0536] Linkers and Additional Sequences
[0537] In some embodiments the antigen-binding molecules and
polypeptides of the present invention comprise a hinge region. In
some embodiments a hinge region is provided between a CH1 region
and a CH2 region. In some embodiments a hinge region is provided
between a CL region and a CH2 region. In some embodiments the hinge
region comprises, or consists of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:120.
[0538] In some embodiments the antigen-binding molecules and
polypeptides of the present invention comprise one or more linker
sequences between amino acid sequences. A linker sequence may be
provided at one or both ends of one or more of a VH, VL, CH1-CH2
hinge region, CH2 region and a CH3 region of the antigen-binding
molecule/polypeptide.
[0539] Linker sequences are known to the skilled person, and are
described, for example in Chen et al., Adv Drug Deliv Rev (2013)
65(10): 1357-1369, which is hereby incorporated by reference in its
entirety. In some embodiments, a linker sequence may be a flexible
linker sequence. Flexible linker sequences allow for relative
movement of the amino acid sequences which are linked by the linker
sequence. Flexible linkers are known to the skilled person, and
several are identified in Chen et al., Adv Drug Deliv Rev (2013)
65(10): 1357-1369. Flexible linker sequences often comprise high
proportions of glycine and/or serine residues.
[0540] In some embodiments, the linker sequence comprises at least
one glycine residue and/or at least one serine residue. In some
embodiments the linker sequence consists of glycine and serine
residues. In some embodiments, the linker sequence has a length of
1-2, 1-3, 1-4, 1-5 or 1-10 amino acids.
[0541] The antigen-binding molecules and polypeptides of the
present invention may additionally comprise further amino acids or
sequences of amino acids. For example, the antigen-binding
molecules and polypeptides may comprise amino acid sequence(s) to
facilitate expression, folding, trafficking, processing,
purification or detection of the antigen-binding
molecule/polypeptide. For example, the antigen-binding
molecule/polypeptide may comprise a sequence encoding a His, (e.g.
6.times.His), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally
at the N- or C-terminus of the antigen-binding
molecule/polypeptide. In some embodiments the antigen-binding
molecule/polypeptide comprises a detectable moiety, e.g. a
fluorescent, lunminescent, immuno-detectable, radio, chemical,
nucleic acid or enzymatic label.
[0542] The antigen-binding molecules and polypeptides of the
present invention may additionally comprise a signal peptide (also
known as a leader sequence or signal sequence). Signal peptides
normally consist of a sequence of 5-30 hydrophobic amino acids,
which form a single alpha helix. Secreted proteins and proteins
expressed at the cell surface often comprise signal peptides.
[0543] The signal peptide may be present at the N-terminus of the
antigen-binding molecule/polypeptide, and may be present in the
newly synthesised antigen-binding molecule/polypeptide. The signal
peptide provides for efficient trafficking and secretion of the
antigen-binding molecule/polypeptide. Signal peptides are often
removed by cleavage, and thus are not comprised in the mature
antigen-binding molecule/polypeptide secreted from the cell
expressing the antigen-binding molecule/polypeptide.
[0544] Signal peptides are known for many proteins, and are
recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL,
Protein Information Resource, Protein Data Bank, Ensembl, and
InterPro, and/or can be identified/predicted e.g. using amino acid
sequence analysis tools such as SignalP (Petersen et al., 2011
Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008
Bioinformatics 24: 2172-2176).
[0545] In some embodiments, the signal peptide of the
antigen-binding molecule/polypeptide of the present invention
comprises, or consists of, an amino acid sequence having at least
80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to the amino acid sequence
of one of SEQ ID NOs:81 to 86.
[0546] Labels and Conjugates
[0547] In some embodiments the antigen-binding molecules of the
present invention additionally comprise a detectable moiety.
[0548] In some embodiments the antigen-binding molecule comprises a
detectable moiety, e.g. a fluorescent label, phosphorescent label,
luminescent label, immuno-detectable label (e.g. an epitope tag),
radiolabel, chemical, nucleic acid or enzymatic label. The
antigen-binding molecule may be covalently or non-covalently
labelled with the detectable moiety.
[0549] Fluorescent labels include e.g. fluorescein, rhodamine,
allophycocyanin, eosine and NDB, green fluorescent protein (GFP)
chelates of rare earths such as europium (Eu), terbium (Tb) and
samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl
umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5. Radiolabels
include radioisotopes such as lodine.sup.123, lodine.sup.125,
lodine.sup.126, lodine.sup.131, lodine.sup.133, Bromine.sup.77,
Technetium.sup.99m, Indium.sup.111, Indium.sup.113m,
Gallium.sup.67, Gallium.sup.68, Ruthenium.sup.95, Ruthenium.sup.97,
Ruthenium.sup.103, Ruthenium.sup.105, Mercury.sup.207,
Mercury.sup.203, Rhenium.sup.99m, Rhenium.sup.101, Rhenium.sup.105,
Scandium.sup.47, Tellurium.sup.121m, Tellurium.sup.122m,
Tellurium.sup.125m, Thulium.sup.165, Thuliuml.sup.167,
Thulium.sup.168, Copper.sup.67, Fluorine.sup.18, Yttrium.sup.90,
Palladium.sup.100, Bismuth.sup.217 and Antimon.sup.211. Luminescent
labels include as radioluminescent, chemiluminescent (e.g.
acridinium ester, luminol, isoluminol) and bioluminescent labels.
Immuno-detectable labels include haptens, peptides/polypeptides,
antibodies, receptors and ligands such as biotin, avidin,
streptavidin or digoxigenin. Nucleic acid labels include aptamers.
Enzymatic labels include e.g. peroxidase, alkaline phosphatase,
glucose oxidase, beta-galactosidase and luciferase.
[0550] In some embodiments the antigen-binding molecules of the
present invention are conjugated to a chemical moiety. The chemical
moiety may be a moiety for providing a therapeutic effect.
Antibody-drug conjugates are reviewed e.g. in Parslow et al.,
Biomedicines. 2016 September; 4(3):14. In some embodiments, the
chemical moiety may be a drug moiety (e.g. a cytotoxic agent). In
some embodiments, the drug moiety may be a chemotherapeutic agent.
In some embodiments, the drug moiety is selected from
calicheamicin, DM1, DM4, monomethylauristatin E (MMAE),
monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin,
D6.5 and PBD.
[0551] Particular Exemplary Embodiments of the Antigen-Binding
Molecules
[0552] In some embodiments the antigen-binding molecule comprises,
or consists of: [0553] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:107; and [0554] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:108.
[0555] In some embodiments the antigen-binding molecule comprises,
or consists of: [0556] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:109; and [0557] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:110.
[0558] In some embodiments the antigen-binding molecule comprises,
or consists of: [0559] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:111; and [0560] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:112.
[0561] In some embodiments the antigen-binding molecule comprises,
or consists of: [0562] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:113; and [0563] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:114.
[0564] In some embodiments the antigen-binding molecule comprises,
or consists of: [0565] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:159; and [0566] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:160.
[0567] In some embodiments the antigen-binding molecule comprises,
or consists of: [0568] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:161; and [0569] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:160.
[0570] In some embodiments the antigen-binding molecule comprises,
or consists of: [0571] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:162; and [0572] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:160.
[0573] In some embodiments the antigen-binding molecule comprises,
or consists of: [0574] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:163; and [0575] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:160.
[0576] In some embodiments the antigen-binding molecule comprises,
or consists of: [0577] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:164; and [0578] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:160.
[0579] In some embodiments the antigen-binding molecule comprises,
or consists of: [0580] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:163; and [0581] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:165.
[0582] In some embodiments the antigen-binding molecule comprises,
or consists of: [0583] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:164; and [0584] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:165.
[0585] In some embodiments the antigen-binding molecule comprises,
or consists of: [0586] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:163; and [0587] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:166.
[0588] In some embodiments the antigen-binding molecule comprises,
or consists of: [0589] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:164; and [0590] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:166.
[0591] In some embodiments the antigen-binding molecule comprises,
or consists of: [0592] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:164; and [0593] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:167.
[0594] In some embodiments the antigen-binding molecule comprises,
or consists of: [0595] (i) two polypeptides comprising, or
consisting of, an amino acid sequence having at least 70%,
preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% amino acid sequence identity to the amino
acid sequence of SEQ ID NO:164; and [0596] (ii) two polypeptides
comprising, or consisting of, an amino acid sequence having at
least 70%, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity
to the amino acid sequence of SEQ ID NO:168.
[0597] Functional Properties of the Antigen-Binding Molecules
[0598] The antigen-binding molecules described herein may be
characterised by reference to certain functional properties. In
some embodiments, the antigen-binding molecule described herein may
possess one or more of the following properties: [0599] binds to
CD47; [0600] binds to CD47-expressing cells; [0601] inhibits
interaction between CD47 and SIRP.alpha.; [0602] inhibits
SIRP.alpha.-mediated signalling; [0603] increases phagocytosis of
CD47-expressing cells by phagocytic cells (e.g. macrophages);
[0604] increases the number/proportion of cancer antigen-specific
immune cells [0605] does not cause substantial hemagglutination;
[0606] causes less hemagglutination as compared to a reference
anti-CD47 antibody; [0607] increases killing of cancer cells;
[0608] inhibits the development/progression of cancer.
[0609] The antigen-binding molecules and antigen-binding domains
described herein preferably display specific binding to the
relevant target antigen(s) (e.g. CD47). As used herein, "specific
binding" refers to binding which is selective for the antigen, and
which can be discriminated from non-specific binding to non-target
antigen. An antigen-binding molecule/domain that specifically binds
to a target molecule preferably binds the target with greater
affinity, and/or with greater duration than it binds to other,
non-target molecules.
[0610] The ability of a given polypeptide to bind specifically to a
given molecule can be determined by analysis according to methods
known in the art, such as by ELISA, Surface Plasmon Resonance (SPR;
see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442),
Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol
Screen 20(4): 498-507), flow cytometry, or by a radiolabeled
antigen-binding assay (RIA) enzyme-linked immunosorbent assay.
Through such analysis binding to a given molecule can be measured
and quantified. In some embodiments, the binding may be the
response detected in a given assay.
[0611] In some embodiments, the extent of binding of the
antigen-binding molecule to an non-target molecule is less than
about 10% of the binding of the antibody to the target molecule as
measured, e.g. by ELISA, SPR, Bio-Layer Interferometry or by RIA.
Alternatively, binding specificity may be reflected in terms of
binding affinity where the antigen-binding molecule binds with a
dissociation constant (KD) that is at least 0.1 order of magnitude
(i.e. 0.1.times.10.sup.n, where n is an integer representing the
order of magnitude) greater than the KD of the antigen-binding
molecule towards a non-target molecule. This may optionally be one
of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or
2.0.
[0612] In some embodiments, the antigen-binding molecule described
herein binds to CD47 with a K.sub.D of 10 .mu.M or less, preferably
one of .ltoreq.5 .mu.M, .ltoreq.2 .mu.M, .ltoreq.1 .mu.M,
.ltoreq.500 nM, .ltoreq.100 nM, .ltoreq.75 nM, .ltoreq.50 nM,
.ltoreq.40 nM, .ltoreq.30 nM, .ltoreq.20 nM, .ltoreq.15 nM,
.ltoreq.12.5 nM, .ltoreq.10 nM, .ltoreq.9 nM, .ltoreq.8 nM,
.ltoreq.7 nM, .ltoreq.6 nM, .ltoreq.5 nM, .ltoreq.4 nM .ltoreq.3
nM, .ltoreq.2 nM, .ltoreq.1 nM or .ltoreq.500 .mu.M. In some
embodiments, the antigen-binding molecule binds to CD47 with an
affinity of K.sub.D=.ltoreq.10 nM, .ltoreq.9 nM, .ltoreq.8 nM,
.ltoreq.7 nM or .ltoreq.6 nM, e.g. .about.5 nM.
[0613] In some embodiments, the antigen-binding molecule binds to
CD47 with an affinity of binding (e.g. as determined by ELISA) of
EC50=100 .mu.g/ml or less, preferably one of .ltoreq.90 .mu.g/ml,
.ltoreq.80 .mu.g/ml, .ltoreq.70 .mu.g/ml, .ltoreq.60 .mu.g/ml,
.ltoreq.50 .mu.g/ml, .ltoreq.40 .mu.g/ml, .ltoreq.30 .mu.g/ml,
.ltoreq.20 .mu.g/ml, .ltoreq.10 .mu.g/ml, .ltoreq.9 .mu.g/ml,
.ltoreq.8 .mu.g/ml, .ltoreq.7 .mu.g/ml, .ltoreq.6 .mu.g/ml,
.ltoreq.5 .mu.g/ml, .ltoreq.4 .mu.g/ml, .ltoreq.3 .mu.g/ml,
.ltoreq.2 .mu.g/ml, .ltoreq.1.5 .mu.g/ml, .ltoreq.1 .mu.g/ml,
.ltoreq.0.5 .mu.g/ml, .ltoreq.0.25 .mu.g/ml, or .ltoreq.0.1
.mu.g/ml.
[0614] The antigen-binding molecules of the present invention may
bind to a particular region of interest of the target antigen(s).
The antigen-binding region of an antigen-binding molecule according
to the present domain may bind to linear epitope of a target
antigen (e.g. CD47), consisting of a contiguous sequence of amino
acids (i.e. an amino acid primary sequence). In some embodiments,
the antigen-binding region molecule may bind to a conformational
epitope of a target antigen (i.e. CD47), consisting of a
discontinuous sequence of amino acids of the amino acid
sequence.
[0615] In some embodiments, the antigen-binding molecule of the
present invention is capable of binding to CD47. In some
embodiments, the antigen-binding molecule is capable of binding to
CD47 in an extracellular region of CD47. In some embodiments, the
antigen-binding molecule is capable of binding to CD47 in
extracellular region 1 of CD47 (e.g. the region shown in SEQ ID
NO:10). In some embodiments, the antigen-binding molecule is
capable of binding to the V-type Ig-like domain of CD47 (e.g. the
region shown in SEQ ID NO:9).
[0616] In some embodiments the antigen-binding molecule is capable
of binding to a polypeptide comprising or consisting of the amino
acid sequence shown in SEQ ID NO:10. In some embodiments the
antigen-binding molecule is capable of binding to a polypeptide
comprising or consisting of the amino acid sequence shown in SEQ ID
NO:9. In some embodiments the antigen-binding molecule is capable
of binding to a polypeptide comprising or consisting of the amino
acid sequence shown in SEQ ID NO:9. In some embodiments the
antigen-binding molecule is capable of binding to a peptide or
polypeptide comprising or consisting of the amino acid sequence
shown in SEQ ID NO:21. In some embodiments the antigen-binding
molecule is capable of binding to a peptide or polypeptide
comprising or consisting of the amino acid sequence shown in SEQ ID
NO:22.
[0617] As used herein, a "peptide" refers to a chain of two or more
amino acid monomers linked by peptide bonds. A peptide typically
has a length in the region of about 2 to 50 amino acids. A
"polypeptide" is a polymer chain of two or more peptides.
Polypeptides typically have a length greater than about 50 amino
acids.
[0618] The ability of an antigen-binding molecule to bind to a
given peptide/polypeptide can be analysed by methods well known to
the skilled person, including analysis by ELISA, immunoblot (e.g.
western blot), immunoprecipitation, surface plasmon resonance and
biolayer interferometry.
[0619] In some embodiments the antigen-binding molecule is capable
of binding the same region of CD47, or an overlapping region of
CD47, to the region of CD47 which is bound by an antibody
comprising the VH and VL sequences of one of clones 1-1-A1_BM,
1-1-A1, 5-48-A6, 5-48-D2, 11A1H1, 11A1H2, 11A1H3, 11A1H4, 11A1H5,
11A1H6, 11A1H7, 11A1H8, 11A1H9, 11A1H10 or 11A1H11.
[0620] The region of a peptide/polypeptide to which an antibody
binds can be determined by the skilled person using various methods
well known in the art, including X-ray co-crystallography analysis
of antibody-antigen complexes, peptide scanning, mutagenesis
mapping, hydrogen-deuterium exchange analysis by mass spectrometry,
phage display, competition ELISA and proteolysis-based `protection`
methods. Such methods are described, for example, in Gershoni et
al., BioDrugs, 2007, 21(3):145-156, which is hereby incorporated by
reference in its entirety.
[0621] In some embodiments the antigen-binding molecule of the
present invention displays cross-reactivity with CD47 of a
non-human primate. That is, in some embodiments the antigen-binding
molecule binds to both human CD47 and CD47 from a non-human
primate. In some embodiments the non-human primate is rhesus
macaque (Macaca mulatta).
[0622] In some embodiments the antigen-binding molecule of the
present invention binds to CD47 in a region which is accessible to
an antigen-binding molecule (i.e., an extracellular antigen-binding
molecule) when CD47 is expressed at the cell surface (i.e. in or at
the cell membrane). In some embodiments the antigen-binding
molecule is capable of binding to CD47 expressed at the cell
surface of a cell expressing CD47. In some embodiments the
antigen-binding molecule is capable of binding to CD47-expressing
cells (e.g. myeloid cells, myeloid leukemia cells, HL-60 cells,
HMC-1 cells, HEL cells or Raji cells).
[0623] The ability of an antigen-binding molecule to bind to a
given cell type can be analysed by contacting cells with the
antigen-binding molecule, and detecting antigen-binding molecule
bound to the cells, e.g. after a washing step to remove unbound
antigen-binding molecule. The ability of an antigen-binding
molecule to bind to immune cell surface molecule-expressing cells
and/or cancer cell antigen-expressing cells can be analysed by
methods such as flow cytometry and immunofluorescence
microscopy.
[0624] The antigen-binding molecule of the present invention may be
an antagonist of CD47. In some embodiments, the antigen-binding
molecule is capable of inhibiting a function or process (e.g.
interaction, signalling or other activity) mediated by CD47.
Herein, `inhibition` refers to a reduction, decrease or lessening
relative to a control condition.
[0625] In some embodiments the antigen-binding molecule of the
present invention is capable of inhibiting interaction between CD47
and a ligand for CD47. In some embodiments the antigen-binding
molecule of the present invention is capable of inhibiting
interaction between CD47 and SIRP.alpha..
[0626] The ability of an antigen-binding molecule to inhibit
interaction between two factors can be determined for example by
analysis of interaction in the presence of, or following incubation
of one or both of the interaction partners with, the
antibody/fragment. An example of a suitable assay to determine
whether a given antigen-binding molecule is capable of inhibiting
interaction between two interaction partners is a competition ELISA
assay.
[0627] An antigen-binding molecule which is capable of inhibiting a
given interaction (e.g. between CD47 and SIRP.alpha.) is identified
by the observation of a reduction/decrease in the level of
interaction between the interaction partners in the presence of--or
following incubation of one or both of the interaction partners
with--the antigen-binding molecule, as compared to the level of
interaction in the absence of the antigen-binding molecule (or in
the presence of an appropriate control antigen-binding molecule).
Suitable analysis can be performed in vitro, e.g. using recombinant
interaction partners or using cells expressing the interaction
partners. Cells expressing interaction partners may do so
endogenously, or may do so from nucleic acid introduced into the
cell. For the purposes of such assays, one or both of the
interaction partners and/or the antigen-binding molecule may be
labelled or used in conjunction with a detectable entity for the
purposes of detecting and/or measuring the level of
interaction.
[0628] The ability of an antigen-binding molecule to inhibit
interaction between two binding partners can also be determined by
analysis of the downstream functional consequences of such
interaction. For example, downstream functional consequences of
interaction between CD47 and SIRP.alpha. may include
SIRP.alpha.-mediated signalling. For example, the ability of an
antigen-binding molecule to inhibit interaction of CD47 and
SIRP.alpha. may be determined by analysis of SIRP.alpha. ITIM
phosphorylation, or analysis of phagocytosis of CD47-expressing
cell by a SIRP.alpha.-expressing cell.
[0629] In some embodiments, the antigen-binding molecule of the
present invention is capable of inhibiting interaction between CD47
and SIRP.alpha. to less than less than 1 times, e.g. .ltoreq.0.99
times, .ltoreq.0.95 times, .ltoreq.0.9 times, .ltoreq.0.85 times,
.ltoreq.0.8 times, .ltoreq.0.75 times, .ltoreq.0.7 times,
.ltoreq.0.65 times, .ltoreq.0.6 times, .ltoreq.0.55 times,
.ltoreq.0.5 times, .ltoreq.0.45 times, .ltoreq.0.4 times,
.ltoreq.0.35 times, .ltoreq.0.3 times, .ltoreq.0.25 times,
.ltoreq.0.2 times, .ltoreq.0.15 times, .ltoreq.0.1 times,
.ltoreq.0.05 times, or .ltoreq.0.01 times the level of interaction
between CD47 and SIRP.alpha. in the absence of the antigen-binding
molecule (or in the presence of an appropriate control
antigen-binding molecule).
[0630] In some embodiments, the antigen-binding molecule inhibits
interaction between CD47 and SIRP.alpha. with an IC50 (e.g. as
determined by ELISA) of 100 .mu.g/ml or less, preferably one of
.ltoreq.90 .mu.g/ml, .ltoreq.80 .mu.g/ml, .ltoreq.70 .mu.g/ml,
.ltoreq.60 .mu.g/ml, .ltoreq.50 .mu.g/ml, .ltoreq.40 .mu.g/ml,
.ltoreq.30 .mu.g/ml, .ltoreq.20 .mu.g/ml, .ltoreq.10 .mu.g/ml,
.ltoreq.9 .mu.g/ml, .ltoreq.8 .mu.g/ml, .ltoreq.7 .mu.g/ml,
.ltoreq.6 .mu.g/ml, .ltoreq.5 .mu.g/ml, .ltoreq.4 .mu.g/ml,
.ltoreq.3 .mu.g/ml, .ltoreq.2 .mu.g/ml, .ltoreq.1.5 .mu.g/ml,
.ltoreq.1 .mu.g/ml, .ltoreq.0.5 .mu.g/ml, .ltoreq.0.25 .mu.g/ml, or
.ltoreq.0.1 .mu.g/ml.
[0631] In some embodiments the antigen-binding molecule inhibits
SIRP.alpha.-mediated signalling. SIRP.alpha.-mediated signalling
can be analysed using SIRP.alpha.-expressing cells e.g. using an
assay for detecting and/or quantifying SIRP.alpha. ITIM
phosphorylation, or using in vitro assay of phagocytosis of
CD47-expressing cells (e.g. Raji cells) by SIRP.alpha.-expressing
cells (e.g. macrophages). For example, an in vitro assay of
phagocytosis of CD47-expressing cells by SIRP.alpha.-expressing
cells may be performed as described in Feng et al., Proc Natl Acad
Sci USA. (2015) 112(7): 2145-2150 (hereby incorporated by reference
in its entirety), or as described in the experimental examples
herein.
[0632] In some embodiments, the antigen-binding molecule of the
present invention is capable of inhibiting SIRP.alpha.-mediated
signalling to less than 1 times, e.g. .ltoreq.0.99 times,
.ltoreq.0.95 times, .ltoreq.0.9 times, .ltoreq.0.85 times,
.ltoreq.0.8 times, .ltoreq.0.75 times, .ltoreq.0.7 times,
.ltoreq.0.65 times, .ltoreq.0.6 times, .ltoreq.0.55 times,
.ltoreq.0.5 times, .ltoreq.0.45 times, .ltoreq.0.4 times,
.ltoreq.0.35 times, .ltoreq.0.3 times, .ltoreq.0.25 times,
.ltoreq.0.2 times, .ltoreq.0.15 times, .ltoreq.0.1 times,
.ltoreq.0.05 times, or .ltoreq.0.01 times the level of
SIRP.alpha.-mediated signalling in the absence of the
antigen-binding molecule (or in the presence of an appropriate
control antigen-binding molecule).
[0633] In some embodiments, the antigen-binding molecule of the
present invention is capable of increasing phagocytosis of
CD47-expressing cells. In some embodiments, the antigen-binding
molecule of the present invention is capable of increasing
phagocytosis of CD47-expressing cells (e.g. Raji cells) by
SIRP.alpha.-expressing cells (e.g. macrophages).
[0634] An antigen-binding molecule which is capable of increasing
phagocytosis of CD47-expressing cells by SIRP.alpha.-expressing
cells is identified by the observation of an increased level of
phagocytosis of the CD47-expressing cells by the
SIRP.alpha.-expressing cells in the presence of--or following
incubation of the CD47-expressing cells with--the antigen-binding
molecule, as compared to the level of phagocytosis detected in the
absence of the antigen-binding molecule (or in the presence of an
appropriate control antigen-binding molecule).
[0635] In some embodiments, the antigen-binding molecule of the
present invention is capable of increasing phagocytosis of
CD47-expressing cells (e.g. Raji cells) by SIRP.alpha.-expressing
cells (e.g. macrophages) to more than 1 times, e.g. .gtoreq.1.01
times, .gtoreq.1.02 times, .gtoreq.1.03 times, .gtoreq.1.04 times,
.gtoreq.1.05 times, .gtoreq.1.1 times, .gtoreq.1.2 times,
.gtoreq.1.3 times, .gtoreq.1.4 times, .gtoreq.1.5 times,
.gtoreq.1.6 times, .gtoreq.1.7 times, .gtoreq.1.8 times,
.gtoreq.1.9 times, .gtoreq.2 times, .gtoreq.3 times, .gtoreq.4
times, .gtoreq.5 times, .gtoreq.6 times, .gtoreq.7 times, .gtoreq.8
times, .gtoreq.9 times or .gtoreq.10 times the level phagocytosis
of the CD47-expressing cells by the SIRP.alpha.-expressing cells in
the absence of the antigen-binding molecule (or in the presence of
an appropriate control antigen-binding molecule).
[0636] In some embodiments, the antigen-binding molecule of the
present invention is capable of increasing the number/proportion of
cancer antigen-specific immune cells (e.g. CD8+ T cells or CD8+
CTLs) relative to a negative control condition, e.g. in an
appropriate in vitro assay, or in vivo. Tseng et al., Proc Natl
Acad Sci USA. (2013) 110(27): 11103-11108 (hereby incorporated by
reference in its entirety) demonstrated that increased phagocytosis
of CD47-expressing cancer cells by macrophages in the presence of
an anti-CD47 antibody was associated with increased priming of
cancer antigen-specific CD8+ T cells. Antigen-binding molecules
capable of causing an increase in the number/proportion of cancer
antigen-specific immune cells can be identified using a T cell
priming assay e.g. as described in Tseng et al., Proc Natl Acad Sci
USA. (2013) 110(27): 11103-11108.
[0637] In some embodiments, the antigen-binding molecule of the
present invention does not cause substantial hemagglutination (e.g.
at concentrations of up to 400 .mu.g/ml). Hemagglutination refers
to agglutination of red blood cells (erythrocytes).
[0638] An agent which causes hemagglutination may be referred to as
a hemagglutinin. In some embodiments the antigen-binding molecule
of the present invention is not a hemagglutinin.
[0639] The ability of an antibody to cause hemagglutination can be
analysed e.g. using an in vitro hemagglutination assay. A suitable
assay of hemagglutination for the purposes of such analysis is
described e.g. in Example 5 of WO 2013/119714 A1 (hereby
incorporated by reference in its entirety), or the assay of
hemagglutination described in the experimental examples herein.
"Substantial" hemagglutination may be a level of hemagglutination
which is more than 2 times, e.g. more than 3, 4, 5, 6, 7, 8, 9 or
10 times the level of hemagglutination detected in the absence of
the antigen-binding molecule (or in the presence of an appropriate
control antigen-binding molecule which does not cause
hemagglutination).
[0640] In some embodiments, the antigen-binding molecule of the
present invention causes less hemagglutination as compared to a
reference anti-CD47 antibody (e.g. a prior art anti-CD47 antibody).
In some embodiments, the antigen-binding molecule of the present
invention causes less than 1 times, e.g. .ltoreq.0.99 times,
.ltoreq.0.95 times, .ltoreq.0.9 times, .ltoreq.0.85 times,
.ltoreq.0.8 times, .ltoreq.0.75 times, .ltoreq.0.7 times,
.ltoreq.0.65 times, .ltoreq.0.6 times, .ltoreq.0.55 times,
.ltoreq.0.5 times, .ltoreq.0.45 times, .ltoreq.0.4 times,
.ltoreq.0.35 times, .ltoreq.0.3 times, .ltoreq.0.25 times,
.ltoreq.0.2 times, .ltoreq.0.15 times, .ltoreq.0.1 times,
.ltoreq.0.05 times, or .ltoreq.0.01 times the level of
hemagglutination as compared to a reference anti-CD47 antibody
(e.g. a prior art anti-CD47 antibody), e.g. as determined using an
in vitro assay of hemagglutination.
[0641] In some embodiments the antigen-binding molecule of the
present invention increases killing of cancer cells. In some
embodiments the antigen-binding molecule of the present invention
causes a reduction in the number of cancer cells in vivo, e.g. as
compared to an appropriate control condition. The cancer may be a
cancer expressing CD47, or may comprise cells expressing CD47 (e.g.
the CD47+ AML cell line, HL-60). The antigen-binding molecule of
the present invention may be analysed for anticancer activity in an
appropriate in vivo model, e.g. an AML cell line-derived xenograft
model.
[0642] In some embodiments the antigen-binding molecule of the
present invention causes a greater reduction of the number of
cancer cells in vivo in a AML cell line-derived xenograft model as
compared to a reference anti-CD47 antibody (e.g. a prior art
anti-CD47 antibody).
[0643] In some embodiments, administration of an antigen-binding
molecule according to the present invention may cause one or more
of: inhibition of the development/progression of the cancer, a
delay to/prevention of onset of the cancer, a reduction in/delay
to/prevention of tumor growth, a reduction in/delay to/prevention
of metastasis, a reduction in the severity of the symptoms of the
cancer, a reduction in the number of cancer cells, a reduction in
tumour size/volume, and/or an increase in survival (e.g.
progression free survival), e.g. as determined in an AML cell
line-derived xenograft model.
[0644] Chimeric Antigen Receptors (CARs)
[0645] The present invention also provides Chimeric Antigen
Receptors (CARs) comprising the antigen-binding polypeptides or
polypeptides of the present invention.
[0646] CARs are recombinant receptors that provide both
antigen-binding and T cell activating functions. CAR structure and
engineering is reviewed, for example, in Dotti et al., Immunol Rev
(2014) 257(1), hereby incorporated by reference in its entirety.
CARs comprise an antigen-binding region linked to a cell membrane
anchor region and a signalling region. An optional hinge region may
provide separation between the antigen-binding region and cell
membrane anchor region, and may act as a flexible linker.
[0647] The CAR of the present invention comprises an
antigen-binding region which comprises or consists of the
antigen-binding molecule of the present invention, or which
comprises or consists of a polypeptide according to the
invention.
[0648] The cell membrane anchor region is provided between the
antigen-binding region and the signalling region of the CAR and
provides for anchoring the CAR to the cell membrane of a cell
expressing a CAR, with the antigen-binding region in the
extracellular space, and signalling region inside the cell. In some
embodiments, the CAR comprises a cell membrane anchor region
comprising or consisting of an amino acid sequence which comprises,
consists of, or is derived from, the transmembrane region amino
acid sequence for one of CD3-.zeta., CD4, CD8 or CD28. As used
herein, a region which is `derived from` a reference amino acid
sequence comprises an amino acid sequence having at least 60%, e.g.
one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference
sequence.
[0649] The signalling region of a CAR allows for activation of the
T cell. The CAR signalling regions may comprise the amino acid
sequence of the intracellular domain of CD3-, which provides
immunoreceptor tyrosine-based activation motifs (ITAMs) for
phosphorylation and activation of the CAR-expressing T cell.
Signalling regions comprising sequences of other ITAM-containing
proteins such as Fc.gamma.RI have also been employed in CARs
(Haynes et al., 2001 J Immunol 166(1):182-187). Signalling regions
of CARs may also comprise co-stimulatory sequences derived from the
signalling region of co-stimulatory molecules, to facilitate
activation of CAR-expressing T cells upon binding to the target
protein. Suitable co-stimulatory molecules include CD28, OX40,
4-1BB, ICOS and CD27. In some cases CARs are engineered to provide
for co-stimulation of different intracellular signalling pathways.
For example, signalling associated with CD28 costimulation
preferentially activates the phosphatidylinositol 3-kinase (P13K)
pathway, whereas the 4-1BB-mediated signalling is through TNF
receptor associated factor (TRAF) adaptor proteins. Signalling
regions of CARs therefore sometimes contain co-stimulatory
sequences derived from signalling regions of more than one
co-stimulatory molecule. In some embodiments, the CAR of the
present invention comprises one or more co-stimulatory sequences
comprising or consisting of an amino acid sequence which comprises,
consists of, or is derived from, the amino acid sequence of the
intracellular domain of one or more of CD28, OX40, 4-1BB, ICOS and
CD27.
[0650] An optional hinge region may provide separation between the
antigen-binding domain and the transmembrane domain, and may act as
a flexible linker. Hinge regions may be derived from IgG1. In some
embodiments, the CAR of the present invention comprises a hinge
region comprising or consisting of an amino acid sequence which
comprises, consists of, or is derived from, the amino acid sequence
of the hinge region of IgG1.
[0651] Also provided is a cell comprising a CAR according to the
invention. The CAR according to the present invention may be used
to generate CAR-expressing immune cells, e.g. CAR-T or CAR-NK
cells. Engineering of CARs into immune cells may be performed
during culture, in vitro.
[0652] The antigen-binding region of the CAR of the present
invention may be provided with any suitable format, e.g. scFv,
scFab, etc.
[0653] Nucleic Acids and Vectors
[0654] The present invention provides a nucleic acid, or a
plurality of nucleic acids, encoding an antigen-binding molecule,
polypeptide or CAR according to the present invention.
[0655] In some embodiments, the nucleic acid is purified or
isolated, e.g. from other nucleic acid, or naturally-occurring
biological material. In some embodiments the nucleic acid(s)
comprise or consist of DNA and/or RNA.
[0656] The present invention also provides a vector, or plurality
of vectors, comprising the nucleic acid or plurality of nucleic
acids according to the present invention.
[0657] The nucleotide sequence may be contained in a vector, e.g.
an expression vector. A "vector" as used herein is a nucleic acid
molecule used as a vehicle to transfer exogenous nucleic acid into
a cell. The vector may be a vector for expression of the nucleic
acid in the cell. Such vectors may include a promoter sequence
operably linked to the nucleotide sequence encoding the sequence to
be expressed. A vector may also include a termination codon and
expression enhancers. Any suitable vectors, promoters, enhancers
and termination codons known in the art may be used to express a
peptide or polypeptide from a vector according to the
invention.
[0658] The term "operably linked" may include the situation where a
selected nucleic acid sequence and regulatory nucleic acid sequence
(e.g. promoter and/or enhancer) are covalently linked in such a way
as to place the expression of nucleic acid sequence under the
influence or control of the regulatory sequence (thereby forming an
expression cassette). Thus a regulatory sequence is operably linked
to the selected nucleic acid sequence if the regulatory sequence is
capable of effecting transcription of the nucleic acid sequence.
The resulting transcript(s) may then be translated into a desired
peptide(s)/polypeptide(s).
[0659] Suitable vectors include plasmids, binary vectors, DNA
vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors
(e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral
vectors, adenovirus vectors, adeno-associated virus vectors,
vaccinia virus vectors and herpesvirus vectors), transposon-based
vectors, and artificial chromosomes (e.g. yeast artificial
chromosomes).
[0660] In some embodiments, the vector may be a eukaryotic vector,
e.g. a vector comprising the elements necessary for expression of
protein from the vector in a eukaryotic cell. In some embodiments,
the vector may be a mammalian vector, e.g. comprising a
cytomegalovirus (CMV) or SV40 promoter to drive protein
expression.
[0661] Constituent polypeptides of an antigen-binding molecule
according to the present invention may be encoded by different
nucleic acids of the plurality of nucleic acids, or by different
vectors of the plurality of vectors.
[0662] Cells Comprising/Expressing the Antigen-Binding Molecules
and Polypeptides
[0663] The present invention also provides a cell comprising or
expressing an antigen-binding molecule, polypeptide or CAR
according to the present invention. Also provided is a cell
comprising or expressing a nucleic acid, a plurality of nucleic
acids, a vector or a plurality of vectors according to the
invention.
[0664] The cell may be a eukaryotic cell, e.g. a mammalian cell.
The mammal may be a primate (rhesus, cynomolgous, non-human primate
or human) or a non-human mammal (e.g. rabbit, guinea pig, rat,
mouse or other rodent (including any animal in the order Rodentia),
cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy
cows, or any animal in the order Bos), horse (including any animal
in the order Equidae), donkey, and non-human primate).
[0665] The present invention also provides a method for producing a
cell comprising a nucleic acid(s) or vector(s) according to the
present invention, comprising introducing a nucleic acid, a
plurality of nucleic acids, a vector or a plurality of vectors
according to the present invention into a cell. In some
embodiments, introducing an isolated nucleic acid(s) or vector(s)
according to the invention into a cell comprises transformation,
transfection, electroporation or transduction (e.g. retroviral
transduction).
[0666] The present invention also provides a method for producing a
cell expressing/comprising an antigen-binding molecule, polypeptide
or CAR according to the present invention, comprising introducing a
nucleic acid, a plurality of nucleic acids, a vector or a plurality
of vectors according to the present invention in a cell. In some
embodiments, the methods additionally comprise culturing the cell
under conditions suitable for expression of the nucleic acid(s) or
vector(s) by the cell. In some embodiments, the methods are
performed in vitro.
[0667] The present invention also provides cells obtained or
obtainable by the methods according to the present invention.
[0668] Producing the Antigen-Binding Molecules and Polypeptides
[0669] Antigen-binding molecules and polypeptides according to the
invention may be prepared according to methods for the production
of polypeptides known to the skilled person.
[0670] Polypeptides may be prepared by chemical synthesis, e.g.
liquid or solid phase synthesis. For example, peptides/polypeptides
can by synthesised using the methods described in, for example,
Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby
incorporated by reference in its entirety.
[0671] Alternatively, antigen-binding molecules and polypeptides
may be produced by recombinant expression. Molecular biology
techniques suitable for recombinant production of polypeptides are
well known in the art, such as those set out in Green and Sambrook,
Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring
Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both
of which are hereby incorporated by reference in their entirety.
Methods for the recombinant production of antigen-binding molecules
are also described in Frenzel et al., Front Immunol. (2013); 4: 217
and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:
3451-3461, both of which are hereby incorporated by reference in
their entirety.
[0672] In some cases the antigen-binding molecule of the present
invention are comprised of more than one polypeptide chain. In such
cases, production of the antigen-binding molecules may comprise
transcription and translation of more than one polypeptide, and
subsequent association of the polypeptide chains to form the
antigen-binding molecule.
[0673] For recombinant production according to the invention, any
cell suitable for the expression of polypeptides may be used. The
cell may be a prokaryote or eukaryote. In some embodiments the cell
is a prokaryotic cell, such as a cell of archaea or bacteria. In
some embodiments the bacteria may be Gram-negative bacteria such as
bacteria of the family Enterobacteriaceae, for example Escherichia
coli. In some embodiments, the cell is a eukaryotic cell such as a
yeast cell, a plant cell, insect cell or a mammalian cell, e.g.
CHO, HEK (e.g. HEK293), HeLa or COS cells.
[0674] In some cases the cell is not a prokaryotic cell because
some prokaryotic cells do not allow for the same folding or
post-translational modifications as eukaryotic cells. In addition,
very high expression levels are possible in eukaryotes and proteins
can be easier to purify from eukaryotes using appropriate tags.
Specific plasmids may also be utilised which enhance secretion of
the protein into the media.
[0675] In some embodiments polypeptides may be prepared by
cell-free-protein synthesis (CFPS), e.g. according using a system
described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431,
which is hereby incorporated by reference in its entirety.
[0676] Production may involve culture or fermentation of a
eukaryotic cell modified to express the polypeptide(s) of interest.
The culture or fermentation may be performed in a bioreactor
provided with an appropriate supply of nutrients, air/oxygen and/or
growth factors. Secreted proteins can be collected by partitioning
culture media/fermentation broth from the cells, extracting the
protein content, and separating individual proteins to isolate
secreted polypeptide(s). Culture, fermentation and separation
techniques are well known to those of skill in the art, and are
described, for example, in Green and Sambrook, Molecular Cloning: A
Laboratory Manual (4th Edition; incorporated by reference herein
above).
[0677] Bioreactors include one or more vessels in which cells may
be cultured. Culture in the bioreactor may occur continuously, with
a continuous flow of reactants into, and a continuous flow of
cultured cells from, the reactor. Alternatively, the culture may
occur in batches. The bioreactor monitors and controls
environmental conditions such as pH, oxygen, flow rates into and
out of, and agitation within the vessel such that optimum
conditions are provided for the cells being cultured.
[0678] Following culturing the cells that express the
antigen-binding molecule/polypeptide(s), the polypeptide(s) of
interest may be isolated. Any suitable method for separating
proteins from cells known in the art may be used. In order to
isolate the polypeptide it may be necessary to separate the cells
from nutrient medium. If the polypeptide(s) are secreted from the
cells, the cells may be separated by centrifugation from the
culture media that contains the secreted polypeptide(s) of
interest. If the polypeptide(s) of interest collect within the
cell, protein isolation may comprise centrifugation to separate
cells from cell culture medium, treatment of the cell pellet with a
lysis buffer, and cell disruption e.g. by sonification, rapid
freeze-thaw or osmotic lysis.
[0679] It may then be desirable to isolate the polypeptide(s) of
interest from the supernatant or culture medium, which may contain
other protein and non-protein components. A common approach to
separating protein components from a supernatant or culture medium
is by precipitation. Proteins of different solubilities are
precipitated at different concentrations of precipitating agent
such as ammonium sulfate. For example, at low concentrations of
precipitating agent, water soluble proteins are extracted. Thus, by
adding different increasing concentrations of precipitating agent,
proteins of different solubilities may be distinguished. Dialysis
may be subsequently used to remove ammonium sulfate from the
separated proteins.
[0680] Other methods for distinguishing different proteins are
known in the art, for example ion exchange chromatography and size
chromatography. These may be used as an alternative to
precipitation, or may be performed subsequently to
precipitation.
[0681] Once the polypeptide(s) of interest have been isolated from
culture it may be desired or necessary to concentrate the
polypeptide(s). A number of methods for concentrating proteins are
known in the art, such as ultrafiltration or lyophilisation.
[0682] Compositions
[0683] The present invention also provides compositions comprising
the antigen-binding molecules, polypeptides, CARs, nucleic acids,
expression vectors and cells described herein.
[0684] The antigen-binding molecules, polypeptides, CARs, nucleic
acids, expression vectors and cells described herein may be
formulated as pharmaceutical compositions or medicaments for
clinical use and may comprise a pharmaceutically acceptable
carrier, diluent, excipient or adjuvant. The composition may be
formulated for topical, parenteral, systemic, intracavitary,
intravenous, intra-arterial, intramuscular, intrathecal,
intraocular, intraconjunctival, intratumoral, subcutaneous,
intradermal, intrathecal, oral or transdermal routes of
administration which may include injection or infusion.
[0685] Suitable formulations may comprise the antigen-binding
molecule in a sterile or isotonic medium. Medicaments and
pharmaceutical compositions may be formulated in fluid, including
gel, form. Fluid formulations may be formulated for administration
by injection or infusion (e.g. via catheter) to a selected region
of the human or animal body.
[0686] In some embodiments the composition is formulated for
injection or infusion, e.g. into a blood vessel or tumor.
[0687] In accordance with the invention described herein methods
are also provided for the production of pharmaceutically useful
compositions, such methods of production may comprise one or more
steps selected from: producing an antigen-binding molecule,
polypeptide, CAR, nucleic acid (or plurality thereof), expression
vector (or plurality thereof) or cell described herein; isolating
an antigen-binding molecule, polypeptide, CAR, nucleic acid (or
plurality thereof), expression vector (or plurality thereof) or
cell described herein; and/or mixing antigen-binding molecule,
polypeptide, CAR, nucleic acid (or plurality thereof), expression
vector (or plurality thereof) or cell described herein with a
pharmaceutically acceptable carrier, adjuvant, excipient or
diluent.
[0688] For example, a further aspect the invention described herein
relates to a method of formulating or producing a medicament or
pharmaceutical composition for use in the treatment of a
disease/condition (e.g. a cancer), the method comprising
formulating a pharmaceutical composition or medicament by mixing an
antigen-binding molecule, polypeptide, CAR, nucleic acid (or
plurality thereof), expression vector (or plurality thereof) or
cell described herein with a pharmaceutically acceptable carrier,
adjuvant, excipient or diluent.
[0689] Therapeutic and Prophylactic Applications
[0690] The antigen-binding molecules, polypeptides, CARs, nucleic
acids, expression vectors, cells and compositions described herein
find use in therapeutic and prophylactic methods.
[0691] The present invention provides an antigen-binding molecule,
polypeptide, CAR, nucleic acid (or plurality thereof), expression
vector (or plurality thereof), cell or composition described herein
for use in a method of medical treatment or prophylaxis. Also
provided is the use of an antigen-binding molecule, polypeptide,
CAR, nucleic acid (or plurality thereof), expression vector (or
plurality thereof), cell or composition described herein in the
manufacture of a medicament for treating or preventing a disease or
condition. Also provided is a method of treating or preventing a
disease or condition, comprising administering to a subject a
therapeutically or prophylactically effective amount of an
antigen-binding molecule, polypeptide, CAR, nucleic acid (or
plurality thereof), expression vector (or plurality thereof), cell
or composition described herein.
[0692] The methods may be effective to reduce the development or
progression of a disease/condition, alleviation of the symptoms of
a disease/condition or reduction in the pathology of a
disease/condition. The methods may be effective to prevent
progression of the disease/condition, e.g. to prevent worsening of,
or to slow the rate of development of, the disease/condition. In
some embodiments the methods may lead to an improvement in the
disease/condition, e.g. a reduction in the symptoms of the
disease/condition or reduction in some other correlate of the
severity/activity of the disease/condition. In some embodiments the
methods may prevent development of the disease/condition a later
stage (e.g. a chronic stage or metastasis).
[0693] It will be appreciated that the articles of the present
invention may be used for the treatment/prevention of any
disease/condition that would derived therapeutic or prophylactic
benefit from a reduction in the number and/or activity of cells
expressing CD47. For example, the disease/condition may be a
disease/condition in which cells expressing CD47 are pathologically
implicated, e.g. a disease/condition in which an increased
number/proportion of cells expressing CD47 is positively associated
with the onset, development or progression of the
disease/condition, and/or severity of one or more symptoms of the
disease/condition, or for which an increased number/proportion of
cells expressing CD47, is a risk factor for the onset, development
or progression of the disease/condition.
[0694] In some embodiments, the disease/condition to be
treated/prevented in accordance with the present invention is a
disease/condition characterised by an increase in the
number/proportion/activity of cells expressing CD47, e.g. as
compared to the number/proportion/activity of cells expressing CD47
in the absence of the disease/condition.
[0695] In some embodiments the disease/condition to be
treated/prevented is a cancer. CD47 has been proposed to be a
cell-surface marker expressed by all human cancers (Willingham et
al. Proc Natl Acad Sci USA. (2012) 109(17): 6662-6667)
[0696] The cancer may be any unwanted cell proliferation (or any
disease manifesting itself by unwanted cell proliferation),
neoplasm or tumor. The cancer may be benign or malignant and may be
primary or secondary (metastatic). A neoplasm or tumor may be any
abnormal growth or proliferation of cells and may be located in any
tissue. The cancer may be of tissues/cells derived from e.g. the
adrenal gland, adrenal medulla, anus, appendix, bladder, blood,
bone, bone marrow, brain, breast, cecum, central nervous system
(including or excluding the brain) cerebellum, cervix, colon,
duodenum, endometrium, epithelial cells (e.g. renal epithelia),
gallbladder, oesophagus, glial cells, heart, ileum, jejunum,
kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node,
lymphoblast, maxilla, mediastinum, mesentery, myometrium,
nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland,
peripheral nervous system, peritoneum, pleura, prostate, salivary
gland, sigmoid colon, skin, small intestine, soft tissues, spleen,
stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea,
uterus, vulva, white blood cells.
[0697] Tumors to be treated may be nervous or non-nervous system
tumors. Nervous system tumors may originate either in the central
or peripheral nervous system, e.g. glioma, medulloblastoma,
meningioma, neurofibroma, ependymoma, Schwannoma,
neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous
system cancers/tumors may originate in any other non-nervous
tissue, examples include melanoma, mesothelioma, lymphoma, myeloma,
leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic
myelogenous leukemia (CML), acute myeloid leukemia (AML),
myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL),
chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma,
prostate carcinoma, breast cancer, lung cancer, colon cancer,
ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC,
hematologic cancer and sarcoma.
[0698] The treatment/prevention may be aimed at one or more of:
delaying/preventing the onset/progression of symptoms of the
cancer, reducing the severity of symptoms of the cancer, reducing
the survival/growth/invasion/metastasis of cells of the cancer,
reducing the number of cells of the cancer and/or increasing
survival of the subject.
[0699] In some embodiments, the cancer to be treated/prevented
comprises cells expressing CD47. In some embodiments, the cancer to
be treated/prevented is a cancer which is positive for CD47. In
some embodiments, the cancer over-expresses CD47. Overexpression of
CD47 can be determined by detection of a level of expression of
CD47 which is greater than the level of expression by equivalent
non-cancerous cells/non-tumor tissue.
[0700] CD47 expression may be determined by any suitable means.
Expression may be gene expression or protein expression. Gene
expression can be determined e.g. by detection of mRNA encoding
CD47, for example by quantitative real-time PCR (qRT-PCR). Protein
expression can be determined e.g. by detection of CD47, for example
by antibody-based methods, for example by western blot,
immunohistochemistry, immunocytochemistry, flow cytometry, or
ELISA.
[0701] In some embodiments, a patient may be selected for treatment
described herein based on the detection of a cancer expressing
CD47, or overexpressing CD47, e.g. in a sample obtained from the
subject.
[0702] The role of CD47 in the development and progression of
various cancers is reviewed e.g. in Sick et al. Br J Pharmacol.
(2012) 167(7): 1415-1430 and Chao et al., Curr Opin Immunol. 2012
April; 24(2): 225-232 (hereby incorporated by reference in its
entirety). Elevated CD47 expression is a negative prognostic
indicator for several cancers, and may contribute to cancer
development/progression by reducing killing of cancer cells and by
increasing proliferation, migration and/or invasion of cancer
cells. CD47 has been shown to suppress innate macrophage and NK
cell-mediated anticancer responses (Soto-Pantoja et al., Expert
Opin Ther Targets. (2013) 17(1): 89-103, which is hereby
incorporated by reference in its entirety).
[0703] CD47 is expressed by acute myeloid leukemia (AML), chronic
myeloid leukemia (CML), acute lymphoblastic leukemia (ALL),
non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder
cancer, brain cancer and ovarian cancer cells. Willingham et al.
Proc Natl Acad Sci USA. (2012) 109(17): 6662-6667 reported
expression of CD47 on cells of ovarian, breast, colon, bladder,
glioblastoma, hepatocellular carcinoma, and prostate tumors, and
CD47 has recently been shown to promote tumor invasion and
metastasis in Non-small Cell Lung Cancer (NSCLC; Zhao et al., Sci
Rep. (2016) 6: 29719) and melanoma (Ngo et al., Cell Reports (2016)
16, 1701-1716).
[0704] Accordingly, in some embodiments the cancer to be
treated/prevented in accordance with the present invention is
selected from: a hematologic malignancy, a myeloid hematologic
malignancy, a lymphoblastic hematologic malignancy, myelodysplastic
syndrome (MDS), acute myeloid leukemia (AML), chronic myeloid
leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's
lymphoma (NHL), multiple myeloma (MM), bladder cancer, brain
cancer, glioblastoma, ovarian cancer, breast cancer, colon cancer,
liver cancer, hepatocellular carcinoma, prostate cancer, lung
cancer, Non-small Cell Lung Cancer (NSCLC), skin cancer and
melanoma.
[0705] CD47 is a particularly attractive therapeutic targets for
AML because it is highly expressed in all characterised AML cell
lines, and play functional roles which therefore reduce risk of
antigen loss. The large population of tissue-resident macrophages
in the liver (Kupffer cells) represents an attractive therapeutic
mechanism for hematological malignancies, and macrophage-driven
clearance of malignant cells offers a further route for neo-antigen
presentation to adaptive immune system.
[0706] CD47 is also implicated in the pathogenesis of autoimmune
diseases, inflammatory diseases, ischemia-reperfusion injury (IRI)
and cardiovascular diseases (see e.g. Soto-Pantoja et al., Expert
Opin Ther Targets. (2013) 17(1): 89-103). The CD47-SIRP.alpha. axis
has been implicated in type I diabetes (Dugas et al., J Autoimmun.
(2010) 35(1):23-32). Thrombospondin-1 has been shown to act via
CD47 to inhibit nitric oxide signaling throughout the vascular
system, and blocking TSP1-CD47 interaction alleviates tissue
ischemia (Isenberg et al., Arterioscler Thromb Vasc Biol. (2008)
28(4): 615-621) and reduces ischemia-reperfusion injury (IRI) (Xiao
et al., Liver Transpl. (2015) 21(4): 468-477).
[0707] Accordingly, in some embodiments the disease/disorder to be
treated/prevented is a cancer, an autoimmune disease (e.g. type I
diabetes), an inflammatory disease, ischemia-reperfusion injury
(IRI) or cardiovascular disease.
[0708] Administration of the articles of the present invention is
preferably in a "therapeutically effective" or "prophylactically
effective" amount, this being sufficient to show therapeutic or
prophylactic benefit to the subject. The actual amount
administered, and rate and time-course of administration, will
depend on the nature and severity of the disease/condition and the
particular article administered. Prescription of treatment, e.g.
decisions on dosage etc., is within the responsibility of general
practitioners and other medical doctors, and typically takes
account of the disease/disorder to be treated, the condition of the
individual subject, the site of delivery, the method of
administration and other factors known to practitioners. Examples
of the techniques and protocols mentioned above can be found in
Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub.
Lippincott, Williams & Wilkins.
[0709] Administration may be alone or in combination with other
treatments, either simultaneously or sequentially dependent upon
the condition to be treated. The antigen-binding molecule or
composition described herein and a therapeutic agent may be
administered simultaneously or sequentially.
[0710] In some embodiments, the methods comprise additional
therapeutic or prophylactic intervention, e.g. for the
treatment/prevention of a cancer. In some embodiments, the
therapeutic or prophylactic intervention is selected from
chemotherapy, immunotherapy, radiotherapy, surgery, vaccination
and/or hormone therapy. In some embodiments, the therapeutic or
prophylactic intervention comprises leukapheresis. In some
embodiments the therapeutic or prophylactic intervention comprises
a stem cell transplant.
[0711] The antigen-binding molecules of the present invention are
particularly suitable for use in conjunction with radiotherapy.
Antagonism of CD47 has previously been shown to help maintain the
viability of normal tissues after irradiation, while increasing the
radiosensitivity of tumors (Maxhimer et al., Science Translational
Medicine (2009) 1(3): 3ra7).
[0712] Simultaneous administration refers to administration of the
antigen-binding molecule, polypeptide, CAR, nucleic acid (or
plurality thereof), expression vector (or plurality thereof), cell
or composition and therapeutic agent together, for example as a
pharmaceutical composition containing both agents (combined
preparation), or immediately after each other and optionally via
the same route of administration, e.g. to the same artery, vein or
other blood vessel. Sequential administration refers to
administration of one of the antigen-binding molecule/composition
or therapeutic agent followed after a given time interval by
separate administration of the other agent. It is not required that
the two agents are administered by the same route, although this is
the case in some embodiments. The time interval may be any time
interval.
[0713] Chemotherapy and radiotherapy respectively refer to
treatment of a cancer with a drug or with ionising radiation (e.g.
radiotherapy using X-rays or .gamma.-rays). The drug may be a
chemical entity, e.g. small molecule pharmaceutical, antibiotic,
DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a
biological agent, e.g. antibody, antibody fragment, aptamer,
nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein. The
drug may be formulated as a pharmaceutical composition or
medicament. The formulation may comprise one or more drugs (e.g.
one or more active agents) together with one or more
pharmaceutically acceptable diluents, excipients or carriers.
[0714] A treatment may involve administration of more than one
drug. A drug may be administered alone or in combination with other
treatments, either simultaneously or sequentially dependent upon
the condition to be treated. For example, the chemotherapy may be a
co-therapy involving administration of two drugs, one or more of
which may be intended to treat the cancer.
[0715] The chemotherapy may be administered by one or more routes
of administration, e.g. parenteral, intravenous injection, oral,
subcutaneous, intradermal or intratumoral.
[0716] The chemotherapy may be administered according to a
treatment regime. The treatment regime may be a pre-determined
timetable, plan, scheme or schedule of chemotherapy administration
which may be prepared by a physician or medical practitioner and
may be tailored to suit the patient requiring treatment. The
treatment regime may indicate one or more of: the type of
chemotherapy to administer to the patient; the dose of each drug or
radiation; the time interval between administrations; the length of
each treatment; the number and nature of any treatment holidays, if
any etc. For a co-therapy a single treatment regime may be provided
which indicates how each drug is to be administered.
[0717] Chemotherapeutic drugs may be selected from: Abemaciclib,
Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane
(Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD,
ABVE, ABVE-PC, AC, Acalabrutinib, AC-T, Adcetris (Brentuximab
Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin
Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo
(Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod),
Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta
(Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran
for Injection (Melphalan Hydrochloride), Alkeran Tablets
(Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig
(Brigatinib), Ambochlorin (Chlorambucil), Amboclorin
(Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole,
Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole),
Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide,
Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi,
Atezolizumab, Avastin (Bevacizumab), Avelumab, Axicabtagene
Ciloleucel, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP,
Becenum (Carmustine), Beleodaq (Belinostat), Belinostat,
Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin),
Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131
Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin,
Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif
(Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel,
Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx
(Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, Calquence
(Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan
Hydrochloride), Capecitabine, CAPDX, Carac (Fluorouracil--Topical),
Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris
(Carmustine), Carmustine, Carmustine Implant, Casodex
(Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin
Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine),
Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP,
Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine,
Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib,
Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC,
COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib),
Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza
(Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U
(Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine,
Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex
(Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin
Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide
Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin
Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone,
Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil
(Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride,
Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin
Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab,
Efudex (Fluorouracil--Topical), Elitek (Rasburicase), Ellence
(Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin),
Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab),
Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH,
Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib),
Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia
chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide
Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin
Hydrochloride Liposome), Everolimus, Evista (Raloxifene
Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU
(Fluorouracil Injection), 5-FU (Fluorouracil--Topical), Fareston
(Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC,
Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate),
Fludarabine Phosphate, Fluoroplex (Fluorouracil--Topical),
Fluorouracil Injection, Fluorouracil--Topical, Flutamide, Folex
(Methotrexate), Folex PFS (Methotrexate), FOLFIRI,
FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn
(Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV
Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent
Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine
Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN,
Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif
(Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel
(Carmustine Implant), Gliadel wafer (Carmustine Implant),
Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate),
Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), HPV
Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant,
HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan
Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD,
Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE,
Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin
Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa
(Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum
(Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica
(Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene
Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin,
Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin),
Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab
and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan
Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax
(Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone),
Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla
(Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride),
Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali
(Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib),
Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab),
Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate),
Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide
Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid),
Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride
Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil
Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot
(Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate),
Lynparza (Olaparib), Marciibo (Vincristine Sulfate Liposome),
Matulane (Procarbazine Hydrochloride), Mechlorethamine
Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan,
Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna),
Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF
(Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate),
Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone
Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil
(Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin
(Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg
(Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel
Albumin-stabilized Nanoparticle Formulation), Navelbine
(Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar
(Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate),
Netupitant and Palonosetron Hydrochloride, Neulasta
(Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib
Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro
(Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab,
Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab,
Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab,
Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron
Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak
(Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib,
Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle
Formulation, PAD, Palbociclib, Palifermin, Palonosetron
Hydrochloride, Palonosetron Hydrochloride and Netupitant,
Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat
(Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride,
PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b,
PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed
Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin),
Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst
(Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab),
Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin
(Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine),
Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol
(Mercaptopurine), Purixan (Mercaptopurine), [No Entries], Radium
223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase,
R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent
Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine,
Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine,
Recombinant Interferon Alfa-2b, Regorafenib, Relistor
(Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide),
Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab),
Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab,
Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride,
Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride),
Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib
Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol
(Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide
Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib),
STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga
(Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate),
Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synribo
(Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar
(Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene
Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine),
Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna
(Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq
(Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus,
Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa,
Tisagenlecleucel, Tolak (Fluorouracil--Topical), Topotecan
Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and
Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF,
Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine
Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox
(Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin
(Dinutuximab), Uridine Triacetate, VAC, Valrubicin, Valstar
(Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride),
Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade
(Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta
(Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur
(Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate,
Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate,
Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP,
Vismodegib, Vistogard (Uridine Triacetate), Voraxaze
(Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride),
Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome),
Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda
(Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium
223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab),
Yescarta (Axicabtagene Ciloleucel), Yondelis (Trabectedin), Zaltrap
(Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate
Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab
Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept,
Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate),
Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid),
Zydelig (Idelalisib), Zykadia (Ceritinib) and Zytiga (Abiraterone
Acetate).
[0718] In some embodiments the chemotherapeutic agent is selected
from one or more of: cytarabine, 5-azacytidine (5-AZA), valproic
acid (VPA), all-trans retinoic acid (ATRA), decitabine, sodium
phenylbutyrate, hydrozyurea, 6-mercaptopurine (6-MP), 6-thioguanine
(6-TG), Mocetinostat (MGCD0103), Panobinostat (LBH-589),
romidepsin, an antracycline, daunorubicin, daunomycin, idarubicin,
cladribine (Leustatin, 2-CdA), midostaurin, fludarabine (Fludara)
and topotecan.
[0719] In some embodiments the chemotherapeutic agent is histone
deacetylase (HDAC) inhibitor, e.g. a HDAC inhibitor described in
Fredly et al., Clin Epigenetics. (2013) 5(1):12 (hereby
incorporated by reference in its entirety). In some embodiments the
chemotherapeutic agent is cytarabine.
[0720] Multiple doses of the producing an antigen-binding molecule,
polypeptide, CAR, nucleic acid (or plurality thereof), expression
vector (or plurality thereof), cell or composition may be provided.
One or more, or each, of the doses may be accompanied by
simultaneous or sequential administration of another therapeutic
agent.
[0721] Multiple doses may be separated by a predetermined time
interval, which may be selected to be one of 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months.
By way of example, doses may be given once every 7, 14, 21 or 28
days (plus or minus 3, 2, or 1 days).
[0722] Methods of Detection
[0723] The invention also provides the articles of the present
invention for use in methods for detecting, localizing or imaging
CD47, or cells expressing CD47.
[0724] The antigen-binding molecules described herein may be used
in methods that involve the antigen-binding molecule to CD47. Such
methods may involve detection of the bound complex of the
antigen-binding molecule and CD47.
[0725] As such, a method is provided, comprising contacting a
sample containing, or suspected to contain, CD47, and detecting the
formation of a complex of the antigen-binding molecule and CD47.
Also provided is a method comprising contacting a sample
containing, or suspected to contain, a cell expressing CD47, and
detecting the formation of a complex of the antigen-binding
molecule and a cell expressing CD47.
[0726] Suitable method formats are well known in the art, including
immunoassays such as sandwich assays, e.g. ELISA. The methods may
involve labelling the antigen-binding molecule, or target(s), or
both, with a detectable moiety, e.g. a fluorescent label,
phosphorescent label, luminescent label, immuno-detectable label,
radiolabel, chemical, nucleic acid or enzymatic label as described
herein. Detection techniques are well known to those of skill in
the art and can be selected to correspond with the labelling
agent.
[0727] Methods of this kind may provide the basis of methods for
the diagnostic and/or prognostic evaluation of a disease or
condition, e.g. a cancer. Such methods may be performed in vitro on
a patient sample, or following processing of a patient sample. Once
the sample is collected, the patient is not required to be present
for the in vitro method to be performed, and therefore the method
may be one which is not practised on the human or animal body. In
some embodiments the method is performed in vivo.
[0728] Detection in a sample may be used for the purpose of
diagnosis of a disease/condition (e.g. a cancer), predisposition to
a disease/condition, or for providing a prognosis (prognosticating)
for a disease/condition, e.g. a disease/condition described herein.
The diagnosis or prognosis may relate to an existing (previously
diagnosed) disease/condition.
[0729] Such methods may involve detecting or quantifying one or
more of CD47 or cells expressing CD47, e.g. in a patient sample.
Where the method comprises quantifying the relevant factor, the
method may further comprise comparing the determined amount against
a standard or reference value as part of the diagnostic or
prognostic evaluation. Other diagnostic/prognostic tests may be
used in conjunction with those described herein to enhance the
accuracy of the diagnosis or prognosis or to confirm a result
obtained by using the tests described herein.
[0730] A sample may be taken from any tissue or bodily fluid. The
sample may comprise or may be derived from: a quantity of blood; a
quantity of serum derived from the individual's blood which may
comprise the fluid portion of the blood obtained after removal of
the fibrin clot and blood cells; a tissue sample or biopsy; pleural
fluid; cerebrospinal fluid (CSF); or cells isolated from said
individual. In some embodiments, the sample may be obtained or
derived from a tissue or tissues which are affected by the
disease/condition (e.g. tissue or tissues in which symptoms of the
disease manifest, or which are involved in the pathogenesis of the
disease/condition).
[0731] The present invention also provides methods for
selecting/stratifying a subject for treatment with a CD47-targeted
agent. In some embodiments a subject is selected for
treatment/prevention in accordance with the invention, or is
identified as a subject which would benefit from such
treatment/prevention, based on detection/quantification of CD47, or
cells expressing CD47, e.g. in a sample obtained from the
individual.
[0732] Subjects
[0733] The subject in accordance with aspects the invention
described herein may be any animal or human. The subject is
preferably mammalian, more preferably human. The subject may be a
non-human mammal, but is more preferably human. The subject may be
male or female. The subject may be a patient. A subject may have
been diagnosed with a disease or condition requiring treatment
(e.g. a cancer), may be suspected of having such a
disease/condition, or may be at risk of developing/contracting such
a disease/condition.
[0734] In embodiments according to the present invention the
subject is preferably a human subject. In some embodiments, the
subject to be treated according to a therapeutic or prophylactic
method of the invention herein is a subject having, or at risk of
developing, a cancer. In embodiments according to the present
invention, a subject may be selected for treatment according to the
methods based on characterisation for certain markers of such
disease/condition.
[0735] Kits
[0736] In some aspects of the invention described herein a kit of
parts is provided. In some embodiments the kit may have at least
one container having a predetermined quantity of an antigen-binding
molecule, polypeptide, CAR, nucleic acid (or plurality thereof),
expression vector (or plurality thereof), cell or composition
described herein.
[0737] In some embodiments, the kit may comprise materials for
producing an antigen-binding molecule, polypeptide, CAR, nucleic
acid (or plurality thereof), expression vector (or plurality
thereof), cell or composition described herein.
[0738] The kit may provide the antigen-binding molecule,
polypeptide, CAR, nucleic acid (or plurality thereof), expression
vector (or plurality thereof), cell or composition together with
instructions for administration to a patient in order to treat a
specified disease/condition.
[0739] In some embodiments the kit may further comprise at least
one container having a predetermined quantity of another
therapeutic agent (e.g. anti-infective agent or chemotherapy
agent). In such embodiments, the kit may also comprise a second
medicament or pharmaceutical composition such that the two
medicaments or pharmaceutical compositions may be administered
simultaneously or separately such that they provide a combined
treatment for the specific disease or condition. The therapeutic
agent may also be formulated so as to be suitable for injection or
infusion to a tumor or to the blood.
[0740] Sequence Identity
[0741] As used herein, "sequence identity" refers to the percent of
nucleotides/amino acid residues in a subject sequence that are
identical to nucleotides/amino acid residues in a reference
sequence, after aligning the sequences and, if necessary,
introducing gaps, to achieve the maximum percent sequence identity
between the sequences. Pairwise and multiple sequence alignment for
the purposes of determining percent sequence identity between two
or more amino acid or nucleic acid sequences can be achieved in
various ways known to a person of skill in the art, for instance,
using publicly available computer software such as ClustalOmega
(Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame
et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann
and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh
and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780
software. When using such software, the default parameters, e.g.
for gap penalty and extension penalty, are preferably used.
TABLE-US-00002 SEQUENCES SEQ ID NO: DESCRIPTION SEQUENCE 1 Human
CD47
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDI
isoform
YTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELK
OA3-323
YRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAI
(UniProt:
LFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSL- CI
Q08722-1, v1)
AACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE 2
Human CD47
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDI
isoform
YTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELK
OA3-293
YRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAI
(UniProt:
LFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSL- CI
Q08722-2) AACIPMHGPLLISGLSILALAQLLGLVYMKFV 3 Human CD47
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDI
isoform
YTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELK
OA3-305
YRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAI
(UniProt:
LFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSL- CI
Q08722-3) AACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRNN 4 Human
CD47
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDI
isoform
YTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELK
OA3-312
YRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAI
(UniProt:
LFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSL- CI
Q08722-4) AACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLN 5
Mature human
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSA
CD47 isoform
KIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPI
OA3-323
FAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVT-
S (UniProt:
TGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILA-
LAQ Q08722-1, v1 LLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE
positions 19-323) 6 Mature human
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSA
CD47 isoform
KIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPI
OA3-293
FAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVT-
S (UniProt:
TGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILA-
LAQ Q08722-2 LLGLVYMKFV positions 19-292) 7 Mature human
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSA
CD47 isoform
KIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPI
OA3-305
FAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVT-
S (UniProt:
TGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILA-
LAQ Q08722-3, LLGLVYMKFVASNQKTIQPPRNN positions 19-305) 8 Mature
human
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSA
CD47 isoform
KIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPI
OA3-312
FAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVT-
S (UniProt:
TGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILA-
LAQ Q08722-4 LLGLVYMKFVASNQKTIQPPRKAVEEPLN positions 19-311) 9
V-type
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSS-
A Ig-like KIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETII domain
(UniProt: Q08722-1 positions 19-127) 10 Human CD47
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSA
extracellular
KIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNE region 1
(UniProt: Q08722-1 positions 19-141) 11 Human CD47
NILIVIFPIFAILLFWGQFGI transmembrane region 1 (UniProt: Q08722-1
positions 142-162) 12 Human CD47 KTLKYRSGGMDEKT cytoplasmic region
1 (UniProt: Q08722-1 positions 163-176) 13 Human CD47
IALLVAGLVITVIVIVGAILF transmembrane region 2 (UniProt: Q08722-1
positions 177-197) 14 Human CD47 VPGEYSLKNA extracellular region 2
(UniProt: Q08722-1 positions 198-207) 15 Human CD47
TGLGLIVTSTGILILLHYYVF transmembrane region 3 (UniProt: Q08722-1
positions 208-228) 16 Human CD47 STAIGLT cytoplasmic region 2
(UniProt: Q08722-1 positions 229-235) 17 Human CD47
SFVIAILVIQVIAYILAVVGL transmembrane region 4 (UniProt: Q08722-1
positions 236-256) 18 Human CD47 SLCIAACIPMHG extracellular region
3 (UniProt: Q08722-1 positions 257-268) 19 Human CD47
PLLISGLSILALAQLLGLVYM transmembrane region 5 (UniProt: Q08722-1
positions 269-289) 20 Human CD47 KFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE
cytoplasmic region 3 (UniProt: Q08722-1 positions 290-323) 21
Region of VKWKFKGRDI human CD47 targeted by 1-1-A1 and 1-1-A1_BM
(UniProt: Q08722-1 positions 56-65) 22 Region of KTKSVEFTFCN human
CD47 targeted by 5-48-A6 and 5-48-D2 (UniProt: Q08722-1 positions
24-34) 23 1-1-A1_BM
QVQLQQSGPDLKKPGASVKVSCKVSGYTFTNYVIHWVRQKPGQGLEWMGYINPYNDGTKSNEKFKG
heavy chain KATLTSDKSSTSAYMELSSLTSEDTAVYYCASGGYYTMDYWGQGTSVTVSS
variable region 24 1-1-A1_BM, GYTFTNYV 1-1-A1, 11A1H1, 11A1H2,
11A1H3, 11A1H4, 11A1H6, 11A1 H8 heavy chain CDR1 25 1-1-A1_BM,
INPYNDGT 1-1-A1, 11A1H1, 11A1H2, 11A1H3, 11A1H4, 11A1H6, 11A1 H8
heavy chain CDR2 26 1-1-A1_BM, ASGGYYTMDY 1-1-A1, 11A1H1, 11A1H2,
11A1H3, 11A1H4, 11A1H6, 11A1H8, 11A1H5, 11A1H7, 11A1H9, 11A1H10,
11A1H11 heavy chain CDR3 27 1-1-A1_BM heavy
QVQLQQSGPDLKKPGASVKVSCKVS chain FR1 28 1-1-A1_BM heavy
IHWVRQKPGQGLEWMGY chain FR2 29 1-1-A1_BM heavy
KSNEKFKGKATLTSDKSSTSAYMELSSLTSEDTAVYYC
chain FR3 30 1-1-A1_BM heavy WGQGTSVTVSS chain FR4 31 1-1-A1_BM
light
DVVMTQTPLSLPVTLGDQASISCRSSQHLEYSNGYSYLHWYQQRPGQSPQLLIYKISNRFSGVPDR
chain variable FSGSGSGTDFTLKISRVEAEDLGVYYCSQSTHVPYTFGGGTKLEIK
region 32 1-1-A1_BM, QHLEYSNGYSY 1-1-A1, 11A1H1, 11A1H2, 11A1H3,
11A1H4, 11A1H5, 11A1H10, 11A1H11 light chain CDR1 33 1-1-A1_13M,
KIS 1-1-A1, 11A1H1, 11A1H2, 11A1H3, 11A1H4, 11A1H5, 11A1H11 light
chain CDR2 34 1-1-A1_BM, SQSTHVPYT 1-1-A1, 11A1H1, 11A1H2, 11A1H3,
11A1H4, 11A1H5 11A1H6, 11A1H7 11A1H8, 11A1H9 11A1H10 light chain
CDR3 35 1-1-A1_BM light DVVMTQTPLSLPVTLGDQASISCRSS chain FR1 36
1-1-A1_BM light LHWYQQRPGQSPQLLIY chain FR2 37 1-1-A1_BM light
NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC chain FR3 38 1-1-A1_BM light
FGGGTKLEIK chain FR4 39 1-1-A1 heavy
EVQLQQSGPDLVKPGASVKMSCKASGYTFTNYVIHWVKQKPGQGLEWIGYINPYNDGTKSNEKFKG
chain variable KATLTSDKSSTSAYMELSSLTSEDSAVYYCASGGYYTMDYWGQGTSVTVSS
region 40 1-1-A1 heavy EVQLQQSGPDLVKPGASVKMSCKAS chain FR1 41
1-1-A1 heavy IHWVKQKPGQGLEWIGY chain FR2 42 1-1-A1 heavy
KSNEKFKGKATLTSDKSSTSAYMELSSLTSEDSAVYYC chain FR3 43 1-1-A1 heavy
WGQGTSVTVSS chain FR4 44 1-1-A1
DVVMTQTPLSLPVSLGDQASISCRSSQHLEYSNGYSYLHWYLQKPGQSPQLLIYKISNRFSGVP-
DR light chain FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK
variable region 45 1-1-A1 light DVVMTQTPLSLPVSLGDQASISCRSS chain
FR1 46 1-1-A1 light LHWYLQKPGQSPQLLIY chain FR2 47 1-1-A1 light
NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC chain FR3 48 1-1-A1 light
FGGGTKLEIK chain FR4 49 5-48-A6
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSAL-
MSR heavy chain
LSISKDNSKSQVFLKMNSLQTDDTAMYYCARVPTGRIKSYFYAMDYWGQGTSVTVSS variable
region 50 5-48-A6 heavy GFSLTSYG chain CDR1 51 5-48-A6 heavy
IWAGGST chain CDR2 52 5-48-A6 heavy ARVPTGRIKSYFYAMDY chain CDR3 53
5-48-A6 heavy QVQLKESGPGLVAPSQSLSITCTVS chain FR1 54 5-48-A6 heavy
VHWVRQPPGKGLEWLGV chain FR2 55 5-48-A6 heavy
NYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYC chain FR3 56 5-48-A6 heavy
WGQGTSVTVSS chain FR4 57 5-48-A6 light
DIKMTQSPSSMYSSLGERVTITCKASQDISSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSG
chain variable SGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK region 58
5-48-A6 light QDISSY chain CDR1 59 5-48-A6 light RAN chain CDR2 60
5-48-A6 light LQYDEFPYT chain CDR3 61 5-48-A6 light
DIKMTQSPSSMYSSLGERVTITCKAS chain FR1 62 5-48-A6 light
LSWFQQKPGKSPKTLIY chain FR2 63 5-48-A6 light
RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC chain FR3 64 5-48-A6 light
FGGGTKLEIK chain FR4 65 5-48-D2 heavy
EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKD
chain variable KFIISRDNAKNTLYLQMSKVRSEDTALYYCATGTGFAYWGQGTLVTVSA
region 66 5-48-D2 heavy GFDFSRYW chain CDR1 67 5-48-D2 heavy
INPDSSTI chain CDR2 68 5-48-D2 heavy ATGTGFAY chain CDR3 69 5-48-D2
heavy EVKLLESGGGLVQPGGSLKLSCAAS chain FR1 70 5-48-D2 heavy
MSWVRQAPGKGLEWIGE chain FR2 71 5-48-D2 heavy NYTPSLKDKFI
ISRDNAKNTLYLQMSKVRSEDTALYYC chain FR3 72 5-48-D2 heavy WGQGTLVTVSA
chain FR4 73 5-48-D2 light
DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSG
chain variable SGTQFSLKINSLQPEDFGSYYCQHHYVTPWTFGGVTKLEIK region 74
5-48-D2 light ENIYSY chain CDR1 75 5-48-D2 light NAK chain CDR2 76
5-48-D2 light QHHYVTPWT chain CDR3 77 5-48-D2 light
DIQMTQSPASLSASVGETVTITCRAS chain FR1 78 5-48-D2 light
LAWYQQKQGKSPQLLVY chain FR2 79 5-48-D2 light
TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC chain FR3 80 5-48-D2 light
FGGVTKLEIK chain FR4 81 1-1-A1 heavy MEWSWIFLFLLSGTAGVHS chain
SignalP 82 1-1-A1 light MKLPVRLLVLMFWIPASSS chain SignalP 83
5-48-A6 heavy MAVLVLFLCLVAFPSCVLS chain SignalP 84 5-48-A6 light
MRTPAQFLGILLLWFPGIKC chain SignalP 85 5-48-D2 heavy
MDFGLIFFIVALLKGVQC chain SignalP 86 5-48-D2 light
MSVPTQVLGLLLLWLTGARC chain SignalP 87 1-1-A1_BM
CAGGTGCAGCTGCAGCAGTCTGGACCAGACCTGAAGAAGCCTGGAGCCAGCGTGAAGGTGTCCTGT
heavy chain
AAGGTGTCCGGCTACACCTTCACAAACTATGTGATCCACTGGGTGAGGCAGAAGCCAGGACAGGGC
DNA
CTGGAGTGGATGGGCTACATCAACCCCTATAATGACGGCACCAAGTCTAATGAGAAGTTTAAGGGC
AAGGCCACCCTGACATCTGATAAGAGCAGCACCAGCGCCTACATGGAGCTGTCTAGCCTGACCAGC
GAGGACACAGCCGTGTACTATTGCGCTTCCGGCGGCTACTATACAATGGATTATTGGGGCCAGGGC
ACCAGCGTGACAGTGTCCTCT 88 1-1-A1_BM
GACGTGGTCATGACCCAGACACCACTGTCCCTGCCTGTGACCCTGGGCGATCAGGCCTCTATCAGC
light chain
TGTAGAAGCTCCCAGCACCTGGAGTACAGCAACGGCTACTCCTATCTGCACTGGTATCAGCAGCGC
DNA
CCAGGACAGTCTCCACAGCTGCTGATCTACAAGATCTCTAATCGGTTCAGCGGCGTGCCTGACAGG
TTTTCCGGCTCTGGCAGCGGCACCGATTTCACACTGAAGATCAGCAGAGTGGAGGCTGAGGACCTG
GGCGTGTACTATTGCTCCCAGTCTACCCACGTGCCCTATACATTTGGCGGCGGCACCAAGCTGGAG
ATCAAG 89 1-1-A1 heavy
GAGGTCCAGCTGCAGCAGTCTGGACCTGACCTAGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGC
chain DNA
AAGGCTTCTGGATACACATTCACTAATTATGTTATACACTGGGTGAAGCAGAAGCCTGGGCAG-
GGC
CTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTAAGTCCAATGAGAAGTTCAAAGGC
AAGGCCACACTGACTTCAGACAAATCCTCCACCTCAGCCTACATGGAGCTCAGCAGCCTGACCTCT
GAGGACTCTGCGGTCTATTACTGTGCAAGCGGAGGGTACTATACTATGGACTATTGGGGTCAAGGA
ACCTCAGTCACCGTCTCCTCG 90 1-1-A1
GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCT-
CT light chain
TGCAGATCTAGTCAACACCTTGAATACAGTAATGGATACTCCTATTTGCATTGGTACCTGCAGAAG
DNA
CCAGGCCAGTCTCCACAGCTCCTGATCTACAAAATTTCCAACCGATTTTCTGGGGTCCCAGACAGG
TTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTG
GGGGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTACACATTCGGAGGGGGGACCAAGCTGGAA
ATAAAA 91 5-48-A6
CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACT-
TGC heavy chain
ACTGTCTCTGGGTTTTCATTAACCAGTTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGT
DNA
CTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGA
CTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGAT
GACACAGCCATGTACTACTGTGCCAGAGTTCCGACAGGTCGGATTAAATCTTATTTCTATGCTATG
GACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCG 92 5-48-A6
GACATCAAGATGACCCAGTCTCCATCTTCCATGTATTCATCTCTTGGAGAGAGAGTCACTATC-
ACT
light chain
TGCAAGGCGAGTCAGGACATTAGTAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAGTCTCCT
DNA
AAGACCCTGATCTATCGTGCAAACAGATTGGTGGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGA
TCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGT
CTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 93
5-48-D2
GAGGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCC-
TGT heavy chain
GCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGG
DNA
CTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCTAAAGGAT
AAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCT
GAGGACACAGCCCTTTATTACTGTGCAACTGGGACGGGGTTTGCTTACTGGGGCCAAGGGACTCTG
GTCACTGTCTCTGCG 94 5-48-D2
GACATCCAGATGACTCAGTCTCCAGCTTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATC-
ACA light chain
TGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCT
DNA
CAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCCTCAAGGTTCAGTGGCAGTGGA
TCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGT
CAACATCATTATGTTACTCCGTGGACGTTCGGTGGAGTCACCAAGCTGGAAATCAAA 95 Human
SIRPA
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPI
isoform 1
QWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSP-
DDV (UniProt:
EFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQ-
TNV P78324-1,
DPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQ-
PVR v2)
AENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLT
CQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRI
RQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQP
ASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK 96 Human SIRPA
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPI
isoform 2
QWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSP- DD
(UniProt:
VEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDF- QT
P78324-2)
NVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVT-
QQP VRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRD
DVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAA
LYLVRIRQKKAQGSTSSTRLHEPEKNAREITQVQSLDTNDITYADLNLPKGKKPAPQAAEPNNHTE
YASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK 97 Human SIRPA
MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPI
isoform 4
QWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSP-
DVE (UniProt:
FKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQT-
NVD P78324-4)
PVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQP-
VRA
ENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTC
QVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIR
QKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPA
SEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK 98 Mature
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVS-
DL human SIRPA
TKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATP
isoform 1
QHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQ-
VIC (UniProt:
EVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENG-
NVS P20138-1
RTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGS-
NT positions
AAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQD-
TND 31-504)
ITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEP-
S FSEYASVQVPRK 99 Mature
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVS-
DL human SIRPA
TKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATP
isoform 2
QHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQ-
VIC (UniProt:
EVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENG-
NVS P78324-2
RTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGS-
NT positions
AAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQV-
QSL 31-478)
DTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAP-
K PEPSFSEYASVQVPRK 100 Mature
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTV-
SDL human SIRPA
TKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQ
isoform 4
HTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQV-
ICE (UniProt:
VAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGN-
VSR P78324-4
TETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSN-
TA positions
AENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDT-
NDI 31-473)
TYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPS-
F SEYASVQVPRK 101 Human SIRPA
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDL
extra
TKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATP
cellular
QHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQV-
IC domain
EVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVS
(UniProt:
RTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQG-
SNT P78324-1 AAENTGSNERNIY positions 31-373) 102 Human SIRPA
IVVGVVCTLLVALLMAALYLV transmembrane domain (UniProt: P78324-1
positions 374-394) 103 Human SIRPA
RIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSP
cytoplasmic QPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK domain
(UniProt: P78324-1 positions 395-504) 104 Human SIRPA
EELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLT
V-type KRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSG Ig-like domain
(UniProt: P78324-1 positions 32-137) 105 Human SIRPA
PSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTA
C1-type KVVLTREDVHSQVICEVAHVTLQGDPLRGTANLS Ig-like domain 1
(UniProt: P78324-1 positions 148-247) 106 Human SIRPA
PTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVN
C1-type VSAHRDDVKLTCQVEHDGQPAVSKSHDLK Ig-like domain 2 (UniProt:
P78324-1 positions 254-348) 107 1-1-A1_BM
QVQLQQSGPDLKKPGASVKVSCKVSGYTFTNYVI HWVRQKPGQGLEWMGYINPYNDGTKSNEKFK
VH-CH1-CH2-
GKATLTSDKSSTSAYMELSSLTSEDTAVYYCASGGYYTMDYWGQGTSVTVSSASTKGPSVFPLAPS
CH3
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 108 1-1-A1_BM
DVVMTQTPLSLPVTLGDQASISCRSSQHLEYSNGYSYLHWYQQRPGQSPQLLIYKISNRFSGVPDR
VL-C.kappa.
FSGSGSGTDFTLKISRVEAEDLGVYYCSQSTHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 109 1-1-A1
EVQLQQSGPDLVKPGASVKMSCKASGYTFTNYVIHWVKQKPGQGLEWIGYINPYNDGTKSNEK-
FKG VH-CH1-CH2-
KATLTSDKSSTSAYMELSSLTSEDSAVYYCASGGYYTMDYWGQGTSVTVSSASTKGPSVFPLAPSS
CH3
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 110 1-1-A1
DVVMTQTPLSLPVSLGDQASISCRSSQHLEYSNGYSYLHWYLQKPGQSPQLLIYKISNRFSGV-
PDR VL-C.kappa.
FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 111 5-48-A6
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSR
VH-CH1-CH2-
LSISKDNSKSQVFLKMNSLQTDDTAMYYCARVPTGRIKSYFYAMDYWGQGTSVTVSSASTKGPSVF
CH3
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 112
5-48-A6
DIKMTQSPSSMYSSLGERVTITCKASQDISSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSG
VL-C.kappa.
SGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGEC 113 5-48-D2
EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKD
VH-CH1-CH2-
KFIISRDNAKNTLYLQMSKVRSEDTALYYCATGTGFAYWGQGTLVTVSAASTKGPSVFPLAPSSKS
CH3
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 114 5-48-D2
DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSG
VL-C.kappa.
SGTQFSLKINSLQPEDFGSYYCQHHYVTPWTFGGVTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGEC 115 anti-CD33
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKS
VH-CH1-CH2-
KATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK
CH3
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 116 anti-CD33
DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRF
VL-C.kappa.
SGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC 117 Rhesus
MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGR-
DIY macaque
TFDGALNKSTAPANFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKY-
R CD47
VVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLMITVIVIVGAILFV
(UniProt:
PGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIA-
ACI F7F5Y9-1,
PMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRNDNFRLKNEEKFILN v2) 118
Human IgG1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
constant
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP-
KD region
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
(IGHG1;
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE-
W UniProt:
ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP-
GK P01857-1, v1) 119 CH1 IgG1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
(positions SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV 1-98 of P01857-1, v1)
120 Hinge IgG1 EPKSCDKTHTCP (positions 99-110 of P01857-1, v1) 121
CH2 IgG1
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
(positions EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 111-223
of P01857-1, v1) 122 CH3 IgG1
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
(positions YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 224-330 of
P01857-1, v1) 123 CH3 (D356E,
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
L358M; YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK positions numbered
according to EU numbering) 124 C.kappa. CL (IGCK;
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
UniProt: SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC P01834-1, v2)
125 J6M0
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFK-
G VH-CH1-CH2-
RVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFPL
CH3
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 126 J6M0
VL-C.kappa.
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGEC 127 11A1H1 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGKGLEWMGYINPYNDGTKSNEKFKG
RVTLTSDKSSTSAYMELSSLRSEDTAVYYCASGGYYTMDYWGQGTLVTVSS 128 11A1H1,
11A1H2,
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSNGYSYLHWYQQRPGQSPRLLIYKISNRFSGVPDR
11A1H3, 11A1H4, FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIK
11A1H5 VL 129 11A1H2 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGKGLEWMGYINPYNDGTKSNEKFKG
RVTLTSDTSTTTAYMELSSLRSEDTAVYYCASGGYYTMDYWGQGTLVTVSS 130 11A1 H3 VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDGTKSNEKFQG
RVTLTSDTSTSTAYMELSSLRSEDTAVYYCASGGYYTMDYWGQGTLV 131 11A1H4, 11A1H6,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYINPYNDGTKYNQKFKG
11A1H8 VH RVTLTSDTSTTTAYMELSRLRSDDTAVYYCASGGYYTMDYWGQGTLV 132
11A1H5, 11A1H7,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYVIHWVRQAPGQGLEWMGYINPYNGGTNYAQKFKG
11A1H9, 11A1H10,
RVTLTSDTSTTTAYMELSRLRSEDTAVYYCASGGYYTMDYWGQGTLVTVSS 11A1H11 VH 133
11A1H6, 11A1H7
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSQGYSYLHWYQQRPGQSPRLLIYKVSNRDSGVPDR
VL FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIK 134 11A1H8,
11A1H9
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSTGYSYLHWYQQRPGQSPRLLIYKVSNRDSGVPDR
VL FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIK 135 11A1H10 VL
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSNGYSYLHWYQQRPGQSPRLLIYKVSNRDSGVPDR
FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIK 136 11A1H11 VL
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSNGYSYLHWYQQRPGQSPRLLIYKISNRFSGVPDR
FSGSGSGTDFTLKISRVEAEDVGVYYCSQGTHVPYTFGGGTKVEIK 137 11A1H5, 11A1H7,
GYTFTGYV 11A1H9, 11A1H10, 11A1H11 HC-CDR1 138 11A1H5, 11A1H7,
INPYNGGT 11A1H9, 11A1H10, 11A1H11 HC-CDR2 139 11A1H6, 11A1H7
QHLEYSQGYSY LC-CDR1 140 11A1 H8, 11A1H9 QHLEYSTGYSY LC-CDR1 141
11A1H6, 11A1H7, KVS 11A1H8, 11A1H9, 11A1H10 LC-CDR2 142 11A1H11
LC-CDR3 SQGTHVPYT 143 11A1H1, 11A1H2, QVQLVQSGAEVKKPGASVKVSCKAS
11A1H3, 11A1H4, 11A1H5, 11A1H6, 11A1H7, 11A1H8, 11A1H9, 11A1H10,
11A1H11 HC-FR1 144 11A1H1, 11A1H2, IHWVRQAPGKGLEWMGY HC-FR2 145
11A1H3 HC-FR2 MHWVRQAPGQGLEWMGY 146 11A1H4, 11A1H5,
IHWVRQAPGQGLEWMGY 11A1H6, 11A1H7, 11A1H8, 11A1H9, 11A1H10, 11A1H11
HC-FR2 147 11A1H1 HC-FR3 KSNEKFKGRVTLTSDKSSTSAYMELSSLRSEDTAVYYC 148
11A1H2 HC-FR3 KSNEKFKGRVTLTSDTSTTTAYMELSSLRSEDTAVYYC 149 11A1H3
HC-FR3 KSNEKFQGRVTLTSDTSTSTAYMELSSLRSEDTAVYYC 150 11A1H4, 11A1H6,
KYNQKFKGRVTLTSDTSTTTAYMELSRLRSDDTAVYYC 11A1H8 HC-FR3 151 11A1H5,
11A1H7, NYAQKFKGRVTLTSDTSTTTAYMELSRLRSEDTAVYYC 11A1H9, 11A1H10,
11A1H11 HC-FR3 152 11A1H1, 11A1H2, WGQGTLVTVSS 11A1H5, 11A1H7,
11A1H9, 11A1H10, 11A1H11 HC-FR4 153 11A1H3, 11A1H4, WGQGTLV 11A1H6,
11A1H8 HC-FR4 154 11A1H1, 11A1H2, DVVMTQSPLSLPVTLGQPASISCRSS
11A1H3, 11A1H4, 11A1H5, 11A1H6, 11A1H7, 11A1H8, 11A1H9, 11A1H10,
11A1H11 LC-FR1 155 11A1H1, 11A1H2, LHWYQQRPGQSPRLLIY 11A1H3,
11A1H4, 11A1H5, 11A1H6, 11A1H7, 11A1H8, 11A1H9, 11A1H10, 11A1H11
LC-FR2 156 11A1H1, 11A1H2, NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
11A1H3, 11A1H4, 11A1H5, 11A1H11 LC-FR3 157 11A1H6, 11A1H7,
NRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 11A1H8, 11A1H9, 11A1H10 LC-FR3
158 11A1H1, 11A1H2, FGGGTKVEIK 11A1H3, 11A1H4, 11A1H5, 11A1H6,
11A1H7, 11A1H8, 11A1H9, 11A1H10, 11A1H11 LC-FR4 159 11A1H1
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGKGLEWMGYINPYNDGTKSNEK-
FKG VH-CH1-CH2-
RVTLTSDKSSTSAYMELSSLRSEDTAVYYCASGGYYTMDYWGQGTLVTVSSASTKGPSVFPLAPSS
CH3
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 160 11A1H1,
11A1H2,
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSNGYSYLHWYQQRPGQSPRLLIYKISNRFSGVPDR
11A1H3, 11A1H4,
FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS
11A1H5 VL-CK
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 161 11A1H2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGKGLEWMGYINPYNDGTKSNEK-
FKG VH-CH1-CH2-
RVTLTSDTSTTTAYMELSSLRSEDTAVYYCASGGYYTMDYWGQGTLVTVSSASTKGPSVFPLAPSS
CH3
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 162 11A1H3
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVMHWVRQAPGQGLEWMGYINPYNDGTKSNEK-
FQG VH-CH1-CH2-
RVTLTSDTSTSTAYMELSSLRSEDTAVYYCASGGYYTMDYWGQGTLVASTKGPSVFPLAPSSKSTS
CH3
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 163 11A1H4, 11A1H6,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYINPYNDGTKYNQKFKG
11A1H8 VH-CH1-
RVTLTSDTSTTTAYMELSRLRSDDTAVYYCASGGYYTMDYWGQGTLVASTKGPSVFPLAPSSKSTS
CH2-CH3
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN-
V
NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 164 11A1H5, 11A1H7,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYVIHWVRQAPGQGLEWMGYINPYNGGTNYAQKFKG
11A1H9, 11A1H10,
RVTLTSDTSTTTAYMELSRLRSEDTAVYYCASGGYYTMDYWGQGTLVTVSSASTKGPSVFPLAPSS
11A1H11
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT-
Y VH-CH1-CH2-
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
CH3
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 165 11A1H6,
11A1H7
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSQGYSYLHWYQQRPGQSPRLLIYKVSNRDSGVPDR
VL-CK
FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 166 11A1H8, 11A1H9
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSTGYSYLHWYQQRPGQSPRLLIYKVSNRDSGVPDR
VL-CK
FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 167 11A1H10 VL-CK
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSNGYSYLHWYQQRPGQSPRLLIYKVSNRDSGVPDR
FSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 168 11A1H11 VL-CK
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSNGYSYLHWYQQRPGQSPRLLIYKISNRFSGVPDR
FSGSGSGTDFTLKISRVEAEDVGVYYCSQGTHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 169 11A1H_CHC-CDR1 GYTFTX.sub.1YV X.sub.1 = N
or G 170 11A1H_CHC-CDR2 INPYNX.sub.2GT X.sub.2 = D or G 171
11A1H_CLC-CDR1 QHLEYSX.sub.3GYSY X.sub.3 = N, Q or T 172
11A1H_CLC-CDR2 KX.sub.4S X.sub.4 = I or V 173 11A1H_CLC-CDR3
SQX.sub.5THVPYT X.sub.5 = S or G 174 11A1H_CHC-FR2
X.sub.6HWVRQAPGX.sub.7GLEWMGY X.sub.6 = I or M X.sub.7 = Q or K 175
11A1H_CHC-FR3
X.sub.8X.sub.9X.sub.10X.sub.11KFX.sub.12GRVTLTSDX.sub.13SX.sub.14SX.sub.1-
5AYMELSX.sub.16LRSX.sub.17DTAVYYC X.sub.8 = K or N X.sub.9 = S or Y
X.sub.10 = N or A X.sub.11 = E or Q X.sub.12 = K or Q X.sub.13 = T
or K X.sub.14 = T or S X.sub.15 = T or S X.sub.16 = S or R X.sub.17
= E or D 176 11A1H_CHC-FR4 WGQGTLVX.sub.18X.sub.19X.sub.20X.sub.21
X.sub.18 = T or absent X.sub.19 = V or absent X.sub.20 = S or
absent X.sub.21 = S or absent 177 11A1H_CLC-FR3
NRX.sub.22SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC X.sub.22 = D or F 178
11A1H_CVH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTX.sub.22YVX.sub.23HVVVRQAPGX.sub.24GLEWMGYI-
NPYNX.sub.25G
TX.sub.26X.sub.27X.sub.28X.sub.29KFX.sub.30GRVTLTSDX.sub.3iSX.sub.32SX.s-
ub.33AYMELSX.sub.34LRSX.sub.35DTAVYYCASGGYYT
MDYWGQGTLVX.sub.36X.sub.37X.sub.38X.sub.39 X.sub.22 = N or G
X.sub.23 = I or M X.sub.24 = Q or K X.sub.25 = D or G X.sub.26 = K
or N X.sub.27 = S or Y X.sub.28 = N or A X.sub.29 = E or Q X.sub.30
= K or Q X.sub.31 = T or K X.sub.32 = T or S X.sub.33 = T or S
X.sub.34 = S or R X.sub.35 = E or D X.sub.36 = T or absent X.sub.37
= V or absent X.sub.38 = S or absent X.sub.39 = S or absent 179
11A1H_CVL
DVVMTQSPLSLPVTLGQPASISCRSSQHLEYSX.sub.40GYSYLHWYQQRPGQSPRLLIYKX.sub.41SNR-
X.sub.42
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQX.sub.43THVPYTFGGGTKVEIK
X.sub.40 = N, Q or T X.sub.41 = I or V X.sub.42 = D or F X.sub.43 =
S or G
[0742] The invention includes the combination of the aspects and
preferred features described except where such a combination is
clearly impermissible or expressly avoided.
[0743] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
[0744] Aspects and embodiments of the present invention will now be
illustrated, by way of example, with reference to the accompanying
figures. Further aspects and embodiments will be apparent to those
skilled in the art. All documents mentioned in this text are
incorporated herein by reference.
[0745] Throughout this specification, including the claims which
follow, unless the context requires otherwise, the word "comprise,"
and variations such as "comprises" and "comprising," will be
understood to imply the inclusion of a stated integer or step or
group of integers or steps but not the exclusion of any other
integer or step or group of integers or steps.
[0746] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Ranges may be expressed herein as from "about" one particular
value, and/or to "about" another particular value. When such a
range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by the use of the
antecedent "about," it will be understood that the particular value
forms another embodiment.
[0747] Where a nucleic acid sequence is disclosed herein, the
reverse complement thereof is also expressly contemplated.
[0748] Methods described herein may preferably performed in vitro.
The term "in vitro" is intended to encompass procedures performed
with cells in culture whereas the term "in vivo" is intended to
encompass procedures with/on intact multi-cellular organisms.
BRIEF DESCRIPTION OF THE FIGURES
[0749] Embodiments and experiments illustrating the principles of
the invention will now be discussed with reference to the
accompanying figures.
[0750] FIG. 1. Ribbon diagram showing the 3D structure of
interacting SIPRP.alpha. and CD47 domains, with regions used as
immunogens for raising anti-CD47 antibodies overlain with
spheres.
[0751] FIGS. 2A and 2B. Sensorgrams showing affinity of binding of
anti-CD47 antibodies to human CD47. (2A) Sensorgram for 1-1-A1.
(2B) Sensorgram for 1-1-A1_BM.
[0752] FIGS. 3A to 3D. Histograms showing staining of
CD47-expressing cells by anti-CD47 antibodies as determined by flow
cytometry. (3A) Histogram showing staining of HEK293T cells (which
express CD47), or HEK293T-derived CD47 knockout cells, by anti-CD47
antibody clone 1-1-A1 or isotype control antibody. (3B) Histogram
showing staining of HEK293T cells, or HEK293T-derived CD47 knockout
cells, by anti-CD47 antibody clone 1-1-A1_BM or isotype control
antibody. (3C) Histogram showing staining of HEK293T cells, or
HEK293T-derived CD47 knockout cells, by anti-CD47 antibody clone
B6H12 or isotype control antibody. (3D) Histogram showing staining
of MM.1S cells, H929 cells, U226 cells, 8226 cells and RAJI cells
by anti-CD47 antibody clone B6H12.
[0753] FIG. 4. Bar chart showing inhibition of interaction between
human CD47 and human SIRP.alpha. by antigen-binding molecules as
determined by ELISA.
[0754] FIG. 5. Graph showing binding to human CD47 (hCD47) and
rhesus macaque CD47 (RhCD47) by the indicated antigen-binding
molecules, as determined by ELISA.
[0755] FIG. 6. Histogram showing phagocytosis of CFSE-labelled Raji
cells by macrophages in the presence of the indicated
antigen-binding molecules or PBS, as determined by flow
cytometry.
[0756] FIGS. 7A to 7C. Fluorescence microscopy images and bar chart
showing phagocytosis of CFSE-labelled HL-60 cells by macrophages in
the presence of the indicated antigen-binding molecules. (7A and
7B) Images showing binding phagocytosis in the presence of (7A)
isotype control antibody (negative control), (7B) anti-CD47 clone
1-1-A1_BM IgG1, (7C) Bar chart summarising phagocytic indices for
CFSE-labelled HL-60 cells by macrophages in the presence of the
indicated antigen-binding molecules.
[0757] FIG. 8. Sensorgram showing affinity of binding of anti-CD47
antibody 1-1-A1_BM to human CD47.
[0758] FIG. 9. Graph showing binding to human CD47 by the indicated
antigen-binding molecules, as determined by ELISA.
[0759] FIG. 10. Graph showing binding to human VISTA by the
indicated antigen-binding molecules, as determined by ELISA.
[0760] FIGS. 11A to 11H. Sensorgrams showing affinity of binding of
anti-CD47 antibodies to human CD47. (11A) Sensorgram for 11A1_BM.
(11B) Sensorgram for 11A1H3. (11C) Sensorgram for 11A1H5. (11D)
Sensorgram for 11A1H6. (11E) Sensorgram for 11A1H7. (11F)
Sensorgram for 11A1H9. (11G) Sensorgram for 11A1H10. (11H)
Sensorgram for 11A1H11.
[0761] FIG. 12. Graph showing inhibition of interaction between
human CD47 and SIRP.alpha. by the indicated antigen-binding
molecules, as determined by ELISA.
[0762] FIG. 13. Images showing the results of analysis of
hemagglutination by the indicated antigen-binding molecules.
Positive control=anti-red blood cells antibody (ANTI RBC), negative
control=isotype matched antibody specific for an irrelevant target
antigen (Irrelevant Ag), and buffer only (BUFFER).
EXAMPLES
[0763] In the following Examples, the inventors describe the
generation of novel CD47-specific antibody clones targeted to
specific regions of interest in the CD47 molecule, the biophysical
and functional characterisation and the therapeutic evaluation of
these antigen-binding molecules.
Example 1: CD47 Target Design and Anti-CD47 Antibody Hybridoma
Production
[0764] The inventors selected two regions in the Ig-like V region
(SEQ ID NO:9) of the extracellular region 1 of human CD47 (SEQ ID
NO:10) for raising CD47-binding monoclonal antibodies. The
inventors focused on regions of CD47 known to be involved in the
interaction between CD47 and SIRP.alpha. (FIG. 1).
[0765] 1.1 Hybridoma Production
[0766] Approximately 6 week old female BALB/c mice were obtained
from InVivos (Singapore). Animals were housed under specific
pathogen-free conditions and were treated in compliance with the
Institutional Animal Care and Use Committee (IACUC) guidelines.
[0767] For hybridoma production, mice were immunized with
proprietary mixtures of antigenic peptide for a total of 4
intraperitoneal injections with a 2 week interval between each
injection. Antigen for immunizations included one of the following:
[0768] i) Up to 50 .mu.g of synthetic peptide conjugated with KLH
(China Peptides Co. Ltd, China) [0769] ii) Up to 50 .mu.g of
commercially available recombinant Fc-tagged human CD47
(Sinobiological Inc, China) [0770] iii) Up to 20.times.10.sup.6
isogenic cells overexpressing human CD47.
[0771] Prior to harvesting the spleen for fusion, mice were boosted
with antigen mixture for three consecutive days. 24 h after the
final boost total splenocytes were isolated and fused with the
myeloma cell line P3X63.Ag8.653 (ATCC, USA), with PEG using
ClonaCell-HY Hybridoma Cloning Kit, in accordance with the
manufacturer's instructions (Stemcell Technologies, Canada).
[0772] Fused cells were cultured in ClonaCell-HY Medium C (Stemcell
Technologies, Canada) overnight at 37.degree. C. in a 5% CO.sub.2
incubator. The next day, fused cells were centrifuged and
resuspended in 10 ml of ClonaCell-HY Medium C and then gently mixed
with 90 ml of semisolid methylcellulose-based ClonaCell-HY Medium D
(StemCell Technologies, Canada) containing HAT components, which
combines the hybridoma selection and cloning into one step.
[0773] The fused cells were then plated into 96 well plates and
allowed to grow at 37.degree. C. in a 5% CO.sub.2 incubator. After
7-10 days, single hybridoma clones were isolated and antibody
producing hybridomas were selected by screening the supernatants by
Enzyme-linked immunosorbent assay (ELISA) and
Fluorescence-activated cell sorting (FACs).
[0774] 1.2 Antibody Variable Region Amplification and
Sequencing
[0775] Total RNA was extracted from hybridoma cells using TRIzol
reagent (Life Technologies, Inc., USA) using manufacturer's
protocol. Double-stranded cDNA was synthesized using SMARTer RACE
573' Kit (Clontech.TM., USA) in accordance with the manufacturer's
instructions. Briefly, 1 .mu.g total RNA was used to generate
full-length cDNA using 5'-RACE CDS primer (provided in the kit),
and the 5' adaptor (SMARTer II A primer) was then incorporated into
each cDNA according to manufacturer's instructions. cDNA synthesis
reactions contained: 5.times. First-Strand Buffer, DTT (20 mM),
dNTP Mix (10 mM), RNase Inhibitor (40 U/.mu.l) and SMARTScribe
Reverse Transcriptase (100 U/.mu.l).
[0776] The race-ready cDNAs were amplified using SeqAmp DNA
Polymerase (Clontech.TM., USA). Amplification reactions contained
SeqAmp DNA Polymerase, 2.times.Seq AMP buffer, 5' universal primer
provided in the 5' SMARTer Race kit, that is complement to the
adaptor sequence, and 3' primers that anneal to respective heavy
chain or light chain constant region primer. The 5' constant region
were designed based on previously reported primer mix either by
Krebber et al. J. Immunol. Methods 1997; 201: 35-55, Wang et al.
Journal of Immunological Methods 2000, 233; 167-177 or Tiller et
al. Journal of Immunological Methods 2009; 350:183-193. The
following thermal protocol was used: pre-denature cycle at
94.degree. C. for 1 min; 35 cycles of 94.degree. C., 30 s,
55.degree. C., 30 s and 72.degree. C., 45 s; final extension at
72.degree. C. for 3 min.
[0777] The resulting VH and VL PCR products, approximately 550 bp,
were cloned into pJET1.2/blunt vector using CloneJET PCR Cloning
Kit (Thermo Scientific, USA) and used to transform highly competent
E. coli DH5.alpha.. From the resulting transformants, plasmid DNA
was prepared using Miniprep Kit (Qiagene, Germany) and sequenced.
DNA sequencing was carried out by AlTbiotech. These sequencing data
were analyzed using the international IMGT (ImMunoGeneTics)
information system (LeFranc et al., Nucleic Acids Res. (2015) 43
(Database issue):D413-22) to characterize the individual CDRs and
framework sequences. The signal peptide at 5' end of the VH and VL
was identified by SignalP (v 4.1; Nielsen, in Kihara, D (ed):
Protein Function Prediction (Methods in Molecular Biology vol.
1611) 59-73, Springer 2017).
[0778] Three monoclonal anti-CD47 antibody clones were selected for
further development: 1-1-A1, 5-48-A6 and 5-48-D2.
[0779] A humanised version of antibody clone 1-1-A1 was also
prepared according to standard methods by cloning the CDRs of
antibody clone 1-1-A1 into VH and VL comprising human antibody
framework regions. This antibody clone was designated antibody
clone 1-1-A1_BM.
TABLE-US-00003 Peptide immunogen used Antibody clone VH/VL sequence
to raise the antibody 1-1-A1_BM VH = SEQ ID NO: 23 SEQ ID NO: 21 VL
= SEQ ID NO: 31 1-1-A1 VH = SEQ ID NO: 39 VL = SEQ ID NO: 44
5-48-A6 VH = SEQ ID NO: 49 SEQ ID NO: 22 VL = SEQ ID NO: 57 5-48-D2
VH = SEQ ID NO: 65 VL = SEQ ID NO: 73
Example 2: Antibody Production and Purification
[0780] 2.1 Cloning VH and VL into Expression Vectors:
[0781] DNA sequence encoding the heavy and light chain variable
regions of the anti-CD47 antibody clones were subcloned into the
pFUSE-CHIg-hG1 and pFUSE2ss-CLIg-hk (InvivoGen, USA) eukaryotic
expression vectors for construction of human-mouse chimeric
antibodies. Human IgG1 constant region encoded by pFUSE-CHIg-hG1
comprises the substitutions D356E, L358M (positions numbered
according to EU numbering) in the CH3 region relative to Human IgG1
constant region (IGHG1; UniProt:P01857-1, v1). pFUSE2ss-CLIg-hk
encodes human IgG1 light chain kappa constant region (IGCK;
UniProt: P01834-1, v2).
[0782] Variable regions along with the signal peptides were
amplified from the cloning vector using SeqAmp enzyme
(Clontech.TM., USA) following the manufacturer's protocol. Forward
and reverse primers having 15-20 bp overlap with the appropriate
regions within VH or VL plus 6 bp at 5' end as restriction sites
were used. The DNA insert and the pFuse vector were digested with
restriction enzyme recommended by the manufacturer to ensure no
frameshift was introduced (e.g., EcoRI and NheI for VH, AgeI and
BsiWI for VL,) and ligated into its respective plasmid using T4
ligase enzyme (Thermo Scientific, USA). The molar ratio of 3:1 of
DNA insert to vector was used for ligation.
[0783] 2.2 Expression of Antibodies in Mammalian Cells
[0784] Antibodies were expressed using either 1) Expi293 Transient
Expression System Kit (Life Technologies, USA), or 2) HEK293-6E
Transient Expression System (CNRC-NRC, Canada) following the
manufacturer's instructions.
[0785] 1) Expi293 Transient Expression System:
[0786] Cell Line Maintenance:
[0787] HEK293F cells (Expi293F) were obtained from Life
Technologies, Inc (USA). Cells were cultured in serum-free,
protein-free, chemically defined medium (Expi293 Expression Medium,
Thermo Fisher, USA), supplemented with 50 IU/ml penicillin and 50
.mu.g/ml streptomycine (Gibco, USA) at 37.degree. C., in 8%
CO.sub.2 and 80% humidified incubators with shaking platform.
[0788] Transfection:
[0789] Expi293F cells were transfected with expression plasmids
using ExpiFectamine 293 Reagent kit (Gibco, USA) according to its
manufacturer's protocol. Briefly, cells at maintenance were
subjected to a media exchange to remove antibiotics by spinning
down the culture, cell pellets were re-suspended in fresh media
without antibiotics at 1 day before transfection. On the day of
transfection, 2.5.times.10.sup.6/ml of viable cells were seeded in
shaker flasks for each transfection. DNA-ExpiFectamine complexes
were formed in serum-reduced medium, Opti-MEM (Gibco, USA), for 25
min at room temperature before being added to the cells. Enhancers
were added to the transfected cells at 16-18 h post transfection.
An equal amount of media was topped up to the transfectants at day
4 post-transfection to prevent cell aggregation. Transfectants were
harvested at day 7 by centrifugation at 4000.times.g for 15 min,
and filtered through 0.22 .mu.m sterile filter units.
[0790] 2) HEK293-6E Transient Expression System
[0791] Cell Line Maintenance:
[0792] HEK293-6E cells were obtained from National Research Council
Canada. Cells were cultured in serum-free, protein-free, chemically
defined Freestyle F17 Medium (Invitrogen, USA), supplemented with
0.1% Kolliphor-P188 and 4 mM L-Glutamine (Gibco, USA) and 25
.mu.g/ml G-418 at 37.degree. C., in 5% CO.sub.2 and 80% humidified
incubators with shaking platform.
[0793] Transfection:
[0794] HEK293-6E cells were transfected with expression plasmids
using PEIpro.TM. (Polyplus, USA) according to its manufacturer's
protocol. Briefly, cells at maintenance were subjected to a media
exchange to remove antibiotics by centrifugation, cell pellets were
re-suspended with fresh media without antibiotics at 1 day before
transfection. On the day of transfection, 1.5-2.times.10.sup.6
cells/ml of viable cells were seeded in shaker flasks for each
transfection. DNA and PEIpro.TM. were mixed to a ratio of 1:1 and
the complexes were allowed to form in F17 medium for 5 min at RT
before adding to the cells. 0.5% (w/v) of Tryptone N1 was fed to
transfectants at 24-48 h post transfection. Transfectants were
harvested at day 6-7 by centrifugation at 4000.times.g for 15 min
and the supernatant was filtered through 0.22 .mu.m sterile filter
units.
[0795] Cells were transfected with vectors encoding the following
combinations of polypeptides:
TABLE-US-00004 Antigen- binding molecule Polypeptides Antibody [1]
1-1-A1_BM anti-CD47 clone VH-CH1-CH2-CH3 1-1-A1_BM IgG1 (SEQ ID NO:
107) + 1-1-A1_BM VL-C.sub.K (SEQ ID NO: 108) [2] 1-1-A1 anti-CD47
clone VH-CH1-CH2-CH3 1-1-A1 IgG1 (SEQ ID NO: 109) + 1-1-A1
VL-C.sub.K (SEQ ID NO: 110) [3] 5-48-A6 anti-CD47 clone
VH-CH1-CH2-CH3 5-48-A6 IgG1 (SEQ ID NO: 111) + 5-48-A6 VL-C.sub.K
(SEQ ID NO: 112) [4] 5-48-D2 anti-CD47 clone VH-CH1-CH2-CH3 5-48-D2
IgG1 (SEQ ID NO: 113) + 5-48-D2 VL-C.sub.K (SEQ ID NO: 114) [5]
anti-CD33 anti-CD33 IgG1 VH-CH1-CH2-CH3 (SEQ ID NO: 115) +
anti-CD33 VL-C.sub.K (SEQ ID NO: 116)
[0796] 2.3 Antibody Purification
[0797] Affinity Purification, Buffer Exchange and Storage:
[0798] Antibodies secreted by the transfected cells into the
culture supernatant were purified using liquid chromatography
system AKTA Start (GE Healthcare, UK). Specifically, supernatants
were loaded onto HiTrap Protein G column (GE Healthcare, UK) at a
binding rate of 5 ml/min, followed by washing the column with 10
column volumes of washing buffer (20 mM sodium phosphate, pH 7.0).
Bound mAbs were eluted with elution buffer (0.1 M glycine, pH 2.7)
and the eluents were fractionated to collection tubes which contain
appropriate amount of neutralization buffer (1 M Tris, pH 9).
Neutralised elution buffer containing purified mAb were exchanged
into PBS using 30K MWCO protein concentrators (Thermo Fisher, USA)
or 3.5K MWCO dialysis cassettes (Thermo Fisher, USA). Monoclonal
antibodies were sterilized by passing through 0.22 .mu.m filter,
aliquoted and snap-frozen in -80.degree. C. for storage.
[0799] 2.4 Antibody-Purity Analysis
[0800] Size Exclusion Chromatography (SEC):
[0801] Antibody purity was analyzed by size exclusion
chromatography (SEC) using HiLoad 16/600 Superdex 200 .mu.g column
(GE Healthcare, UK) on a AKTA Explorer liquid chromatography system
(GE Healthcare, UK). Protein samples are injected to SEC column at
concentrations ranging between 0.2-1.5 mg/ml and 1.times.PBS was
pumped to the column at a flow rate of 1 ml/min. Proteins were
eluted according to their molecular weights.
[0802] Sodium-Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
(SDS-PAGE):
[0803] Antibody purity was also analysed by SDS-PAGE under reducing
and non-reducing conditions according to standard methods. Briefly,
4%-20% TGX protein gels (Bio-Rad, USA) were used to resolve
proteins using a Mini-Protean Electrophoresis System (Bio-Rad,
USA). For non-reducing condition, protein samples were denatured by
mixing with 2.times. Laemmli sample buffer (Bio-Rad, USA) and
boiled at 95.degree. C. for 5-10 min before loading to the gel. For
reducing conditions, 2.times. sample buffer containing 5% of
.beta.-mercaptoethanol (.beta.ME), or 40 mM DTT (dithiothreitol)
was used. Electrophoresis was carried out at a constant voltage of
150V for 1 h in SDS running buffer (25 mM Tris, 192 mM glycine, 1%
SDS, pH 8.3).
Example 3: Biophysical Characterisation
[0804] 3.1 Global Affinity Study Using BLITz System
[0805] Bio-Layer Interferometry (BLI) experiments were performed
using a single channel BLItz system (ForteBio, Menlo Park, Calif.)
using Anti-human immunoglobulin G (IgG) Fc (AHC) coated biosensor
tips (Pall ForteBio, Menlo Park, Calif.) for capturing human IgGs.
Biosensors were first hydrated for at least 10 m in assay buffer
(phosphate buffered saline) followed by buffer baseline for 30 s
and loading of the human IgGs onto the biosensor tips at
concentrations ranging from 25-50 nM for 120 s. The tips were then
washed briefly for 30 s with the assay buffer to remove
nonspecifically bound proteins or unbound IgGs for obtaining a
second buffer baseline. The association phase of the IgGs with
antigens (500 nM-0 nM) was set up at 120 s which was followed by a
dissociation phase (assay buffer alone) for 120 s. All the BLITz
runs were measured at room temperature at a stirring speed of 1000
rpm and AHC biosensors were regenerated using 10 mM of glycine (pH
2.7) after the assay. Binding affinity between the immobilized
antibodies on the AHC sensors and human CD47 were determined by
analyzing the binding kinetic curves using the software BLItz Pro.
All the sensorgrams were reference subtracted and globally fitted
into a 1:1 model which analysed the binding curves at different
concentrations of antigens and generated kinetic constants
(KD/Ka/Kd) for the globally fitted data. All the binding curves
were subjected to step correction which corrects the misalignment
between association and dissociation steps and only the curves with
R.sup.2 values greater than 0.9 were used for analysis.
[0806] The anti-CD47 antibody clones in IgG1 format were analyzed
for binding affinity to human CD47.
[0807] Representative sensorgrams for the analysis are shown in
FIGS. 2A and 2B. Clone 1-1-A1 was found to have a K.sub.D of 9 nM,
and 1-1-A1_BM was found to have a K.sub.D of 16.1 nM.
[0808] In a separate experiment, the affinity of 1-1-A1_BM ([1] of
Example 2.2) for human CD47 was analysed by BLI using an
anti-Penta-HIS (HIS1K) Octet sensors. Buffer baseline was obtained
for 30 s, and then sensors were loaded with his-tagged human CD47
(1.2 .mu.M) for 120 s. A second buffer baseline was obtained for 60
s, followed by an association phase with 1-1-A1_BM at
concentrations ranging from 15.6 M to 500 nM for 120 s, and a
dissociation phase in buffer for 120 s.
[0809] The results are shown in FIG. 8. 1-1-A1_BM was found to bind
to human CD47 in this assay with a K.sub.D=10.4 nM.
[0810] 3.2 Analysis of Cell Surface Antigen-Binding by Flow
Cytometry
[0811] HEK293T cells (which express high levels of CD47) and cells
of a HEK293T cell-derived CD47 knockout cell line were incubated
with 20 .mu.g/ml of anti-CD47 antibody or isotype control antibody
at 4.degree. C. for 1 hr. The anti-CD47 antibody clone B6H12 (Santa
Cruz Biotechnology, cat no. sc-12730) was included in the analysis
as a positive control.
[0812] The cells were washed thrice with FACS buffer (PBS with 5 mM
EDTA and 0.5% BSA) and resuspended in FITC-conjugated anti-FC
antibody (Invitrogen, USA) for 40 min at 2-8.degree. C. Cells were
washed again and resuspended in 200 .mu.L of FACS flow buffer (PBS
with 5 mM EDTA) for flow cytometric analysis using MACSQuant 10
(Miltenyi Biotec, Germany). After acquisition, all raw data were
analyzed using Flowlogic software. Cells were gated using forward
and side scatter profile and Median of Fluorescence Intensity (MFI)
value was determined for native and overexpressing cell
populations.
[0813] The anti-CD47 antibodies were shown to bind to human CD47
with high specificity. FIGS. 3A and 3B show the results obtained
using clones 1-1-A1 and 1-1-A1_BM, and FIG. 3C shows results
obtained using the commercially-available anti-CD47 antibody clone
B6H12 (positive control).
[0814] Multiple myeloma and Burkitt's lymphoma cell lines were
analysed for CD47 expression by flow cytometry using anti-CD47
antibody clone B6H12. Briefly, 0.5.times.10.sup.6 cells were fixed
by treatment with 4% paraformaldehyde for 10 min at room
temperature, and subsequently stained with APC-conjugated anti-CD47
antibody at a 1:11 dilution, for 30 min at 4.degree. C. The results
of the analysis are shown in FIG. 3D and in the table below:
TABLE-US-00005 Cell Line % cells positive for CD47 MM.1S 99.9 H929
2.23 U226 93.3 8226 99.4 RAJI 97.9
[0815] 3.3 ELISAs for Determining Antibody Specificity
[0816] ELISAs were used to determine the binding specificity of the
antibodies. The antibodies were tested against target peptide and
protein as well as respective mouse, rat and monkey homologues
(Sino Biological Inc., China).
[0817] ELISAs were carried out according to standard protocols.
Briefly, 96-well plates (Nunc, Denmark) were coated with 1 .mu.g/ml
of Fc-tagged human CD47 in phosphate-buffered saline (PBS) for 16 h
at 4.degree. C. After blocking for 1 h with 1% BSA in Tris buffer
saline (TBS) at room temperature, the candidate antigen-binding
molecule was serially diluted with the highest conc. being 10
.mu.g/ml and added to the plate. Post 1 h incubation at RT, plates
were washed three times with TBS containing 0.05% Tween 20 (TBS-T)
and were then incubated with a HRP-conjugated anti-His antibody
(Life Technologies, Inc., USA) for 1 h at room temperature. After
washing, plates were developed with colorimetric detection
substrate 3,3',5,5'-tetramethylbenzidine (Turbo-TMB; Pierce, USA).
The reaction was stopped with 2M H.sub.2SO.sub.4, and OD was
measured at 450 nM.
[0818] Binding of anti-CD47 clone 1-1-A1_BM IgG1 ([1] of Example
2.2) to rhesus macaque CD47 (RhCD47) was compared to binding to
human CD47 (hCD47).
[0819] The results are shown in FIG. 5.
Example 4: Functional Characterisation
[0820] 4.1 Analysis of Ability to Block CD47-SIRP.alpha.
Interaction
[0821] 96-well plates (Nunc, Denmark) were coated with 1 .mu.g/ml
of untagged human CD47 protein (Sinobiological Inc, China) in
1.times.PBS for 16 h at 4.degree. C. After blocking for 1 h with 1%
BSA in TBS at room temperature, 1 .mu.g/ml of SIRP.alpha./human His
tagged fusion protein (Sinobiological Inc, China) was added either
in the absence of antibody, or in the presence of increasing
concentrations of anti-CD47 antibody at room temperature for 1 hr.
Plates were subsequently washed three times with TBS-T and
incubated with an HRP-conjugated anti-his secondary antibody
(Thermo Scientific, USA) for 1 h at room temperature. After
washing, plates were developed with colorimetric detection
substrate Turbo-TMB (Pierce, USA). The reaction was stopped with 2M
H2504, and OD was measured at 450 nM.
[0822] Percent inhibition of CD47-SIRP.alpha. interaction
calculated relative to the signal in the absence of SIRP.alpha.
(100%).
[0823] In a first experiment, inhibition of interaction between
CD47 and SIRP.alpha. was evaluated for the following
antigen-binding molecules: [0824] anti-CD47 clone 1-1-A1 IgG1 ([2]
of Example 2.2) [0825] anti-CD47 clone 5-48-A6 IgG1 ([3] of Example
2.2) [0826] anti-CD47 clone 5-48-D2 IgG1 ([4] of Example 2.2)
[0827] The results are shown in FIG. 4. Several of the anti-CD47
binding antibodies were found to be potent inhibitors of
CD47-SIRP.alpha. interaction.
[0828] 4.2 In Vitro Phagocytosis Assay
[0829] In vitro phagocytosis assays were performed according to
standard protocols. Briefly, Raji or HL60 cells were cultured in
RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1%
Pen/Strep at 37.degree. C. in a 5% CO.sub.2 incubator. HL-60 or
Raji cells were then harvested and CFSE-labelled using CellTrace
CFSE Cell Proliferation Kit (Thermo Scientific, USA), in accordance
with the manufacturer's protocol. The labelled cells were then
incubated with human peripheral blood-derived macrophages (Stemcell
Technologies, Canada) in the presence of 20 .mu.g/ml of anti-CD47
antibody, or an isotype control antibody for 2 h at 37.degree. C.
Cells were washed thrice with 1.times.PBS to remove all the
non-phagocytosed labelled cells and resuspended in 200 .mu.L of
FACS flow buffer (PBS with 5 mM EDTA) for flow cytometric analysis
using MACSQuant 10 (Miltenyi Biotec, Germany). After acquisition,
all raw data were analyzed using Flowlogic software. Cells were
gated using forward and side scatter profile and percentage of the
engulfed effector cells were calculated.
[0830] In a first experiment, antigen-binding molecules were
analysed for their ability to promote phagocytosis of CSFE-labelled
Raji cells by macrophages, compared to a negative control condition
in which PBS was added instead of antibodies.
[0831] The following antigen-binding molecules were analysed in the
experiment: [0832] anti-CD47 clone 1-1-A1_BM IgG1 ([1] of Example
2.2)
[0833] Anti-CD47 antibody clone B6H12 (Santa Cruz Biotechnology,
cat no. sc-12730) was included as a positive control condition.
[0834] The results are shown in FIG. 6. Anti-CD47 clone 1-1-A1_BM
IgG1 was found to be extremely potent at promoting phagocytosis of
Raji cells by macrophages.
[0835] In a separate experiment, antigen-binding molecules were
analysed for their ability to promote phagocytosis of CSFE-labelled
HL-60 cells by macrophages, as determined by fluorescence
microscopy. Phagocytic index was calculated as the number of
engulfed CFSE-labelled HL-60 cells per phagocyte, for 200 cells
using the fluorescence microscope.
[0836] The anti-CD47 clone 1-1-A1_BM IgG1 ([1] of Example 2.2) was
analysed in the experiment, and an isotype control condition was
included as a negative control.
[0837] The results are shown in FIGS. 7A to 7C. Anti-CD47 clone
1-1-A1_BM IgG1 was shown to be potent at inducing phagocytosis of
HL-60 cells by macrophages.
Example 5: Production of Humanised Versions of Anti-CD47 Clone
1-1-A1
[0838] Humanised versions of anti-CD47 antibody clone 1-1-A1 were
produced and purified as described in Example 2.
TABLE-US-00006 Antigen- binding molecule Polypeptides Antibody [6]
SEQ ID NO: 159 + SEQ ID NO: 160 11A1H1-IgG1 [7] SEQ ID NO: 161 +
SEQ ID NO: 160 11A1H2-IgG1 [8] SEQ ID NO: 162 + SEQ ID NQ: 160
11A1H3-IgG1 [9] SEQ ID NO: 163 + SEQ ID NQ: 160 11A1H4-IgG1 [10]
SEQ ID NO: 164 + SEQ ID NQ: 160 11A1H5-IgG1 [11] SEQ ID NO: 163 +
SEQ ID NO: 165 11A1H6-IgG1 [12] SEQ ID NO: 164 + SEQ ID NO: 165
11A1H7-IgG1 [13] SEQ ID NO: 163 + SEQ ID NO: 166 11A1H8-IgG1 [14]
SEQ ID NO: 164 + SEQ ID NO: 166 11A1H9-IgG1 [15] SEQ ID NO: 164 +
SEQ ID NO: 167 11A1H10-IgG1 [16] SEQ ID NO: 164 + SEQ ID NO: 168
11A1H11-IgG1
[0839] The CDRs of the humanised versions of anti-CD47 antibody
clone 1-1-A1 are shown below:
TABLE-US-00007 Clone HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2
LC-CDR3 1A11H1 GYTFTNYV INPYNDGT ASGGYYTMDY QHLEYSNGYSY KIS
SQSTHVPYT (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID (SEQ ID NO:
24) NO: 25) NO: 26) 32) NO: 33) NO: 34) 1A11H2 1A11H3 1A11H4 1A11H5
GYTFTGYV INPYNGGT (SEQ ID (SEQ ID NO: 137) NO: 138) 1A11H6 GYTFTNYV
INPYNDGT QHLEYSQGYSY KVS (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID NO:
24) NO: 25) 139) NO: 141) 1A11H7 GYTFTGYV INPYNGGT (SEQ ID (SEQ ID
NO: 137) NO: 138) 1A11H8 GYTFTNYV INPYNDGT QHLEYSTGYSY (SEQ ID (SEQ
ID (SEQ ID NO: NO: 24) NO: 25) 140) 1A11H9 GYTFTGYV INPYNGGT (SEQ
ID (SEQ ID NO: 137) NO: 138) 1A11H10 QHLEYSNGYSY (SEQ ID NO: 32)
1A11H11 KIS SQGTHVPYT (SEQ ID (SEQ ID NO: 33) NO: 142) Consensus
GYTFTX.sub.1YV INPYNX.sub.2GT ASGGYYTMDY QHLEYSX.sub.3GYSY
KX.sub.4S SQX.sub.5THVPYT X.sub.1 = N or X.sub.2 = D or (SEQ ID
X.sub.3 = N, Q X.sub.4 = I X.sub.5 = S or G (SEQ G (SEQ NO: 26) or
T (SEQ or V G (SEQ ID ID NO: ID NO: ID NO: 171) (SEQ ID NO: 173)
169) 170) NO: 172)
[0840] The FRs of the humanised versions of anti-CD47 antibody
clone 1-1-A1 are shown below:
TABLE-US-00008 Clone HC-FR1 HC-FR2 HC-FR3 HC-FR4 1A11H1
QVQLVQSGAEVKK IHWVRQAPGKG KSNEKFKGRVTLTS WGQGTLVTVSS PGASVKVSCKAS
LEWMGY DKSSTSAYMELSSL (SEQ ID NO: (SEQ ID NO: (SEQ ID RSEDTAVYYC
152) 143) NO: 144) (SEQ ID NO: 147) 1A11H2 KSNEKFKGRVTLTS
DTSTTTAYMELSSL RSEDTAVYYC (SEQ ID NO: 148) 1A11H3 MHWVRQAPGQG
KSNEKFQGRVTLTS WGQGTLV LEWMGY DTSTSTAYMELSSL (SEQ ID NO: (SEQ ID
RSEDTAVYYC 153) NO: 145) (SEQ ID NO: 149) 1A11H4 IHWVRQAPGQG
KYNQKFKGRVTLTS LEWMGY DTSTTTAYMELSRL (SEQ ID RSDDTAVYYC NO: 146)
(SEQ ID NO: 150) 1A11H5 NYAQKFKGRVTLTS WGQGTLVTVSS DTSTTTAYMELSRL
(SEQ ID NO: RSEDTAVYYC 152) (SEQ ID NO: 151) 1A11H6 KYNQKFKGRVTLTS
WGQGTLV DTSTTTAYMELSRL (SEQ ID NO: RSDDTAVYYC 153) (SEQ ID NO: 150)
1A11H7 NYAQKFKGRVTLTS WGQGTLVTVSS DTSTTTAYMELSRL (SEQ ID NO:
RSEDTAVYYC 152) (SEQ ID NO: 151) 1A11H8 KYNQKFKGRVTLTS WGQGTLV
DTSTTTAYMELSRL (SEQ ID NO: RSDDTAVYYC 153) (SEQ ID NO: 150) 1A11H9
NYAQKFKGRVTLTS WGQGTLVTVSS DTSTTTAYMELSRL (SEQ ID NO: RSEDTAVYYC
152) (SEQ ID NO: 151) 1A11H10 1A11H11 Consensus QVQLVQSGAEVKK
X.sub.6HWVRQAPG X.sub.8X.sub.9X.sub.10X.sub.11KFX.sub.12 WGQGTLVX18
PGASVKVSCKAS X.sub.7GLEWMGY GRVTLTSDX.sub.13SX.sub.14 X19X20X21
(SEQ ID NO: X.sub.6 = I or M SX.sub.15AYMELSX.sub.16LR X18 = T or
143) X.sub.7 = Q or K SX.sub.17DTAVYYC absent (SEQ ID NO: X.sub.8 =
K or N X19 = V or 174) X.sub.9 = S or Y absent X.sub.10 = N or A
X20 = S or X.sub.11 = E or Q absent X.sub.12 = K or Q X21 = S or
X.sub.13 = T or K absent X.sub.14 = T or S (SEQ ID NO: X.sub.15 = T
or S 176) X.sub.16 = S or R X.sub.17 = E or D (SEQ ID NO: 175)
Clone LC-FR1 LC-FR2 LC-FR3 LC-FR4 1A11H1 DVVMTQSPLSLP LHWYQQRPGQS
NRFSGVPDRFSGSG VTLGQPASISCR PRLLIY (SEQ SGTDFTLKISRVEA SS (SEQ ID
ID NO: 155) EDVGVYYC (SEQ NO: 154) ID NO: 156) 1A11H2 1A11H3 1A11H4
1A11H5 1A11H6 NRDSGVPDRFSGSG FGGGTKVEIK SGTDFTLKISRVEA (SEQ ID NO:
EDVGVYYC (SEQ 158) ID NO: 157) 1A11H7 1A11H8 1A11H9 1A11H10 1A11H11
NRFSGVPDRFSGSG SGTDFTLKISRVEA EDVGVYYC (SEQ ID NO: 156) Consensus
DVVMTQSPLSLP LHWYQQRPGQS NRX.sub.22SGVPDRFSG FGGGTKVEIK
VTLGQPASISCR PRLLIY SGSGTDFTLKISRV (SEQ ID NO: SS (SEQ ID (SEQ ID
NO: EAEDVGVYYC 158) NO: 154) 155) X.sub.22 = D or F (SEQ ID NO:
177)
Example 6: Biophysical Characterisation of Humanised Versions of
Anti-CD47 Antibody Clone 1-1-A1
[0841] 6.1 ELISAs for Determining Antibody Specificity
[0842] The binding specificity of the humanised versions of
anti-CD47 clone 1-1-A1 was analysed be ELISA.
[0843] 96-well plates (Nunc, Denmark) were coated with 1 .mu.g/ml
of human CD47 or VISTA protein in PBS, for 1 h at room temperature.
Plates were blocked for 1 h at room temperature with 1% BSA in Tris
buffer saline containing 0.05% Tween 20 (TBS-T). The test
antigen-binding molecules were added at concentrations ranging from
to 0.002 .mu.g/ml to 200 .mu.g/ml, and the plates were incubated at
room temperature for 1 h. Plates were then washed three times with
TBS-T, and were then incubated with a HRP-conjugated secondary
antibody for 1 h at room temperature. After washing, plates were
developed with colorimetric detection substrate
3,3',5,5'-tetramethylbenzidine (Turbo-TMB; Pierce, USA). The
reaction was stopped after 3.5 min with 2M H2504, and OD was
measured at 450 nM.
[0844] The following antigen-binding molecules were analysed in the
experiment: [0845] 11A1H3-IgG1 ([8] of Example 5). [0846]
11A1H4-IgG1 ([9] of Example 5). [0847] 11A1H5-IgG1 ([10] of Example
5). [0848] 11A1H6-IgG1 ([11] of Example 5). [0849] 11A1H7-IgG1
([12] of Example 5). [0850] 11A1H9-IgG1 ([14] of Example 5). [0851]
11A1H10-IgG1 ([15] of Example 5). [0852] 11A1H11-IgG1 ([16] of
Example 5). [0853] anti-CD47 clone 1-1-A1_BM IgG1 ([1] of Example
2.2). [0854] anti-CD33 IgG1 ([5] of Example 2.2) (negative
control)--referred to as `M195` in FIG. 9.
[0855] The results are shown in FIG. 9. The humanised antibodies
displayed binding to human CD47. EC50 values were calculated, and
the fold increase in EC50 value relative to EC50 for 1-1-A1_BM are
shown below.
TABLE-US-00009 Fold increase in EC.sub.50 relative Antibody
EC.sub.50 (.mu.g/mL) to 1-1-A1_BM 11A1H3 0.00022 0.12 11A1H4
0.00018 0.10 11A1H5 0.00015 0.08 11A1H6 13.4 7444 11A1H7 36.8 20444
11A1H9 23.5 13056 11A1H10 38.1 21167 11A1H11 0.0021 1.17 11A1BM
0.0018 1.0
[0856] In a separate ELISA, the antigen-binding molecules were
evaluated for binding to human VISTA. Anti-human VISTA antibody
VSTB112 (described e.g. in WO 2015/097536) was included as a
positive control.
[0857] The results are shown in FIG. 10. The humanised antibodies
were found not to cross-react with human VISTA.
[0858] 6.2 Global Affinity Study Using BLITz System
[0859] The affinity of binding of humanised versions of anti-CD47
clone 1-1-A1 to human CD47 was in BLI experiments performed using a
single channel BLItz system (ForteBio, Menlo Park, Calif.) using
Anti-human immunoglobulin G (IgG) Fc (AHC) coated biosensor tips
(Pall ForteBio, Menlo Park, Calif.) for capturing human IgGs.
Biosensors were first hydrated for at least 10 m in assay buffer
(phosphate buffered saline) followed by buffer baseline for 60 s
and loading of the human IgGs onto the biosensor tips at 25 nM for
120 s. The tips were then washed briefly for 60 s with the assay
buffer to remove nonspecifically bound proteins or unbound IgGs for
obtaining a second buffer baseline. The association phase of the
IgGs with antigens (250 nM to 62.5 nM) was set up at 120 s which
was followed by a dissociation phase (assay buffer alone) for 120
s. All the BLITz runs were measured at room temperature at a
stirring speed of 1000 rpm and AHC biosensors were regenerated
using 10 mM of glycine (pH 2.7) after the assay. Binding affinity
between the immobilized antibodies on the AHC sensors and human
CD47 were determined by analyzing the binding kinetic curves using
the software BLItz Pro. All the sensorgrams were reference
subtracted and globally fitted into a 1:1 model which analysed the
binding curves at different concentrations of antigens and
generated kinetic constants (KD/Ka/Kd) for the globally fitted
data. All the binding curves were subjected to step correction
which corrects the misalignment between association and
dissociation steps and only the curves with R.sup.2 values greater
than 0.9 were used for analysis.
[0860] Representative sensorgrams are shown in FIGS. 11A to 11H,
and the calculated kinetic and thermodynamic constants are shown
below.
TABLE-US-00010 Antibody K.sub.D (nM) K.sub.on (M.sup.-1s.sup.-1)
K.sub.dis (s.sup.-1) 11A1BM 9.31 1.30 .times. 10.sup.5 1.21 .times.
10.sup.-3 11A1H3 3.39 2.66 .times. 10.sup.5 9.04 .times. 10.sup.-4
11A1H5 9.28 1.29 .times. 10.sup.5 1.20 .times. 10.sup.-3 11A1H6 134
1.08 .times. 10.sup.5 1.44 .times. 10.sup.-2 11A1H7 232 3.24
.times. 10.sup.5 7.50 .times. 10.sup.-3 11A1H9 23.3 2.73 .times.
10.sup.5 6.35 .times. 10.sup.-3 11A1H10 111 8.09 .times. 10.sup.4
8.98 .times. 10.sup.-3 11A1H11 13.8 1.18 .times. 10.sup.5 4.28
.times. 10.sup.-3
Example 7: Functional Characterisation of Humanised Versions of
Anti-CD47 Antibody Clone 1-1-A1
[0861] 7.1 Analysis of Ability to Block CD47-SIRP.alpha.
Interaction
[0862] The ability of humanised versions of anti-CD47 antibody
clone 1-1-A1 to inhibit interaction between human CD47 and
SIRP.alpha. was investigated by ELISA, as described in Example
4.1.
[0863] The following antigen-binding molecules were analysed in the
experiment: [0864] 11A1H4-IgG1 ([9] of Example 5). [0865]
11A1H5-IgG1 ([10] of Example 5). [0866] 11A1H6-IgG1 ([11] of
Example 5). [0867] 11A1H7-IgG1 ([12] of Example 5). [0868]
11A1H9-IgG1 ([14] of Example 5). [0869] 11A1H10-IgG1 ([15] of
Example 5). [0870] 11A1H11-IgG1 ([16] of Example 5). [0871]
anti-CD47 clone 1-1-A1_BM IgG1 ([1] of Example 2.2). [0872]
anti-CD33 IgG1 ([5] of Example 2.2) (negative control)--referred to
as `M195` in FIG. 12. [0873] J6M0-IgG1 ([17] below) (negative
control). [0874] Isotype control hlgG (negative control).
TABLE-US-00011 [0874] Antigen- binding molecule Polypeptides
Antibody [17] J6M0 VH-CH1-CH2-CH3 J6M0-IgG1 (SEQ ID NO: 125) + J6M0
VL-C.sub.K (SEQ ID NO: 126),
[0875] The results are shown in FIG. 12. IC50 values were
calculated, and the fold increase in IC50 value for the inhibition
of interaction between CD47 and SIRP.alpha. relative to IC50 for
1-1-A1_BM are shown below.
TABLE-US-00012 Fold increase in IC.sub.50 relative Antibody
IC.sub.50 (.mu.g/mL) to 1-1-A1_BM 11A1H4 0.150 0.32 11A1H5 0.201
0.42 11A1H6 >100 >200 11A1H7 >100 >200 11A1H9 >100
>200 11A1H10 >100 >200 11A1H11 0.483 1.02 11A1BM 0.474
1.00
[0876] 7.2 In Vitro Hemagglutination Assay
[0877] The hemagglutinating capacity of the humanised versions of
anti-CD47 antibody clone 1-1-A1 was investigated using an in vitro
hemagglutination assay.
[0878] To evaluate the hemagglutinating capacity of the test
antigen-binding molecules, human RBCs were prepared by extensively
washing blood with 1.times.PBS and centrifuging at 1500 rpm for 5
min, until a clear supernatant was observed. For the assay, 1%
human RBCs were incubated for 1 hr at RT in presence or absence of
increasing concentrations of the test antigen-binding molecules in
a round bottom 96 well plate. Presence of hemagglutination was
accessed by the presence of non-settled RBCs, appearing as a haze
compared to a punctuated red dot of non-hemagglutinated RBCs.
[0879] An anti-red blood cells antibody (AbCam, cat. no. ab34858)
condition was included as a positive control for hemagglutination,
and an isotype control antibody condition was included as a
negative control.
[0880] The following antigen-binding molecules were analysed in the
experiment: [0881] 11A1H1-IgG1 ([6] of Example 5). [0882]
11A1H2-IgG1 ([7] of Example 5). [0883] 11A1H3-IgG1 ([7] of Example
5). [0884] 11A1H4-IgG1 ([8] of Example 5). [0885] 11A1H5-IgG1 ([10]
of Example 5). [0886] 11A1H6-IgG1 ([11] of Example 5). [0887]
11A1H7-IgG1 ([12] of Example 5). [0888] 11A1H9-IgG1 ([14] of
Example 5). [0889] 11A1H10-IgG1 ([15] of Example 5). [0890]
11A1H11-IgG1 ([16] of Example 5). [0891] anti-CD47 clone 1-1-A1_BM
IgG1 ([1] of Example 2.2). [0892] anti-CD33 IgG1 ([5] of Example
2.2) (negative control)--referred to as `M195` in FIG. 13. [0893]
J6M0-IgG1 ([17] of Example 7.1) (negative control)--referred to as
`Irrelevant Ag` in FIG. 13. [0894] An anti-red blood cells antibody
(AbCam, cat. no. ab34858)-- referred to as `ANTI RBC` in FIG.
13.
[0895] The results are shown in FIG. 13.
Sequence CWU 1
1
1791323PRTHomo sapiensCD47 isoform OA3-323 1Met 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 Glu2292PRTHomo sapiensCD47 isoform OA3-293 2Met 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 2903305PRTHomo sapiensCD47 isoform OA3-305 3Met 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 Asn 290 295
300Asn3054311PRTHomo sapiensCD47 isoform OA3-312 4Met 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 Asn305 3105305PRTHomo sapiensmature CD47 isoform
OA3-323 5Gln 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 Asn Glu Asn Ile Leu Ile Val 115 120 125Ile Phe Pro
Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe Gly Ile 130 135 140Lys
Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr Ile Ala145 150
155 160Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val Gly
Ala 165 170 175Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala
Thr Gly Leu 180 185 190Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile
Leu Leu His Tyr Tyr 195 200 205Val Phe Ser Thr Ala Ile Gly Leu Thr
Ser Phe Val Ile Ala Ile Leu 210 215 220Val Ile Gln Val Ile Ala Tyr
Ile Leu Ala Val Val Gly Leu Ser Leu225 230 235 240Cys Ile Ala Ala
Cys Ile Pro Met His Gly Pro Leu Leu Ile Ser Gly 245 250 255Leu Ser
Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr Met Lys 260 265
270Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys Ala Val
275 280 285Glu Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met
Asn Asp 290 295 300Glu3056274PRTHomo sapiensmature CD47 isoform
OA3-293 6Gln 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 Asn Glu Asn Ile Leu Ile Val 115 120 125Ile Phe Pro
Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe Gly Ile 130 135 140Lys
Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr Ile Ala145 150
155 160Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val Gly
Ala 165 170 175Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala
Thr Gly Leu 180 185 190Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile
Leu Leu His Tyr Tyr 195 200 205Val Phe Ser Thr Ala Ile Gly Leu Thr
Ser Phe Val Ile Ala Ile Leu 210 215 220Val Ile Gln Val Ile Ala Tyr
Ile Leu Ala Val Val Gly Leu Ser Leu225 230 235 240Cys Ile Ala Ala
Cys Ile Pro Met His Gly Pro Leu Leu Ile Ser Gly 245 250 255Leu Ser
Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr Met Lys 260 265
270Phe Val7287PRTHomo sapiensmature CD47 isoform OA3-305 7Gln 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
Asn Glu Asn Ile Leu Ile Val 115 120 125Ile Phe Pro Ile Phe Ala Ile
Leu Leu Phe Trp Gly Gln Phe Gly Ile 130 135 140Lys Thr Leu Lys Tyr
Arg Ser Gly Gly Met Asp Glu Lys Thr Ile Ala145 150 155 160Leu Leu
Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val Gly Ala 165 170
175Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr Gly Leu
180 185 190Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His
Tyr Tyr 195 200 205Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val
Ile Ala Ile Leu 210 215 220Val Ile Gln Val Ile Ala Tyr Ile Leu Ala
Val Val Gly Leu Ser Leu225 230 235 240Cys Ile Ala Ala Cys Ile Pro
Met His Gly Pro Leu Leu Ile Ser Gly 245 250 255Leu Ser Ile Leu Ala
Leu Ala Gln Leu Leu Gly Leu Val Tyr Met Lys 260 265 270Phe Val Ala
Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Asn Asn 275 280
2858293PRTHomo sapiensmature CD47 isoform OA3-312 8Gln 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 Asn Glu Asn
Ile Leu Ile Val 115 120 125Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe
Trp Gly Gln Phe Gly Ile 130 135 140Lys Thr Leu Lys Tyr Arg Ser Gly
Gly Met Asp Glu Lys Thr Ile Ala145 150 155 160Leu Leu Val Ala Gly
Leu Val Ile Thr Val Ile Val Ile Val Gly Ala 165 170 175Ile Leu Phe
Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr Gly Leu 180 185 190Gly
Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His Tyr Tyr 195 200
205Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala Ile Leu
210 215 220Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu
Ser Leu225 230 235 240Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro
Leu Leu Ile Ser Gly 245 250 255Leu Ser Ile Leu Ala Leu Ala Gln Leu
Leu Gly Leu Val Tyr Met Lys 260 265 270Phe Val Ala Ser Asn Gln Lys
Thr Ile Gln Pro Pro Arg Lys Ala Val 275 280 285Glu Glu Pro Leu Asn
2909109PRTArtificial SequenceSynthetic-V-type Ig-like domain 9Gln
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 100 10510123PRTHomo sapiensCD47 extracellular region 1
10Gln 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 Asn Glu 115 1201121PRTHomo sapiensCD47 transmembrane region
1 11Asn Ile Leu Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe
Trp1 5 10 15Gly Gln Phe Gly Ile 201214PRTHomo sapiensCD47
cytoplasmic region 1 12Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp
Glu Lys Thr1 5 101321PRTHomo sapiensCD47 transmembrane region 2
13Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val1
5 10 15Gly Ala Ile Leu Phe 201410PRTHomo sapiensCD47 extracellular
region 2 14Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala1 5 101521PRTHomo
sapiensCD47 transmembrane region 3 15Thr Gly Leu Gly Leu Ile Val
Thr Ser Thr Gly Ile Leu Ile Leu Leu1 5 10 15His Tyr Tyr Val Phe
20167PRTHomo sapiensCD47 cytoplasmic region 2 16Ser Thr Ala Ile Gly
Leu Thr1 51721PRTHomo sapiensCD47 transmembrane region 4 17Ser Phe
Val Ile Ala Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu1 5 10 15Ala
Val Val Gly Leu 201812PRTHomo sapiensCD47 extracellular region 3
18Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly1 5 101921PRTHomo
sapiensCD47 transmembrane region 5 19Pro Leu Leu Ile Ser Gly Leu
Ser Ile Leu Ala Leu Ala Gln Leu Leu1 5 10 15Gly Leu Val Tyr Met
202034PRTHomo sapiensCD47 cytoplasmic region 3 20Lys Phe Val Ala
Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys Ala1 5 10 15Val Glu Glu
Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met Asn 20 25 30Asp
Glu2110PRTHomo sapiensCD47 region targeted by 1-1-A1 and 1-1-A1_BM
21Val Lys Trp Lys Phe Lys Gly Arg Asp Ile1 5 102211PRTHomo
sapiensCD47 region targeted by 5-48-A6 and 5-48-D2 22Lys Thr Lys
Ser Val Glu Phe Thr Phe Cys Asn1 5 1023117PRTArtificial
SequenceSynthetic-1-1-A1_BM heavy chain variable region 23Gln Val
Gln Leu Gln Gln Ser Gly Pro Asp Leu Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25
30Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Ser Asn Glu Lys
Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Ser
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp
Gly Gln Gly Thr Ser 100 105 110Val Thr Val Ser Ser
115248PRTArtificial SequenceSynthetic-1-1-A1_BM, Synthetic-1-1-A1,
Synthetic-11A1H1, Synthetic-11A1H2, Synthetic-11A1H3, Synthetic-
11A1H4, Synthetic-11A1H6, Synthetic-11A1H8 heavy chain CDR1 24Gly
Tyr Thr Phe Thr Asn Tyr Val1 5258PRTArtificial
SequenceSynthetic-1-1-A1_BM, Synthetic-1-1-A1, Synthetic-11A1H1,
Synthetic-11A1H2, Synthetic-11A1H3, Synthetic- 11A1H4,
Synthetic-11A1H6, Synthetic-11A1H8 heavy chain CDR2 25Ile Asn Pro
Tyr Asn Asp Gly Thr1 52610PRTArtificial
SequenceSynthetic-1-1-A1_BM, Synthetic-1-1-A1, Synthetic-11A1H1,
Synthetic-11A1H2, Synthetic-11A1H3, Synthetic- 11A1H4,
Synthetic-11A1H6, Synthetic-11A1H8, Synthetic-11A1H5,
Synthetic-11A1H7, Synthetic-11A1H9, Synthetic-11A1H10, Synthetic-
11A1H11 heavy chain CDR3 26Ala Ser Gly Gly Tyr Tyr Thr Met Asp Tyr1
5 102725PRTArtificial SequenceSynthetic-1-1-A1_BM heavy chain FR1
27Gln Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser 20 252817PRTArtificial
SequenceSynthetic-1-1-A1_BM heavy chain FR2 28Ile His Trp Val Arg
Gln Lys Pro Gly Gln Gly Leu Glu Trp Met Gly1 5 10
15Tyr2938PRTArtificial SequenceSynthetic-1-1-A1_BM heavy chain FR3
29Lys Ser Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys1
5 10 15Ser Ser Thr Ser Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu
Asp 20 25 30Thr Ala Val Tyr Tyr Cys 353011PRTArtificial
SequenceSynthetic-1-1-A1_BM heavy chain FR4 30Trp Gly Gln Gly Thr
Ser Val Thr Val Ser Ser1 5 1031112PRTArtificial
SequenceSynthetic-1-1-A1_BM light chain variable region 31Asp Val
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Asp
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln His Leu Glu Tyr Ser 20 25
30Asn Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser
35 40 45Pro Gln Leu Leu Ile Tyr Lys Ile 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 Tyr
Cys Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 1103211PRTArtificial
SequenceSynthetic-1-1-A1_BM, Synthetic-1-1-A1, Synthetic-11A1H1,
Synthetic-11A1H2, Synthetic-11A1H3, Synthetic- 11A1H4,
Synthetic-11A1H5, Synthetic-11A1H10, Synthetic-11A1H11 light chain
CDR1 32Gln His Leu Glu Tyr Ser Asn Gly Tyr Ser Tyr1 5
10333PRTArtificial SequenceSynthetic-1-1-A1_BM, Synthetic-1-1-A1,
Synthetic-11A1H1, Synthetic-11A1H2, Synthetic-11A1H3, Synthetic-
11A1H4, Synthetic-11A1H5, Synthetic-11A1H11 light chain CDR2 33Lys
Ile Ser1349PRTArtificial SequenceSynthetic-1-1-A1_BM,
Synthetic-1-1-A1, Synthetic-11A1H1, Synthetic-11A1H2,
Synthetic-11A1H3, Synthetic- 11A1H4, Synthetic-11A1H5,
Synthetic-11A1H6, Synthetic-11A1H7 11A1H8, Synthetic-11A1H9 11A1H10
light chain CDR3 34Ser Gln Ser Thr His Val Pro Tyr Thr1
53526PRTArtificial SequenceSynthetic-1-1-A1_BM light chain FR1
35Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly1
5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 20
253617PRTArtificial SequenceSynthetic-1-1-A1_BM light chain FR2
36Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile1
5 10 15Tyr3736PRTArtificial SequenceSynthetic-1-1-A1_BM light chain
FR3 37Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly1 5 10 15Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
Leu Gly 20 25 30Val Tyr Tyr Cys 353810PRTArtificial
SequenceSynthetic-1-1-A1_BM light chain FR4 38Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys1 5 1039117PRTArtificial
SequenceSynthetic-1-1-A1 heavy chain variable region 39Glu Val Gln
Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala1 5 10 15Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Val
Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Ser Asn Glu Lys Phe
50 55 60Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Ser Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly
Gln Gly Thr Ser 100 105 110Val Thr Val Ser Ser 1154025PRTArtificial
SequenceSynthetic-1-1-A1 heavy chain FR1 40Glu Val Gln Leu Gln Gln
Ser Gly Pro Asp Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser
Cys Lys Ala Ser 20 254117PRTArtificial SequenceSynthetic-1-1-A1
heavy chain FR2 41Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
Glu Trp Ile Gly1 5 10 15Tyr4238PRTArtificial
SequenceSynthetic-1-1-A1 heavy chain FR3 42Lys Ser Asn Glu Lys Phe
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys1 5 10 15Ser Ser Thr Ser Ala
Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp 20 25 30Ser Ala Val Tyr
Tyr Cys 354311PRTArtificial SequenceSynthetic-1-1-A1 heavy chain
FR4 43Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser1 5
1044112PRTArtificial SequenceSynthetic-1-1-A1 light chain variable
region 44Asp 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 His Leu
Glu Tyr Ser 20 25 30Asn Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Ile 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 Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 1104526PRTArtificial
SequenceSynthetic-1-1-A1 light chain FR1 45Asp 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 20 254617PRTArtificial SequenceSynthetic-1-1-A1
light chain FR2 46Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro
Gln Leu Leu Ile1 5 10 15Tyr4736PRTArtificial
SequenceSynthetic-1-1-A1 light chain FR3 47Asn Arg Phe Ser Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu
Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 20 25 30Val Tyr Phe Cys
354810PRTArtificial SequenceSynthetic-1-1-A1 light chain FR4 48Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1049123PRTArtificial
SequenceSynthetic-5-48-A6 heavy chain variable region 49Gln Val Gln
Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu
Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly
Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe
Leu65 70 75 80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr
Tyr Cys Ala 85 90 95Arg Val Pro Thr Gly Arg Ile Lys Ser Tyr Phe Tyr
Ala Met Asp Tyr 100 105 110Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser 115 120508PRTArtificial SequenceSynthetic-5-48-A6 heavy chain
CDR1 50Gly Phe Ser Leu Thr Ser Tyr Gly1 5517PRTArtificial
SequenceSynthetic-5-48-A6 heavy chain CDR2 51Ile Trp Ala Gly Gly
Ser Thr1 55217PRTArtificial SequenceSynthetic-5-48-A6 heavy chain
CDR3 52Ala Arg Val Pro Thr Gly Arg Ile Lys Ser Tyr Phe Tyr Ala Met
Asp1 5 10 15Tyr5325PRTArtificial SequenceSynthetic-5-48-A6 heavy
chain FR1 53Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro
Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser 20
255417PRTArtificial SequenceSynthetic-5-48-A6 heavy chain FR2 54Val
His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly1 5 10
15Val5538PRTArtificial SequenceSynthetic-5-48-A6 heavy chain FR3
55Asn Tyr Asn Ser Ala Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn1
5 10 15Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp
Asp 20 25 30Thr Ala Met Tyr Tyr Cys 355611PRTArtificial
SequenceSynthetic-5-48-A6 heavy chain FR4 56Trp Gly Gln Gly Thr Ser
Val Thr Val Ser Ser1 5 1057107PRTArtificial
SequenceSynthetic-5-48-A6 light chain variable region 57Asp Ile Lys
Met Thr Gln Ser Pro Ser Ser Met Tyr Ser Ser Leu Gly1 5 10 15Glu Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30Leu
Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40
45Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu
Tyr65 70 75 80Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu
Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105586PRTArtificial SequenceSynthetic-5-48-A6 light chain CDR1
58Gln Asp Ile Ser Ser Tyr1 5593PRTArtificial
SequenceSynthetic-5-48-A6 light chain CDR2 59Arg Ala
Asn1609PRTArtificial SequenceSynthetic-5-48-A6 light chain CDR3
60Leu Gln Tyr Asp Glu Phe Pro Tyr Thr1 56126PRTArtificial
SequenceSynthetic-5-48-A6 light chain FR1 61Asp Ile Lys Met Thr Gln
Ser Pro Ser Ser Met Tyr Ser Ser Leu Gly1 5 10 15Glu Arg Val Thr Ile
Thr Cys Lys Ala Ser 20 256217PRTArtificial
SequenceSynthetic-5-48-A6 light chain FR2 62Leu Ser Trp Phe Gln Gln
Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile1 5 10
15Tyr6336PRTArtificial SequenceSynthetic-5-48-A6 light chain FR3
63Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly1
5 10 15Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr Glu Asp Met
Gly 20 25 30Ile Tyr Tyr Cys 356410PRTArtificial
SequenceSynthetic-5-48-A6 light chain FR4 64Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys1 5 1065115PRTArtificial SequenceSynthetic-5-48-D2
heavy chain variable region 65Glu Val Lys Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asn Pro Asp
Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60Lys Asp Lys Phe Ile
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Thr
Gly Thr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ala
115668PRTArtificial SequenceSynthetic-5-48-D2 heavy chain CDR1
66Gly Phe Asp Phe Ser Arg Tyr Trp1 5678PRTArtificial
SequenceSynthetic-5-48-D2 heavy chain CDR2 67Ile Asn Pro Asp Ser
Ser Thr Ile1 5688PRTArtificial SequenceSynthetic-5-48-D2 heavy
chain CDR3 68Ala Thr Gly Thr Gly Phe Ala Tyr1 56925PRTArtificial
SequenceSynthetic-5-48-D2 heavy chain FR1 69Glu Val Lys Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser
Cys Ala Ala Ser 20 257017PRTArtificial SequenceSynthetic-5-48-D2
heavy chain FR2 70Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile Gly1 5 10 15Glu7138PRTArtificial
SequenceSynthetic-5-48-D2 heavy chain FR3 71Asn Tyr Thr Pro Ser Leu
Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn1 5 10 15Ala Lys Asn Thr Leu
Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp 20 25 30Thr Ala Leu Tyr
Tyr Cys 357211PRTArtificial SequenceSynthetic-5-48-D2 heavy chain
FR4 72Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala1 5
1073107PRTArtificial SequenceSynthetic-5-48-D2 light chain variable
region 73Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile
Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro
Gln Leu Leu Val 35 40 45Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys
Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Gly Ser Tyr Tyr Cys
Gln His His Tyr Val Thr Pro Trp 85 90 95Thr Phe Gly Gly Val Thr Lys
Leu Glu Ile Lys 100 105746PRTArtificial SequenceSynthetic-5-48-D2
light chain CDR1 74Glu Asn Ile Tyr Ser Tyr1 5753PRTArtificial
SequenceSynthetic-5-48-D2 light chain CDR2 75Asn Ala
Lys1769PRTArtificial SequenceSynthetic-5-48-D2 light chain CDR3
76Gln His His Tyr Val Thr Pro Trp Thr1 57726PRTArtificial
SequenceSynthetic-5-48-D2 light chain FR1 77Asp Ile Gln Met Thr Gln
Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile
Thr Cys Arg Ala Ser 20 257817PRTArtificial
SequenceSynthetic-5-48-D2 light chain FR2 78Leu Ala Trp Tyr Gln Gln
Lys Gln Gly Lys Ser Pro Gln Leu Leu Val1 5 10
15Tyr7936PRTArtificial SequenceSynthetic-5-48-D2 light chain FR3
79Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly1
5 10 15Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro Glu Asp Phe
Gly 20 25 30Ser Tyr Tyr Cys 358010PRTArtificial
SequenceSynthetic-5-48-D2 light chain FR4 80Phe Gly Gly Val Thr Lys
Leu Glu Ile Lys1 5 108119PRTArtificial SequenceSynthetic-1-1-A1
heavy chain SignalP 81Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu
Ser Gly Thr Ala Gly1 5 10 15Val His Ser8219PRTArtificial
SequenceSynthetic-1-1-A1 light chain SignalP 82Met Lys Leu Pro Val
Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala1 5 10 15Ser Ser
Ser8319PRTArtificial SequenceSynthetic-5-48-A6 heavy chain SignalP
83Met Ala Val Leu Val Leu Phe Leu Cys Leu Val Ala Phe Pro Ser Cys1
5 10 15Val Leu Ser8420PRTArtificial SequenceSynthetic-5-48-A6 light
chain SignalP 84Met Arg Thr Pro Ala Gln Phe Leu Gly Ile Leu Leu Leu
Trp Phe Pro1 5 10 15Gly Ile Lys Cys 208518PRTArtificial
SequenceSynthetic-5-48-D2 heavy chain SignalP 85Met Asp Phe Gly Leu
Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val1 5 10 15Gln
Cys8620PRTArtificial SequenceSynthetic-5-48-D2 light chain SignalP
86Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr1
5 10 15Gly Ala Arg Cys 2087351DNAArtificial
SequenceSynthetic-1-1-A1_BM heavy chain DNA 87caggtgcagc tgcagcagtc
tggaccagac ctgaagaagc ctggagccag cgtgaaggtg 60tcctgtaagg tgtccggcta
caccttcaca aactatgtga tccactgggt gaggcagaag 120ccaggacagg
gcctggagtg gatgggctac atcaacccct ataatgacgg caccaagtct
180aatgagaagt ttaagggcaa ggccaccctg acatctgata agagcagcac
cagcgcctac 240atggagctgt ctagcctgac cagcgaggac acagccgtgt
actattgcgc ttccggcggc 300tactatacaa tggattattg gggccagggc
accagcgtga cagtgtcctc t 35188336DNAArtificial
SequenceSynthetic-1-1-A1_BM light chain DNA 88gacgtggtca tgacccagac
accactgtcc ctgcctgtga ccctgggcga tcaggcctct 60atcagctgta gaagctccca
gcacctggag tacagcaacg gctactccta tctgcactgg 120tatcagcagc
gcccaggaca gtctccacag ctgctgatct acaagatctc taatcggttc
180agcggcgtgc ctgacaggtt ttccggctct ggcagcggca ccgatttcac
actgaagatc 240agcagagtgg aggctgagga cctgggcgtg tactattgct
cccagtctac ccacgtgccc 300tatacatttg gcggcggcac caagctggag atcaag
33689351DNAArtificial SequenceSynthetic-1-1-A1 heavy chain DNA
89gaggtccagc tgcagcagtc tggacctgac ctagtaaagc ctggggcttc agtgaagatg
60tcctgcaagg cttctggata cacattcact aattatgtta tacactgggt gaagcagaag
120cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg
tactaagtcc 180aatgagaagt tcaaaggcaa ggccacactg acttcagaca
aatcctccac ctcagcctac 240atggagctca gcagcctgac ctctgaggac
tctgcggtct attactgtgc aagcggaggg 300tactatacta tggactattg
gggtcaagga acctcagtca ccgtctcctc g 35190336DNAArtificial
SequenceSynthetic-1-1-A1 light chain DNA 90gatgttgtga tgacccaaac
tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca
acaccttgaa tacagtaatg gatactccta tttgcattgg 120tacctgcaga
agccaggcca gtctccacag ctcctgatct acaaaatttc caaccgattt
180tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac
actcaagatc 240agcagagtgg aggctgagga tctgggggtt tatttctgct
ctcaaagtac acatgttccg 300tacacattcg gaggggggac caagctggaa ataaaa
33691369DNAArtificial SequenceSynthetic-5-48-A6 heavy chain DNA
91caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc
60acttgcactg tctctgggtt ttcattaacc agttatggtg tacactgggt tcgccagcct
120ccaggaaagg gtctggagtg gctgggagta atatgggctg gtggaagcac
aaattataat 180tcggctctca tgtccagact gagcatcagc aaagacaact
ccaagagcca agttttctta 240aaaatgaaca gtctgcaaac tgatgacaca
gccatgtact actgtgccag agttccgaca 300ggtcggatta aatcttattt
ctatgctatg gactactggg gtcaaggaac ctcagtcacc 360gtctcctcg
36992321DNAArtificial SequenceSynthetic-5-48-A6 light chain DNA
92gacatcaaga tgacccagtc tccatcttcc atgtattcat ctcttggaga gagagtcact
60atcacttgca aggcgagtca ggacattagt agctatttaa gctggttcca gcagaaacca
120gggaagtctc ctaagaccct gatctatcgt gcaaacagat tggtggatgg
ggtcccatca 180aggttcagtg gcagtggatc tgggcaagat tattctctca
ccatcagcag cctggagtat 240gaagatatgg gaatttatta ttgtctacag
tatgatgagt ttccgtacac gttcggaggg 300gggaccaagc tggaaataaa a
32193345DNAArtificial SequenceSynthetic-5-48-D2 heavy chain DNA
93gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc ctggaggatc cctgaaactc
60tcctgtgcag cctcaggatt cgattttagt agatactgga tgagttgggt ccggcaggct
120ccagggaaag ggctagaatg gattggagaa attaatccag atagcagtac
gataaactat 180acgccatctc taaaggataa attcatcatc tccagagaca
acgccaaaaa tacgctgtac 240ctgcaaatga gcaaagtgag atctgaggac
acagcccttt attactgtgc aactgggacg 300gggtttgctt actggggcca
agggactctg gtcactgtct ctgcg 34594321DNAArtificial
SequenceSynthetic-5-48-D2 light chain DNA 94gacatccaga tgactcagtc
tccagcttcc ctatctgcat ctgtgggaga aactgtcacc 60atcacatgtc gagcaagtga
gaatatttac agttatttag catggtatca gcagaaacag 120ggaaaatctc
ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccctca
180aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag
cctgcagcct 240gaagattttg ggagttatta ctgtcaacat cattatgtta
ctccgtggac gttcggtgga 300gtcaccaagc tggaaatcaa a 32195504PRTHomo
sapiens 95Met 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 50096508PRTHomo sapiens 96Met 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 Val Gln Ser Leu Asp Thr Asn Asp Ile Thr 420 425 430Tyr
Ala Asp Leu Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala 435 440
445Ala Glu Pro Asn Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro
450 455 460Gln Pro Ala Ser Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp
Met Val465 470 475 480His Leu Asn Arg Thr Pro Lys Gln Pro Ala Pro
Lys Pro Glu Pro Ser 485 490 495Phe Ser Glu Tyr Ala Ser Val Gln Val
Pro Arg Lys 500 50597503PRTHomo sapiens 97Met 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 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 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
50098474PRTHomo sapiens 98Glu 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 Ala Gly Thr Glu Leu 100 105 110Ser
Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala 115 120
125Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly
130 135 140Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
Asn Glu145 150 155 160Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val
Gly Glu Ser Val Ser 165 170 175Tyr Ser Ile His Ser Thr Ala Lys Val
Val Leu Thr Arg Glu Asp Val 180 185 190His Ser Gln Val Ile Cys Glu
Val Ala His Val Thr Leu Gln Gly Asp 195 200 205Pro Leu Arg Gly Thr
Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro 210 215 220Thr Leu Glu
Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn225 230 235
240Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr
245 250 255Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser
Thr Val 260 265 270Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser
Trp Leu Leu Val 275 280 285Asn Val Ser Ala His Arg Asp Asp Val Lys
Leu Thr Cys Gln Val Glu 290 295 300His Asp Gly Gln Pro Ala Val Ser
Lys Ser His Asp Leu Lys Val Ser305 310 315 320Ala His Pro Lys Glu
Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly 325 330 335Ser Asn Glu
Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu 340 345 350Leu
Val Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln 355 360
365Lys Lys Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu
370 375 380Lys Asn Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr
Tyr Ala385 390 395 400Asp Leu Asn Leu Pro Lys Gly Lys Lys Pro Ala
Pro Gln Ala Ala Glu 405 410 415Pro Asn Asn His Thr Glu Tyr Ala Ser
Ile Gln Thr Ser Pro Gln Pro 420 425 430Ala Ser Glu Asp Thr Leu Thr
Tyr Ala Asp Leu Asp Met Val His Leu 435 440 445Asn Arg Thr Pro Lys
Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser 450 455 460Glu Tyr Ala
Ser Val Gln Val Pro Arg Lys465 47099478PRTHomo sapiens 99Glu 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
Ala Gly Thr Glu Leu 100 105 110Ser Val Arg Ala Lys Pro Ser Ala Pro
Val Val Ser Gly Pro Ala Ala 115 120 125Arg Ala Thr Pro Gln His Thr
Val Ser Phe Thr Cys Glu Ser His Gly 130 135 140Phe Ser Pro Arg Asp
Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu145 150 155 160Leu Ser
Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser 165 170
175Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val
180 185 190His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
Gly Asp 195 200 205Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile
Arg Val Pro Pro 210 215 220Thr Leu Glu Val Thr Gln Gln Pro Val Arg
Ala Glu Asn Gln Val Asn225 230 235 240Val Thr Cys Gln Val Arg Lys
Phe Tyr Pro Gln Arg Leu Gln Leu Thr 245 250 255Trp Leu Glu Asn Gly
Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val 260 265 270Thr Glu Asn
Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val 275 280 285Asn
Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu 290 295
300His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val
Ser305 310 315 320Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala
Glu Asn Thr Gly 325 330 335Ser Asn Glu Arg Asn Ile Tyr Ile Val Val
Gly Val Val Cys Thr Leu 340 345 350Leu Val Ala Leu Leu Met Ala Ala
Leu Tyr Leu Val Arg Ile Arg Gln 355 360 365Lys Lys Ala Gln Gly Ser
Thr Ser Ser Thr Arg Leu His Glu Pro Glu 370 375 380Lys Asn Ala Arg
Glu Ile Thr Gln Val Gln Ser Leu Asp Thr Asn Asp385 390 395 400Ile
Thr Tyr Ala Asp Leu Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro 405 410
415Gln Ala Ala Glu Pro Asn Asn His Thr Glu Tyr Ala Ser Ile Gln Thr
420 425 430Ser Pro Gln Pro Ala Ser Glu Asp Thr Leu Thr Tyr Ala Asp
Leu Asp 435 440 445Met Val His Leu Asn Arg Thr Pro Lys Gln Pro Ala
Pro Lys Pro Glu 450 455 460Pro Ser Phe Ser Glu Tyr Ala Ser Val Gln
Val Pro Arg Lys465 470 475100473PRTHomo sapiens 100Glu 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 Val Glu Phe Lys Ser Gly Ala Gly Thr Glu
Leu Ser 100 105 110Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly
Pro Ala Ala Arg 115 120 125Ala Thr Pro Gln His Thr Val Ser Phe Thr
Cys Glu Ser His Gly Phe 130 135 140Ser Pro Arg Asp Ile Thr Leu Lys
Trp Phe Lys Asn Gly Asn Glu Leu145 150 155 160Ser Asp Phe Gln Thr
Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr 165 170 175Ser Ile His
Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His 180 185 190Ser
Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro 195 200
205Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr
210 215 220Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val
Asn Val225 230 235 240Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg
Leu Gln Leu Thr Trp 245 250 255Leu Glu Asn Gly Asn Val Ser Arg Thr
Glu Thr Ala Ser Thr Val Thr 260 265 270Glu Asn Lys Asp Gly Thr Tyr
Asn Trp Met Ser Trp Leu Leu Val Asn 275 280 285Val Ser Ala His Arg
Asp Asp Val Lys Leu Thr Cys Gln Val Glu His 290 295 300Asp Gly Gln
Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala305 310 315
320His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser
325 330 335Asn Glu Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr
Leu Leu 340 345 350Val Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg
Ile Arg Gln Lys 355 360 365Lys Ala Gln Gly Ser Thr Ser Ser Thr Arg
Leu His Glu Pro Glu Lys 370 375 380Asn Ala Arg Glu Ile Thr Gln Asp
Thr Asn Asp Ile Thr Tyr Ala Asp385 390 395 400Leu Asn Leu Pro Lys
Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro 405 410 415Asn Asn His
Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala 420 425 430Ser
Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn 435 440
445Arg Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu
450 455 460Tyr Ala Ser Val Gln Val Pro Arg Lys465 470101343PRTHomo
sapiens 101Glu 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 Ala Gly Thr Glu Leu 100 105 110Ser Val Arg Ala Lys
Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala 115 120 125Arg Ala Thr
Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly 130 135 140Phe
Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu145 150
155 160Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val
Ser 165 170 175Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg
Glu Asp Val 180 185 190His Ser Gln Val Ile Cys Glu Val Ala His Val
Thr Leu Gln Gly Asp 195 200 205Pro Leu Arg Gly Thr Ala Asn Leu Ser
Glu Thr Ile Arg Val Pro Pro 210 215 220Thr Leu Glu Val Thr Gln Gln
Pro Val Arg Ala Glu Asn Gln Val Asn225 230 235 240Val Thr Cys Gln
Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr 245 250 255Trp Leu
Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val 260 265
270Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val
275 280 285Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln
Val Glu 290 295 300His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp
Leu Lys Val Ser305 310 315 320Ala His Pro Lys Glu Gln Gly Ser Asn
Thr Ala Ala Glu Asn Thr Gly 325 330 335Ser Asn Glu Arg Asn Ile Tyr
34010221PRTHomo sapiens 102Ile Val Val Gly Val Val Cys Thr Leu Leu
Val Ala Leu Leu Met Ala1 5 10 15Ala Leu Tyr Leu Val 20103110PRTHomo
sapiens 103Arg Ile Arg Gln Lys Lys Ala Gln Gly Ser Thr Ser Ser Thr
Arg Leu1 5 10 15His Glu Pro Glu Lys Asn Ala Arg Glu Ile Thr Gln Asp
Thr Asn Asp 20 25 30Ile Thr Tyr Ala Asp Leu Asn Leu Pro Lys Gly Lys
Lys Pro Ala Pro 35 40 45Gln Ala Ala Glu Pro Asn Asn His Thr Glu Tyr
Ala Ser Ile Gln Thr 50 55 60Ser Pro Gln Pro Ala Ser Glu Asp Thr Leu
Thr Tyr Ala Asp Leu Asp65 70 75 80Met Val His Leu Asn Arg Thr Pro
Lys Gln Pro Ala Pro Lys Pro Glu 85 90 95Pro Ser Phe Ser Glu Tyr Ala
Ser Val Gln Val Pro Arg Lys 100 105 110104106PRTHomo sapiens 104Glu
Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala1 5 10
15Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val
20 25 30Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
Ile 35 40 45Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val
Ser Asp 50 55 60Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile
Gly Asn Ile65 70 75 80Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val
Lys Phe Arg Lys Gly 85 90 95Ser Pro Asp Asp Val Glu Phe Lys Ser Gly
100 105105100PRTHomo sapiens 105Pro Ser Ala Pro Val Val Ser Gly Pro
Ala Ala Arg Ala Thr Pro Gln1 5 10 15His Thr Val Ser Phe Thr Cys Glu
Ser His Gly Phe Ser Pro Arg Asp 20 25 30Ile Thr Leu Lys Trp Phe Lys
Asn Gly Asn Glu Leu Ser Asp Phe Gln 35 40 45Thr Asn Val Asp Pro Val
Gly Glu Ser Val Ser Tyr Ser Ile His Ser 50 55 60Thr Ala Lys Val Val
Leu Thr Arg Glu Asp Val His Ser Gln Val Ile65 70 75 80Cys Glu Val
Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr 85 90 95Ala Asn
Leu Ser 10010695PRTHomo sapiens 106Pro Thr Leu Glu Val Thr Gln Gln
Pro Val Arg Ala Glu Asn Gln Val1 5 10 15Asn Val Thr Cys Gln Val Arg
Lys Phe Tyr Pro Gln Arg Leu Gln Leu 20 25 30Thr Trp Leu Glu Asn Gly
Asn Val Ser Arg Thr Glu Thr Ala Ser Thr 35 40 45Val Thr Glu Asn Lys
Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu 50 55 60Val Asn Val Ser
Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val65 70 75 80Glu His
Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys 85 90
95107447PRTArtificial SequenceSynthetic-1-1-A1_BM VH-CH1-CH2-CH3
107Gln Val Gln Leu Gln Gln Ser
Gly Pro Asp Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Val Ile His Trp Val
Arg Gln Lys Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn
Pro Tyr Asn Asp Gly Thr Lys Ser Asn Glu Lys Phe 50 55 60Lys Gly Lys
Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Ser Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215
220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330
335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445108219PRTArtificial SequenceSynthetic-1-1-A1_BM VL-Ckappa 108Asp
Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10
15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln His Leu Glu Tyr Ser
20 25 30Asn Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Ile 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
Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215109447PRTArtificial SequenceSynthetic-1-1-A1 VH-CH1-CH2-CH3
109Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala1
5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Ser Asn
Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser
Thr Ser Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Gly Tyr Tyr Thr Met Asp
Tyr Trp Gly Gln Gly Thr Ser 100 105 110Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155
160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 445110219PRTArtificial SequenceSynthetic-1-1-A1
VL-Ckappa 110Asp 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 His
Leu Glu Tyr Ser 20 25 30Asn Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln
Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Ile 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 Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215111453PRTArtificial
SequenceSynthetic-5-48-A6 VH-CH1-CH2-CH3 111Gln Val Gln Leu Lys Glu
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val His Trp
Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile
Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60Ser Arg
Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65 70 75
80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95Arg Val Pro Thr Gly Arg Ile Lys Ser Tyr Phe Tyr Ala Met Asp
Tyr 100 105 110Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser
Thr Lys Gly 115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200
205Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu225 230 235 240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu305 310 315
320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 355 360 365Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440
445Leu Ser Pro Gly Lys 450112214PRTArtificial
SequenceSynthetic-5-48-A6 VL-Ckappa 112Asp Ile Lys Met Thr Gln Ser
Pro Ser Ser Met Tyr Ser Ser Leu Gly1 5 10 15Glu Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30Leu Ser Trp Phe Gln
Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45Tyr Arg Ala Asn
Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr65 70 75 80Glu
Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90
95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 210113445PRTArtificial
SequenceSynthetic-5-48-D2 VH-CH1-CH2-CH3 113Glu Val Lys Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60Lys Asp
Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95Ala Thr Gly Thr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200
205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315
320Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser 340 345 350Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His
Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445114214PRTArtificial
SequenceSynthetic-5-48-D2 VL-Ckappa 114Asp Ile Gln Met Thr Gln Ser
Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr
Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn Ala Lys
Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Val Thr Pro Trp 85 90
95Thr Phe Gly Gly Val Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 210115446PRTArtificial
SequenceSynthetic-anti-CD33 VH-CH1-CH2-CH3 115Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Asn Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr
Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55 60Lys
Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn 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 Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu 130 135 140Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200
205Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445116218PRTArtificial SequenceSynthetic-anti-CD33 VL-Ckappa 116Asp
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 Glu Ser Val Asp Asn Tyr
20 25 30Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala
Pro 35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val
Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser65 70 75 80Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Lys 85 90 95Glu Val Pro Trp Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170
175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215117318PRTArtificial SequenceSynthetic-Rhesus macaque CD47
(UniProt F7F5Y9-1, v2) 117Met 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 Ala Pro Ala Asn65 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 Met
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 Asn 290 295 300Asp Asn Phe Arg Leu Lys Asn Glu
Glu Lys Phe Ile Leu Asn305 310 315118330PRTArtificial
SequenceSynthetic-Human IgG1 constant region (IGHG1;
UniProtP01857-1, v1) 118Ala 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 33011998PRTArtificial SequenceSynthetic-CH1
IgG1 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 Val12012PRTArtificial
SequenceSynthetic-Hinge IgG1 (positions 99-110 of P01857-1, v1)
120Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro1 5
10121113PRTArtificial SequenceSynthetic-CH2 IgG1 (positions 111-223
of P01857-1, v1) 121Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe1 5 10 15Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 20 25 30Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe 35 40 45Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro 50 55 60Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr65 70 75 80Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 100 105
110Lys122107PRTArtificial SequenceSynthetic-CH3 IgG1 (positions
224-330 of P01857-1, v1) 122Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 100 105123107PRTArtificial
SequenceSynthetic-CH3 (D356E, L358M; positions numbered according
to EU numbering) 123Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu1 5 10 15Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly65 70 75 80Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr 85 90 95Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 100 105124107PRTArtificial
SequenceSynthetic-Ckappa CL (IGCK; UniProt P01834-1, v2) 124Arg 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 105125451PRTArtificial SequenceSynthetic-J6M0 VH-CH1-CH2-CH3
125Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn
Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ala Thr Tyr Arg Gly His Ser Asp Thr Tyr Tyr Asn
Gln Lys Phe 50 55 60Lys Gly Arg Val 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 Gly Ala Ile Tyr Asp Gly Tyr
Asp Val Leu Asp Asn Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260
265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450126214PRTArtificial SequenceSynthetic-J6M0 VL-Ckappa 126Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Tyr Thr Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg
Lys Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210127117PRTArtificial
SequenceSynthetic-11A1H1 VH 127Gln 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 Tyr Thr Phe Thr Asn Tyr 20 25 30Val Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr
Asn Asp Gly Thr Lys Ser Asn Glu Lys Phe 50 55 60Lys Gly Arg Val Thr
Leu Thr Ser Asp Lys Ser Ser Thr Ser Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser
Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser 115128112PRTArtificial
SequenceSynthetic-11A1H1, Synthetic-11A1H2, Synthetic-11A1H3,
Synthetic-11A1H4, Synthetic-11A1H5 VL 128Asp Val Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln His Leu Glu Tyr Ser 20 25 30Asn Gly Tyr Ser
Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Arg Leu
Leu Ile Tyr Lys Ile 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 Val Gly Val Tyr Tyr Cys Ser Gln Ser
85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 110129117PRTArtificial SequenceSynthetic-11A1H2 VH
129Gln 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 Tyr Thr Phe Thr Asn
Tyr 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Ser Asn
Glu Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ser Asp Thr Ser Thr
Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Gly Tyr Tyr Thr Met Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115130113PRTArtificial SequenceSynthetic-11A1H3 VH 130Gln 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 Tyr Thr Phe Thr Asn Tyr 20 25 30Val
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Ser Asn Glu Lys Phe
50 55 60Gln Gly Arg Val Thr Leu Thr Ser Asp Thr 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 Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val131113PRTArtificial
SequenceSynthetic-11A1H4, Synthetic-11A1H6, Synthetic-11A1H8 VH
131Gln 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 Tyr Thr Phe Thr Asn
Tyr 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ser Asp Thr Ser Thr
Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Gly Tyr Tyr Thr Met Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val132117PRTArtificial
SequenceSynthetic-11A1H5, Synthetic-11A1H7, Synthetic-11A1H9,
Synthetic-11A1H10, Synthetic-11A1H11 VH 132Gln 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 Tyr Thr Phe Thr Gly Tyr 20 25 30Val Ile His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile
Asn Pro Tyr Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Lys Gly
Arg Val Thr Leu Thr Ser Asp Thr Ser Thr Thr Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser 115133112PRTArtificial
SequenceSynthetic-11A1H6, Synthetic-11A1H7 VL 133Asp Val Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln His Leu Glu Tyr Ser 20 25 30Gln Gly
Tyr Ser Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro
Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Asp 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 Val Gly Val Tyr Tyr Cys Ser Gln
Ser 85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 110134112PRTArtificial SequenceSynthetic-11A1H8,
Synthetic-11A1H9 VL 134Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln His Leu Glu Tyr Ser 20 25 30Thr Gly Tyr Ser Tyr Leu His Trp Tyr
Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val
Ser Asn Arg Asp 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 Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
110135112PRTArtificial SequenceSynthetic-11A1H10 VL 135Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln His Leu Glu Tyr Ser 20 25 30Asn
Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40
45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Asp 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 Val Gly Val Tyr Tyr Cys
Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 110136112PRTArtificial
SequenceSynthetic-11A1H11 VL 136Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln His Leu Glu Tyr Ser 20 25 30Asn Gly Tyr Ser Tyr Leu His
Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Arg Leu Leu Ile Tyr
Lys Ile 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 Val Gly Val Tyr Tyr Cys Ser Gln Gly 85 90 95Thr His
Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
1101378PRTArtificial SequenceSynthetic-11A1H5, Synthetic-11A1H7,
Synthetic-11A1H9, Synthetic-11A1H10, Synthetic-11A1H11 HC-CDR1
137Gly Tyr Thr Phe Thr Gly Tyr Val1 51388PRTArtificial
SequenceSynthetic-11A1H5, Synthetic-11A1H7, Synthetic-11A1H9,
Synthetic-11A1H10, Synthetic-11A1H11 HC-CDR2 138Ile Asn Pro Tyr Asn
Gly Gly Thr1 513911PRTArtificial SequenceSynthetic-11A1H6,
Synthetic-11A1H7 LC-CDR1 139Gln His Leu Glu Tyr Ser Gln Gly Tyr Ser
Tyr1 5 1014011PRTArtificial SequenceSynthetic-11A1H8,
Synthetic-11A1H9 LC-CDR1 140Gln His Leu Glu Tyr Ser Thr Gly Tyr Ser
Tyr1 5 101413PRTArtificial SequenceSynthetic-11A1H6,
Synthetic-11A1H7, Synthetic-11A1H8, Synthetic-11A1H9,
Synthetic-11A1H10 LC-CDR2 141Lys Val Ser11429PRTArtificial
SequenceSynthetic-11A1H11 LC-CDR3 142Ser Gln Gly Thr His Val Pro
Tyr Thr1 514325PRTArtificial SequenceSynthetic-11A1H1,
Synthetic-11A1H2, Synthetic-11A1H3, Synthetic-11A1H4,
Synthetic-11A1H5, Synthetic- 11A1H6, Synthetic-11A1H7,
Synthetic-11A1H8, Synthetic-11A1H9, Synthetic-11A1H10,
Synthetic-11A1H11 HC-FR1 143Gln 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
20 2514417PRTArtificial SequenceSynthetic-11A1H1, Synthetic-11A1H2,
Synthetic-HC-FR2 144Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met Gly1 5 10 15Tyr14517PRTArtificial
SequenceSynthetic-11A1H3 HC-FR2 145Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met Gly1 5 10 15Tyr14617PRTArtificial
SequenceSynthetic-11A1H4, Synthetic-11A1H5, Synthetic-11A1H6,
Synthetic-11A1H7, Synthetic-11A1H8, Synthetic- 11A1H9,
Synthetic-11A1H10, Synthetic-11A1H11 HC-FR2 146Ile His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly1 5 10
15Tyr14738PRTArtificial SequenceSynthetic-11A1H1 HC-FR3 147Lys Ser
Asn Glu Lys Phe Lys Gly Arg Val Thr Leu Thr Ser Asp Lys1 5 10 15Ser
Ser Thr Ser Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25
30Thr Ala Val Tyr Tyr Cys 3514838PRTArtificial
SequenceSynthetic-11A1H2 HC-FR3 148Lys Ser Asn Glu Lys Phe Lys Gly
Arg Val Thr Leu Thr Ser Asp Thr1 5 10 15Ser Thr Thr Thr Ala Tyr Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30Thr Ala Val Tyr Tyr Cys
3514938PRTArtificial SequenceSynthetic-11A1H3 HC-FR3 149Lys Ser Asn
Glu Lys Phe Gln Gly Arg Val Thr Leu Thr Ser Asp Thr1 5 10 15Ser Thr
Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 20 25 30Thr
Ala Val Tyr Tyr Cys 3515038PRTArtificial SequenceSynthetic-11A1H4,
Synthetic-11A1H6, Synthetic-11A1H8 HC-FR3 150Lys Tyr Asn Gln Lys
Phe Lys Gly Arg Val Thr Leu Thr Ser Asp Thr1 5 10 15Ser Thr Thr Thr
Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp 20 25 30Thr Ala Val
Tyr Tyr Cys 3515138PRTArtificial SequenceSynthetic-11A1H5,
Synthetic-11A1H7, Synthetic-11A1H9, Synthetic-11A1H10,
Synthetic-11A1H11 HC-FR3 151Asn Tyr Ala Gln Lys Phe Lys Gly Arg Val
Thr Leu Thr Ser Asp Thr1 5 10 15Ser Thr Thr Thr Ala Tyr Met Glu Leu
Ser Arg Leu Arg Ser Glu Asp 20 25 30Thr Ala Val Tyr Tyr Cys
3515211PRTArtificial SequenceSynthetic-11A1H1, Synthetic-11A1H2,
Synthetic-11A1H5, Synthetic-11A1H7, Synthetic-11A1H9, Synthetic-
11A1H10, Synthetic-11A1H11 HC-FR4 152Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser1 5 101537PRTArtificial SequenceSynthetic-11A1H3,
Synthetic-11A1H4, Synthetic-11A1H6, Synthetic-11A1H8 HC-FR4 153Trp
Gly Gln Gly Thr Leu Val1 515426PRTArtificial
SequenceSynthetic-11A1H1, Synthetic-11A1H2, Synthetic-11A1H3,
Synthetic-11A1H4, Synthetic-11A1H5, Synthetic- 11A1H6,
Synthetic-11A1H7, Synthetic-11A1H8, Synthetic-11A1H9, 11A1H10,
11A1H11 LC-FR1 154Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser 20
2515517PRTArtificial SequenceSynthetic-11A1H1, Synthetic-11A1H2,
Synthetic-11A1H3, Synthetic-11A1H4, Synthetic-11A1H5, Synthetic-
11A1H6, Synthetic-11A1H7, Synthetic-11A1H8, Synthetic-11A1H9,
Synthetic-11A1H10, Synthetic-11A1H11 LC-FR2 155Leu His Trp Tyr Gln
Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile1 5 10
15Tyr15636PRTArtificial SequenceSynthetic-11A1H1, Synthetic-11A1H2,
Synthetic-11A1H3, Synthetic-11A1H4, Synthetic-11A1H5, Synthetic-
11A1H11 LC-FR3 156Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu
Ala Glu Asp Val Gly 20 25 30Val Tyr Tyr Cys 3515736PRTArtificial
SequenceSynthetic-11A1H6, Synthetic-11A1H7, Synthetic-11A1H8,
Synthetic-11A1H9, Synthetic-11A1H10 LC-FR3 157Asn Arg Asp Ser Gly
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1 5 10 15Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly 20 25 30Val Tyr Tyr
Cys
3515810PRTArtificial SequenceSynthetic-11A1H1, Synthetic-11A1H2,
Synthetic-11A1H3, Synthetic-11A1H4, Synthetic-11A1H5, Synthetic-
11A1H6, Synthetic-11A1H7, Synthetic-11A1H8, Synthetic-11A1H9,
Synthetic-11A1H10, Synthetic-11A1H11 LC-FR4 158Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys1 5 10159447PRTArtificial
SequenceSynthetic-11A1H1 VH-CH1-CH2-CH3 159Gln 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 Tyr Thr Phe Thr Asn Tyr 20 25 30Val Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile
Asn Pro Tyr Asn Asp Gly Thr Lys Ser Asn Glu Lys Phe 50 55 60Lys Gly
Arg Val Thr Leu Thr Ser Asp Lys Ser Ser Thr Ser Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445160219PRTArtificial SequenceSynthetic-11A1H1, Synthetic-11A1H2,
Synthetic-11A1H3, Synthetic-11A1H4, Synthetic-11A1H5 VL-Ckappa
160Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1
5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln His Leu Glu Tyr
Ser 20 25 30Asn Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly
Gln Ser 35 40 45Pro Arg Leu Leu Ile Tyr Lys Ile 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 Val Gly Val
Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215161447PRTArtificial SequenceSynthetic-11A1H2
VH-CH1-CH2-CH3 161Gln 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 Tyr
Thr Phe Thr Asn Tyr 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly
Thr Lys Ser Asn Glu Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ser
Asp Thr Ser Thr Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Gly Tyr
Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 445162443PRTArtificial
SequenceSynthetic-11A1H3 VH-CH1-CH2-CH3 162Gln 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 Tyr Thr Phe Thr Asn Tyr 20 25 30Val Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile
Asn Pro Tyr Asn Asp Gly Thr Lys Ser Asn Glu Lys Phe 50 55 60Gln Gly
Arg Val Thr Leu Thr Ser Asp Thr 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 Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser 115 120 125Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp 130 135 140Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr145 150 155 160Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200
205Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
210 215 220Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro225 230 235 240Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr 245 250 255Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn 260 265 270Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser305 310 315
320Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
325 330 335Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp 340 345 350Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe 355 360 365Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu 370 375 380Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe385 390 395 400Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440163443PRTArtificial
SequenceSynthetic-11A1H4, Synthetic-11A1H6, Synthetic-11A1H8
VH-CH1-CH2-CH3 163Gln 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 Tyr
Thr Phe Thr Asn Tyr 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly
Thr Lys Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ser
Asp Thr Ser Thr Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Gly Tyr
Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135
140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250
255Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
260 265 270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 275 280 285Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val 290 295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser305 310 315 320Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 340 345 350Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375
380Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe385 390 395 400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 405 410 415Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr 420 425 430Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440164447PRTArtificial SequenceSynthetic-11A1H5,
Synthetic-11A1H7, Synthetic-11A1H9, Synthetic-11A1H10,
Synthetic-11A1H11 VH-CH1-CH2- CH3 164Gln 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 Tyr Thr Phe Thr Gly Tyr 20 25 30Val Ile His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn
Pro Tyr Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Lys Gly Arg
Val Thr Leu Thr Ser Asp Thr Ser Thr Thr Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215
220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330
335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 445165219PRTArtificial
SequenceSynthetic-11A1H6, Synthetic-11A1H7 VL-Ckappa 165Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln His Leu Glu Tyr Ser 20 25 30Gln
Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40
45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Asp 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 Val Gly Val Tyr Tyr Cys
Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185
190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215166219PRTArtificial SequenceSynthetic-11A1H8, Synthetic-11A1H9
VL-Ckappa 166Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val
Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln His
Leu Glu Tyr Ser 20 25 30Thr Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln
Arg Pro Gly Gln Ser 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn
Arg Asp 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
Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215167219PRTArtificial
SequenceSynthetic-11A1H10 VL-Ckappa 167Asp Val Val Met Thr Gln Ser
Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln His Leu Glu Tyr Ser 20 25 30Asn Gly Tyr Ser Tyr
Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Arg Leu Leu
Ile Tyr Lys Val Ser Asn Arg Asp 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 Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90
95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215168219PRTArtificial
SequenceSynthetic-11A1H11 VL-Ckappa 168Asp Val Val Met Thr Gln Ser
Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln His Leu Glu Tyr Ser 20 25 30Asn Gly Tyr Ser Tyr
Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Arg Leu Leu
Ile Tyr Lys Ile 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 Val Gly Val Tyr Tyr Cys Ser Gln Gly 85 90
95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 2151698PRTArtificial
SequenceSynthetic-11A1H_C HC-CDR1MISC_FEATURE(6)..(6)Xaa = N or G
169Gly Tyr Thr Phe Thr Xaa Tyr Val1 51708PRTArtificial
SequenceSynthetic-11A1H_C HC-CDR2MISC_FEATURE(6)..(6)Xaa = D or G
170Ile Asn Pro Tyr Asn Xaa Gly Thr1 517111PRTArtificial
SequenceSynthetic-11A1H_C LC-CDR1MISC_FEATURE(7)..(7)Xaa = N, Q or
T 171Gln His Leu Glu Tyr Ser Xaa Gly Tyr Ser Tyr1 5
101723PRTArtificial SequenceSynthetic-11A1H_C
LC-CDR2MISC_FEATURE(2)..(2)Xaa = I or V 172Lys Xaa
Ser11739PRTArtificial SequenceSynthetic-11A1H_C
LC-CDR3MISC_FEATURE(3)..(3)Xaa = S or G 173Ser Gln Xaa Thr His Val
Pro Tyr Thr1 517417PRTArtificial SequenceSynthetic-11A1H_C
HC-FR2MISC_FEATURE(1)..(1)Xaa = I or MMISC_FEATURE(10)..(10)Xaa = Q
or K 174Xaa His Trp Val Arg Gln Ala Pro Gly Xaa Gly Leu Glu Trp Met
Gly1 5 10 15Tyr17538PRTArtificial SequenceSynthetic-11A1H_C
HC-FR3MISC_FEATURE(1)..(1)Xaa = K or NMISC_FEATURE(2)..(2)Xaa = S
or YMISC_FEATURE(3)..(3)Xaa = N or AMISC_FEATURE(4)..(4)Xaa = E or
QMISC_FEATURE(7)..(7)Xaa = K or QMISC_FEATURE(16)..(16)Xaa = T or
KMISC_FEATURE(18)..(18)Xaa = T or SMISC_FEATURE(20)..(20)Xaa = T or
SMISC_FEATURE(27)..(27)Xaa = S or RMISC_FEATURE(31)..(31)Xaa = E or
D 175Xaa Xaa Xaa Xaa Lys Phe Xaa Gly Arg Val Thr Leu Thr Ser Asp
Xaa1 5 10 15Ser Xaa Ser Xaa Ala Tyr Met Glu Leu Ser Xaa Leu Arg Ser
Xaa Asp 20 25 30Thr Ala Val Tyr Tyr Cys 3517611PRTArtificial
SequenceSynthetic-11A1H_C HC-FR4MISC_FEATURE(8)..(8)Xaa = T or
absentMISC_FEATURE(9)..(9)Xaa = V or
absentMISC_FEATURE(10)..(10)Xaa = S or
absentMISC_FEATURE(11)..(11)Xaa = S or absent 176Trp Gly Gln Gly
Thr Leu Val Xaa Xaa Xaa Xaa1 5 1017736PRTArtificial
SequenceSynthetic-11A1H_C LC-FR3MISC_FEATURE(3)..(3)Xaa = D or F
177Asn Arg Xaa Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly1
5 10 15Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val
Gly 20 25 30Val Tyr Tyr Cys 35178117PRTArtificial
SequenceSynthetic-11A1H_C VHMISC_FEATURE(31)..(31)Xaa = N or
GMISC_FEATURE(34)..(34)Xaa = I or MMISC_FEATURE(43)..(43)Xaa = Q or
KMISC_FEATURE(56)..(56)Xaa = D or GMISC_FEATURE(59)..(59)Xaa = K or
NMISC_FEATURE(60)..(60)Xaa = S or YMISC_FEATURE(61)..(61)Xaa = N or
AMISC_FEATURE(62)..(62)Xaa = E or QMISC_FEATURE(65)..(65)Xaa = K or
QMISC_FEATURE(74)..(74)Xaa = T or KMISC_FEATURE(76)..(76)Xaa = T or
SMISC_FEATURE(78)..(78)Xaa = T or SMISC_FEATURE(85)..(85)Xaa = S or
RMISC_FEATURE(89)..(89)Xaa = E or DMISC_FEATURE(114)..(114)Xaa = T
or absentMISC_FEATURE(115)..(115)Xaa = V or
absentMISC_FEATURE(116)..(116)Xaa = S or
absentMISC_FEATURE(117)..(117)Xaa = S or absent 178Gln 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 Tyr Thr Phe Thr Xaa Tyr 20 25 30Val Xaa
His Trp Val Arg Gln Ala Pro Gly Xaa Gly Leu Glu Trp Met 35 40 45Gly
Tyr Ile Asn Pro Tyr Asn Xaa Gly Thr Xaa Xaa Xaa Xaa Lys Phe 50 55
60Xaa Gly Arg Val Thr Leu Thr Ser Asp Xaa Ser Xaa Ser Xaa Ala Tyr65
70 75 80Met Glu Leu Ser Xaa Leu Arg Ser Xaa Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ser Gly Gly Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Xaa Xaa Xaa Xaa 115179112PRTArtificial
SequenceSynthetic-11A1H_C VLMISC_FEATURE(33)..(33)Xaa = N, Q or
TMISC_FEATURE(56)..(56)Xaa = I or VMISC_FEATURE(60)..(60)Xaa = D or
FMISC_FEATURE(96)..(96)Xaa = S or G 179Asp Val Val Met Thr Gln Ser
Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln His Leu Glu Tyr Ser 20 25 30Xaa Gly Tyr Ser Tyr
Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Arg Leu Leu
Ile Tyr Lys Xaa Ser Asn Arg Xaa 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 Val Gly Val Tyr Tyr Cys Ser Gln Xaa 85 90
95Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
* * * * *