U.S. patent application number 17/047656 was filed with the patent office on 2021-07-01 for anti-cd27 and anti-pd-l1 antibodies and bispecific constructs.
This patent application is currently assigned to Celldex Therapeutics, Inc.. The applicant listed for this patent is Celldex Therapeutics, Inc.. Invention is credited to Joel GOLDSTEIN, Lizhen HE, Tibor KELER, Laura A. VITALE.
Application Number | 20210198374 17/047656 |
Document ID | / |
Family ID | 1000005407025 |
Filed Date | 2021-07-01 |
United States Patent
Application |
20210198374 |
Kind Code |
A1 |
KELER; Tibor ; et
al. |
July 1, 2021 |
ANTI-CD27 AND ANTI-PD-L1 ANTIBODIES AND BISPECIFIC CONSTRUCTS
Abstract
Provided herein are novel anti-CD27 and anti-PD-L1 antibodies,
and binding domains thereof, as well as bispecific constructs and
anti-CD27 binding domain linked to an anti-PD-L1 binding domain.
Also provided herein are methods of stimulating T cell activity,
methods of inducing or enhancing an immune response, and methods of
treating a disease or condition (e.g., cancer) by administering the
bispecific constructs, antibodies, or antigen binding fragments
thereof, or compositions described herein to a patient in need
thereof.
Inventors: |
KELER; Tibor; (Pipersville,
PA) ; GOLDSTEIN; Joel; (Hopewell, NJ) ;
VITALE; Laura A.; (Doylestown, PA) ; HE; Lizhen;
(Allentown, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Celldex Therapeutics, Inc. |
Hampton |
NJ |
US |
|
|
Assignee: |
Celldex Therapeutics, Inc.
Hampton
NJ
|
Family ID: |
1000005407025 |
Appl. No.: |
17/047656 |
Filed: |
April 17, 2019 |
PCT Filed: |
April 17, 2019 |
PCT NO: |
PCT/US2019/027897 |
371 Date: |
October 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62826091 |
Mar 29, 2019 |
|
|
|
62658899 |
Apr 17, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/34 20130101;
C07K 2317/21 20130101; C07K 2317/90 20130101; C07K 16/2827
20130101; C07K 2317/35 20130101; A61P 35/00 20180101; C07K 2317/622
20130101; A61K 2039/505 20130101; C07K 2317/76 20130101; C07K
2317/31 20130101; C07K 16/2878 20130101; C07K 2317/74 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Claims
1. A bispecific construct comprising an anti-CD27 binding domain
linked to an anti-PD-L1 binding domain, wherein: (i) the anti-CD27
binding domain comprises: a. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:1, 2, and 3, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4,
5, and 6, respectively, or conservative sequence modifications
thereof, or b. heavy chain variable region CDR1, CDR2 and CDR3 as
set forth in SEQ ID NOs:7, 8, and 9, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:10, 11, and 12,
respectively, or conservative sequence modifications thereof; and
(ii) the anti-PD-L1 binding domain comprises: a. a heavy chain
variable region CDR1 comprising an amino acid sequence selected
from the consensus sequence: (T,S)(S,Y,H)WMS (SEQ ID NO:167) or
conservative sequence modifications thereof; b. a heavy chain
variable region CDR2 comprising SEQ ID NO:168 or conservative
sequence modifications thereof; c. a heavy chain variable region
CDR3 comprising SEQ ID NO:169 or conservative sequence
modifications thereof; d. a light chain variable region CDR1
comprising SEQ ID NO:170 or conservative sequence modifications
thereof; e. a light chain variable region CDR2 comprising SEQ ID
NO:171 or conservative sequence modifications thereof; and f. a
light chain variable region CDR3 comprising SEQ ID NO:172 or
conservative sequence modifications thereof.
2. A bispecific construct comprising an anti-CD27 binding domain
linked to an anti-PD-L1 binding domain, wherein: (i) the anti-CD27
binding domain comprises: a. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:1, 2, and 3, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4,
5, and 6, respectively, or conservative sequence modifications
thereof, or b. heavy chain variable region CDR1, CDR2 and CDR3 as
set forth in SEQ ID NOs:7, 8, and 9, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:10, 11, and 12,
respectively; or conservative sequence modifications thereof and
(ii) the anti-PD-L1 binding domain comprises: a. a heavy chain
variable region comprising SEQ ID NO:77 and a light chain variable
region comprising SEQ ID NO:78 or sequences at least 95% identical
thereto; b. a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80 or
sequences at least 95% identical thereto; c. a heavy chain variable
region comprising SEQ ID NO:81 and a light chain variable region
comprising SEQ ID NO:82 or sequences at least 95% identical
thereto; d. a heavy chain variable region comprising SEQ ID NO:83
and a light chain variable region comprising SEQ ID NO:84 or
sequences at least 95% identical thereto; e. a heavy chain variable
region comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86 or sequences at least 95% identical
thereto; or f. a heavy chain variable region comprising SEQ ID
NO:87 and a light chain variable region comprising SEQ ID NO:88 or
sequences at least 95% identical thereto.
3. The bispecific construct of claim 1 or 2, wherein the anti-CD27
binding domain comprises a heavy chain variable region comprising
SEQ ID NO:17 and a light chain variable region comprising SEQ ID
NO:18.
4. The bispecific construct of claim 1 or 2, wherein the anti-CD27
binding domain comprises a heavy chain variable region comprising
SEQ ID NO:19 and a light chain variable region comprising SEQ ID
NO:20.
5. The bispecific construct of claim 1 or 2, wherein the anti-PD-L1
binding domain comprises heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs: 29, 30, and 31, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:32,
33, and 34, respectively, or conservative sequence modifications
thereof.
6. The bispecific construct of claim 1 or 2, wherein the anti-PD-L1
binding domain comprises a heavy chain variable region comprising
SEQ ID NO:77 and a light chain variable region comprising SEQ ID
NO:78.
7. The bispecific construct of claim 1 or 2, wherein the anti-PD-L1
binding domain comprises heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs: 35, 36, and 37, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38,
39, and 40, respectively, or conservative sequence modifications
thereof.
8. The bispecific construct of claim 1 or 2, wherein the anti-PD-L1
binding domain comprises a heavy chain variable region comprising
SEQ ID NO:79 and a light chain variable region comprising SEQ ID
NO:80.
9. The bispecific construct of claim 1 or 2, wherein the anti-PD-L1
binding domain comprises heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs: 41, 42, and 43, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44,
45, and 46, respectively, or conservative sequence modifications
thereof.
10. The bispecific construct of claim 1 or 2, wherein the
anti-PD-L1 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:81 and a light chain variable region
comprising SEQ ID NO:82.
11. The bispecific construct of claim 1 or 2, wherein the
anti-PD-L1 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:50, 51, and 52, respectively, or conservative sequence
modifications thereof.
12. The bispecific construct of claim 1 or 2, wherein the
anti-PD-L1 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84.
13. The bispecific construct of claim 1 or 2, wherein the
anti-PD-L1 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 53, 54, and 55,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:56, 57, and 58, respectively, or conservative sequence
modifications thereof.
14. The bispecific construct of claim 1 or 2, wherein the
anti-PD-L1 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86.
15. The bispecific construct of claim 1 or 2, wherein the
anti-PD-L1 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 59, 60, and 61,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:62, 63, and 64, respectively, or conservative sequence
modifications thereof.
16. The bispecific construct of claim 1 or 2, wherein the
anti-PD-L1 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88.
17. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively; or conservative sequence
modifications thereof, and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:29, 30, and 31, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 32, 33, and 34,
respectively, or conservative sequence modifications thereof.
18. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:77 and
a light chain variable region comprising SEQ ID NO:78.
19. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1,2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:35, 36, and 37, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 38, 39, and 40,
respectively, or conservative sequence modifications thereof.
20. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:79 and
a light chain variable region comprising SEQ ID NO:80.
21. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:41, 42, and 43, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44, 45, and 46,
respectively, or conservative sequence modifications thereof.
22. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:17and a light chain variable region comprising
SEQ ID NO:18; and b. the anti-PD-L1 binding domain comprises a
heavy chain variable region comprising SEQ ID NO:81 and a light
chain variable region comprising SEQ ID NO:82.
23. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:47, 48, and 49, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof.
24. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:83 and
a light chain variable region comprising SEQ ID NO:84.
25. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:53, 54, and 55, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof.
26. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:85 and
a light chain variable region comprising SEQ ID NO:86.
27. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:59, 60, and 61, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 62, 63, and 64,
respectively, or conservative sequence modifications thereof.
28. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:87 and
a light chain variable region comprising SEQ ID NO:88.
29. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:29, 30, and 31, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:32, 33, and 34,
respectively, or conservative sequence modifications thereof.
30. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:77 and
a light chain variable region comprising SEQ ID NO:78.
31. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof ,and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:35, 36, and 37, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and 40,
respectively, or conservative sequence modifications thereof.
32. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:79 and
a light chain variable region comprising SEQ ID NO:80.
33. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:41, 42, and 43, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44, 45, and 46,
respectively, or conservative sequence modifications thereof.
34. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:81 and
a light chain variable region comprising SEQ ID NO:82.
35. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:47, 48, and 49, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof.
36. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:83 and
a light chain variable region comprising SEQ ID NO:84.
37. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:53, 54, and 55, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof.
38. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:85 and
a light chain variable region comprising SEQ ID NO:86.
39. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof ,and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof; and b. the anti-PD-L1 binding domain
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:59, 60, and 61, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:62, 63, and 64,
respectively, or conservative sequence modifications thereof.
40. The bispecific construct of claim 1 or 2, wherein: a. the
anti-CD27 binding domain comprises a heavy chain variable region
comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and b. the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:87 and
a light chain variable region comprising SEQ ID NO:88.
41. The bispecific construct of any one of the preceding claims
wherein (a) the anti-PD-L1 binding domain further comprises a human
IgG.sub.1 constant domain or (b) the anti-CD27 binding domain
further comprises a human IgG.sub.1 constant domain.
42. The bispecific construct of any one of the preceding claims,
wherein (a) the anti-CD27 binding domain is linked to the
C-terminus of the heavy chain of the anti-PD-L1 binding domain or
(b) the anti-PD-L1 binding domain is linked to the C-terminus of
the heavy chain of the anti-CD27 binding domain.
43. The bispecific construct of any one of the preceding claims,
wherein the anti-CD27 binding domain is a scFv or (b) the
anti-PD-L1 binding domain is a scFv.
44. The bispecific construct of any one of the preceding claims,
wherein the anti-PD-L1 binding domain and the anti-CD27 binding
domain are genetically fused.
45. The bispecific construct of any one of the preceding claims,
wherein the anti-PD-L1 binding domain and the anti-CD27 binding
domain are chemically conjugated.
46. A bispecific construct comprising an anti-PD-L1 antibody linked
to an anti-CD27 scFv, wherein: (i) the anti-CD27 scFv comprises: a.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:1, 2, and 3, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:4, 5, and 6, respectively, or
conservative sequence modifications thereof, or b. heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7,
8, and 9, respectively, or conservative sequence modifications
thereof, and light chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:10, 11, and 12, respectively, or conservative
sequence modifications thereof; and (ii) the anti-PD-L1 antibody
comprises: a. heavy chain variable region CDR1, CDR2 and CDR3 as
set forth in SEQ ID NOs: 29, 30, and 31, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:32,
33, and 34, respectively, or conservative sequence modifications
thereof; b. heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 35, 36, and 37, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and 40,
respectively, or conservative sequence modifications thereof; c.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs: 41, 42, and 43, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:44, 45, and 46, respectively,
or conservative sequence modifications thereof; d. heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 47,
48, and 49, respectively, or conservative sequence modifications
thereof, and light chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:50, 51, and 52, respectively, or conservative
sequence modifications thereof; e. heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 53, 54, and 55,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:56, 57, and 58, respectively, or conservative sequence
modifications thereof; or f. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 59, 60, and 61, respectively,
or conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:62,
63, and 64, respectively, or conservative sequence modifications
thereof; and g. a human IgG.sub.1 constant domain;.
47. A bispecific construct comprising an anti-PD-L1 antibody linked
to an anti-CD27 scFv, wherein: (i) the anti-CD27 scFv comprises: a.
a heavy chain variable region comprising SEQ ID NO:17 and a light
chain variable region comprising SEQ ID NO:18; or b. a heavy chain
variable region comprising SEQ ID NO:19 and a light chain variable
region comprising SEQ ID NO:20; and (ii) the anti-PD-L1 antibody
comprises: a. a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78; b. a
heavy chain variable region comprising SEQ ID NO:79 and a light
chain variable region comprising SEQ ID NO:80; c. a heavy chain
variable region comprising SEQ ID NO:81 and a light chain variable
region comprising SEQ ID NO:82; d. a heavy chain variable region
comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84; e. a heavy chain variable region
comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86; or f. a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88; and g. a human IgG.sub.1 constant
domain.
48. A bispecific construct comprising an anti-PD-L1 antibody linked
to an anti-CD27 scFv, wherein: (i) the anti-CD27 scFv comprises
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:7, 8, and 9, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:10, 11, and 12, respectively,
or conservative sequence modifications thereof; and (ii) the
anti-PD-L1 antibody comprises a heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:50, 51, and 52, respectively, or conservative sequence
modifications thereof; and a human IgG.sub.1 constant domain.
49. A bispecific construct comprising an anti-PD-L1 antibody linked
to an anti-CD27 scFv, wherein: (i) the anti-CD27 scFv comprises a
heavy chain variable region comprising SEQ ID NO:19, a light chain
variable region comprising SEQ ID NO:20; and (ii) the anti-PD-L1
antibody comprises a heavy chain variable region comprising SEQ ID
NO:83 and a light chain variable region comprising SEQ ID NO:84,
and a human IgG.sub.1 constant domain.
50. A bispecific construct comprising an anti-PD-L1 antibody linked
to an anti-CD27 scFv, wherein: (i) the anti-CD27 scFv comprises
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:7, 8, and 9, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:10, 11, and 12, respectively,
or conservative sequence modifications thereof; and (ii) the
anti-PD-L1 antibody comprises heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:59, 60, and 61,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:62, 63, and 64, respectively or conservative sequence
modifications thereof, and a human IgG.sub.1 constant domain.
51. A bispecific construct comprising an anti-PD-L1 antibody linked
to an anti-CD27 scFv, wherein: (i) the anti-CD27 scFv comprises a
heavy chain variable region comprising SEQ ID NO:19, a light chain
variable region comprising SEQ ID NO:20; and (ii) the anti-PD-L1
antibody comprises a heavy chain variable region comprising SEQ ID
NO:87 and a light chain variable region comprising SEQ ID NO:88,
and a human IgG.sub.1 constant domain.
52. A bispecific construct comprising an anti-CD27 antibody linked
to an anti-PD-L1 scFv, wherein: (i) the anti-CD27 antibody
comprises: a. heavy chain variable region CDR1, CDR2 and CDR3 as
set forth in SEQ ID NOs:1, 2, and 3, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4, 5, and 6,
respectively, or conservative sequence modifications thereof, or b.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:7, 8, and 9, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:10, 11, and 12, respectively,
or conservative sequence modifications thereof; and c. a human
IgG.sub.1 constant domain; and (ii) the anti-PD-L1 scFv comprises:
a. heavy chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs: 29, 30, and 31, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:32, 33, and 34, respectively,
or conservative sequence modifications thereof; b. heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 35,
36, and 37, respectively, or conservative sequence modifications
thereof, and light chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:38, 39, and 40, respectively, or conservative
sequence modifications thereof; c. heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 41, 42, and 43,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:44, 45, and 46, respectively, or conservative sequence
modifications thereof; d. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49, respectively,
or conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50,
51, and 52, respectively, or conservative sequence modifications
thereof; e. heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 53, 54, and 55, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof; or f.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs: 59, 60, and 61, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:62, 63, and 64, respectively,
or conservative sequence modifications thereof.
53. A bispecific construct comprising an anti-CD27 antibody linked
to an anti-PD-L1 scFv, wherein: (i) the anti-CD27 antibody
comprises: a. a heavy chain variable region comprising SEQ ID NO:17
and a light chain variable region comprising SEQ ID NO:18; or b. a
heavy chain variable region comprising SEQ ID NO:19 and a light
chain variable region comprising SEQ ID NO:20; and c. a human
IgG.sub.1 constant domain; and (ii) the anti-PD-L1 scFv comprises:
a. a heavy chain variable region comprising SEQ ID NO:77 and a
light chain variable region comprising SEQ ID NO:78; b. a heavy
chain variable region comprising SEQ ID NO:79 and a light chain
variable region comprising SEQ ID NO:80; c. a heavy chain variable
region comprising SEQ ID NO:81 and a light chain variable region
comprising SEQ ID NO:82; d. a heavy chain variable region
comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84; e. a heavy chain variable region
comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86; or f. a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88.
54. A bispecific construct comprising an anti-CD27 antibody linked
to an anti-PD-L1 scFv, wherein: (i) the anti-CD27 antibody
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:7, 8, and 9, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:10, 11, and 12,
respectively, or conservative sequence modifications thereof, and a
human IgG.sub.1 constant domain; and (ii) the anti-PD-L1 scFv
comprises a heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 47, 48, and 49, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof.
55. A bispecific construct comprising an anti-CD27 antibody linked
to an anti-PD-L1 scFv, wherein: (i) the anti-CD27 antibody
comprises a heavy chain variable region comprising SEQ ID NO:19, a
light chain variable region comprising SEQ ID NO:20, and a human
IgG.sub.1 constant domain; and (ii) the anti-PD-L1 scFv comprises a
heavy chain variable region comprising SEQ ID NO:83 and a light
chain variable region comprising SEQ ID NO:84.
56. A bispecific construct comprising an anti-CD27 antibody linked
to an anti-PD-L1 scFv, wherein: (i) the anti-CD27 antibody
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:7, 8, and 9, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:10, 11, and 12,
respectively, or conservative sequence modifications thereof, and a
human IgG.sub.1 constant domain; and (ii) the anti-PD-L1 scFv
comprises heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:59, 60, and 61, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:62, 63, and 64,
respectively, or conservative sequence modifications thereof.
57. A bispecific construct comprising an anti-CD27 antibody linked
to an anti-PD-L1 scFv, wherein: (i) the anti-CD27 antibody
comprises a heavy chain variable region comprising SEQ ID NO:19, a
light chain variable region comprising SEQ ID NO:20, and a human
IgG.sub.1 constant domain; and (ii) the anti-PD-L1 scFv comprises a
heavy chain variable region comprising SEQ ID NO:87 and a light
chain variable region comprising SEQ ID NO:88.
58. An anti-CD27 antibody, or antigen-binding fragment thereof,
wherein the antibody binds to one or more residues within amino
acid residues 85-89, of the extracellular domain (ECD) of human
CD27 (SEQ ID NO: 183).
59. An anti-CD27 antibody, or antigen-binding fragment thereof,
wherein the antibody binds to the wildtype ECD of human CD27 (SEQ
ID NO: 183), but does not bind to a mutated version of the wildtype
ECD of human CD27 having the following amino acid substitutions:
A85S, R87A, N88A, and G89A.
60. An anti-CD27 antibody, or antigen-binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:1, 2, and 3, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4, 5, and 6,
respectively, or conservative sequence modifications thereof.
61. An anti-CD27 antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:17
and a light chain variable region comprising SEQ ID NO:18 or
sequences at least 95% identical thereto.
62. An anti-CD27 antibody, or antigen-binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:7, 8, and 9, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:10, 11, and 12,
respectively, or conservative sequence modifications thereof.
63. An anti-CD27 antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:19
and a light chain variable region comprising SEQ ID NO:20 or
sequences at least 95% identical thereto.
64. The anti-CD27 antibody, or antigen-binding fragment thereof, of
any one of claims 58-63, wherein the anti-CD27 antibody, or
antigen-binding fragment thereof, has one or more of the following
characteristics: a. induces or enhances a T cell-mediated immune
response; b. blocks binding of sCD70 to CD27; c. binds to human
CD27 with an equilibrium dissociation constant Kd of 10.sup.-9 M or
less, or alternatively, an equilibrium association constant Ka of
10.sup.+9 M.sup.-1 or greater; d. induces specific complement
mediated cytotoxicity (CDC) of CD27 expressing cells; e. induces
antibody dependent cell-mediated cytotoxicity (ADCC) specific lysis
of CD27 expressing cells; f. induces or enhances antigen-specific
immune responses in vivo in combination with a vaccine or
endogenous antigen; g. induces or enhances antigen-specific TH1
immune responses in vivo in combination with a vaccine or
endogenous antigen; h. induces or enhances antigen-specific T-cell
proliferation or activation in vivo in combination with a vaccine
or endogenous antigen; and/or i. induces or enhances T-cell
activity when combined with simultaneous, separate or sequential
TCR activation.
65. A bispecific construct comprising the anti-CD27 antibody, or
antigen binding fragment thereof, of any one of claims 58-63 linked
to an anti-PD-L1 binding domain.
66. The bispecific construct of claim 64, wherein the anti-PD-L1
binding domain is selected from the group consisting of: a. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 29, 30, and 31, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:32, 33, and 34,
respectively, or conservative sequence modifications thereof; b. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 35, 36, and 37, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and 40,
respectively, or conservative sequence modifications thereof; c. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 41, 42, and 43, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44, 45, and 46,
respectively, or conservative sequence modifications thereof; d. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 47, 48, and 49, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof; e.
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 53, 54, and 55, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof; and
f. anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 59, 60, and 61, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:62, 63, and 64,
respectively, or conservative sequence modifications thereof.
67. The bispecific construct of claim 65 or 66, wherein the
anti-PD-L1 binding domain is selected from the group consisting of:
a. a heavy chain variable region comprising SEQ ID NO:77 and a
light chain variable region comprising SEQ ID NO:78; b. a heavy
chain variable region comprising SEQ ID NO:79 and a light chain
variable region comprising SEQ ID NO:80; c. a heavy chain variable
region comprising SEQ ID NO:81 and a light chain variable region
comprising SEQ ID NO:82; d. a heavy chain variable region
comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84; e. a heavy chain variable region
comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86; and f. a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88.
68. The bispecific construct of any one of claims 64-66, wherein
the anti-PD-L1 binding domain is an scFv.
69. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:29, 30, and 31, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:32, 33, and 34,
respectively, or conservative sequence modifications thereof.
70. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78 or
sequences at least 95% identical thereto.
71. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:35, 36, and 37, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and 40,
respectively, or conservative sequence modifications thereof.
72. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80 or
sequences at least 95% identical thereto.
73. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:41, 42, and 43, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44, 45, and 46,
respectively, or conservative sequence modifications thereof.
74. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:81
and a light chain variable region comprising SEQ ID NO:82 or
sequences at least 95% identical thereto.
75. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:47, 48, and 49, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof.
76. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:83
and a light chain variable region comprising SEQ ID NO:84 or
sequences at least 95% identical thereto.
77. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:53, 54, and 55, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof.
78. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:85
and a light chain variable region comprising SEQ ID NO:86 or
sequences at least 95% identical thereto.
79. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:59, 60, and 61, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:62, 63, and 64,
respectively, or conservative sequence modifications thereof.
80. An anti-PD-L1 antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:87
and a light chain variable region comprising SEQ ID NO:88 or
sequences at least 95% identical thereto.
81. The anti-PD-L1 antibody, or antigen-binding fragment thereof,
of any one of claims 69-80 wherein the anti-PD-L1 antibody, or
antigen-binding fragment thereof, has one or more of the following
characteristics: a. blocks binding of PD1 to PD-L1; b. induces NFAT
pathway activation; and/or c. induces a mixed lymphocyte
response.
82. A bispecific construct comprising the anti-PD-L1
antigen-binding fragment of any one of claims 69-80 linked to an
anti-CD27 binding domain.
83. The bispecific construct of claim 82, wherein the anti-CD27
binding domain is selected from the group consisting of: a. an
anti-CD27 antibody comprising heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3, respectively,
or conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4,
5, and 6, respectively, or conservative sequence modifications
thereof, and b. an anti-CD27 antibody comprising heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7,
8, and 9, respectively, or conservative sequence modifications
thereof, and light chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:10, 11, and 12, respectively, or conservative
sequence modifications thereof.
84. The bispecific construct of claim 82 or 83, wherein the
anti-CD27 binding domain is selected from the group consisting of:
a. an anti-CD27 antibody comprising a heavy chain variable region
comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18; and b. an anti-CD27 antibody comprising a
heavy chain variable region comprising SEQ ID NO:19 and a light
chain variable region comprising SEQ ID NO:20.
85. The bispecific construct of any one of claims 82-84, wherein
the anti-CD27 binding domain further comprises a human IgG.sub.1
constant domain.
86. A multispecific antibody comprising an anti-PD-L1 antibody
having an Fc domain wherein at least one anti-CD27 antibody binding
domain is bound to the Fc domain.
87. A multispecific antibody comprising an anti-PD-L1 antibody
having an Fc domain wherein at least one anti-CD27 scFv peptide is
bound to the Fc domain.
88. A multispecific antibody comprising an anti-PD-L1 antibody
having an Fc domain wherein an anti-CD27 scFv peptide is bound to a
carboxy terminus of at least one of the antibody heavy chains.
89. A multispecific antibody comprising an anti-PD-L1 antibody
having an Fc domain wherein an anti-CD27 scFv peptide is bound to
the carboxy terminus of one of the antibody heavy chains and a
further scFv peptide is bound to the carboxy terminus of the other
heavy chain.
90. A composition comprising the bispecific construct of any one of
claims 1-57, 65-68, and 82-85 or the antibody, or antigen binding
fragment thereof, of any one of claims 58-64 and 69-81, or the
multispecific antibody of any one of claims 86-89 and a
pharmaceutically acceptable carrier.
91. A kit comprising the bispecific construct of any one of claims
1-57, 66-69, and 82-85, the antibody, or antigen binding fragment
thereof, of any one of claims 58-64 and 69-81, the multispecific
antibody of any one of claims 86-89, or the composition of claim
90, and instructions for use.
92. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding an antibody variable region, wherein the antibody
variable region comprises the amino acid sequence depicted in SEQ
ID NO:17, 18, 19, 20, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
or 88.
93. An isolated nucleic acid molecule comprising a nucleotide
sequence as set forth in SEQ ID NO:25, 26, 27, 28, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, or 112.
94. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding heavy and light chain variable regions of an
antibody, wherein the heavy and light chain variable regions
comprise the amino acid sequences depicted in SEQ ID NOs:17 and 18,
SEQ ID NOs:19 and 20, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80,
SEQ ID NOs:81 and 82, SEQ ID NOs: 83 and 84, SEQ ID NOs:85 and 86,
or SEQ ID NOs:87 and 88, respectively.
95. A nucleic acid molecule coding for the bispecific construct as
set forth in any one of claims 1-57, 65-68, and 82-85.
96. A nucleic acid molecule as set forth in any one of claims 92-95
in the form of an expression vector.
97. A nucleic acid molecule as set forth in any one of claims 92-96
in the form of an expression vector which expresses the bispecific
construct when administered to a subject in vivo.
98. A method of stimulating T cell activity comprising contacting T
cells with the bispecific construct of any one of claims 1-57,
65-68, and 82-85, the antibody, or antigen binding fragment
thereof, of any one of claims 58-64 and 69-81, the multispecific
antibody of any one of claims 86-89, or the composition of claim
90.
99. A method for inducing or enhancing an immune response in a
subject comprising administering to the subject the bispecific
construct of any one of claims 1-57, 65-68, and 82-85, the
antibody, or antigen binding fragment thereof, of any one of claims
58-64 and 69-81, the multispecific antibody of any one of claims
86-89, or the composition of claim 90 in an amount effective to
induce or enhance an immune response in the subject.
100. A method for treating a condition or disease in a subject, the
method comprising administering to the subject the bispecific
construct of any one of claims 1-57, 65-68, and 82-85, the
antibody, or antigen binding fragment thereof, of any one of claims
58-64 and 69-81, the multispecific antibody of any one of claims
86-89, or the composition of claim 88 in an amount effective to
treat the condition or disease.
101. A method for treating a condition or disease in a subject, the
method comprising administering to the subject an anti-CD27
antibody, or antigen binding fragment thereof, in combination with
an anti-PD-L1 antibody, or antigen binding fragment thereof,
wherein: (i) the anti-CD27 antibody, or antigen binding fragment
thereof, is selected from the group consisting of: a. an anti-CD27
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:1, 2, and 3, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:4, 5, and 6, respectively, or
conservative sequence modifications thereof, and b. an anti-CD27
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:7, 8, and 9, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:10, 11, and 12, respectively,
or conservative sequence modifications thereof; and (ii) the
anti-PD-L1 antibody, or antigen binding fragment thereof, is
selected from the group consisting of: a. an anti-PD-L1 antibody,
or antigen binding fragment thereof, comprising heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 29,
30, and 31, respectively, or conservative sequence modifications
thereof, and light chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:32, 33, and 34, respectively, or conservative
sequence modifications thereof; b. an anti-PD-L1 antibody, or
antigen binding fragment thereof, comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 35, 36, and
37, respectively, or conservative sequence modifications thereof,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:38, 39, and 40, respectively, or conservative sequence
modifications thereof; c. an anti-PD-L1 antibody, or antigen
binding fragment thereof, comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 41, 42, and 43,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:44, 45, and 46, respectively, or conservative sequence
modifications thereof; d. an anti-PD-L1 antibody, or antigen
binding fragment thereof, comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:50, 51, and 52, respectively;, or conservative sequence
modifications thereof e. anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 53, 54, and 55, respectively,
or conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56,
57, and 58, respectively, or conservative sequence modifications
thereof; and f. anti-PD-L1 antibody, or antigen binding fragment
thereof, comprising heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs: 59, 60, and 61, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:62,
63, and 64, respectively, or conservative sequence modifications
thereof.
102. A method for treating a condition or disease in a subject, the
method comprising administering to the subject an anti-CD27
antibody, or antigen binding fragment thereof, in combination with
an anti-PD-L1 antibody, or antigen binding fragment thereof,
wherein: (i) the anti-CD27 antibody, or antigen binding fragment
thereof, is selected from the group consisting of: a. an anti-CD27
antibody, or antigen-binding fragment thereof, comprising a heavy
chain variable region comprising SEQ ID NO:17 and a light chain
variable region comprising SEQ ID NO:18 and b. an anti-CD27
antibody, or antigen-binding fragment thereof, comprising SEQ ID
NO:19 and a light chain variable region comprising SEQ ID NO:20 or
sequences; and (ii) the anti-PD-L1 antibody, or antigen binding
fragment thereof, is selected from the group consisting of: a. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78; b. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80; c. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:81
and a light chain variable region comprising SEQ ID NO:82; d. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:83
and a light chain variable region comprising SEQ ID NO:84; e. an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:85
and a light chain variable region comprising SEQ ID NO:86; and f.
an anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:87
and a light chain variable region comprising SEQ ID NO:88.
103. The method of claim 101 or 102, wherein the anti-CD27
antibody, or antigen binding fragment thereof, and the anti-PD-L1
antibody, or antigen binding fragment thereof, are administered
separately.
104. The method of claim 101 or 102, wherein the anti-CD27
antibody, or antigen binding fragment thereof, and the anti-PD-L1
antibody, or antigen binding fragment thereof, are administered
together.
105. The method of any one of claims 98-104, wherein the subject
suffers from a condition or disease in which stimulation of an
immune response is desired.
106. The method of claim 105, wherein the condition or disease is
cancer.
107. The method of claim 106, wherein the cancer is selected from
the group consisting of colorectal cancer, ovarian cancer, renal
cell carcinoma, head and neck squamous cell carcinoma and
glioblastoma.
108. A bispecific tetravalent antibody, comprising: i) two IgG
heavy chains; ii) two light chains; and iii) two single chain Fv
(scFv) domains; wherein the two IgG heavy chains and two light
chains form an IgG moiety which binds specifically human PD-L1 and
wherein the two scFv domains each bind specifically to human CD27,
and wherein each scFv domain is connected to the C-terminal residue
of one of the IgG heavy chains by a connector sequence.
109. A bispecific tetravalent antibody as claimed in claim 108
wherein the IgG heavy chains are IgG.sub.1 heavy chains.
110. A bispecific tetravalent antibody as claimed in claim 108 or
claim 109 wherein the light chains are kappa light chains.
111. A bispecific tetravalent antibody as claimed in any one of
claims 108 to 110 wherein each scFv domain has a structure order of
either i) N terminus-variable heavy domain-linker-variable light
domain-C terminus or ii) N-terminus-variable light
domain-linker-variable heavy domain-C terminus, and wherein in each
case the linker comprises an amino acid sequence of
(G.sub.4S).sub.m; wherein m is an integer of at least 3.
112. A bispecific tetravalent antibody as claimed in any one of
claims 108 to 111 wherein the connector sequence comprises an amino
acid sequence of G.sub.4S.
113. A bispecific tetravalent antibody as claimed in any one of
claims 108 to 112 wherein the connector sequence comprises an amino
acid sequence of GS.sub.2G.sub.4S.
114. A bispecific tetravalent antibody as claimed in any one of
claims 108 to 113 wherein the linker sequence comprises an amino
acid sequence of (G.sub.4S).sub.4.
115. A bispecific tetravalent antibody as claimed in any one of
claims 108 to 114 wherein at least one of the anti-CD27 scFv
domains comprises heavy chain CDR1, CDR2 and CDR3 domains having
the sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively,
or conservative sequence modifications thereof, and light chain
CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ
ID NOs: 10, 11, and 12, respectively, or conservative sequence
modifications thereof.
116. A bispecific tetravalent antibody as claimed in any one of
claims 108 to 115 wherein at least one of the anti-PD-L1 IgG heavy
chains comprises heavy chain CDR1, CDR2 and CDR3 domains having the
sequences set forth in SEQ ID NOs: 59, 60, and 61, respectively, or
conservative sequence modifications thereof, and at least one of
the anti-PD-L1 light chains comprises light chain CDR1, CDR2 and
CDR3 domains having the sequences set forth in SEQ ID NOs:62, 63,
and 64, respectively, or conservative sequence modifications
thereof.
117. A composition comprising the bispecific tetravalent antibody
of any one of claims 108 to 116 and a pharmaceutically acceptable
carrier.
118. A kit comprising the bispecific tetravalent antibody of any
one of claims 108 to 116, or the composition of claim 117, and
instructions for use.
119. A method of stimulating T cell activity comprising contacting
T cells with the bispecific tetravalent antibody of any one of
claims 108 to 116, or the composition of claim 117.
120. A method for inducing or enhancing an immune response in a
subject comprising administering to the subject the bispecific
tetravalent antibody of any one of claims 108 to 116, or the
composition of claim 117, in an amount effective to induce or
enhance an immune response in the subject.
121. A method for treating a condition or disease in a subject, the
method comprising administering to the subject the bispecific
tetravalent antibody of any one of claims 108 to 116, or the
composition of claim 117, in an amount effective to treat the
condition or disease.
122. The method of any one of claims 119 to 121, wherein the
subject suffers from a condition or disease in which stimulation of
an immune response is desired.
123. The method of claim 122, wherein the condition or disease is
cancer.
124. The method of claim 123, wherein the cancer is selected from
the group consisting of colorectal cancer, ovarian cancer, renal
cell carcinoma, head and neck squamous cell carcinoma and
glioblastoma.
Description
RELATED APPLICATIONS
[0001] This application is a 35 U.S.C. 371 national stage filing of
International Application No. PCT/US2019/027897, filed Apr. 17,
2019, which claims the benefit of priority of U.S. Provisional
Application No. 62/658,899 (filed on Apr. 17, 2018) and U.S.
Provisional Application No. 62/826,091 (filed on Mar. 29, 2019).
The contents of the aforementioned applications are hereby
incorporated by reference in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 14, 2020, is named CDJ_400_US_SL.txt and is 119,376 bytes
in size.
I. BACKGROUND OF THE INVENTION
[0003] Interactions between T cells and antigen-presenting cells
involve a variety of accessory molecules that facilitate in the
generation of an immune response. One such molecule is CD27, which
binds CD70 and belongs to the tumor necrosis factor receptor
(TNF-R) superfamily (Ranheim, E. A. et al. (1995) Blood,
85(12):3556-65). CD27 typically exists as a glycosylated, type I
transmembrane protein, frequently in the form of homodimers with a
disulfide bridge linking the two monomers. The disulfide bridge is
in the extracellular domain close to the membrane (Camerini et al.
(1991) J. Immunol., 147:3165-69). CD27 may also be expressed in a
soluble form (see, e.g., van Oers, M. H. et al. (1993) Blood
82(11):3430-6 and Loenen, W. A. et al. (1992) Eur. J. Immunol.,
22:447). Cross-linking the CD27 antigen on T cells provides a
costimulatory signal that, in concert with T-cell receptor
crosslinking, can induce T-cell proliferation and cellular immune
activation.
[0004] CD27 is expressed on mature thymocytes, on most CD4+ and
CD8+ peripheral blood T cells, natural killer cells and B cells
(Kobata, T. et al. (1995) Proc. Natl. Acad. Sci. USA,
92(24):11249-53). CD27 is also highly expressed on B cell
non-Hodgkin's lymphomas and B cell chronic lymphocytic leukemias
(Ranheim, E. A. et al. (1995) Blood, 85(12):3556-65). Additionally,
increased levels of soluble CD27 protein have been identified in
sera or sites of disease activity in parasitic infection,
cytomegalovirus (CMV) infection, sarcoidosis, multiple sclerosis,
and B-cell chronic lymphocytic leukemia (Loenen, W. A. et al.
(1992) Eur. J. Immunol, 22:447).
[0005] Programmed death-ligand 1 (PD-L1) is a 40 kDa type 1
transmembrane protein that has been speculated to play a major role
in suppressing the immune system during particular events such as
pregnancy, tissue allografts, autoimmune disease, and other disease
states such as hepatitis. Normally the immune system reacts to
foreign antigens that are associated with exogenous or endogenous
danger signals, which triggers a proliferation of antigen-specific
CD8+ T cells and/or CD4+ helper cells. The binding of PD-L1 to PD-1
transmits an inhibitory signal that reduces the proliferation of
these T cells and can also induce apoptosis, which is further
mediated by a lower regulation of the gene Bc1-2. PD-L1 is abundant
in a variety of human cancers (Dong et al. (2002) Nat. Med.
8:787-9). The interaction between PD-1 and PD-L1 results in a
decrease in tumor infiltrating lymphocytes, a decrease in T-cell
receptor mediated proliferation, and immune evasion by the
cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et
al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al.
(2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be
reversed by inhibiting the local interaction of PD-1 with PD-L1,
and the effect is additive when the interaction of PD-1 with PD-L2
is blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA
99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).
[0006] Despite advances in multimodal therapy, there is a need in
the art for new and improved therapeutic agents to treat conditions
or diseases (e.g., in which stimulation of an immune response is
desired). Accordingly, it is an object of the present invention to
provide improved methods for treating subjects with such conditions
or diseases (e.g., cancer).
II. SUMMARY OF THE INVENTION
[0007] Provided herein are novel anti-CD27 and anti-PD-L1
antibodies, and binding domains thereof, as well as bispecific
constructs and multispecific constructs comprising an anti-CD27
binding domain linked to an anti-PD-L1 binding domain. Also
provided herein are methods of stimulating T cell activity, methods
of inducing or enhancing an immune response, and methods of
treating a disease or condition (e.g., cancer) by administering the
bispecific or multispecific constructs, antibodies, or antigen
binding fragments thereof, or compositions described herein to a
patient in need thereof.
[0008] An exemplary anti-CD27 antibody is antibody 3C2 as described
herein. In one embodiment, the anti-CD27 antibody or binding domain
thereof comprises the heavy and light chain CDRs or variable
regions of antibody 3C2. In another embodiment, the antibody or
binding domain thereof comprises the CDR1, CDR2, and CDR3 domains
of the heavy chain variable region of antibody 3C2 having the
sequence set forth in SEQ ID NO:17, and the CDR1, CDR2 and CDR3
domains of the light chain variable region of antibody 3C2 having
the sequence set forth in SEQ ID NO:18. In another embodiment, the
antibody or binding domain thereof comprises heavy chain CDR1, CDR2
and CDR3 domains having the sequences set forth in SEQ ID NOs:1, 2,
and 3, respectively, or conservative sequence modifications
thereof, and light chain CDR1, CDR2 and CDR3 domains having the
sequences set forth in SEQ ID NOs:4, 5, and 6, respectively, or
conservative sequence modifications thereof. In another embodiment,
the antibody or binding domain thereof comprises a heavy chain
variable region having the amino acid sequence set forth in SEQ ID
NO:17. In another embodiment, the antibody or binding domain
thereof comprises a light chain variable region having the amino
acid sequence set forth in SEQ ID NO:18. In another embodiment, the
antibody or binding domain thereof comprises heavy and light chain
variable regions having the amino acid sequences set forth in SEQ
ID NO:17 and SEQ ID NO:18, respectively.
[0009] Another exemplary anti-CD27 antibody is antibody 2B3 as
described herein. In one embodiment, the anti-CD27 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 2B3. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 2B3
having the sequence set forth in SEQ ID NO:19, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 3C2
having the sequence set forth in SEQ ID NO:20. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:7, 8, and 9, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs: 10, 11, and 12,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequences set forth in SEQ ID NO:19. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequences set forth in SEQ ID NO:20.
In another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:19 and SEQ ID NO:20,
respectively.
[0010] An exemplary anti-PD-L1 antibody is antibody 7H7 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 7H7. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 7H7
having the sequence set forth in SEQ ID NO:77, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 7H7
having the sequence set forth in SEQ ID NO:78. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:29, 30, and 31, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:32, 33, and 34,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO:77. In another embodiment, the
antibody or binding domain thereof comprises a heavy chain variable
region having the amino acid sequence set forth in SEQ ID NO:77. In
another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:77 and SEQ ID NO:78,
respectively.
[0011] Another exemplary anti-PD-L1 antibody is antibody 1B3 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 1B3. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 1B3
having the sequence set forth in SEQ ID NO:79, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 1B3
having the sequence set forth in SEQ ID NO:80. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:35, 36, and 37, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:38, 39, and 40,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequences set forth in SEQ ID NO:79. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequences set forth in SEQ ID NO:80.
In another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:79 and SEQ ID NO:80,
respectively.
[0012] Another exemplary anti-PD-L1 antibody is antibody 3B6 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 3B6. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 3B6
having the sequence set forth in SEQ ID NO:81, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 3B6
having the sequence set forth in SEQ ID NO:82. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:41, 42, and 43, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:44, 45, and 46,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO:81. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequence set forth in SEQ ID NO:82. In
another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:81 and SEQ ID NO:82,
respectively.
[0013] Another exemplary anti-PD-L1 antibody is antibody 8B1 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 8B1. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 8B1
having the sequence set forth in SEQ ID NO:83, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 8B1
having the sequence set forth in SEQ ID NO:84. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:47, 48, and 49, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequences set forth in SEQ ID NO:83. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequences set forth in SEQ ID NO:84.
In another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:83 and SEQ ID NO:84,
respectively.
[0014] Another exemplary anti-PD-L1 antibody is antibody 4A3 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 4A3. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 4A3
having the sequence set forth in SEQ ID NO:85, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 4A3
having the sequence set forth in SEQ ID NO:86. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:53, 54, and 55, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO:85. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequence set forth in SEQ ID NO:86. In
another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:85 and SEQ ID NO:86,
respectively.
[0015] Another exemplary anti-PD-L1 antibody is antibody 9H9 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 9H9. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 9H9
having the sequence set forth in SEQ ID NO:87, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 9H9
having the sequence set forth in SEQ ID NO:88. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:59, 60, and 61, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:62, 63, and 64,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO:87. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequence set forth in SEQ ID NO:88. In
another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:87 and SEQ ID NO:88,
respectively.
[0016] In one embodiment, the CDR1, 2, and/or 3 regions of the
anti-C27 or anti-PD-L1 binding domains described herein can
comprise the exact amino acid sequences as those of antibodies 3C2,
2B3, 7H7, 1B3, 3B6, 8B1, 4A3 and 9H9 disclosed herein. In another
embodiment, the antibodies comprise derivatives from the exact CDR
sequences of 3C2, 2B3, 7H7, 1B3, 3B6, 8B1, 4A3 and 9H9, yet still
retain the ability of to bind either CD27 or PD-L1 effectively.
Such sequence modifications may include one or more (e.g., 1, 2, 3,
4, 5, or 6) amino acid additions, deletions, or substitutions,
e.g., conservative sequence modifications.
[0017] In another embodiment, the anti-C27 or anti-PD-L1 binding
domains described herein can be composed of one or more CDRs that
are, for example, 90%, 95%, 98% or 99.5% identical to one or more
CDRs of antibodies 3C2, 2B3, 7H7, 1B3, 3B6, 8B1, 4A3 and 9H9.
Ranges intermediate to the above-recited values, e.g., CDRs that
are 90-95%, 95-98%, or 98-100% identical identity to one or more of
the above sequences are also intended to be encompassed by the
present invention.
[0018] The antibody sequences can also be consensus sequences of
several antibodies. For example, in one embodiment, the anti-PD-L1
binding domain comprises a heavy chain variable region CDR1
comprising an amino acid sequence selected from the consensus
sequence: (T,S)(S,Y,H)WMS (SEQ ID NO:167). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region CDR2 comprising SEQ ID NO:168. In another embodiment, the
anti-PD-L1 binding domain comprises a heavy chain variable region
CDR3 comprising SEQ ID NO:169. In another embodiment, the
anti-PD-L1 binding domain comprises a light chain variable region
CDR1 comprising SEQ ID NO:170. In another embodiment, the
anti-PD-L1 binding domain comprises a light chain variable region
CDR2 comprising SEQ ID NO:171. In another embodiment, the
anti-PD-L1 binding domain comprises a light chain variable region
CDR3 comprising SEQ ID NO:172.
[0019] Sequences substantially identical to the anti-C27 and/or
anti-PD-L1 binding domains described herein (e.g., at least 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical
to the aforementioned sequences), are also encompassed by the
invention. In one embodiment, the anti-CD27 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:17,
SEQ ID NO: 19 or a sequence at least 90% identical thereto (e.g.,
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-CD27 binding domain comprises a light chain variable
region comprising SEQ ID NO:18, SEQ ID NO:20, or a sequence at
least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned
sequences). In another embodiment, the anti-CD27 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:17 and
a light chain variable region comprising SEQ ID NO:18 or sequences
at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned
sequences). In another embodiment, the anti-CD27 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:19 and
a light chain variable region comprising SEQ ID NO:20 or sequences
at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned
sequences).
[0020] In another embodiment, the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:77,
SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID
NO:87, or a sequence at least 90% identical thereto (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
aforementioned sequences). In another embodiment, the anti-PD-L1
binding domain comprises a light chain variable region comprising
SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID
NO:86, SEQ ID NO:88 or a sequence at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:77 and a light chain variable region
comprising SEQ ID NO:78 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:79 and a light chain variable region
comprising SEQ ID NO:80 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:81 and a light chain variable region
comprising SEQ ID NO:82 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences).
[0021] Anti-CD27 and/or anti-PD-L1 antibodies or binding domains
thereof that compete for binding with any of the antibodies or
binding domains thereof described herein or that bind the same
epitope as any of the antibodies or binding domains thereof
described herein are also suitable for use and are encompassed by
the invention. For example, in one embodiment, the anti-CD27
antibody or binding domain thereof competes for binding to CD27
with antibody 3C2 and/or antibody 2B3, as described herein. For
example, as described in Example 28, antibodies of the invention
(e.g., antibody 2B3) bind to one or more residues within amino
acids 80-95 of the ECD of human CD27 (SEQ ID NO: 183), e.g., one or
more residues within amino acids 85-89, e.g., amino acids 85, 87,
88, and/or 89 of the ECD of human CD27 (SEQ ID NO: 183).
[0022] In another embodiment, the antibodies bind to the wildtype
ECD of human CD27, but not to a mutated version of the ECD having
amino acid substitutions at one or more positions within amino acid
residues 85-89 (e.g., A85S, R87A, N88A, and/or G89A) of the ECD of
human CD27 (SEQ ID NO: 183). For example, the anti-CD27 antibody,
or antigen-binding fragment thereof, binds to the wildtype ECD of
human CD27 (SEQ ID NO: 183), but does not bind to a mutated version
of the wildtype ECD of human CD27 having the following amino acid
substitutions: A85S, R87A, N88A, and G89A.
[0023] In another embodiment, the anti-CD27 antibody or binding
domain binds to the same epitope on CD27 as antibody 3C2 and/or
antibody 2B3, as described herein. In another embodiment, the
antibody or anti-PD-L1 binding domain competes for binding to PD-L1
with antibody 7H7, 1B3, 3B6, 8B1, 4A3 and/or 9H9, as described
herein. In another embodiment, the anti-PD-L1 antibody or binding
domain binds to the same epitope on PD-L1 as antibody 7H7, 1B3,
3B6, 8B1, 4A3 and/or 9H9, as described herein. In one aspect, a
bispecific construct (or multispecific construct) comprising an
anti-CD27 binding domain linked to an anti-PD-L1 binding domain is
provided, wherein: [0024] (i) the anti-CD27 binding domain
comprises: [0025] a. heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:1, 2, and 3, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4,
5, and 6, respectively, or conservative sequence modifications
thereof, or [0026] b. heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively, or
conservative sequence modifications thereof, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:10,
11, and 12, respectively, or conservative sequence modifications
thereof; and [0027] (ii) the anti-PD-L1 binding domain comprises:
[0028] a. a heavy chain variable region CDR1 comprising an amino
acid sequence selected from the consensus sequence: (T,S)(S,Y,H)WMS
(SEQ ID NO:167); [0029] b. a heavy chain variable region CDR2
comprising SEQ ID NO:168; [0030] c. a heavy chain variable region
CDR3 comprising SEQ ID NO:169; [0031] d. a light chain variable
region CDR1 comprising SEQ ID NO:170; [0032] e. a light chain
variable region CDR2 comprising SEQ ID NO:171; and [0033] f. a
light chain variable region CDR3 comprising SEQ ID NO:172.
[0034] In another embodiment, a bispecific construct comprising an
anti-CD27 binding domain linked to an anti-PD-L1 binding domain,
wherein: [0035] (i) the anti-CD27 binding domain comprises: [0036]
a. heavy chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:1, 2, and 3, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:4, 5, and 6, respectively, or
conservative sequence modifications thereof, or [0037] b. heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:7, 8, and 9, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:10, 11, and 12, respectively,
or conservative sequence modifications thereof; and [0038] (ii) the
anti-PD-L1 binding domain comprises: [0039] a. a heavy chain
variable region comprising SEQ ID NO:77 and a light chain variable
region comprising SEQ ID NO:78 or sequences at least 95% identical
thereto; [0040] b. a heavy chain variable region comprising SEQ ID
NO:79 and a light chain variable region comprising SEQ ID NO:80 or
sequences at least 95% identical thereto; [0041] c. a heavy chain
variable region comprising SEQ ID NO:81 and a light chain variable
region comprising SEQ ID NO:82 or sequences at least 95% identical
thereto; [0042] d. a heavy chain variable region comprising SEQ ID
NO:83 and a light chain variable region comprising SEQ ID NO:84 or
sequences at least 95% identical thereto; [0043] e. a heavy chain
variable region comprising SEQ ID NO:85 and a light chain variable
region comprising SEQ ID NO:86 or sequences at least 95% identical
thereto; or [0044] f. a heavy chain variable region comprising SEQ
ID NO:87 and a light chain variable region comprising SEQ ID NO:88
or sequences at least 95% identical thereto.
[0045] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof, and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:29, 30, and 31, respectively, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 32,
33, and 34, respectively.
[0046] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78.
[0047] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1,2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:35, 36, and 37, respectively, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 38,
39, and 40, respectively.
[0048] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80.
[0049] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:41, 42, and 43, respectively, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44,
45, and 46, respectively.
[0050] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:81
and a light chain variable region comprising SEQ ID NO:82.
[0051] In another embodiment, the bispecific construct comprises
(a) an the anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:47, 48, and 49, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof.
[0052] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:83
and a light chain variable region comprising SEQ ID NO:84.
[0053] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof, and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:53, 54, and 55, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof.
[0054] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:85
and a light chain variable region comprising SEQ ID NO:86.
[0055] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or conservative sequence
modifications thereof, and an anti-PD-L1 binding domain comprising
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:59, 60, and 61, respectively, or conservative sequence
modifications thereof, and light chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 62, 63, and 64, respectively,
or conservative sequence modifications thereof.
[0056] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:87
and a light chain variable region comprising SEQ ID NO:88.
[0057] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof, and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:29, 30, and 31, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:32, 33, and 34,
respectively, or conservative sequence modifications thereof.
[0058] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78.
[0059] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof, and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:35, 36, and 37, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and 40,
respectively, or conservative sequence modifications thereof.
[0060] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) and anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80.
[0061] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof, and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:41, 42, and 43, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44, 45, and 46,
respectively, or conservative sequence modifications thereof.
[0062] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and an anti-PD-L1 binding domain comprising
a heavy chain variable region comprising SEQ ID NO:81 and a light
chain variable region comprising SEQ ID NO:82.
[0063] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof and (b) an anti-PD-L1 binding domain
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:47, 48, and 49, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof.
[0064] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) an anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:83 and
a light chain variable region comprising SEQ ID NO:84.
[0065] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:53, 54, and 55, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof.
[0066] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:85
and a light chain variable region comprising SEQ ID NO:86.
[0067] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, or conservative sequence modifications thereof, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively, or conservative sequence
modifications thereof, and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:59, 60, and 61, respectively, or conservative
sequence modifications thereof, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:62, 63, and 64,
respectively, or conservative sequence modifications thereof.
[0068] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) an anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:87 and
a light chain variable region comprising SEQ ID NO:88.
[0069] In one embodiment, the anti-PD-L1 binding domain and the
anti-CD27 binding domain are genetically fused. The bispecific
construct can be, for example, a fusion protein, which can be made
by genetic engineering using standard recombinant DNA techniques to
operatively link nucleic acid encoding the anti-CD27 and anti-PD-L1
binding domains. In another embodiment, the anti-PD-L1 binding
domain and the anti-CD27 binding domain are chemically
conjugated.
[0070] For example, the bispecific construct can be a chemical
conjugate, which can be made by chemical conjugation of the
anti-CD27 and anti-PD-L1 binding domains. In one embodiment, the
anti-PD-L1 binding domain further comprises a human IgG.sub.1
constant domain. In another embodiment, the anti-CD27 binding
domain is linked to the C-terminus of the heavy chain of the
anti-PD-L1 binding domain. In another embodiment, the anti-CD27
binding domain is a scFv.
[0071] In another embodiment, the anti-CD27 binding domain further
comprises a human IgG.sub.1 constant domain. In another embodiment,
the anti-PD-L1 binding domain is linked to the C-terminus of the
heavy chain of the anti-CD27 binding domain. In another embodiment,
the anti-PD-L1 binding domain is a scFv.
[0072] In a particular embodiment, the bispecific construct
comprises an anti-PD-L1 antibody linked to an anti-CD27 scFv,
wherein: [0073] (i) the anti-CD27 scFv comprises: [0074] a. heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:1, 2, and 3, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4, 5, and 6,
respectively, or [0075] b. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively; and [0076] (ii) the anti-PD-L1
antibody comprises: [0077] a. heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs: 29, 30, and 31,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:32, 33, and 34, respectively; [0078] b.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs: 35, 36, and 37, respectively, and light chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and
40, respectively; [0079] c. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 41, 42, and 43, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:44, 45, and 46, respectively; [0080] d. heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 47,
48, and 49, respectively, and light chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively; [0081] e. heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs: 53, 54, and 55, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:56, 57, and 58, respectively; or [0082] f. heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 59,
60, and 61, respectively, and light chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:62, 63, and 64,
respectively; and [0083] g. a human IgG.sub.1 constant domain.
[0084] In another particular embodiment, the bispecific construct
comprises an anti-CD27 antibody linked to an anti-PD-L1 scFv,
wherein: [0085] (i) the anti-CD27 antibody comprises: [0086] a.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:1, 2, and 3, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4, 5, and 6,
respectively, or [0087] b. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively; and [0088] c. a human
IgG.sub.1 constant domain; and [0089] (ii) the anti-PD-L1 scFv
comprises: [0090] a. heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs: 29, 30, and 31, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:32, 33, and 34, respectively; [0091] b. heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 35, 36, and
37, respectively, and light chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:38, 39, and 40, respectively;
[0092] c. heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 41, 42, and 43, respectively, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44,
45, and 46, respectively; [0093] d. heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:50, 51, and 52, respectively; [0094] e.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs: 53, 54, and 55, respectively, and light chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and
58, respectively; or [0095] f. heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs: 59, 60, and 61,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:62, 63, and 64, respectively.
[0096] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0097]
(i) the anti-CD27 scFv comprises: [0098] a. a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18; or [0099] b. a heavy chain variable region
comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and [0100] (ii) the anti-PD-L1 antibody
comprises: [0101] a. a heavy chain variable region comprising SEQ
ID NO:77 and a light chain variable region comprising SEQ ID NO:78;
[0102] b. a heavy chain variable region comprising SEQ ID NO:79 and
a light chain variable region comprising SEQ ID NO:80; [0103] c. a
heavy chain variable region comprising SEQ ID NO:81 and a light
chain variable region comprising SEQ ID NO:82; [0104] d. a heavy
chain variable region comprising SEQ ID NO:83 and a light chain
variable region comprising SEQ ID NO:84; [0105] e. a heavy chain
variable region comprising SEQ ID NO:85 and a light chain variable
region comprising SEQ ID NO:86; or [0106] f. a heavy chain variable
region comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88; and [0107] g. a human IgG.sub.1 constant
domain.
[0108] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0109]
(i) the anti-CD27 antibody comprises: [0110] a. a heavy chain
variable region comprising SEQ ID NO:17 and a light chain variable
region comprising SEQ ID NO:18; or [0111] b. a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and [0112] c. a human IgG.sub.1 constant
domain; and [0113] (ii) the anti-PD-L1 scFv comprises: [0114] a. a
heavy chain variable region comprising SEQ ID NO:77 and a light
chain variable region comprising SEQ ID NO:78; [0115] b. a heavy
chain variable region comprising SEQ ID NO:79 and a light chain
variable region comprising SEQ ID NO:80; [0116] c. a heavy chain
variable region comprising SEQ ID NO:81 and a light chain variable
region comprising SEQ ID NO:82; [0117] d. a heavy chain variable
region comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84; [0118] e. a heavy chain variable region
comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86; or [0119] f. a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88.
[0120] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0121]
(i) the anti-CD27 scFv comprises heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:10, 11, and 12, respectively; and [0122] (ii) the
anti-PD-L1 antibody comprises a heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:50, 51, and 52, respectively, and a
human IgG.sub.1 constant domain.
[0123] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0124]
(i) the anti-CD27 antibody comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively, and a
human IgG.sub.1 constant domain; and [0125] (ii) the anti-PD-L1
scFv comprises a heavy chain variable region CDR1, CDR2 and CDR3 as
set forth in SEQ ID NOs: 47, 48, and 49, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:50, 51, and 52, respectively.
[0126] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0127]
(i) the anti-CD27 scFv comprises a heavy chain variable region
comprising SEQ ID NO:19, a light chain variable region comprising
SEQ ID NO:20; and [0128] (ii) the anti-PD-L1 antibody comprises a
heavy chain variable region comprising SEQ ID NO:83 and a light
chain variable region comprising SEQ ID NO:84, and a human
IgG.sub.1 constant domain.
[0129] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0130]
(i) the anti-CD27 antibody comprises a heavy chain variable region
comprising SEQ ID NO:19, a light chain variable region comprising
SEQ ID NO:20, and a human IgG.sub.1 constant domain; and [0131]
(ii) the anti-PD-L1 scFv comprises a heavy chain variable region
comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84.
[0132] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0133]
(i) the anti-CD27 scFv comprises heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:10, 11, and 12, respectively; and [0134] (ii) the
anti-PD-L1 antibody comprises heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:59, 60, and 61,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:62, 63, and 64, respectively, and a
human IgG.sub.1 constant domain.
[0135] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0136]
(i) the anti-CD27 antibody comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively, and a
human IgG.sub.1 constant domain; and [0137] (ii) the anti-PD-L1
scFv comprises heavy chain variable region CDR1, CDR2 and CDR3 as
set forth in SEQ ID NOs:59, 60, and 61, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:62, 63, and 64, respectively.
[0138] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0139]
(i) the anti-CD27 scFv comprises a heavy chain variable region
comprising SEQ ID NO:19, a light chain variable region comprising
SEQ ID NO:20; and [0140] (ii) the anti-PD-L1 antibody comprises a
heavy chain variable region comprising SEQ ID NO:87 and a light
chain variable region comprising SEQ ID NO:88, and a human
IgG.sub.1 constant domain.
[0141] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0142]
(i) the anti-CD27 antibody comprises a heavy chain variable region
comprising SEQ ID NO:19, a light chain variable region comprising
SEQ ID NO:20, and a human IgG.sub.1 constant domain; and [0143]
(ii) the anti-PD-L1 scFv comprises a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88.
[0144] In another embodiment, the bispecific construct has one or
more of the following functional features: induces NF.kappa.B
activation, increases T cell proliferation, induces a CD8 T cell
response, and/or increases IL-2 production. In another embodiment,
the bispecific construct increases IL-2 production by at least
about 1.5-fold (e.g., at least 2-fold, 2.5 fold, 3-fold, 3.5 fold,
or 4-fold) compared to an anti-CD27 monoclonal antibody or
anti-PD-L1 monoclonal antibody alone. In another embodiment, the
bispecific construct induces a CD8 T cell response by at least
about 2-fold greater (e.g., at least 2-fold, 2.5 fold, 3-fold, 3.5
fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold,
7.5-fold, 8.0-fold, 8.5-fold, or 9-fold) than an anti-CD27
monoclonal antibody alone. In another embodiment, the bispecific
construct increases survival by at least about 1.5-fold longer
(e.g., at least 1.5-fold, 2.0-fold, 2.5 fold, 3-fold, 3.5 fold,
4-fold, 4.5-fold, or 5-fold) compared to an anti-CD27 monoclonal
antibody or anti-PD-L1 monoclonal antibody alone. In another
embodiment, the bispecific construct decreases tumor weight by at
least about 1.5-fold (e.g., at least 1.5-fold, 2.0-fold, 2.5 fold,
3-fold, 3.5 fold, 4-fold, 4.5-fold, or 5-fold) compared to
anti-CD27 monoclonal antibodies or anti-PD-L1 monoclonal antibodies
alone or in combination. In another embodiment, the bispecific
construct increases T cell production by at least about 1.5-fold
(e.g., at least 1.5-fold, 2.0-fold, 2.5 fold, 3-fold, 3.5 fold,
4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold,
7.5-fold, 8.0-fold, 8.5-fold, or 9-fold) compared to anti-CD27
monoclonal antibodies or anti-PD-L1 monoclonal antibodies alone or
in combination.
[0145] In certain embodiments, the bispecific constructs described
herein exhibit synergistic effects (e.g., in enhancing immune
responses in vivo) as compared to use of anti-CD27 binding domains
and anti-PD-L1 binding domains in combination (i.e.,
co-administration of unlinked antibodies).
[0146] In another aspect, novel anti-CD27 antibodies, or
antigen-binding portions thereof, are provided which comprise any
of the anti-CD27 binding domains described herein. In one
embodiment, the anti-CD27 antibody, or antigen-binding fragment
thereof, comprises heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:4, 5, and 6, respectively. In another embodiment, the anti-CD27
antibody, or antigen-binding fragment thereof, comprises a heavy
chain variable region comprising SEQ ID NO:17 and a light chain
variable region comprising SEQ ID NO:18 or sequences at least 90%
identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned
sequences).
[0147] In another embodiment, the anti-CD27 antibody, or
antigen-binding fragment thereof, comprises heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively. In another
embodiment, the anti-CD27 antibody, or antigen-binding fragment
thereof, comprises a heavy chain variable region comprising SEQ ID
NO:19 and a light chain variable region comprising SEQ ID NO:20 or
sequences at least 95% identical thereto.
[0148] In another embodiment, the anti-CD27 antibody, or
antigen-binding fragment thereof, has one or more of the following
functional features: induces or enhances a T cell-mediated immune
response, blocks binding of sCD70 to CD27 (e.g., partially or
completely), induces NF.kappa.B activation, increases T cell
proliferation, binds to human CD27 with an equilibrium dissociation
constant Kd of 10.sup.-9 M or less, or alternatively, an
equilibrium association constant Ka of 10.sup.+9 M.sup.-1 or
greater, induces specific complement mediated cytotoxicity (CDC) of
CD27 expressing cells, induces antibody dependent cell-mediated
cytotoxicity (ADCC) specific lysis of CD27 expressing cells,
induces or enhances antigen-specific immune responses in vivo in
combination with a vaccine or endogenous antigen, induces or
enhances antigen-specific TH1 immune responses in vivo in
combination with a vaccine or endogenous antigen, induces or
enhances antigen-specific T-cell proliferation or activation in
vivo in combination with a vaccine or endogenous antigen; and/or
induces or enhances T-cell activity when combined with
simultaneous, separate or sequential TCR activation.
[0149] In another aspect, a bispecific construct is provided,
wherein the bispecific construct comprises any one of the anti-CD27
antibodies described herein linked to an anti-PD-L1 binding domain.
In one embodiment, the anti-PD-L1 binding domain is selected from
the group consisting of: [0150] a. an anti-PD-L1 antibody, or
antigen binding fragment thereof, comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 29, 30, and
31, respectively, and light chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:32, 33, and 34, respectively;
[0151] b. an anti-PD-L1 antibody, or antigen binding fragment
thereof, comprising heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs: 35, 36, and 37, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:38, 39, and 40, respectively; [0152] c. an anti-PD-L1 antibody,
or antigen binding fragment thereof, comprising heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 41,
42, and 43, respectively, and light chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:44, 45, and 46,
respectively; [0153] d. an anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:50, 51, and 52, respectively; [0154] e. anti-PD-L1
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs: 53, 54, and 55, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and 58,
respectively; and [0155] f. anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 59, 60, and 61, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:62, 63, and 64, respectively.
[0156] In another embodiment, the anti-PD-L1 binding domain is
selected from the group consisting of: (a) a heavy chain variable
region comprising SEQ ID NO:77 and a light chain variable region
comprising SEQ ID NO:78; (b) a heavy chain variable region
comprising
[0157] SEQ ID NO:79 and a light chain variable region comprising
SEQ ID NO:80; (c) a heavy chain variable region comprising SEQ ID
NO:81 and a light chain variable region comprising SEQ ID NO:82;
(d) a heavy chain variable region comprising SEQ ID NO:83 and a
light chain variable region comprising SEQ ID NO:84; (e) a heavy
chain variable region comprising SEQ ID NO:85 and a light chain
variable region comprising SEQ ID NO:86; and (f) a heavy chain
variable region comprising SEQ ID NO:87 and a light chain variable
region comprising SEQ ID NO:88. In a particular embodiment, the
anti-PD-L1 binding domain is an scFv.
[0158] In certain embodiments, the bispecific constructs described
herein exhibit synergistic effects (e.g., in enhancing immune
responses in vivo) as compared to use of anti-CD27 binding domains
and anti-PD-L1 binding domains in combination (i.e.,
co-administration of unlinked antibodies).
[0159] In another aspect, novel anti-PD-L1 antibodies, or
antigen-binding portions thereof, are provided, which comprise any
of the anti-PD-L1 binding domains described herein. In one
embodiment, the anti-PD-L1 antibody, or antigen-binding fragment
thereof, comprises heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:29, 30, and 31, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:32, 33, and 34, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:77 and a light
chain variable region comprising SEQ ID NO:78 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:35, 36, and 37, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:38, 39, and 40, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:79 and a light
chain variable region comprising SEQ ID NO:80 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:41, 42, and 43, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:44, 45, and 46, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:81 and a light
chain variable region comprising SEQ ID NO:82 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:47, 48, and 49, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:50, 51, and 52, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:83 and a light
chain variable region comprising SEQ ID NO:84 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:53, 54, and 55, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:56, 57, and 58, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:85 and a light
chain variable region comprising SEQ ID NO:86 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:59, 60, and 61, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:62, 63, and 64, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:87 and a light
chain variable region comprising SEQ ID NO:88 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned
sequences).
[0160] In another embodiment, the anti-PD-L1 antibody, or
antigen-binding fragment thereof, has one or more of the following
functional features: (a) blocks binding of PD1 to PD-L1 (e.g.,
partially or completely), (b) induces NFAT pathway activation,
and/or (c) induces a mixed lymphocyte response.
[0161] In another aspect, a bispecific construct is provided,
wherein the bispecific construct comprises any one of the
anti-PD-L1 antibodies, or antigen binding fragments thereof,
described herein, linked to an anti-CD27 binding domain. In one
embodiment, the anti-C27 binding domain comprises an anti-CD27
antibody comprising heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:4, 5, and 6, respectively,. In another embodiment, the
anti-CD27 binding domain comprises an antibody comprising a heavy
chain variable region comprising SEQ ID NO:17 and a light chain
variable region comprising SEQ ID NO:18, or sequences at least 90%
identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-C27 binding domain comprises an
anti-CD27 antibody comprising heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:10, 11, and 12, respectively. In another embodiment, the
anti-C27 binding domain comprises an anti-CD27 antibody comprising
a heavy chain variable region comprising SEQ ID NO:19 and a light
chain variable region comprising SEQ ID NO:20, or sequences at
least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned
sequences). In one embodiment, the anti-CD27 binding domain further
comprises a human IgG.sub.1 constant domain.
[0162] In certain embodiments, the bispecific constructs described
herein exhibit synergistic effects (e.g., in enhancing immune
responses in vivo) as compared to use of anti-CD27 binding domains
and anti-PD-L1 binding domains in combination (i.e.,
co-administration of unlinked antibodies).
[0163] In another aspect, provided herein are compositions
including any of the bispecific constructs (multispecific
constructs), antibodies, or antigen binding fragments thereof,
described herein and a pharmaceutically acceptable carrier. Also
provided are kits comprising any of the bispecific constructs
(multispecific constructs), antibodies, or antigen binding
fragments thereof, described herein and instructions for use.
[0164] In a further aspect, isolated nucleic acid molecules
encoding the binding domains, antibodies, or antigen-binding
portions thereof, and bispecific or multispecific constructs
described herein are also provided, as well as expression vectors
comprising such nucleic acids and host cells comprising such
expression vectors. In another embodiment, a nucleic acid molecule
coding for any of the binding domains, antibodies, or
antigen-binding portions thereof, or bispecific constructs
described herein is provided. In another embodiment, the nucleic
acid molecule is in the form of an expression vector. In another
embodiment, the nucleic acid molecule is in the form of an
expression vector which expresses the binding domain, antibody, or
antigen-binding portion thereof, or bispecific construct when
administered to a subject in vivo.
[0165] In one embodiment, the nucleic acid molecule comprises a
nucleotide sequence encoding an antibody variable region, wherein
the antibody variable region comprises the amino acid sequence
depicted in SEQ ID NO:17, 18, 19, 20, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, or an amino acid sequence at least 90%
identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to one or more of the aforementioned
sequences). In another embodiment, the nucleic acid molecule
comprises a nucleotide sequence as set forth in SEQ ID NO:25, 26,
27, 28, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
or a nucleotide sequence at least 90% identical thereto (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical
to one or more of the aforementioned sequences).
[0166] In another embodiment, the nucleic acid molecule comprises a
nucleotide sequence encoding heavy and light chain variable regions
of an antibody, wherein the heavy and light chain variable regions
comprise the amino acid sequences depicted in SEQ ID NOs:17 and 18,
SEQ ID NOs:19 and 20, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80,
SEQ ID NOs:81 and 82, SEQ ID NOs: 83 and 84, SEQ ID NOs:85 and 86,
or SEQ ID NOs:87 and 88, respectively, or amino acids sequences at
least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical the aforementioned
sequences).
[0167] In another aspect, methods of stimulating T cell activity
are provided, which comprise contacting T cells with any one of the
antibodies, or antigen binding fragments thereof, bispecific
constructs, multispecific constructs, or the compositions of
described herein. Stimulating T cell activity can comprise, for
example, stimulating IFN-gamma production.
[0168] In yet another aspect, methods for inducing or enhancing an
immune response (e.g., against an antigen) in a subject comprising
administering to the subject any one of the antibodies, or antigen
binding fragments thereof, bispecific constructs, multispecific
constructs, or the compositions described herein, in an amount
effective to induce or enhance an immune response in the subject
(e.g., against an antigen).
[0169] In a further aspect, methods of for treating a condition or
disease in a subject are provided, the method comprising
administering to the subject any one of the antibodies, or antigen
binding fragments thereof, bispecific constructs, multispecific
constructs, or the compositions described herein, in an amount
effective to treat the condition or disease.
[0170] In another aspect, methods for treating a condition or
disease in a subject are provided, wherein the method comprises
administering to the subject any one of the anti-CD27 antibodies,
or antigen binding fragments thereof, described herein in
combination with any one of the anti-PD-L1 antibodies, or antigen
binding fragments thereof, described herein. For example, in one
embodiment: [0171] (i) the anti-CD27 antibody, or antigen binding
fragment thereof, is selected from the group consisting of: (a) an
anti-CD27 antibody, or antigen binding fragment thereof, comprising
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:1, 2, and 3, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4, 5, and 6,
respectively, and (b) an anti-CD27 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively; and [0172] (ii) the anti-PD-L1
antibody, or antigen binding fragment thereof, is selected from the
group consisting of: (a) an anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 29, 30, and 31, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:32, 33, and 34, respectively; (b) an anti-PD-L1
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs: 35, 36, and 37, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and 40,
respectively; (c) an anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 41, 42, and 43, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:44, 45, and 46, respectively; (d) an anti-PD-L1
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs: 47, 48, and 49, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively; (e) anti-PD-L1 antibody, or antigen binding fragment
thereof, comprising heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs: 53, 54, and 55, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:56, 57, and 58, respectively; and (f) anti-PD-L1 antibody, or
antigen binding fragment thereof, comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 59, 60, and
61, respectively, and light chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:62, 63, and 64, respectively.
[0173] In another embodiment, [0174] (i) the anti-CD27 antibody, or
antigen binding fragment thereof, is selected from the group
consisting of: (a) an anti-CD27 antibody, or antigen-binding
fragment thereof, comprising a heavy chain variable region
comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-CD27 antibody, or
antigen-binding fragment thereof, comprising SEQ ID NO:19 and a
light chain variable region comprising SEQ ID NO:20 or sequences;
and [0175] (ii) the anti-PD-L1 antibody, or antigen binding
fragment thereof, is selected from the group consisting of: (a) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78; (b) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80; (c) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:81
and a light chain variable region comprising SEQ ID NO:82; (d) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:83
and a light chain variable region comprising SEQ ID NO:84; (e) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:85
and a light chain variable region comprising SEQ ID NO:86; and (f)
an anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:87
and a light chain variable region comprising SEQ ID NO:88.
[0176] In one embodiment, the anti-CD27 antibody, or antigen
binding fragment thereof, and the anti-PD-L1 antibody, or antigen
binding fragment thereof, are administered separately. In one
embodiment, the anti-CD27 antibody, or antigen binding fragment
thereof, and the anti-PD-L1 antibody, or antigen binding fragment
thereof, are administered sequentially. For example, the anti-CD27
antibody, or antigen binding fragment thereof, can be administered
first followed by (e.g., immediately followed by) administration of
the anti-PD-L1 antibody, or antigen binding fragment thereof, or
vice versa. In another embodiment, the anti-CD27 antibody, or
antigen binding fragment thereof, and the anti-PD-L 1 antibody, or
antigen binding fragment thereof, are administered together. In
another embodiment, the anti-CD27 antibody, or antigen binding
fragment thereof, and the anti-PD-L 1 antibody, or antigen binding
fragment thereof, are administered simultaneously. In another
embodiment, the anti-CD27 antibody, or antigen binding fragment
thereof, and the anti-PD-L 1 antibody, or antigen binding fragment
thereof, are simultaneously administered in a single formulation.
Alternatively, the anti-CD27 antibody, or antigen binding fragment
thereof, and the anti-PD-L1 antibody, or antigen binding fragment
thereof, are formulated for separate administration and are
administered concurrently or sequentially. Such concurrent or
sequential administration preferably results in both antibodies
being simultaneously present in treated patients.
[0177] In certain embodiments, administration of any of the
anti-CD27 antibodies, or antigen binding fragment thereof,
described herein in combination with any of the anti-PD-L1
antibodies, or antigen binding fragments thereof, described herein
results in synergistic effects (e.g., in enhancing immune responses
in vivo) as compared to use of either antibody alone.
[0178] The subject can be, for example, one who suffers from a
condition or disease in which stimulation of an immune response is
desired. In one embodiment, the condition or disease in which
stimulation of an immune response is desired is cancer. The method
of inducing or enhancing an immune response (e.g., against an
antigen) in a subject can further comprise administering the
antigen to the subject. Preferred antigens to be co-administered
with the antibodies, or antigen binding fragments thereof,
bispecific constructs, multispecific constructs, or the
compositions of described herein are tumor antigens.
III. BRIEF DESCRIPTION OF THE DRAWINGS
[0179] FIG. 1 is a graph showing binding of CD27 antibodies 2B3 and
3C2 to recombinant human CD27, as a function of antibody
concentration.
[0180] FIG. 2 is a graph showing binding of CD27 antibodies 2B3 and
3C2 to recombinant cynomolgus CD27.
[0181] FIG. 3 is a graph showing high level binding of CD27
antibodies 2B3 and 3C2 to Ramos cells expressing CD27 on their
surface.
[0182] FIG. 4 is a graph showing high level binding of CD27
antibodies 2B3 and 3C2 to T cells.
[0183] FIG. 5 is a graph showing antibodies 2B3 and 3C2
significantly block CD70 binding to CD27.
[0184] FIG. 6 is a graph showing antibodies 2B3 and 3C2 induced
significant NF.kappa.B activation, as a function of antibody
concentration.
[0185] FIG. 7 is a graph showing antibodies 2B3 and 3C2 increase T
cell proliferation.
[0186] FIG. 8 is a graph showing anti-PD-L1 antibodies bind human
PD-L1 as a function of antibody concentration.
[0187] FIG. 9 is a graph showing binding of anti-PD-L1 antibodies
to recombinant cynomolgus PD-L1.
[0188] FIG. 10 is a graph showing anti-PD-L1 antibodies
significantly block PD-L1 binding to PD1 as a function of antibody
concentration.
[0189] FIG. 11 is a graph showing high level of binding of
anti-PD-L1 antibodies to cells that express human PD-L1 as a
function of antibody concentration.
[0190] FIG. 12 is a graph showing high level of binding of
anti-PD-L1 antibodies to human dendritic cells as a function of
antibody concentration.
[0191] FIG. 13 is a graph showing anti-PD-L1 antibodies block
PD1/PD-L1 interaction between cells resulting in NFAT pathway
activation.
[0192] FIG. 14 is a graph showing anti-PD-L1 antibodies induce a
mixed lymphocyte response as a function of antibody
concentration.
[0193] FIG. 15A is a representative DNA expression vector
containing the anti-CD27 light chain, anti-CD27 heavy chain, and
the c-terminal anti-PD-L1 single chain (VL+VH) peptide.
[0194] FIG. 15B is a drawing of the CD27/PD-L1 bispecific antibody
protein wherein an anti-PD-L1 antibody is linked to an anti-CD27
scFv.
[0195] FIG. 15C is a drawing of the CD27/PD-L1 bispecific antibody
protein wherein an anti-CD27 antibody is linked to an anti-PD-L1
scFv.
[0196] FIG. 15D is a table of representative anti-CD27/anti-PD-L1
bispecific constructs.
[0197] FIG. 15E shows characterization of the bispecific antibody
CDX-527 by HPLC and gel electrophoresis.
[0198] FIG. 16 is a graph showing binding of anti-CD27/antiPD-L1
bispecific constructs (BsAbs) to CD27 and PD-L1 using a
bifunctional ELISA.
[0199] FIG. 17 is a graph showing increased NF.kappa.B activation
induced by anti-CD27/anti-PD-L1 bispecific constructs as compared
to antibodies 1F5 or 2B3 alone.
[0200] FIG. 18 is a graph showing anti-CD27/anti-PD-L1 bispecific
constructs block PD1/PD-L1 interaction and induce NFAT pathway
activation as a function of antibody concentration.
[0201] FIG. 19 is a graph showing anti-CD27/anti-PD-L1 bispecific
constructs increase IL-2 production/secretion in a mixed lymphocyte
reaction as compared to antibodies AbX (a known anti-PD-L1
monoclonal antibody), 8B1, or 9H9 alone.
[0202] FIGS. 20A and 20B are graphs showing an anti-CD27/anti-PD-L1
bispecific construct (e.g., CD27xAbX) induces a higher CD8 T cell
response as compared to CD27 monoclonal antibody alone.
[0203] FIG. 21 is a Kaplan-Meier curve showing improved survival of
mice treated with a bispecific construct (e.g., CD27xAbX) compared
to CD27 and PD-L1 antibodies administered alone or in combination
in a mouse tumor model.
[0204] FIG. 22A is a graph showing decreased tumor weight in mice
treated with a bispecific construct (e.g., CD27xAbX) compared to
CD27 and PD-L1 antibodies administered alone or in combination.
FIG. 22B is a graph showing increased percent CD8 T cells in mice
treated with a bispecific construct (e.g., CD27xAbX) compared to
CD27 and PD-L1 antibodies administered alone or in combination.
FIG. 22C is a graph showing increased percent CD4 T cells in mice
treated with a bispecific construct (e.g., CD27xAbX) compared to
CD27 and PD-L1 antibodies administered alone or in combination.
FIG. 22D is a graph showing increased activated CD8 T cells in mice
treated with a bispecific construct (e.g., CD27xAbX) compared to
CD27 and PD-L1 antibodies administered alone or in combination.
[0205] FIGS. 23A and 23B are graphs showing anti-CD27 Ab (e.g.,
CDX-1127) upregulates PD-L1 expression in tumor cells and tumor
infiltrated cells.
[0206] FIG. 24 is a graph showing binding of the
anti-CD27/antiPD-L1 bispecific construct CDX-527 to CD27 and PD-L1
using a bifunctional ELISA.
[0207] FIG. 25 are graphs showing increased NF.kappa.B activation
induced by anti-CD27/anti-PD-L1 bispecific construct CDX-527 as
compared to antibodies 1F5 or 2B3 alone (left graph) and also in
presence of soluble Fc.gamma.R1 (right graph).
[0208] FIG. 26 is a graph showing increased IL-2
production/secretion in a mixed lymphocyte reaction by
anti-CD27/anti-PD-L1 bispecific construct CDX-527 as compared to
antibodies 2B3 or 9H9 alone or in combination.
[0209] FIG. 27 is a graph showing increased IL-2
production/secretion in T-cells by anti-CD27/anti-PD-L1 bispecific
construct CDX-527 as compared to antibodies 2B3 and 9H9 in
combination.
[0210] FIG. 28 is a graph showing serum levels of CDX-527 in a NHP
pharmacokinetic study.
[0211] FIG. 29 is a graph showing increased blockade of PD-1
signaling by the 9H9x2B3 configuration as compared to the 2B3x9H9
configuration.
[0212] FIG. 30 is a graph showing increased T-cell activation by
the 9H9x2B3 configuration as compared to the 2B3x9H9
configuration.
[0213] FIG. 31 is a graph showing increased stimulation of a
vaccine induced CD8+ T-cell response by AbXx2B3 as compared to
2B3.times.AbX.
[0214] FIG. 32 is a graph showing increased anti-tumor activity by
AbXx2B3 as compared to 2B3.times.AbX.
[0215] FIG. 33 is a graph showing blocking of PD-L1 Binding to CD80
by anti-PD-L1 antibodies AbX and 9H9.
[0216] FIG. 34 (SEQ ID NO: 183) shows the sequence of the
extracellular domain (ECD) of wild type huCD27 and a mutated
version with substitutions at positions 85, 87, 88 and 89.
[0217] FIG. 35 is a graph showing binding of anti-CD27 antibody 2B3
to both wild type huCD27 and mutated huCD27 with sequences as shown
in FIG. 34.
[0218] FIG. 36 is a graph showing improved production (expression)
of a modified 9H9x2B3 (DD) construct compared to the original
(unmodified) 9H9x2B3 construct.
IV. DETAILED DESCRIPTION OF THE INVENTION
[0219] CD27 is an important co-stimulatory receptor that can be
exploited for immunotherapy using agonist molecules. CD27 plays a
key role in diverse immunological processes, including the
survival, activation and effector functions of T cells, as well as
the proliferation and cytotoxic activity of natural killer (NK)
cells. These events occur in response to appropriate interaction of
the ligand (CD70) with CD27 resulting in intracellular signaling
events that lead to an activation of NF-kB and expression of
relevant genes. As with most co-stimulatory molecules, effective
stimulation of T cells with either the ligand or agonist antibodies
also requires simultaneous stimulation through the T cell receptor
(TCR).
[0220] Most CD27 agonists molecules require multimerization or
crosslinking for their activity. For example, anti-CD27 antibodies
that are agonists are cross-linked through interaction of their Fc
domains with Fc receptors through cis or trans interactions. In
vitro this can also be performed artificially using a secondary
anti-Ig antibody or by adsorbing the antibody to a solid phase
matrix. The requirement for FcR interaction of anti-CD27 agonist
antibodies implies that the T cell stimulating activity is at least
partially dependent on the number FcR expressing cells.
[0221] To eliminate the requirement for FcR interactions, CD27
agonists have been invented that 1) eliminate the need for
interaction with receptors other than CD27 (herein termed CD27
hyper-crosslinking agents), or 2) can provide the crosslinking
through interaction of alternate receptors that may be on different
specific cell types not expressing FcR, and which alternate
receptors may also provide additional function (herein termed CD27
multi-specific agents).
[0222] In one aspect, the invention provides bispecific constructs
(or multispecific constructs) that comprise an anti-CD27 antibody,
or antigen-binding fragment thereof, linked to an anti-PD-L1
antibody, or antigen-binding fragment thereof. Such
anti-CD27.times.anti-PD-L1 bispecific agents of the invention have
now been shown to exhibit synergistic effects in vivo, such as in
enhancing immune parameters and inhibiting tumor growth, as
compared to co-administration of an anti-CD27 antibody with an
anti-PD-L1 antibody (see Examples 9 and 10).
[0223] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0224] A. Definitions
[0225] As used herein, the term "subject" includes any human or
non-human animal. For example, the methods and compositions of the
present invention can be used to treat a subject with an immune
disorder. The term "non-human animal" includes all vertebrates,
e.g., mammals and non-mammals, such as non-human primates, sheep,
dog, cow, chickens, amphibians, reptiles, etc.
[0226] As used herein, the term "binding domain" refers to the
portion of a protein or antibody which comprises the amino acid
residues that interact with an antigen. Binding domains include,
but are not limited to, antibodies (e.g., full length antibodies),
as well as antigen-binding portions thereof. The binding domain
confers on the binding agent its specificity and affinity for the
antigen. The term also covers any protein having a binding domain
which is homologous or largely homologous to an
immunoglobulin-binding domain. Such proteins may be derived from
natural sources, or partly or wholly synthetically produced.
[0227] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chain version thereof. An "antibody" refers, in
one preferred embodiment, to a glycoprotein comprising at least two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds, or an antigen binding portion thereof. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as V.sub.H) and a heavy chain constant region. The heavy
chain constant region is comprised of three domains, CH1, CH2 and
CH3. Each light chain is comprised of a light chain variable region
(abbreviated herein as V.sub.L) and a light chain constant region.
The light chain constant region is comprised of one domain, CL. The
V.sub.H and V.sub.L regions can be further subdivided into regions
of hypervariability, termed complementarity determining regions
(CDR), interspersed with regions that are more conserved, termed
framework regions (FR). Each V.sub.H and V.sub.L is composed of
three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (e.g., effector cells) and the first component
(Clq) of the classical complement system.
[0228] The term "antigen-binding fragment" of an antibody (or
simply "antibody fragment"), as used herein, refers to one or more
fragments or portions of an antibody that retain the ability to
specifically bind to an antigen (e.g., human CD27). Such
"fragments" are, for example between about 8 and about 1500 amino
acids in length, suitably between about 8 and about 745 amino acids
in length, suitably about 8 to about 300, for example about 8 to
about 200 amino acids, or about 10 to about 50 or 100 amino acids
in length. It has been shown that the antigen-binding function of
an antibody can be performed by fragments of a full-length
antibody. Examples of binding fragments encompassed within the term
"antigen-binding fragment" of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent
fragment comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) a Fd fragment consisting of the V.sub.H
and CH1 domains; (iv) a Fv fragment consisting of the V.sub.L and
V.sub.H domains of a single arm of an antibody, (v) a dAb fragment
(Ward et al., (1989) Nature 341:544-546), which consists of a
V.sub.H domain; and (vi) an isolated complementarity determining
region (CDR) or (vii) a combination of two or more isolated CDRs
which may optionally be joined by a synthetic linker. Furthermore,
although the two domains of the Fv fragment, V.sub.L and V.sub.H,
are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the V.sub.L and V.sub.H
regions pair to form monovalent molecules (known as single chain Fv
(sFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston
et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single
chain antibodies are also intended to be encompassed within the
term "antigen-binding fragment" of an antibody. These antibody
fragments are obtained using conventional techniques known to those
with skill in the art, and the fragments are screened for utility
in the same manner as are intact antibodies. Antigen-binding
portions can be produced by recombinant DNA techniques, or by
enzymatic or chemical cleavage of intact immunoglobulins.
[0229] The term "monoclonal antibody," as used herein, refers to an
antibody that displays a single binding specificity and affinity
for a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to an antibody which displays a single binding
specificity and which has variable and optional constant regions
derived from human germline immunoglobulin sequences. In one
embodiment, human monoclonal antibodies are produced by a hybridoma
that includes a B cell obtained from a transgenic non-human animal,
e.g., a transgenic mouse, having a genome comprising a human heavy
chain transgene and a light chain transgene fused to an
immortalized cell.
[0230] The term "recombinant human antibody," as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared therefrom, (b) antibodies isolated from a host cell
transformed to express the antibody, e.g., from a transfectoma, (c)
antibodies isolated from a recombinant, combinatorial human
antibody library, and (d) antibodies prepared, expressed, created
or isolated by any other means that involve splicing of human
immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies comprise variable and constant regions
that utilize particular human germline immunoglobulin sequences are
encoded by the germline genes, but include subsequent
rearrangements and mutations which occur, for example, during
antibody maturation. As known in the art (see, e.g., Lonberg (2005)
Nature Biotech. 23(9):1117-1125), the variable region contains the
antigen binding domain, which is encoded by various genes that
rearrange to form an antibody specific for a foreign antigen. In
addition to rearrangement, the variable region can be further
modified by multiple single amino acid changes (referred to as
somatic mutation or hypermutation) to increase the affinity of the
antibody to the foreign antigen. The constant region will change in
further response to an antigen (i.e., isotype switch). Therefore,
the rearranged and somatically mutated nucleic acid molecules that
encode the light chain and heavy chain immunoglobulin polypeptides
in response to an antigen may not have sequence identity with the
original nucleic acid molecules, but instead will be substantially
identical or similar (i.e., have at least 80% identity).
[0231] The term "human antibody" includes antibodies having
variable and constant regions (if present) of human germline
immunoglobulin sequences. Human antibodies of the invention can
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo)
(see, Lonberg, N. et al. (1994) Nature 368(6474): 856-859);
Lonberg, N. (1994) Handbook of Experimental Pharmacology
113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol.
Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.
Acad. Sci 764:536-546). However, the term "human antibody" does not
include antibodies in which CDR sequences derived from the germline
of another mammalian species, such as a mouse, have been grafted
onto human framework sequences (i.e., chimeric and humanized
antibodies).
[0232] An "isolated antibody," as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to human CD27 is substantially
free of antibodies that specifically bind antigens other than human
CD27; an isolated antibody that specifically binds to human PD-L1
is substantially free of antibodies that specifically bind antigens
other than human PD-L1). An isolated antibody that specifically
binds to an epitope may, however, have cross-reactivity to the same
antigen from different species. In addition, an isolated antibody
is typically substantially free of other cellular material and/or
chemicals.
[0233] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody
specifically binds. Epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods for determining what epitopes are
bound by a given antibody (i.e., epitope mapping) are well known in
the art and include, for example, immunoblotting and
immunoprecipitation assays, wherein overlapping or contiguous
peptides from the antigen (e.g., CD27 or PD-L1) are tested for
reactivity with the given antibody (e.g., an anti-CD27 or
anti-PD-L1 antibody. Methods of determining spatial conformation of
epitopes include techniques in the art and those described herein,
for example, x-ray crystallography and 2-dimensional nuclear
magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods
in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).
[0234] The term "antibody that binds the same epitope" as another
antibody is intended to encompass antibodies that interact with,
i.e., bind to, the same structural region on human CD27 as a
reference anti-CD27 antibody. The "same epitope" to which the
antibodies bind may be a linear epitope or a conformational epitope
formed by tertiary folding of the antigen.
[0235] The term "competing antibody" refers to an antibody that
competes for binding to human CD27 with a reference anti-CD27
antibody, i.e., competitively inhibits binding of the reference
anti-CD27 antibody to CD27. A "competing antibody" may bind the
same epitope on CD27 as the reference anti-CD27 antibody, may bind
to an overlapping epitope or may sterically hinder the binding of
the reference anti-CD27 antibody to CD27.
[0236] Antibodies that recognize the same epitope or compete for
binding can be identified using routine techniques. Such techniques
include, for example, an immunoassay, which shows the ability of
one antibody to block the binding of another antibody to a target
antigen, i.e., a competitive binding assay. Competitive binding is
determined in an assay in which the immunoglobulin under test
inhibits specific binding of a reference antibody to a common
antigen, such as CD27. Numerous types of competitive binding assays
are known, for example: solid phase direct or indirect
radioimmunoassay (RIA), solid phase direct or indirect enzyme
immunoassay (EIA), sandwich competition assay (see Stahli et al.,
Methods in Enzymology 9:242 (1983)); solid phase direct
biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614
(1986)); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase
direct label RIA using I-125 label (see Morel et al., Mol. Immunol.
25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et
al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer
et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabeled test immunoglobulin and
a labeled reference immunoglobulin. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin.
Usually the test immunoglobulin is present in excess. Usually, when
a competing antibody is present in excess, it will inhibit specific
binding of a reference antibody to a common antigen by at least
50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
[0237] Other techniques include, for example, epitope mapping
methods, such as, x-ray analyses of crystals of antigen:antibody
complexes which provides atomic resolution of the epitope. Other
methods monitor the binding of the antibody to antigen fragments or
mutated variations of the antigen where loss of binding due to a
modification of an amino acid residue within the antigen sequence
is often considered an indication of an epitope component. In
addition, computational combinatorial methods for epitope mapping
can also be used. These methods rely on the ability of the antibody
of interest to affinity isolate specific short peptides from
combinatorial phage display peptide libraries. The peptides are
then regarded as leads for the definition of the epitope
corresponding to the antibody used to screen the peptide library.
For epitope mapping, computational algorithms have also been
developed which have been shown to map conformational discontinuous
epitopes.
[0238] As used herein, the terms "specific binding," "selective
binding," "selectively binds," and "specifically binds," refer to
antibody binding to an epitope on a predetermined antigen.
Typically, the antibody binds with an equilibrium dissociation
constant (K.sub.D) of approximately less than 10.sup.-7 M, such as
approximately less than 10 .sup.-8 M, 10.sup.-9 M or 10.sup.-10 M
or even lower when determined by surface plasmon resonance (SPR)
technology in a BIACORE 2000 instrument (e.g., using recombinant
human CD27 as the analyte and the antibody as the ligand) and binds
to the predetermined antigen with an affinity that is at least
two-fold greater than its affinity for binding to a non-specific
antigen (e.g., BSA, casein) other than the predetermined antigen or
a closely-related antigen. The phrases "an antibody recognizing an
antigen" and "an antibody specific for an antigen" are used
interchangeably herein with the term "an antibody which binds
specifically to an antigen."
[0239] The term "K.sub.D," as used herein, is intended to refer to
the dissociation equilibrium constant of a particular
antibody-antigen interaction. Typically, the human antibodies of
the invention bind to CD27 with a dissociation equilibrium constant
(K.sub.D) of approximately 10.sup.-8 M or less, such as less than
10.sup.-9 M or 10.sup.-10 M or even lower when determined by
surface plasmon resonance (SPR) technology in a BIACORE 2000
instrument (e.g., using recombinant human CD27 as the analyte and
the antibody as the ligand).
[0240] The term "kd" as used herein, is intended to refer to the
off rate constant for the dissociation of an antibody from the
antibody/antigen complex.
[0241] The term "ka" as used herein, is intended to refer to the on
rate constant for the association of an antibody with the
antigen.
[0242] The term "EC50," as used herein, refers to the concentration
of an antibody or an antigen-binding portion thereof, which induces
a response, either in an in vitro or an in vivo assay, which is 50%
of the maximal response, i.e., halfway between the maximal response
and the baseline.
[0243] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG.sub.1) that is encoded by heavy chain constant
region genes. In one embodiment, a human monoclonal antibody of the
invention is of the IgG.sub.1 isotype. In another embodiment, a
human monoclonal antibody of the invention is of the IgG2
isotype.
[0244] As used herein, the terms "inhibits" or "blocks" (e.g.,
referring to inhibition/blocking of binding of CD70 to CD27 and/or
PD1 to PD-L1) are used interchangeably and encompass both partial
and complete inhibition/blocking. The inhibition/blocking
preferably reduces or alters the normal level or type of activity
that occurs when binding occurs without inhibition or blocking.
Inhibition and blocking are also intended to include any measurable
decrease in the binding affinity of CD70 when in contact with an
anti-CD27 antibody as compared to CD70 not in contact with an
anti-CD27 antibody, e.g., inhibits binding of CD70 by at least
about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75% , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In one
embodiment, the anti-CD27 antibody inhibits binding of CD70 by at
least about 70%. In another embodiment, the anti-CD27 antibody
inhibits binding of CD70 by at least 80%. Inhibition and blocking
are also intended to include any measurable decrease in the binding
affinity of PD1 when in contact with an anti-PD-L1 antibody as
compared to PD1 not in contact with an anti-PD-L1 antibody, e.g.,
inhibits binding of PD1 by at least about 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or 100%. In one embodiment, the anti-PD-L1
antibody inhibits binding of PD1 by at least about 70%. In another
embodiment, the anti-PD-L1 antibody inhibits binding of PD1 by at
least 80%.
[0245] The term "cross-reacts," as used herein, refers to the
ability of an anti-CD27 binding domain or an anti-PD-L1 binding
domain of the invention to bind to CD27 or PD-L1, respectively,
from a different species. For example, a CD27 binding domain of the
invention that binds human CD27 may also bind another species of
CD27. Similarly, an anti-PD-L1 binding domain of the invention that
binds human PD-L1 may also bind another species of PD-L1. As used
herein, cross-reactivity is measured by detecting a specific
reactivity with purified antigen in binding assays (e.g., SPR,
ELISA) or binding to, or otherwise functionally interacting with,
cells physiologically expressing CD27. Methods for determining
cross-reactivity include standard binding assays as described
herein, for example, by Biacore.TM. surface plasmon resonance (SPR)
analysis using a Biacore.TM. 2000 SPR instrument (Biacore AB,
Uppsala, Sweden), or flow cytometric techniques.
[0246] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0247] The present invention also encompasses "conservative
sequence modifications" of any of the sequences set forth in SEQ ID
NOs: 1-160, i.e., nucleotide and amino acid sequence modifications
which do not abrogate the binding of the VH and VL sequences
encoded by the nucleotide sequence or containing the amino acid
sequence, to the antigen. Such conservative sequence modifications
include conservative nucleotide and amino acid substitutions, as
well as, nucleotide and amino acid additions and deletions. For
example, modifications can be introduced into SEQ ID NOs:1-160 by
standard techniques known in the art, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Conservative amino acid
substitutions include ones in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid residue in an anti-CD27 antibody is
preferably replaced with another amino acid residue from the same
side chain family. Methods of identifying nucleotide and amino acid
conservative substitutions which do not eliminate antigen binding
are well-known in the art (see, e.g., Brummell et al., Biochem.
32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884
(1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417
(1997)).
[0248] In certain embodiments, conservative amino acid sequence
modifications refer to at most 1, 2, 3, 4 or 5 conservative amino
acid substitutions to the CDR sequences described herein. For
example, each such CDR may contain up to 5 conservative amino acid
substitutions, e.g., up to (i.e., not more than) 4 conservative
amino acid substitutions, e.g.,up to (i.e., not more than) 3
conservative amino acid substitutions, e.g., up to (i.e., not more
than) 2 conservative amino acid substitutions, or no more than 1
conservative amino acid substitution.
[0249] Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of an anti-CD27 or anti-PD-L1
binding domain coding sequence, such as by saturation mutagenesis,
and the resulting modified anti-CD27 or anti-PD-L1 antibodies can
be screened for binding activity.
[0250] For nucleic acids, the term "substantial homology" indicates
that two nucleic acids, or designated sequences thereof, when
optimally aligned and compared, are identical, with appropriate
nucleotide insertions or deletions, in at least about 80% of the
nucleotides, usually at least about 90% to 95%, and more preferably
at least about 98% to 99.5% of the nucleotides. Alternatively,
substantial homology exists when the segments will hybridize under
selective hybridization conditions, to the complement of the
strand.
[0251] For amino acids, the term "substantial homology" indicates
that two amino acid sequences, or designated sequences thereof,
when optimally aligned and compared, are identical, with
appropriate amino acid insertions or deletions, in at least about
80% of the amino acids, usually at least about 90% to 95%, and more
preferably at least about 98% to 99% or 99.5% of the amino
acids.
[0252] The percent identity between two sequences is a function of
the number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of positions.times.100),
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences. The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm, as described in the non-limiting examples
below.
[0253] The percent identity between two nucleotide sequences can be
determined using the GAP program in the GCG software package
(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and
a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5, or 6. The percent identity between two nucleotide or amino
acid sequences can also be determined using the algorithm of E.
Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. In addition, the percent identity between two amino acid
sequences can be determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6.
[0254] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score =100, wordlength=12 to obtain nucleotide
sequences identical to the nucleic acid molecules of the invention.
BLAST protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences identical to
the protein molecules of the invention. To obtain gapped alignments
for comparison purposes, Gapped BLAST can be utilized as described
in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov.
[0255] B. Anti-CD27 Antibodies and Binding Domains Thereof
[0256] Provided herein are novel anti-CD27 antibodies and binding
domains thereof. The term "CD27" (also referred to as "CD27
molecule", "CD27L receptor", "S1521", "T cell activation antigen
CD27", "TNFRSF7," "MGC20393," "tumor necrosis factor receptor
superfamily, member 7", "T cell activation antigen S152" "Tp55",
"Tumor necrosis factor receptor superfamily member 7", "CD27
antigen", and "T-cell activation antigen CD27") refers to a
receptor that is a member of the TNF-receptor superfamily, which
binds to ligand CD70. CD27 is required for generation and long-term
maintenance of T cell immunity and plays a key role in regulating
B-cell activation and immunoglobulin synthesis. The term "CD27"
includes any variants or isoforms of CD27 which are naturally
expressed by cells (e.g., human CD27 deposited with GENBANK.RTM.
having accession no. AAH12160.1 as set forth in SEQ ID NO:173).
Accordingly, CD27 binding domains of the invention may cross-react
with CD27 from species other than human. Alternatively, the CD27
binding domains may be specific for human CD27 and may not exhibit
any cross-reactivity with other species. CD27 or any variants and
isoforms thereof, may either be isolated from cells or tissues that
naturally express them or be recombinantly produced using
well-known techniques in the art and/or those described herein.
Preferably the CD27 binding domains are targeted to human CD27,
which has a normal glycosylation pattern.
[0257] The term "CD70" (also referred to as "CD70 molecule",
"CD27L", "CD27LG", "TNFSF7," "tumor necrosis factor (ligand)
superfamily member 7," "CD27 ligand," "CD70 antigen," "surface
antigen CD70," "tumor necrosis factor ligand superfamily, member
7," "Ki-24 antigen," and "CD27-L") refers to the ligand for CD27
(see, for example, Bowman MR et al., J. Immunol. 1994 Feb. 15;
152(4):1756-61). CD70 is a type II transmembrane protein that
belongs to the tumor necrosis factor (TNF) ligand family. It is a
surface antigen on activated T and B lymphocytes that induces
proliferation of co-stimulated T cells, enhances the generation of
cytolytic T cells, and contributes to T cell activation. It has
also been suggested that CD70 plays a role in regulating B-cell
activation, cytotoxic function of natural killer cells, and
immunoglobulin synthesis (Hintzen RQ et al., J. Immunol. 1994 Feb
15;152(4):1762-73). Genbank.RTM. Accession No. NP_001243 reports
the amino acid sequence of human CD70 (SEQ ID NO:174).
[0258] An exemplary anti-CD27 antibody is antibody 3C2 as described
herein. In one embodiment, the anti-CD27 antibody or binding domain
thereof comprises the heavy and light chain CDRs or variable
regions of antibody 3C2. In another embodiment, the antibody or
binding domain thereof comprises the CDR1, CDR2, and CDR3 domains
of the heavy chain variable region of antibody 3C2 having the
sequence set forth in SEQ ID NO:17, and the CDR1, CDR2 and CDR3
domains of the light chain variable region of antibody 3C2 having
the sequence set forth in SEQ ID NO:18. In another embodiment, the
antibody or binding domain thereof comprises heavy chain CDR1, CDR2
and CDR3 domains having the sequences set forth in SEQ ID NOs:1, 2,
and 3, respectively, or conservative sequence modifications
thereof, and light chain CDR1, CDR2 and CDR3 domains having the
sequences set forth in SEQ ID NOs:4, 5, and 6, respectively, or
conservative sequence modifications thereof. In another embodiment,
the antibody or binding domain thereof comprises a heavy chain
variable region having the amino acid sequence set forth in SEQ ID
NO:17. In another embodiment, the antibody or binding domain
thereof comprises a light chain variable region having the amino
acid sequence set forth in SEQ ID NO:18. In another embodiment, the
antibody or binding domain thereof comprises heavy and light chain
variable regions having the amino acid sequences set forth in SEQ
ID NO:17 and SEQ ID NO:18, respectively.
[0259] Another exemplary anti-CD27 antibody is antibody 2B3 as
described herein. In one embodiment, the anti-CD27 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 2B3. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 2B3
having the sequence set forth in SEQ ID NO:19, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 3C2
having the sequence set forth in SEQ ID NO:20. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:7, 8, and 9, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs: 10, 11, and 12,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequences set forth in SEQ ID NO:19. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequences set forth in SEQ ID NO:20.
In another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:19 and SEQ ID NO:20,
respectively.
[0260] Sequences substantially identical to the anti-C27 binding
domains described herein (e.g., at least 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identical (identical) to the
aforementioned sequences), are also provided. In one embodiment,
the anti-CD27 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:17, SEQ ID NO: 19 or a sequence at
least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned
sequences). In another embodiment, the anti-CD27 binding domain
comprises a light chain variable region comprising SEQ ID NO:18,
SEQ ID NO:20, or a sequence at least 90% identical thereto (e.g.,
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-CD27 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-CD27 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences).
[0261] Anti-CD27 antibodies and binding domains thereof that that
compete for binding with any of the anti-CD27 antibody or binding
domain thereof described herein or that bind the same epitope as
any of the anti-CD27 antibody or binding domain thereof described
herein are also suitable for use and provided herein. For example,
in one embodiment, the anti-CD27 antibody or binding domain thereof
competes for binding to CD27 with antibody 3C2 (or an antibody
having the heavy and light chain CDRs and/or heavy and light chain
variable region sequences corresponding to antibody 3C2). In
another embodiment, the anti-CD27 antibody or binding domain
thereof competes for binding to CD27 with antibody 2B3 (or an
antibody having the heavy and light chain CDRs and/or heavy and
light chain variable region sequences corresponding to antibody
2B3). In another embodiment, the antibody or anti-CD27 binding
domain thereof binds to the same epitope on CD27 as antibody 3C2
(or an antibody having the heavy and light chain CDRs and/or heavy
and light chain variable region sequences corresponding to antibody
3C2). In another embodiment, the anti-CD27 antibody or binding
domain thereof binds to the same epitope on CD27 as antibody 2B3
(or an antibody having the heavy and light chain CDRs and/or heavy
and light chain variable region sequences corresponding to antibody
2B3).
[0262] In one embodiment, the anti-CD27 antibody, or
antigen-binding fragment thereof, comprises heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:4, 5, and 6, respectively. In another
embodiment, the anti-CD27 antibody, or antigen-binding fragment
thereof, comprises a heavy chain variable region comprising SEQ ID
NO:17 and a light chain variable region comprising SEQ ID NO:18 or
sequences at least 90% identical thereto (e.g., at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
aforementioned sequences).
[0263] In another embodiment, the anti-CD27 antibody, or
antigen-binding fragment thereof, comprises heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID
[0264] NOs:7, 8, and 9, respectively, and light chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:10, 11, and
12, respectively. In another embodiment, the anti-CD27 antibody, or
antigen-binding fragment thereof, comprises a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 or sequences at least 95% identical
thereto.
[0265] In another embodiment, the anti-CD27 antibody, or
antigen-binding fragment thereof, has one or more of the following
functional features: induces or enhances a T cell-mediated immune
response, blocks binding of sCD70 to CD27 (e.g., partially or
completely), induces NF.kappa.B activation, increases T cell
proliferation, binds to human CD27 with an equilibrium dissociation
constant Kd of 10.sup.-9 M or less, or alternatively, an
equilibrium association constant Ka of 10.sup.+9 M.sup.-1 or
greater, induces specific complement mediated cytotoxicity (CDC) of
CD27 expressing cells, induces antibody dependent cell-mediated
cytotoxicity (ADCC) specific lysis of CD27 expressing cells,
induces or enhances antigen-specific immune responses in vivo in
combination with a vaccine or endogenous antigen, induces or
enhances antigen-specific TH1 immune responses in vivo in
combination with a vaccine or endogenous antigen, induces or
enhances antigen-specific T-cell proliferation or activation in
vivo in combination with a vaccine or endogenous antigen; and/or
induces or enhances T-cell activity when combined with
simultaneous, separate or sequential TCR activation.
[0266] C. Anti-PD-L1 Antibodies and Binding Domains
[0267] Provided herein are novel anti-PD-L1 antibodies binding
domains thereof. As used herein, the terms "Programmed cell death 1
ligand 1", "PD-L1", "PDCD1 ligand 1", "Programmed death ligand 1",
"B7 homolog 1", "B7-H1" and "CD274" are used interchangeably, and
include variants, isoforms, species homologs of human PD-L1, and
analogs having at least one common epitope with PD-L1. The complete
PD-L1 sequence can be found under GenBank Accession No.
NP_001254635 as set forth in SEQ ID NO:176.
[0268] Programmed death-ligand 1 (PD-L1) is a 40 kDa type 1
transmembrane protein that has been speculated to play a major role
in suppressing the immune system during particular events such as
pregnancy, tissue allografts, autoimmune disease, and other disease
states such as hepatitis. Normally the immune system reacts to
foreign antigens that are associated with exogenous or endogenous
danger signals, which triggers a proliferation of antigen-specific
CD8+ T cells and/or CD4+ helper cells. The binding of PD-L1 to PD-1
transmits an inhibitory signal that reduces the proliferation of
these T cells and can also induce apoptosis, which is further
mediated by a lower regulation of the gene Bc1-2. As used herein,
the terms "Programmed Death 1," "Programmed Cell Death 1," "Protein
PD-1," "PD-1," PD1," "PDCD1," "hPD-1" and "hPD-I" are used
interchangeably, and include variants, isoforms, species homologs
of human PD-1, and analogs having at least one common epitope with
PD-1. The complete PD-1 sequence can be found under GenBank
Accession No. NP_005009 as set forth in SEQ ID NO:175.
[0269] PD-L1 is abundant in a variety of human cancers (Dong et al.
(2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1
results in a decrease in tumor infiltrating lymphocytes, a decrease
in T-cell receptor mediated proliferation, and immune evasion by
the cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7;
Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi
et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression
can be reversed by inhibiting the local interaction of PD-1 with
PD-L1, and the effect is additive when the interaction of PD-1 with
PD-L2 is blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad.
Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol.
170:1257-66).
[0270] An exemplary anti-PD-L1 antibody is antibody 7H7 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 7H7. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 7H7
having the sequence set forth in SEQ ID NO:77, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 7H7
having the sequence set forth in SEQ ID NO:78. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:29, 30, and 31, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:32, 33, and 34,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO:77. In another embodiment, the
antibody or binding domain thereof comprises a heavy chain variable
region having the amino acid sequence set forth in SEQ ID NO:77. In
another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:77 and SEQ ID NO:78,
respectively.
[0271] Another exemplary anti-PD-L1 antibody is antibody 1B3 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 1B3. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 1B3
having the sequence set forth in SEQ ID NO:79, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 1B3
having the sequence set forth in SEQ ID NO:80. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:35, 36, and 37, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:38, 39, and 40,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequences set forth in SEQ ID NO:79. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequences set forth in SEQ ID NO:80.
In another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:79 and SEQ ID NO:80,
respectively.
[0272] Another exemplary anti-PD-L1 antibody is antibody 3B6 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 3B6. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 3B6
having the sequence set forth in SEQ ID NO:81, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 3B6
having the sequence set forth in SEQ ID NO:82. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:41, 42, and 43, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:44, 45, and 46,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO:81. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequence set forth in SEQ ID NO:82. In
another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:81 and SEQ ID NO:82,
respectively.
[0273] Another exemplary anti-PD-L1 antibody is antibody 8B1 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 8B1. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 8B1
having the sequence set forth in SEQ ID NO:83, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 8B1
having the sequence set forth in SEQ ID NO:84. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:47, 48, and 49, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:50, 51, and 52,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequences set forth in SEQ ID NO:83. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequences set forth in SEQ ID NO:84.
In another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:83 and SEQ ID NO:84,
respectively.
[0274] Another exemplary anti-PD-L1 antibody is antibody 4A3 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 4A3. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 4A3
having the sequence set forth in SEQ ID NO:85, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 4A3
having the sequence set forth in SEQ ID NO:86. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:53, 54, and 55, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:56, 57, and 58,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO:85. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequence set forth in SEQ ID NO:86. In
another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:85 and SEQ ID NO:86,
respectively.
[0275] Another exemplary anti-PD-L1 antibody is antibody 9H9 as
described herein. In one embodiment, the anti-PD-L1 antibody or
binding domain thereof comprises the heavy and light chain CDRs or
variable regions of antibody 9H9. In another embodiment, the
antibody or binding domain thereof comprises the CDR1, CDR2, and
CDR3 domains of the heavy chain variable region of antibody 9H9
having the sequence set forth in SEQ ID NO:87, and the CDR1, CDR2
and CDR3 domains of the light chain variable region of antibody 9H9
having the sequence set forth in SEQ ID NO:88. In another
embodiment, the antibody or binding domain thereof comprises heavy
chain CDR1, CDR2 and CDR3 domains having the sequences set forth in
SEQ ID NOs:59, 60, and 61, respectively, or conservative sequence
modifications thereof, and light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs:62, 63, and 64,
respectively, or conservative sequence modifications thereof. In
another embodiment, the antibody or binding domain thereof
comprises a heavy chain variable region having the amino acid
sequence set forth in SEQ ID NO:87. In another embodiment, the
antibody or binding domain thereof comprises a light chain variable
region having the amino acid sequence set forth in SEQ ID NO:88. In
another embodiment, the antibody or binding domain thereof
comprises heavy and light chain variable regions having the amino
acid sequences set forth in SEQ ID NO:87 and SEQ ID NO:88,
respectively.
[0276] The antibody sequences can also be consensus sequences of
several antibodies. For example, in one embodiment, the anti-PD-L1
binding domain comprises a heavy chain variable region CDR1
comprising an amino acid sequence selected from the consensus
sequence: (T,S)(S,Y,H)WMS (SEQ ID NO:167). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region CDR2 comprising SEQ ID NO:168. In another embodiment, the
anti-PD-L1 binding domain comprises a heavy chain variable region
CDR3 comprising SEQ ID NO:169. In another embodiment, the
anti-PD-L1 binding domain comprises a light chain variable region
CDR1 comprising SEQ ID NO:170. In another embodiment, the
anti-PD-L1 binding domain comprises a light chain variable region
CDR2 comprising SEQ ID NO:171. In another embodiment, the
anti-PD-L1 binding domain comprises a light chain variable region
CDR3 comprising SEQ ID NO:172.
[0277] Sequences substantially identical to the anti-PD-L1
antibodies and binding domains thereof described herein (e.g., at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences), are also encompassed by
the invention. In one embodiment, the anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:77,
SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID
NO:87, or a sequence at least 90% identical thereto (e.g., at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
aforementioned sequences). In another embodiment, the anti-PD-L1
binding domain comprises a light chain variable region comprising
SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID
NO:86, SEQ ID NO:88 or a sequence at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:77 and a light chain variable region
comprising SEQ ID NO:78 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:79 and a light chain variable region
comprising SEQ ID NO:80 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:81 and a light chain variable region
comprising SEQ ID NO:82 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). In another embodiment,
the anti-PD-L1 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88 or sequences at least 90% identical thereto
(e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences). Anti-PD-L1 antibodies
and binding domains thereof that compete for binding with any of
the anti-PD-L1 antibodies or binding domains thereof as described
herein or that bind the same epitope as any of the anti-PD-L1
antibodies or binding domains thereof as described herein are also
suitable for use and provided herein. For example, in one
embodiment, the anti-PD-L1 antibody or binding domain thereof
competes for binding to PD-L1 with antibody 7H7 (or an antibody
having the heavy and light chain CDRs and/or heavy and light chain
variable region sequences corresponding to antibody 7H7). In
another embodiment, the anti-PD-L1 antibody or binding domain
thereof binds to the same epitope on PD-L1 as antibody 7H7 (or an
antibody having the heavy and light chain CDRs and/or heavy and
light chain variable region sequences corresponding to antibody
7H7).
[0278] In another embodiment, the anti-PD-L1 antibody or binding
domain thereof competes for binding to PD-L1 with antibody 1B3 (or
an antibody having the heavy and light chain CDRs and/or heavy and
light chain variable region sequences corresponding to antibody
1B3). In another embodiment, the anti-PD-L1 antibody or binding
domain thereof binds to the same epitope on PD-L1 as antibody 1B3
(or an antibody having the heavy and light chain CDRs and/or heavy
and light chain variable region sequences corresponding to antibody
1B3).
[0279] In another embodiment, the anti-PD-L1 antibody or binding
domain thereof competes for binding to PD-L1 with antibody 3B6 (or
an antibody having the heavy and light chain CDRs and/or heavy and
light chain variable region sequences corresponding to antibody
3B6). In another embodiment, the anti-PD-L1 antibody or binding
domain thereof binds to the same epitope on PD-L1 as antibody 3B6
(or an antibody having the heavy and light chain CDRs and/or heavy
and light chain variable region sequences corresponding to antibody
3B6).
[0280] In another embodiment, the anti-PD-L1 antibody or binding
domain thereof competes for binding to PD-L1 with antibody 8B1 (or
an antibody having the heavy and light chain CDRs and/or heavy and
light chain variable region sequences corresponding to antibody
8B1). In another embodiment, the anti-PD-L1 antibody or binding
domain thereof binds to the same epitope on PD-L1 as antibody 8B1
(or an antibody having the heavy and light chain
[0281] CDRs and/or heavy and light chain variable region sequences
corresponding to antibody 8B1).
[0282] In another embodiment, the anti-PD-L1 antibody or binding
domain thereof competes for binding to PD-L1 with antibody 4A3 (or
an antibody having the heavy and light chain CDRs and/or heavy and
light chain variable region sequences corresponding to antibody
4A3). In another embodiment, the anti-PD-L1 antibody or binding
domain thereof binds to the same epitope on PD-L1 as antibody 4A3
(or an antibody having the heavy and light chain CDRs and/or heavy
and light chain variable region sequences corresponding to antibody
4A3).
[0283] In another embodiment, the anti-PD-L1 antibody or binding
domain thereof competes for binding to PD-L1 with antibody 9H9 (or
an antibody having the heavy and light chain CDRs and/or heavy and
light chain variable region sequences corresponding to antibody
9H9). In another embodiment, the anti-PD-L1 antibody or binding
domain thereof binds to the same epitope on PD-L1 as antibody 9H9
(or an antibody having the heavy and light chain CDRs and/or heavy
and light chain variable region sequences corresponding to antibody
9H9).
[0284] In another embodiment, the anti-PD-L1 binding domain is an
anti-PD-L1 antibody, or antigen-binding portion thereof. In one
embodiment, the anti-PD-L1 antibody, or antigen-binding fragment
thereof, comprises heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:29, 30, and 31, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:32, 33, and 34, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:77 and a light
chain variable region comprising SEQ ID NO:78 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:35, 36, and 37, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:38, 39, and 40, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:79 and a light
chain variable region comprising SEQ ID NO:80 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:41, 42, and 43, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:44, 45, and 46, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:81 and a light
chain variable region comprising SEQ ID NO:82 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:47, 48, and 49, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:50, 51, and 52, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:83 and a light
chain variable region comprising SEQ ID NO:84 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:53, 54, and 55, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:56, 57, and 58, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:85 and a light
chain variable region comprising SEQ ID NO:86 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-PD-L1 antibody, or antigen-binding
fragment thereof, comprises heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:59, 60, and 61, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:62, 63, and 64, respectively. In another embodiment, the
anti-PD-L1 antibody, or antigen-binding fragment thereof, comprises
a heavy chain variable region comprising SEQ ID NO:87 and a light
chain variable region comprising SEQ ID NO:88 or sequences at least
90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned
sequences).
[0285] In another embodiment, the anti-PD-L1 antibody, or
antigen-binding fragment thereof, has one or more of the following
functional features: (a) blocks binding of PD1 to PD-L1 (e.g.,
partially or completely), (b) induces NFAT pathway activation,
and/or (c) induces a mixed lymphocyte reaction.
[0286] D. Bispecific Constructs
[0287] Provided herein are bispecific constructs comprising an
anti-CD27 binding domain linked to an anti-PD-L1 binding domain.
Such bispecific constructs linked to one or more additional binding
agents to form multispecific constructs also are provided.
[0288] A "bispecific" or "bifunctional" construct is an artificial
hybrid having two different binding domain (e.g., heavy/light
chain) pairs and two different binding sites. Bispecific constructs
can be produced by a variety of methods including fusion of
hybridomas or linking of Fab' fragments. See, e.g., Songsivilai
& Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et
al., J. Immunol. 148, 1547-1553 (1992).
[0289] As used herein, the term "linked" refers to the association
of two or more molecules. The linkage can be covalent or
non-covalent. The linkage also can be genetic (i.e., recombinantly
fused). Such linkages can be achieved using a wide variety of art
recognized techniques, such as chemical conjugation and recombinant
protein production.
[0290] For chemical conjugation, suitable reagents and methods are
known in the art for coupling two or more moieties, in particular
two or more antibodies, or fragments thereof, together. A variety
of coupling or crosslinking agents are commercially available and
can be used to conjugate the anti-CD27 binding domain and
anti-PD-L1 binding domain. Non-limiting examples include
Sulfo-SMCC, protein A, carboiimide, dimaleimide,
dithio-bis-nitrobenzoic acid (DTNB), and
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Sulfo-SMCC,
SPDP and DTNB are preferred agents, with Sulfo-SMCC being
particularly preferred. Other suitable procedures for crosslinking
components (e.g., binding domains) with cross-linking agents are
known in the art. See e.g., Karpovsky, B. et al., (1984) J. Exp.
Med. 160:1686; Liu, M. A. et al., (1985) Proc. Natl. Acad. Sci USA
82:8648; Segal, D. M. and Perez, P., U.S. Pat. No. 4,676,980; and
Brennan, M. (1986) Biotechniques 4:424.
[0291] For genetic engineering, nucleic acid molecules encoding the
anti-CD27 binding domain can be inserted into an appropriate
expression vector using standard recombinant DNA techniques. A
nucleic acid molecule(s) encoding the anti-PD-L1 binding domain
also can be inserted into the same expression vector, such that it
is operatively linked (e.g., in-frame cloning) to the CD27 binding
domain, thereby resulting in an expression vector that encodes a
fusion protein that is the bispecific construct. Preferably, the
anti-PD-L1 binding domain is operatively linked to the C-terminal
region of the heavy chain of the anti-CD27 binding domain. Other
suitable expression vectors and cloning strategies for preparing
the bispecific constructs described herein are known in the
art.
[0292] For expression of the bispecific constructs in host cells,
the coding regions of the binding domains are combined with cloned
promoter, leader sequence, translation initiation, leader sequence,
constant region, 3' untranslated, polyadenylation, and
transcription termination, sequences to form expression vector
constructs. These constructs can be used to express, for example,
full length human IgG.sub.1.kappa. or IgG.sub.4.kappa. antibodies.
Fully human, humanized and chimeric antibodies used in the
bispecific constructs described herein also include IgG2, IgG3,
IgE, IgA, IgM, and IgD antibodies. Similar plasmids can be
constructed for expression of other heavy chain isotypes, or for
expression of antibodies comprising lambda light chains.
[0293] Following preparation of an expression vector encoding the
bispecific construct, the bispecific construct can be expressed
recombinantly in a host cell using standard transfection methods.
For example, in one embodiment, nucleic acid encoding the
bispecific construct can be ligated into an expression vector, such
as a eukaryotic expression plasmid, such as used by GS gene
expression system disclosed in WO 87/04462, WO 89/01036 and EP 338
841 or other expression systems well known in the art. The purified
plasmid with the cloned bispecific construct gene can be introduced
in eukaryotic host cells, such as CHO-cells or NSO-cells or
alternatively other eukaryotic cells like a plant derived cells,
fungi or yeast cells. The method used to introduce these genes
could be methods described in the art, such as electroporation,
lipofectin, lipofectamine or other. After introducing the
expression vector in the host cells, cells expressing the
bispecific construct can be identified and selected. These cells
represent the transfectomas that can then be amplified for their
expression level and upscaled to produce bispecific constructs.
Alternatively these cloned bispecific constructs can be expressed
in other expression systems, such as E. coli or in complete
organisms or can be synthetically expressed. Recombinant bispecific
constructs can be isolated and purified from these culture
supernatants and/or cells.
[0294] A bispecific construct of the invention, whether prepared by
chemical conjugation or by genetic engineering, can be isolated and
purified using one or more methodologies for protein purification
well established in the art. Preferred methods for isolation and
purification include, but are not limited to, gel filtration
chromatography, affinity chromatography, anion-exchange
chromatography and the like. A particularly preferred method is gel
filtration chromatography, e.g., using a Superdex 200 column.
Isolated and purified bispecific constructs can be evaluated using
standard methods such as SDS-PAGE analysis.
[0295] Accordingly, in one embodiment, the anti-PD-L1 binding
domain and the anti-CD27 binding domain are genetically fused. In
another embodiment, the anti-PD-L1 binding domain and the anti-CD27
binding domain are chemically conjugated. In one embodiment, the
anti-PD-L1 binding domain further comprises a human IgG.sub.1
constant domain. In another embodiment, the anti-CD27 binding
domain is linked to the C-terminus of the heavy chain of the
anti-PD-L1 binding domain. In another embodiment, the anti-CD27
binding domain is a scFv. In another embodiment, the anti-CD27
binding domain further comprises a human IgG.sub.1 constant domain.
In another embodiment, the anti-PD-L1 binding domain is linked to
the C-terminus of the heavy chain of the anti-CD27 binding domain.
In another embodiment, the anti-PD-L1 binding domain is a scFv.
[0296] Exemplary bispecific constructs are set forth below in
Tables 1-2, wherein the binding domains are defined by CDR
sequences (Table 1) or variable region sequences (Table 2).
TABLE-US-00001 TABLE 1 Exemplary Bispecific Constructs (CDRs)
Bispecific CD27 Heavy Chain CD27 Light Chain PD-L1 Heavy Chain
PD-L1 Light Chain Construct Variable CDRs Variable CDRs Variable
CDRs Variable CDRs 2B3x7H7 CDR1: SEQ ID NO: 7 CDR1: SEQ ID NO: 10
CDR1: SEQ ID NO: 29 CDR1: SEQ ID NO: 32 CDR2: SEQ ID NO: 8 CDR2:
SEQ ID NO: 11 CDR2: SEQ ID NO: 30 CDR2: SEQ ID NO: 33 CDR3: SEQ ID
NO: 9 CDR3: SEQ ID NO: 12 CDR3: SEQ ID NO: 31 CDR3: SEQ ID NO: 34
2B3x1B3 CDR1: SEQ ID NO: 7 CDR1: SEQ ID NO: 10 CDR1: SEQ ID NO: 35
CDR1: SEQ ID NO: 38 CDR2: SEQ ID NO: 8 CDR2: SEQ ID NO: 11 CDR2:
SEQ ID NO: 36 CDR2: SEQ ID NO: 39 CDR3: SEQ ID NO: 9 CDR3: SEQ ID
NO: 12 CDR3: SEQ ID NO: 37 CDR3: SEQ ID NO: 40 2B3x3B6 CDR1: SEQ ID
NO: 7 CDR1: SEQ ID NO: 10 CDR1: SEQ ID NO: 41 CDR1: SEQ ID NO: 44
CDR2: SEQ ID NO: 8 CDR2: SEQ ID NO: 11 CDR2: SEQ ID NO: 42 CDR2:
SEQ ID NO: 45 CDR3: SEQ ID NO: 9 CDR3: SEQ ID NO: 12 CDR3: SEQ ID
NO: 43 CDR3: SEQ ID NO: 46 2B3x8B1 CDR1: SEQ ID NO: 7 CDR1: SEQ ID
NO: 10 CDR1: SEQ ID NO: 47 CDR1: SEQ ID NO: 50 CDR2: SEQ ID NO: 8
CDR2: SEQ ID NO: 11 CDR2: SEQ ID NO: 48 CDR2: SEQ ID NO: 51 CDR3:
SEQ ID NO: 9 CDR3: SEQ ID NO: 12 CDR3: SEQ ID NO: 49 CDR3: SEQ ID
NO: 52 2B3x4A3 CDR1: SEQ ID NO: 7 CDR1: SEQ ID NO: 10 CDR1: SEQ ID
NO: 53 CDR1: SEQ ID NO: 56 CDR2: SEQ ID NO: 8 CDR2: SEQ ID NO: 11
CDR2: SEQ ID NO: 54 CDR2: SEQ ID NO: 57 CDR3: SEQ ID NO: 9 CDR3:
SEQ ID NO: 12 CDR3: SEQ ID NO: 55 CDR3: SEQ ID NO: 58 2B3x9H9 CDR1:
SEQ ID NO: 7 CDR1: SEQ ID NO: 10 CDR1: SEQ ID NO: 59 CDR1: SEQ ID
NO: 62 CDR2: SEQ ID NO: 8 CDR2: SEQ ID NO: 11 CDR2: SEQ ID NO: 60
CDR2: SEQ ID NO: 63 CDR3: SEQ ID NO: 9 CDR3: SEQ ID NO: 12 CDR3:
SEQ ID NO: 61 CDR3: SEQ ID NO: 64 3C2x7H7 CDR1: SEQ ID NO: 1 CDR1:
SEQ ID NO: 4 CDR1: SEQ ID NO: 29 CDR1: SEQ ID NO: 32 CDR2: SEQ ID
NO: 2 CDR2: SEQ ID NO: 5 CDR2: SEQ ID NO: 30 CDR2: SEQ ID NO: 33
CDR3: SEQ ID NO: 3 CDR3: SEQ ID NO: 6 CDR3: SEQ ID NO: 31 CDR3: SEQ
ID NO: 34 3C2x1B3 CDR1: SEQ ID NO: 1 CDR1: SEQ ID NO: 4 CDR1: SEQ
ID NO: 35 CDR1: SEQ ID NO: 38 CDR2: SEQ ID NO: 2 CDR2: SEQ ID NO: 5
CDR2: SEQ ID NO: 36 CDR2: SEQ ID NO: 39 CDR3: SEQ ID NO: 3 CDR3:
SEQ ID NO: 6 CDR3: SEQ ID NO: 37 CDR3: SEQ ID NO: 40 3C2x3B6 CDR1:
SEQ ID NO: 1 CDR1: SEQ ID NO: 4 CDR1: SEQ ID NO: 41 CDR1: SEQ ID
NO: 44 CDR2: SEQ ID NO: 2 CDR2: SEQ ID NO: 5 CDR2: SEQ ID NO: 42
CDR2: SEQ ID NO: 45 CDR3: SEQ ID NO: 3 CDR3: SEQ ID NO: 6 CDR3: SEQ
ID NO: 43 CDR3: SEQ ID NO: 46 3C2x8B1 CDR1: SEQ ID NO: 1 CDR1: SEQ
ID NO: 4 CDR1: SEQ ID NO: 47 CDR1: SEQ ID NO: 50 CDR2: SEQ ID NO: 2
CDR2: SEQ ID NO: 5 CDR2: SEQ ID NO: 48 CDR2: SEQ ID NO: 51 CDR3:
SEQ ID NO: 3 CDR3: SEQ ID NO: 6 CDR3: SEQ ID NO: 49 CDR3: SEQ ID
NO: 52 3C2x4A3 CDR1: SEQ ID NO: 1 CDR1: SEQ ID NO: 4 CDR1: SEQ ID
NO: 53 CDR1: SEQ ID NO: 56 CDR2: SEQ ID NO: 2 CDR2: SEQ ID NO: 5
CDR2: SEQ ID NO: 54 CDR2: SEQ ID NO: 57 CDR3: SEQ ID NO: 3 CDR3:
SEQ ID NO: 6 CDR3: SEQ ID NO: 55 CDR3: SEQ ID NO: 58 3C2x9H9 CDR1:
SEQ ID NO: 1 CDR1: SEQ ID NO: 4 CDR1: SEQ ID NO: 59 CDR1: SEQ ID
NO: 62 CDR2: SEQ ID NO: 2 CDR2: SEQ ID NO: 5 CDR2: SEQ ID NO: 60
CDR2: SEQ ID NO: 63 CDR3: SEQ ID NO: 3 CDR3: SEQ ID NO: 6 CDR3: SEQ
ID NO: 61 CDR3: SEQ ID NO: 64
TABLE-US-00002 TABLE 2 Exemplary Bispecific Constructs (VRs) CD27
CD27 PD-L1 PD-L1 Bispecific Heavy Chain Light Chain Heavy Chain
Light Chain Constructs Variable Region Variable Region Variable
Region Variable Region 2B3x7H7 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID
NO: 77 SEQ ID NO: 78 2B3x1B3 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO:
79 SEQ ID NO: 80 2B3x3B6 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 81
SEQ ID NO: 82 2B3x8B1 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 83 SEQ
ID NO: 84 2B3x4A3 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 85 SEQ ID
NO: 86 2B3x9H9 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 87 SEQ ID NO:
88 3C2x7H7 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 77 SEQ ID NO: 78
3C2x1B3 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 79 SEQ ID NO: 80
3C2x3B6 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 81 SEQ ID NO: 82
3C2x8B1 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 83 SEQ ID NO: 84
3C2x4A3 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 85 SEQ ID NO: 86
3C2x9H9 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 87 SEQ ID NO: 88
[0297] In one embodiment, a bispecific construct comprising an
anti-CD27 binding domain linked to an anti-PD-L1 binding domain is
provided, wherein: [0298] (i) the anti-CD27 binding domain
comprises: [0299] a. heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:1, 2, and 3, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or [0300] b. heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively; and [0301]
(ii) the anti-PD-L1 binding domain comprises: [0302] a. a heavy
chain variable region CDR1 comprising an amino acid sequence
selected from the consensus sequence: (T,S)(S,Y,H)WMS (SEQ ID
NO:167); [0303] b. a heavy chain variable region CDR2 comprising
SEQ ID NO:168; [0304] c. a heavy chain variable region CDR3
comprising SEQ ID NO:169; [0305] d. a light chain variable region
CDR1 comprising SEQ ID NO:170; [0306] e. a light chain variable
region CDR2 comprising SEQ ID NO:171; and [0307] f. a light chain
variable region CDR3 comprising SEQ ID NO:172.
[0308] In another embodiment, a bispecific construct comprising an
anti-CD27 binding domain linked to an anti-PD-L1 binding domain is
provided, wherein: [0309] (i) the anti-CD27 binding domain
comprises: [0310] a. heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:1, 2, and 3, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:4, 5, and 6, respectively, or [0311] b. heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively; and [0312]
(ii) the anti-PD-L1 binding domain comprises: [0313] a. a heavy
chain variable region comprising SEQ ID NO:77 and a light chain
variable region comprising SEQ ID NO:78 or sequences at least 95%
identical thereto (e.g., at least 95%, 96%, 97%, 98% or 99%
identical to the aforementioned sequences); [0314] b. a heavy chain
variable region comprising SEQ ID NO:79 and a light chain variable
region comprising SEQ ID NO:80 or sequences at least 95% identical
thereto (e.g., at least 95%, 96%, 97%, 98% or 99% identical to the
aforementioned sequences); [0315] c. a heavy chain variable region
comprising SEQ ID NO:81 and a light chain variable region
comprising SEQ ID NO:82 or sequences at least 95% identical thereto
(e.g., at least 95%, 96%, 97%, 98% or 99% identical to the
aforementioned sequences); [0316] d. a heavy chain variable region
comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84 or sequences at least 95% identical thereto
(e.g., at least 95%, 96%, 97%, 98% or 99% identical to the
aforementioned sequences); [0317] e. a heavy chain variable region
comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86 or sequences at least 95% identical thereto
(e.g., at least 95%, 96%, 97%, 98% or 99% identical to the
aforementioned sequences); or [0318] f. a heavy chain variable
region comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88 or sequences at least 95% identical thereto
(e.g., at least 95%, 96%, 97%, 98% or 99% identical to the
aforementioned sequences).
[0319] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ
[0320] ID NOs:1, 2, and 3, respectively, and light chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4, 5, and 6,
respectively and (b) an anti-PD-L1 binding domain comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:29, 30, and 31, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 32, 33, and 34,
respectively.
[0321] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78.
[0322] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1,2, and 3,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:4, 5, and 6, respectively and (b) an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:35, 36, and 37,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs: 38, 39, and 40, respectively.
[0323] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80.
[0324] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:4, 5, and 6, respectively and (b) an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:41, 42, and 43,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:44, 45, and 46, respectively.
[0325] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:81
and a light chain variable region comprising SEQ ID NO:82.
[0326] In another embodiment, the bispecific construct comprises
(a) an the anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:4, 5, and 6, respectively and (b) an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:47, 48, and 49,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:50, 51, and 52, respectively.
[0327] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:83
and a light chain variable region comprising SEQ ID NO:84.
[0328] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:4, 5, and 6, respectively; and (b) an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:53, 54, and 55,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:56, 57, and 58, respectively.
[0329] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:85
and a light chain variable region comprising SEQ ID NO:86.
[0330] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:1, 2, and 3,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:4, 5, and 6, respectively and an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:59, 60, and 61,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs: 62, 63, and 64, respectively.
[0331] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:87
and a light chain variable region comprising SEQ ID NO:88.
[0332] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively and (b) an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:29, 30, and 31,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:32, 33, and 34, respectively.
[0333] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78.
[0334] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively and an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:35, 36, and 37,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:38, 39, and 40, respectively.
[0335] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) and anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80.
[0336] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively and an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID
[0337] NOs:41, 42, and 43, respectively, and light chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44, 45, and
46, respectively.
[0338] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprises a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and an anti-PD-L1 binding domain comprising
a heavy chain variable region comprising SEQ ID NO:81 and a light
chain variable region comprising SEQ ID NO:82.
[0339] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively and (b) an
anti-PD-L1 binding domain comprising heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:47, 48, and 49,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:50, 51, and 52, respectively.
[0340] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) an anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:83 and
a light chain variable region comprising SEQ ID NO:84.
[0341] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively and (b) an
anti-PD-L1 binding domain comprising a heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:53, 54, and 55,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:56, 57, and 58, respectively.
[0342] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) an anti-PD-L1 binding domain
comprising a heavy chain variable region comprising SEQ ID NO:85
and a light chain variable region comprising SEQ ID NO:86.
[0343] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively and (b) an
anti-PD-L1 binding domain comprising a heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:59, 60, and 61,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:62, 63, and 64, respectively.
[0344] In another embodiment, the bispecific construct comprises
(a) an anti-CD27 binding domain comprising a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20 and (b) an anti-PD-L1 binding domain
comprises a heavy chain variable region comprising SEQ ID NO:87 and
a light chain variable region comprising SEQ ID NO:88.
[0345] In a particular embodiment, the bispecific construct
comprises an anti-PD-L1 antibody linked to an anti-CD27 scFv,
wherein: [0346] (i) the anti-CD27 scFv comprises: [0347] a. heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:1, 2, and 3, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4, 5, and 6,
respectively, or [0348] b. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively; and [0349] (ii) the anti-PD-L1
antibody comprises: [0350] a. heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs: 29, 30, and 31,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:32, 33, and 34, respectively; [0351] b.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs: 35, 36, and 37, respectively, and light chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and
40, respectively; [0352] c. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 41, 42, and 43, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:44, 45, and 46, respectively; [0353] d. heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 47,
48, and 49, respectively, and light chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively; [0354] e. heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs: 53, 54, and 55, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:56, 57, and 58, respectively; or [0355] f. heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 59,
60, and 61, respectively, and light chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:62, 63, and 64,
respectively; and [0356] g. a human IgG.sub.1 constant domain.
[0357] In another particular embodiment, the bispecific construct
comprises an anti-CD27 antibody linked to an anti-PD-L1 scFv,
wherein: [0358] (i) the anti-CD27 antibody comprises: [0359] a.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:1, 2, and 3, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:4, 5, and 6,
respectively, or [0360] b. heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:10, 11, and 12, respectively; and [0361] c. a human
IgG.sub.1 constant domain; and [0362] (ii) the anti-PD-L1 scFv
comprises: [0363] a. heavy chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs: 29, 30, and 31, respectively, and
light chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:32, 33, and 34, respectively; [0364] b. heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 35, 36, and
37, respectively, and light chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:38, 39, and 40, respectively;
[0365] c. heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs: 41, 42, and 43, respectively, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:44,
45, and 46, respectively; [0366] d. heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:50, 51, and 52, respectively; [0367] e.
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs: 53, 54, and 55, respectively, and light chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:56, 57, and
58, respectively; or [0368] f. heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs: 59, 60, and 61,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:62, 63, and 64, respectively.
[0369] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0370]
(i) the anti-CD27 scFv comprises: [0371] a. a heavy chain variable
region comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18; or [0372] b. a heavy chain variable region
comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and [0373] (ii) the anti-PD-L1 antibody
comprises: [0374] a. a heavy chain variable region comprising SEQ
ID NO:77 and a light chain variable region comprising SEQ ID NO:78;
[0375] b. a heavy chain variable region comprising SEQ ID NO:79 and
a light chain variable region comprising SEQ ID NO:80; [0376] c. a
heavy chain variable region comprising SEQ ID NO:81 and a light
chain variable region comprising SEQ ID NO:82; [0377] d. a heavy
chain variable region comprising SEQ ID NO:83 and a light chain
variable region comprising SEQ ID NO:84; [0378] e. a heavy chain
variable region comprising SEQ ID NO:85 and a light chain variable
region comprising SEQ ID NO:86; or [0379] f. a heavy chain variable
region comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88; and [0380] g. a human IgG.sub.1 constant
domain.
[0381] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0382]
(i) the anti-CD27 antibody comprises: [0383] a. a heavy chain
variable region comprising SEQ ID NO:17 and a light chain variable
region comprising SEQ ID NO:18; or [0384] b. a heavy chain variable
region comprising SEQ ID NO:19 and a light chain variable region
comprising SEQ ID NO:20; and [0385] c. a human IgG.sub.1 constant
domain; and [0386] (ii) the anti-PD-L1 scFv comprises: [0387] a. a
heavy chain variable region comprising SEQ ID NO:77 and a light
chain variable region comprising SEQ ID NO:78; [0388] b. a heavy
chain variable region comprising SEQ ID NO:79 and a light chain
variable region comprising SEQ ID NO:80; [0389] c. a heavy chain
variable region comprising SEQ ID NO:81 and a light chain variable
region comprising SEQ ID NO:82; [0390] d. a heavy chain variable
region comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84; [0391] e. a heavy chain variable region
comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86; or [0392] f. a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88.
[0393] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0394]
(i) the anti-CD27 scFv comprises heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:10, 11, and 12, respectively; and [0395] (ii) the
anti-PD-L1 antibody comprises a heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs: 47, 48, and 49,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:50, 51, and 52, respectively, and a
human IgG.sub.1 constant domain.
[0396] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0397]
(i) the anti-CD27 antibody comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively, and a
human IgG.sub.1 constant domain; and [0398] (ii) the anti-PD-L1
scFv comprises a heavy chain variable region CDR1, CDR2 and CDR3 as
set forth in SEQ ID NOs: 47, 48, and 49, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:50, 51, and 52, respectively.
[0399] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0400]
(i) the anti-CD27 scFv comprises a heavy chain variable region
comprising SEQ ID NO:19, a light chain variable region comprising
SEQ ID NO:20; and [0401] (ii) the anti-PD-L1 antibody comprises a
heavy chain variable region comprising SEQ ID NO:83 and a light
chain variable region comprising SEQ ID NO:84, and a human
IgG.sub.1 constant domain.
[0402] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0403]
(i) the anti-CD27 antibody comprises a heavy chain variable region
comprising SEQ ID NO:19, a light chain variable region comprising
SEQ ID NO:20, and a human IgG.sub.1 constant domain; and [0404]
(ii) the anti-PD-L1 scFv comprises a heavy chain variable region
comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84.
[0405] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0406]
(i) the anti-CD27 scFv comprises heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:10, 11, and 12, respectively; and [0407] (ii) the
anti-PD-L1 antibody comprises heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:59, 60, and 61,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:62, 63, and 64, respectively, and a
human IgG.sub.1 constant domain.
[0408] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0409]
(i) the anti-CD27 antibody comprises heavy chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9,
respectively, and light chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:10, 11, and 12, respectively, and a
human IgG.sub.1 constant domain; and [0410] (ii) the anti-PD-L1
scFv comprises heavy chain variable region CDR1, CDR2 and CDR3 as
set forth in SEQ ID NOs:59, 60, and 61, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:62, 63, and 64, respectively.
[0411] In another embodiment, the bispecific construct comprises an
anti-PD-L1 antibody linked to an anti-CD27 scFv, wherein: [0412]
(i) the anti-CD27 scFv comprises a heavy chain variable region
comprising SEQ ID NO:19, a light chain variable region comprising
SEQ ID NO:20; and [0413] (ii) the anti-PD-L1 antibody comprises a
heavy chain variable region comprising SEQ ID NO:87 and a light
chain variable region comprising SEQ ID NO:88, and a human
IgG.sub.1 constant domain.
[0414] In another embodiment, the bispecific construct comprises an
anti-CD27 antibody linked to an anti-PD-L1 scFv, wherein: [0415]
(i) the anti-CD27 antibody comprises a heavy chain variable region
comprising SEQ ID NO:19, a light chain variable region comprising
SEQ ID NO:20, and a human IgG.sub.1 constant domain; and [0416]
(ii) the anti-PD-L1 scFv comprises a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88.
[0417] In another aspect, the bispecific construct is provided,
wherein the bispecific construct comprises any one of the anti-CD27
antibodies, or antigen binding fragments thereof, described herein
linked to an anti-PD-L1 binding domain. In one embodiment, the
anti-PD-L1 binding domain is selected from the group consisting of:
[0418] a. an anti-PD-L1 antibody, or antigen binding fragment
thereof, comprising heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs: 29, 30, and 31, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:32, 33, and 34, respectively; [0419] b. an anti-PD-L1 antibody,
or antigen binding fragment thereof, comprising heavy chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 35,
36, and 37, respectively, and light chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and 40,
respectively; [0420] c. an anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 41, 42, and 43, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:44, 45, and 46, respectively; [0421] d. an anti-PD-L1
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs: 47, 48, and 49, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively; [0422] e. anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 53, 54, and 55, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:56, 57, and 58, respectively; and [0423] f. anti-PD-L1
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs: 59, 60, and 61, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:62, 63, and 64,
respectively.
[0424] In another embodiment, the anti-PD-L1 binding domain is
selected from the group consisting of: (a) a heavy chain variable
region comprising SEQ ID NO:77 and a light chain variable region
comprising SEQ ID NO:78; (b) a heavy chain variable region
comprising SEQ ID NO:79 and a light chain variable region
comprising SEQ ID NO:80; (c) a heavy chain variable region
comprising SEQ ID NO:81 and a light chain variable region
comprising SEQ ID NO:82; (d) a heavy chain variable region
comprising SEQ ID NO:83 and a light chain variable region
comprising SEQ ID NO:84; (e) a heavy chain variable region
comprising SEQ ID NO:85 and a light chain variable region
comprising SEQ ID NO:86; and (f) a heavy chain variable region
comprising SEQ ID NO:87 and a light chain variable region
comprising SEQ ID NO:88. In a particular embodiment, the anti-PD-L1
binding domain further comprises a human IgG.sub.1 constant domain.
In another embodiment, the anti-PD-L1 binding domain is an
scFv.
[0425] In another aspect, a bispecific construct is provided,
wherein the bispecific construct comprises any one of the
anti-PD-L1 antibodies, or antigen binding fragments thereof,
described herein, linked to an anti-CD27 binding domain. In one
embodiment, the anti-C27 binding domain comprises an anti-CD27
antibody comprising heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:4, 5, and 6, respectively,. In another embodiment, the
anti-CD27 binding domain comprises an antibody comprising a heavy
chain variable region comprising SEQ ID NO:17 and a light chain
variable region comprising SEQ ID NO:18, or sequences at least 90%
identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to the aforementioned sequences). In
another embodiment, the anti-C27 binding domain comprises an
anti-CD27 antibody comprising heavy chain variable region CDR1,
CDR2 and CDR3 as set forth in SEQ ID NOs:7, 8, and 9, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:10, 11, and 12, respectively. In another embodiment, the
anti-C27 binding domain comprises an anti-CD27 antibody comprising
a heavy chain variable region comprising SEQ ID NO:19 and a light
chain variable region comprising SEQ ID NO:20, or sequences at
least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the aforementioned
sequences). In one embodiment, the anti-CD27 binding domain is an
scFv. In another embodiment, the anti-CD27 binding domain further
comprises a human IgG.sub.1 constant domain.
[0426] In another embodiment, the bispecific construct has one or
more of the following functional features: induces NF.kappa.B
activation, increases T cell proliferation, induces a CD8 T cell
response, and/or increases IL-2 production. In another embodiment,
the bispecific construct increases IL-2 production by at least
about 1.5-fold (e.g., at least 2-fold, 2.5 fold, 3-fold, 3.5 fold,
or 4-fold) compared to an anti-CD27 monoclonal antibody or
anti-PD-L1 monoclonal antibody alone. In another embodiment, the
bispecific construct induces a CD8 T cell response by at least
about 2-fold greater (e.g., at least 2-fold, 2.5 fold, 3-fold, 3.5
fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold,
7.5-fold, 8.0-fold, 8.5-fold, or 9-fold) than an anti-CD27
monoclonal antibody alone. In another embodiment, the bispecific
construct increases survival by at least about 1.5-fold longer
(e.g., at least 1.5-fold, 2.0-fold, 2.5 fold, 3-fold, 3.5 fold,
4-fold, 4.5-fold, or 5-fold) compared to an anti-CD27 monoclonal
antibody or anti-PD-L1 monoclonal antibody alone. In another
embodiment, the bispecific construct decreases tumor weight by at
least about 1.5-fold (e.g., at least 1.5-fold, 2.0-fold, 2.5 fold,
3-fold, 3.5 fold, 4-fold, 4.5-fold, or 5-fold) compared to
anti-CD27 monoclonal antibodies or anti-PD-L1 monoclonal antibodies
alone or in combination. In another embodiment, the bispecific
construct increases T cell production by at least about 1.5-fold
(e.g., at least 1.5-fold, 2.0-fold, 2.5 fold, 3-fold, 3.5 fold,
4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold,
7.5-fold, 8.0-fold, 8.5-fold, or 9-fold) compared to anti-CD27
monoclonal antibodies or anti-PD-L1 monoclonal antibodies alone or
in combination.
[0427] In certain embodiments, the bispecific constructs described
herein exhibit synergistic effects (e.g., in enhancing immune
responses in vivo) as compared to use of anti-CD27 binding domains
and anti-PD-L1 binding domains in combination (i.e.,
co-administration of unlinked antibodies).
[0428] E. Compositions
[0429] Also provided herein are compositions, e.g., a composition
comprising one or a combination of any of the binding domains,
antibodies, or antigen binding fragments thereof, the bispecific
constructs, or the multispecific constructs described herein,
formulated together with a carrier (e.g., a pharmaceutically
acceptable carrier).
[0430] As used herein, the terms "carrier" and "pharmaceutically
acceptable carrier" includes any and all solvents, salts,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like that are
physiologically compatible. Preferably, the carrier is suitable for
intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal administration (e.g., by injection or infusion).
Depending on the route of administration, the active compound
(i.e., any of the binding domains, antibodies, or antigen binding
fragments thereof, the bispecific constructs, or the multispecific
constructs described herein), may be coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0431] Examples of adjuvants which may be used with the binding
domains, antibodies, or antigen binding fragments thereof, the
bispecific constructs, or the multispecific constructs described
here include, but are not limited to : Freund's Incomplete Adjuvant
and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck
Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2
(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as
aluminum hydroxide gel (alum) or aluminum phosphate; salts of
calcium, iron or zinc; an insoluble suspension of acylated
tyrosine; acylated sugars; cationically or anionically derivatised
polysaccharides; polyphosphazenes; biodegradable microspheres;
cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like
factors; 3D-MPL; CpG oligonucleotide; and monophosphoryl lipid A,
for example 3-de-O-acylated monophosphoryl lipid A.
[0432] MPL adjuvants are available from Corixa Corporation
(Seattle, Wash; see, for example, U.S. Pat. Nos. 4,436,727;
4,877,611; 4,866,034 and 4,912,094). CpG-containing
oligonucleotides (in which the CpG dinucleotide is unmethylated)
are well known and are described, for example, in WO 96/02555, WO
99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.
Immunostimulatory DNA sequences are also described, for example, by
Sato et al., Science 273:352, 1996.
[0433] Further alternative adjuvants include, for example,
saponins, such as Quil A, or derivatives thereof, including QS21
and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin;
Digitonin; or Gypsophila or Chenopodium quinoa saponins; Montanide
ISA 720 (Seppic, France); SAF (Chiron, California, United States);
ISCOMS (CSL), MF-59 (Chiron); the SBAS series of adjuvants (e.g.,
SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart,
Belgium); Detox (Enhanzyn.TM.) (Corixa, Hamilton, Mont.); RC-529
(Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide
4-phosphates (AGPs); polyoxyethylene ether adjuvants such as those
described in WO 99/52549A1; synthetic imidazoquinolines such as
imiquimod [S-26308, R-837], (Harrison, et al., Vaccine 19:
1820-1826, 2001; and resiquimod [S-28463, R-848] (Vasilakos, et
al., Cellular immunology 204: 64-74, 2000; Schiff bases of
carbonyls and amines that are constitutively expressed on antigen
presenting cell and T-cell surfaces, such as tucaresol (Rhodes, J.
et al., Nature 377: 71-75, 1995); cytokine, chemokine and
co-stimulatory molecules as either protein or peptide, including
for example pro-inflammatory cytokines such as Interferon, GM-CSF,
IL-1 alpha, IL-1 beta, TGF-alpha and TGF-beta, Th1 inducers such as
interferon gamma, IL-2, IL-12, IL-15, IL-18 and IL-21, Th2 inducers
such as IL-4, IL-5, IL-6, IL-10 and IL-13 and other chemokine and
co-stimulatory genes such as MCP-1, MIP-1 alpha, MIP-1 beta,
RANTES,
[0434] TCA-3, CD80, CD86 and CD4OL; immunostimulatory agents
targeting ligands such as CTLA-4 and L-selectin, apoptosis
stimulating proteins and peptides such as Fas; synthetic lipid
based adjuvants, such as vaxfectin, (Reyes et al., Vaccine 19:
3778-3786, 2001) squalene, alpha-tocopherol, polysorbate 80, DOPC
and cholesterol; endotoxin, [LPS], (Beutler, B., Current Opinion in
Microbiology 3: 23-30, 2000); ligands that trigger Toll receptors
to produce Thl-inducing cytokines, such as synthetic Mycobacterial
lipoproteins, Mycobacterial protein p19, peptidoglycan, teichoic
acid and lipid A; and CT (cholera toxin, subunits A and B) and LT
(heat labile enterotoxin from E. coli, subunits A and B), heat
shock protein family (HSPs), and LLO (listeriolysin 0; WO
01/72329). These and various further Toll-like Receptor (TLR)
agonists are described for example in Kanzler et al, Nature
Medicine, May 2007, Vol 13, No 5.
[0435] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0436] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. The active compounds can
be prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J.R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0437] To administer a compound of the invention by certain routes
of administration, it may be necessary to coat the compound with,
or co-administer the compound with, a material to prevent its
inactivation. For example, the compound may be administered to a
subject in an appropriate carrier, for example, liposomes, or a
diluent. Acceptable diluents include saline and aqueous buffer
solutions. Liposomes include water-in-oil-in-water CGF emulsions as
well as conventional liposomes (Strejan et al. (1984) J.
Neuroimmunol. 7:27).
[0438] Carriers include sterile aqueous solutions or dispersions
and sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media and
agents for pharmaceutically active substances is known in the art.
Except insofar as any conventional media or agent is incompatible
with the active compound, use thereof in the pharmaceutical
compositions of the invention is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0439] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0440] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0441] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. For example, the antibodies of the invention may be
administered once or twice weekly by subcutaneous or intramuscular
injection or once or twice monthly by subcutaneous or intramuscular
injection.
[0442] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0443] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0444] For the therapeutic compositions, formulations of the
present invention include those suitable for oral, nasal, topical
(including buccal and sublingual), rectal, vaginal and/or
parenteral administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods
known in the art of pharmacy. The amount of active ingredient which
can be combined with a carrier material to produce a single dosage
form will vary depending upon the subject being treated, and the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the composition which
produces a therapeutic effect. Generally, out of one hundred per
cent, this amount will range from about 0.001 per cent to about
ninety percent of active ingredient, preferably from about 0.005
per cent to about 70 per cent, most preferably from about 0.01 per
cent to about 30 per cent.
[0445] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate. Dosage forms for the
topical or transdermal administration of compositions of this
invention include powders, sprays, ointments, pastes, creams,
lotions, gels, solutions, patches and inhalants. The active
compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0446] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0447] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0448] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents that delay absorption such
as aluminum monostearate and gelatin.
[0449] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given alone
or as a pharmaceutical composition containing, for example, 0.001
to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) of
active ingredient in combination with a pharmaceutically acceptable
carrier.
[0450] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0451] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient that is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts. A physician or veterinarian having ordinary skill in
the art can readily determine and prescribe the effective amount of
the pharmaceutical composition required. For example, the physician
or veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved. In general, a suitable daily dose of a composition of the
invention will be that amount of the compound that is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above. It is
preferred that administration be intravenous, intramuscular,
intraperitoneal, or subcutaneous, preferably administered proximal
to the site of the target. If desired, the effective daily dose of
a therapeutic composition may be administered as two, three, four,
five, six or more sub-doses administered separately at appropriate
intervals throughout the day, optionally, in unit dosage forms.
While it is possible for a compound of the present invention to be
administered alone, it is preferable to administer the compound as
a pharmaceutical formulation (composition).
[0452] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335,
5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Patent No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Patent No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Patent No.
4,447,233, which discloses a medication infusion pump for
delivering medication at a precise infusion rate; U.S. Patent No.
4,447,224, which discloses a variable flow implantable infusion
apparatus for continuous drug delivery; U.S. Patent No. 4,439,196,
which discloses an osmotic drug delivery system having
multi-chamber compartments; and U.S. Patent No. 4,475,196, which
discloses an osmotic drug delivery system. Many other such
implants, delivery systems, and modules are known to those skilled
in the art.
[0453] In certain embodiments, the antibodies of the invention can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention cross the BBB (if desired), they can be formulated, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties that are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233:134), different species of which may comprise the
formulations of the inventions, as well as components of the
invented molecules; p120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In
one embodiment of the invention, the therapeutic compounds of the
invention are formulated in liposomes; in a more preferred
embodiment, the liposomes include a targeting moiety. In a most
preferred embodiment, the therapeutic compounds in the liposomes
are delivered by bolus injection to a site proximal to the tumor or
infection. The composition must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and
fungi.
[0454] The ability of a compound to inhibit cancer can be evaluated
in an animal model system predictive of efficacy in human tumors.
Alternatively, this property of a composition can be evaluated by
examining the ability of the compound to inhibit, such inhibition
in vitro by assays known to the skilled practitioner. A
therapeutically effective amount of a therapeutic compound can
decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity
of the subject's symptoms, and the particular composition or route
of administration selected.
[0455] The composition must be sterile and fluid to the extent that
the composition is deliverable by syringe. In addition to water,
the carrier can be an isotonic buffered saline solution, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), and suitable mixtures thereof.
Proper fluidity can be maintained, for example, by use of coating
such as lecithin, by maintenance of required particle size in the
case of dispersion and by use of surfactants. In many cases, it is
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition. Long-term absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0456] When the active compound is suitably protected, as described
above, the compound may be orally administered, for example, with
an inert diluent or an assimilable edible carrier.
[0457] F. Nucleic Acids
[0458] The term "nucleic acid molecule," as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0459] The term "isolated nucleic acid molecule," as used herein in
reference to nucleic acids encoding binding domains, antibodies, or
antibody portions (e.g., V.sub.H, V.sub.L, CDR3) that bind to CD27
and/or PD-L1, is intended to refer to a nucleic acid molecule in
which the nucleotide sequences encoding the binding domain,
antibodies, or antibody portions are free of other nucleotide
sequences encoding the binding domain, antibodies, or antibody
portions that bind antigens other than CD27 and/or PD-L1, which
other sequences may naturally flank the nucleic acid in human
genomic DNA.
[0460] The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form. A
nucleic acid is "isolated" or "rendered substantially pure" when
purified away from other cellular components or other contaminants,
e.g., other cellular nucleic acids or proteins, by standard
techniques, including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York (1987).
[0461] The nucleic acid molecules of the present invention, while
often in a native sequence (except for modified restriction sites
and the like), from either cDNA, genomic or mixtures thereof may be
mutated, in accordance with standard techniques to provide gene
sequences.
[0462] For coding sequences, these mutations, may affect amino acid
sequence as desired. In particular, DNA sequences substantially
identical to or derived from native V, D, J, constant, switches and
other such sequences described herein are contemplated (where
"derived" indicates that a sequence is identical or modified from
another sequence).
[0463] A nucleic acid is "operably linked" or "operatively linked"
when it is placed into a functional relationship with another
nucleic acid sequence. For instance, a promoter or enhancer is
operably linked to a coding sequence if it affects the
transcription of the sequence. With respect to transcription
regulatory sequences, operably linked means that the DNA sequences
being linked are contiguous and, where necessary to join two
protein coding regions, contiguous and in reading frame. For switch
sequences, operably linked indicates that the sequences are capable
of effecting switch recombination.
[0464] Isolated nucleic acid molecules encoding the binding
domains, antibodies, or antigen-binding portions thereof,
bispecific constructs, and multispecific constructs described
herein are also provided, as well as expression vectors comprising
such nucleic acids and host cells comprising such expression
vectors. In another embodiment, a nucleic acid molecule coding for
any of the binding domains, antibodies, or antigen-binding portions
thereof, bispecific constructs, or multispecific constructs
described herein is provided. In another embodiment, the nucleic
acid molecule is in the form of an expression vector. In another
embodiment, the nucleic acid molecule is in the form of an
expression vector which expresses the binding domain, antibody, or
antigen-binding portion thereof, bispecific construct, or the
multispecific construct when administered to a subject in vivo.
[0465] In one embodiment, the nucleic acid molecule comprises a
nucleotide sequence encoding an antibody variable region, wherein
the antibody variable region comprises the amino acid sequence
depicted in SEQ ID NO:17, 18, 19, 20, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, or an amino acid sequence at least 90%
identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to one or more of the aforementioned
sequences). In another embodiment, the nucleic acid molecule
comprises a nucleotide sequence as set forth in SEQ ID NO:25, 26,
27, 28, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
or a nucleotide sequence at least 90% identical thereto (e.g., at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical
to one or more of the aforementioned sequences).
[0466] In another embodiment, the nucleic acid molecule comprises a
nucleotide sequence encoding heavy and light chain variable regions
of an antibody, wherein the heavy and light chain variable regions
comprise the amino acid sequences depicted in SEQ ID NOs:17 and 18,
SEQ ID NOs:19 and 20, SEQ ID NOs:77 and 78, SEQ ID NOs:79 and 80,
SEQ ID NOs:81 and 82, SEQ ID NOs: 83 and 84, SEQ ID NOs:85 and 86,
or SEQ ID NOs:87 and 88, respectively, or amino acids sequences at
least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identical the aforementioned
sequences).
[0467] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid,"
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors"(or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0468] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant expression vector has been introduced. It should be
understood that such terms are intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
[0469] G. Combination Therapies
[0470] Any of the binding domains, antibodies, antigen binding
fragments thereof, bispecific constructs, and/or multispecific
constructs described herein, can be administered in combination
with an additional therapy, i.e., combined with other agents. The
term "coadministered" as used herein includes any or all of
simultaneous, separate, or sequential administration of the binding
domains, antibodies, antigen binding fragments thereof, bispecific
constructs, or multispecific constructs described herein with
adjuvants and other agents, including administration as part of a
dosing regimen. For example, the combination therapy can include
administering any of the binding domains, antibodies, antigen
binding fragments thereof, bispecific constructs, and/or
multispecific constructs described herein with at least one or more
additional therapeutic agents, such as anti-inflammatory agents,
DMARDs (disease-modifying anti-rheumatic drugs), immunosuppressive
agents, chemotherapeutics, radiation therapy, other antibodies,
cytotoxins and/or drugs, as well as adjuvants, immunostimulatory
agents and/or immunosuppressive agents.
[0471] Chemotherapeutic agents suitable for coadministration with
the binding domains, antibodies, antigen binding fragments thereof,
bispecific constructs, and/or multispecific constructs described
herein in the treatment of tumors include, for example: taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Further agents include, for example,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine) and temozolomide.
[0472] Agents that delete or inhibit immunosuppressive activities,
for example, by immune cells (for example regulatory T-cells, NKT
cells, macrophages, myeloid-derived suppressor cells, immature or
suppressive dendritic cells) or suppressive factors produced by the
tumor or host cells in the local microenvironment of the tumor (for
example, TGF.beta., indoleamine 2,3 dioxygenase--IDO), may also be
administered with the binding domains, antibodies, antigen binding
fragments thereof, bispecific constructs, and/or multispecific
constructs described herein. Such agents include antibodies and
small molecule drugs such as IDO inhibitors such as 1 methyl
tryptophan or derivatives.
[0473] Suitable agents for coadministration with the binding
domains, antibodies, antigen binding fragments thereof, bispecific
constructs, and/or multispecific constructs described herein for
treatment of such immune disorders include for example,
immunosuppressive agents such as rapamycin, cyclosporin and FK506;
anti-TNF agents such as etanercept, adalimumab and infliximab; and
steroids. Examples of specific natural and synthetic steroids
include, for example: aldosterone, beclomethasone, betamethasone,
budesonide, cloprednol, cortisone, cortivazol, deoxycortone,
desonide, desoximetasone, dexamethasone, difluorocortolone,
fluclorolone, flumethasone, flunisolide, fluocinolone,
fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone,
fluorometholone, flurandrenolone, fluticasone, halcinonide,
hydrocortisone, icomethasone, meprednisone, methylprednisolone,
paramethasone, prednisolone, prednisone, tixocortol and
triamcinolone.
[0474] Suitable agents for coadministration with the binding
domains, antibodies, antigen binding fragments thereof, bispecific
constructs, and/or multispecific constructs described herein for
inducement or enhancement of an immune response include, for
example, adjuvants and/or immunostimulatory agents, non-limiting
examples of which have been disclosed hereinbefore. In one
embodiment, the immunostimulatory agent is a TLR3 agonist, such as
Poly IC.
[0475] As used herein, the term "immunostimulatory agent" includes,
but is not limited to, compounds capable of stimulating antigen
presenting cells (APCs), such as dendritic cells (DCs) and
macrophages. For example, suitable immunostimulatory agents for use
in the present invention are capable of stimulating APCs, so that
the maturation process of the APCs is accelerated, the
proliferation of APCs is increased, and/or the recruitment or
release of co-stimulatory molecules (e.g., CD80, CD86, ICAM-1, MHC
molecules and CCR7) and pro-inflammatory cytokines (e.g.,
IL-1.beta., IL-6, IL-12, IL-15, and IFN-.gamma.) is upregulated.
Suitable immunostimulatory agents are also capable of increasing T
cell proliferation. Such immunostimulatory agents include, but are
not be limited to, CD40 ligand; FLT 3 ligand; cytokines, such as
IFN-.alpha., IFN-.beta., IFN-.gamma. and IL-2; colony-stimulating
factors, such as G-CSF (granulocyte colony-stimulating factor) and
GM-CSF (granulocyte-macrophage colony-stimulating factor); an
anti-CTLA-4 antibody, anti-PD1 antibody, anti-41BB antibody, or
anti-OX-40 antibody; LPS (endotoxin); ssRNA; dsRNA; Bacille
Calmette-Guerin (BCG);
[0476] Levamisole hydrochloride; and intravenous immune globulins.
In one embodiment an immunostimulatory agant may be a Toll-like
Receptor (TLR) agonist. For example the immunostimulatory agent may
be a TLR3 agonist such as double-stranded inosine:cytosine
polynucleotide (Poly I:C, for example available as Ampligen.TM.
from Hemispherx Bipharma, Pa., US or Poly IC:LC from Oncovir) or
Poly A:U; a TLR4 agonist such as monophosphoryl lipid A (MPL) or
RC-529 (for example as available from GSK, UK); a TLRS agonist such
as flagellin; a TLR7 or TLR8 agonist such as an imidazoquinoline
TLR7 or TLR 8 agonist, for example imiquimod (eg Aldara.TM.) or
resiquimod and related imidazoquinoline agents (for example as
available from 3M Corporation); or a TLR 9 agonist such as a
deoxynucleotide with unmethylated CpG motifs (so-called "CpGs", for
example as available from Coley Pharmaceutical). Such
immunostimulatory agents may be administered simultaneously,
separately or sequentially with the binding domains, antibodies,
antigen binding fragments thereof, bispecific constructs, and/or
multispecific constructs described herein.
[0477] H. Uses and Methods of the Invention
[0478] Provided herein are methods of stimulating T cell activity,
methods of inducing or enhancing an immune response, and methods of
treating a disease or condition (e.g., cancer) by administering the
bispecific constructs, multispecific constructs, antibodies, or
antigen binding fragments thereof, or compositions described herein
to a patient in need thereof.
[0479] As used herein, the term "T cell-mediated response" or "T
cell activity" refers to any response mediated by T cells,
including effector T cells (e.g., CD8+ cells) and helper T cells
(e.g., CD4+ cells). T cell mediated responses include, for example,
T cell cytotoxicity and proliferation. Stimulation of T cell
activity can be evaluated using any of a number of indicators of T
cell activity known in the art. For example, enhancement of
interferon-gamma production by OKT3-stimulated T cells can be used
as a measure of T cell activation. Stimulation of T cell activity
also can be evaluated using an NF.kappa.B-driven reporter gene
system in a CD27-expressing cell. Other suitable assays of T cell
activation are well established in the art.
[0480] The terms "inducing an immune response" and "enhancing an
immune response" are used interchangeably and refer the stimulation
of an immune response (i.e., either passive or adaptive) to a
particular antigen.
[0481] The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative measures described
herein. The methods of "treatment" employ administration to a
subject, in need of such treatment, a bispecific construct,
multispecific construct, antibody, antigen binding fragment
thereof, or composition as described herein, for example, a subject
in need of an enhanced immune response against a particular antigen
or a subject who ultimately may acquire such a disorder, in order
to prevent, cure, delay, reduce the severity of, or ameliorate one
or more symptoms of the disorder or recurring disorder, or in order
to prolong the survival of a subject beyond that expected in the
absence of such treatment.
[0482] The term "effective dose" or "effective dosage" is defined
as an amount sufficient to achieve or at least partially achieve
the desired effect. The term "therapeutically effective dose" is
defined as an amount sufficient to cure or at least partially
arrest the disease and its complications in a patient already
suffering from the disease. Amounts effective for this use will
depend upon the severity of the disorder being treated and the
general state of the patient's own immune system.
[0483] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0484] As used herein, the term "inhibits growth" (e.g., referring
to cells) is intended to include any measurable decrease in the
growth of a cell, e.g., the inhibition of growth of a cell by at
least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or
100%.
[0485] In one aspect, methods of stimulating T cell activity are
provided, which comprise contacting T cells with any one of the
antibodies, or antigen binding fragments thereof, bispecific
constructs, multispecific constructs, or the compositions of
described herein. Stimulating T cell activity can comprise, for
example, stimulating IFN-gamma production.
[0486] In another aspect, methods for inducing or enhancing an
immune response (e.g., against an antigen) in a subject comprising
administering to the subject any one of the antibodies, or antigen
binding fragments thereof, bispecific constructs, multispecific
constructs, or the compositions described herein, in an amount
effective to induce or enhance an immune response in the subject
(e.g., against an antigen).
[0487] In another aspect, methods of for treating a condition or
disease in a subject are provided, the method comprising
administering to the subject any one of the antibodies, or antigen
binding fragments thereof, bispecific constructs, multispecific
constructs, or the compositions described herein, in an amount
effective to treat the condition or disease.
[0488] In another aspect, methods for treating a condition or
disease in a subject are provided, wherein the method comprises
administering to the subject any one of the anti-CD27 antibodies,
or antigen binding fragments thereof, described herein in
combination with any one of the anti-PD-L1 antibodies, or antigen
binding fragments thereof, described herein. For example, in one
embodiment: [0489] (i) the anti-CD27 antibody, or antigen binding
fragment thereof, is selected from the group consisting of: (a) an
anti-CD27 antibody, or antigen binding fragment thereof, comprising
heavy chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ
ID NOs:1, 2, and 3, respectively, and light chain variable region
CDR1, CDR2 and
[0490] CDR3 as set forth in SEQ ID NOs:4, 5, and 6, respectively,
and (b) an anti-CD27 antibody, or antigen binding fragment thereof,
comprising heavy chain variable region CDR1, CDR2 and CDR3 as set
forth in SEQ ID NOs:7, 8, and 9, respectively, and light chain
variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:10,
11, and 12, respectively; and [0491] (ii) the anti-PD-L1 antibody,
or antigen binding fragment thereof, is selected from the group
consisting of: (a) an anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 29, 30, and 31, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:32, 33, and 34, respectively; (b) an anti-PD-L1
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs: 35, 36, and 37, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:38, 39, and 40,
respectively; (c) an anti-PD-L1 antibody, or antigen binding
fragment thereof, comprising heavy chain variable region CDR1, CDR2
and CDR3 as set forth in SEQ ID NOs: 41, 42, and 43, respectively,
and light chain variable region CDR1, CDR2 and CDR3 as set forth in
SEQ ID NOs:44, 45, and 46, respectively; (d) an anti-PD-L1
antibody, or antigen binding fragment thereof, comprising heavy
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs: 47, 48, and 49, respectively, and light chain variable region
CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:50, 51, and 52,
respectively; (e) anti-PD-L1 antibody, or antigen binding fragment
thereof, comprising heavy chain variable region CDR1, CDR2 and CDR3
as set forth in SEQ ID NOs: 53, 54, and 55, respectively, and light
chain variable region CDR1, CDR2 and CDR3 as set forth in SEQ ID
NOs:56, 57, and 58, respectively; and (f) anti-PD-L1 antibody, or
antigen binding fragment thereof, comprising heavy chain variable
region CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 59, 60, and
61, respectively, and light chain variable region CDR1, CDR2 and
CDR3 as set forth in SEQ ID NOs:62, 63, and 64, respectively.
[0492] In Another Embodiment, [0493] (i) the anti-CD27 antibody, or
antigen binding fragment thereof, is selected from the group
consisting of: (a) an anti-CD27 antibody, or antigen-binding
fragment thereof, comprising a heavy chain variable region
comprising SEQ ID NO:17 and a light chain variable region
comprising SEQ ID NO:18 and (b) an anti-CD27 antibody, or
antigen-binding fragment thereof, comprising SEQ ID NO:19 and a
light chain variable region comprising SEQ ID NO:20 or sequences;
and [0494] (ii) the anti-PD-L1 antibody, or antigen binding
fragment thereof, is selected from the group consisting of: (a) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:77
and a light chain variable region comprising SEQ ID NO:78; (b) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:79
and a light chain variable region comprising SEQ ID NO:80; (c) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:81
and a light chain variable region comprising SEQ ID NO:82; (d) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:83
and a light chain variable region comprising SEQ ID NO:84; (e) an
anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:85
and a light chain variable region comprising SEQ ID NO:86; and (f)
an anti-PD-L1 antibody, or antigen binding fragment thereof,
comprising a heavy chain variable region comprising SEQ ID NO:87
and a light chain variable region comprising SEQ ID NO:88.
[0495] In one embodiment, the anti-CD27 antibody, or antigen
binding fragment thereof, and the anti-PD-L1 antibody, or antigen
binding fragment thereof, are administered separately. In one
embodiment, the anti-CD27 antibody, or antigen binding fragment
thereof, and the anti-PD-L1 antibody, or antigen binding fragment
thereof, are administered sequentially. For example, the anti-CD27
antibody, or antigen binding fragment thereof, can be administered
first followed by (e.g., immediately followed by) administration of
the anti-PD-L1 antibody, or antigen binding fragment thereof, or
vice versa. In another embodiment, the anti-CD27 antibody, or
antigen binding fragment thereof, and the anti-PD-L 1 antibody, or
antigen binding fragment thereof, are administered together. In
another embodiment, the anti-CD27 antibody, or antigen binding
fragment thereof, and the anti-PD-L 1 antibody, or antigen binding
fragment thereof, are administered simultaneously. In another
embodiment, the anti-CD27 antibody, or antigen binding fragment
thereof, and the anti-PD-L 1 antibody, or antigen binding fragment
thereof, are simultaneously administered in a single formulation.
Alternatively, the anti-CD27 antibody, or antigen binding fragment
thereof, and the anti-PD-L1 antibody, or antigen binding fragment
thereof, are formulated for separate administration and are
administered concurrently or sequentially. Such concurrent or
sequential administration preferably results in both antibodies
being simultaneously present in treated patients.
[0496] In certain embodiments, administration of any of the
anti-CD27 antibodies, or antigen binding fragment thereof,
described herein in combination with any of the anti-PD-L1
antibodies, or antigen binding fragments thereof, described herein
results in synergistic effects (e.g., in enhancing immune responses
in vivo) as compared to use of either antibody alone.
[0497] The subject can be, for example, one who suffers from a
condition or disease in which stimulation of an immune response is
desired. In one embodiment, the condition or disease is cancer.
Types of cancers include, but are not limited to, leukemia, acute
lymphocytic leukemia, acute myelocytic leukemia, myeloblasts
promyelocyte myelomonocytic monocytic erythroleukemia, chronic
leukemia, chronic myelocytic (granulocytic) leukemia, chronic
lymphocytic leukemia, mantle cell lymphoma, primary central nervous
system lymphoma, Burkitt's lymphoma and marginal zone B cell
lymphoma, Polycythemia vera Lymphoma, Hodgkin's disease,
non-Hodgkin's disease, multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and
carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,
chrondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal
carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, uterine cancer,
testicular tumor, lung carcinoma, small cell lung carcinoma, non
small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma,
basal cell carcinoma, biliary tract cancer, bladder cancer, bone
cancer, brain and central nervous system (CNS) cancer, cervical
cancer, choriocarcinoma, colorectal cancers, connective tissue
cancer, cancer of the digestive system, endometrial cancer,
esophageal cancer, eye cancer, head and neck cancer, gastric
cancer, intraepithelial neoplasm, kidney cancer, larynx cancer,
liver cancer, lung cancer (small cell, large cell), melanoma,
neuroblastoma; oral cavity cancer(for example lip, tongue, mouth
and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma,
rhabdomyosarcoma, rectal cancer; cancer of the respiratory system,
sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid
cancer, uterine cancer, and cancer of the urinary system.
Particular cancers include CD27-expressing tumors selected from the
group consisting of chronic lymphocytic leukemia, mantle cell
lymphoma, primary central nervous system lymphoma, Burkitt's
lymphoma and marginal zone B cell lymphoma. [0498] Other disease
indications include bacterial, fungal, viral and parasitic
infectious diseases.
[0499] The methods of inducing or enhancing an immune response
(e.g., against an antigen) in a subject described herein can
further comprise administering the antigen to the subject.
[0500] As used herein, the term "antigen" refers to any natural or
synthetic immunogenic substance, such as a protein, peptide,
hapten, polysaccharide and/or lipid. The bispecific construct,
multispecific construct, antibody, antigen binding fragment
thereof, or composition described herein and antigen can be
administered at the same time or, alternatively, the bispecific
construct, multispecific construct, antibody, antigen binding
fragment thereof, or composition can be administered before or
after the antigen is administered.
[0501] In one embodiment, a bispecific construct, multispecific
construct, antibody, antigen binding fragment thereof, or
composition described herein is administered in combination with a
vaccine, to enhance the immune response against the vaccine
antigen, for example a tumor antigen (to thereby enhance the immune
response against the tumor) or an antigen from an infectious
disease pathogen (to thereby enhance the immune response against
the infectious disease pathogen). Accordingly, in one embodiment, a
vaccine antigen can comprise, for example, an antigen or antigenic
composition capable of eliciting an immune response against a tumor
or against an infectious disease pathogen such as a virus, a
bacteria, a parasite or a fungus. The antigen or antigens be
derived from tumors, such as the various tumor antigens previously
disclosed herein. Alternatively, the antigen or antigens can be
derived from pathogens such as viruses, bacteria, parasites and/or
fungi.
[0502] Preferred antigens to be co-administered with the
antibodies, or antigen binding fragments thereof, bispecific
constructs, multispecific constructs, or the compositions of
described herein include tumor antigens and vaccine antigens (e.g.,
bacterial, viral or other pathogen antigens against which
protective immunity is desired to be raised in a subject for
purposes of vaccination). Additional examples of suitable pathogen
antigens include tumor-associated antigens (TAAs), including but
not limited to, sequences comprising all or part of the sequences
of EGFR, EGFRvIII, gp100 or Pme117, HER2/neu, mesothelin, CEA,
MART1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MUC-1, GPNMB, HMW-MAA,
TIM1, ROR1, CD19 and germ cell derived tumor antigens.
[0503] Other suitable antigens include viral antigens for the
prevention or treatment of viral diseases. Examples of viral
antigens include, but are not limited to, HIV-1 env, HBsAg, HPV,
FAS, HSV-1, HSV-2, p17, ORF2 and ORF3 antigens. In addition, viral
antigens or antigenic determinants can be derived from, for
example,: Cytomegalovirus (especially Human, such as gB or
derivatives thereof); Epstein Barr virus (such as gp350);
flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne
encephalitis virus, Japanese Encephalitis Virus); hepatitis virus
such as hepatitis B virus (for example Hepatitis B Surface antigen
such as the PreS1, PreS2 and S antigens described in EP-A-414 374;
EP-A-0304 578, and EP-A-198474), hepatitis A virus, hepatitis C
virus and hepatitis E virus; HIV-1, (such as tat, nef, gp120 or
gp160); human herpes viruses, such as gD or derivatives thereof or
Immediate Early protein such as ICP27 from HSV1 or HSV2; human
papilloma viruses (for example HPV6, 11, 16, 18); Influenza virus
(whole live or inactivated virus, split influenza virus, grown in
eggs or MDCK cells, or Vero cells or whole flu virosomes (as
described by Gluck, Vaccine, 1992,10, 915-920) or purified or
recombinant proteins thereof, such as NP, NA, HA, or M proteins);
measles virus; mumps virus; parainfluenza virus; rabies virus;
Respiratory Syncytial virus (such as F and G proteins); rotavirus
(including live attenuated viruses); smallpox virus; Varicella
Zoster Virus (such as gpl, II and 1E63); and the HPV viruses
responsible for cervical cancer (for example the early proteins E6
or E7 in fusion with a protein D carrier to form Protein D-E6 or E7
fusions from HPV 16, or combinations thereof; or combinations of E6
or E7 with L2 (see for example WO 96/26277).
[0504] Examples of bacterial antigens include, but are not limited
to, Toxoplasma gondii or Treponema pallidum. The bacterial antigens
can be in the treatment or prevention of various bacterial diseases
such as Anthrax, Botulism, Tetanus, Chlamydia, Cholera, Diphtheria,
Lyme Disease, Syphilis and Tuberculosis. Bacterial antigens or
antigenic determinants can be derived from, for example: Bacillus
spp., including B. anthracis (e.g., botulinum toxin); Bordetella
spp, including B. pertussis (for example pertactin, pertussis
toxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae);
Borrelia spp., including B. burgdorferi (eg OspA, OspC, DbpA,
DbpB), B. garinii (eg OspA, OspC, DbpA, DbpB), B. afzelii (eg OspA,
OspC, DbpA, DbpB), B. andersonii (eg OspA, OspC, DbpA, DbpB), B.
hermsii; Campylobacter spp, including C. jejuni (for example
toxins, adhesins and invasins) and C. coli; Chlamydia spp.,
including C. trachomatis (eg MOMP, heparin-binding proteins), C.
pneumonie (eg MOMP, heparin-binding proteins), C. psittaci;
Clostridium spp., including C. tetani (such as tetanus toxin), C.
botulinum (for example botulinum toxin), C. difficile (eg
clostridium toxins A or B); Corynebacterium spp., including C.
diphtheriae (eg diphtheria toxin); Ehrlichia spp., including E.
equi and the agent of the Human Granulocytic Ehrlichiosis;
Rickettsia spp, including R. rickettsii; Enterococcus spp.,
including E. faecalis, E. faecium; Escherichia spp, including
enterotoxic E. coli (for example colonization factors, heat-labile
toxin or derivatives thereof, or heat-stable toxin),
enterohemorragic E. coli, enteropathogenic E. coli (for example
shiga toxin-like toxin); Haemophilus spp., including H. influenzae
type B (eg PRP), non-typable H. influenzae, for example OMP26, high
molecular weight adhesins, P5, P6, protein D and lipoprotein D, and
fimbrin and fimbrin derived peptides (see for example U.S. Pat. No.
5,843,464); Helicobacter spp, including H. pylori (for example
urease, catalase, vacuolating toxin); Pseudomonas spp, including P.
aeruginosa; Legionella spp, including L. pneumophila; Leptospira
spp., including L. interrogans; Listeria spp., including L.
monocytogenes; Moraxella spp, including M catarrhalis, also known
as Branhamella catarrhalis (for example high and low molecular
weight adhesins and invasins); Morexella Catarrhalis (including
outer membrane vesicles thereof, and OMP106 (see for example
W097/41731)); Mycobacterium spp., including M. tuberculosis (for
example ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M.
avium, M. paratuberculosis, M. smegmatis; Neisseria spp, including
N. gonorrhea and N. meningitidis (for example capsular
polysaccharides and conjugates thereof, transferrin-binding
proteins, lactoferrin binding proteins, PilC, adhesins); Neisseria
mengitidis B (including outer membrane vesicles thereof, and NspA
(see for example WO 96/29412); Salmonella spp, including S. typhi,
S. paratyphi, S. choleraesuis, S. enteritidis; Shigella spp,
including S. sonnei, S. dysenteriae, S. flexnerii; Staphylococcus
spp., including S. aureus, S. epidermidis; Streptococcus spp,
including S. pneumonie (e.g., capsular polysaccharides and
conjugates thereof, PsaA, PspA, streptolysin, choline-binding
proteins) and the protein antigen Pneumolysin (Biochem Biophys
Acta, 1989,67,1007; Rubins et al., Microbial Pathogenesis,
25,337-342), and mutant detoxified derivatives thereof (see for
example WO 90/06951; WO 99/03884); Treponema spp., including T.
pallidum (eg the outer membrane proteins), T. denticola, T.
hyodysenteriae; Vibrio spp, including V. cholera (for example
cholera toxin); and Yersinia spp, including Y. enterocolitica (for
example a Yop protein), Y. pestis, Y. pseudotuberculosis.
[0505] Parasitic/fungal antigens or antigenic determinants can be
derived from, for example,: Babesia spp., including B. microti;
Candida spp., including C. albicans; Cryptococcus spp., including
C. neoformans; Entamoeba spp., including E. histolytica; Giardia
spp., including; G. lamblia; Leshmania spp., including L. major;
Plasmodium. faciparum (MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2,
Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25,
Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and their analogues in
Plasmodium spp.); Pneumocystis spp., including P. carinii;
Schisostoma spp., including S. mansoni; Trichomonas spp., including
T. vaginalis; Toxoplasma spp., including T. gondii (for example
SAG2, SAGS, Tg34); Trypanosoma spp., including T. cruzi.
[0506] It will be appreciated that in accordance with this aspect
of the present invention antigens and antigenic determinants can be
used in many different forms. For example, antigens or antigenic
determinants can be present as isolated proteins or peptides (for
example in so-called "subunit vaccines") or, for example, as
cell-associated or virus-associated antigens or antigenic
determinants (for example in either live or killed pathogen
strains). Live pathogens will preferably be attenuated in known
manner. Alternatively, antigens or antigenic determinants may be
generated in situ in the subject by use of a polynucleotide coding
for an antigen or antigenic determinant (as in so-called "DNA
vaccination"), although it will be appreciated that the
polynucleotides which can be used with this approach are not
limited to DNA, and may also include RNA and modified
polynucleotides as discussed above.
[0507] In one embodiment, a vaccine antigen can also be targeted,
for example to particular cell types or to particular tissues. For
example, the vaccine antigen can be targeted to Antigen Presenting
Cells (APCs), for example by use of agents such as antibodies
targeted to APC-surface receptors such as DEC-205, for example as
discussed in WO 2009/061996 (Celldex Therapeutics, Inc), or the
Mannose Receptor (CD206) for example as discussed in WO 03040169
(Medarex, Inc.).
[0508] I. Kits
[0509] Also provided are kits (e.g., diagnostic kits) comprising
one or more anti-CD27 binding domains, anti-PD-L1 binding domains,
bispecific constructs, multispecific constructs, or compositions as
described herein, optionally with instructions for use. Kits may
also include informative pamphlets, for example, pamphlets
informing one how to use reagents to practice a method disclosed
herein. The term "pamphlet" includes any writing, marketing
materials or recorded material supplied on or with the kit, or
which otherwise accompanies the kit.
[0510] The present invention is further illustrated by the
following examples, which should not be construed as further
limiting. The contents of figures and all references, patents and
published patent applications cited throughout this application are
expressly incorporated herein by reference.
V. EXAMPLES
Example 1
Generation of CD27-Specific Human Monoclonal Antibodies
[0511] Human anti-CD27 monoclonal antibodies were generated by
immunizing the H2L2 strain of Harbour.RTM. transgenic mice with a
soluble human CD27 antigen. Harbour.RTM. transgenic mice have had
the endogenous mouse heavy chain (HC) and kappa light chain
(.kappa.-chain) DNA sequences knocked out and have had sequences
for the human variable (V) regions and rat constant (C) regions
stably incorporated into the mouse genome.
[0512] Antigen and Immunization: The antigen was a soluble fusion
protein comprising a CD27 extracellular domain with an Fc tag
(R&D Systems). The antigen was mixed with MPL plus TDM adjuvant
system (Sigma) for immunizations. 5-25 micrograms soluble
recombinant CD27 antigen in PBS were mixed 1:1 with the adjuvant.
Mice were injected with 200 microliters of the prepared antigen
into the peritoneal cavity every 14 days. Animals that developed
anti-CD27 titers were given an i.v. injection of 5-10 micrograms
soluble recombinant CD27 antigen three to four days prior to
fusion. Mouse spleens were harvested, and the isolated splenocytes
used for hybridoma preparation.
[0513] Hybridoma Preparation: The P3x63Ag8.653 murine myeloma cell
line (ATCC CRL 1580) was used for the fusions. RPMI 1640
(Invitrogen) containing 10% FBS was used to culture the myeloma
cells. Additional media supplements were added to the Hybridoma
growth media, which included: up to 10% Hybridoma Enhancing
Supplement (Sigma), 10% FBS (Sigma), L-glutamine (Gibco), 0.1%
gentamycin (Gibco), 2-mercaptoethanol (Gibco), with HAT (Sigma;
1.0.times.10.sup.4 M hypoxanthine, 4.0.times.10.sup.-7 M
aminopterin, 1.6.times.10.sup.-5 M thymidine media).
[0514] Spleen cells were mixed with the P3x63Ag8.653 myeloma cells
in a 6:1 ratio and pelleted by centrifugation. Polyethylene glycol
was added dropwise with careful mixing to facilitate fusion.
Hybridomas were allowed to grow out for one to two weeks until
visible colonies become established. Supernatant was harvested and
used for initial screening for rat IgG via ELISA using a human
soluble CD27 fusion protein and a rat Fc specific detection. IgG
positive supernatants were then assayed for CD27 specificity via
flow cytometry. The hybridomas were also screened for
cross-reactivity with cynomolgus macaque CD27 and all were positive
for binding.
[0515] Hybridoma cells were expanded and cell pellets were frozen
for RNA isolation and sequencing. The V.sub.H and V.sub.L coding
regions of human monoclonal antibodies were identified using RNA
from the corresponding hybridomas. RNA was reverse transcribed to
cDNA, the V coding regions were amplified by PCR and the PCR
products were sequenced, inserted into a human IgG1kappa vector,
transiently expressed and purified by protein A column
chromatography which led to the isolation of antibodies of
particular interest, which were designated as 2B3 and 3C2.
Example 2
Assays to Determine Human Monoclonal Antibody Binding
Characteristics to CD27
[0516] Microtiter plates were coated with recombinant human
CD27-FLAG-HIS in PBS, and then blocked with 5% bovine serum albumin
in PBS. Protein A purified human monoclonal antibodies were added
at various concentrations and incubated at 37.degree. C. The plates
were washed with PBS/Tween and then incubated with a
goat-anti-human IgG Fc-specific polyclonal reagent conjugated to
horseradish peroxidase at 37.degree. C. After washing, the plates
were developed with HRP substrate, and analyzed at OD 450-650 nm
using a microtiter plate reader. FIG. 1 shows that anti-CD27
antibodies 2B3 and 3C2 bind human CD27.
[0517] To establish that cynomolgus macaques are a relevant model
for testing anti-CD27 monoclonal antibodies, microtiter plates were
coated with recombinant cynomolgus CD27-FLAG-HIS in PBS, and then
blocked with 5% bovine serum albumin in PBS. Hybridoma supernatants
or rat IgG control were added and incubated at 37.degree. C. The
plates were washed with PBS/Tween and then incubated with a
mouse-anti-rat IgG Fc-specific polyclonal reagent conjugated to
horseradish peroxidase at 37.degree. C. After washing, the plates
were developed with HRP substrate, and analyzed at OD 450-650 nm
using a microtiter plate reader. FIG. 2 shows that anti-CD27
antibodies 2B3 and 3C2 bind cynomolgus CD27.
Example 3
Binding to CD27 Cells
[0518] The ability of anti-CD27 human monoclonal antibodies to bind
to CD27 on cells expressing human CD27 on their surface was
investigated by flow cytometry as follows:
[0519] Antibodies were tested for binding to a human cell line
expressing human CD27 on their surface. Protein A purified human
monoclonal antibodies (3 .mu.g/ml) were incubated with Ramos cells
expressing human CD27 at room temperature on a plate shaker. After
20 minutes, the cells were washed with PBS containing 0.1% BSA and
0.05% NaN.sub.3 (PBA) and the bound antibodies were detected by
incubating the cells with a PE labeled goat anti-human IgG
Fc-specific probe. The excess probe was washed from the cells with
PBA and the cell associated fluorescence was determined by analysis
using a FACSCanto II.TM. instrument (BD Biosciences, N.J., USA)
according to the manufacturer's directions.
[0520] As shown in FIG. 3, the anti-CD27 human monoclonal
antibodies demonstrated high levels of binding to cells expressing
human CD27.
Example 4
Binding to Human T Cells
[0521] The ability of anti-CD27 human monoclonal antibodies to bind
to CD27 on human T cells was investigated by flow cytometry as
follows:
[0522] Antibodies were tested for binding to human CD3.sup.+ T
cells which express human CD27 on their surface. Human peripheral
blood mononuclear cells were isolated from buffy coats using Ficoll
separation, and CD3.sup.+ cells were further isolated from the
PBMC's using magnetic bead separation technology from Miltenyi
Biotec. Protein A purified human monoclonal antibodies (3 .mu.g/ml)
were incubated with the T cells at room temperature on a plate
shaker. After 20 minutes, the cells were washed with PBS containing
0.1% BSA and 0.05% NaN.sub.3 (PBA) and the bound antibodies were
detected by incubating the cells with a PE labeled goat anti-human
IgG Fc-specific probe. The excess probe was washed from the cells
with PBA and the cell associated fluorescence was determined by
analysis using a FACSCanto II.TM. instrument (BD Biosciences, N.J.,
USA) according to the manufacturer's directions.
[0523] As shown in FIG. 4, the anti-CD27 human monoclonal
antibodies demonstrated high levels of binding to human T
cells.
Example 5
Blocking of CD70 Binding
[0524] The effect of the human monoclonal antibodies on the binding
of soluble CD70 to CD27 on the cell surface was measured by flow
cytometry. Ramos cells expressing CD27 were incubated for 5 minutes
at room temperature with the antibodies (50 .mu.g/ml), followed by
the addition of human CD70-biotin ([final]=0.5 .mu.g/mL) for 20
minutes at room temperature on a plate shaker. CD27 captured CD70
was detected with streptavidin PE and analyzed on a FACSCanto
II.TM. instrument (BD Biosciences, N.J., USA). FIG. 5 shows that
anti-CD27 antibodies 2B3 and 3C2 block CD70 binding to CD27 on
cells.
Example 6
NF.kappa.B Activation
[0525] A luciferase reporter cell line expressing CD27 was
incubated for 6 hours at 37.degree. C., 6% CO.sub.2 with various
concentrations of the human anti-CD27 antibodies. Luciferase is
expressed upon activation and was detected with the Luciferase
Assay System by Promega according to the manufacturer's guidelines.
FIG. 6 shows the high level of NF.kappa.B activation induced by 2B3
and 3C2 antibodies as a function of antibody concentration.
Example 7
T Cell Proliferation
[0526] Human Peripheral Blood Mononuclear Cells (PBMCs) isolated
from buffy coat preparations and CD3.sup.+ cells were further
isolated from the PBMCs using magnetic bead separation technology
from Miltenyi Biotec. The T cells were labeled with 1 mM
carboxyfluorescein succinimidyl ester (CFSE) at room temperature
while rotating for 5 minutes. The CFSE labeled PBMCs
(1.times.10.sup.6) were dispensed into wells dry coated with
anti-CD3 antibody (OKT3, eBioscience) at 1 .mu.g/mL and the
anti-CD27 antibodies or human IgG.sub.1 control at 10 .mu.g/mL. The
plates were incubated at 37.degree. C., 5% CO.sub.2 for 72 hours.
The cells were harvested and analyzed by flow cytometry on a
FACSCanto II.TM. instrument (BD Biosciences, N.J., USA) according
to the manufacturer's directions. FIG. 7 shows that antibodies 2B3
and 3C2 significantly increase T cell proliferation.
Example 8
Generation of PD-L1-Specific Human Monoclonal Antibodies
[0527] Human anti-PD-L1 monoclonal antibodies were generated by
immunizing the H2L2 strain of Harbour.RTM. transgenic mice with a
soluble human PD-L1 antigen. Harbour.RTM. transgenic mice have had
the endogenous mouse heavy chain (HC) and kappa light chain
(.kappa.-chain) DNA sequences knocked out and have had sequences
for the human variable (V) regions and rat constant (C) regions
stably incorporated into the mouse genome.
[0528] Antigen and Immunization: The antigen was a soluble fusion
protein comprising a PD-L1 extracellular domain with a HIS tag
(R&D Systems), or a recombinant human PD-L1-msG2a chimeric
protein (made in-house). The antigen was mixed with MPL plus TDM
adjuvant system (Sigma) for immunizations. 5-25 micrograms soluble
recombinant PD-L1 antigen in PBS were mixed 1:1 with the adjuvant.
Mice were injected with 200 microliters of the prepared antigen
into the peritoneal cavity every 14 days. Animals that developed
anti-PD-L1 titers were given an iv injection of 5-10 micrograms
soluble recombinant PD-L1 antigen three to four days prior to
fusion. Mouse spleens were harvested, and the isolated splenocytes
used for hybridoma preparation.
[0529] Hybridoma Preparation: The P3x63Ag8.653 murine myeloma cell
line (ATCC CRL 1580) was used for the fusions. RPMI 1640
(Invitrogen) containing 10% FBS was used to culture the myeloma
cells. Additional media supplements were added to the Hybridoma
growth media, which included: up to 10% Hybridoma Enhancing
Supplement (Sigma), 10% FBS (Sigma), L-glutamine (Gibco), 0.1%
gentamycin (Gibco), 2-mercaptoethanol (Gibco), with HAT (Sigma;
1.0.times.10.sup.4 M hypoxanthine, 4.0.times.10.sup.-7 M
aminopterin, 1.6.times.10.sup.-5 M thymidine media).
[0530] Spleen cells were mixed with the P3x63Ag8.653 myeloma cells
in a 6:1 ratio and pelleted by centrifugation. Polyethylene glycol
was added dropwise with careful mixing to facilitate fusion.
Hybridomas were allowed to grow out for one to two weeks until
visible colonies become established. Supernatant was harvested and
used for initial screening for rat IgG via ELISA using a human
soluble PD-L1 fusion protein and a rat Fc specific detection. IgG
positive supernatants were then assayed for PD-L1 specificity via
flow cytometry. The hybridomas were also screened for
cross-reactivity with cynomolgus macaque PD-L1 and all were
positive for binding.
[0531] Hybridoma cells were expanded and cell pellets were frozen
for RNA isolation and sequencing. The V.sub.H and V.sub.L coding
regions of human monoclonal antibodies were identified using RNA
from the corresponding hybridomas. RNA was reverse transcribed to
cDNA, the V coding regions were amplified by PCR and the PCR
products were sequenced, inserted into human IgG1kappa vector,
transiently expressed and purified by protein A column
chromatography which led to the isolation of a number of antibodies
of particular interest, which were designated as 1B3, 3B6, 4A3,
7H7, 8B1 and 9H9.
Example 9
Assays to Determine Human Monoclonal Antibody Binding
Characteristics to PD-L1
[0532] Microtiter plates were coated with recombinant human
PD-L1-msFc in PBS, and then blocked with 5% bovine serum albumin in
PBS. Protein A purified human monoclonal antibodies were added at
various concentrations and incubated at 37.degree. C. The plates
were washed with PBS/Tween and then incubated with a
goat-anti-human IgG Fc-specific polyclonal reagent conjugated to
horseradish peroxidase at 37.degree. C. After washing, the plates
were developed with HRP substrate, and analyzed at OD 450-650 nm
using a microtiter plate reader. FIG. 8 shows that the anti-PD-L1
antibodies strongly bind human PD-L1 as a function of antibody
concentration.
[0533] To establish that cynomolgus macaques are a relevant model
for testing anti-PD-L1 monoclonal antibodies, microtiter plates
were coated with recombinant cynomolgus PD-L1-FLAG-HIS in PBS, and
then blocked with 5% bovine serum albumin in PBS. Hybridoma
supernatants or rat IgG control were added and incubated at
37.degree. C. The plates were washed with PBS/Tween and then
incubated with a mouse-anti-rat IgG Fc-specific polyclonal reagent
conjugated to horseradish peroxidase at 37.degree. C. After
washing, the plates were developed with HRP substrate, and analyzed
at OD 450-650 nm using a microtiter plate reader. FIG. 9 shows that
the anti-PD-L1 antibodies bind cynomolgus PD-L1.
Example 10
Blocking of PD1 Binding
[0534] The effect of the human monoclonal antibodies on the binding
of soluble PD1 to PD-L1 on the cell surface was measured by flow
cytometry. 293 cells expressing PD-L1 were incubated for 5 minutes
at room temperature with the antibodies, followed by the addition
of human PD1-biotin ([final]=0.5 mg/mL). PD-L1 captured PD1 was
detected with streptavidin PE and analyzed on a FACSCanto II.TM.
instrument (BD Biosciences, N.J., USA). FIG. 10 shows that the
anti-PD-L1 antibodies block PD-L1 binding to PD1 as a function of
antibody concentration.
Example 11
Binding to PD-L1 Cells
[0535] The ability of anti-PD-L1 human monoclonal antibodies to
bind to PD-L1 on cells expressing human PD-L1 on their surface was
investigated by flow cytometry as follows:
[0536] Antibodies were tested for binding to a human cell line
expressing human PD-L1 on their surface. Protein A purified human
monoclonal antibodies were incubated with 293 cells expressing
human PD-L1 at room temperature on a plate shaker. After 20
minutes, the cells were washed with PBS containing 0.1% BSA and
0.05% NaN.sub.3 (PBA) and the bound antibodies were detected by
incubating the cells with a PE labeled goat anti-human IgG
Fc-specific probe. The excess probe was washed from the cells with
PBA and the cell associated fluorescence was determined by analysis
using a FACSCanto II.TM. instrument (BD Biosciences, N.J., USA)
according to the manufacturer's directions.
[0537] As shown in FIG. 11, the anti-PD-L1 human monoclonal
antibodies demonstrated high levels of binding to cells expressing
human PD-L1 as a function of antibody concentration.
Example 12
Binding to Human Dendritic Cells
[0538] The ability of anti-PD-L1 human monoclonal antibodies to
bind to PD-L1 on human dendritic cells was investigated by flow
cytometry as follows:
[0539] Antibodies were tested for binding to human dendritic cells
which express human PD-L1 on their surface. Dendritic cells were
generated as follows: PMBC's were added to a T175cm.sup.2 flasks
and monocytes allowed to adhere for .about.2 hours at 37.degree.
C., 6% CO.sub.2. The non-adherent cells were removed and the
monocytes cultured for 7 days in RPMI containing 10% FBS, 10 ng/mL
IL-4 (R&D Systems) and 100 ng/mL GM-CSF (R&D Systems).
Non-adherent cells were harvested and confirmed to be dendritic
cells by expression of CD11c (not shown). Protein A purified human
monoclonal antibodies were incubated with the dendritic cells at
room temperature on a plate shaker. After 20 minutes, the cells
were washed with PBS containing 0.1% BSA and 0.05% NaN.sub.3 (PBA)
and the bound antibodies were detected by incubating the cells with
a PE labeled goat anti-human IgG Fc-specific probe. The excess
probe was washed from the cells with PBA and the cell associated
fluorescence was determined by analysis using a FACSCanto II.TM.
instrument (BD Biosciences, N.J., USA) according to the
manufacturer's directions.
[0540] As shown in FIG. 12, the anti-PD-L1 human monoclonal
antibodies demonstrated high levels of binding to human dendritic
cells as a function of antibody concentration.
Example 13
T cell PD1/PD-L1 Blockade Bioassay
[0541] The effect of the PD-L1 antibodies on blockade of the
PD1/PD-L1 interaction was determined using the PD1/PD-L1 Blockade
Assay from Promega. Two engineered cell lines, PD1 Effector cells
and PD-L1 aAPC/CHO-K1 cells were co-cultured in the presence of the
antibodies for 6 hours. Blocking of the PD1/PD-L1 interaction
results in TCR activation and induces luminescence via the NFAT
pathway. Luminescence was detected by the addition of Bio-Glo
reagent and quantitated on a Perkin Elmer Victor X luminometer. As
shown on FIG. 13, the anti-PD-L1 antibodies effectively block the
PD1/PD-L1 interaction between cells leading to activation of the
NFAT pathway.
Example 14
Mixed Lymphocyte Reaction
[0542] Human peripheral blood mononuclear cells were isolated from
buffy coats using Ficoll separation, and CD4.sup.+ cells were
further isolated from the PBMC's using magnetic bead separation
technology from Miltenyi Biotec. Allogeneic dendritic cells were
generated as follows: PMBC's were added to a T175cm.sup.2 flasks
and monocytes allowed to adhere for .about.2 hours at 37.degree.
C., 6% CO.sub.2. The non-adherent cells were removed and the
monocytes cultured for 7 days in RPMI containing 10% FBS, 10 ng/mL
IL-4 (R&D Systems) and 100 ng/mL GM-CSF (R&D Systems).
Non-adherent cells were harvested and confirmed to be dendritic
cells by expression of CD11c (not shown). The CD4.sup.+ cells and
DC's were co-incubated at a 10:1 ratio in the presence of the
antibody dilutions for 3 days. Supernatants were harvested and
analyzed for IL-2 production by ELISA (R&D Systems). As shown
on FIG. 14, the anti-PD-L1 antibodies were able to induce a
significant mixed lymphocyte response.
Example 15
Development and Functional Testing of Bispecific Constructs
[0543] Tetravalent bispecific constructs were developed using a
fully human IgG.sub.1 backbone for the PD-L1 monoclonal antibody
and the scFv of the CD27 monoclonal antibody genetically linked to
the c-terminus of the heavy chain. Alternative bispecific
constructs also were developed using a fully human IgG.sub.1
backbone for the CD27 monoclonal antibody and the scFv of the PD-L1
monoclonal antibody. FIG. 15A shows a representative vector
containing the CD27 light chain, CD27 heavy chain, and the
c-terminal PD-L1 single chain Fv (VL+VH) polypeptide. FIGS. 15B and
15C are two alternative depictions of the CD27/PD-L1 bispecific
format. FIG. 15B shows the CD27/PD-L1 bispecific antibody protein
wherein an anti-PD-L1 antibody is linked to an anti-CD27 scFv
(referred to herein as "CDX-527" when the anti-PD-L1 antibody is
9H9 and the anti-CD27 scFv is derived from 2B3. Otherwise AbXx2B3)
and FIG. 15C shows the CD27/PD-L1 bispecific antibody protein
wherein an anti-CD27 antibody is linked to an anti-PD-L1 scFv. FIG.
15D is a table of representative anti-CD27/anti-PD-L1 bispecific
constructs generated.
[0544] The full 9H9x2B3 (CDX-527) heavy chain sequence was as
follows (with the IgG.sub.1 constant region sequence shown in
bold):
TABLE-US-00003 (SEQ ID NO: 179)
EVQLVESGGGLVQPGGSLRLSCAASGGIISTYWMSWVRQAPGKGLE
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRVEDTA
MYYCARDRPVAGASALWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGGGSEIV
MTQSPATLSVSPGERATLSCRASQSIRSNLAWYQQKPGQAPRLLIY
GASTRATGIPARFSGSGSGTEFTLTISSLQSENFAVYYCQQYNNWP
LTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKP
GASVKVSCKASGYTFTGYYIHWVRQAPGQCLEWMGWINPNSGGTNS
AQKFQDRVTITRVTSINTAYMELSRLRSDDTAVYFCARDRLVLPWF
GEIFPDAFDIWGQGTLVTVSS
[0545] The 9H9x2B3 (CDX-527) light chain sequence was as follows
(with the constant region sequence shown in bold):
TABLE-US-00004 (SEQ ID NO: 180)
DIQMTOSPSTLSASVGDRVTITCRASOSISGWLAWYQQKPGKAPKLL
IYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGS
SRTFGQGTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKSFNRGEC
Example 16
Assays to Determine Bispecific Monoclonal Antibody Binding
Characteristics and Functional Activity
[0546] Binding of bispecific constructs to CD27 and PD-L1 was
assessed using a bifunctional ELISA. Antibody AbX is a known
anti-PD-L1 monoclonal antibody. In brief, a microtiter plate was
coated with human CD27-FLAG-HIS. Dilutions of the bispecific
constructs were allowed to bind before adding human PD-L1-msFc that
was detected with an HRP labeled goat anti-mouse IgG (Fc specific)
antibody. Representative binding curves for three bispecific
constructs (CD27xAbX, CD27x8B1, and CD27x9H9) are shown in FIG. 16.
All three antibodies demonstrated significant binding to CD27 and
PD-L1.
[0547] CD27 pathway activation was assessed by measuring NF.kappa.B
activation. In brief, CD27 was transfected into a
NF.kappa.B-luciferase reporter cell line (Signosis). The cells were
incubated for 6 hours with each bispecific construct or antibody
(1F5xAbX, 2B3x8B1, 2B3x9H9, 1F5, 2B3, or huIgG.sub.1) and
luciferase expression was detected with the Brite-Glo.TM. system
(Promega). Note: the reporter cell line is positive for human
PD-L1. FIG. 17 shows the level of NF.kappa.B activation induced by
the antibodies as a function of concentration. Additionally, the
bispecific constructs 1F5xAbX, 2B3x8B1, and 2B3x9H9 showed
significantly higher NF.kappa.B activation than 1F5 or 2B3
alone.
[0548] PD-1 signaling blockade was assessed by measuring NFAT
pathway activation. In brief, PD-1 Effector cells and PD-L1 aAPC
cells were co-cultured in the presence of dilutions of each
bispecific construct or the control antibody (CD27xAbX, CD27x8B1,
CD27x9H9, or huIgG.sub.1). Activation of the NFAT pathway via
PD-L1/PD-1 blockade is detected by addition of Bio-Glo.TM. reagent.
(Commercially available kit from Promega). As shown in FIG. 18, the
bispecific constructs induced strong NFAT pathway activation as a
function of antibody concentration.
[0549] IL-2 production/secretion was also measured in a mixed
lymphocyte reaction. In brief, CD4 cells were incubated in the
presence of allogeneic dendritic cells and dilutions of each
bispecific constructs or antibody (CD27xAbX, CD27x8B1, CD27x9H9,
huIgG1, AbX, 8B1, or 9H9) for 3 days. Supernatants were harvest and
IL-2 levels were assessed by ELISA (R&D Systems).
Representative IL-2 concentration curves are shown in FIG. 19. The
bispecific constructs CD27xAbX, CD27x8B1 and CD27x9H9 showed
significantly higher IL-2 production/secretion than AbX (a known
anti-PD-L1 monoclonal antibody), 8B1, or 9H9 alone (e.g., about
2.times. higher IL-2 production).
Example 17
Assays to Determine In vivo Activity of CD27/PD-L1 Bispecific
Constructs
[0550] HuCD27-Tg mice were injected with 0.1 mg of bispecific
CD27xAbX construct (BsAb) or CD27 monospecific antibody (mAb), and
5 mg of Ovalbumin on day 0, as indicated in the x-axis of each
graph. On Day 7 spleen cells were harvested and intracellular
cytokine IFN.gamma. and IL2, and cytolysis enzyme granzyme B (GrB)
were detected by flow cytometry analysis with and without ex-vivo
stimulation of SIINFEKL peptide. Percentage of SIINFEKL-specific
IFN-.gamma..sup.+ and IL2.sup.+ in CD8 T cells (FIG. 20A), and
percentage of GrB+in CD8 T cells without SIINFEKL stimulation (FIG.
20B) are shown, indicating the CD27xAbX bispecific construct
induces a significant CD8 T cell response as compared to CD27
monoclonal antibody alone (e.g., about 2.5.times. to about 8.times.
higher response).
[0551] Tumor growth, survival, and tumor infiltrate numbers were
also measured in mice treated with the BsAb or mAbs. HuCD27-Tg mice
were injected i.v. with BCL1 cells (5.times.10.sup.6) on day 0.
Antibodies or the bispecific construct were injected i.p (0.2 mg)
on day 5. Mice were divided into two groups. One group was used to
measure survival (n=8). FIG. 21 shows percent survival over time of
mice treated with either CD27 monoclonal antibody, PD-L1 monoclonal
antibody, combination of CD27+PD-L1 monoclonal antibodies, and
CD27xAbX BsAb. Mice treated with CD27+PD-L1 antibodies survived
significantly longer than mice treated with either CD27 monoclonal
antibody, PD-L1 monoclonal antibody, or Hu IgG.sub.1 alone (e.g.,
1.5.times.-2.times. longer). Moreover, mice treated with the
CD27xAbX BsAb survived significantly longer than mice treated with
any of the other antibodies tested either alone or in combination.
In fact, 70% to 80% of mice treated with CD27xAbX BsAb were still
alive after 80 days post tumor inoculation whereas mice in other
groups had all died. Three surviving mice were re-challenged with
same number of BCL1 cells after 180 days follow-up, and they were
protected from the re-challenge.
[0552] The second group of mice was used to measure tumor weight
and T cell levels on day 11. FIGS. 22A-22D show tumor weight,
percent CD8 T cells, percent CD4 T cells, and IFN.gamma. and GrB
double positive CD8 T cells, respectively. Similar to the survival
data, CD27xAbX BsAb significantly reduced tumor weight (e.g.,
1.5.times. to 3.times. less), and significantly increased T cell
numbers and activity (e.g., 1.5.times. to 4.times. more), as
compared to any of the other antibodies tested either alone or in
combination. FIGS. 23A and 23B show the upregulation of PD-L1
expression level on the surface of BCL1 lymphoma cells (FIG. 23A)
and tumor microenvironment infiltrated cells (FIG. 23B) upon CD27
mAb treatment (CDX-1127), providing rationale for the combination
or BsAb of anti-CD27 and anti-PD-L1.
Example 18
Bifunctional ELISA
[0553] Characterization and binding of the bispecific construct
CDX-527 (prepared as in Example 15) was analyzed. FIG. 15E shows
characterization of the bispecific antibody CDX-527 by HPLC and gel
electrophoresis (reducing conditions). Binding to both CD27 and
PD-L1 was determined using a bifunctional ELISA generally as
described in Example 16. Results are shown in FIG. 24 from which it
can be seen that CDX-527 demonstrated significant binding to CD27
and PD-L1.
Example 19
Activation of NFkB by Bispecific Construct CDX-527
[0554] Activation of NF.kappa.B by the bispecific construct CDX-527
(prepared as in Example 15) was determined generally as described
in Example 16 except that SteadyGlo.TM. reagent from Promega was
used instead of BriteGlo.TM..
[0555] Results are shown in FIG. 25, which shows the higher level
of activation by CDX-527 compared to the monospecific anti-CD27
antibodies 1F5 and 2B3 alone. Activation was also measured in the
presence of soluble Fc.gamma.R1, which further increased NF.kappa.B
activation as shown.
Example 20
Mixed Lymphocyte Reaction
[0556] The ability of the bispecific construct CDX-527 (prepared as
in Example 15) to induce a mixed lymphocyte response was determined
by the method described generally in Example 14, with testing for
IL-2.
[0557] Results are shown in FIG. 26, from which it can be seen that
CDX-527 was able to induce a significant mixed lymphocyte response
which was also significantly greater than that of the monospecific
antibodies 2B3 and 9H9 when used either alone or in
combination.
Example 21
T-Cell Activation
[0558] Human Peripheral Blood Mononuclear Cells (PBMCs) were
isolated from buffy coat preparations and CD3+ cells were further
isolated from the PBMCs using magnetic bead separation technology
from Miltenyi Biotec. CD3+ cells (1.times.10.sup.5) were dispensed
into wells coated with anti-CD3 antibody (OKT3, eBioscience) and
soluble human PD-L1. Antibodies 2B3 and 9H9 or CDX-527 (prepared as
in Example 15) were added to the cells at concentrations between
0.1 nM and 10 nM. The plates were incubated at 37.degree. C., 5%
CO.sub.2 for 72 hours at which time IL-2 levels in the supernatants
were measured.
[0559] Results are shown in FIG. 27 from which it can be seen that
CDX-527 significantly activated T-cells and did so to a
significantly greater extent than the monospecific antibodies 2B3
and 9H9 when used in combination.
Example 22
Pharmacokinetics
[0560] Pharmacokinetics of CDX-527 was studied in non-human
primates (NHPs) at a dose level of 7.0 mg/kg and volume of 3.0
ml/kg i.v.. No significant change was observed in any clinical
parameters during the 21 day study. Serum levels of CDX-527 were
determined by ELISA.
[0561] Results are shown in FIG. 28. Pharmacokinetic analysis
resulted in T1/2 of approximately 110 hours.
Example 23
Cell based PD1/PDL1 Blockade Assay
[0562] Bispecific antibodies 9H9x2B3 and the opposite configuration
2B3x9H9 were compared in a cell based PD1/PDL1 blockade assay
generally as described in Example 13.
[0563] Results are shown in FIG. 29 from which it can be seen that
the 9H9x2B3 configuration was more potent at blockade of PD-1
signaling than the 2B3x9H9 configuration.
Example 24
Mixed Lymphocyte Reaction
[0564] Bispecific antibodies 9H9x2B3 and the opposite configuration
2B3x9H9 were compared in a mixed lymphocyte reaction generally as
described in Example 14.
[0565] Results are shown in FIG. 30 from which it can be seen that
the 9H9x2B3 configuration was more potent at T-cell activation than
the 2B3x9H9 configuration.
Example 25
Vaccine Induced CD8 T Cell Response
[0566] Bispecific antibodies AbXx2B3 and the opposite configuration
2B3xAbX were compared in a vaccine model of T-cell response
generally as described in Example 17.
[0567] Results are shown in FIG. 31 from which it can be seen that
AbXx2B3 was more potent than 2B3xAbX in stimulating a vaccine
induced CD8+ T-cell response.
Example 26
BCL1 Tumor Model
[0568] Bispecific antibodies AbXx2B3 and the opposite configuration
2B3xAbX were compared in a BCL1 Tumor Model generally as described
in Example 17.
[0569] Results are shown in FIG. 32 from which it can be seen that
AbXx2B3 had greater anti-tumor activity than 2B3xAbX.
Example 27
Blocking of PD-L1 Binding to CD80
[0570] A microtiter plate was coated with recombinant human CD80,
then blocked. Biotinylated huPD-L1 was pre-incubated for 1 hour at
room temperature with 50 ug/mL of the huIgG.sub.1 control or the
anti-PD-L1 antibodies (AbX or 9H9), then added to the plate.
Streptavidin-HRP was used to detect the binding of the PD-L1 to
CD80.
[0571] Results are shown in FIG. 33 from which it can be seen that
the anti-PD-L1 antibodies completely blocked PD-L1 binding to
CD80.
Example 28
2B3 Binding to huCD27
[0572] The full length wild type extracellular domain (ECD) of
human CD27 or human CD27 with mutated amino acids 85, 87, 88, and
89 (A85S, R87A, N88A and G89A; see FIG. 34) were coated to a plate,
then blocked. Supernatant from transiently transfected cells
expressing mAb 2B3 was added and binding was detected with an HRP
conjugated goat anti-human IgG Fc polyclonal antibody.
[0573] Results are shown in FIG. 35 from which it can be seen that
the anti-CD27 antibody 2B3 did not bind to the mutated CD27 ECD.
Accordingly, this indicates that antibody 2B3 binds to an epitope
on human CD27 involving or including one or more residues within
amino acids 80-95, e.g., one or more residues within amino acids
85-89, of the extracellular domain (ECD) of human CD27 (SEQ ID NO:
183), e.g., amino acids 85, 87, 88, and/or 89 of the ECD of human
CD27 (SEQ ID NO: 183).
Example 29
Increasing 9H9x2B3 expression through protein engineering: 9H9-2B3
(DD)
[0574] To increase expression of the bispecific product the rare
valine (V) residue at H72 in FR3 of the 2B3 heavy chain and the
rare asparagine (N) residue at L82 in FR3 of the 2B3 light chain
were each changed to aspartic acid (D) residues (ie V72D and N82D
respectively).
[0575] The modified 2B3 heavy chain sequence was as follows (the
CDRs are underlined and the modified residue in FR3 is double
underlined):
TABLE-US-00005 (SEQ ID NO: 177)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWM
GWINPNSGGTNSAQKFQDRVTITRDTSINTAYMELSRLRSDDTAVYFC
ARDRLVLPWFGEIFPDAFDIWGQGTLVTVSS
[0576] The modified 2B3 light chain sequence was as follows (the
CDRs are underlined and the modified residue in FR3 is double
underlined):
TABLE-US-00006 (SEQ ID NO: 178)
EIVMTQSPATLSVSPGERATLSCRASQSIRSNLAWYQQKPGQAPRLLI
YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPL TFGGGTKVEIK
[0577] These sequences (designated 2B3(DD)) were used in an scFv
format which was genetically linked to the C-terminus of the heavy
chains of the 9H9 antibody. The resulting 9H9x2B3(DD) bispecific
antibody was designated 9H9x2B3(DD).
[0578] The full 9H9x2B3(DD) heavy chain sequence was as follows
(with the IgG.sub.1 constant "backbone" sequence shown in
bold):
TABLE-US-00007 (SEQ ID NO: 181)
EVQLVESGGGLVQPGGSLRLSCAASGGIISTYWMSWVRQAPGKGLEW
VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRVEDTAMY
YCARDRPVAGASALWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGKGSSGGGGSEIVMTQSPATLSVS
PGERATLSCRASQSIRSNLAWYQQKPGQAPRLLIYGASTRATGIPAR
FSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGCGTKVEIKG
GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYT
FTGYYIHWVRQAPGQCLEWMGWINPNSGGTNSAQKFQDRVTITRDTS
INTAYMELSRLRSDDTAVYFCARDRLVLPWFGEIFPDAFDIWGQGTL VTVSS
[0579] The 9H9x2B3(DD) light chain sequence was as follows (with
the constant region shown in bold):
TABLE-US-00008 (SEQ ID NO: 182)
DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLI
YKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSR
TFGQGTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC
[0580] The DNA sequence of the 9H9x2B3(DD) variable domain sequence
was as follows:
TABLE-US-00009 (SEQ ID NO: 185)
GAAGTGCAACTGGTGGAGTCGGGTGGTGGACTCGTGCAGCCCGGCGG
ATCCCTGAGACTCTCTTGTGCCGCATCGGGCGGCATTATTAGCACTT
ACTGGATGTCATGGGTCAGACAGGCACCGGGAAAGGGCTTGGAATGG
GTGGCGAATATCAAGCAGGATGGATCCGAGAAGTACTACGTGGACTC
CGTGAAGGGCAGATTCACCATTTCCCGGGACAACGCCAAGAACTCGC
TCTATCTGCAAATGAACTCGTTGCGGGTGGAAGATACTGCCATGTAC
TACTGCGCCCGGGACCGGCCTGTGGCCGGGGCGTCGGCCCTCTGGGG
CCAGGGCACTCTGGTCACCGTGTCCTCT
[0581] The DNA sequence of the 9H9x2B3(DD) scFv domain sequence was
as follows (connector and linker sequences are shown in bold):
TABLE-US-00010 (SEQ ID NO: 186)
GGCTCCAGCGGGGGTGGCGGTTCCGAGATCGTGATGACTCAGAGCCC
GGCAACCCTGTCCGTGTCTCCGGGGGAGCGGGCTACTCTTTCCTGCC
GGGCATCCCAGTCCATCCGGTCGAACCTTGCGTGGTACCAACAGAAG
CCTGGACAGGCGCCCCGCCTGCTGATCTACGGGGCGTCGACTAGGGC
CACCGGCATCCCGGCCCGCTTCTCCGGGTCCGGATCCGGCACCGAAT
TCACCCTCACCATCTCGAGCCTGCAGTCCGAAAACTTCGCCGTCTAC
TACTGCCAGCAGTACAACAACTGGCCGCTGACATTCGGATGCGGAAC
CAAAGTGGAAATCAAGGGCGGCGGCGGATCCGGCGGTGGCGGCAGCG
GCGGTGGAGGATCCGGTGGCGGCGGTTCACAAGTGCAGCTGGTGCAG
TCAGGCGCCGAAGTCAAGAAGCCCGGGGCCAGCGTGAAAGTCAGCTG
CAAGGCTTCCGGATACACCTTCACGGGTTACTACATTCACTGGGTTC
GCCAAGCGCCCGGGCAGTGTCTGGAGTGGATGGGATGGATCAACCCT
AACTCGGGGGGAACCAACTCGGCCCAAAAGTTCCAGGACCGGGTCAC
CATTACAAGAGTCACGTCCATCAACACTGCCTACATGGAGTTGAGCC
GGCTGCGATCAGACGACACCGCCGTGTACTTCTGCGCGAGGGACCGC
CTCGTCCTCCCGTGGTTTGGAGAGATCTTCCCGGATGCCTTCGACAT
TTGGGGACAGGGGACCCTCGTGACTGTGTCCAGC
[0582] The DNA sequence of the 9H9x2B3(DD) light chain variable
sequence was as follows:
TABLE-US-00011 (SEQ ID NO: 187)
GATATCCAGATGACCCAGAGCCCGTCCACCCTTTCCGCGAGCGTCGGC
GACAGAGTGACCATTACTTGTCGGGCCTCGCAAAGCATCTCCGGCTGG
CTGGCTTGGTACCAGCAAAAGCCTGGAAAGGCCCCTAAGCTGCTGATC
TACAAGGCCTCATCCCTGGAGTCCGGAGTGCCTTCACGCTTTTCGGGG
AGCGGATCGGGGACTGAGTTCACCCTCACCATTTCCTCCCTGCAACCC
GACGATTTCGCGACATACTACTGCCAGCAGTACTACGGTTCCTCGCGC
ACGTTCGGACAGGGCACTAACGTCGAGATCAAG
[0583] As can be seen from FIG. 36 transient transfections showed
improved production (expression) of the 9H9-2B3(DD) modified
construct compared to the original (unmodified) 9H9-2B3
construct.
TABLE-US-00012 SUMMARY OF SEQUENCE LISTING SEQ ID NO: 1 GYYWS 3C2
V.sub.H CDR1 SEQ ID NO: 2 YNYYSGSTNYNPSLKS 3C2 V.sub.H CDR2 SEQ ID
NO: 3 YPLIRGAFDY 3C2 V.sub.H CDR3 SEQ ID NO: 4 RSSQNLLHTNGYNYLD 3C2
V.sub.L CDR1 SEQ ID NO: 5 LGSNRAS 3C2 V.sub.L CDR2 SEQ ID NO: 6
MQALQTPLT 3C2 V.sub.L CDR3 SEQ ID NO: 7 GYYIH 2B3 V.sub.H CDR1 SEQ
ID NO: 8 WINPNSGGTNSAQKFQD 2B3 V.sub.H CDR2 SEQ ID NO: 9
DRLVLPWFGEIFPDAFDI 2B3 V.sub.H CDR3 SEQ ID NO: 10 RASQSIRSNLA 2B3
V.sub.L CDR1 SEQ ID NO: 11 GASTRAT 2B3 V.sub.L CDR2 SEQ ID NO: 12
QQYNNWPLT 2B3 V.sub.L CDR3 SEQ ID NO: 13
MKHLWFCLLLVAAPRWVLSQAQLQESGPGLVKPSETLSLTCTVSTGSIS 3C2 V.sub.H-with
GYYWSWIRQPPGKGLEWIGYNYYSGSTNYNPSLKSRVTISIDTSKNQFS signal sequence
LKLNSVTAADTAVYYCARYPLIRGAFDYWGQGTLVTVSS SEQ ID NO: 14
MRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQNL 3C2 V.sub.L-with
LHTNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFT signal sequence
LKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK SEQ ID NO: 15
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGY 2B3 V.sub.H-with
TFTGYYIHWVRQAPGQGLEWMGWINPNSGGTNSAQKFQDRVTITRVT signal sequence
SINTAYMELSRLRSDDTAVYFCARDRLVLPWFGEIFPDAFDIWGQGTLV TVSS SEQ ID NO:
16 MEAPAQLLFLLLLWLPDSTGEIVMTQSPATLSVSPGERATLSCRASQSIR 2B3
V.sub.L-with SNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS
signal sequence ENFAVYYCQQYNNWPLTFGGGTKVEIK SEQ ID NO: 17
QAQLQESGPGLVKPSETLSLTCTVSTGSISGYYWSWIRQPPGKGLEWIG 3C2 V.sub.H-
YNYYSGSTNYNPSLKSRVTISIDTSKNQFSLKLNSVTAADTAVYYCARY without signal
PLIRGAFDYWGQGTLVTVSS sequence SEQ ID NO: 18
DIVMTQSPLSLPVTPGEPASISCRSSQNLLHTNGYNYLDWYLQKPGQSP 3C2 V.sub.L-
QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAL without signal
QTPLTFGGGTKVEIK sequence SEQ ID NO: 19
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLE 2B3 V.sub.H-
WMGWINPNSGGTNSAQKFQDRVTITRVTSINTAYMELSRLRSDDTAVY without signal
FCARDRLVLPWFGEIFPDAFDIWGQGTLVTVSS sequence SEQ ID NO: 20
EIVMTQSPATLSVSPGERATLSCRASQSIRSNLAWYQQKPGQAPRLLIY 2B3 V.sub.L-
GASTRATGIPARFSGSGSGTEFTLTISSLQSENFAVYYCQQYNNWPLTFG without signal
GGTKVEIK sequence SEQ ID NO: 21
ATGAAACATCTGTGGTTCTGCCTTCTCCTGGTGGCAGCTCCCAGATG 3C2 V.sub.H DNA
GGTCCTGTCCCAGGCGCAGCTGCAGGAGTCGGGCCCAGGACTGGTG Sequence-with
AAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTACTGGCTC signal sequence
CATCAGTGGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAG
GGACTGGAGTGGATTGGGTATAATTATTACAGTGGGAGCACCAACT
ACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAATAGACACGTC
CAAGAACCAGTTCTCCCTGAAGCTGAATTCTGTGACCGCTGCGGAC
ACGGCCGTATATTACTGTGCGAGATATCCTCTGATTCGGGGAGCTTT
TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 22
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTC 3C2 V.sub.L DNA
TGGATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGC Sequence-with
CCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGTAGGTCTAGTCAG signal sequence
AACCTCCTGCATACTAATGGCTACAACTATTTGGATTGGTACCTGCA
GAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATC
GGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCAC
AGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGG
GTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCACTTTCGGCGG
AGGGACCAAGGTGGAGATCAAA SEQ ID NO: 23
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAG 2B3 V.sub.H DNA
GAGCCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAA Sequence-with
GAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATAC signal sequence
ACCTTCACCGGCTACTATATACACTGGGTGCGACAGGCCCCTGGAC
AAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCAC
AAACTCTGCACAGAAGTTTCAGGACAGGGTCACCATCACCAGGGTC
ACGTCCATCAACACAGCCTACATGGAGCTGAGCAGACTGAGATCTG
ACGACACGGCCGTGTATTTCTGTGCGAGAGATCGGCTCGTATTACCA
TGGTTCGGGGAAATATTCCCAGATGCTTTTGATATCTGGGGCCAAGG
GACATTGGTCACCGTCTCTTCA SEQ ID NO: 24
ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCC 2B3 V.sub.L DNA
AGATTCCACTGGAGAAATAGTGATGACGCAGTCTCCAGCCACCCTG Sequence-with
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTC signal sequence
AGAGTATTAGGAGCAACTTAGCCTGGTATCAGCAGAAACCTGGCCA
GGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTA
TCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTC
ACCATCAGCAGCCTGCAGTCTGAAAATTTTGCAGTTTATTACTGTCA
GCAGTATAATAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTG GAGATCAAA SEQ ID NO:
25 CAGGCGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG 3C2 V.sub.H DNA
AGACCCTGTCCCTCACCTGCACTGTCTCTACTGGCTCCATCAGTGGT Sequence-
TACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGT without signal
GGATTGGGTATAATTATTACAGTGGGAGCACCAACTACAACCCCTC sequence
CCTCAAGAGTCGAGTCACCATATCAATAGACACGTCCAAGAACCAG
TTCTCCCTGAAGCTGAATTCTGTGACCGCTGCGGACACGGCCGTATA
TTACTGTGCGAGATATCCTCTGATTCGGGGAGCTTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 26
GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGG 3C2 V.sub.L DNA
AGAGCCGGCCTCCATCTCCTGTAGGTCTAGTCAGAACCTCCTGCATA Sequence-
CTAATGGCTACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCA without signal
GTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGG sequence
TCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTG
AAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCA
TGCAAGCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGT GGAGATCAAA SEQ ID
NO: 27 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG 2B3 V.sub.H
DNA CCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGC Sequence-
TACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGT without signal
GGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTCTGCACA sequence
GAAGTTTCAGGACAGGGTCACCATCACCAGGGTCACGTCCATCAAC
ACAGCCTACATGGAGCTGAGCAGACTGAGATCTGACGACACGGCCG
TGTATTTCTGTGCGAGAGATCGGCTCGTATTACCATGGTTCGGGGAA
ATATTCCCAGATGCTTTTGATATCTGGGGCCAAGGGACATTGGTCAC CGTCTCTTCA SEQ ID
NO: 28 GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGG 2B3 V.sub.L
DNA GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGGAGC Sequence-
AACTTAGCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCTCC without signal
TCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTC sequence
AGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCC
TGCAGTCTGAAAATTTTGCAGTTTATTACTGTCAGCAGTATAATAAC
TGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 29 TSWMS 7H7
V.sub.H CDR1 SEQ ID NO: 30 NIKQDGSEKYYVDSVKG 7H7 V.sub.H CDR2 SEQ
ID NO: 31 DRPVAGASAL 7H7 V.sub.H CDR3 SEQ ID NO: 32 RASQSISGWLA 7H7
V.sub.L CDR1 SEQ ID NO: 33 KASSLES 7H7 V.sub.L CDR2 SEQ ID NO: 34
QQYYGSSRT 7H7 V.sub.L CDR3 SEQ ID NO: 35 TSWMS 1B3 V.sub.H CDR1 SEQ
ID NO: 36 NIKQDGSEKYYVDSVKG 1B3 V.sub.H CDR2 SEQ ID NO: 37
DRPVAGASAL 1B3 V.sub.H CDR3 SEQ ID NO: 38 RASQSISGWLA 1B3 V.sub.L
CDR1 SEQ ID NO: 39 KASSLES 1B3 V.sub.L CDR2 SEQ ID NO: 40 QQYYGSSRT
1B3 V.sub.L CDR3 SEQ ID NO: 41 TYWMS 3B6 V.sub.H CDR1 SEQ ID NO: 42
NIKQDGSEKYYVDSVKG 3B6 V.sub.H CDR2 SEQ ID NO: 43 DRPVAGASAL 3B6
V.sub.L CDR3 SEQ ID NO: 44 RASQSISGWLA 3B6 V.sub.L CDR1 SEQ ID NO:
45 KASSLES 3B6 V.sub.L CDR2 SEQ ID NO: 46 QQYYGSSRT 3B6 V.sub.L
CDR3 SEQ ID NO: 47 THWMS 8B1 V.sub.H CDR1 SEQ ID NO: 48
NIKQDGSEKYYVDSVKG 8B1 V.sub.H CDR2 SEQ ID NO: 49 DRPVAGASAL 8B1
V.sub.H CDR3 SEQ ID NO: 50 RASQSISGWLA 8B1 V.sub.L CDR1 SEQ ID N0:
51 KASSLES 8B1 V.sub.L CDR2 SEQ ID NO: 52 QQYYGSSRT 8B1 V.sub.L
CDR3 SEQ ID NO: 53 SSWMS 4A3 V.sub.H CDR1 SEQ ID NO: 54
NIKQDGSEKYYVDSVKG 4A3 V.sub.H CDR2 SEQ ID NO: 55 DRPVAGASAL 4A3
V.sub.H CDR3 SEQ ID NO: 56 RASQSISGWLA 4A3 V.sub.L CDR1 SEQ ID NO:
57 KASSLES 4A3 V.sub.L CDR2 SEQ ID NO: 58 QQYYGSSRT 4A3 V.sub.L
CDR3
SEQ ID NO: 59 TYWMS 9H9 V.sub.H CDR1 SEQ ID NO: 60
NIKQDGSEKYYVDSVKG 9H9 V.sub.H CDR2 SEQ ID NO: 61 DRPVAGASAL 9H9
V.sub.H CDR3 SEQ ID NO: 62 RASQSISGWLA 9H9 V.sub.L CDR1 SEQ ID NO:
63 KASSLES 9H9 V.sub.L CDR2 SEQ ID NO: 64 QQYYGSSRT 9H9 V.sub.L
CDR3 SEQ ID NO: 65
MELGLSXVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGGTIS 7H7 V.sub.H-with
TSWMS WVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAK signal sequence
NSLYLQMNSLRVEDTAIYYCARDRPVAGASALWGQGTLVTVSS SEQ ID NO: 66
MRVPAQEEGEEEEWEPGAKCDIQMTQSPSTESASVGDRVTITCRASQSI 7H7 V.sub.L-with
SGWLAWYQQKQGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSL signal sequence
QPDDFATYYCQQYYGSSRTFGQGTNVEIK SEQ ID NO: 67
MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGGTI 1B3 V.sub.H-with
STSWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAK signal sequence
NSLYLQMNSLRVEDTAMYYCARDRPVAGASALWGQGTLVTVSS SEQ ID NO: 68
MRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQSI 1B3 V.sub.L-with
SGWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQ signal sequence
PDDFATYYCQQYYGSSRTFGQGTNVEIK SEQ ID NO: 69
MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGGTT 3B6 V.sub.H-with
STYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNA signal sequence
KNSLNLQMNSLRVEDTAIYYCARDRPVAGASALWGQGTLVTVSS SEQ ID NO: 70
MRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQSI 3B6 V.sub.L-with
SGWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQ signal sequence
PDDFATYYCQQYYGSSRTFGQGTNVEIK SEQ ID N0: 71
MELGLSWVFLVAILEGVKCEVRLVESGGGLVQPGGSLRLSCAASGDIIS 8B1 V.sub.H-with
THWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAK signal sequence
NSLYLQMNTLRVEDTAIYYCTRDRPVAGASALWGQGTLVTVSS SEQ ID NO: 72
MRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQSI 8B1 V.sub.L-with
SGWLAWYQQKPGKAPKLLIYKASSLESGVPLRFSGSGSGTEFTLTISSL signal sequence
QPDDFATYYCQQYYGSSRTFGQGTNVEIK SEQ ID NO: 73
MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGGIIS 4A3 V.sub.H-with
SSWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAK signal sequence
DLLYLQMNSLRVEDTALYYCARDRPVAGASALWGQGTLVTVSS SEQ ID NO: 74
MRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQSI 4A3 V.sub.L-with
SGWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQ signal sequence
PDDFATYYCQQYYGSSRTFGQGTNVEIK SEQ ID NO: 75
MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGGIIS 9H9 V.sub.H-with
TYWMS WVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAK signal sequence
NSLYLQMNSLRVEDTAMYYCARDRPVAGASALWGQGTLVTVSS SEQ ID NO: 76
MRVPAQLLGLLLLWLPGAKCDIQMTQSPSTLSASVGDRVTITCRASQSI 9H9 V.sub.L-with
SGWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQ signal sequence
PDDFATYYCQQYYGSSRTFGQGTNVEIK SEQ ID NO: 77
EVQLVESGGGLVQPGGSLRLSCAASGGTISTSWMSWVRQAPGKGLEW 7H7 V.sub.H-
VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRVEDTAIYY without signal
CARDRPVAGASALWGQGTLVTVSS sequence SEQ ID NO: 78
DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKQGKAPKLLIY 7H7 V.sub.L-
KASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTFG without signal
QGTNVEIK sequence SEQ ID NO: 79
EVQLVESGGGLVQPGGSLRLSCAASGGTISTSWMSWVRQAPGKGLEW 1B3 V.sub.H-
VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRVEDTAMY without signal
YCARDRPVAGASALWGQGTLVTVSS sequence SEQ ID NO: 80
DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIY 1B3 V.sub.L-
KASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTFG without signal
QGTNVEIK sequence SEQ ID NO: 81
EVQLVESGGGLVQPGGSLRLSCAASGGTTSTYWMSWVRQAPGKGLE 3B6 V.sub.H -
WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLNLQMNSLRVEDTAIY without signal
YCARDRPVAGASALWGQGTLVTVSS sequence SEQ ID NO: 82
DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIY 3B6 V.sub.L-
KASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTFG without signal
QGTNVEIK sequence SEQ ID NO: 83
EVRLVESGGGLVQPGGSLRLSCAASGDIISTHWMSWVRQAPGKGLEW 8B1 V.sub.H-
VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNTLRVEDTAIYY without signal
CTRDRPVAGASALWGQGTLVTVSS sequence SEQ ID NO: 84
DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIY 8B1 V.sub.L-
KASSLESGVPLRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTFG without signal
QGTNVEIK sequence SEQ ID NO: 85
EVQLVESGGGLVQPGGSLRLSCAASGGIISSSWMSWVRQAPGKGLEWV 4A3 V.sub.H-
ANIKQDGSEKYYVDSVKGRFTISRDNAKDLLYLQMNSLRVEDTALYYC without signal
ARDRPVAGASALWGQGTLVTVSS sequence SEQ ID NO: 86
DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIY 4A3 V.sub.L-
KASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTFG without signal
QGTNVEIK sequence SEQ ID NO: 87
EVQLVESGGGLVQPGGSLRLSCAASGGIISTYWMSWVRQAPGKGLEW 9H9 V.sub.H-
VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRVEDTAMY without signal
YCARDRPVAGASALWGQGTLVTVSS sequence SEQ ID NO: 88
DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIY 9H9 V.sub.L-
KASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTFG without signal
QGTNVEIK sequence SEQ ID NO: 89
ATGGAATTGGGGCTGAGCTGNGTTTTCCTTGTTGCTATTTTAGAAGG 7H7 V.sub.H DNA
TGTCCAGTGTGAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTC Sequence-with
CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCA signal sequence
CCATTAGTACCTCTTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAA
GGGGCTGGAATGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAA
ATATTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACA
ACGCCAAGAACTCACTGTATTTGCAAATGAACAGCCTGAGAGTCGA
GGACACGGCTATATATTACTGTGCGAGAGATCGTCCAGTGGCTGGT
GCGTCGGCCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 90
ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCC 7H7 V.sub.L DNA
AGGTGCCAAATGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGT Sequence-with
CTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCA signal sequence
GAGTATAAGTGGCTGGTTGGCCTGGTATCAGCAGAAACAAGGGAAA
GCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGT
CCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTC
ACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCA
ACAGTATTATGGTTCTTCTCGGACGTTCGGCCAAGGGACCAATGTGG AAATCAAA SEQ ID NO:
91 ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGG 1B3 V.sub.H DNA
TGTCCAGTGTGAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTC Sequence-with
CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCA signal sequence
CCATTAGTACCTCTTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAA
GGGGCTGGAATGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAA
ATATTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACA
ACGCCAAGAACTCACTGTATTTGCAAATGAACAGCCTGAGAGTCGA
AGACACGGCTATGTATTACTGTGCGAGAGATCGTCCAGTGGCTGGT
GCGTCGGCCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 92
ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCC 1B3 V.sub.L DNA
AGGTGCCAAATGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGT Sequence-with
CTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCA signal sequence
GAGTATTAGTGGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA
GCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGT
CCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTC
ACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCA
ACAGTATTATGGTTCTTCTCGGACGTTCGGCCAAGGGACCAATGTGG AAATCAAA SEQ ID NO:
93 ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGG 3B6 V.sub.H DNA
TGTCCAGTGTGAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTC Sequence-with
CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCA signal sequence
CAACCAGTACCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAA
GGGGCTGGAATGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAA
ATATTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACA
ACGCCAAGAACTCACTGAATTTGCAAATGAACAGCCTGAGAGTCGA
GGACACGGCTATATATTACTGTGCGAGAGATCGTCCAGTGGCTGGT
GCGTCGGCCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 94
ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCC 3B6 V.sub.L DNA
AGGTGCCAAATGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGT Sequence-with
CTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCA signal sequence
GAGTATTAGTGGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA
GCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGT
CCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTC
ACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCA
ACAGTATTATGGTTCTTCTCGGACGTTCGGCCAAGGGACCAATGTGG AAATCAAA SEQ ID NO:
95 ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGG 8B1 V.sub.H DNA
TGTCAAGTGTGAGGTGCGACTGGTGGAGTCTGGGGGAGGCTTGGTC Sequence-with
CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGACAT signal sequence
AATTAGTACCCATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAATGGGTGGCCAACATAAAACAAGATGGAAGTGAGAAG
TATTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAA
CGCCAAGAACTCACTGTATTTGCAAATGAACACCCTGAGAGTCGAG
GACACGGCTATATATTACTGTACGAGAGATCGTCCAGTGGCTGGTG
CGTCGGCCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 96
ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCC 8B1 V.sub.L DNA
AGGTGCCAAATGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGT Sequence-with
CTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCA signal sequence
GAGTATTAGTGGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA
GCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGT
CCCATTAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTC
ACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCA
ACAGTATTATGGTTCTTCTCGGACGTTCGGCCAAGGGACCAATGTGG AAATCAAA SEQ ID NO:
97 ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGG 4A3 V.sub.H DNA
TGTCCAGTGTGAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTC Sequence-with
CAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCA signal sequence
TCATTAGTTCCTCTTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAATGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAA
TATTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAA
CGCCAAAGACTTACTGTATTTGCAAATGAACAGCCTGAGAGTCGAG
GACACGGCTTTATATTACTGTGCGAGAGATCGTCCAGTGGCTGGTGC
GTCGGCCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCT SEQ ID NO: 98
ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCC 4A3 V.sub.L DNA
AGGTGCCAAATGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGT Sequence-with
CTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCA signal sequence
GAGTATTAGTGGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA
GCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGT
CCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTC
ACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCA
ACAGTATTATGGTTCTTCTCGGACGTTCGGCCAAGGGACCAATGTGG AAATCAAA SEQ ID NO:
99 ATGGAATTGGGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGG 9H9 V.sub.H DNA
TGTCCAGTGTGAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTC Sequence-with
CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCAT signal sequence
CATTAGTACCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAATGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAA
TATTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAA
CGCCAAGAACTCACTGTATTTGCAAATGAACAGCCTGAGAGTCGAG
GACACGGCTATGTATTACTGTGCGAGAGATCGTCCAGTGGCTGGTG
CGTCGGCCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 100
ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCC 9H9 V.sub.L DNA
AGGTGCCAAATGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGT Sequence-with
CTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCA signal sequence
GAGTATTAGTGGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA
GCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGT
CCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTC
ACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCA
ACAGTATTATGGTTCTTCTCGGACGTTCGGCCAAGGGACCAATGTGG AAATCAAA SEQ ID NO:
101 GAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG 7H7 V.sub.H DNA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACCATTAGTACC Sequence-
TCTTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT without signal
GGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATATTATGTGGA sequence
CTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC
TCACTGTATTTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTA
TATATTACTGTGCGAGAGATCGTCCAGTGGCTGGTGCGTCGGCCCTC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 102
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGG 7H7 V.sub.L DNA
AGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATAAGTGGC Sequence-
TGGTTGGCCTGGTATCAGCAGAAACAAGGGAAAGCCCCTAAGCTCC without signal
TGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTC sequence
AGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCC
TGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATTATGGT
TCTTCTCGGACGTTCGGCCAAGGGACCAATGTGGAAATCAAA SEQ ID NO: 103
GAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG 1B3 V.sub.H DNA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACCATTAGTACC Sequence-
TCTTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT without signal
GGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATATTATGTGGA sequence
CTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC
TCACTGTATTTGCAAATGAACAGCCTGAGAGTCGAAGACACGGCTA
TGTATTACTGTGCGAGAGATCGTCCAGTGGCTGGTGCGTCGGCCCTC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 104
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGG 1B3 V.sub.L DNA
AGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGGC Sequence-
TGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCC without signal
TGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTC sequence
AGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCC
TGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATTATGGT
TCTTCTCGGACGTTCGGCCAAGGGACCAATGTGGAAATCAAA SEQ ID NO: 105
GAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG 3B6 V.sub.H DNA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACAACCAGTACC Sequence-
TATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT without signal
GGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATATTATGTGGA sequence
CTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC
TCACTGAATTTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTA
TATATTACTGTGCGAGAGATCGTCCAGTGGCTGGTGCGTCGGCCCTC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 106
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGG 3B6 V.sub.L DNA
AGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGGC Sequence-
TGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCC without signal
TGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTC sequence
AGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCC
TGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATTATGGT
TCTTCTCGGACGTTCGGCCAAGGGACCAATGTGGAAATCAAA SEQ ID NO: 107
GAGGTGCGACTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG 8B1 V.sub.H DNA
GGTCCCTCjAGACTCTCCTGTGCAGCCTCTGGAGACATAATTAGTACC Sequence-
CATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT without signal
GGGTGGCCAACATAAAACAAGATGGAAGTGAGAAGTATTATGTGGA sequence
CTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC
TCACTGTATTTGCAAATGAACACCCTGAGAGTCGAGGACACGGCTA
TATATTACTGTACGAGAGATCGTCCAGTGGCTGGTGCGTCGGCCCTC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 108
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGG 8B1 V.sub.L DNA
AGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGGC Sequence-
TGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCC without signal
TGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATTAAGGTTC sequence
AGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCC
TGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATTATGGT
TCTTCTCGGACGTTCGGCCAAGGGACCAATGTGGAAATCAAA SEQ ID NO: 109
GAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGG 4A3 V.sub.H DNA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCATCATTAGTTCC Sequence-
TCTTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT without signal
GGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATATTATGTGGA sequence
CTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAAGAC
TTACTGTATTTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTTT
ATATTACTGTGCGAGAGATCGTCCAGTGGCTGGTGCGTCGGCCCTCT
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCT SEQ ID NO: 110
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGG 4A3 V.sub.L DNA
AGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGGC Sequence-
TGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCC without signal
TGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTC sequence
AGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCC
TGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATTATGGT
TCTTCTCGGACGTTCGGCCAAGGGACCAATGTGGAAATCAAA SEQ ID NO: 111
GAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG 9H9 V.sub.H DNA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAGGCATCATTAGTACC Sequence-
TATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT without signal
GGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATATTATGTGGA sequence
CTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC
TCACTGTATTTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTA
TGTATTACTGTGCGAGAGATCGTCCAGTGGCTGGTGCGTCGGCCCTC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 112
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGG 9H9 V.sub.L DNA
AGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGGC Sequence-
TGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCC without signal
TGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTC sequence
AGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCC
TGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATTATGGT
TCTTCTCGGACGTTCGGCCAAGGGACCAATGTGGAAATCAAA SEQ ID NO: 113 TGSISGY
3C2 V.sub.H CDR1 (Chothia) SEQ ID NO: 114 YYSGS 3C2 V.sub.H CDR2
(Chothia) SEQ ID NO: 115 YPLIRGAFDY 3C2 V.sub.H CDR3 (Chothia) SEQ
ID NO: 116 RSSQNLLHTNGYNYLD 3C2 Vi.CDRl (Chothia) SEQ ID NO: 117
LGSNRAS 3C2 V.sub.L CDR2 (Chothia) SEQ ID NO: 118 MQALQTPLT 3C2
V.sub.L CDR3 (Chothia) SEQ ID NO: 119 GYTFTGY 2B3V.sub.H CDR1
(Chothia) SEQ ID NO: 120 NPNSGG 2B3 V.sub.H CDR2 (Chothia) SEQ ID
NO: 121 DRLVLPWFGEIFPDAFDI 2B3 V.sub.H CDR3 (Chothia) SEQ ID NO:
122 RASQSIRSNLA 2B3 V.sub.L CDR1 (Chothia) SEQ ID NO: 123 GASTRAT
2B3 V.sub.L CDR2 (Chothia) SEQ ID NO: 124 QQYNNWPLT 2B3 V.sub.L
CDR3 (Chothia) SEQ ID NO: 125 GGTISTS 7H7 V.sub.H CDR1 (Chothia)
SEQ ID NO: 126 KQDGSE 7H7 V.sub.H CDR2 (Chothia) SEQ ID NO: 127
DRPVAGASAL 7H7 V.sub.H CDR3 (Chothia) SEQ ID NO: 128 RASQSISGWLA
7H7 V.sub.L CDR1 (Chothia) SEQ ID NO: 129 KASSLES 7H7 V.sub.L CDR2
(Chothia) SEQ ID NO: 130 QQYYGSSRT 7H7 V.sub.L CDR3 (Chothia) SEQ
ID NO: 131 GGTISTS 1B3 V.sub.H CDR1 (Chothia) SEQ ID NO: 132 KQDGSE
1B3 V.sub.H CDR2 (Chothia) SEQ ID NO: 133 DRPVAGASAL 1B3 V.sub.H
CDR3 (Chothia) SEQ ID NO: 134 RASQSISGWLA 1B3 V.sub.L CDR1
(Chothia) SEQ ID NO: 135 KASSLES 1B3 V.sub.L CDR2 (Chothia) SEQ ID
NO: 136 QQYYGSSRT 1B3 V.sub.L CDR3 (Chothia) SEQ ID NO: 137 GGTTSTY
3B6 V.sub.H CDR1 (Chothia) SEQ ID NO: 138 KQDGSE 3B6 V.sub.H CDR2
(Chothia) SEQ ID NO: 139 DRPVAGASAL 3B6 V.sub.H CDR3 (Chothia) SEQ
ID NO: 140 RASQSISGWLA 3B6 V.sub.L CDR1 (Chothia) SEQ ID NO: 141
KASSLES 3B6 V.sub.L CDR2 (Chothia) SEQ ID NO: 142 QQYYGSSRT 3B6
V.sub.L CDR3 (Chothia) SEQ ID NO: 143 GDIISTH 8B1 V.sub.H CDR1
(Chothia) SEQ ID NO: 144 KQDGSE 8B1 V.sub.H CDR2 (Chothia) SEQ ID
NO: 145 DRPVAGASAL 8B1 V.sub.H CDR3 (Chothia) SEQ ID NO: 146
RASQSISGWLA 8B1 V.sub.L CDR1 (Chothia) SEQ ID NO: 147 KASSLES 8B1
V.sub.L CDR2 (Chothia) SEQ ID NO: 148 QQYYGSSRT 8B1 V.sub.L CDR3
(Chothia) SEQ ID NO: 149 GGIISSS 4A3 V.sub.H CDR1 (Chothia) SEQ ID
NO: 150 KQDGSE 4A3 V.sub.H CDR2 (Chothia) SEQ ID NO: 151 DRPVAGASAL
4A3 V.sub.H CDR3
(Chothia) SEQ ID NO: 152 RASQSISGWLA 4A3 V.sub.L CDR1 (Chothia) SEQ
ID NO: 153 KASSLES 4A3 V.sub.L CDR2 (Chothia) SEQ ID NO: 154
QQYYGSSRT 4A3 V.sub.L CDR3 (Chothia) SEQ ID NO: 155 GGIISTY 9H9
V.sub.H CDR1 (Chothia) SEQ ID NO: 156 KQDGSE 9H9 V.sub.H CDR2
(Chothia) SEQ ID NO: 157 DRPVAGASAL 9H9 V.sub.H CDR3 (Chothia) SEQ
ID NO: 158 RASQSISGWLA 9H9 V.sub.L CDR1 (Chothia) SEQ ID NO: 159
KASSLES 9H9 V.sub.L CDR2 (Chothia) SEQ ID NO: 160 QQYYGSSRT 9H9
V.sub.L CDR3 (Chothia) SEQ ID NO: 161 GYY(W,I)(S,H) CD27 antibody
V.sub.H CDR1 consensus sequence (Kabat) SEQ ID NO: 162 (-, W)(Y,
I)N(Y, P)(Y, N)SG(S, G)TN(Y, S) CD27 antibody (N, A)(P, Q)(S, K)(L,
F)(K, Q) V.sub.H CDR2 (S, D) consensus sequence (Kabat) SEQ ID NO:
163 (-, D)(-, R)(-, L)(-, V)(-, L)(-, P) CD27 antibody (-, W)(-,
F)(-, G)(-, E)(-, I)(-Y, F) V.sub.H CDR3 P(L,-)(I,-)(R,-)(G,
D)AFD(Y, I) consensus sequence (Kabat) SEQ ID NO: 164 R(S, A)SQ(-,
S)(-, I)(-, R)(-, S)NL(L, A) CD27 antibody
(H,-)(T,-)(N,-)(G,-)(Y,-)(N,-)(Y,-) V.sub.L CDR1 (L,-)(D,-)
consensus sequence (Kabat) SEQ ID NO: 165 (L, G)(G, A)S(N, T)RA(S,
T) CD27 antibody V.sub.L CDR2 consensus sequence (Kabat) SEQ ID NO:
166 (M, Q)Q(A, Y)(L, N)(Q, N)(T, W)PLT CD27 antibody V.sub.L CDR3
consensus sequence (Kabat) SEQ ID NO: 167 (T, S)(S, Y, H)WMS PD-L1
antibody V.sub.H CDR1 consensus sequence (Kabat) SEQ ID NO: 168
NIKQDGSEKYYVDSVKG PD-L1 antibody V.sub.H CDR2 consensus sequence
(Kabat) SEQ ID NO: 169 DRPVAGASAL PD-L1 antibody V.sub.H CDR3
consensus sequence (Kabat) SEQ ID NO: 170 RASQSISGWLA PD-L1
antibody V.sub.L CDR1 consensus sequence (Kabat) SEQ ID NO: 171
KASSLES PD-L1 antibody V.sub.L CDR2 consensus sequence (Kabat) SEQ
ID NO: 172 QQYYGSSRT PD-L1 antibody V.sub.L CDR3 consensus sequence
(Kabat) SEQ ID NO: 173
MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPG CD27 amino
TFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVR acid sequence
NCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT (Accession No.
HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRI AAH12160.1)
LVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCRYSCPREEE
GSTIPIQEDYRKPEPACSP SEQ ID NO: 174
MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQL CD70 amino
PLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLR acid sequence
IHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRL (Accession No.
SFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP NP_001243) SEQ ID
NO: 175 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEG PD1 amino
acid DNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRF sequence
RVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELR (Accession No.
VTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI NP_005009)
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP
CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL SEQ ID NO: 176
MRIFAVFIFMTYWHLLNAPYNKINQRILVVDPVTSEHELTCQAEGYPKA PD-L1 amino
EVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRR acid sequence
LDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRK (isoform b
GRMMDVKKCGIQDTNSKKQSDTHLEET precursor) (Accession No. NP_001254635)
SEQ ID NO: 177 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLE 2B3
V.sub.H WMGWINPNSGGTNSAQKFQDRVTITRDTSINTAYMELSRLRSDDTAVY (modified)
FCARDRLVLPWFGEIFPDAFDIWGQGTLVTVSS SEQ ID NO: 178
EIVMTQSPATLSVSPGERATLSCRASQSIRSNLAWYQQKPGQAPRLLIY 2B3 V.sub.L
GASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFG (modified)
GGTKVEIK SEQ ID NO: 179
EVQLVESGGGLVQPGGSLRLSCAASGGIISTYWMSWVRQAPGKGLEW Full 9H9-2B3
VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRVEDTAMY (CDX-527)
YCARDRPVAGASALWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT heavy chain
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV sequence was as
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA follows (with the
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW IgG1 constant
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC region sequence
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL shown in bold)
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGGGSEIVMT
QSPATLSVSPGERATLSCRASQSIRSNLAWYQQKPGQAPRLLIYGASTR
ATGIPARFSGSGSGTEFTLTISSLQSENFAVYYCQQYNNWPLTFGCGTK
VEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTGYYIHWVRQAPGQCLEWMGWINPNSGGTNSAQKFQDRVTIT
RVTSINTAYMELSRLRSDDTAVYFCARDRLVLPWFGEIFPDAFDIWGQ GTLVTVSS SEQ ID
NO: 180 DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIY 9H9-2B3
(CDX- KASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTF 527) light
chain GQGTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV sequence was
as QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY follows (with the
ACEVTHQGLSSPVTKSFNRGEC constant region sequence shown in bold) SEQ
ID NO: 181 EVQLVESGGGLVQPGGSLRLSCAASGGIISTYWMSWVRQAPGKGLEW Full
9H9- VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRVEDTAMY 2B3(DD) heavy
YCARDRPVAGASALWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT chain sequence
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV was as follows
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA (with the IgG1
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW constant
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC "backbone"
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL sequence shown
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK in bold)
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSSGGGGSEIVMT
QSPATLSVSPGERATLSCRASQSIRSNLAWYQQKPGQAPRLLIYGASTR
ATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGCGTK
VEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTGYYIHWVRQAPGQCLEWMGWINPNSGGTNSAQKFQDRVTI
TRDTSINTAYMELSRLRSDDTAVYFCARDRLVLPWFGEIFPDAFDIWGQ GTLVTVSS SEQ ID
NO: 182 DIQMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIY
9H9-2B3(DD) KASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYGSSRTF light
chain GQGTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV sequence was
as QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY follows (with the
ACEVTHQGLSSPVTKSFNRGEC constant region shown in hold) SEQ ID NO:
183 TPAPKSCPER HYWAQGKLCC QMCEPGTFLV KDCDQHRKAA ECD of huCD27
QCDPCIPGVS FSPDHHTRPH CESCRHCNSG LLVRNCTITA NAECACRNGW QCRDKECTEC
DPLPNPSLTA RSSQALSPHP QPTHLPYVSE MLEARTAGHM QTLADFRQLP ARTLSTHWPP
QRSLCSSD SEQ ID NO: 184 TPAPKSCPER HYWAQGKLCC QMCEPGTFLV KDCDQHRKAA
ECD of huCD27 QCDPCIPGVS FSPDHHTRPH CESCRHCNSG LLVRNCTITA mutated
NAECSCAAAW QCRDKECTEC DPLPNPSLTA RSSQALSPHP QPTHLPYVSE MLEARTAGHM
QTLADFRQLP ARTLSTHWPP QRSLCSSD SEQ ID NO: 185
GAAGTGCAACTGGTGGAGTCGGGTGGTGGACTCGTGCAGCCCGGCG NA sequence of
GATCCCTGAGACTCTCTTGTGCCGCATCGGGCGGCATTATTAGCACT 9H9x2B3(DD)
TACTGGATGTCATGGGTCAGACAGGCACCGGGAAAGGGCTTGGAAT variable domain
GGGTGGCGAATATCAAGCAGGATGGATCCGAGAAGTACTACGTGGA
CTCCGTGAAGGGCAGATTCACCATTTCCCGGGACAACGCCAAGAAC
TCGCTCTATCTGCAAATGAACTCGTTGCGGGTGGAAGATACTGCCAT
GTACTACTGCGCCCGGGACCGGCCTGTGGCCGGGGCGTCGGCCCTC
TGGGGCCAGGGCACTCTGGTCACCGTGTCCTCT SEQ ID NO: 186
GGCTCCAGCGGGGGTGGCGGTTCCGAGATCGTGATGACTCAGAG NA sequence of
CCCGGCAACCCTGTCCGTGTCTCCGGGGGAGCGGGCTACTCTTTCCT 9H9x2B3(DD)
GCCGGGCATCCCAGTCCATCCGGTCGAACCTTGCGTGGTACCAACA scFv domain
GAAGCCTGGACAGGCGCCCCGCCTGCTGATCTACGGGGCGTCGACT (connector and
AGGGCCACCGGCATCCCGGCCCGCTTCTCCGGGTCCGGATCCGGCA linker sequences
CCGAATTCACCCTCACCATCTCGAGCCTGCAGTCCGAAAACTTCGCC are shown in
GTCTACTACTGCCAGCAGTACAACAACTGGCCGCTGACATTCGGAT hold)
GCGGAACCAAAGTGGAAATCAAGGGCGGCGGCGGATCCGGCGGT
GGCGGCAGCGGCGGTGGAGGATCCGGTGGCGGCGGTTCACAAG
TGCAGCTGGTGCAGTCAGGCGCCGAAGTCAAGAAGCCCGGGGCCAG
CGTGAAAGTCAGCTGCAAGGCTTCCGGATACACCTTCACGGGTTACT
ACATTCACTGGGTTCGCCAAGCGCCCGGGCAGTGTCTGGAGTGGAT
GGGATGGATCAACCCTAACTCGGGGGGAACCAACTCGGCCCAAAAG
TTCCAGGACCGGGTCACCATTACAAGAGTCACGTCCATCAACACTG
CCTACATGGAGTTGAGCCGGCTGCGATCAGACGACACCGCCGTGTA
CTTCTGCGCGAGGGACCGCCTCGTCCTCCCGTGGTTTGGAGAGATCT
TCCCGGATGCCTTCGACATTTGGGGACAGGGGACCCTCGTGACTGTG TCCAGC SEQ ID NO:
187 GATATCCAGATGACCCAGAGCCCGTCCACCCTTTCCGCGAGCGTCG NA sequence of
GCGACAGAGTGACCATTACTTGTCGGGCCTCGCAAAGCATCTCCGG
the CTGGCTGGCTTGGTACCAGCAAAAGCCTGGAAAGGCCCCTAAGCTG 9H9x2B3(DD)
CTGATCTACAAGGCCTCATCCCTGGAGTCCGGAGTGCCTTCACGCTT light chain
TTCGGGGAGCGGATCGGGGACTGAGTTCACCCTCACCATTTCCTCCC variable domain
TGCAACCCGACGATTTCGCGACATACTACTGCCAGCAGTACTACGGT
TCCTCGCGCACGTTCGGACAGGGCACTAACGTCGAGATCAAG (SEQ ID NO: 187)
Equivalents
[0584] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
18715PRTArtificial SequenceSynthetic 3C2 VH CDR1 1Gly Tyr Tyr Trp
Ser1 5216PRTArtificial SequenceSynthetic 3C2 VH CDR2 2Tyr Asn Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10
15310PRTArtificial SequenceSynthetic 3C2 VH CDR3 3Tyr Pro Leu Ile
Arg Gly Ala Phe Asp Tyr1 5 10416PRTArtificial SequenceSynthetic 3C2
VL CDR1 4Arg Ser Ser Gln Asn Leu Leu His Thr Asn Gly Tyr Asn Tyr
Leu Asp1 5 10 1557PRTArtificial SequenceSynthetic 3C2 VL CDR2 5Leu
Gly Ser Asn Arg Ala Ser1 569PRTArtificial SequenceSynthetic 3C2 VL
CDR3 6Met Gln Ala Leu Gln Thr Pro Leu Thr1 575PRTArtificial
SequenceSynthetic 2B3 VH CDR1 7Gly Tyr Tyr Ile His1
5817PRTArtificial SequenceSynthetic 2B3 VH CDR2 8Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Ser Ala Gln Lys Phe Gln1 5 10
15Asp918PRTArtificial SequenceSynthetic 2B3 VH CDR3 9Asp Arg Leu
Val Leu Pro Trp Phe Gly Glu Ile Phe Pro Asp Ala Phe1 5 10 15Asp
Ile1011PRTArtificial SequenceSynthetic 2B3 VL CDR1 10Arg Ala Ser
Gln Ser Ile Arg Ser Asn Leu Ala1 5 10117PRTArtificial
SequenceSynthetic 2B3 VL CDR2 11Gly Ala Ser Thr Arg Ala Thr1
5129PRTArtificial SequenceSynthetic 2B3 VL CDR3 12Gln Gln Tyr Asn
Asn Trp Pro Leu Thr1 513137PRTArtificial SequenceSynthetic 3C2 VH
with signal sequence 13Met Lys His Leu Trp Phe Cys Leu Leu Leu Val
Ala Ala Pro Arg Trp1 5 10 15Val Leu Ser Gln Ala Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys 20 25 30Pro Ser Glu Thr Leu Ser Leu Thr Cys
Thr Val Ser Thr Gly Ser Ile 35 40 45Ser Gly Tyr Tyr Trp Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60Glu Trp Ile Gly Tyr Asn Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro65 70 75 80Ser Leu Lys Ser Arg
Val Thr Ile Ser Ile Asp Thr Ser Lys Asn Gln 85 90 95Phe Ser Leu Lys
Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110Tyr Cys
Ala Arg Tyr Pro Leu Ile Arg Gly Ala Phe Asp Tyr Trp Gly 115 120
125Gln Gly Thr Leu Val Thr Val Ser Ser 130 13514132PRTArtificial
SequenceSynthetic 3C2 VL with signal sequence 14Met Arg Leu Pro Ala
Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser1 5 10 15Gly Ser Ser Gly
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30Val Thr Pro
Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn 35 40 45Leu Leu
His Thr Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60Pro
Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala65 70 75
80Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
85 90 95Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr 100 105 110Cys Met Gln Ala Leu Gln Thr Pro Leu Thr Phe Gly Gly
Gly Thr Lys 115 120 125Val Glu Ile Lys 13015146PRTArtificial
SequenceSynthetic 2B3 VH with signal sequence 15Met Asp Trp Thr Trp
Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1 5 10 15Ala His Ser Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Gly
Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60Glu
Trp Met Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Ser Ala65 70 75
80Gln Lys Phe Gln Asp Arg Val Thr Ile Thr Arg Val Thr Ser Ile Asn
85 90 95Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val 100 105 110Tyr Phe Cys Ala Arg Asp Arg Leu Val Leu Pro Trp Phe
Gly Glu Ile 115 120 125Phe Pro Asp Ala Phe Asp Ile Trp Gly Gln Gly
Thr Leu Val Thr Val 130 135 140Ser Ser14516127PRTArtificial
SequenceSynthetic 2B3 VL with signal sequence 16Met Glu Ala Pro Ala
Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Ser Thr Gly
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30Val Ser Pro
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45Ile Arg
Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60Arg
Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Ser Glu Asn Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Asn 100 105 110Asn Trp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 115 120 12517118PRTArtificial SequenceSynthetic 3C2 VH
without signal sequence 17Gln Ala Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser
Thr Gly Ser Ile Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Asn Tyr Tyr Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser
Ile Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Asn Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Pro
Leu Ile Arg Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser 11518112PRTArtificial SequenceSynthetic 3C2 VL
without signal sequence 18Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Asn Leu Leu His Thr 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala 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 Met Gln Ala 85 90 95Leu Gln Thr
Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11019127PRTArtificial SequenceSynthetic 2B3 VH without signal
sequence 19Gln 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 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Ser Ala Gln Lys Phe 50 55 60Gln Asp Arg Val Thr Ile Thr Arg Val Thr
Ser Ile Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Asp Arg Leu Val Leu
Pro Trp Phe Gly Glu Ile Phe Pro Asp 100 105 110Ala Phe Asp Ile Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
12520107PRTArtificial SequenceSynthetic 2B3 VL without signal
sequence 20Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile
Arg Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asn Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Asn Asn Trp Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 10521411DNAArtificial SequenceSynthetic 3C2 VH
DNA Sequence with signal sequence 21atgaaacatc tgtggttctg
ccttctcctg gtggcagctc ccagatgggt cctgtcccag 60gcgcagctgc aggagtcggg
cccaggactg gtgaagcctt cggagaccct gtccctcacc 120tgcactgtct
ctactggctc catcagtggt tactactgga gctggatccg gcagccccca
180gggaagggac tggagtggat tgggtataat tattacagtg ggagcaccaa
ctacaacccc 240tccctcaaga gtcgagtcac catatcaata gacacgtcca
agaaccagtt ctccctgaag 300ctgaattctg tgaccgctgc ggacacggcc
gtatattact gtgcgagata tcctctgatt 360cggggagctt ttgactactg
gggccaggga accctggtca ccgtctcctc a 41122396DNAArtificial
SequenceSynthetic 3C2 VL DNA Sequence with signal sequence
22atgaggctcc ctgctcagct cctggggctg ctaatgctct gggtctctgg atccagtggg
60gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc
120atctcctgta ggtctagtca gaacctcctg catactaatg gctacaacta
tttggattgg 180tacctgcaga agccagggca gtctccacag ctcctgatct
atttgggttc taatcgggcc 240tccggggtcc ctgacaggtt cagtggcagt
ggatcaggca cagattttac actgaaaatc 300agcagagtgg aggctgagga
tgttggggtt tattactgca tgcaagctct acaaactccg 360ctcactttcg
gcggagggac caaggtggag atcaaa 39623438DNAArtificial
SequenceSynthetic 2B3 VH DNA Sequence with signal sequence
23atggactgga cctggaggat cctcttcttg gtggcagcag ccacaggagc ccactcccag
60gtgcagctgg tgcagtctgg ggctgaggtg aagaagcctg gggcctcagt gaaggtctcc
120tgcaaggctt ctggatacac cttcaccggc tactatatac actgggtgcg
acaggcccct 180ggacaagggc ttgagtggat gggatggatc aaccctaaca
gtggtggcac aaactctgca 240cagaagtttc aggacagggt caccatcacc
agggtcacgt ccatcaacac agcctacatg 300gagctgagca gactgagatc
tgacgacacg gccgtgtatt tctgtgcgag agatcggctc 360gtattaccat
ggttcgggga aatattccca gatgcttttg atatctgggg ccaagggaca
420ttggtcaccg tctcttca 43824381DNAArtificial SequenceSynthetic 2B3
VL DNA Sequence with signal sequence 24atggaagccc cagcgcagct
tctcttcctc ctgctactct ggctcccaga ttccactgga 60gaaatagtga tgacgcagtc
tccagccacc ctgtctgtgt ctccagggga aagagccacc 120ctctcctgca
gggccagtca gagtattagg agcaacttag cctggtatca gcagaaacct
180ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg
tatcccagcc 240aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 300gaaaattttg cagtttatta ctgtcagcag
tataataact ggcctctcac tttcggcgga 360gggaccaagg tggagatcaa a
38125354DNAArtificial SequenceSynthetic 3C2 VH DNA Sequence without
signal sequence 25caggcgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcggagac cctgtccctc 60acctgcactg tctctactgg ctccatcagt ggttactact
ggagctggat ccggcagccc 120ccagggaagg gactggagtg gattgggtat
aattattaca gtgggagcac caactacaac 180ccctccctca agagtcgagt
caccatatca atagacacgt ccaagaacca gttctccctg 240aagctgaatt
ctgtgaccgc tgcggacacg gccgtatatt actgtgcgag atatcctctg
300attcggggag cttttgacta ctggggccag ggaaccctgg tcaccgtctc ctca
35426336DNAArtificial SequenceSynthetic 3C2 VL DNA Sequence without
signal sequence 26gatattgtga tgactcagtc tccactctcc ctgcccgtca
cccctggaga gccggcctcc 60atctcctgta ggtctagtca gaacctcctg catactaatg
gctacaacta tttggattgg 120tacctgcaga agccagggca gtctccacag
ctcctgatct atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt
cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg
aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg
300ctcactttcg gcggagggac caaggtggag atcaaa 33627381DNAArtificial
SequenceSynthetic 2B3 VH DNA Sequence without signal sequence
27caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttctggata caccttcacc ggctactata tacactgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg
cacaaactct 180gcacagaagt ttcaggacag ggtcaccatc accagggtca
cgtccatcaa cacagcctac 240atggagctga gcagactgag atctgacgac
acggccgtgt atttctgtgc gagagatcgg 300ctcgtattac catggttcgg
ggaaatattc ccagatgctt ttgatatctg gggccaaggg 360acattggtca
ccgtctcttc a 38128321DNAArtificial SequenceSynthetic 2B3 VL DNA
Sequence without signal sequence 28gaaatagtga tgacgcagtc tccagccacc
ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtattagg
agcaacttag cctggtatca gcagaaacct 120ggccaggctc ccaggctcct
catctatggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg
gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct
240gaaaattttg cagtttatta ctgtcagcag tataataact ggcctctcac
tttcggcgga 300gggaccaagg tggagatcaa a 321295PRTArtificial
SequenceSynthetic 7H7 VH CDR1 29Thr Ser Trp Met Ser1
53017PRTArtificial SequenceSynthetic 7H7 VH CDR2 30Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15Gly3110PRTArtificial SequenceSynthetic 7H7 VH CDR3 31Asp Arg Pro
Val Ala Gly Ala Ser Ala Leu1 5 103211PRTArtificial
SequenceSynthetic 7H7 VL CDR1 32Arg Ala Ser Gln Ser Ile Ser Gly Trp
Leu Ala1 5 10337PRTArtificial SequenceSynthetic 7H7 VL CDR2 33Lys
Ala Ser Ser Leu Glu Ser1 5349PRTArtificial SequenceSynthetic 7H7 VL
CDR3 34Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1 5355PRTArtificial
SequenceSynthetic 1B3 VH CDR1 35Thr Ser Trp Met Ser1
53617PRTArtificial SequenceSynthetic 1B3 VH CDR2 36Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15Gly3710PRTArtificial SequenceSynthetic 1B3 VH CDR3 37Asp Arg Pro
Val Ala Gly Ala Ser Ala Leu1 5 103811PRTArtificial
SequenceSynthetic 1B3 VL CDR1 38Arg Ala Ser Gln Ser Ile Ser Gly Trp
Leu Ala1 5 10397PRTArtificial SequenceSynthetic 1B3 VL CDR2 39Lys
Ala Ser Ser Leu Glu Ser1 5409PRTArtificial SequenceSynthetic 1B3 VL
CDR3 40Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1 5415PRTArtificial
SequenceSynthetic 3B6 VH CDR1 41Thr Tyr Trp Met Ser1
54217PRTArtificial SequenceSynthetic 3B6 VH CDR2 42Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15Gly4310PRTArtificial SequenceSynthetic 3B6 VH CDR3 43Asp Arg Pro
Val Ala Gly Ala Ser Ala Leu1 5 104411PRTArtificial
SequenceSynthetic 3B6 VL CDR1 44Arg Ala Ser Gln Ser Ile Ser Gly Trp
Leu Ala1 5 10457PRTArtificial SequenceSynthetic 3B6 VL CDR2 45Lys
Ala Ser Ser Leu Glu Ser1 5469PRTArtificial SequenceSynthetic 3B6 VL
CDR3 46Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1 5475PRTArtificial
SequenceSynthetic 8B1 VH CDR1 47Thr His Trp Met Ser1
54817PRTArtificial SequenceSynthetic 8B1 VH CDR2 48Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15Gly4910PRTArtificial SequenceSynthetic 8B1 VH CDR3 49Asp Arg Pro
Val Ala Gly Ala Ser Ala Leu1 5 105011PRTArtificial
SequenceSynthetic 8B1 VL CDR1 50Arg Ala Ser Gln Ser Ile Ser Gly Trp
Leu Ala1 5 10517PRTArtificial SequenceSynthetic 8B1 VL CDR2 51Lys
Ala Ser Ser Leu Glu Ser1 5529PRTArtificial SequenceSynthetic 8B1 VL
CDR3 52Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1 5535PRTArtificial
SequenceSynthetic 4A3 VH CDR1 53Ser Ser Trp Met Ser1
55417PRTArtificial SequenceSynthetic 4A3 VH CDR2 54Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15Gly5510PRTArtificial SequenceSynthetic 4A3 VH CDR3 55Asp Arg Pro
Val Ala Gly Ala Ser Ala Leu1 5 105611PRTArtificial
SequenceSynthetic 4A3 VL CDR1 56Arg Ala Ser Gln Ser Ile Ser Gly Trp
Leu Ala1 5 10577PRTArtificial SequenceSynthetic 4A3 VL CDR2 57Lys
Ala Ser Ser Leu Glu Ser1 5589PRTArtificial SequenceSynthetic 4A3 VL
CDR3 58Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1 5595PRTArtificial
SequenceSynthetic 9H9 VH CDR1 59Thr Tyr Trp Met Ser1
56017PRTArtificial SequenceSynthetic 9H9 VH CDR2 60Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15Gly6110PRTArtificial SequenceSynthetic 9H9 VH CDR3 61Asp Arg Pro
Val Ala Gly Ala Ser Ala Leu1 5 106211PRTArtificial
SequenceSynthetic 9H9 VL CDR1 62Arg Ala Ser Gln Ser Ile Ser Gly Trp
Leu Ala1 5
10637PRTArtificial SequenceSynthetic 9H9 VL CDR2 63Lys Ala Ser Ser
Leu Glu Ser1 5649PRTArtificial SequenceSynthetic 9H9 VL CDR3 64Gln
Gln Tyr Tyr Gly Ser Ser Arg Thr1 565138PRTArtificial
SequenceSynthetic 7H7 VH with signal
sequencemisc_feature(7)..(7)Xaa can be any naturally occurring
amino acid 65Met Glu Leu Gly Leu Ser Xaa Val Phe Leu Val Ala Ile
Leu Glu Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Gly Thr Ile 35 40 45Ser Thr Ser Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Asn Ile Lys Gln Asp
Gly Ser Glu Lys Tyr Tyr Val65 70 75 80Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Tyr Leu Gln Met
Asn Ser Leu Arg Val Glu Asp Thr Ala Ile 100 105 110Tyr Tyr Cys Ala
Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp 115 120 125Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 130 13566127PRTArtificial
SequenceSynthetic 7H7 VL with signal sequence 66Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro1 5 10 15Gly Ala Lys Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser 20 25 30Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 35 40 45Ile Ser
Gly Trp Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ala Pro 50 55 60Lys
Leu Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr 100 105 110Gly Ser Ser Arg Thr Phe Gly Gln Gly Thr Asn Val Glu
Ile Lys 115 120 12567138PRTArtificial SequenceSynthetic 1B3 VH with
signal sequence 67Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala
Ile Leu Glu Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Gly Thr Ile 35 40 45Ser Thr Ser Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val65 70 75 80Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Tyr Leu Gln
Met Asn Ser Leu Arg Val Glu Asp Thr Ala Met 100 105 110Tyr Tyr Cys
Ala Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp 115 120 125Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 130 13568127PRTArtificial
SequenceSynthetic 1B3 VL with signal sequence 68Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro1 5 10 15Gly Ala Lys Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser 20 25 30Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 35 40 45Ile Ser
Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys
Leu Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr 100 105 110Gly Ser Ser Arg Thr Phe Gly Gln Gly Thr Asn Val Glu
Ile Lys 115 120 12569138PRTArtificial SequenceSynthetic 3B6 VH with
signal sequence 69Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala
Ile Leu Glu Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Gly Thr Thr 35 40 45Ser Thr Tyr Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val65 70 75 80Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Asn Leu Gln
Met Asn Ser Leu Arg Val Glu Asp Thr Ala Ile 100 105 110Tyr Tyr Cys
Ala Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp 115 120 125Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 130 13570127PRTArtificial
SequenceSynthetic 3B6 VL with signal sequence 70Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro1 5 10 15Gly Ala Lys Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser 20 25 30Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 35 40 45Ile Ser
Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys
Leu Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr 100 105 110Gly Ser Ser Arg Thr Phe Gly Gln Gly Thr Asn Val Glu
Ile Lys 115 120 12571138PRTArtificial SequenceSynthetic 8B1 VH with
signal sequence 71Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala
Ile Leu Glu Gly1 5 10 15Val Lys Cys Glu Val Arg Leu Val Glu Ser Gly
Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Asp Ile Ile 35 40 45Ser Thr His Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val65 70 75 80Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Tyr Leu Gln
Met Asn Thr Leu Arg Val Glu Asp Thr Ala Ile 100 105 110Tyr Tyr Cys
Thr Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp 115 120 125Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 130 13572127PRTArtificial
SequenceSynthetic 8B1 VL with signal sequence 72Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro1 5 10 15Gly Ala Lys Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser 20 25 30Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 35 40 45Ile Ser
Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys
Leu Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Leu65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr 100 105 110Gly Ser Ser Arg Thr Phe Gly Gln Gly Thr Asn Val Glu
Ile Lys 115 120 12573138PRTArtificial SequenceSynthetic 4A3 VH with
signal sequence 73Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala
Ile Leu Glu Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Gly Ile Ile 35 40 45Ser Ser Ser Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val65 70 75 80Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp 85 90 95Leu Leu Tyr Leu Gln
Met Asn Ser Leu Arg Val Glu Asp Thr Ala Leu 100 105 110Tyr Tyr Cys
Ala Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp 115 120 125Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 130 13574127PRTArtificial
SequenceSynthetic 4A3 VL with signal sequence 74Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro1 5 10 15Gly Ala Lys Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser 20 25 30Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 35 40 45Ile Ser
Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys
Leu Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr 100 105 110Gly Ser Ser Arg Thr Phe Gly Gln Gly Thr Asn Val Glu
Ile Lys 115 120 12575138PRTArtificial SequenceSynthetic 9H9 VH with
signal sequence 75Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala
Ile Leu Glu Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Gly Ile Ile 35 40 45Ser Thr Tyr Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val65 70 75 80Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Tyr Leu Gln
Met Asn Ser Leu Arg Val Glu Asp Thr Ala Met 100 105 110Tyr Tyr Cys
Ala Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp 115 120 125Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 130 13576127PRTArtificial
SequenceSynthetic 9H9 VL with signal sequence 76Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro1 5 10 15Gly Ala Lys Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser 20 25 30Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 35 40 45Ile Ser
Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60Lys
Leu Leu Ile Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Tyr 100 105 110Gly Ser Ser Arg Thr Phe Gly Gln Gly Thr Asn Val Glu
Ile Lys 115 120 12577119PRTArtificial SequenceSynthetic 7H7 VH
without signal sequence 77Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Gly Thr Ile Ser Thr Ser 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Val Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Asp
Arg Pro Val Ala Gly Ala Ser Ala Leu Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 11578107PRTArtificial SequenceSynthetic 7H7
VL without signal sequence 78Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Gly Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Gln Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu
Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Ser Ser Arg 85 90 95Thr Phe
Gly Gln Gly Thr Asn Val Glu Ile Lys 100 10579119PRTArtificial
SequenceSynthetic 1B3 VH without signal sequence 79Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Gly Thr Ile Ser Thr Ser 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp Gly
Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11580107PRTArtificial SequenceSynthetic 1B3 VL without signal
sequence 80Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Gly Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Tyr Gly Ser Ser Arg 85 90 95Thr Phe Gly Gln Gly Thr Asn
Val Glu Ile Lys 100 10581119PRTArtificial SequenceSynthetic 3B6 VH
without signal sequence 81Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Gly Thr Thr Ser Thr Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Asn65 70 75 80Leu Gln Met Asn
Ser Leu Arg Val Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Asp
Arg Pro Val Ala Gly Ala Ser Ala Leu Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 11582107PRTArtificial SequenceSynthetic 3B6
VL without signal sequence 82Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Gly Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu
Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Ser Ser Arg 85 90 95Thr Phe
Gly Gln Gly Thr Asn Val Glu Ile Lys 100
10583119PRTArtificial SequenceSynthetic 8B1 VH without signal
sequence 83Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Ile Ile
Ser Thr His 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr
Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Thr Leu Arg Val
Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Thr Arg Asp Arg Pro Val Ala
Gly Ala Ser Ala Leu Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 11584107PRTArtificial SequenceSynthetic 8B1 VL without
signal sequence 84Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Gly Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Glu Ser Gly
Val Pro Leu Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Tyr Tyr Gly Ser Ser Arg 85 90 95Thr Phe Gly Gln Gly
Thr Asn Val Glu Ile Lys 100 10585119PRTArtificial SequenceSynthetic
4A3 VH without signal sequence 85Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Gly Ile Ile Ser Ser Ser 20 25 30Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asp Leu Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Val Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala
Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser 11586107PRTArtificial
SequenceSynthetic 4A3 VL without signal sequence 86Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Trp 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Ser Ser
Arg 85 90 95Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys 100
10587119PRTArtificial SequenceSynthetic 9H9 VH without signal
sequence 87Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Ile Ile
Ser Thr Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr
Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Val
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Asp Arg Pro Val Ala
Gly Ala Ser Ala Leu Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 11588107PRTArtificial SequenceSynthetic 9H9 VL without
signal sequence 88Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Gly Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Tyr Tyr Gly Ser Ser Arg 85 90 95Thr Phe Gly Gln Gly
Thr Asn Val Glu Ile Lys 100 10589414DNAArtificial SequenceSynthetic
7H7 VH DNA Sequence with signal sequencemisc_feature(21)..(21)n is
a, c, g, or t 89atggaattgg ggctgagctg ngttttcctt gttgctattt
tagaaggtgt ccagtgtgag 60gtgcaactgg tggagtctgg gggaggcttg gtccagcctg
gggggtccct gagactctcc 120tgtgcagcct ctggaggcac cattagtacc
tcttggatga gctgggtccg ccaggctcca 180gggaaggggc tggaatgggt
ggccaacata aagcaagatg gaagtgagaa atattatgtg 240gactctgtga
agggccgatt caccatctcc agagacaacg ccaagaactc actgtatttg
300caaatgaaca gcctgagagt cgaggacacg gctatatatt actgtgcgag
agatcgtcca 360gtggctggtg cgtcggccct ctggggccag ggaaccctgg
tcaccgtctc ctca 41490381DNAArtificial SequenceSynthetic 7H7 VL DNA
Sequence with signal sequence 90atgagggtcc ccgctcagct cctggggctc
ctgctgctct ggctcccagg tgccaaatgt 60gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgcc gggccagtca
gagtataagt ggctggttgg cctggtatca gcagaaacaa 180gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
240aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 300gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 360gggaccaatg tggaaatcaa a
38191414DNAArtificial SequenceSynthetic 1B3 VH DNA Sequence with
signal sequence 91atggaattgg ggctgagctg ggttttcctt gttgctattt
tagaaggtgt ccagtgtgag 60gtgcaactgg tggagtctgg gggaggcttg gtccagcctg
gggggtccct gagactctcc 120tgtgcagcct ctggaggcac cattagtacc
tcttggatga gctgggtccg ccaggctcca 180gggaaggggc tggaatgggt
ggccaacata aagcaagatg gaagtgagaa atattatgtg 240gactctgtga
agggccgatt caccatctcc agagacaacg ccaagaactc actgtatttg
300caaatgaaca gcctgagagt cgaagacacg gctatgtatt actgtgcgag
agatcgtcca 360gtggctggtg cgtcggccct ctggggccag ggaaccctgg
tcaccgtctc ctca 41492381DNAArtificial SequenceSynthetic 1B3 VL DNA
Sequence with signal sequence 92atgagggtcc ccgctcagct cctggggctc
ctgctgctct ggctcccagg tgccaaatgt 60gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 180gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
240aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 300gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 360gggaccaatg tggaaatcaa a
38193414DNAArtificial SequenceSynthetic 3B6 VH DNA Sequence with
signal sequence 93atggaattgg ggctgagctg ggttttcctt gttgctattt
tagaaggtgt ccagtgtgag 60gtgcaactgg tggagtctgg gggaggcttg gtccagcctg
gggggtccct gagactctcc 120tgtgcagcct ctggaggcac aaccagtacc
tattggatga gctgggtccg ccaggctcca 180gggaaggggc tggaatgggt
ggccaacata aagcaagatg gaagtgagaa atattatgtg 240gactctgtga
agggccgatt caccatctcc agagacaacg ccaagaactc actgaatttg
300caaatgaaca gcctgagagt cgaggacacg gctatatatt actgtgcgag
agatcgtcca 360gtggctggtg cgtcggccct ctggggccag ggaaccctgg
tcaccgtctc ctca 41494381DNAArtificial SequenceSynthetic 3B6 VL DNA
Sequence with signal sequence 94atgagggtcc ccgctcagct cctggggctc
ctgctgctct ggctcccagg tgccaaatgt 60gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 180gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
240aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 300gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 360gggaccaatg tggaaatcaa a
38195414DNAArtificial SequenceSynthetic 8B1 VH DNA Sequence with
signal sequence 95atggaattgg ggctgagctg ggttttcctt gttgctattt
tagaaggtgt caagtgtgag 60gtgcgactgg tggagtctgg gggaggcttg gtccagcctg
gggggtccct gagactctcc 120tgtgcagcct ctggagacat aattagtacc
cattggatga gctgggtccg ccaggctcca 180gggaaggggc tggaatgggt
ggccaacata aaacaagatg gaagtgagaa gtattatgtg 240gactctgtga
agggccgatt caccatctcc agagacaacg ccaagaactc actgtatttg
300caaatgaaca ccctgagagt cgaggacacg gctatatatt actgtacgag
agatcgtcca 360gtggctggtg cgtcggccct ctggggccag ggaaccctgg
tcaccgtctc ctca 41496381DNAArtificial SequenceSynthetic 8B1 VL DNA
Sequence with signal sequence 96atgagggtcc ccgctcagct cctggggctc
ctgctgctct ggctcccagg tgccaaatgt 60gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 180gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatta
240aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 300gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 360gggaccaatg tggaaatcaa a
38197414DNAArtificial SequenceSynthetic 4A3 VH DNA Sequence with
signal sequence 97atggaattgg ggctgagctg ggttttcctt gttgctattt
tagaaggtgt ccagtgtgag 60gtgcaactgg tggagtctgg gggaggcttg gtccagccgg
gggggtccct gagactctcc 120tgtgcagcct ctggaggcat cattagttcc
tcttggatga gctgggtccg ccaggctcca 180gggaaggggc tggaatgggt
ggccaacata aagcaagatg gaagtgagaa atattatgtg 240gactctgtga
agggccgatt caccatctcc agagacaacg ccaaagactt actgtatttg
300caaatgaaca gcctgagagt cgaggacacg gctttatatt actgtgcgag
agatcgtcca 360gtggctggtg cgtcggccct ctggggccag ggaaccctgg
tcaccgtctc ctct 41498381DNAArtificial SequenceSynthetic 4A3 VL DNA
Sequence with signal sequence 98atgagggtcc ccgctcagct cctggggctc
ctgctgctct ggctcccagg tgccaaatgt 60gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 180gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
240aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 300gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 360gggaccaatg tggaaatcaa a
38199414DNAArtificial SequenceSynthetic 9H9 VH DNA Sequence with
signal sequence 99atggaattgg ggctgagctg ggttttcctt gttgctattt
tagaaggtgt ccagtgtgag 60gtgcaactgg tggagtctgg gggaggcttg gtccagcctg
gggggtccct gagactctcc 120tgtgcagcct ctggaggcat cattagtacc
tattggatga gctgggtccg ccaggctcca 180gggaaggggc tggaatgggt
ggccaacata aagcaagatg gaagtgagaa atattatgtg 240gactctgtga
agggccgatt caccatctcc agagacaacg ccaagaactc actgtatttg
300caaatgaaca gcctgagagt cgaggacacg gctatgtatt actgtgcgag
agatcgtcca 360gtggctggtg cgtcggccct ctggggccag ggaaccctgg
tcaccgtctc ctca 414100381DNAArtificial SequenceSynthetic 9H9 VL DNA
Sequence with signal sequence 100atgagggtcc ccgctcagct cctggggctc
ctgctgctct ggctcccagg tgccaaatgt 60gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 120atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 180gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
240aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 300gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 360gggaccaatg tggaaatcaa a
381101357DNAArtificial SequenceSynthetic 7H7 VH DNA Sequence
without signal sequence 101gaggtgcaac tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggagg caccattagt
acctcttgga tgagctgggt ccgccaggct 120ccagggaagg ggctggaatg
ggtggccaac ataaagcaag atggaagtga gaaatattat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240ttgcaaatga acagcctgag agtcgaggac acggctatat attactgtgc
gagagatcgt 300ccagtggctg gtgcgtcggc cctctggggc cagggaaccc
tggtcaccgt ctcctca 357102321DNAArtificial SequenceSynthetic 7H7 VL
DNA Sequence without signal sequence 102gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gagtataagt ggctggttgg cctggtatca gcagaaacaa 120gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 240gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 300gggaccaatg tggaaatcaa a
321103357DNAArtificial SequenceSynthetic 1B3 VH DNA Sequence
without signal sequence 103gaggtgcaac tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggagg caccattagt
acctcttgga tgagctgggt ccgccaggct 120ccagggaagg ggctggaatg
ggtggccaac ataaagcaag atggaagtga gaaatattat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240ttgcaaatga acagcctgag agtcgaagac acggctatgt attactgtgc
gagagatcgt 300ccagtggctg gtgcgtcggc cctctggggc cagggaaccc
tggtcaccgt ctcctca 357104321DNAArtificial SequenceSynthetic 1B3 VL
DNA Sequence without signal sequence 104gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 240gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 300gggaccaatg tggaaatcaa a
321105357DNAArtificial SequenceSynthetic 3B6 VH DNA Sequence
without signal sequence 105gaggtgcaac tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggagg cacaaccagt
acctattgga tgagctgggt ccgccaggct 120ccagggaagg ggctggaatg
ggtggccaac ataaagcaag atggaagtga gaaatattat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgaat
240ttgcaaatga acagcctgag agtcgaggac acggctatat attactgtgc
gagagatcgt 300ccagtggctg gtgcgtcggc cctctggggc cagggaaccc
tggtcaccgt ctcctca 357106321DNAArtificial SequenceSynthetic 3B6 VL
DNA Sequence without signal sequence 106gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 240gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 300gggaccaatg tggaaatcaa a
321107357DNAArtificial SequenceSynthetic 8B1 VH DNA Sequence
without signal sequence 107gaggtgcgac tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggaga cataattagt
acccattgga tgagctgggt ccgccaggct 120ccagggaagg ggctggaatg
ggtggccaac ataaaacaag atggaagtga gaagtattat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240ttgcaaatga acaccctgag agtcgaggac acggctatat attactgtac
gagagatcgt 300ccagtggctg gtgcgtcggc cctctggggc cagggaaccc
tggtcaccgt ctcctca 357108321DNAArtificial SequenceSynthetic 8B1 VL
DNA Sequence without signal sequence 108gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatta
180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 240gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 300gggaccaatg tggaaatcaa a
321109357DNAArtificial SequenceSynthetic 4A3 VH DNA Sequence
without signal sequence 109gaggtgcaac tggtggagtc tgggggaggc
ttggtccagc cgggggggtc cctgagactc 60tcctgtgcag cctctggagg catcattagt
tcctcttgga tgagctgggt ccgccaggct 120ccagggaagg ggctggaatg
ggtggccaac ataaagcaag atggaagtga gaaatattat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaaaga cttactgtat
240ttgcaaatga acagcctgag agtcgaggac acggctttat attactgtgc
gagagatcgt 300ccagtggctg gtgcgtcggc cctctggggc cagggaaccc
tggtcaccgt ctcctct 357110321DNAArtificial SequenceSynthetic 4A3 VL
DNA Sequence without signal sequence 110gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca
gagtattagt ggctggttgg cctggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag
cctgcagcct 240gatgattttg caacttatta ctgccaacag tattatggtt
cttctcggac gttcggccaa 300gggaccaatg tggaaatcaa a
321111357DNAArtificial SequenceSynthetic 9H9 VH DNA Sequence
without signal sequence 111gaggtgcaac tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggagg catcattagt
acctattgga tgagctgggt ccgccaggct 120ccagggaagg ggctggaatg
ggtggccaac ataaagcaag
atggaagtga gaaatattat 180gtggactctg tgaagggccg attcaccatc
tccagagaca acgccaagaa ctcactgtat 240ttgcaaatga acagcctgag
agtcgaggac acggctatgt attactgtgc gagagatcgt 300ccagtggctg
gtgcgtcggc cctctggggc cagggaaccc tggtcaccgt ctcctca
357112321DNAArtificial SequenceSynthetic 9H9 VL DNA Sequence
without signal sequence 112gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagt
ggctggttgg cctggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct
240gatgattttg caacttatta ctgccaacag tattatggtt cttctcggac
gttcggccaa 300gggaccaatg tggaaatcaa a 3211137PRTArtificial
SequenceSynthetic 3C2 VH CDR1 (Chothia) 113Thr Gly Ser Ile Ser Gly
Tyr1 51145PRTArtificial SequenceSynthetic 3C2 VH CDR2 (Chothia)
114Tyr Tyr Ser Gly Ser1 511510PRTArtificial SequenceSynthetic 3C2
VH CDR3 (Chothia) 115Tyr Pro Leu Ile Arg Gly Ala Phe Asp Tyr1 5
1011616PRTArtificial SequenceSynthetic 3C2 VL CDR1 (Chothia) 116Arg
Ser Ser Gln Asn Leu Leu His Thr Asn Gly Tyr Asn Tyr Leu Asp1 5 10
151177PRTArtificial SequenceSynthetic 3C2 VL CDR2 (Chothia) 117Leu
Gly Ser Asn Arg Ala Ser1 51189PRTArtificial SequenceSynthetic 3C2
VL CDR3 (Chothia) 118Met Gln Ala Leu Gln Thr Pro Leu Thr1
51197PRTArtificial SequenceSynthetic 2B3 VH CDR1 (Chothia) 119Gly
Tyr Thr Phe Thr Gly Tyr1 51206PRTArtificial SequenceSynthetic 2B3
VH CDR2 (Chothia) 120Asn Pro Asn Ser Gly Gly1 512118PRTArtificial
SequenceSynthetic 2B3 VH CDR3 (Chothia) 121Asp Arg Leu Val Leu Pro
Trp Phe Gly Glu Ile Phe Pro Asp Ala Phe1 5 10 15Asp
Ile12211PRTArtificial SequenceSynthetic 2B3 VL CDR1 (Chothia)
122Arg Ala Ser Gln Ser Ile Arg Ser Asn Leu Ala1 5
101237PRTArtificial SequenceSynthetic 2B3 VL CDR2 (Chothia) 123Gly
Ala Ser Thr Arg Ala Thr1 51249PRTArtificial SequenceSynthetic 2B3
VL CDR3 (Chothia) 124Gln Gln Tyr Asn Asn Trp Pro Leu Thr1
51257PRTArtificial SequenceSynthetic 7H7 VH CDR1 (Chothia) 125Gly
Gly Thr Ile Ser Thr Ser1 51266PRTArtificial SequenceSynthetic 7H7
VH CDR2 (Chothia) 126Lys Gln Asp Gly Ser Glu1 512710PRTArtificial
SequenceSynthetic 7H7 VH CDR3 (Chothia) 127Asp Arg Pro Val Ala Gly
Ala Ser Ala Leu1 5 1012811PRTArtificial SequenceSynthetic 7H7 VL
CDR1 (Chothia) 128Arg Ala Ser Gln Ser Ile Ser Gly Trp Leu Ala1 5
101297PRTArtificial SequenceSynthetic 7H7 VL CDR2 (Chothia) 129Lys
Ala Ser Ser Leu Glu Ser1 51309PRTArtificial SequenceSynthetic 7H7
VL CDR3 (Chothia) 130Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1
51316PRTArtificial SequenceSynthetic 1B3 VH CDR1 (Chothia) 131Gly
Gly Thr Ser Thr Ser1 51326PRTArtificial SequenceSynthetic 1B3 VH
CDR2 (Chothia) 132Lys Gln Asp Gly Ser Glu1 513310PRTArtificial
SequenceSynthetic 1B3 VH CDR3 (Chothia) 133Asp Arg Pro Val Ala Gly
Ala Ser Ala Leu1 5 1013411PRTArtificial SequenceSynthetic 1B3 VL
CDR1 (Chothia) 134Arg Ala Ser Gln Ser Ile Ser Gly Trp Leu Ala1 5
101357PRTArtificial SequenceSynthetic 1B3 VL CDR2 (Chothia) 135Lys
Ala Ser Ser Leu Glu Ser1 51369PRTArtificial SequenceSynthetic 1B3
VL CDR3 (Chothia) 136Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1
51377PRTArtificial SequenceSynthetic 3B6 VH CDR1 (Chothia) 137Gly
Gly Thr Thr Ser Thr Tyr1 51386PRTArtificial SequenceSynthetic 3B6
VH CDR2 (Chothia) 138Lys Gln Asp Gly Ser Glu1 513910PRTArtificial
SequenceSynthetic 3B6 VH CDR3 (Chothia) 139Asp Arg Pro Val Ala Gly
Ala Ser Ala Leu1 5 1014011PRTArtificial SequenceSynthetic 3B6 VL
CDR1 (Chothia) 140Arg Ala Ser Gln Ser Ile Ser Gly Trp Leu Ala1 5
101417PRTArtificial SequenceSynthetic 3B6 VL CDR2 (Chothia) 141Lys
Ala Ser Ser Leu Glu Ser1 51429PRTArtificial SequenceSynthetic 3B6
VL CDR3 (Chothia) 142Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1
51437PRTArtificial SequenceSynthetic 8B1 VH CDR1 (Chothia) 143Gly
Asp Ile Ile Ser Thr His1 51446PRTArtificial SequenceSynthetic 8B1
VH CDR2 (Chothia) 144Lys Gln Asp Gly Ser Glu1 514510PRTArtificial
SequenceSynthetic 8B1 VH CDR3 (Chothia) 145Asp Arg Pro Val Ala Gly
Ala Ser Ala Leu1 5 1014611PRTArtificial SequenceSynthetic 8B1 VL
CDR1 (Chothia) 146Arg Ala Ser Gln Ser Ile Ser Gly Trp Leu Ala1 5
101477PRTArtificial SequenceSynthetic 8B1 VL CDR2 (Chothia) 147Lys
Ala Ser Ser Leu Glu Ser1 51489PRTArtificial SequenceSynthetic 8B1
VL CDR3 (Chothia) 148Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1
51497PRTArtificial SequenceSynthetic 4A3 VH CDR1 (Chothia) 149Gly
Gly Ile Ile Ser Ser Ser1 51506PRTArtificial SequenceSynthetic 4A3
VH CDR2 (Chothia) 150Lys Gln Asp Gly Ser Glu1 515110PRTArtificial
SequenceSynthetic 4A3 VH CDR3 (Chothia) 151Asp Arg Pro Val Ala Gly
Ala Ser Ala Leu1 5 1015211PRTArtificial SequenceSynthetic 4A3 VL
CDR1 (Chothia) 152Arg Ala Ser Gln Ser Ile Ser Gly Trp Leu Ala1 5
101537PRTArtificial SequenceSynthetic 4A3 VL CDR2 (Chothia) 153Lys
Ala Ser Ser Leu Glu Ser1 51549PRTArtificial SequenceSynthetic 4A3
VL CDR3 (Chothia) 154Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1
51557PRTArtificial SequenceSynthetic 9H9 VH CDR1 (Chothia) 155Gly
Gly Ile Ile Ser Thr Tyr1 51566PRTArtificial SequenceSynthetic 9H9
VH CDR2 (Chothia) 156Lys Gln Asp Gly Ser Glu1 515710PRTArtificial
SequenceSynthetic 9H9 VH CDR3 (Chothia) 157Asp Arg Pro Val Ala Gly
Ala Ser Ala Leu1 5 1015811PRTArtificial SequenceSynthetic 9H9 VL
CDR1 (Chothia) 158Arg Ala Ser Gln Ser Ile Ser Gly Trp Leu Ala1 5
101597PRTArtificial SequenceSynthetic 9H9 VL CDR2 (Chothia) 159Lys
Ala Ser Ser Leu Glu Ser1 51609PRTArtificial SequenceSynthetic 9H9
VL CDR3 (Chothia) 160Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1
51615PRTArtificial SequenceSynthetic CD27 antibody VH CDR1
consensus sequence (Kabat)misc_feature(4)..(4)X is W or
Imisc_feature(5)..(5)X is S or H 161Gly Tyr Tyr Xaa Xaa1
516217PRTArtificial SequenceSynthetic CD27 antibody VH CDR2
consensus sequence (Kabat)misc_feature(1)..(1)X is Absent or
Wmisc_feature(2)..(2)X is Y or Imisc_feature(4)..(4)X is Y or
Pmisc_feature(5)..(5)X is Y or Nmisc_feature(8)..(8)X is S or
Gmisc_feature(11)..(11)X is Y or Smisc_feature(12)..(12)X is N or
Amisc_feature(13)..(13)X is P or Qmisc_feature(14)..(14)X is S or
Kmisc_feature(15)..(15)X is L or Fmisc_feature(16)..(16)X is K or
Qmisc_feature(17)..(17)X is S or D 162Xaa Xaa Asn Xaa Xaa Ser Gly
Xaa Thr Asn Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa16321PRTArtificial
SequenceSynthetic CD27 antibody VH CDR3 consensus sequence
(Kabat)misc_feature(1)..(1)X is Absent or Dmisc_feature(2)..(2)X is
Absent or Rmisc_feature(3)..(3)X is Absent or
Lmisc_feature(4)..(4)X is Absent or Vmisc_feature(5)..(5)X is
Absent or Lmisc_feature(6)..(6)X is Absent or
Pmisc_feature(7)..(7)X is Absent or Wmisc_feature(8)..(8)X is
Absent or Fmisc_feature(9)..(9)X is Absent or
Gmisc_feature(10)..(10)X is Absent or Emisc_feature(11)..(11)X is
Absent or Imisc_feature(12)..(12)X is AbsentY or
Fmisc_feature(14)..(14)X is L or Absentmisc_feature(15)..(15)X is I
or Absentmisc_feature(16)..(16)X is R or
Absentmisc_feature(17)..(17)X is G or Dmisc_feature(21)..(21)X is Y
or I S or A 163Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
Xaa Xaa Xaa1 5 10 15Xaa Ala Phe Asp Xaa 2016420PRTArtificial
SequenceSynthetic CD27 antibody VL CDR1 consensus sequence
(Kabat)misc_feature(2)..(2)X is S or Amisc_feature(5)..(5)X is
Absent or Smisc_feature(6)..(6)X is Absent or
Imisc_feature(7)..(7)X is Absent or Rmisc_feature(8)..(8)X is
Absent or Smisc_feature(11)..(11)X is L or Amisc_feature(12)..(12)X
is H or Absentmisc_feature(13)..(13)X is T or
Absentmisc_feature(14)..(14)X is N or Absentmisc_feature(15)..(15)X
is G or Absentmisc_feature(16)..(16)X is Y or
Absentmisc_feature(17)..(17)X is N or Absentmisc_feature(18)..(18)X
is Y or Absentmisc_feature(19)..(19)X is L or
Absentmisc_feature(20)..(20)X is D or Absent 164Arg Xaa Ser Gln Xaa
Xaa Xaa Xaa Asn Leu Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa
201657PRTArtificial SequenceSynthetic CD27 antibody VL CDR2
consensus sequence (Kabat)misc_feature(1)..(1)X is L or
Gmisc_feature(2)..(2)X is G or Amisc_feature(4)..(4)X is N or
Tmisc_feature(7)..(7)X is S or T 165Xaa Xaa Ser Xaa Arg Ala Xaa1
51669PRTArtificial SequenceSynthetic CD27 antibody VL CDR3
consensus sequence (Kabat)misc_feature(1)..(1)X is M or
Qmisc_feature(3)..(3)X is A or Ymisc_feature(4)..(4)X is L or
Nmisc_feature(5)..(5)X is Q or Nmisc_feature(6)..(6)X is T or W
166Xaa Gln Xaa Xaa Xaa Xaa Pro Leu Thr1 51675PRTArtificial
SequenceSynthetic PD-L1 antibody VH CDR1 consensus sequence
(Kabat)misc_feature(1)..(1)X is T or Smisc_feature(2)..(2)X is S or
Y or H 167Xaa Xaa Trp Met Ser1 516817PRTArtificial
SequenceSynthetic PD-L1 antibody VH CDR2 consensus sequence (Kabat)
168Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1
5 10 15Gly16910PRTArtificial SequenceSynthetic PD-L1 antibody VH
CDR3 consensus sequence (Kabat) 169Asp Arg Pro Val Ala Gly Ala Ser
Ala Leu1 5 1017011PRTArtificial SequenceSynthetic PD-L1 antibody VL
CDR1 consensus sequence (Kabat) 170Arg Ala Ser Gln Ser Ile Ser Gly
Trp Leu Ala1 5 101717PRTArtificial SequenceSynthetic PD-L1 antibody
VL CDR2 consensus sequence (Kabat) 171Lys Ala Ser Ser Leu Glu Ser1
51729PRTArtificial SequenceSynthetic PD-L1 antibody VL CDR3
consensus sequence (Kabat) 172Gln Gln Tyr Tyr Gly Ser Ser Arg Thr1
5173260PRTHomo sapiensmisc_feature(1)..(260)CD27 amino acid
sequence 173Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr
Leu Val1 5 10 15Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu
Arg His Tyr 20 25 30Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu
Pro Gly Thr Phe 35 40 45Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala
Ala Gln Cys Asp Pro 50 55 60Cys Ile Pro Gly Val Ser Phe Ser Pro Asp
His His Thr Arg Pro His65 70 75 80Cys Glu Ser Cys Arg His Cys Asn
Ser Gly Leu Leu Val Arg Asn Cys 85 90 95Thr Ile Thr Ala Asn Ala Glu
Cys Ala Cys Arg Asn Gly Trp Gln Cys 100 105 110Arg Asp Lys Glu Cys
Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu 115 120 125Thr Ala Arg
Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His 130 135 140Leu
Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met145 150
155 160Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser
Thr 165 170 175His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe
Ile Arg Ile 180 185 190Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe
Thr Leu Ala Gly Ala 195 200 205Leu Phe Leu His Gln Arg Arg Lys Tyr
Arg Ser Asn Lys Gly Glu Ser 210 215 220Pro Val Glu Pro Ala Glu Pro
Cys Arg Tyr Ser Cys Pro Arg Glu Glu225 230 235 240Glu Gly Ser Thr
Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro 245 250 255Ala Cys
Ser Pro 260174193PRTHomo sapiensmisc_feature(1)..(193)CD70 amino
acid sequence 174Met Pro Glu Glu Gly Ser Gly Cys Ser Val Arg Arg
Arg Pro Tyr Gly1 5 10 15Cys Val Leu Arg Ala Ala Leu Val Pro Leu Val
Ala Gly Leu Val Ile 20 25 30Cys Leu Val Val Cys Ile Gln Arg Phe Ala
Gln Ala Gln Gln Gln Leu 35 40 45Pro Leu Glu Ser Leu Gly Trp Asp Val
Ala Glu Leu Gln Leu Asn His 50 55 60Thr Gly Pro Gln Gln Asp Pro Arg
Leu Tyr Trp Gln Gly Gly Pro Ala65 70 75 80Leu Gly Arg Ser Phe Leu
His Gly Pro Glu Leu Asp Lys Gly Gln Leu 85 90 95Arg Ile His Arg Asp
Gly Ile Tyr Met Val His Ile Gln Val Thr Leu 100 105 110Ala Ile Cys
Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu 115 120 125Ala
Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg 130 135
140Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr
Pro145 150 155 160Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr
Gly Thr Leu Leu 165 170 175Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe
Gly Val Gln Trp Val Arg 180 185 190Pro175288PRTHomo
sapiensmisc_feature(1)..(288)PD1 amino acid sequence 175Met Gln Ile
Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly
Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn
Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40
45Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu
Ala65 70 75 80Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys
Arg Phe Arg 85 90 95Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met
Ser Val Val Arg 100 105 110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu
Cys Gly Ala Ile Ser Leu 115 120 125Ala Pro Lys Ala Gln Ile Lys Glu
Ser Leu Arg Ala Glu Leu Arg Val 130 135 140Thr Glu Arg Arg Ala Glu
Val Pro Thr Ala His Pro Ser Pro Ser Pro145 150 155 160Arg Pro Ala
Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175Leu
Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185
190Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
195 200 205Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp
Tyr Gly 210 215 220Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu
Pro Pro Val Pro225 230 235 240Cys Val Pro Glu Gln Thr Glu Tyr Ala
Thr Ile Val Phe Pro Ser Gly 245 250 255Met Gly Thr Ser Ser Pro Ala
Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270Ser Ala Gln Pro Leu
Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280
285176176PRTHomo sapiensmisc_feature(1)..(176)PD-L1 amino acid
sequence (isoform b precursor) 176Met Arg Ile Phe Ala Val Phe Ile
Phe Met Thr Tyr Trp His Leu Leu1 5 10 15Asn Ala Pro Tyr Asn Lys Ile
Asn Gln Arg Ile Leu Val Val Asp Pro 20 25 30Val Thr Ser Glu His Glu
Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 35 40 45Ala Glu Val Ile Trp
Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 50 55 60Thr Thr Thr Thr
Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr65 70 75 80Ser Thr
Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 85 90 95Phe
Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 100 105
110Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His Leu Val
115 120 125Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr
Phe Ile 130 135 140Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys
Lys Cys Gly Ile145 150 155 160Gln Asp Thr Asn Ser Lys Lys Gln Ser
Asp Thr His Leu Glu Glu Thr 165 170 175177127PRTArtificial
SequenceSynthetic 2B3 VH (modified) 177Gln 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 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Ser Ala Gln Lys Phe 50 55 60Gln Asp Arg Val Thr Ile Thr Arg Asp Thr
Ser Ile Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Asp Arg Leu Val Leu
Pro Trp Phe Gly Glu Ile Phe Pro Asp 100 105 110Ala Phe Asp Ile Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
125178107PRTArtificial SequenceSynthetic 2B3 VL (modified) 178Glu
Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Arg Ser Asn
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Asn Asn Trp Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105179711PRTArtificial SequenceSynthetic Full 9H9-2B3
(CDX-527) heavy chain sequence 179Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Gly Ile Ile Ser Thr Tyr 20 25 30Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Val Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala
Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230
235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Lys Gly Ser
Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser 450 455 460Pro
Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys465 470
475 480Arg Ala Ser Gln Ser Ile Arg Ser Asn Leu Ala Trp Tyr Gln Gln
Lys 485 490 495Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser
Thr Arg Ala 500 505 510Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe 515 520 525Thr Leu Thr Ile Ser Ser Leu Gln Ser
Glu Asn Phe Ala Val Tyr Tyr 530 535 540Cys Gln Gln Tyr Asn Asn Trp
Pro Leu Thr Phe Gly Cys Gly Thr Lys545 550 555 560Val Glu Ile Lys
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 565 570 575Gly Gly
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly 580 585
590Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala
595 600 605Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Ile His Trp Val Arg
Gln Ala 610 615 620Pro Gly Gln Cys Leu Glu Trp Met Gly Trp Ile Asn
Pro Asn Ser Gly625 630 635 640Gly Thr Asn Ser Ala Gln Lys Phe Gln
Asp Arg Val Thr Ile Thr Arg 645 650 655Val Thr Ser Ile Asn Thr Ala
Tyr Met Glu Leu Ser Arg Leu Arg Ser 660 665 670Asp Asp Thr Ala Val
Tyr Phe Cys Ala Arg Asp Arg Leu Val Leu Pro 675 680 685Trp Phe Gly
Glu Ile Phe Pro Asp Ala Phe Asp Ile Trp Gly Gln Gly 690 695 700Thr
Leu Val Thr Val Ser Ser705 710180214PRTArtificial SequenceSynthetic
9H9-2B3 (CDX-527) light chain sequence 180Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly Ser Ser Arg
85 90 95Thr Phe Gly Gln Gly Thr Asn Val 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 210181711PRTArtificial SequenceSynthetic
Full 9H9-2B3(DD) heavy chain sequence 181Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Gly Ile Ile Ser Thr Tyr 20 25 30Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile
Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Asp Arg Pro Val Ala Gly Ala Ser Ala Leu Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys Gly Ser Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser
450 455 460Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu
Ser Cys465 470 475 480Arg Ala Ser Gln Ser Ile Arg Ser Asn Leu Ala
Trp Tyr Gln Gln Lys 485 490 495Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr Gly Ala Ser Thr Arg Ala 500 505 510Thr Gly Ile Pro Ala Arg Phe
Ser Gly Ser Gly Ser Gly Thr Glu Phe 515 520 525Thr Leu Thr Ile Ser
Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr 530 535 540Cys Gln Gln
Tyr Asn Asn Trp Pro Leu Thr Phe Gly Cys Gly Thr Lys545 550 555
560Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
565 570 575Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln
Ser Gly 580 585 590Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val
Ser Cys Lys Ala 595 600 605Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Ile
His Trp Val Arg Gln Ala 610 615 620Pro Gly Gln Cys Leu Glu Trp Met
Gly Trp Ile Asn Pro Asn Ser Gly625 630 635 640Gly Thr Asn Ser Ala
Gln Lys Phe Gln Asp Arg Val Thr Ile Thr Arg 645 650 655Asp Thr Ser
Ile Asn Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser 660 665 670Asp
Asp Thr Ala Val Tyr Phe Cys Ala Arg Asp Arg Leu Val Leu Pro 675 680
685Trp Phe Gly Glu Ile Phe Pro Asp Ala Phe Asp Ile Trp Gly Gln Gly
690 695 700Thr Leu Val Thr Val Ser Ser705 710182214PRTArtificial
SequenceSynthetic 9H9-2B3(DD) light chain sequence 182Asp Ile Gln
Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Trp 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Gly
Ser Ser Arg 85 90 95Thr Phe Gly Gln Gly Thr Asn Val 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 210183168PRTArtificial
SequenceSynthetic ECD of huCD27 183Thr Pro Ala Pro Lys Ser Cys Pro
Glu Arg His Tyr Trp Ala Gln Gly1 5 10 15Lys Leu Cys Cys Gln Met Cys
Glu Pro Gly Thr Phe Leu Val Lys Asp 20 25 30Cys Asp Gln His Arg Lys
Ala Ala Gln Cys Asp Pro Cys Ile Pro Gly 35 40 45Val Ser Phe Ser Pro
Asp His His Thr Arg Pro His Cys Glu Ser Cys 50 55 60Arg His Cys Asn
Ser Gly Leu Leu Val Arg Asn Cys Thr Ile Thr Ala65 70 75 80Asn Ala
Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp Lys Glu 85 90 95Cys
Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala Arg Ser 100 105
110Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro Tyr Val
115 120 125Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr
Leu Ala 130 135 140Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr
His Trp Pro Pro145 150 155 160Gln Arg Ser Leu Cys Ser Ser Asp
165184168PRTArtificial SequenceSynthetic ECD of huCD27 mutated
184Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala Gln Gly1
5 10 15Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val Lys
Asp 20 25 30Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile
Pro Gly 35 40 45Val Ser Phe Ser Pro Asp His His Thr Arg Pro His Cys
Glu Ser Cys 50 55 60Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys
Thr Ile Thr Ala65 70 75 80Asn Ala Glu Cys Ser Cys Ala Ala Ala Trp
Gln Cys Arg Asp Lys Glu 85 90 95Cys Thr Glu Cys Asp Pro Leu Pro Asn
Pro Ser Leu Thr Ala Arg Ser 100 105 110Ser Gln Ala Leu Ser Pro His
Pro Gln Pro Thr His Leu Pro Tyr Val 115 120 125Ser Glu Met Leu Glu
Ala Arg Thr Ala Gly His Met Gln Thr Leu Ala 130 135 140Asp Phe Arg
Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp Pro Pro145 150 155
160Gln Arg Ser Leu Cys Ser Ser Asp 165185357DNAArtificial
SequenceSynthetic NA sequence of 9H9x2B3(DD) variable domain
185gaagtgcaac tggtggagtc gggtggtgga ctcgtgcagc ccggcggatc
cctgagactc 60tcttgtgccg catcgggcgg cattattagc acttactgga tgtcatgggt
cagacaggca 120ccgggaaagg gcttggaatg ggtggcgaat atcaagcagg
atggatccga gaagtactac 180gtggactccg tgaagggcag attcaccatt
tcccgggaca acgccaagaa ctcgctctat 240ctgcaaatga actcgttgcg
ggtggaagat actgccatgt actactgcgc ccgggaccgg 300cctgtggccg
gggcgtcggc cctctggggc cagggcactc tggtcaccgt gtcctct
357186786DNAArtificial SequenceSynthetic NA sequence of 9H9x2B3(DD)
scFv domain 186ggctccagcg ggggtggcgg ttccgagatc gtgatgactc
agagcccggc aaccctgtcc 60gtgtctccgg gggagcgggc tactctttcc tgccgggcat
cccagtccat ccggtcgaac 120cttgcgtggt accaacagaa gcctggacag
gcgccccgcc tgctgatcta cggggcgtcg 180actagggcca ccggcatccc
ggcccgcttc tccgggtccg gatccggcac cgaattcacc 240ctcaccatct
cgagcctgca gtccgaaaac ttcgccgtct actactgcca gcagtacaac
300aactggccgc tgacattcgg atgcggaacc aaagtggaaa tcaagggcgg
cggcggatcc 360ggcggtggcg gcagcggcgg tggaggatcc ggtggcggcg
gttcacaagt gcagctggtg 420cagtcaggcg ccgaagtcaa gaagcccggg
gccagcgtga aagtcagctg caaggcttcc 480ggatacacct tcacgggtta
ctacattcac tgggttcgcc aagcgcccgg gcagtgtctg 540gagtggatgg
gatggatcaa ccctaactcg gggggaacca actcggccca aaagttccag
600gaccgggtca ccattacaag agtcacgtcc atcaacactg cctacatgga
gttgagccgg 660ctgcgatcag acgacaccgc cgtgtacttc tgcgcgaggg
accgcctcgt cctcccgtgg 720tttggagaga tcttcccgga tgccttcgac
atttggggac aggggaccct cgtgactgtg 780tccagc 786187321DNAArtificial
SequenceSynthetic NA sequence of the 9H9x2B3(DD) light chain
variable domain 187gatatccaga tgacccagag cccgtccacc ctttccgcga
gcgtcggcga cagagtgacc 60attacttgtc gggcctcgca aagcatctcc ggctggctgg
cttggtacca gcaaaagcct 120ggaaaggccc ctaagctgct gatctacaag
gcctcatccc tggagtccgg agtgccttca 180cgcttttcgg ggagcggatc
ggggactgag ttcaccctca ccatttcctc cctgcaaccc 240gacgatttcg
cgacatacta ctgccagcag tactacggtt cctcgcgcac gttcggacag
300ggcactaacg tcgagatcaa g 321
* * * * *
References