U.S. patent application number 16/687107 was filed with the patent office on 2020-03-05 for antibodies to cd40 with enhanced agonist activity.
This patent application is currently assigned to The Rockefeller University. The applicant listed for this patent is BRISTOL-MYERS SQUIBB COMPANY, The Rockefeller University. Invention is credited to Bryan C. Barnhart, Rony Dahan, Brigitte Devaux, Shannon L. Okada, Jeffrey V. Ravetch, Brenda L. Stevens, Aaron P. Yamniuk.
Application Number | 20200071412 16/687107 |
Document ID | / |
Family ID | 56497854 |
Filed Date | 2020-03-05 |
United States Patent
Application |
20200071412 |
Kind Code |
A1 |
Ravetch; Jeffrey V. ; et
al. |
March 5, 2020 |
ANTIBODIES TO CD40 WITH ENHANCED AGONIST ACTIVITY
Abstract
Provided herein are agonistic antibodies, or antigen binding
portions thereof, that bind to human CD40. Such antibodies
optionally comprise Fc regions with enhanced specificity for
Fc.gamma.RIIb. The invention also provides methods of treatment of
cancer or chronic infection by administering the antibodies of the
invention to a subject in need thereof.
Inventors: |
Ravetch; Jeffrey V.; (New
York, NY) ; Dahan; Rony; (New York, NY) ;
Barnhart; Bryan C.; (San Francisco, CA) ; Devaux;
Brigitte; (Palo Alto, CA) ; Yamniuk; Aaron P.;
(Lawrenceville, NJ) ; Okada; Shannon L.; (Seattle,
WA) ; Stevens; Brenda L.; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Rockefeller University
BRISTOL-MYERS SQUIBB COMPANY |
New York
Princeton |
NY
NJ |
US
US |
|
|
Assignee: |
The Rockefeller University
New York
NY
BRISTOL-MYERS SQUIBB COMPANY
Princeton
NJ
|
Family ID: |
56497854 |
Appl. No.: |
16/687107 |
Filed: |
November 18, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15195098 |
Jun 28, 2016 |
10479838 |
|
|
16687107 |
|
|
|
|
62303838 |
Mar 4, 2016 |
|
|
|
62252615 |
Nov 9, 2015 |
|
|
|
62186076 |
Jun 29, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/72 20130101;
A61P 37/04 20180101; A61K 2039/505 20130101; C07K 2317/75 20130101;
A61P 35/00 20180101; C07K 2317/21 20130101; C07K 2317/34 20130101;
C07K 2317/73 20130101; C07K 2317/52 20130101; C07K 16/2878
20130101; A61P 35/02 20180101; A61P 31/12 20180101; C07K 2317/92
20130101; C07K 2317/24 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. An isolated antibody, or antigen binding portion thereof, that
specifically binds to human CD40 and competes for binding to human
CD40 in a cross-blocking assay with one or more of antibodies
selected from the group consisting of 12D6 (SEQ ID NOs: 3 and 4),
5F11 (SEQ ID NOs: 23 and 24), 8E8 (SEQ ID NOs: 40 and 41), 5G7 (SEQ
ID NOs: 52 and 53), and 19G3 (SEQ ID NOs: 58 and 59).
2. The isolated antibody or antigen binding portion thereof of
claim 1, wherein the competition in a cross-blocking assay
comprises the ability to reduce binding of the selected antibody to
human CD40 in a competition ELISA by at least 20% when used at an
equal molar concentration with the selected antibody.
3. An isolated antibody, or antigen binding portion thereof, that
specifically binds to human CD40 at: a. an epitope comprising or
consisting of the sequence EPPTACREKQYLINS (residues 21-35 of SEQ
ID NO: 1) (antibodies 12D6, 5G7 and 19G3); or b. an epitope
comprising or consisting of the sequence ECLPCGESE (residues 58-66
of SEQ ID NO: 1) (antibody 5F11).
4. An isolated antibody, or antigen binding portion thereof, that
specifically binds to human CD40 comprising: a) heavy chain CDR
sequences derived at least in part from murine V region germline
VH1-39_01 and J region germline IGHJ4 and light chain CDR sequences
derived at least in part from murine V region germline VK1-110_01
and J region germline IGKJ1 (12D6); b) heavy chain CDR sequences
derived at least in part from murine V region germline VH1-4_02 and
J region germline IGHJ3 and light chain CDR sequences derived at
least in part from murine V region germline VK3-5_01 and J region
germline IGKJ5 (5F11); c) heavy chain CDR sequences derived at
least in part from murine V region germline VH1-80_01 and J region
germline IGHJ2 and light chain CDR sequences derived at least in
part from murine V region germline VK1-110_01 and J region germline
IGKJ2 (8E8); d) heavy chain CDR sequences derived at least in part
from murine V region germline VH1-18_01 and J region germline IGHJ4
and light chain CDR sequences derived at least in part from murine
V region germline VK10-96_01 and J region germline IGKJ2 (5G7); or
e) heavy chain CDR sequences derived at least in part from murine V
region germline VH5-9-4_01 and J region germline IGHJ3 and light
chain CDR sequences derived at least in part from murine V region
germline VK1-117_01 and J region germline IGKJ2 (19G3).
5. The isolated antibody or antigen binding portion thereof of
claim 1 comprising a heavy chain and a light chain, wherein the
heavy chain comprises CDRH1, CDRH2 and CDRH3 sequences and the
light chain comprises CDRL1, CDRL2 and CDRL3 sequences selected
from the group consisting of: a) the CDRs of antibody 12D6-03
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-108, respectively, of SEQ ID NO:5 and CDRL1, CDRL2 and CDRL3
comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID
NO:6; b) the CDRs of antibody 12D6-22 wherein CDRH1, CDRH2 and
CDRH3 comprise residues 31-35, 50-66 and 99-108, respectively, of
SEQ ID NO:7 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39,
55-61 and 94-102, respectively, of SEQ ID NO:9; c) the CDRs of
antibody 12D6-23 wherein CDRH1, CDRH2 and CDRH3 comprise residues
31-35, 50-66 and 99-108, respectively, of SEQ ID NO:10 and CDRL1,
CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102,
respectively, of SEQ ID NO:11; d) the CDRs of antibody 12D6-24
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-108, respectively, of SEQ ID NO:12 and CDRL1, CDRL2 and CDRL3
comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID
NO:9; e) the CDRs of antibody 5F11-17 wherein CDRH1, CDRH2 and
CDRH3 comprise residues 31-35, 50-66 and 99-106, respectively, of
SEQ ID NO:25 and CDRL1, CDRL2 and CDRL3 comprise residues 24-38,
54-60 and 93-101, respectively, of SEQ ID NO:26; f) the CDRs of
antibody 5F11-23 wherein CDRH1, CDRH2 and CDRH3 comprise residues
31-35, 50-66 and 99-106, respectively, of SEQ ID NO:27 and CDRL1,
CDRL2 and CDRL3 comprise residues 24-38, 54-60 and 93-101,
respectively, of SEQ ID NO:28; g) the CDRs of antibody 5F11-45
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-106, respectively, of SEQ ID NO:29 and CDRL1, CDRL2 and CDRL3
comprise residues 24-38, 54-60 and 93-101, respectively, of SEQ ID
NO:30; h) the CDRs of antibody 8E8-56 wherein CDRH1, CDRH2 and
CDRH3 comprise residues 31-35, 50-66 and 99-111, respectively, of
SEQ ID NO:42 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39,
55-61 and 94-102, respectively, of SEQ ID NO:43; i) the CDRs of
antibody 8E8-62 wherein CDRH1, CDRH2 and CDRH3 comprise residues
31-35, 50-66 and 99-111, respectively, of SEQ ID NO:44 and CDRL1,
CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102,
respectively, of SEQ ID NO:45; j) the CDRs of antibody 8E8-67
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-111, respectively, of SEQ ID NO:46 and CDRL1, CDRL2 and CDRL3
comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID
NO:47; k) the CDRs of antibody 8E8-70 wherein CDRH1, CDRH2 and
CDRH3 comprise residues 31-35, 50-66 and 99-111, respectively, of
SEQ ID NO:48 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39,
55-61 and 94-102, respectively, of SEQ ID NO:49; l) the CDRs of
antibody 8E8-71 wherein CDRH1, CDRH2 and CDRH3 comprise residues
31-35, 50-66 and 99-111, respectively, of SEQ ID NO:50 and CDRL1,
CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102,
respectively, of SEQ ID NO:51; m) the CDRs of antibody 5G7-22
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-102, respectively, of SEQ ID NO:54 and CDRL1, CDRL2 and CDRL3
comprise residues 24-34, 50-56 and 89-97, respectively, of SEQ ID
NO:55; n) the CDRs of antibody 5G7-25 wherein CDRH1, CDRH2 and
CDRH3 comprise residues 31-35, 50-66 and 99-102, respectively, of
SEQ ID NO:56 and CDRL1, CDRL2 and CDRL3 comprise residues 24-34,
50-56 and 89-97, respectively, of SEQ ID NO:57; o) the CDRs of
antibody 19G3-11 wherein CDRH1, CDRH2 and CDRH3 comprise residues
31-35, 50-66 and 99-101, respectively, of SEQ ID NO:60 and CDRL1,
CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102,
respectively, of SEQ ID NO:62; and p) the CDRs of antibody 19G3-22
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-101, respectively, of SEQ ID NO:63 and CDRL1, CDRL2 and CDRL3
comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID
NO:64.
6. The antibody of claim 5 comprising heavy and light chain
variable domain sequences selected from the groups consisting of:
a) the heavy and light chain variable regions of antibody 12D6-03
comprising residues 1-119 and 1-112 of SEQ ID NO:5 and SEQ ID NO:6,
respectively; b) the heavy and light chain variable regions of
antibody 12D6-22 comprising residues 1-119 and 1-112 of SEQ ID NO:7
and SEQ ID NO:9, respectively; c) the heavy and light chain
variable regions of antibody 12D6-23 comprising residues 1-119 and
1-112 of SEQ ID NO:10 and SEQ ID NO:11, respectively; d) the heavy
and light chain variable regions of antibody 12D6-24 comprising
residues 1-119 and 1-112 of SEQ ID NO:12 and SEQ ID NO:9,
respectively; e) the heavy and light chain variable regions of
antibody 5F11-17 comprising residues 1-117 and 1-111 of SEQ ID
NO:25 and SEQ ID NO:26, respectively; f) the heavy and light chain
variable regions of antibody 5F11-23 comprising residues 1-117 and
1-111 of SEQ ID NO:27 and SEQ ID NO:28, respectively; g) the heavy
and light chain variable regions of antibody 5F11-45 comprising
residues 1-117 and 1-111 of SEQ ID NO:29 and SEQ ID NO:30,
respectively; h) the heavy and light chain variable regions of
antibody 8E8-56 comprising residues 1-122 and 1-112 of SEQ ID NO:42
and SEQ ID NO:43, respectively; i) the heavy and light chain
variable regions of antibody 8E8-62 comprising residues 1-122 and
1-112 of SEQ ID NO:44 and SEQ ID NO:45, respectively; j) the heavy
and light chain variable regions of antibody 8E8-67 comprising
residues 1-122 and 1-112 of SEQ ID NO:46 and SEQ ID NO:47,
respectively; k) the heavy and light chain variable regions of
antibody 8E8-70 comprising residues 1-122 and 1-112 of SEQ ID NO:45
and SEQ ID NO:49, respectively; l) the heavy and light chain
variable regions of antibody 8E8-71 comprising residues 1-122 and
1-112 of SEQ ID NO:50 and SEQ ID NO:51, respectively; m) the heavy
and light chain variable regions of antibody 5G7-22 comprising
residues 1-113 and 1-107 of SEQ ID NO:54 and SEQ ID NO:55,
respectively; n) the heavy and light chain variable regions of
antibody 5G7-25 comprising residues 1-113 and 1-107 of SEQ ID NO:56
and SEQ ID NO:57, respectively; o) the heavy and light chain
variable regions of antibody 19G3-11 comprising residues 1-112 and
1-112 of SEQ ID NO:60 and SEQ ID NO:62, respectively; and p) the
heavy and light chain variable regions of antibody 19G3-22
comprising residues 1-112 and 1-112 of SEQ ID NO:63 and SEQ ID
NO:64, respectively.
7. The antibody of claim 1 wherein the antibody further comprises
an Fc region modified to enhance specificity of binding to
Fc.gamma.RIIb.
8. The antibody of claim 7 exhibiting an A/I ratio of less than
5.
9. The antibody of claim 8 exhibiting an A/I ratio of less than
1.
10. The antibody of claim 7 wherein the modified Fc region is in a
heavy chain constant region selected from the group consisting of
SE (SEQ ID NO: 66), SELF (SEQ ID NO: 67), P238D (SEQ ID NO: 68), V4
(SEQ ID NO: 69), V4 D270E (SEQ ID NO: 70), V7 (SEQ ID NO: 71), V8
(SEQ ID NO: 72), V9 (SEQ ID NO: 73), V9 D270E (SEQ ID NO: 74), V11
(SEQ ID NO: 75), and V12 (SEQ ID NO: 76).
11. The antibody of claim 5 comprising heavy and light chain
sequences selected from the groups consisting of: a) the heavy and
light chains of antibody 12D6-24-P238D comprising the sequences of
SEQ ID NO:13 and SEQ ID NO:9, respectively; b) the heavy and light
chains of antibody 12D6-24-SE comprising the sequences of SEQ ID
NO:14 and SEQ ID NO:9, respectively; c) the heavy and light chains
of antibody 12D6-24-SELF comprising the sequences of SEQ ID NO:15
and SEQ ID NO:9, respectively; d) the heavy and light chains of
antibody 12D6-24-V4 comprising the sequences of SEQ ID NO:16 and
SEQ ID NO:9, respectively; e) the heavy and light chains of
antibody 12D6-24-V4 D270E comprising the sequences of SEQ ID NO:17
and SEQ ID NO:9, respectively; f) the heavy and light chains of
antibody 12D6-24-V8 comprising the sequences of SEQ ID NO:18 and
SEQ ID NO:9, respectively; g the heavy and light chains of antibody
12D6-24-V9 comprising the sequences of SEQ ID NO:19 and SEQ ID
NO:9, respectively; h) the heavy and light chains of antibody
12D6-24-V9 D270E comprising the sequences of SEQ ID NO:20 and SEQ
ID NO:9, respectively; i) the heavy and light chains of antibody
12D6-24-V11 comprising the sequences of SEQ ID NO:21 and SEQ ID
NO:9, respectively; j) the heavy and light chains of antibody
12D6-24-V12 comprising the sequences of SEQ ID NO:22 and SEQ ID
NO:9, respectively; k) the heavy and light chains of antibody
5F11-45-SE comprising the sequences of SEQ ID NO:31 and SEQ ID
NO:30, respectively; l) the heavy and light chains of antibody
5F11-45-SELF comprising the sequences of SEQ ID NO:32 and SEQ ID
NO:30, respectively; m) the heavy and light chains of antibody
5F11-45-V4 comprising the sequences of SEQ ID NO:33 and SEQ ID
NO:30, respectively; n) the heavy and light chains of antibody
5F11-45-V4 D270E comprising the sequences of SEQ ID NO:34 and SEQ
ID NO:30, respectively; o) the heavy and light chains of antibody
5F11-45 V8 comprising the sequences of SEQ ID NO:35 and SEQ ID
NO:30, respectively; p) the heavy and light chains of antibody
5F11-45-V9 comprising the sequences of SEQ ID NO:36 and SEQ ID
NO:30, respectively; q) the heavy and light chains of antibody
5F11-45-V9 D270E comprising the sequences of SEQ ID NO:37 and SEQ
ID NO:30, respectively; r) the heavy and light chains of antibody
5F11-45-V11 comprising the sequences of SEQ ID NO:38 and SEQ ID
NO:30, respectively; and s) the heavy and light chains of antibody
5F11-45-V12 comprising the sequences of SEQ ID NO:39 and SEQ ID
NO:30, respectively.
12. A nucleic acid encoding the heavy and/or light chain variable
region of the antibody, or antigen binding portion thereof, of
claim 1.
13. An expression vector comprising the nucleic acid molecule of
claim 12.
14. A cell transformed with an expression vector of claim 13.
15. A method of preparing an anti-human CD40 antibody, or antigen
binding portion thereof, comprising: a) expressing the antibody, or
antigen binding portion thereof, in the cell of claim 14; and b)
isolating the antibody, or antigen binding portion thereof, from
the cell.
16. A pharmaceutical composition comprising: a) the antibody, or
antigen binding portion thereof, of claim 1; and b) a carrier.
17. A method of stimulating an immune response in a subject
comprising administering to the subject the pharmaceutical
composition of claim 16.
18. The method of claim 17, wherein the subject has a tumor and an
immune response against the tumor is stimulated.
19. The method of claim 17, wherein the subject has a chronic viral
infection and an immune response against the viral infection is
stimulated.
20. A method of treating cancer comprising administering to a
subject in need thereof a therapeutically effective amount of the
pharmaceutical composition of claim 16.
21. The method of claim 20, wherein the cancer is selected from the
group consisting of: bladder cancer, breast cancer,
uterine/cervical cancer, ovarian cancer, prostate cancer,
testicular cancer, esophageal cancer, gastrointestinal cancer,
pancreatic cancer, colorectal cancer, colon cancer, kidney cancer,
head and neck cancer, lung cancer, stomach cancer, germ cell
cancer, bone cancer, liver cancer, thyroid cancer, skin cancer,
neoplasm of the central nervous system, lymphoma, leukemia,
myeloma, sarcoma, and virus-related cancer.
22. A method of treating a chronic viral infection comprising
administering to a subject in need thereof a therapeutically
effective amount of the pharmaceutical composition of claim 16.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 15/195,098, filed Jun. 28, 2016, which claims priority to
U.S. Provisional Application Nos. 62/303,838, filed Mar. 4, 2016,
62/252,615, filed Nov. 9, 2015 and 62/186,076, filed Jun. 29, 2015,
the disclosures of which are all incorporated herein by
reference.
BACKGROUND
[0002] Recent research has revealed that human cancers and chronic
infections may be treated with agents that modulate the patient's
immune response to malignant or infected cells. See, e.g., Reck
& Paz-Ares (2015) Semin. Oncol. 42:402. Agonistic anti-CD40
antibodies, such as CP-870893 and dacetuzumab (SGN-40) have been
tried for treating cancer based on the belief that they may enhance
such an immune response. See, e.g., Kirkwood et al. (2012) CA
Cancer J. Clin. 62:309; Vanderheide & Glennie (2013) Clin.
Cancer Res. 19:1035. Recent experiments in mice have revealed that
anti-CD40 antibodies with enhanced specificity for the inhibitory
Fc receptor Fc.gamma.RIIb have increased anti-tumor efficacy. See,
e.g., WO 2012/087928; Li & Ravetch (2011) Science 333:1030; Li
& Ravetch (2012) Proc. Nat'l Acad. Sci (USA) 109:10966; Wilson
et al. (2011) Cancer Cell 19:101; White et al. (2011) J. Immunol.
187:1754.
[0003] The need exists for improved agonistic anti-human CD40
antibodies for treatment of cancer and chronic infections in human
subjects. Such antibodies will preferably have enhanced specificity
for the inhibitory Fc receptor Fc.gamma.RIIb as compared to
activating Fc receptors, and will exhibit enhanced anti-tumor
and/or anti-infective activity.
SUMMARY OF THE INVENTION
[0004] Provided herein are isolated humanized murine monoclonal
antibodies that specifically bind to human CD40 (the mature
sequence of SEQ ID NO: 1), optionally having modified Fc regions
that enhance specificity for binding to Fc.gamma.RIIb receptor.
[0005] In certain embodiments, the invention relates to anti-huCD40
antibodies or antigen binding fragments thereof that compete for
binding with, cross-block, or bind to the same epitope as, one or
more of antibodies 12D6 (SEQ ID NOs: 3 and 4), 5F11 (SEQ ID NOs: 23
and 24), 8E8 (SEQ ID NOs: 40 and 41), 5G7 (SEQ ID NOs: 52 and 53),
and 19G3 (SEQ ID NOs: 58 and 59), including human or humanized
antibodies.
[0006] In certain embodiments, the anti-human CD40 antibodies of
the present invention, or antigen binding fragments thereof, bind
at an epitope comprising or consisting of one or more sequences
selected from the group consisting of WGCLLTAVHPEPPTACRE (residues
11-28 of SEQ ID NO: 1) (antibody 12D6), EPPTACREKQYLINS (residues
21-35 of SEQ ID NO: 1) (antibodies 12D6, 5G7 and 19G3), and
ECLPCGESE (residues 58-66 of SEQ ID NO: 1) (antibody 5F11).
[0007] In some embodiments the antibody of the present invention
comprises a heavy chain and a light chain, wherein the heavy chain
comprises CDRH1, CDRH2 and CDRH3 sequences and the light chain
comprises CDRL1, CDRL2 and CDRL3 sequences derived at least in part
from the same mouse germline V region gene segments and J region
gene segments as anti-huCD40 antibody 12D6, 5F11, 8E8, 5G7 or 19G3,
as disclosed at Table 3. Specifically, the antibody may comprise
CDR sequences derived from the same murine germlines as antibody
12D6 (heavy chain CDR sequences derived at least in part from
murine V region germline VH1-39_01 and J region germline IGHJ4 and
light chain CDR sequences derived at least in part from murine V
region germline VK1-110_01 and J region germline IGKJ1), antibody
5F11 (heavy chain CDR sequences derived at least in part from
murine V region germline VH1-4_02 and J region germline IGHJ3 and
light chain CDR sequences derived at least in part from murine V
region germline VK3-5_01 and J region germline IGKJ5), antibody 8E8
(heavy chain CDR sequences derived at least in part from murine V
region germline VH1-80_01 and J region germline IGHJ2 and light
chain CDR sequences derived at least in part from murine V region
germline VK1-110_01 and J region germline IGKJ2), antibody 5G7
(heavy chain CDR sequences derived at least in part from murine V
region germline VH1-18_01 and J region germline IGHJ4 and light
chain CDR sequences derived at least in part from murine V region
germline VK10-96_01 and J region germline IGKJ2), or antibody 19G3
(heavy chain CDR sequences derived at least in part from murine V
region germline VH5-9-4_01 and J region germline IGHJ3 and light
chain CDR sequences derived at least in part from murine V region
germline VK1-117_01 and J region germline IGKJ2).
[0008] In various embodiments the antibody of the present invention
comprises a heavy chain and a light chain, wherein the heavy chain
comprises CDRH1, CDRH2 and CDRH3 sequences and the light chain
comprises CDRL1, CDRL2 and CDRL3 sequences selected from the group
consisting of: the CDRs of antibody 12D6-03 wherein CDRH1, CDRH2
and CDRH3 comprise residues 31-35, 50-66 and 99-108, respectively,
of SEQ ID NO:5 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39,
55-61 and 94-102, respectively, of SEQ ID NO:6; the CDRs of
antibody 12D6-22 wherein CDRH1, CDRH2 and CDRH3 comprise residues
31-35, 50-66 and 99-108, respectively, of SEQ ID NO:7 and CDRL1,
CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102,
respectively, of SEQ ID NO:9; the CDRs of antibody 12D6-23 wherein
CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-108,
respectively, of SEQ ID NO:10 and CDRL1, CDRL2 and CDRL3 comprise
residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:11;
the CDRs of antibody 12D6-24 wherein CDRH1, CDRH2 and CDRH3
comprise residues 31-35, 50-66 and 99-108, respectively, of SEQ ID
NO:12 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and
94-102, respectively, of SEQ ID NO:9; the CDRs of antibody 5F11-17
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-106, respectively, of SEQ ID NO:25 and CDRL1, CDRL2 and CDRL3
comprise residues 24-38, 54-60 and 93-101, respectively, of SEQ ID
NO:26; the CDRs of antibody 5F11-23 wherein CDRH1, CDRH2 and CDRH3
comprise residues 31-35, 50-66 and 99-106, respectively, of SEQ ID
NO:27 and CDRL1, CDRL2 and CDRL3 comprise residues 24-38, 54-60 and
93-101, respectively, of SEQ ID NO:28; the CDRs of antibody 5F11-45
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-106, respectively, of SEQ ID NO:29 and CDRL1, CDRL2 and CDRL3
comprise residues 24-38, 54-60 and 93-101, respectively, of SEQ ID
NO:30; the CDRs of antibody 8E8-56 wherein CDRH1, CDRH2 and CDRH3
comprise residues 31-35, 50-66 and 99-111, respectively, of SEQ ID
NO:42 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and
94-102, respectively, of SEQ ID NO:43; the CDRs of antibody 8E8-62
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-111, respectively, of SEQ ID NO:44 and CDRL1, CDRL2 and CDRL3
comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID
NO:45; the CDRs of antibody 8E8-67 wherein CDRH1, CDRH2 and CDRH3
comprise residues 31-35, 50-66 and 99-111, respectively, of SEQ ID
NO:46 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and
94-102, respectively, of SEQ ID NO:47; the CDRs of antibody 8E8-70
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-111, respectively, of SEQ ID NO:48 and CDRL1, CDRL2 and CDRL3
comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID
NO:49; the CDRs of antibody 8E8-71 wherein CDRH1, CDRH2 and CDRH3
comprise residues 31-35, 50-66 and 99-111, respectively, of SEQ ID
NO:50 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and
94-102, respectively, of SEQ ID NO:51; the CDRs of antibody 5G7-22
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-102, respectively, of SEQ ID NO:54 and CDRL1, CDRL2 and CDRL3
comprise residues 24-34, 50-56 and 89-97, respectively, of SEQ ID
NO:55; the CDRs of antibody 5G7-25 wherein CDRH1, CDRH2 and CDRH3
comprise residues 31-35, 50-66 and 99-102, respectively, of SEQ ID
NO:56 and CDRL1, CDRL2 and CDRL3 comprise residues 24-34, 50-56 and
89-97, respectively, of SEQ ID NO:57; the CDRs of antibody 19G3-11
wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and
99-101, respectively, of SEQ ID NO:60 and CDRL1, CDRL2 and CDRL3
comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID
NO:62; and the CDRs of antibody 19G3-22 wherein CDRH1, CDRH2 and
CDRH3 comprise residues 31-35, 50-66 and 99-101, respectively, of
SEQ ID NO:63 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39,
55-61 and 94-102, respectively, of SEQ ID NO:64.
[0009] In various embodiments the antibody of the present invention
comprises a heavy chain comprising a variable domain selected from
the group consisting of 12D6 (residues 1-119 of SEQ ID NO: 3), 5F11
(residues 1-117 of SEQ ID NO: 23), 8E8 (residues 1-122 of SEQ ID
NO: 40), 5G7 (residues 1-113 of SEQ ID NO: 52) and 19G3 (residues
1-112 of SEQ ID NO: 58) with constant regions comprising
Fc.gamma.RIIb-specific Fc region selected from the group consisting
of IgG1f (SEQ ID NO: 65), SE (SEQ ID NO: 66), SELF (SEQ ID NO: 67),
P238D (SEQ ID NO: 68), V4 (SEQ ID NO: 69), V4 D270E (SEQ ID NO:
70), V7 (SEQ ID NO: 71), V8 (SEQ ID NO: 72), V9 (SEQ ID NO: 73), V9
D270E (SEQ ID NO: 74), V11 (SEQ ID NO: 75), and V12 (SEQ ID NO:
76).
[0010] In some embodiments the antibody comprises specific heavy
chain variable domains and light chain variable domains selected
from the group consisting of 12D6-03 (residues 1-119 and 1-112 of
SEQ ID NO:5 and SEQ ID NO:6, respectively), 12D6-22 (residues 1-119
and 1-112 of SEQ ID NO:7 and SEQ ID NO:9, respectively), 12D6-23
(residues 1-119 and 1-112 of SEQ ID NO:10 and SEQ ID NO:11,
respectively), 12D6-24 (residues 1-119 and 1-112 of SEQ ID NO:12
and SEQ ID NO:9, respectively), 5F11-17 (residues 1-117 and 1-111
of SEQ ID NO:25 and SEQ ID NO:26, respectively), 5F11-23 (residues
1-117 and 1-111 of SEQ ID NO:27 and SEQ ID NO:28, respectively),
5F11-45 (residues 1-117 and 1-111 of SEQ ID NO:29 and SEQ ID
NO:30), 8E8-56 (residues 1-122 and 1-112 of SEQ ID NO:42 and SEQ ID
NO:43, respectively), 8E8-62 (residues 1-122 and 1-112 of SEQ ID
NO:44 and SEQ ID NO:45, respectively), 8E8-67 (residues 1-122 and
1-112 of SEQ ID NO:46 and SEQ ID NO:47, respectively), 8E8-70
(residues 1-122 and 1-112 of SEQ ID NO:48 and SEQ ID NO:49), 8E8-71
(residues 1-122 and 1-112 of SEQ ID NO:50 and SEQ ID NO:51,
respectively), 5G7-22 (residues 1-113 and 1-107 of SEQ ID NO:54 and
SEQ ID NO:55, respectively), 5G7-25 (residues 1-113 and 1-107 of
SEQ ID NO:56 and SEQ ID NO:57, respectively), 19G3-11 (residues
1-112 and 1-112 of SEQ ID NO:60 and SEQ ID NO:62, respectively),
and 9G3-22 (residues 1-112 and 1-112 of SEQ ID NO:63 and SEQ ID
NO:64, respectively). Any of these antibodies may further comprise
a heavy chain constant region comprising an Fc.gamma.RIIb-specific
Fc region, said heavy chain constant region selected from the group
consisting of IgG1f (SEQ ID NO: 65), SE (SEQ ID NO: 66), SELF (SEQ
ID NO: 67), P238D (SEQ ID NO: 68), V4 (SEQ ID NO: 69), V4 D270E
(SEQ ID NO: 70), V7 (SEQ ID NO: 71), V8 (SEQ ID NO: 72), V9 (SEQ ID
NO: 73), V9 D270E (SEQ ID NO: 74), V11 (SEQ ID NO: 75), and V12
(SEQ ID NO: 76). Any of these antibodies may further comprise the
light chain kappa constant region of SEQ ID NO: 77.
[0011] In specific embodiments, the antibody of the present
invention comprises a humanized 12D6-24 antibody comprising a light
chain of SEQ ID NO: 9 and a heavy chain selected from the group
consisting of any of SEQ ID NOs: 13-22, or a humanized 5F11-45
antibody comprising a light chain of SEQ ID NO: 30 and a heavy
chain selected from the group consisting of any of SEQ ID NOs:
31-39. Specific antibodies include 12D6-24 SE (SEQ ID NOs: 9 and
14), 12D6-24 SELF (SEQ ID NOs: 9 and 15), 12D6-24 P238D (SEQ ID
NOs: 9 and 13), 12D6-24 V4 (SEQ ID NOs: 9 and 16), 12D6-24 V4 D270E
(SEQ ID NOs: 9 and 17), 12D6-24 V8 (SEQ ID NOs: 9 and 18), 12D6-24
V9 (SEQ ID NOs: 9 and 19), 12D6-24 V9 D270E (SEQ ID NOs: 9 and 20),
12D6-24 V11 (SEQ ID NOs: 9 and 21), 12D6-24 V12 (SEQ ID NOs: 9 and
22), 5F11-45 SE (SEQ ID NOs: 30 and 31), 5F11-45 SELF (SEQ ID NOs:
30 and 32), 5F11-45 V4 (SEQ ID NOs: 30 and 33), 5F11-45 V4 D270E
(SEQ ID NOs: 30 and 34), 5F11-45 V8 (SEQ ID NOs: 30 and 35),
5F11-45 V9 (SEQ ID NOs: 30 and 36), 5F11-45 V9 D270E (SEQ ID NOs:
30 and 37), 5F11-45 V11 (SEQ ID NOs: 30 and 38), and 5F11-45 V12
(SEQ ID NOs: 30 and 39), where sequences are provided for light and
heavy chains, respectively.
[0012] In further embodiments the anti-huCD40 antibodies of the
present comprise heavy and light chains sharing at least 80%, 85%,
90% and 95% sequence identity with the sequences of the heavy and
light chains of 12D6-24 SE (SEQ ID NOs: 9 and 14), 12D6-24 SELF
(SEQ ID NOs: 9 and 15), 12D6-24 P238D (SEQ ID NOs: 9 and 13),
12D6-24 V4 (SEQ ID NOs: 9 and 16), 12D6-24 V4 D270E (SEQ ID NOs: 9
and 17), 12D6-24 V8 (SEQ ID NOs: 9 and 18), 12D6-24 V9 (SEQ ID NOs:
9 and 19), 12D6-24 V9 D270E (SEQ ID NOs: 9 and 20), 12D6-24 V11
(SEQ ID NOs: 9 and 21), 12D6-24 V12 (SEQ ID NOs: 9 and 22), 5F11-45
SE (SEQ ID NOs: 30 and 31), 5F11-45 SELF (SEQ ID NOs: 30 and 32),
5F11-45 V4 (SEQ ID NOs: 30 and 33), 5F11-45 V4 D270E (SEQ ID NOs:
30 and 34), 5F11-45 V8 (SEQ ID NOs: 30 and 35), 5F11-45 V9 (SEQ ID
NOs: 30 and 36), 5F11-45 V9 D270E (SEQ ID NOs: 30 and 37), 5F11-45
V11 (SEQ ID NOs: 30 and 38), or 5F11-45 V12 (SEQ ID NOs: 30 and
39).
[0013] In yet further embodiments the anti-huCD40 antibodies of the
present comprise heavy and light chains consisting essentially of
the sequences of the heavy and light chains of 12D6-24 SE (SEQ ID
NOs: 9 and 14), 12D6-24 SELF (SEQ ID NOs: 9 and 15), 12D6-24 P238D
(SEQ ID NOs: 9 and 13), 12D6-24 V4 (SEQ ID NOs: 9 and 16), 12D6-24
V4 D270E (SEQ ID NOs: 9 and 17), 12D6-24 V8 (SEQ ID NOs: 9 and 18),
12D6-24 V9 (SEQ ID NOs: 9 and 19), 12D6-24 V9 D270E (SEQ ID NOs: 9
and 20), 12D6-24 V11 (SEQ ID NOs: 9 and 21), 12D6-24 V12 (SEQ ID
NOs: 9 and 22), 5F11-45 SE (SEQ ID NOs: 30 and 31), 5F11-45 SELF
(SEQ ID NOs: 30 and 32), 5F11-45 V4 (SEQ ID NOs: 30 and 33),
5F11-45 V4 D270E (SEQ ID NOs: 30 and 34), 5F11-45 V8 (SEQ ID NOs:
30 and 35), 5F11-45 V9 (SEQ ID NOs: 30 and 36), 5F11-45 V9 D270E
(SEQ ID NOs: 30 and 37), 5F11-45 V11 (SEQ ID NOs: 30 and 38), or
5F11-45 V12 (SEQ ID NOs: 30 and 39).
[0014] In some embodiments, anti-huCD40 antibodies of the present
invention that comprise V4 or V9 Fc sequence variants further
comprise the D270E sequence variant. Such antibodies include a
humanized 12D6-24 V4 D270E (SEQ ID NOs: 9 and 17), 12D6-24 V9 D270E
(SEQ ID NOs: 9 and 20), 5F11-45 V4 D270E (SEQ ID NOs: 30 and 34),
and 5F11-45 V9 D270E (SEQ ID NOs: 30 and 37), where sequences are
provided for light and heavy chains, respectively. In alternative
embodiments, anti-human CD40 antibodies of the present invention
include antibodies comprising heavy and light chains consisting
essentially of the sequences of these heavy and light chains, or
comprise heavy and light chains sharing at least 80%, 85%, 90% and
95% sequence identity with these sequences. In some embodiments,
the anti-huCD40 antibodies of the present invention comprise
modified Fc regions with greater specificity for binding to
Fc.gamma.RIIb as opposed to binding to activating receptors than
antibodies with naturally occurring Fc regions. In certain
embodiments the A/I ratio for the anti-huCD40 antibody of the
present invention is less than 5, and in preferred embodiments,
less than 1.
[0015] In some embodiments the anti-huCD40 antibody of the present
invention comprises one or more heavy chains and one or more light
chains, such as two heavy chains and two light chains.
[0016] The present invention further provides nucleic acids
encoding the heavy and/or light chain variable regions, of the
anti-CD40 antibodies of the present invention, or antigen binding
fragments thereof, expression vectors comprising the nucleic acid
molecules, cells transformed with the expression vectors, and
methods of producing the antibodies by expressing the antibodies
from cells transformed with the expression vectors and recovering
the antibody.
[0017] The present invention also provides pharmaceutical
compositions comprising anti-huCD40 antibodies of the present
invention, or antigen binding fragments thereof, and a carrier.
[0018] The present invention provides a method of enhancing an
immune response in a subject comprising administering an effective
amount of an anti-huCD40 antibody of the present invention, or
antigen binding fragment thereof, to the subject such that an
immune response in the subject is enhanced. In certain embodiments,
the subject has a tumor and an immune response against the tumor is
enhanced. In another embodiment, the subject has a viral infection,
e.g. a chronic viral infection, and an anti-viral immune response
is enhanced.
[0019] The present invention also provides a method of inhibiting
the growth of tumors in a subject comprising administering to the
subject an anti-huCD40 antibody of the present invention, or
antigen binding fragment thereof, such that growth of the tumor is
inhibited.
[0020] The present invention further provides a method of treating
cancer, e.g., by immunotherapy, comprising administering to a
subject in need thereof a therapeutically effective amount an
anti-huCD40 antibody of the present invention, or antigen binding
fragment thereof, e.g. as a pharmaceutical composition, thereby
treating the cancer. In certain embodiments, the cancer is bladder
cancer, breast cancer, uterine/cervical cancer, ovarian cancer,
prostate cancer, testicular cancer, esophageal cancer,
gastrointestinal cancer, pancreatic cancer, colorectal cancer,
colon cancer, kidney cancer, head and neck cancer, lung cancer,
stomach cancer, germ cell cancer, bone cancer, liver cancer,
thyroid cancer, skin cancer, neoplasm of the central nervous
system, lymphoma, leukemia, myeloma, sarcoma, and virus-related
cancer. In certain embodiments, the cancer is a metastatic cancer,
refractory cancer, or recurrent cancer.
[0021] In certain embodiments, the methods of modulating immune
function and methods of treatment described herein comprise
administering an anti-huCD40 antibody of the present invention in
combination with, or as a bispecific reagent with, one or more
additional therapeutics, for example, an anti-PD1 antibody, an
anti-PD-L1 antibody, an anti-LAG3 antibody, an anti-GITR antibody,
an anti-OX40 antibody, an anti-CD73 antibody, an anti-TIGIT
antibody, an anti-CD137 antibody, an anti-CD27 antibody, an
anti-CSF-1R antibody, an anti-CTLA-4 antibody, a TLR agonist, or a
small molecule antagonist of IDO or TGF.beta.. In specific
embodiments, anti-huCD40 therapy is combined with anti-PD1 and/or
anti-PD-L1 therapy, e.g. treatment with an antibody or antigen
binding fragment thereof that binds to human PD1 or an antibody or
antigen binding fragment thereof that binds to human PD-L1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows the sequence of human IgG1f constant domain
(SEQ ID NO: 65) renumbered 118-446 to better illustrate the Fc
sequence variants disclosed herein (Table 4). Residues subject to
variation are in bold, and the altered amino acid is provided in
bold below the residue. The D270E substitution is underlined. A
C-terminal lysine (K) residue has been removed in FIG. 1 and SEQ ID
NO: 65, as well as all other heavy chain and heavy chain constant
domain sequences disclosed in the Sequence Listing. However, in
other embodiments, especially nucleic acid constructs encoding the
heavy chains and heavy chain constant domains of the anti-huCD40
antibodies of the present invention, these sequences include an
additional lysine residue at the C-terminus of the protein or
nucleotides encoding the extra lysine at the 3' end of the nucleic
acid.
[0023] FIG. 2 is a Venn diagram illustrating the epitope groups
("bins") on human CD40 bound by the antibodies of the present
invention, as well as blocking of CD40L binding. Antibodies with
overlapping ovals or circles compete for binding to human CD40, and
antibodies falling within the rectangle block CD40L binding to
human CD40.
[0024] FIGS. 3A and 3B show activation of dendritic cells, as
measured by IL-6 secretion, by agonist anti-CD40 antibodies as a
function of Fc sequence. See Example 7. A series of antibodies was
constructed comprising an mAb 12D6-24 variable domain and various
human IgG1f constant regions, including IgG1f, SE, SELF, P238D, V4,
V8, V9 and V12 variants. FIG. 3A presents data obtained using cells
from one donor, and FIG. 3B presents data obtained using cells from
a different donor.
[0025] FIG. 4 shows activation of cells, as measured by cell
surface CD54, by agonist anti-CD40 antibodies as a function of
variable domain sequence. See Example 7. A series of antibodies was
constructed comprising a human IgG1f-V12 constant region and
variable domains from parental (murine) anti-CD40 mAbs 12D6, 5G7,
8E8, 19G3 and 5F11. Results are plotted as median fluorescence
intensity (MFI) as a function of antibody concentration.
[0026] FIG. 5 shows percent Fc.gamma.R binding by various
antibodies of the present invention, including antibodies having
D270 substitutions. See Example 8. Antibody names including "-sup"
represent supernatants from antibody producing cells, whereas
others are purified antibodies. Data are presented as percentages
of a maximum receptor binding value for each combination of
antibody and receptor, as measured in a FORTEBIO Octet system. See,
e.g., Example 3. Each cluster of three bars represents, from left
to right, binding to hCD32a/Fc.gamma.RIIa-H131 (10 .mu.M) (hatched
bars), hCD32b/Fc.gamma.RIIa-R131 (10 .mu.M) (black bars), and
hCD32b/Fc.gamma.RIIb (1 .mu.M) (white bars).
[0027] FIGS. 6A and 6B show the effects of selected anti-CD40
antibodies of the present invention on T cell activation and change
in platelet count, respectively, in transgenic mice expressing
human CD40 and human Fey receptors. See Example 9. FIG. 6A shows
the percent of Tet-OVA reactive CD8+ T cells in animals treated
with selected anti-CD40 antibodies of the present invention, as
indicated. FIG. 6B shows the platelet count as a percentage of
pre-treatment platelet count at 24 hours post-injection with
antibody. Comparison of the figures shows that the level of
activation correlates with reduction in platelet count, with
antibody 12D6-V11 exhibiting the highest activation but also the
greatest reduction in platelet count. See Example 9.
[0028] FIG. 6C shows the antitumor response of humanized
CD40/Fc.gamma.R mice that were inoculated with MC38 tumor cells and
treated with Fc variants of anti-CD40 12D6-24 and 5F11-45 clones.
Results presented as means+/-SEM. n=7 (12D6-24) or 6 (5F11-45). See
Example 9.
[0029] FIG. 6D shows tumor free mice from the 12D6-24 group in the
experiment described in C were re-challenged with MC38 cells
subcutaneously and followed for tumor growth. Control group
consists of naive mice. Results presented as means+/-SEM. n=4. See
Example 9.
DETAILED DESCRIPTION
[0030] The present invention provides isolated antibodies,
particularly monoclonal antibodies, e.g., humanized or human
monoclonal antibodies, that specifically bind to human CD40
("huCD40") and have agonist activity. Sequences are provided for
various humanized murine anti-huCD40 monoclonal antibodies. In
certain embodiments, the antibodies described herein are derived
from particular murine heavy and light chain germline sequences
and/or comprise particular structural features such as CDR regions
comprising particular amino acid sequences. In other embodiments
antibodies compete for CD40 binding with, or bind to the same
epitope as, the anti-CD40 antibodies for which sequences are
provided herein. In some embodiments the sequence of the heavy
chain Fc region is modified to specifically enhance binding to
Fc.gamma.RIIb.
[0031] Further provided herein are methods of making such
antibodies, immunoconjugates and bispecific molecules comprising
such antibodies or antigen-binding fragments thereof, and
pharmaceutical compositions formulated to contain the antibodies or
fragments. Also provided herein are methods of using the antibodies
for immune response enhancement, alone or in combination with other
immunostimulatory agents (e.g., antibodies) and/or cancer or
anti-infective therapies. Accordingly, the anti-huCD40 antibodies
described herein may be used in a treatment in a wide variety of
therapeutic applications, including, for example, inhibiting tumor
growth and treating chronic viral infections.
Definitions
[0032] In order that the present description may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0033] CD40 refers to "TNF receptor superfamily member 5"
(TNFRSF5). Unless otherwise indicated, or clear from the context,
references to CD40 herein refer to human CD40 ("huCD40"), and
anti-CD40 antibodies refer to anti-human CD40 antibodies. Human
CD40 is further described at GENE ID NO: 958 and MIM (Mendelian
Inheritance in Man): 109535. The sequence of human CD40
(NP_001241.1), including 20 amino acid signal sequence, is provided
at SEQ ID NO: 1.
[0034] CD40 interacts with CD40 ligand (CD40L), which is also
referred to as TNFSF5, gp39 and CD154. Unless otherwise indicated,
or clear from the context, references to CD40L herein refer to
human CD40L ("huCD40L"). Human CD40L is further described at GENE
ID NO: 959 and MIM: 300386. The sequence of human CD40L
(NP_000065.1) is provided at SEQ ID NO: 2.
[0035] Unless otherwise indicated or clear from the context, the
term "antibody" as used to herein may include whole antibodies and
any antigen-binding fragments (i.e., "antigen-binding portions") or
single chains thereof. An "antibody" refers, in one 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 fragment thereof. Each heavy chain is comprised of a heavy
chain variable region (abbreviated herein as V.sub.H) and a heavy
chain constant region. In certain naturally occurring IgG, IgD and
IgA antibodies, the heavy chain constant region is comprised of
three domains, CH1, CH2 and CH3. In certain naturally occurring
antibodies, 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 framework regions (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.
[0036] Antibodies typically bind specifically to their cognate
antigen with high affinity, reflected by a dissociation constant
(K.sub.D) of 10.sup.-7 to 10.sup.-11 M or less. Any K.sub.D greater
than about 10.sup.-6M is generally considered to indicate
nonspecific binding. As used herein, an antibody that "binds
specifically" to an antigen refers to an antibody that binds to the
antigen and substantially identical antigens with high affinity,
which means having a K.sub.D of 10.sup.-7 M or less, preferably
10.sup.-8 M or less, even more preferably 5.times.10.sup.-9 M or
less, and most preferably between 10.sup.-8 M and 10.sup.-10 M or
less, but does not bind with high affinity to unrelated antigens.
An antigen is "substantially identical" to a given antigen if it
exhibits a high degree of sequence identity to the given antigen,
for example, if it exhibits at least 80%, at least 90%, preferably
at least 95%, more preferably at least 97%, or even more preferably
at least 99% sequence identity to the sequence of the given
antigen. By way of example, an antibody that binds specifically to
human CD40 might also cross-react with CD40 from certain non-human
primate species (e.g., cynomolgus monkey), but might not
cross-react with CD40 from other species, or with an antigen other
than CD40.
[0037] Unless otherwise indicated, an immunoglobulin may be from
any of the commonly known isotypes, including but not limited to
IgA, secretory IgA, IgG and IgM. The IgG isotype is divided in
subclasses in certain species: IgG1, IgG2, IgG3 and IgG4 in humans,
and IgG1, IgG2a, IgG2b and IgG3 in mice. Immunoglobulins, e.g.,
human IgG1, exist in several allotypes, which differ from each
other in at most a few amino acids. Unless otherwise indicated,
antibodies of the present invention comprise the IgG1f constant
domain (SEQ ID NO: 65). Unless otherwise indicated, "antibody" may
include, by way of example, monoclonal and polyclonal antibodies;
chimeric and humanized antibodies; human and non-human antibodies;
wholly synthetic antibodies; and single chain antibodies.
[0038] The term "antigen-binding portion" or "antigen binding
fragment" of an antibody, as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., human CD40). Examples of binding
fragments encompassed within the term "antigen-binding
portion/fragment" of an antibody include (i) a Fab fragment--a
monovalent fragment consisting of the VL, VH, CL and CH1 domains;
(ii) a F(ab')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 VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, and (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546) consisting of a VH domain. An isolated complementarity
determining region (CDR), or a combination of two or more isolated
CDRs joined by a synthetic linker, may comprise and antigen binding
domain of an antibody if able to bind antigen.
[0039] Single chain antibody constructs are also included in the
invention. Although the two domains of the Fv fragment, VL and VH,
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 VL and VH regions pair
to form monovalent molecules known as single chain Fv (scFv); 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 portion/fragment" of an antibody. These and
other potential constructs are described at Chan & Carter
(2010) Nat. Rev. Immunol. 10:301. 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/fragments
can be produced by recombinant DNA techniques, or by enzymatic or
chemical cleavage of intact immunoglobulins.
[0040] Unless otherwise indicated, the word "fragment" when used
with reference to an antibody, such as in a claim, refers to an
antigen binding fragment of the antibody, such that "antibody or
fragment" has the same meaning as "antibody or antigen binding
fragment thereof."
[0041] A "bispecific" or "bifunctional antibody" is an artificial
hybrid antibody having two different heavy/light chain pairs,
giving rise to two antigen binding sites with specificity for
different antigens. Bispecific antibodies can be produced by a
variety of methods including fusion of hybridomas or linking of
Fab' fragments. See, e.g., Songsivilai & Lachmann, (1990) Clin.
Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148,
1547-1553.
[0042] The term "monoclonal antibody," as used herein, refers to an
antibody that displays a single binding specificity and affinity
for a particular epitope or a composition of antibodies in which
all antibodies display a single binding specificity and affinity
for a particular epitope. Typically such monoclonal antibodies will
be derived from a single cell or nucleic acid encoding the
antibody, and will be propagated without intentionally introducing
any sequence alterations. Accordingly, the term "human monoclonal
antibody" refers to a monoclonal antibody that has variable and
optional constant regions derived from human germline
immunoglobulin sequences. In one embodiment, human monoclonal
antibodies are produced by a hybridoma, for example, obtained by
fusing a B cell obtained from a transgenic or transchromosomal
non-human animal (e.g., a transgenic mouse having a genome
comprising a human heavy chain transgene and a light chain
transgene), to an immortalized cell.
[0043] 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 that 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 sequences that
encode the light chain and heavy chain immunoglobulin polypeptides
in response to an antigen may not be identical to the original
germline sequences, but instead will be substantially identical or
similar (i.e., have at least 80% identity).
[0044] A "human" antibody (HuMAb) refers to an antibody having
variable regions in which both the framework and CDR regions are
derived from human germline immunoglobulin sequences. Furthermore,
if the antibody contains a constant region, the constant region
also is derived from human germline immunoglobulin sequences. Human
antibodies of the present invention may 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). However, the term "human
antibody," as used herein, is not intended to 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. The terms "human" antibodies and "fully human"
antibodies are used synonymously.
[0045] A "humanized" antibody refers to an antibody in which some,
most or all of the amino acids outside the CDR domains of a
non-human antibody, e.g. a mouse antibody, are replaced with
corresponding amino acids derived from human immunoglobulins. In
one embodiment of a humanized form of an antibody, some, most or
all of the amino acids outside the CDR domains have been replaced
with amino acids from human immunoglobulins, whereas some, most or
all amino acids within one or more CDR regions are unchanged. Small
additions, deletions, insertions, substitutions or modifications of
amino acids are permissible as long as they do not abrogate the
ability of the antibody to bind to a particular antigen. A
"humanized" antibody retains an antigenic specificity similar to
that of the original antibody.
[0046] A "chimeric antibody" refers to an antibody in which the
variable regions are derived from one species and the constant
regions are derived from another species, such as an antibody in
which the variable regions are derived from a mouse antibody and
the constant regions are derived from a human antibody. A "hybrid"
antibody refers to an antibody having heavy and light chains of
different types, such as a mouse (parental) heavy chain and a
humanized light chain, or vice versa.
[0047] As used herein, "isotype" refers to the antibody class
(e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE
antibody) that is encoded by the heavy chain constant region
genes.
[0048] "Allotype" refers to naturally occurring variants within a
specific isotype group, which variants differ in one or a few amino
acids. See, e.g., Jefferis et al. (2009) mAbs 1:1.
[0049] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody that binds specifically to an
antigen."
[0050] An "isolated antibody," as used herein, refers to an
antibody that is substantially free of other antibodies having
different antigenic specificities (e.g., an isolated antibody that
specifically binds to CD40 is substantially free of antibodies that
specifically bind antigens other than CD40). An isolated antibody
that specifically binds to an epitope of CD40 may, however, have
cross-reactivity to other CD40 proteins from different species.
[0051] "Effector functions," deriving from the interaction of an
antibody Fc region with certain Fc receptors, include but are not
necessarily limited to Clq binding, complement dependent
cytotoxicity (CDC), Fc receptor binding, Fc.gamma.R-mediated
effector functions such as ADCC and antibody dependent
cell-mediated phagocytosis (ADCP), and down regulation of a cell
surface receptor (e.g., the B cell receptor; BCR). Such effector
functions generally require the Fc region to be combined with an
antigen binding domain (e.g., an antibody variable domain).
[0052] An "Fc receptor" or "FcR" is a receptor that binds to the Fc
region of an immunoglobulin. FcRs that bind to an IgG antibody
comprise receptors of the Fc.gamma.R family, including allelic
variants and alternatively spliced forms of these receptors. The
Fc.gamma.R family consists of three activating (Fc.gamma.RI,
Fc.gamma.RIII, and Fc.gamma.RIV in mice; Fc.gamma.RIA,
Fc.gamma.RIIA, and Fc.gamma.RIIIA in humans) and one inhibitory
(Fc.gamma.RIIb, or equivalently Fc.gamma.RIIB) receptor. Various
properties of human Fc.gamma.Rs are summarized in Table 1. The
majority of innate effector cell types co-express one or more
activating Fc.gamma.R and the inhibitory Fc.gamma.RIIb, whereas
natural killer (NK) cells selectively express one activating Fc
receptor (Fc.gamma.RIII in mice and Fc.gamma.RIIIA in humans) but
not the inhibitory Fc.gamma.RIIb in mice and humans. Human IgG1
binds to most human Fc receptors and is considered equivalent to
murine IgG2a with respect to the types of activating Fc receptors
that it binds to.
TABLE-US-00001 TABLE 1 Properties of Human Fc.gamma.Rs Allelic
Affinity for Fc.gamma. variants human IgG Isotype preference
Cellular distribution Fc.gamma.RI None High (K.sub.D ~10 IgG1 = 3
> 4 >> 2 Monocytes, macrophages, described nM) activated
neutrophils, dendritic cells? Fc.gamma.RIIA H131 Low to medium IgG1
> 3 > 2 > 4 Neutrophils, monocytes, R131 Low IgG1 > 3
> 4 > 2 macrophages, eosinophils, dendritic cells, platelets
Fc.gamma.RIIIA V158 Medium IgG1 = 3 >> 4 > 2 NK cells,
monocytes, F158 Low IgG1 = 3 >> 4 > 2 macrophages, mast
cells, eosinophils, dendritic cells? Fc.gamma.RIIb I232 Low IgG1 =
3 = 4 > 2 B cells, monocytes, T232 Low IgG1 = 3 = 4 > 2
macrophages, dendritic cells, mast cells
[0053] An "Fc region" (fragment crystallizable region) or "Fc
domain" or "Fc" refers to the C-terminal region of the heavy chain
of an antibody that mediates the binding of the immunoglobulin to
host tissues or factors, including binding to Fc receptors located
on various cells of the immune system (e.g., effector cells) or to
the first component (C1q) of the classical complement system. Thus,
an Fc region comprises the constant region of an antibody excluding
the first constant region immunoglobulin domain (e.g., CH1 or CL).
In IgG, IgA and IgD antibody isotypes, the Fc region comprises
C.sub.H2 and C.sub.H3 constant domains in each of the antibody's
two heavy chains; IgM and IgE Fc regions comprise three heavy chain
constant domains (C.sub.H domains 2-4) in each polypeptide chain.
For IgG, the Fc region comprises immunoglobulin domains C.gamma.2
and C.gamma.3 and the hinge between C.gamma.1 and C.gamma.2.
Although the boundaries of the Fc region of an immunoglobulin heavy
chain might vary, the human IgG heavy chain Fc region is usually
defined to stretch from an amino acid residue at position C226 or
P230 (or an amino acid between these two amino acids) to the
carboxy-terminus of the heavy chain, wherein the numbering is
according to the EU index as in Kabat. Kabat et al. (1991)
Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, Md.; see also FIGS. 3c-3f of U.S.
Pat. App. Pub. No. 2008/0248028. The C.sub.H2 domain of a human IgG
Fc region extends from about amino acid 231 to about amino acid
340, whereas the C.sub.H3 domain is positioned on C-terminal side
of a C.sub.H2 domain in an Fc region, i.e., it extends from about
amino acid 341 to about amino acid 447 of an IgG (including a
C-terminal lysine). As used herein, the Fc region may be a native
sequence Fc, including any allotypic variant, or a variant Fc
(e.g., a non-naturally occurring Fc). Fc may also refer to this
region in isolation or in the context of an Fc-comprising protein
polypeptide such as a "binding protein comprising an Fc region,"
also referred to as an "Fc fusion protein" (e.g., an antibody or
immunoadhesin).
[0054] A "native sequence Fc region" or "native sequence Fc"
comprises an amino acid sequence that is identical to the amino
acid sequence of an Fc region found in nature. Native sequence
human Fc regions include a native sequence human IgG1 Fc region;
native sequence human IgG2 Fc region; native sequence human IgG3 Fc
region; and native sequence human IgG4 Fc region as well as
naturally occurring variants thereof. Native sequence Fc include
the various allotypes of Fcs. See, e.g., Jefferis et al. (2009)
mAbs 1:1.
[0055] The term "epitope" or "antigenic determinant" refers to a
site on an antigen (e.g., huCD40) to which an immunoglobulin or
antibody specifically binds. Epitopes within protein antigens can
be formed both from contiguous amino acids (usually a linear
epitope) or noncontiguous amino acids juxtaposed by tertiary
folding of the protein (usually a conformational epitope). Epitopes
formed from contiguous amino acids are typically, but not always,
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.
[0056] The term "epitope mapping" refers to the process of
identification of the molecular determinants on the antigen
involved in antibody-antigen recognition. Methods for determining
what epitopes are bound by a given antibody are well known in the
art and include, for example, immunoblotting and
immunoprecipitation assays, wherein overlapping or contiguous
peptides from (e.g., from CD40) are tested for reactivity with a
given antibody (e.g., anti-CD40 antibody); x-ray crystallography;
2-dimensional nuclear magnetic resonance; yeast display (see
Example 6); and HDX-MS (see, e.g., Epitope Mapping Protocols in
Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996))
(see Example 5).
[0057] The term "binds to the same epitope" with reference to two
or more antibodies means that the antibodies bind to the same
segment of amino acid residues, as determined by a given method.
Techniques for determining whether antibodies bind to the "same
epitope on CD40" with the antibodies described herein include, for
example, epitope mapping methods, such as, x-ray analyses of
crystals of antigen:antibody complexes, which provides atomic
resolution of the epitope, and hydrogen/deuterium exchange mass
spectrometry (HDX-MS). Other methods monitor the binding of the
antibody to antigen fragments (e.g. proteolytic fragments) or to
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, such as
alanine scanning mutagenesis (Cunningham & Wells (1985) Science
244:1081) or yeast display of mutant target sequence variants (see
Example 6). 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.
Antibodies having the same or closely related VH and VL or the same
CDR sequences are expected to bind to the same epitope.
[0058] Antibodies that "compete with another antibody for binding
to a target" refer to antibodies that inhibit (partially or
completely) the binding of the other antibody to the target.
Whether two antibodies compete with each other for binding to a
target, i.e., whether and to what extent one antibody inhibits the
binding of the other antibody to a target, may be determined using
known competition experiments. In certain embodiments, an antibody
competes with, and inhibits binding of another antibody to a target
by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
The level of inhibition or competition may be different depending
on which antibody is the "blocking antibody" (i.e., the cold
antibody that is incubated first with the target). Competition
assays can be conducted as described, for example, in Ed Harlow and
David Lane, Cold Spring Harb. Protoc.; 2006;
doi:10.1101/pdb.prot4277 or in Chapter 11 of "Using Antibodies" by
Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., (USA) 1999. Competing antibodies bind to the
same epitope, an overlapping epitope or to adjacent epitopes (e.g.,
as evidenced by steric hindrance).
[0059] Other competitive binding assays include: solid phase direct
or indirect radioimmunoassay (RIA), solid phase direct or indirect
enzyme immunoassay (EIA), sandwich competition assay (see Stahli et
al. (1983) Methods in Enzymology 9:242); solid phase direct
biotin-avidin EIA (see Kirkland et al. (1986) J. Immunol.
137:3614); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane (1988), Antibodies: A
Laboratory Manual, Cold Spring Harbor Press); solid phase direct
label RIA using I-125 label (see Morel et al. (1988) Mol. Immunol.
25(1):7); solid phase direct biotin-avidin EIA (Cheung et al.
(1990) Virology 176:546); and direct labeled RIA. (Moldenhauer et
al. (1990) Scand. J. Immunol. 32:77).
[0060] 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 but not
to other antigens. Typically, the antibody (i) 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, e.g.,
surface plasmon resonance (SPR) technology in a BIACORE.RTM. 2000
surface plasmon resonance instrument using the predetermined
antigen, e.g., recombinant human CD40, as the analyte and the
antibody as the ligand, or Scatchard analysis of binding of the
antibody to antigen positive cells, and (ii) 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. Accordingly, an antibody that
"specifically binds to human CD40" refers to an antibody that binds
to soluble or cell bound human CD40 with a K.sub.D of 10.sup.-7 M
or less, such as approximately less than 10.sup.-8 M, 10.sup.-9 M
or 10.sup.-10 M or even lower. An antibody that "cross-reacts with
cynomolgus CD40" refers to an antibody that binds to cynomolgus
CD40 with a K.sub.D of 10.sup.-7 M or less, such as approximately
less than 10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or even
lower.
[0061] The term "kassoc" or "K.sub.A", as used herein, refers to
the association rate constant of a particular antibody-antigen
interaction, whereas the term "kdis" or "K.sub.D," as used herein,
refers to the dissociation rate constant of a particular
antibody-antigen interaction. The term "K.sub.D", as used herein,
refers to the equilibrium dissociation constant, which is obtained
from the ratio of K.sub.D to K.sub.A (i.e., K.sub.D/K.sub.A) and is
expressed as a molar concentration (M). K.sub.D values for
antibodies can be determined using methods well established in the
art. A preferred method for determining the K.sub.D of an antibody
is biolayer interferometry (BLI) analysis, preferably using a
FORTEBIO Octet RED device (see Example 3), surface plasmon
resonance, preferably using a biosensor system such as a
BIACORE.RTM. surface plasmon resonance system (see Example 4), or
flow cytometry and Scatchard analysis.
[0062] The term "EC50" in the context of an in vitro or in vivo
assay using an antibody or antigen binding fragment thereof, refers
to the concentration of an antibody or an antigen-binding fragment
thereof that induces a response that is 50% of the maximal
response, i.e., halfway between the maximal response and the
baseline.
[0063] The term "binds to immobilized CD40" refers to the ability
of an antibody described herein to bind to CD40, for example,
expressed on the surface of a cell or attached to a solid
support.
[0064] The term "cross-reacts," as used herein, refers to the
ability of an antibody described herein to bind to CD40 from a
different species. For example, an antibody described herein that
binds human CD40 may also bind CD40 from another species (e.g.,
cynomolgus CD40). As used herein, cross-reactivity may be 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 CD40. Methods
for determining cross-reactivity include standard binding assays as
described herein, for example, by BIACORE.RTM. surface plasmon
resonance (SPR) analysis using a BIACORE.RTM. 2000 SPR instrument
(BIACORE AB, Uppsala, Sweden), or flow cytometric techniques.
[0065] 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.
[0066] A "polypeptide" refers to a chain comprising at least two
consecutively linked amino acid residues, with no upper limit on
the length of the chain. One or more amino acid residues in the
protein may contain a modification such as, but not limited to,
glycosylation, phosphorylation or a disulfide bond. A "protein" may
comprise one or more polypeptides.
[0067] 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, and may be
cDNA.
[0068] Also provided are "conservative sequence modifications" to
the antibody sequence provided herein, i.e. nucleotide and amino
acid sequence modifications that do not abrogate the binding of the
antibody encoded by the nucleotide sequence or containing the amino
acid sequence, to the antigen. For example, modifications can be
introduced by standard techniques known in the art, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative sequence modifications include conservative amino acid
substitutions, 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-CD40 antibody is preferably replaced with another amino acid
residue from the same side chain family. Methods of identifying
nucleotide and amino acid conservative substitutions that 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).
[0069] Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of an anti-CD40 antibody
coding sequence, such as by saturation mutagenesis, and the
resulting modified anti-CD40 antibodies can be screened for
improved binding activity.
[0070] 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.
[0071] For polypeptides, the term "substantial homology" indicates
that two polypeptides, 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 99.5% of the amino acids.
[0072] The percent identity between two sequences is a function of
the number of identical positions shared by the sequences when the
sequences are optimally aligned (i.e., % homology=# of identical
positions/total # of positions.times.100), with optimal alignment
determined 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.
[0073] The percent identity between two nucleotide sequences can be
determined using the GAP program in the GCG software package, 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, 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.
[0074] The nucleic acid and protein sequences described herein 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 homologous
to the nucleic acid molecules described herein. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to the
protein molecules described herein. 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.
[0075] 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 (e.g., the other parts of the
chromosome) 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).
[0076] 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, also included
are other forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0077] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell that comprises a
nucleic acid that is not naturally present in the cell, and may be
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.
[0078] An "immune response" refers to a biological response within
a vertebrate against foreign agents, which response protects the
organism against these agents and diseases caused by them. An
immune response is mediated by the action of a cell of the immune
system (for example, a T lymphocyte, B lymphocyte, natural killer
(NK) cell, macrophage, eosinophil, mast cell, dendritic cell or
neutrophil) and soluble macromolecules produced by any of these
cells or the liver (including antibodies, cytokines, and
complement) that results in selective targeting, binding to, damage
to, destruction of, and/or elimination from the vertebrate's body
of invading pathogens, cells or tissues infected with pathogens,
cancerous or other abnormal cells, or, in cases of autoimmunity or
pathological inflammation, normal human cells or tissues. An immune
reaction includes, e.g., activation or inhibition of a T cell,
e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+ T
cell, or the inhibition or depletion of a Treg cell. "T effector"
("Teff") cells refers to T cells (e.g., CD4+ and CD8+ T cells) with
cytolytic activities as well as T helper (Th) cells, which secrete
cytokines and activate and direct other immune cells, but does not
include regulatory T cells (Treg cells).
[0079] As used herein, the term "T cell-mediated response" refers
to a 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.
[0080] As used herein, the term "cytotoxic T lymphocyte (CTL)
response" refers to an immune response induced by cytotoxic T
cells. CTL responses are mediated primarily by CD8+ T cells.
[0081] An "immunomodulator" or "immunoregulator" refers to an
agent, e.g., a component of a signaling pathway that may be
involved in modulating, regulating, or modifying an immune
response. "Modulating," "regulating," or "modifying" an immune
response refers to any alteration in a cell of the immune system or
in the activity of such cell (e.g., an effector T cell). Such
modulation includes stimulation or suppression of the immune system
which may be manifested by an increase or decrease in the number of
various cell types, an increase or decrease in the activity of
these cells, or any other changes which can occur within the immune
system. Both inhibitory and stimulatory immunomodulators have been
identified, some of which may have enhanced function in a tumor
microenvironment. In preferred embodiments, the immunomodulator is
located on the surface of a T cell. An "immunomodulatory target" or
"immunoregulatory target" is an immunomodulator that is targeted
for binding by, and whose activity is altered by the binding of, a
substance, agent, moiety, compound or molecule. Immunomodulatory
targets include, for example, receptors on the surface of a cell
("immunomodulatory receptors") and receptor ligands
("immunomodulatory ligands").
[0082] "Immunotherapy" refers to the treatment of a subject
afflicted with, or at risk of contracting or suffering a recurrence
of, a disease by a method comprising inducing, enhancing,
suppressing or otherwise modifying an immune response.
[0083] "Immunostimulating therapy" or "immunostimulatory therapy"
refers to a therapy that results in increasing (inducing or
enhancing) an immune response in a subject for, e.g., treating
cancer.
[0084] "Potentiating an endogenous immune response" means
increasing the effectiveness or potency of an existing immune
response in a subject. This increase in effectiveness and potency
may be achieved, for example, by overcoming mechanisms that
suppress the endogenous host immune response or by stimulating
mechanisms that enhance the endogenous host immune response.
[0085] 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.
[0086] As used herein, "administering" refers to the physical
introduction of a composition comprising a therapeutic agent to a
subject, using any of the various methods and delivery systems
known to those skilled in the art. Preferred routes of
administration for antibodies described herein include intravenous,
intraperitoneal, intramuscular, subcutaneous, spinal or other
parenteral routes of administration, for example by injection or
infusion. The phrase "parenteral administration" as used herein
means modes of administration other than enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intraperitoneal, intramuscular,
intraarterial, intrathecal, intralymphatic, intralesional,
intracapsular, intraorbital, intracardiac, intradermal,
transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal, epidural and intrasternal
injection and infusion, as well as in vivo electroporation.
Alternatively, an antibody described herein can be administered via
a non-parenteral route, such as a topical, epidermal or mucosal
route of administration, for example, intranasally, orally,
vaginally, rectally, sublingually or topically. Administering can
also be performed, for example, once, a plurality of times, and/or
over one or more extended periods.
[0087] As used herein, the terms "inhibits" or "blocks" are used
interchangeably and encompass both partial and complete
inhibition/blocking by at least about 50%, for example, at least
about 60%, 70%, 80%, 90%, 95%, 99%, or 100%.
[0088] As used herein, "cancer" refers a broad group of diseases
characterized by the uncontrolled growth of abnormal cells in the
body. Unregulated cell division may result in the formation of
malignant tumors or cells that invade neighboring tissues and may
metastasize to distant parts of the body through the lymphatic
system or bloodstream.
[0089] The terms "treat," "treating," and "treatment," as used
herein, refer to any type of intervention or process performed on,
or administering an active agent to, the subject with the objective
of reversing, alleviating, ameliorating, inhibiting, or slowing
down or preventing the progression, development, severity or
recurrence of a symptom, complication, condition or biochemical
indicia associated with a disease. Prophylaxis refers to
administration to a subject who does not have a disease, to prevent
the disease from occurring or minimize its effects if it does.
[0090] The term "effective dose" or "effective dosage" is defined
as an amount sufficient to achieve or at least partially achieve a
desired effect. A "therapeutically effective amount" or
"therapeutically effective dosage" of a drug or therapeutic agent
is any amount of the drug that, when used alone or in combination
with another therapeutic agent, promotes disease regression
evidenced by a decrease in severity of disease symptoms, an
increase in frequency and duration of disease symptom-free periods,
or a prevention of impairment or disability due to the disease
affliction. A "prophylactically effective amount" or a
"prophylactically effective dosage" of a drug is an amount of the
drug that, when administered alone or in combination with another
therapeutic agent to a subject at risk of developing a disease or
of suffering a recurrence of disease, inhibits the development or
recurrence of the disease. The ability of a therapeutic or
prophylactic agent to promote disease regression or inhibit the
development or recurrence of the disease can be evaluated using a
variety of methods known to the skilled practitioner, such as in
human subjects during clinical trials, in animal model systems
predictive of efficacy in humans, or by assaying the activity of
the agent in in vitro assays.
[0091] By way of example, an anti-cancer agent is a drug that slows
cancer progression or promotes cancer regression in a subject. In
preferred embodiments, a therapeutically effective amount of the
drug promotes cancer regression to the point of eliminating the
cancer. "Promoting cancer regression" means that administering an
effective amount of the drug, alone or in combination with an
anti-neoplastic agent, results in a reduction in tumor growth or
size, necrosis of the tumor, a decrease in severity of at least one
disease symptom, an increase in frequency and duration of disease
symptom-free periods, a prevention of impairment or disability due
to the disease affliction, or otherwise amelioration of disease
symptoms in the patient. Pharmacological effectiveness refers to
the ability of the drug to promote cancer regression in the
patient. Physiological safety refers to an acceptably low level of
toxicity, or other adverse physiological effects at the cellular,
organ and/or organism level (adverse effects) resulting from
administration of the drug.
[0092] By way of example for the treatment of tumors, a
therapeutically effective amount or dosage of the drug preferably
inhibits cell growth or tumor growth by at least about 20%, more
preferably by at least about 40%, even more preferably by at least
about 60%, and still more preferably by at least about 80% relative
to untreated subjects. In the most preferred embodiments, a
therapeutically effective amount or dosage of the drug completely
inhibits cell growth or tumor growth, i.e., preferably inhibits
cell growth or tumor growth by 100%. The ability of a compound to
inhibit tumor growth can be evaluated using the assays described
infra. Inhibition of tumor growth may not be immediate after
treatment, and may only occur after a period of time or after
repeated administration. Alternatively, this property of a
composition can be evaluated by examining the ability of the
compound to inhibit cell growth, such inhibition can be measured in
vitro by assays known to the skilled practitioner. In other
preferred embodiments described herein, tumor regression may be
observed and may continue for a period of at least about 20 days,
more preferably at least about 40 days, or even more preferably at
least about 60 days.
[0093] "Combination" therapy, as used herein, unless otherwise
clear from the context, is meant to encompass administration of two
or more therapeutic agents in a coordinated fashion, and includes,
but is not limited to, concurrent dosing. Specifically, combination
therapy encompasses both co-administration (e.g. administration of
a co-formulation or simultaneous administration of separate
therapeutic compositions) and serial or sequential administration,
provided that administration of one therapeutic agent is
conditioned in some way on administration of another therapeutic
agent. For example, one therapeutic agent may be administered only
after a different therapeutic agent has been administered and
allowed to act for a prescribed period of time. See, e.g., Kohrt et
al. (2011) Blood 117:2423.
[0094] The terms "patient" and "subject" refer to any human that
receives either prophylactic or therapeutic treatment. For example,
the methods and compositions described herein can be used to treat
a subject having cancer.
[0095] Various aspects described herein are described in further
detail in the following subsections.
I. Anti-CD40 Antibodies
[0096] The present application discloses agonistic anti-huCD40
antibodies having desirable properties for use as therapeutic
agents in treating diseases such as cancers. These properties
include one or more of the ability to bind to human CD40 with high
affinity, acceptably low immunogenicity in human subjects, the
ability to bind preferentially to Fc.gamma.RIIb, and the absence of
sequence liabilities that might reduce the chemical stability of
the antibody.
[0097] The anti-CD40 antibodies disclosed herein by sequence bind
to specific epitopes on human CD40, as may be determined as
described in Examples 5 and 6. Other antibodies that bind to the
same or closely related epitopes would likely share these desirable
properties, and may be discovered doing competition
experiments.
Anti-huCD40 Antibodies that Compete with Anti-huCD40 Antibodies
Disclosed Herein
[0098] Anti-huCD40 antibodies that compete with the antibodies of
the present invention for binding to huCD40 may be raised using
immunization protocols similar to those described herein (Examples
1 and 2). Antibodies that compete for binding with the anti-huCD40
antibodies disclosed herein by sequence may also be generated by
immunizing mice or other non-human animal with human CD40 or a
construct comprising the extracellular domain thereof (residues
21-193 of SEQ ID NO: 1), or by immunizing with a fragment of human
CD40 containing the epitope bound by the anti-huCD40 antibodies
disclosed herein. The resulting antibodies can be screened for the
ability to block binding of 12D6, 5F11, 8E8, 5G7 and/or 19G3 to
human CD40 by methods well known in the art, for example blocking
binding to fusion protein of the extracellular domain of CD40 and
an immunoglobulin Fc domain in a ELISA, or blocking the ability to
bind to cells expressing huCD40 on their surface, e.g. by FACS. In
various embodiments, the test antibody is contacted with the
CD40-Fc fusion protein (or to cells expressing huCD40 on their
surface) prior to, at the same time as, or after the addition of
12D6, 5F11, 8E8, 5G7 or 19G3. For example, "binning" experiments
may be performed (Example 4) to determine whether a test antibody
falls into the same "bin" as an antibodies disclosed herein by
sequence, with antibodies disclosed herein by sequence as the
"reference" antibodies and the antibodies to be tested as the
"test" antibodies. Antibodies that reduce binding of the antibodies
disclosed herein by sequence to human CD40 (either as an Fc fusion
or on a cell), particularly at roughly stoichiometric
concentrations, are likely to bind at the same, overlapping, or
adjacent epitopes, and thus may share the desirable functional
properties of 12D6, 5F11, 8E8, 5G7 or 19G3.
[0099] Accordingly, provided herein are anti-huCD40 antibodies that
inhibit the binding of an anti-huCD40 antibodies described herein
to huCD40 on cells by at least 10%, 20%, 30%, 40%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or by 100%, and/or whose binding to huCD40 on cells is
inhibited by an anti-huCD40 antibodies described herein by at least
10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or by 100%, e.g., as
measured by ELISA or FACS, such as by using the assay described in
the following paragraph.
[0100] An exemplary competition experiment to determine whether a
test antibody blocks the binding of (i.e., "competes with") a
reference antibody, may be conducted as follows: cells expressing
CD40 are seeded at 10.sup.5 cells per sample well in a 96 well
plate. The plate is set on ice followed by the addition of
unconjugated test antibody at concentrations ranging from 0 to 50
.mu.g/mL (three-fold titration starting from a highest
concentration of 50 .mu.g/mL). An unrelated IgG may be used as an
isotype control for the first antibody and added at the same
concentrations (three-fold titration starting from a highest
concentration of 50 .mu.g/mL). A sample pre-incubated with 50
.mu.g/mL unlabeled reference antibody may be included as a positive
control for complete blocking (100% inhibition) and a sample
without antibody in the primary incubation may be used as a
negative control (no competition; 0% inhibition). After 30 minutes
of incubation, labeled, e.g., biotinylated, reference antibody is
added at a concentration of 2 .mu.g/mL per well without washing.
Samples are incubated for another 30 minutes on ice. Unbound
antibodies are removed by washing the cells with FACS buffer.
Cell-bound labeled reference antibody is detected with an agent
that detects the label, e.g., PE conjugated streptavidin
(INVITROGEN, catalog #521388) for detecting biotin. The samples are
acquired on a FACS Calibur Flow Cytometer (BD, San Jose) and
analyzed with FLOWJO software (TREE STAR, Inc, Ashland, Oreg.). The
results may be represented as the % inhibition.
[0101] Typically, the same experiment is then conducted in the
reverse, i.e., the test antibody is the reference antibody and the
reference antibody is the test antibody. In certain embodiments, an
antibody at least partially (e.g., at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90%) or completely (100%) blocks the binding
of the other antibody to the target, e.g. human CD40 or fragment
thereof, and regardless of whether inhibition occurs when one or
the other antibody is the reference antibody. A reference antibody
and a test antibody "cross-block" binding of each other to the
target when the antibodies compete with each other both ways, i.e.,
in competition experiments in which the reference antibody is added
first and in competition experiments in which the test antibody is
added first.
[0102] Anti-huCD40 antibodies are considered to compete with the
anti-huCD40 antibodies disclosed herein if they inhibit binding of
12D6, 5F11, 8E8, 5G7 and/or 19G3 to human CD40 by at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or by 100%, when present at
roughly equal concentrations, for example in competition
experiments like those described in Example 4. Unless otherwise
indicated, an antibody will be considered to compete with an
antibody selected from the group consisting of the anti-CD40
antibodies of the present invention if it reduces binding of the
selected antibody to human CD40 (SEQ ID NO: 1) by at least 20% when
used at a roughly equal molar concentration with the selected
antibody, as measured in competition ELISA experiments as outlined
in the preceding two paragraphs.
Anti-huCD40 Antibodies that Bind to the Same Epitope
[0103] Anti-huCD40 antibodies that bind to the same or similar
epitopes to the antibodies disclosed herein may be raised using
immunization protocols similar to those described herein (Examples
1 and 2). The resulting antibodies can be screened for high
affinity binding to human CD40 (Example 3). Selected antibodies can
then be studied in yeast display assay in which sequence variants
of huCD40 are presented on the surface of yeast cells (Example 6),
or by hydrogen-deuterium exchange experiments (Example 5), to
determine the precise epitope bound by the antibody.
[0104] Epitope determinations may be made by any method known in
the art. In various embodiments, anti-huCD40 antibodies are
considered to bind to the same epitope as an anti-huCD40 mAb
disclosed herein if they make contact with one or more of the same
residues within at least one region of huCD40; if they make
contacts with a majority of the residues within at least one region
of huCD40; if they make contacts with a majority of the residues
within each region of huCD40; if they make contact with a majority
of contacts along the entire length of huCD40; if they make
contacts within all of the same distinct regions of human CD40; if
they make contact with all of the residues at any one region on
human CD40; or if they make contact with all of the same residues
at all of the same regions. Epitope "regions" are clusters of
residues along the primary sequence.
[0105] Techniques for determining antibodies that bind to the "same
epitope on huCD40" with the antibodies described herein include
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. Methods
may also rely on the ability of an antibody of interest to affinity
isolate specific short peptides (either in native three dimensional
form or in denatured form) from combinatorial phage display peptide
libraries or from a protease digest of the target protein. 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 that have been shown to map conformational
discontinuous epitopes.
[0106] The epitope or region comprising the epitope can also be
identified by screening for binding to a series of overlapping
peptides spanning CD40. Alternatively, the method of Jespers et al.
(1994) Biotechnology 12:899 may be used to guide the selection of
antibodies having the same epitope and therefore similar properties
to the an anti-CD40 antibodies described herein. Using phage
display, first the heavy chain of the anti-CD40 antibody is paired
with a repertoire of (preferably human) light chains to select a
CD40-binding antibody, and then the new light chain is paired with
a repertoire of (preferably human) heavy chains to select a
(preferably human) CD40-binding antibody having the same epitope or
epitope region as an anti-huCD40 antibody described herein.
Alternatively variants of an antibody described herein can be
obtained by mutagenesis of cDNA encoding the heavy and light chains
of the antibody.
[0107] Alanine scanning mutagenesis, as described by Cunningham
& Wells (1989) Science 244: 1081, or some other form of point
mutagenesis of amino acid residues in CD40 (such as the yeast
display method provided at Example 6) may also be used to determine
the functional epitope for an anti-CD40 antibody.
[0108] The epitope or epitope region (an "epitope region" is a
region comprising the epitope or overlapping with the epitope)
bound by a specific antibody may also be determined by assessing
binding of the antibody to peptides comprising fragments of CD40. A
series of overlapping peptides encompassing the sequence of CD40
(e.g., human CD40) may be synthesized and screened for binding,
e.g. in a direct ELISA, a competitive ELISA (where the peptide is
assessed for its ability to prevent binding of an antibody to CD40
bound to a well of a microtiter plate), or on a chip. Such peptide
screening methods may not be capable of detecting some
discontinuous functional epitopes, i.e. functional epitopes that
involve amino acid residues that are not contiguous along the
primary sequence of the CD40 polypeptide chain.
[0109] An epitope may also be identified by MS-based protein
footprinting, such as hydrogen/deuterium exchange mass spectrometry
(HDX-MS) and Fast Photochemical Oxidation of Proteins (FPOP).
HDX-MS may be conducted, e.g., as further described at Wei et al.
(2014) Drug Discovery Today 19:95, the methods of which are
specifically incorporated by reference herein. See also Example 5.
FPOP may be conducted as described, e.g., in Hambley & Gross
(2005) J. American Soc. Mass Spectrometry 16:2057, the methods of
which are specifically incorporated by reference herein.
[0110] The epitope bound by anti-CD40 antibodies may also be
determined by structural methods, such as X-ray crystal structure
determination (e.g., WO 2005/044853), molecular modeling and
nuclear magnetic resonance (NMR) spectroscopy, including NMR
determination of the H-D exchange rates of labile amide hydrogens
in CD40 when free and when bound in a complex with an antibody of
interest (Zinn-Justin et al. (1992) Biochemistry 31:11335;
Zinn-Justin et al. (1993) Biochemistry 32:6884).
[0111] With regard to X-ray crystallography, crystallization may be
accomplished using any of the known methods in the art (e.g. Giege
et al. (1994) Acta Crystallogr. D50:339; McPherson (1990) Eur. J.
Biochem. 189:1), including microbatch (e.g. Chayen (1997) Structure
5:1269), hanging-drop vapor diffusion (e.g. McPherson (1976) J.
Biol. Chem. 251:6300), seeding and dialysis. It is desirable to use
a protein preparation having a concentration of at least about 1
mg/mL and preferably about 10 mg/mL to about 20 mg/mL.
Crystallization may be best achieved in a precipitant solution
containing polyethylene glycol 1000-20,000 (PEG; average molecular
weight ranging from about 1000 to about 20,000 Da), preferably
about 5000 to about 7000 Da, more preferably about 6000 Da, with
concentrations ranging from about 10% to about 30% (w/v). It may
also be desirable to include a protein stabilizing agent, e.g.
glycerol at a concentration ranging from about 0.5% to about 20%. A
suitable salt, such as sodium chloride, lithium chloride or sodium
citrate may also be desirable in the precipitant solution,
preferably in a concentration ranging from about 1 mM to about 1000
mM. The precipitant is preferably buffered to a pH of from about
3.0 to about 5.0, preferably about 4.0. Specific buffers useful in
the precipitant solution may vary and are well-known in the art
(Scopes, Protein Purification: Principles and Practice, Third ed.,
(1994) Springer-Verlag, New York). Examples of useful buffers
include, but are not limited to, HEPES, Tris, MES and acetate.
Crystals may be grow at a wide range of temperatures, including
2.degree. C., 4.degree. C., 8.degree. C. and 26.degree. C.
[0112] Antibody:antigen crystals may be studied using well-known
X-ray diffraction techniques and may be refined using computer
software such as X-PLOR (Yale University, 1992, distributed by
Molecular Simulations, Inc.; see e.g. Blundell & Johnson (1985)
Meth. Enzymol. 114 & 115, H. W. Wyckoff et al., eds., Academic
Press; U.S. Patent Application Publication No. 2004/0014194), and
BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997)
Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et
al. (2000) Acta Cryst. D56:1313-1323), the disclosures of which are
hereby incorporated by reference in their entireties.
[0113] Unless otherwise indicated, and with reference to the
claims, the epitope bound by an antibody is the epitope as
determined by HDX-MS methods, substantially as described in Example
5.
Anti-CD40 Antibodies that Bind with High Affinity
[0114] In some embodiments the anti-huCD40 antibodies of the
present invention bind to huCD40 with high affinity, like the
anti-huCD40 antibodies disclosed herein, increasing their
likelihood of being effective therapeutic agents. In various
embodiments anti-huCD40 antibodies of the present invention bind to
huCD40 with a K.sub.D of less than 10 nM, 5 nM, 2 nM, 1 nM, 300 pM
or 100 pM. In other embodiments, the anti-huCD40 antibodies of the
present invention bind to huCD40 with a K.sub.D between 2 nM and
100 pM. Standard assays to evaluate the binding ability of the
antibodies toward huCD40 include ELISAs, RIAs, Western blots,
biolayer interferometry (BLI) (see Example 3) and BIACORE.RTM. SPR
analysis (see Example 4).
Anti-CD40 Antibody Sequence Variants
[0115] Some variability in the antibody sequences disclosed herein
may be tolerated and still maintain the desirable properties of the
antibody. The CDR regions are delineated using the Kabat system
(Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242). Accordingly, the present
invention further provides anti-huCD40 antibodies comprising CDR
sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to the CDR sequences of the antibodies
disclosed herein (i.e. 12D6, 5F11, 8E8, 5G7 and 19G3 and humanized
derivatives thereof). The present invention also provides
anti-huCD40 antibodies comprising heavy and/or light chain variable
domain sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to the heavy and/or light chain
variable domain sequences of the antibodies disclosed herein (i.e.
12D6, 5F11, 8E8, 5G7 and 19G3 and humanized derivatives
thereof).
Anti-CD40 Antibodies Sharing CDR Sequences or Derived from the Same
Murine Germlines
[0116] Given that antigen-binding specificity is determined
primarily by the CDRs, antibodies sharing CDRs sequences with
antibodies disclosed herein (i.e. 12D6, 5F11, 8E8, 5G7 and 19G3)
are likely to share their desirable properties. In some
embodiments, anti-huCD40 antibodies of the present invention
comprises heavy and light chain variable regions derived from the
same murine V region and J region germline sequences as antibody
12D6, 5F11, 8E8, 5G7 or 19G3. Antibody 12D6 has a heavy chain
derived from murine germlines VH1-39_01 and IGHJ4, and light chain
germlines VK1-110_01 and IGKJ1. Antibody 5F11 has a heavy chain
derived from murine germlines VH1-4_02 and IGHJ3, and light chain
germlines VK3-5_01 and IGKJ5. Antibody 8E8 has a heavy chain
derived from murine germlines VH1-80_01 and IGHJ2, and light chain
germlines VK1-110_01 and IGKJ2. Antibody 5G7 has a heavy chain
derived from murine germlines VH1-18_01 and IGHJ4, and light chain
germlines VK10-96_01 and IGKJ2. Antibody 19G3 has a heavy chain
derived from murine germlines VH5-9-4_01 and IGHJ3, and light chain
germlines VK1-117_01 and IGKJ2. Heavy chain D region germline
sequences (making up part of CDRH3) are not specified, as they are
often difficult to assign given their high variability, and thus
antibodies of the present invention may comprise heavy chains
derived from the listed V and J region germlines and any D region
germline. Other antibodies that bind to human CD40 and are derived
from some or all of these germline sequences are likely to be
closely related in sequence, particularly those derived from the
same V-region genes, and thus would be expected to share the same
desirable properties.
[0117] As used herein, a murine antibody comprises heavy or light
chain variable regions that are "derived from" a particular
germline sequence if the variable regions of the antibody are
obtained from a system that uses murine germline immunoglobulin
genes, and the antibody sequence is sufficiently related to the
germline that it is more likely derived from the given germline
than from any other. Such systems include immunizing a mouse with
the antigen of interest. The murine germline immunoglobulin
sequence(s) from which the sequence of an antibody is "derived" can
be identified by comparing the amino acid sequence of the antibody
to the amino acid sequences of murine germline immunoglobulins and
selecting the germline immunoglobulin sequence that is closest in
sequence (i.e., greatest % identity) to the sequence of the
antibody. A murine antibody that is "derived from" a particular
germline immunoglobulin sequence may contain amino acid differences
as compared to the germline sequence due to, for example,
naturally-occurring somatic mutations or intentional introduction
of site-directed mutation. However, a selected murine antibody
typically is at least 90% identical in amino acids sequence to an
amino acid sequence encoded by a germline immunoglobulin gene (e.g.
V regions). In certain cases, a murine antibody may be at least
95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid
sequence to the amino acid sequence encoded by the germline
immunoglobulin gene (e.g. V regions). Typically, an antibody
derived from a particular murine germline sequence will display no
more than 10 amino acid differences from the amino acid sequence
encoded by the germline immunoglobulin gene (e.g. V regions). In
certain cases, the murine antibody may comprise no more than 5, or
even no more than 4, 3, 2, or 1 amino acid difference from the
amino acid sequence encoded by the germline immunoglobulin gene
(e.g. V regions).
II. Engineered and Modified Antibodies
VH and VL Regions
[0118] Also provided are engineered and modified antibodies that
can be prepared using an antibody having one or more of the V.sub.H
and/or V.sub.L sequences disclosed herein as starting material to
engineer a modified antibody, which modified antibody may have
altered properties from the starting antibody. An antibody can be
engineered by modifying one or more residues within one or both
variable regions (i.e., VH and/or VL), for example within one or
more CDR regions and/or within one or more framework regions.
Additionally or alternatively, an antibody can be engineered by
modifying residues within the constant region(s), for example to
alter the effector function(s) of the antibody.
[0119] One type of variable region engineering that can be
performed is CDR grafting. Such grafting is of particular use in
humanizing non-human anti-CD40 antibodies that compete for binding
with the anti-huCD40 antibodies disclosed herein and/or bind to the
same epitope as the anti-huCD40 antibodies disclosed herein.
Antibodies interact with target antigens predominantly through
amino acid residues that are located in the six heavy and light
chain complementarity determining regions (CDRs). For this reason,
the amino acid sequences within CDRs are more diverse between
individual antibodies than sequences outside of CDRs. Because CDR
sequences are responsible for most antibody-antigen interactions,
it is possible to express recombinant antibodies that mimic the
properties of specific reference antibodies by constructing
expression vectors that include CDR sequences from the specific
reference antibody grafted onto framework sequences from a
different antibody with different properties (see, e.g., Riechmann,
L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature
321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. See. (USA)
86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.)
[0120] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "VBase"
human germline sequence database, as well as in Kabat, E. A., et
al. (1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The
Repertoire of Human Germline VH Sequences Reveals about Fifty
Groups of VH Segments with Different Hypervariable Loops" J. Mol.
Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A Directory of
Human Germ-line VH Segments Reveals a Strong Bias in their Usage"
Eur. J. Immunol. 24:827-836; the contents of each of which are
expressly incorporated herein by reference.
[0121] Preferred framework sequences for use in the antibodies
described herein are those that are structurally similar to the
framework sequences used by antibodies described herein. The VH
CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3 sequences, can be
grafted onto framework regions that have the identical sequence as
that found in the germline immunoglobulin gene from which the
framework sequence derive, or the CDR sequences can be grafted onto
framework regions that contain up to 20, preferably conservative,
amino acid substitutions as compared to the germline sequences. For
example, it has been found that in certain instances it is
beneficial to mutate residues within the framework regions to
maintain or enhance the antigen binding ability of the antibody
(see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and
6,180,370 to Queen et al).
[0122] Engineered antibodies described herein include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.L, e.g. to improve the properties of the
antibody. Often such framework modifications are made to decrease
the immunogenicity of the antibody. For example, one approach is to
"backmutate" one or more framework residues to the corresponding
germline sequence. More specifically, an antibody that has
undergone somatic mutation may contain framework residues that
differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived. To return the framework region sequences to
their germline configuration, the somatic mutations can be
"backmutated" to the germline sequence by, for example,
site-directed mutagenesis or PCR-mediated mutagenesis. Such
"backmutated" antibodies are also intended to be encompassed.
[0123] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 20030153043 by Carr et al.
[0124] Another type of variable region modification is to mutate
amino acid residues within the CDR regions to improve one or more
binding properties (e.g., affinity) of the antibody of interest.
Site-directed mutagenesis or PCR-mediated mutagenesis can be
performed to introduce the mutation(s) and the effect on antibody
binding, or other functional property of interest. Preferably
conservative modifications are introduced. The mutations may be
amino acid additions, deletions, or preferably substitutions.
Moreover, typically no more than one, two, three, four or five
residues within a CDR region are altered.
[0125] Methionine residues in CDRs of antibodies can be oxidized,
resulting in potential chemical degradation and consequent
reduction in potency of the antibody. Accordingly, also provided
are anti-CD40 antibodies that have one or more methionine residues
in the heavy and/or light chain CDRs replaced with amino acid
residues that do not undergo oxidative degradation. Similarly,
deamidation sites may be removed from anti-CD40 antibodies,
particularly in the CDRs. Potential glycosylation sites within the
antigen binding domain are preferably eliminated to prevent
glycosylation that may interfere with antigen binding. See, e.g.,
U.S. Pat. No. 5,714,350.
Targeted Antigen Binding
[0126] In various embodiments, the antibody of the present
invention is modified to selectively block antigen binding in
tissues and environments where antigen binding would be
detrimental, but allow antigen binding where it would be
beneficial. In one embodiment, a blocking peptide "mask" is
generated that specifically binds to the antigen binding surface of
the antibody and interferes with antigen binding, which mask is
linked to each of the binding arms of the antibody by a peptidase
cleavable linker. See, e.g., U.S. Pat. No. 8,518,404 to CytomX.
Such constructs are useful for treatment of cancers in which
protease levels are greatly increased in the tumor microenvironment
compared with non-tumor tissues. Selective cleavage of the
cleavable linker in the tumor microenvironment allows
disassociation of the masking/blocking peptide, enabling antigen
binding selectively in the tumor, rather than in peripheral tissues
in which antigen binding might cause unwanted side effects.
[0127] Alternatively, in a related embodiment, a bivalent binding
compound ("masking ligand") comprising two antigen binding domains
is developed that binds to both antigen binding surfaces of the
(bivalent) antibody and interfere with antigen binding, in which
the two binding domains masks are linked to each other (but not the
antibody) by a cleavable linker, for example cleavable by a
peptidase. See, e.g., Int'l Pat. App. Pub. No. WO 2010/077643 to
Tegopharm Corp. Masking ligands may comprise, or be derived from,
the antigen to which the antibody is intended to bind, or may be
independently generated. Such masking ligands are useful for
treatment of cancers in which protease levels are greatly increased
in the tumor microenvironment compared with non-tumor tissues.
Selective cleavage of the cleavable linker in the tumor
microenvironment allows disassociation of the two binding domains
from each other, reducing the avidity for the antigen-binding
surfaces of the antibody. The resulting dissociation of the masking
ligand from the antibody enables antigen binding selectively in the
tumor, rather than in peripheral tissues in which antigen binding
might cause unwanted side effects.
Fcs and Modified Fcs
[0128] Antibodies of the present invention may comprise the
variable domains of the invention combined with constant domains
comprising different Fc regions, selected based on the biological
activities (if any) of the antibody for the intended use. Salfeld
(2007) Nat. Biotechnol. 25:1369. Human IgGs, for example, can be
classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, and
each these of these comprises an Fc region having a unique profile
for binding to one or more of Fey receptors (activating receptors
Fc.gamma.RI (CD64), Fc.gamma.RIIA, Fc.gamma.RIIC (CD32a,c);
Fc.gamma.RIIIA and Fc.gamma.RIIIB (CD16a,b) and inhibiting receptor
Fc.gamma.RIIB (CD32b), and for the first component of complement
(C1q). Human IgG1 and IgG3 bind to all Fey receptors; IgG2 binds to
Fc.gamma.RIIA.sub.H131, and with lower affinity to
Fc.gamma.RIIA.sub.R131 Fc.gamma.RIIIA.sub.V158; IgG4 binds to
Fc.gamma.RI, Fc.gamma.RIIA, Fc.gamma.RIIB, Fc.gamma.RIIC, and
Fc.gamma.RIIIA.sub.V158; and the inhibitory receptor Fc.gamma.RIIB
has a lower affinity for IgG1, IgG2 and IgG3 than all other Fey
receptors. Bruhns et al. (2009) Blood 113:3716. Studies have shown
that Fc.gamma.RI does not bind to IgG2, and Fc.gamma.RIIIB does not
bind to IgG2 or IgG4. Id. In general, with regard to ADCC activity,
human IgG1 .quadrature. IgG3 .quadrature. IgG4 .quadrature. IgG2.
As a consequence, for example, an IgG1 constant domain, rather than
an IgG2 or IgG4, might be chosen for use in a drug where ADCC is
desired; IgG3 might be chosen if activation of
Fc.gamma.RIIIA-expressing NK cells, monocytes of macrophages; and
IgG4 might be chosen if the antibody is to be used to desensitize
allergy patients. IgG4 may also be selected if it is desired that
the antibody lack all effector function.
[0129] Anti-huCD40 variable regions described herein may be linked
(e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2,
IgG3 or IgG4 Fc, which may be of any allotype or isoallotype, e.g.,
for IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m,
G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1),
G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3),
G3m24(c5), G3m26(u), G3m27(v). See, e.g., Jefferis et al. (2009)
mAbs 1:1). Selection of allotype may be influenced by the potential
immunogenicity concerns, e.g. to minimize the formation of
anti-drug antibodies.
[0130] In preferred embodiments, anti-CD40 antibodies of the
present invention have an Fc that binds to or has enhanced binding
to Fc.gamma.RIIb, which can provide enhanced agonism. See, e.g., WO
2012/087928; Li & Ravetch (2011) Science 333:1030; Wilson et
al. (2011) Cancer Cell 19:101; White et al. (2011) J. Immunol.
187:1754. Variable regions described herein may be linked to Fc
variants that enhance affinity for the inhibitory receptor
Fc.gamma.RIIb, e.g. to enhance apoptosis-inducing or adjuvant
activity. Li & Ravetch (2012) Proc. Nat'l Acad. Sci. (USA)
109:10966; U.S. Pat. App. Pub. 2014/0010812. Such variants may
provide an antibody with immunomodulatory activities related to
Fc.gamma.RIIb+ cells, including for example B cells and monocytes.
In one embodiment, the Fc variants provide selectively enhanced
affinity to Fc.gamma.RIIb relative to one or more activating
receptors. Such variants may also exhibit enhanced FcR-mediated
cross-linking, resulting in enhanced therapeutic efficacy.
Modifications for altering binding to Fc.gamma.RIIb include one or
more modifications at a position selected from the group consisting
of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and
332, according to the EU index. Exemplary substitutions for
enhancing Fc.gamma.RIIb affinity include but are not limited to
234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D,
237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F,
328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D,
239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc
variants for enhancing binding to Fc.gamma.RIIb include 235Y-267E,
236D-267E, 239D-268D, 239D-267E, 267E-268D, 267E-268E, and
267E-328F. Specifically, the S267E, G236D, S239D, L328F and I332E
variants, including the S267E-L328F double variant, of human IgG1
are of particular value in specifically enhancing affinity for the
inhibitory Fc.gamma.RIIb receptor. Chu et al. (2008) Mol. Immunol.
45:3926; U.S. Pat. App. Pub. 2006/024298; WO 2012/087928. Enhanced
specificity for Fc.gamma.RIIb (as distinguished from
Fc.gamma.RIIa.sub.R131) may be obtained by adding the P238D
substitution and other mutations (Mimoto et al. (2013) Protein.
Eng. Des. & Selection 26:589; WO 2012/1152410), as well as
V262E and V264E (Yu et al. (2013) J. Am. Chem. Soc. 135:9723, and
WO 2014/184545. See Table 4 (supra).
Half-Life Extension
[0131] In certain embodiments, the antibody is modified to increase
its biological half-life. Various approaches are possible. For
example, this may be done by increasing the binding affinity of the
Fc region for FcRn. In one embodiment, the antibody is altered
within the CH1 or CL region to contain a salvage receptor binding
epitope taken from two loops of a CH2 domain of an Fc region of an
IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by
Presta et al. Other exemplary Fc variants that increase binding to
FcRn and/or improve pharmacokinetic properties include
substitutions at positions 259, 308, and 434, including for example
2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other
variants that increase Fc binding to FcRn include: 250E, 250Q,
428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8):
6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356),
256A, 272A, 305A, 307A, 31 1A, 312A, 378Q, 380A, 382A, 434A
(Shields et al., Journal of Biological Chemistry, 2001,
276(9):6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R,
434F, 434Y, 252Y/254T/256E, 433K/434F/436H (Dall'Acqua et al.,
Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al.,
2006, Journal of Biological Chemistry 281:23514-23524). See U.S.
Pat. No. 8,367,805.
[0132] Modification of certain conserved residues in IgG Fc (1253,
H310, Q311, H433, N434), such as the N434A variant (Yeung et al.
(2009) J. Immunol. 182:7663), have been proposed as a way to
increase FcRn affinity, thus increasing the half-life of the
antibody in circulation. WO 98/023289. The combination Fc variant
comprising M428L and N434S has been shown to increase FcRn binding
and increase serum half-life up to five-fold. Zalevsky et al.
(2010) Nat. Biotechnol. 28:157. The combination Fc variant
comprising T307A, E380A and N434A modifications also extends
half-life of IgG1 antibodies. Petkova et al. (2006) Int. Immunol.
18:1759. In addition, combination Fc variants comprising
M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A,
M428L-N434M, and M428L-N434S variants have also been shown to
extend half-life. WO 2009/086320.
[0133] Further, a combination Fc variant comprising M252Y, S254T
and T256E, increases half-life-nearly 4-fold. Dall'Acqua et al.
(2006) J. Biol. Chem. 281:23514. A related IgG1 modification
providing increased FcRn affinity but reduced pH dependence
(M252Y-S254T-T256E-H433K-N434F) has been used to create an IgG1
construct ("MST-HN Abdeg") for use as a competitor to prevent
binding of other antibodies to FcRn, resulting in increased
clearance of that other antibody, either endogenous IgG (e.g. in an
autoimmune setting) or another exogenous (therapeutic) mAb. Vaccaro
et al. (2005) Nat. Biotechnol. 23:1283; WO 2006/130834.
[0134] Other modifications for increasing FcRn binding are
described in Yeung et al. (2010) J. Immunol. 182:7663-7671;
6,277,375; 6,821,505; WO 97/34631; WO 2002/060919.
[0135] In certain embodiments, hybrid IgG isotypes may be used to
increase FcRn binding, and potentially increase half-life. For
example, an IgG1/IgG3 hybrid variant may be constructed by
substituting IgG1 positions in the CH2 and/or CH3 region with the
amino acids from IgG3 at positions where the two isotypes differ.
Thus a hybrid variant IgG antibody may be constructed that
comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T,
356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In other
embodiments described herein, an IgG1/IgG2 hybrid variant may be
constructed by substituting IgG2 positions in the CH2 and/or CH3
region with amino acids from IgG1 at positions where the two
isotypes differ. Thus a hybrid variant IgG antibody may be
constructed that comprises one or more substitutions, e.g., one or
more of the following amino acid substitutions: 233E, 234L, 235L,
-236G (referring to an insertion of a glycine at position 236), and
327A. See U.S. Pat. No. 8,629,113. A hybrid of IgG1/IgG2/IgG4
sequences has been generated that purportedly increases serum
half-life and improves expression. U.S. Pat. No. 7,867,491
(sequence number 18 therein).
[0136] The serum half-life of the antibodies of the present
invention can also be increased by pegylation. An antibody can be
pegylated to, for example, increase the biological (e.g., serum)
half-life of the antibody. To pegylate an antibody, the antibody,
or fragment thereof, typically is reacted with a polyethylene
glycol (PEG) reagent, such as a reactive ester or aldehyde
derivative of PEG, under conditions in which one or more PEG groups
become attached to the antibody or antibody fragment. Preferably,
the pegylation is carried out via an acylation reaction or an
alkylation reaction with a reactive PEG molecule (or an analogous
reactive water-soluble polymer). As used herein, the term
"polyethylene glycol" is intended to encompass any of the forms of
PEG that have been used to derivatize other proteins, such as mono
(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide. In certain embodiments, the antibody to be
pegylated is an aglycosylated antibody. Methods for pegylating
proteins are known in the art and can be applied to the antibodies
described herein. See for example, EP 0154316 by Nishimura et al.
and EP 0401384 by Ishikawa et al.
[0137] Alternatively, under some circumstances it may be desirable
to decrease the half-life of an antibody of the present invention,
rather than increase it. Modifications such as I253A (Hornick et
al. (2000) J. Nucl. Med. 41:355) and H435A/R, I253A or H310A (Kim
et al. (2000) Eur. J. Immunol. 29:2819) in Fc of human IgG1 can
decrease FcRn binding, thus decreasing half-life (increasing
clearance) for use in situations where rapid clearance is
preferred, such a medical imaging. See also Kenanova et al. (2005)
Cancer Res. 65:622. Other means to enhance clearance include
formatting the antigen binding domains of the present invention as
antibody fragments lacking the ability to bind FcRn, such as Fab
fragments. Such modification can reduce the circulating half-life
of an antibody from a couple of weeks to a matter of hours.
Selective PEGylation of antibody fragments can then be used to
fine-tune (increase) the half-life of the antibody fragments if
necessary. Chapman et al. (1999) Nat. Biotechnol. 17:780. Antibody
fragments may also be fused to human serum albumin, e.g. in a
fusion protein construct, to increase half-life. Yeh et al. (1992)
Proc. Nat'l Acad. Sci. 89:1904. Alternatively, a bispecific
antibody may be constructed with a first antigen binding domain of
the present invention and a second antigen binding domain that
binds to human serum albumin (HSA). See Int'l Pat. Appl. Pub. WO
2009/127691 and patent references cited therein. Alternatively,
specialized polypeptide sequences can be added to antibody
fragments to increase half-life, e.g. "XTEN" polypeptide sequences.
Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; Int'l Pat.
Appl. Pub. WO 2010/091122.
Additional Fc Variants
[0138] When using an IgG4 constant domain, it is usually preferable
to include the substitution S228P, which mimics the hinge sequence
in IgG1 and thereby stabilizes IgG4 molecules, e.g. reducing
Fab-arm exchange between the therapeutic antibody and endogenous
IgG4 in the patient being treated. Labrijn et al. (2009) Nat.
Biotechnol. 27:767; Reddy et al. (2000) J. Immunol. 164:1925.
[0139] A potential protease cleavage site in the hinge of IgG1
constructs can be eliminated by D221G and K222S modifications,
increasing the stability of the antibody. WO 2014/043344.
[0140] The affinities and binding properties of an Fc variant for
its ligands (Fc receptors) may be determined by a variety of in
vitro assay methods (biochemical or immunological based assays)
known in the art including but not limited to, equilibrium methods
(e.g., enzyme-linked immunosorbent assay (ELISA), or
radioimmunoassay (RIA)), or kinetics (e.g., BIACORE.RTM. SPR
analysis), and other methods such as indirect binding assays,
competitive inhibition assays, fluorescence resonance energy
transfer (FRET), gel electrophoresis and chromatography (e.g., gel
filtration), These and other methods may utilize a label on one or
more of the components being examined and/or employ a variety of
detection methods including but not limited to chromogenic,
fluorescent, luminescent, or isotopic labels. A detailed
description of binding affinities and kinetics can be found in
Paul, W. E., ed., Fundamental Immunology, 4th Ed.,
Lippincott-Raven, Philadelphia (1999), which focuses on
antibody-immunogen interactions.
[0141] In still other embodiments, the glycosylation of an antibody
is modified to increase or decrease effector function. For example,
an aglycoslated antibody can be made that lacks all effector
function by mutating the conserved asparagine residue at position
297 (e.g. N297A), thus abolishing complement and Fc.gamma.RI
binding. Bolt et al. (1993) Eur. J. Immunol. 23:403. See also Tao
& Morrison (1989) J. Immunol. 143:2595 (using N297Q in IgG1 to
eliminate glycosylation at position 297).
[0142] Although aglycosylated antibodies generally lack effector
function, mutations can be introduced to restore that function.
Aglycosylated antibodies, e.g. those resulting from N297A/C/D/or H
mutations or produced in systems (e.g. E. coli) that do not
glycosylate proteins, can be further mutated to restore Fc.gamma.R
binding, e.g. S298G and/or T299A/G/or H (WO 2009/079242), or E382V
and M428I (Jung et al. (2010) Proc. Nat'l Acad. Sci. (USA)
107:604).
[0143] Glycoengineering can also be used to modify the
anti-inflammatory properties of an IgG construct by changing the
.alpha.2,6 sialyl content of the carbohydrate chains attached at
Asn297 of the Fc regions, wherein an increased proportion of
.alpha.2,6 sialylated forms results in enhanced anti-inflammatory
effects. See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513.
Conversely, reduction in the proportion of antibodies having
.alpha.2,6 sialylated carbohydrates may be useful in cases where
anti-inflammatory properties are not wanted. Methods of modifying
.alpha.2,6 sialylation content of antibodies, for example by
selective purification of .alpha.2,6 sialylated forms or by
enzymatic modification, are provided at U.S. Pat. Appl. Pub. No.
2008/0206246. In other embodiments, the amino acid sequence of the
Fc region may be modified to mimic the effect of .alpha.2,6
sialylation, for example by inclusion of an F241A modification. WO
2013/095966.
III. Antibody Physical Properties
[0144] Antibodies described herein can contain one or more
glycosylation sites in either the light or heavy chain variable
region. Such glycosylation sites may result in increased
immunogenicity of the antibody or an alteration of the pK of the
antibody due to altered antigen binding (Marshall et al. (1972)
Ann. Rev. Biochem. 41:673-702; Gala and Morrison (2004) J. Immunol.
172:5489-94; Wallick et al. (1988) J. Exp. Med. 168:1099-109; Spiro
(2002) Glycobiology 12:43R-56R; Parekh et al. (1985) Nature
316:452-7; Mimura et al. (2000) Mol Immunol. 37:697-706).
Glycosylation has been known to occur at motifs containing an
N--X-S/T sequence. In some instances, it is preferred to have an
anti-huCD40 antibody that does not contain variable region
glycosylation. This can be achieved either by selecting antibodies
that do not contain the glycosylation motif in the variable region
or by mutating residues within the glycosylation region.
[0145] In certain embodiments, the antibodies described herein do
not contain asparagine isomerism sites. The deamidation of
asparagine may occur on N-G or D-G sequences and result in the
creation of an isoaspartic acid residue that introduces a kink into
the polypeptide chain and decreases its stability (isoaspartic acid
effect).
[0146] Each antibody will have a unique isoelectric point (pI),
which generally falls in the pH range between 6 and 9.5. The pI for
an IgG1 antibody typically falls within the pH range of 7-9.5 and
the pI for an IgG4 antibody typically falls within the pH range of
6-8. There is speculation that antibodies with a pI outside the
normal range may have some unfolding and instability under in vivo
conditions. Thus, it is preferred to have an anti-CD40 antibody
that contains a pI value that falls in the normal range. This can
be achieved either by selecting antibodies with a pI in the normal
range or by mutating charged surface residues.
[0147] Each antibody will have a characteristic melting
temperature, with a higher melting temperature indicating greater
overall stability in vivo (Krishnamurthy R and Manning M C (2002)
Curr. Pharm. Biotechnol. 3:361-71). Generally, it is preferred that
the TM1 (the temperature of initial unfolding) be greater than
60.degree. C., preferably greater than 65.degree. C., even more
preferably greater than 70.degree. C. The melting point of an
antibody can be measured using differential scanning calorimetry
(Chen et al. (2003) Pharm Res 20:1952-60; Ghirlando et al. (1999)
Immunol. Lett. 68:47-52) or circular dichroism (Murray et al.
(2002) J. Chromatogr. Sci. 40:343-9).
[0148] In a preferred embodiment, antibodies are selected that do
not degrade rapidly. Degradation of an antibody can be measured
using capillary electrophoresis (CE) and MALDI-MS (Alexander A J
and Hughes D E (1995) Anal Chem. 67:3626-32).
[0149] In another preferred embodiment, antibodies are selected
that have minimal aggregation effects, which can lead to the
triggering of an unwanted immune response and/or altered or
unfavorable pharmacokinetic properties. Generally, antibodies are
acceptable with aggregation of 25% or less, preferably 20% or less,
even more preferably 15% or less, even more preferably 10% or less
and even more preferably 5% or less. Aggregation can be measured by
several techniques, including size-exclusion column (SEC), high
performance liquid chromatography (HPLC), and light scattering.
IV. Nucleic Acid Molecules
[0150] Another aspect described herein pertains to nucleic acid
molecules that encode the antibodies described herein. 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 (e.g., other chromosomal DNA, e.g., the
chromosomal DNA that is linked to the isolated DNA in nature) or
proteins, by standard techniques, including alkaline/SDS treatment,
CsCl banding, column chromatography, restriction enzymes, agarose
gel electrophoresis and others well known in the art. See, F.
Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology,
Greene Publishing and Wiley Interscience, New York. A nucleic acid
described herein can be, for example, DNA or RNA and may or may not
contain intronic sequences. In a certain embodiments, the nucleic
acid is a cDNA molecule.
[0151] Nucleic acids described herein can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g., hybridomas prepared from transgenic mice carrying
human immunoglobulin genes as described further below), cDNAs
encoding the light and heavy chains of the antibody made by the
hybridoma can be obtained by standard PCR amplification or cDNA
cloning techniques. For antibodies obtained from an immunoglobulin
gene library (e.g., using phage display techniques), nucleic acid
encoding the antibody can be recovered from the library.
[0152] Once DNA fragments encoding VH and VL segments are obtained,
these DNA fragments can be further manipulated by standard
recombinant DNA techniques, for example to convert the variable
region genes to full-length antibody chain genes, to Fab fragment
genes or to a scFv gene. In these manipulations, a VL- or
VH-encoding DNA fragment is operatively linked to another DNA
fragment encoding another protein, such as an antibody constant
region or a flexible linker. The term "operatively linked", as used
in this context, is intended to mean that the two DNA fragments are
joined such that the amino acid sequences encoded by the two DNA
fragments remain in-frame.
[0153] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (hinge, CH1, CH2 and/or CH3). The sequences of
human heavy chain constant region genes are known in the art (see
e.g., Kabat, E. A., el al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, for
example, an IgG1 region. For a Fab fragment heavy chain gene, the
VH-encoding DNA can be operatively linked to another DNA molecule
encoding only the heavy chain CH1 constant region.
[0154] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region.
[0155] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such
that the VH and VL sequences can be expressed as a contiguous
single-chain protein, with the VL and VH regions joined by the
flexible linker (see e.g., Bird et al. (1988) Science 242:423-426;
Huston et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:5879-5883;
McCafferty et al., (1990) Nature 348:552-554).
V. Antibody Generation
[0156] Various antibodies of the present invention, e.g. those that
compete with or bind to the same epitope as the anti-human CD40
antibodies disclosed herein, can be produced using a variety of
known techniques, such as the standard somatic cell hybridization
technique described by Kohler and Milstein, Nature 256: 495 (1975).
Although somatic cell hybridization procedures are preferred, in
principle, other techniques for producing monoclonal antibodies
also can be employed, e.g., viral or oncogenic transformation of B
lymphocytes, phage display technique using libraries of human
antibody genes.
[0157] The preferred animal system for preparing hybridomas is the
murine system. Hybridoma production in the mouse is a very
well-established procedure. Immunization protocols and techniques
for isolation of immunized splenocytes for fusion are known in the
art. Fusion partners (e.g., murine myeloma cells) and fusion
procedures are also known.
[0158] Chimeric or humanized antibodies described herein can be
prepared based on the sequence of a murine monoclonal antibody
prepared as described above. DNA encoding the heavy and light chain
immunoglobulins can be obtained from the murine hybridoma of
interest and engineered to contain non-murine (e.g., human)
immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, the murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to
Cabilly et al.). To create a humanized antibody, the murine CDR
regions can be inserted into a human framework using methods known
in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.
Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.).
[0159] In one embodiment, the antibodies described herein are human
monoclonal antibodies. Such human monoclonal antibodies directed
against human CD40 can be generated using transgenic or
transchromosomic mice carrying parts of the human immune system
rather than the mouse system. These transgenic and transchromosomic
mice include mice referred to herein as HuMAb mice and KM mice,
respectively, and are collectively referred to herein as "human Ig
mice."
[0160] The HuMAb Mouse.RTM. (MEDAREX, Inc.) contains human
immunoglobulin gene miniloci that encode unrearranged human heavy
(.mu. and .gamma.) and .kappa. light chain immunoglobulin
sequences, together with targeted mutations that inactivate the
endogenous .mu. and .kappa. chain loci (see e.g., Lonberg, et al.
(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit
reduced expression of mouse IgM or .kappa., and in response to
immunization, the introduced human heavy and light chain transgenes
undergo class switching and somatic mutation to generate high
affinity human IgG.kappa. monoclonal (Lonberg, N. et al. (1994),
supra; reviewed in Lonberg, N. (1994) Handbook of Experimental
Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern.
Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995)
Ann. N.Y. Acad. Sci. 764:536-546). The preparation and use of HuMab
mice, and the genomic modifications carried by such mice, is
further described in Taylor, L. et al. (1992) Nucleic Acids
Research 20:6287-6295; Chen, J. et al. (1993) International
Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad.
Sci. (USA) 90:3720-3724; Choi et al. (1993) Nature Genetics
4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et
al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994)
International Immunology 6: 579-591; and Fishwild, D. et al. (1996)
Nature Biotechnology 14: 845-851, the contents of all of which are
hereby specifically incorporated by reference in their entirety.
See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299;
and 5,770,429; all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to
Surani et al.; PCT Publication Nos. WO 92/03918, WO 93/12227, WO
94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all to Lonberg
and Kay; and PCT Publication No. WO 01/14424 to Korman et al.
[0161] In certain embodiments, antibodies described herein are
raised using a mouse that carries human immunoglobulin sequences on
transgenes and transchromosomes, such as a mouse that carries a
human heavy chain transgene and a human light chain
transchromosome. Such mice, referred to herein as "KM mice", are
described in detail in PCT Publication WO 02/43478 to Ishida et
al.
[0162] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-huCD40 antibodies described herein. For
example, an alternative transgenic system referred to as the
Xenomouse (ABGENIX, Inc.) can be used; such mice are described in,
for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,
150,584 and 6,162,963 to Kucherlapati et al.
[0163] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-CD40 antibodies described herein. For
example, mice carrying both a human heavy chain transchromosome and
a human light chain transchromosome, referred to as "TC mice" can
be used; such mice are described in Tomizuka et al. (2000) Proc.
Natl. Acad. Sci. (USA) 97:722-727. Furthermore, cows carrying human
heavy and light chain transchromosomes have been described in the
art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can
be used to raise anti-huCD40 antibodies described herein.
[0164] Additional mouse systems described in the art for raising
human antibodies, e.g., human anti-huCD40 antibodies, include (i)
the VelocImmune.RTM. mouse (REGENERON Pharmaceuticals, Inc.), in
which the endogenous mouse heavy and light chain variable regions
have been replaced, via homologous recombination, with human heavy
and light chain variable regions, operatively linked to the
endogenous mouse constant regions, such that chimeric antibodies
(human V/mouse C) are raised in the mice, and then subsequently
converted to fully human antibodies using standard recombinant DNA
techniques; and (ii) the MeMo.RTM. mouse (Merus Biopharmaceuticals,
Inc.), in which the mouse contains unrearranged human heavy chain
variable regions but a single rearranged human common light chain
variable region. Such mice, and use thereof to raise antibodies,
are described in, for example, WO 2009/15777, US 2010/0069614, WO
2011/072204, WO 2011/097603, WO 2011/163311, WO 2011/163314, WO
2012/148873, US 2012/0070861 and US 2012/0073004.
[0165] Human monoclonal antibodies described herein can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. Such phage display methods for
isolating human antibodies are established in the art. See for
example: U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No.
5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717
to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to
McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;
6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et
al.
[0166] Human monoclonal antibodies described herein can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
Immunizations
[0167] To generate fully human antibodies to human CD40, mice or
transgenic or transchromosomal mice containing human immunoglobulin
genes (e.g., HCo12, HCo7 or KM mice) can be immunized with a
purified or enriched preparation of the CD40 antigen and/or cells
expressing CD40, as described for other antigens, for example, by
Lonberg et al. (1994) Nature 368(6474): 856-859; Fishwild et al.
(1996) Nature Biotechnology 14: 845-851 and WO 98/24884.
Alternatively, mice can be immunized with DNA encoding human CD40.
Preferably, the mice will be 6-16 weeks of age upon the first
infusion. For example, a purified or enriched preparation (5-50
.mu.g) of the recombinant human CD40 antigen can be used to
immunize the mice intraperitoneally. In the event that
immunizations using a purified or enriched preparation of the CD40
antigen do not result in antibodies, mice can also be immunized
with cells expressing CD40, e.g., a cell line, to promote immune
responses.
[0168] Cumulative experience with various antigens has shown that
the HuMAb transgenic mice respond best when initially immunized
intraperitoneally (IP) or subcutaneously (SC) with antigen in
Ribi's adjuvant, followed by every other week IP/SC immunizations
(up to a total of 10) with antigen in Ribi's adjuvant. The immune
response can be monitored over the course of the immunization
protocol with plasma samples being obtained by retroorbital bleeds.
The plasma can be screened by ELISA and FACS (as described below),
and mice with sufficient titers of anti-CD40 human immunoglobulin
can be used for fusions. Mice can be boosted intravenously with
antigen 3 days before sacrifice and removal of the spleen and lymph
nodes. It is expected that 2-3 fusions for each immunization may
need to be performed. Between 6 and 24 mice are typically immunized
for each antigen. Usually, HCo7, HCo12, and KM strains are used. In
addition, both HCo7 and HCo12 transgene can be bred together into a
single mouse having two different human heavy chain transgenes
(HCo7/HCo12).
Generation of Hybridomas Producing Monoclonal Antibodies to
CD40
[0169] To generate hybridomas producing monoclonal antibodies
described herein, splenocytes and/or lymph node cells from
immunized mice can be isolated and fused to an appropriate
immortalized cell line, such as a mouse myeloma cell line. The
resulting hybridomas can be screened for the production of
antigen-specific antibodies. For example, single cell suspensions
of splenic lymphocytes from immunized mice can be fused to Sp2/0
nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG.
Cells are plated at approximately 2.times.10.sup.5 in flat bottom
microtiter plate, followed by a two week incubation in selective
medium containing 10% fetal Clone Serum, 18% "653" conditioned
media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5
mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50
mg/ml streptomycin, 50 mg/ml gentamycin and 1.times.HAT (SIGMA).
After approximately two weeks, cells can be cultured in medium in
which the HAT is replaced with HT. Individual wells can then be
screened by ELISA for human monoclonal IgM and IgG antibodies. Once
extensive hybridoma growth occurs, medium can be observed usually
after 10-14 days. The antibody secreting hybridomas can be
replated, screened again, and if still positive for human IgG, the
monoclonal antibodies can be subcloned at least twice by limiting
dilution. The stable subclones can then be cultured in vitro to
generate small amounts of antibody in tissue culture medium for
characterization.
[0170] To purify monoclonal antibodies, selected hybridomas can be
grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-SEPHAROSE (PHARMACIA,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80.degree.
C.
VI. Antibody Manufacture
Generation of Transfectomas Producing Monoclonal Antibodies to
CD40
[0171] Antibodies of the present invention, including both specific
antibodies for which sequences are provided and other, related
anti-CD40 antibodies, can be produced in a host cell transfectoma
using, for example, a combination of recombinant DNA techniques and
gene transfection methods as is well known in the art (Morrison, S.
(1985) Science 229:1202).
[0172] For example, to express antibodies, or antibody fragments
thereof, DNAs encoding partial or full-length light and heavy
chains, can be obtained by standard molecular biology techniques
(e.g., PCR amplification or cDNA cloning using a hybridoma that
expresses the antibody of interest) and the DNAs can be inserted
into expression vectors such that the genes are operatively linked
to transcriptional and translational control sequences. In this
context, the term "operatively linked" is intended to mean that an
antibody gene is ligated into a vector such that transcriptional
and translational control sequences within the vector serve their
intended function of regulating the transcription and translation
of the antibody gene. The expression vector and expression control
sequences are chosen to be compatible with the expression host cell
used. The antibody light chain gene and the antibody heavy chain
gene can be inserted into separate vector or both genes are
inserted into the same expression vector. The antibody genes are
inserted into the expression vector(s) by standard methods (e.g.,
ligation of complementary restriction sites on the antibody gene
fragment and vector, or blunt end ligation if no restriction sites
are present). The light and heavy chain variable regions of the
antibodies described herein can be used to create full-length
antibody genes of any antibody isotype by inserting them into
expression vectors already encoding heavy chain constant and light
chain constant regions of the desired isotype such that the V.sub.H
segment is operatively linked to the C.sub.H segment(s) within the
vector and the V.sub.L segment is operatively linked to the C.sub.L
segment within the vector. Additionally or alternatively, the
recombinant expression vector can encode a signal peptide that
facilitates secretion of the antibody chain from a host cell. The
antibody chain gene can be cloned into the vector such that the
signal peptide is linked in-frame to the amino terminus of the
antibody chain gene. The signal peptide can be an immunoglobulin
signal peptide or a heterologous signal peptide (i.e., a signal
peptide from a non-immunoglobulin protein).
[0173] In addition to the antibody chain genes, recombinant
expression vectors may carry regulatory sequences that control the
expression of the antibody chain genes in a host cell. The term
"regulatory sequence" is intended to include promoters, enhancers
and other expression control elements (e.g., polyadenylation
signals) that control the transcription or translation of the
antibody chain genes. Such regulatory sequences are described, for
example, in Goeddel (Gene Expression Technology. Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will
be appreciated by those skilled in the art that the design of the
expression vector, including the selection of regulatory sequences,
may depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc.
Preferred regulatory sequences for mammalian host cell expression
include viral elements that direct high levels of protein
expression in mammalian cells, such as promoters and/or enhancers
derived from cytomegalovirus (CMV), Simian Virus 40 (SV40),
adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and
polyoma. Alternatively, nonviral regulatory sequences may be used,
such as the ubiquitin promoter or .beta.-globin promoter. Still
further, regulatory elements composed of sequences from different
sources, such as the SR.alpha. promoter system, which contains
sequences from the SV40 early promoter and the long terminal repeat
of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988)
Mol. Cell. Biol. 8:466-472).
[0174] In addition to the antibody chain genes and regulatory
sequences, recombinant expression vectors may carry additional
sequences, such as sequences that regulate replication of the
vector in host cells (e.g., origins of replication) and selectable
marker genes. The selectable marker gene facilitates selection of
host cells into which the vector has been introduced (see, e.g.,
U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et
al.). For example, typically the selectable marker gene confers
resistance to drugs, such as G418, hygromycin or methotrexate, on a
host cell into which the vector has been introduced. Preferred
selectable marker genes include the dihydrofolate reductase (DHFR)
gene (for use in dhfr-host cells with methotrexate
selection/amplification) and the neo gene (for G418 selection).
[0175] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies described herein in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (1985) Immunology Today 6:12-13). Antibodies of the present
invention can also be produced in glycoengineered strains of the
yeast Pichia pastoris. Li et al. (2006) Nat. Biotechnol.
24:210.
[0176] Preferred mammalian host cells for expressing the
recombinant antibodies described herein include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. (USA) 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells,
COS cells and SP2 cells. In particular, for use with NSO myeloma
cells, another preferred expression system is the GS gene
expression system disclosed in WO 87/04462, WO 89/01036 and EP
338,841. When recombinant expression vectors encoding antibody
genes are introduced into mammalian host cells, the antibodies are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. Antibodies can be
recovered from the culture medium using standard protein
purification methods.
[0177] The N- and C-termini of antibody polypeptide chains of the
present invention may differ from the expected sequence due to
commonly observed post-translational modifications. For example,
C-terminal lysine residues are often missing from antibody heavy
chains. Dick et al. (2008) Biotechnol. Bioeng. 100:1132. N-terminal
glutamine residues, and to a lesser extent glutamate residues, are
frequently converted to pyroglutamate residues on both light and
heavy chains of therapeutic antibodies. Dick et al. (2007)
Biotechnol. Bioeng. 97:544; Liu et al. (2011) JBC 28611211; Liu et
al. (2011) J. Biol. Chem. 286:11211.
[0178] Amino acid sequences for various agonist anti-huCD40
antibodies of the present invention are provided in the Sequence
Listing, which is summarized at Table 8. For the reasons mentioned
above, the C-terminal lysine is not included in any of sequences in
the Sequence Listing for heavy chains or heavy chain constant
domains. However, in an alternative embodiment, each heavy chain
for the anti-huCD40 antibodies of the present invention, and/or
genetic construct encoding such antibodies or the heavy or light
chains thereof, includes this additional lysine residue at the
C-terminus of the heavy chain(s).
VII. Assays
[0179] Antibodies described herein can be tested for binding to
CD40 by, for example, standard ELISA. Briefly, microtiter plates
are coated with purified CD40 at 1-2 .mu.g/ml in PBS, and then
blocked with 5% bovine serum albumin in PBS. Dilutions of antibody
(e.g., dilutions of plasma from CD40-immunized mice) are added to
each well and incubated for 1-2 hours at 37.degree. C. The plates
are washed with PBS/Tween and then incubated with secondary reagent
(e.g., for human antibodies, or antibodies otherwise having a human
heavy chain constant region, a goat-anti-human IgG Fc-specific
polyclonal reagent) conjugated to horseradish peroxidase (HRP) for
1 hour at 37.degree. C. After washing, the plates are developed
with ABTS substrate (MOSS Inc, product: ABTS-1000) and analyzed by
a spectrophotometer at OD 415-495. Sera from immunized mice are
then further screened by flow cytometry for binding to a cell line
expressing human CD40, but not to a control cell line that does not
express CD40. Briefly, the binding of anti-CD40 antibodies is
assessed by incubating CD40 expressing CHO cells with the anti-CD40
antibody at 1:20 dilution. The cells are washed and binding is
detected with a PE-labeled anti-human IgG Ab. Flow cytometric
analyses are performed using a FACScan flow cytometry (BECTON
DICKINSON, San Jose, Calif.). Preferably, mice that develop the
highest titers will be used for fusions. Analogous experiments may
be performed using anti-mouse detection antibodies if mouse
anti-huCD40 antibodies are to be detected.
[0180] An ELISA as described above can be used to screen for
antibodies and, thus, hybridomas that produce antibodies that show
positive reactivity with the CD40 immunogen. Hybridomas that
produce antibodies that bind, preferably with high affinity, to
CD40 can then be subcloned and further characterized. One clone
from each hybridoma, which retains the reactivity of the parent
cells (by ELISA), can then be chosen for making a cell bank, and
for antibody purification.
[0181] To purify anti-CD40 antibodies, selected hybridomas can be
grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-SEPHAROSE (PHARMACIA,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80.degree.
C.
[0182] To determine if the selected anti-CD40 monoclonal antibodies
bind to unique epitopes, each antibody can be biotinylated using
commercially available reagents (PIERCE, Rockford, Ill.).
Biotinylated MAb binding can be detected with a streptavidin
labeled probe. Competition studies using unlabeled monoclonal
antibodies and biotinylated monoclonal antibodies can be performed
using CD40 coated-ELISA plates as described above.
[0183] To determine the isotype of purified antibodies, isotype
ELISAs can be performed using reagents specific for antibodies of a
particular isotype. For example, to determine the isotype of a
human monoclonal antibody, wells of microtiter plates can be coated
with 1 .mu.g/ml of anti-human immunoglobulin overnight at 4.degree.
C. After blocking with 1% BSA, the plates are reacted with 1
.mu.g/ml or less of test monoclonal antibodies or purified isotype
controls, at ambient temperature for one to two hours. The wells
can then be reacted with either human IgG1 or human IgM-specific
alkaline phosphatase-conjugated probes. Plates are developed and
analyzed as described above.
[0184] To test the binding of monoclonal antibodies to live cells
expressing CD40, flow cytometry can be used. Briefly, cell lines
expressing membrane-bound CD40 (grown under standard growth
conditions) are mixed with various concentrations of monoclonal
antibodies in PBS containing 0.1% BSA at 4.degree. C. for 1 hour.
After washing, the cells are reacted with Phycoerythrin
(PE)-labeled anti-IgG antibody under the same conditions as the
primary antibody staining. The samples can be analyzed by FACScan
instrument using light and side scatter properties to gate on
single cells and binding of the labeled antibodies is determined.
An alternative assay using fluorescence microscopy may be used (in
addition to or instead of) the flow cytometry assay. Cells can be
stained exactly as described above and examined by fluorescence
microscopy. This method allows visualization of individual cells,
but may have diminished sensitivity depending on the density of the
antigen.
[0185] Anti-huCD40 antibodies can be further tested for reactivity
with the CD40 antigen by Western blotting. Briefly, cell extracts
from cells expressing CD40 can be prepared and subjected to sodium
dodecyl sulfate polyacrylamide gel electrophoresis. After
electrophoresis, the separated antigens will be transferred to
nitrocellulose membranes, blocked with 20% mouse serum, and probed
with the monoclonal antibodies to be tested. IgG binding can be
detected using anti-IgG alkaline phosphatase and developed with
BCIP/NBT substrate tablets (SIGMA Chem. Co., St. Louis, Mo.).
[0186] Methods for analyzing binding affinity, cross-reactivity,
and binding kinetics of various anti-CD40 antibodies include
standard assays known in the art, for example, Biolayer
Interferometry (BLI) analysis, and BIACORE.RTM. surface plasmon
resonance (SPR) analysis using a BIACORE.RTM. 2000 SPR instrument
(BIACORE AB, Uppsala, Sweden).
[0187] In one embodiment, an antibody specifically binds to the
extracellular region of human CD40. An antibody may specifically
bind to a particular domain (e.g., a functional domain) within the
extracellular domain of CD40. In certain embodiments, the antibody
specifically binds to the extracellular region of human CD40 and
the extracellular region of cynomolgus CD40. Preferably, an
antibody binds to human CD40 with high affinity.
VIII. Bispecific Molecules
[0188] Antibodies described herein may be used for forming
bispecific molecules. An anti-CD40 antibody, or antigen-binding
fragments thereof, can be derivatized or linked to another
functional molecule, e.g., another peptide or protein (e.g.,
another antibody or ligand for a receptor) to generate a bispecific
molecule that binds to at least two different binding sites or
target molecules. The antibody described herein may in fact be
derivatized or linked to more than one other functional molecule to
generate multispecific molecules that bind to more than two
different binding sites and/or target molecules; such multispecific
molecules are also intended to be encompassed by the term
"bispecific molecule" as used herein. To create a bispecific
molecule described herein, an antibody described herein can be
functionally linked (e.g., by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other binding
molecules, such as another antibody, antibody fragment, peptide or
binding mimetic, such that a bispecific molecule results.
[0189] Accordingly, provided herein are bispecific molecules
comprising at least one first binding specificity for CD40 and a
second binding specificity for a second target epitope. In an
embodiment described herein in which the bispecific molecule is
multispecific, the molecule can further include a third binding
specificity.
[0190] In one embodiment, the bispecific molecules described herein
comprise as a binding specificity at least one antibody, or an
antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')2,
Fv, or a single chain Fv. The antibody may also be a light chain or
heavy chain dimer, or any minimal fragment thereof such as a Fv or
a single chain construct as described in Ladner et al. U.S. Pat.
No. 4,946,778, the contents of which is expressly incorporated by
reference.
[0191] While human monoclonal antibodies are preferred, other
antibodies that can be employed in the bispecific molecules
described herein are murine, chimeric and humanized monoclonal
antibodies.
[0192] The bispecific molecules described herein can be prepared by
conjugating the constituent binding specificities using methods
known in the art. For example, each binding specificity of the
bispecific molecule can be generated separately and then conjugated
to one another. When the binding specificities are proteins or
peptides, a variety of coupling or cross-linking agents can be used
for covalent conjugation. Examples of cross-linking agents include
protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate
(SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB),
o-phenylenedimaleimide (oPDM),
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. (USA)
82:8648). Other methods include those described in Paulus (1985)
Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science
229:81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from PIERCE Chemical Co. (Rockford, Ill.).
[0193] When the binding specificities are antibodies, they can be
conjugated via sulfhydryl bonding of the C-terminus hinge regions
of the two heavy chains. In a particularly preferred embodiment,
the hinge region is modified to contain an odd number of sulfhydryl
residues, preferably one, prior to conjugation.
[0194] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab')2 or ligand x Fab
fusion protein. A bispecific molecule described herein can be a
single chain molecule comprising one single chain antibody and a
binding determinant, or a single chain bispecific molecule
comprising two binding determinants. Bispecific molecules may
comprise at least two single chain molecules. Methods for preparing
bispecific molecules are described for example in U.S. Pat. Nos.
5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;
5,013,653; 5,258,498; and 5,482,858.
[0195] Binding of the bispecific molecules to their specific
targets can be confirmed using art-recognized methods, such as
enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
FACS analysis, bioassay (e.g., growth inhibition), or Western Blot
assay. Each of these assays generally detects the presence of
protein-antibody complexes of particular interest by employing a
labeled reagent (e.g., an antibody) specific for the complex of
interest.
IX. Compositions
[0196] Further provided are compositions, e.g., a pharmaceutical
compositions, containing one or more anti-CD40 antibodies, or
antigen-binding fragment(s) thereof, as described herein,
formulated together with a pharmaceutically acceptable carrier.
Such compositions may include one or a combination of (e.g., two or
more different) antibodies, or immunoconjugates or bispecific
molecules described herein. For example, a pharmaceutical
composition described herein can comprise a combination of
antibodies (or immunoconjugates or bispecifics) that bind to
different epitopes on the target antigen or that have complementary
activities.
[0197] In certain embodiments, a composition comprises an anti-CD40
antibody at a concentration of at least 1 mg/ml, 5 mg/ml, 10 mg/ml,
50 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, or at 1-300 mg/ml or
100-300 mg/ml.
[0198] Pharmaceutical compositions described herein also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include an
anti-CD40 antibody described herein combined with at least one
other anti-cancer and/or T-cell stimulating (e.g., activating)
agent. Examples of therapeutic agents that can be used in
combination therapy are described in greater detail below in the
section on uses of the antibodies described herein.
[0199] In some embodiments, therapeutic compositions disclosed
herein can include other compounds, drugs, and/or agents used for
the treatment of cancer. Such compounds, drugs, and/or agents can
include, for example, chemotherapy drugs, small molecule drugs or
antibodies that stimulate the immune response to a given cancer. In
some instances, therapeutic compositions can include, for example,
one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an
anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-OX40 (also
known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibody, an
anti-LAG-3 antibody, an anti-CD73 antibody, an anti-CD137 antibody,
an anti-CD27 antibody, an anti-CSF-1R antibody, a TLR agonist, or a
small molecule antagonist of IDO or TGF.beta..
[0200] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, 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., antibody,
immunoconjugate, or bispecific molecule, may be coated in a
material to protect the compound from the action of acids and other
natural conditions that may inactivate the compound.
[0201] The pharmaceutical compounds described herein may include
one or more pharmaceutically acceptable salts. 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.
[0202] A pharmaceutical composition described herein also may
include a pharmaceutically acceptable anti-oxidant. 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.
[0203] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions described herein
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.
[0204] 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.
[0205] Pharmaceutically acceptable 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 described herein is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0206] 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.
[0207] 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.
[0208] The amount of active ingredient that 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 that can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition that produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0209] 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. 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 described herein 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.
[0210] For administration of the antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 0.3 mg/kg body weight,
1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10
mg/kg body weight or within the range of 1-10 mg/kg. An exemplary
treatment regime entails administration once per week, once every
two weeks, once every three weeks, once every four weeks, once a
month, once every 3 months or once every three to 6 months.
[0211] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. A therapeutic antibody is usually
administered on multiple occasions. Intervals between single
dosages can be, for example, weekly, monthly, every three months or
yearly. Intervals can also be irregular as indicated by measuring
blood levels of antibody to the target antigen in the patient. In
some methods, dosage is adjusted to achieve a plasma antibody
concentration of about 1-1000 .mu.g/ml and in some methods about
25-300 .mu.g/ml.
[0212] An antibody can be administered as a sustained release
formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half-life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can optionally be administered a
prophylactic regime, although in many immune-oncology indications
continued treatment is not necessary.
[0213] Actual dosage levels of the active ingredients in the
pharmaceutical compositions described herein 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 described herein 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.
[0214] A "therapeutically effective dosage" of an anti-CD40
antibody described herein preferably results in a decrease in
severity of disease symptoms, an increase in frequency and duration
of disease symptom-free periods, or a prevention of impairment or
disability due to the disease affliction. In the context of cancer,
a therapeutically effective dose preferably prevents further
deterioration of physical symptoms associated with cancer. Symptoms
of cancer are well-known in the art and include, for example,
unusual mole features, a change in the appearance of a mole,
including asymmetry, border, color and/or diameter, a newly
pigmented skin area, an abnormal mole, darkened area under nail,
breast lumps, nipple changes, breast cysts, breast pain, death,
weight loss, weakness, excessive fatigue, difficulty eating, loss
of appetite, chronic cough, worsening breathlessness, coughing up
blood, blood in the urine, blood in stool, nausea, vomiting, liver
metastases, lung metastases, bone metastases, abdominal fullness,
bloating, fluid in peritoneal cavity, vaginal bleeding,
constipation, abdominal distension, perforation of colon, acute
peritonitis (infection, fever, pain), pain, vomiting blood, heavy
sweating, fever, high blood pressure, anemia, diarrhea, jaundice,
dizziness, chills, muscle spasms, colon metastases, lung
metastases, bladder metastases, liver metastases, bone metastases,
kidney metastases, and pancreatic metastases, difficulty
swallowing, and the like. Therapeutic efficacy may be observable
immediately after the first administration of an agonistic
anti-huCD40 mAb of the present invention, or it may only be
observed after a period of time and/or a series of doses. Such
delayed efficacy my only be observed after several months of
treatment, up to 6, 9 or 12 months. It is critical not to decide
prematurely that an agonistic anti-huCD40 mAb of the present
invention lacks therapeutically efficacy in light of the delayed
efficacy exhibited by some immune-oncology agents.
[0215] A therapeutically effective dose may prevent or delay onset
of cancer, such as may be desired when early or preliminary signs
of the disease are present. Laboratory tests utilized in the
diagnosis of cancer include chemistries (including the measurement
of soluble CD40 or CD40L levels) (Hock et al. (2006) Cancer
106:2148; Chung & Lim (2014) J. Trans. Med. 12:102),
hematology, serology and radiology. Accordingly, any clinical or
biochemical assay that monitors any of the foregoing may be used to
determine whether a particular treatment is a therapeutically
effective dose for treating cancer. 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.
[0216] A composition described herein can be administered via one
or more routes of administration using one or more of 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. Preferred routes of
administration for antibodies described herein include intravenous,
intramuscular, intradermal, intraperitoneal, subcutaneous, spinal
or other parenteral routes of administration, for example by
injection or infusion. The phrase "parenteral administration" 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.
[0217] Alternatively, an antibody described herein can be
administered via a non-parenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically.
[0218] 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.
[0219] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition described herein 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 for use with anti-huCD40 antibodies
described herein include: U.S. Pat. No. 4,487,603, which discloses
an implantable micro-infusion pump for dispensing medication at a
controlled rate; U.S. Pat. No. 4,486,194, which discloses a
therapeutic device for administering medicaments through the skin;
U.S. Pat. No. 4,447,233, which discloses a medication infusion pump
for delivering medication at a precise infusion rate; U.S. Pat. No.
4,447,224, which discloses a variable flow implantable infusion
apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196,
which discloses an osmotic drug delivery system having
multi-chamber compartments; and U.S. Pat. No. 4,475,196, which
discloses an osmotic drug delivery system. These patents are
incorporated herein by reference. Many other such implants,
delivery systems, and modules are known to those skilled in the
art.
[0220] In certain embodiments, the anti-huCD40 antibodies described
herein 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
described herein 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); p 120 (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.
X. Uses and Methods
[0221] The antibodies, antibody compositions and methods described
herein have numerous in vitro and in vivo utilities involving, for
example, enhancement of immune response by agonizing CD40
signaling. In a preferred embodiment, the antibodies described
herein are human or humanized antibodies. For example, anti-huCD40
antibodies described herein can be administered to cells in
culture, in vitro or ex vivo, or to human subjects, e.g., in vivo,
to enhance immunity in a variety of diseases. Accordingly, provided
herein are methods of modifying an immune response in a subject
comprising administering to the subject an antibody, or
antigen-binding fragment thereof, described herein such that the
immune response in the subject is enhanced, stimulated or
up-regulated.
[0222] Preferred subjects include human patients in whom
enhancement of an immune response would be desirable. The methods
are particularly suitable for treating human patients having a
disorder that can be treated by augmenting an immune response
(e.g., the T-cell mediated immune response). In a particular
embodiment, the methods are particularly suitable for treatment of
cancer in vivo. To achieve antigen-specific enhancement of
immunity, anti-huCD40 antibodies described herein can be
administered together with an antigen of interest or the antigen
may already be present in the subject to be treated (e.g., a
tumor-bearing or virus-bearing subject). When antibodies to CD40
are administered together with another agent, the two can be
administered separately or simultaneously.
[0223] Also encompassed are methods for detecting the presence of
human CD40 antigen in a sample, or measuring the amount of human
CD40 antigen, comprising contacting the sample, and a control
sample, with a human monoclonal antibody, or an antigen binding
fragment thereof, that specifically binds to human CD40, under
conditions that allow for formation of a complex between the
antibody or fragment thereof and human CD40. The formation of a
complex is then detected, wherein a difference complex formation
between the sample compared to the control sample is indicative the
presence of human CD40 antigen in the sample. Moreover, the
anti-CD40 antibodies described herein can be used to purify human
CD40 via immunoaffinity purification.
[0224] Given the ability of anti-huCD40 antibodies described herein
to enhance co-stimulation of T cell responses, e.g.,
antigen-specific T cell responses, provided herein are in vitro and
in vivo methods of using the antibodies described herein to
stimulate, enhance or upregulate antigen-specific T cell responses,
e.g., anti-tumor T cell responses.
[0225] CD4+ and CD8+ T cell responses can be enhanced using
anti-CD40 antibodies. The T cells can be Teff cells, e.g., CD4+
Teff cells, CD8+ Teff cells, T helper (Th) cells and T cytotoxic
(Tc) cells.
[0226] Further encompassed are methods of enhancing an immune
response (e.g., an antigen-specific T cell response) in a subject
comprising administering an anti-huCD40 antibody described herein
to the subject such that an immune response (e.g., an
antigen-specific T cell response) in the subject is enhanced. In a
preferred embodiment, the subject is a tumor-bearing subject and an
immune response against the tumor is enhanced. A tumor may be a
solid tumor or a liquid tumor, e.g., a hematological malignancy. In
certain embodiments, a tumor is an immunogenic tumor. In certain
embodiments, a tumor is non-immunogenic. In certain embodiments, a
tumor is PD-L1 positive. In certain embodiments a tumor is PD-L1
negative. A subject may also be a virus-bearing subject and an
immune response against the virus is enhanced.
[0227] Further provided are methods for inhibiting growth of tumor
cells in a subject comprising administering to the subject an
anti-huCD40 antibody described herein such that growth of the tumor
is inhibited in the subject. Also provided are methods of treating
chronic viral infection in a subject comprising administering to
the subject an anti-huCD40 antibody described herein such that the
chronic viral infection is treated in the subject.
[0228] In certain embodiments, an anti-huCD40 antibody is given to
a subject as an adjunctive therapy. Treatments of subjects having
cancer with an anti-huCD40 antibody may lead to a long-term durable
response relative to the current standard of care; long term
survival of at least 1, 2, 3, 4, 5, 10 or more years, recurrence
free survival of at least 1, 2, 3, 4, 5, or 10 or more years. In
certain embodiments, treatment of a subject having cancer with an
anti-huCD40 antibody prevents recurrence of cancer or delays
recurrence of cancer by, e.g., 1, 2, 3, 4, 5, or 10 or more years.
An anti-CD40 treatment can be used as a primary or secondary line
of treatment.
[0229] These and other methods described herein are discussed in
further detail below.
Cancer
[0230] Provided herein are methods for treating a subject having
cancer, comprising administering to the subject an anti-huCD40
antibody described herein, such that the subject is treated, e.g.,
such that growth of cancerous tumors is inhibited or reduced and/or
that the tumors regress. An anti-huCD40 antibody can be used alone
to inhibit the growth of cancerous tumors. Alternatively, an
anti-huCD40 antibody can be used in conjunction with another agent,
e.g., other immunogenic agents, standard cancer treatments, or
other antibodies, as described below. Combination with an inhibitor
of PD-1, such as an anti-PD-1 or anti-PD-L1 antibody, is also
provided. See. e.g., Ellmark et al. (2015) Oncammunology 4:7
e1011484.
[0231] Accordingly, provided herein are methods of treating cancer,
e.g., by inhibiting growth of tumor cells, in a subject, comprising
administering to the subject a therapeutically effective amount of
an anti-huCD40 antibody described herein, e.g., a humanized form of
12D6, 5F11, 8E8, 5G7 or 19G3, or antigen-binding fragment thereof.
The antibody may be a humanized anti-huCD40 antibody (such as any
of the humanized anti-huCD40 antibodies described herein), a human
chimeric anti-huCD40 antibody, or a humanized non-human anti-huCD40
antibody, e.g., a human, chimeric or humanized anti-huCD40 antibody
that competes for binding with, or binds to the same epitope as, at
least one of the anti-huCD40 antibodies specifically described
herein.
[0232] Cancers whose growth may be inhibited using the antibodies
of the invention include cancers typically responsive to
immunotherapy. Non-limiting examples of cancers for treatment
include squamous cell carcinoma, small-cell lung cancer, non-small
cell lung cancer, squamous non-small cell lung cancer (NSCLC), non
NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g. clear
cell carcinoma), ovarian cancer, liver cancer, colorectal cancer,
endometrial cancer, kidney cancer (e.g., renal cell carcinoma
(RCC)), prostate cancer (e.g. hormone refractory prostate
adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer,
glioblastoma (glioblastoma multiforme), cervical cancer, stomach
cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma,
and head and neck cancer (or carcinoma), gastric cancer, germ cell
tumor, pediatric sarcoma, sinonasal natural killer, melanoma (e.g.,
metastatic malignant melanoma, such as cutaneous or intraocular
malignant melanoma), bone cancer, skin cancer, uterine cancer,
cancer of the anal region, testicular cancer, carcinoma of the
fallopian tubes, carcinoma of the endometrium, carcinoma of the
cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of
the esophagus, cancer of the small intestine, cancer of the
endocrine system, cancer of the parathyroid gland, cancer of the
adrenal gland, sarcoma of soft tissue, cancer of the urethra,
cancer of the penis, solid tumors of childhood, cancer of the
ureter, carcinoma of the renal pelvis, neoplasm of the central
nervous system (CNS), primary CNS lymphoma, tumor angiogenesis,
spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's
sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma,
environmentally-induced cancers including those induced by
asbestos, virus-related cancers (e.g., human papilloma virus
(HPV)-related tumor), and hematologic malignancies derived from
either of the two major blood cell lineages, i.e., the myeloid cell
line (which produces granulocytes, erythrocytes, thrombocytes,
macrophages and mast cells) or lymphoid cell line (which produces
B, T, NK and plasma cells), such as all types of leukemias,
lymphomas, and myelomas, e.g., acute, chronic, lymphocytic and/or
myelogenous leukemias, such as acute leukemia (ALL), acute
myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and
chronic myelogenous leukemia (CML), undifferentiated AML (MO),
myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell
maturation), promyelocytic leukemia (M3 or M3 variant [M3V]),
myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),
monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic
leukemia (M7), isolated granulocytic sarcoma, and chloroma;
lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma
(NHL), B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid
lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid
tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large-cell
lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma,
angio immunoblastic T-cell lymphoma, angiocentric lymphoma,
intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma,
precursor T-lymphoblastic lymphoma, T-lymphoblastic; and
lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma,
lymphoblastic lymphoma, post-transplantation lymphoproliferative
disorder, true histiocytic lymphoma, primary central nervous system
lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL),
hematopoietic tumors of lymphoid lineage, acute lymphoblastic
leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma,
follicular lymphoma, diffuse histiocytic lymphoma (DHL),
immunoblastic large cell lymphoma, precursor B-lymphoblastic
lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis
fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma
(LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG
myeloma, light chain myeloma, nonsecretory myeloma, smoldering
myeloma (also called indolent myeloma), solitary plasmocytoma, and
multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell
lymphoma; hematopoietic tumors of myeloid lineage, tumors of
mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;
seminoma, teratocarcinoma, tumors of the central and peripheral
nervous, including astrocytoma, schwannomas; tumors of mesenchymal
origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma;
and other tumors, including melanoma, xeroderma pigmentosum,
keratoacanthoma, seminoma, thyroid follicular cancer and
teratocarcinoma, hematopoietic tumors of lymphoid lineage, for
example T-cell and B-cell tumors, including but not limited to
T-cell disorders such as T-prolymphocytic leukemia (T-PLL),
including of the small cell and cerebriform cell type; large
granular lymphocyte leukemia (LGL) preferably of the T-cell type;
a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell
lymphoma (pleomorphic and immunoblastic subtypes); angiocentric
(nasal) T-cell lymphoma; cancer of the head or neck, renal cancer,
rectal cancer, cancer of the thyroid gland; acute myeloid lymphoma,
as well as any combinations of said cancers. The methods described
herein may also be used for treatment of metastatic cancers,
refractory cancers (e.g., cancers refractory to previous
immunotherapy, e.g., with a blocking CTLA-4 or PD-1 antibody), and
recurrent cancers.
[0233] Notwithstanding the above, the agonist anti-huCD40
antibodies of the present invention will not find use in treating
hematologic cancers with CD40 expression, which might be
exacerbated by treatment with a CD40 agonist. Certain cancers may
be known to express CD40 and thus be subject to such exacerbation,
and thus may be categorically excluded. In other embodiments
specific tumor samples are tested for expression of CD40 and are
excluded from therapy with the agonist anti-huCD40 antibodies of
the present invention based on the test results.
[0234] An anti-huCD40 antibody can be administered as a
monotherapy, or as the only immunostimulating therapy, or it can be
combined with an immunogenic agent in a cancer vaccine strategy,
such as cancerous cells, purified tumor antigens (including
recombinant proteins, peptides, and carbohydrate molecules), cells,
and cells transfected with genes encoding immune stimulating
cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting
examples of tumor vaccines that can be used include peptides of
melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2,
MART1 and/or tyrosinase, or tumor cells transfected to express the
cytokine GM-CSF. Many experimental strategies for vaccination
against tumors have been devised (see Rosenberg, S., 2000,
Development of Cancer Vaccines, ASCO Educational Book Spring:
60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302;
Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K.
2000, ASCO Educational Book Spring: 730-738; see also Restifo, N.
and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et
al. (eds.), 1997, Cancer: Principles and Practice of Oncology,
Fifth Edition). In one of these strategies, a vaccine is prepared
using autologous or allogeneic tumor cells. These cellular vaccines
have been shown to be most effective when the tumor cells are
transduced to express GM-CSF. GM-CSF has been shown to be a potent
activator of antigen presentation for tumor vaccination. Dranoff et
al. (1993) Proc. Natl. Acad. Sci. (USA) 90: 3539-43.
[0235] The study of gene expression and large scale gene expression
patterns in various tumors has led to the definition of so called
tumor specific antigens. Rosenberg, S A (1999) Immunity 10: 281-7.
In many cases, these tumor specific antigens are differentiation
antigens expressed in the tumors and in the cell from which the
tumor arose, for example melanocyte antigens gp100, MAGE antigens,
and Trp-2. More importantly, many of these antigens can be shown to
be the targets of tumor specific T cells found in the host. CD40
agonists can be used in conjunction with a collection of
recombinant proteins and/or peptides expressed in a tumor in order
to generate an immune response to these proteins. These proteins
are normally viewed by the immune system as self antigens and are
therefore tolerant to them. The tumor antigen can include the
protein telomerase, which is required for the synthesis of
telomeres of chromosomes and which is expressed in more than 85% of
human cancers and in only a limited number of somatic tissues (Kim
et al. (1994) Science 266: 2011-2013). Tumor antigen can also be
"neo-antigens" expressed in cancer cells because of somatic
mutations that alter protein sequence or create fusion proteins
between two unrelated sequences (i.e., bcr-abl in the Philadelphia
chromosome), or idiotype from B cell tumors.
[0236] Other tumor vaccines can include the proteins from viruses
implicated in human cancers such a Human Papilloma Viruses (HPV),
Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus
(KHSV). Another form of tumor specific antigen that can be used in
conjunction with CD40 inhibition is purified heat shock proteins
(HSP) isolated from the tumor tissue itself. These heat shock
proteins contain fragments of proteins from the tumor cells and
these HSPs are highly efficient at delivery to antigen presenting
cells for eliciting tumor immunity (Suot & Srivastava (1995)
Science 269:1585-1588; Tamura et al. (1997) Science
278:117-120).
[0237] Dendritic cells (DC) are potent antigen presenting cells
that can be used to prime antigen-specific responses. DC's can be
produced ex vivo and loaded with various protein and peptide
antigens as well as tumor cell extracts (Nestle et al. (1998)
Nature Medicine 4: 328-332). DCs can also be transduced by genetic
means to express these tumor antigens as well. DCs have also been
fused directly to tumor cells for the purposes of immunization
(Kugler et al. (2000) Nature Medicine 6:332-336). As a method of
vaccination, DC immunization can be effectively combined with CD40
agonism to activate (unleash) more potent anti-tumor responses.
[0238] Agonism of CD40 can also be combined with standard cancer
treatments (e.g., surgery, radiation, and chemotherapy). Agonism of
CD40 can be effectively combined with chemotherapeutic regimes. In
these instances, it may be possible to reduce the dose of
chemotherapeutic reagent administered (Mokyr et al. (1998) Cancer
Research 58: 5301-5304). An example of such a combination is an
anti-huCD40 antibody in combination with decarbazine for the
treatment of melanoma. Another example of such a combination is an
anti-huCD40 antibody in combination with interleukin-2 (IL-2) for
the treatment of melanoma. The scientific rationale behind the
combined use of CD40 agonists and chemotherapy is that cell death,
that is a consequence of the cytotoxic action of most
chemotherapeutic compounds, should result in increased levels of
tumor antigen in the antigen presentation pathway. Other
combination therapies that may result in synergy with CD40 agonism
through cell death are radiation, surgery, and hormone deprivation.
Each of these protocols creates a source of tumor antigen in the
host. Angiogenesis inhibitors can also be combined with CD40
agonists. Inhibition of angiogenesis leads to tumor cell death
which may feed tumor antigen into host antigen presentation
pathways.
[0239] The anti-huCD40 antibodies described herein can also be used
in combination with bispecific antibodies that target Fc.alpha. or
Fc.gamma. receptor-expressing effectors cells to tumor cells (see,
e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243). Bispecific
antibodies can be used to target two separate antigens. For example
anti-Fc receptor/anti tumor antigen (e.g., Her-2/neu) bispecific
antibodies have been used to target macrophages to sites of tumor.
This targeting may more effectively activate tumor specific
responses. The T cell arm of these responses would be augmented by
agonism of CD40. Alternatively, antigen may be delivered directly
to DCs by the use of bispecific antibodies that bind to tumor
antigen and a dendritic cell specific cell surface marker.
[0240] Tumors evade host immune surveillance by a large variety of
mechanisms. Many of these mechanisms may be overcome by the
inactivation of immunosuppressive proteins expressed by the tumors.
These include among others TGF-.beta. (Kehrl et al. (1986) J. Exp.
Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology
Today 13: 198-200), and Fas ligand (Hahne et al. (1996) Science
274: 1363-1365). Antibodies to each of these entities can be used
in combination with anti-huCD40 antibodies to counteract the
effects of the immunosuppressive agent and favor tumor immune
responses by the host.
[0241] Anti-CD40 antibodies are able to substitute effectively for
T cell helper activity. Ridge et al. (1998) Nature 393: 474-478.
Activating antibodies to T cell costimulatory molecules such as
CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg et al.
(2000) Immunol. 164: 2160-2169), CD137/4-1BB (Melero et al. (1997)
Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff et al. (1999)
Nature 397: 262-266) may also provide for increased levels of T
cell activation. Inhibitors of PD1 or PD-L1 may also be used in
conjunction with anti-huCD40 antibodies.
[0242] There are also several experimental treatment protocols that
involve ex vivo activation and expansion of antigen specific T
cells and adoptive transfer of these cells into recipients in order
to stimulate antigen-specific T cells against tumor (Greenberg
& Riddell (1999) Science 285: 546-51). These methods can also
be used to activate T cell responses to infectious agents such as
CMV. Ex vivo activation in the presence of anti-CD40 antibodies can
increase the frequency and activity of the adoptively transferred T
cells.
Chronic Viral Infections
[0243] In another aspect, the invention described herein provides a
method of treating an infectious disease in a subject comprising
administering to the subject an anti-huCD40 antibody, or
antigen-binding fragment thereof, such that the subject is treated
for the infectious disease.
[0244] Similar to its application to tumors as discussed above,
antibody-mediated CD40 agonism can be used alone, or as an
adjuvant, in combination with vaccines, to enhance the immune
response to pathogens, toxins, and self-antigens. Examples of
pathogens for which this therapeutic approach can be particularly
useful, include pathogens for which there is currently no effective
vaccine, or pathogens for which conventional vaccines are less than
completely effective. These include, but are not limited to HIV,
Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria,
Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. CD40
agonism is particularly useful against established infections by
agents such as HIV that present altered antigens over the course of
the infections. These novel epitopes are recognized as foreign at
the time of anti-human CD40 antibody administration, thus provoking
a strong T cell response.
[0245] Some examples of pathogenic viruses causing infections
treatable by methods described herein include HIV, hepatitis (A, B,
or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV,
Epstein Barr virus), adenovirus, influenza virus, flaviviruses,
echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory
syncytial virus, mumps virus, rotavirus, measles virus, rubella
virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,
papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus
and arboviral encephalitis virus.
[0246] Some examples of pathogenic bacteria causing infections
treatable by methods described herein include chlamydia,
rickettsial bacteria, mycobacteria, staphylococci, streptococci,
pneumonococci, meningococci and gonococci, klebsiella, proteus,
serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli,
cholera, tetanus, botulism, anthrax, plague, leptospirosis, and
Lymes disease bacteria.
[0247] Some examples of pathogenic fungi causing infections
treatable by methods described herein include Candida (albicans,
krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans,
Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor,
absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis,
Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma
capsulatum.
[0248] Some examples of pathogenic parasites causing infections
treatable by methods described herein include Entamoeba
histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp.,
Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,
Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma
cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus
brasiliensis.
[0249] In all of the above methods, CD40 agonism can be combined
with other forms of immunotherapy such as cytokine treatment (e.g.,
interferons, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy,
which provides for enhanced presentation of tumor antigens. See,
e.g., Holliger (1993) Proc. Natl. Acad. Sci. (USA) 90:6444-6448;
Poljak (1994) Structure 2:1121-1123.
Vaccine Adjuvants
[0250] Anti-huCD40 antibodies described herein can be used to
enhance antigen-specific immune responses by co-administration of
an anti-huCD40 antibody with an antigen of interest, e.g., a
vaccine. Accordingly, provided herein are methods of enhancing an
immune response to an antigen in a subject, comprising
administering to the subject: (i) the antigen; and (ii) an
anti-huCD40 antibody, or antigen-binding fragment thereof, such
that an immune response to the antigen in the subject is enhanced.
The antigen can be, for example, a tumor antigen, a viral antigen,
a bacterial antigen or an antigen from a pathogen. Non-limiting
examples of such antigens include those discussed in the sections
above, such as the tumor antigens (or tumor vaccines) discussed
above, or antigens from the viruses, bacteria or other pathogens
described above.
[0251] Suitable routes of administering the antibody compositions
(e.g., human monoclonal antibodies, multispecific and bispecific
molecules and immunoconjugates) described herein in vivo and in
vitro are well known in the art and can be selected by those of
ordinary skill. For example, the antibody compositions can be
administered by injection (e.g., intravenous or subcutaneous).
Suitable dosages of the molecules used will depend on the age and
weight of the subject and the concentration and/or formulation of
the antibody composition.
[0252] As previously described, anti-huCD40 antibodies described
herein can be co-administered with one or other more therapeutic
agents, e.g., a cytotoxic agent, a radiotoxic agent or an
immunosuppressive agent. The antibody can be linked to the agent
(as an immuno-complex) or can be administered separate from the
agent. In the latter case (separate administration), the antibody
can be administered before, after or concurrently with the agent or
can be co-administered with other known therapies, e.g., an
anti-cancer therapy, e.g., radiation. Such therapeutic agents
include, among others, anti-neoplastic agents such as doxorubicin
(adriamycin), cisplatin bleomycin sulfate, carmustine,
chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which,
by themselves, are only effective at levels which are toxic or
subtoxic to a patient. Cisplatin is intravenously administered as a
100 mg/ml dose once every four weeks and adriamycin is
intravenously administered as a 60-75 mg/ml dose once every 21
days. Co-administration of anti-CD40 antibodies, or antigen binding
fragments thereof, described herein with chemotherapeutic agents
provides two anti-cancer agents which operate via different
mechanisms which yield a cytotoxic effect to human tumor cells.
Such co-administration can solve problems due to development of
resistance to drugs or a change in the antigenicity of the tumor
cells that would render them unreactive with the antibody.
[0253] Also within the scope described herein are kits comprising
the antibody compositions described herein (e.g., human antibodies,
bispecific or multispecific molecules, or immunoconjugates) and
instructions for use. The kit can further contain at least one
additional reagent, or one or more additional human antibodies
described herein (e.g., a human antibody having a complementary
activity that binds to an epitope in CD40 antigen distinct from the
first human antibody). Kits typically include a label indicating
the intended use of the contents of the kit. The term label
includes any writing, or recorded material supplied on or with the
kit, or that otherwise accompanies the kit.
Combination Therapies
[0254] In addition to the combinations therapies provided above,
anti-CD40 antibodies described herein can also be used in
combination therapy, e.g., for treating cancer, as described
below.
[0255] The present invention provides methods of combination
therapy in which an anti-huCD40 antibody is co-administered with
one or more additional agents, e.g., antibodies, that are effective
in stimulating immune responses to thereby further enhance,
stimulate or upregulate immune responses in a subject.
[0256] Generally, an anti-huCD40 antibody described herein can be
combined with (i) an agonist of another co-stimulatory receptor
and/or (ii) an antagonist of an inhibitory signal on T cells,
either of which results in amplifying antigen-specific T cell
responses (immune checkpoint regulators). Most of the
co-stimulatory and co-inhibitory molecules are members of the
immunoglobulin super family (IgSF), and anti-CD40 antibodies
described herein may be administered with an agent that targets a
member of the IgSF family to increase an immune response. One
important family of membrane-bound ligands that bind to
co-stimulatory or co-inhibitory receptors is the B7 family, which
includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L),
B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane
bound ligands that bind to co-stimulatory or co-inhibitory
receptors is the TNF family of molecules that bind to cognate TNF
receptor family members, which include CD40 and CD40L, OX-40,
OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137/4-1BB,
TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG,
RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL,
BCMA, LT.beta.R, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR,
EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin .alpha./TNF.beta., TNFR2,
TNF.alpha., LT.beta.R, Lymphotoxin a 1(32, FAS, FASL, RELT, DR6,
TROY, NGFR (see, e.g., Tansey (2009) Drug Discovery Today
00:1).
[0257] In another aspect, anti-huCD40 antibodies can be used in
combination with antagonists of cytokines that inhibit T cell
activation (e.g., IL-6, IL-10, TGF-.beta., VEGF; or other
"immunosuppressive cytokines," or cytokines that stimulate T cell
activation, for stimulating an immune response, e.g., for treating
proliferative diseases, such as cancer.
[0258] In one aspect, T cell responses can be stimulated by a
combination of the anti-huCD40 mAbs of the present invention and
one or more of (i) an antagonist of a protein that inhibits T cell
activation (e.g., immune checkpoint inhibitors) such as CTLA-4,
PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69,
Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H,
LAIR1, TIM-1, and TIM-4, and (ii) an agonist of a protein that
stimulates T cell activation such as B7-1, B7-2, CD28, 4-1BB
(CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70,
CD27, CD40, DR3 and CD28H.
[0259] Exemplary agents that modulate one of the above proteins and
may be combined with agonist anti-huCD40 antibodies, e.g., those
described herein, for treating cancer, include:
YERVOY.RTM./ipilimumab or tremelimumab (to CTLA-4), galiximab (to
B7.1), BMS-936558 (to PD-1), pidilizumab/CT-011 (to PD-1),
KEYTRUDA.RTM./pembrolizumab/MK-3475 (to PD-1), AMP224 (to
B7-DC/PD-L2), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1), MEDI-570
(to ICOS), AMG557 (to B7H2), MGA271 (to B7H3--WO 11/109400), IMP321
(to LAG-3), urelumab/BMS-663513 and PF-05082566 (to CD137/4-1BB),
varlilumab/CDX-1127 (to CD27), MEDI-6383 and MEDI-6469 (to OX40),
RG-7888 (to OX40L--WO 06/029879), Atacicept (to TACI),
muromonab-CD3 (to CD3), ipilumumab (to CTLA-4).
[0260] Other molecules that can be combined with agonist
anti-huCD40 antibodies for the treatment of cancer include
antagonists of inhibitory receptors on NK cells or agonists of
activating receptors on NK cells. For example, agonist anti-huCD40
antibodies can be combined with antagonists of MR (e.g.,
lirilumab).
[0261] Yet other agents for combination therapies include agents
that inhibit or deplete macrophages or monocytes, including but not
limited to CSF-1R antagonists such as CSF-1R antagonist antibodies
including RG7155 (WO 11/70024, WO 11/107553, WO 11/131407, WO
13/87699, WO 13/119716, WO 13/132044) or FPA-008 (WO 11/140249; WO
13/169264; WO 14/036357).
[0262] Generally, agonist anti-huCD40 antibodies described herein
can be used together with one or more of agonistic agents that
ligate positive co-stimulatory receptors, blocking agents that
attenuate signaling through inhibitory receptors, and one or more
agents that increase systemically the frequency of anti-tumor T
cells, agents that overcome distinct immune suppressive pathways
within the tumor microenvironment (e.g., block inhibitory receptor
engagement (e.g., PD-L1/PD-1 interactions), deplete or inhibit
Tregs (e.g., using an anti-CD25 monoclonal antibody (e.g.,
daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit
metabolic enzymes such as IDO, or reverse/prevent T cell anergy or
exhaustion) and agents that trigger innate immune activation and/or
inflammation at tumor sites.
[0263] Provided herein are methods for stimulating an immune
response in a subject comprising administering to the subject a
CD40 agonist, e.g., an antibody, and one or more additional
immunostimulatory antibodies, such as a PD-1 antagonist, e.g.,
antagonist antibody, a PD-L1 antagonist, e.g., antagonist antibody,
a CTLA-4 antagonist, e.g., antagonist antibody and/or a LAG3
antagonist, e.g., an antagonist antibody, such that an immune
response is stimulated in the subject, for example to inhibit tumor
growth or to stimulate an anti-viral response. In one embodiment,
the subject is administered an agonist anti-huCD40 antibody and an
antagonist anti-PD-1 antibody. In one embodiment, the subject is
administered an agonist anti-huCD40 antibody and an antagonist
anti-PD-L1 antibody. In one embodiment, the subject is administered
an agonist anti-huCD40 antibody and an antagonist anti-CTLA-4
antibody. In one embodiment, the at least one additional
immunostimulatory antibody (e.g., an antagonist anti-PD-1, an
antagonist anti-PD-L1, an antagonist anti-CTLA-4 and/or an
antagonist anti-LAG3 antibody) is a human antibody. Alternatively,
the at least one additional immunostimulatory antibody can be, for
example, a chimeric or humanized antibody (e.g., prepared from a
mouse anti-PD-1, anti-PD-L1, anti-CTLA-4 and/or anti-LAG3
antibody).
[0264] Provided herein are methods for treating a
hyperproliferative disease (e.g., cancer), comprising administering
an agonist anti-huCD40 antibody and an antagonist PD-1 antibody to
a subject. In certain embodiments, the agonist anti-huCD40 antibody
is administered at a subtherapeutic dose, the anti-PD-1 antibody is
administered at a subtherapeutic dose, or both are administered at
a subtherapeutic dose. Also provided herein are methods for
altering an adverse event associated with treatment of a
hyperproliferative disease with an immunostimulatory agent,
comprising administering an agonist anti-huCD40 antibody and a
subtherapeutic dose of anti-PD-1 antibody to a subject. In certain
embodiments, the subject is human. In certain embodiments, the
anti-PD-1 antibody is a human sequence monoclonal antibody and the
agonist anti-huCD40 antibody is a humanized monoclonal antibody,
such as an antibody comprising the CDRs or variable regions of the
antibodies disclosed herein.
[0265] Suitable PD-1 antagonists for use in the methods described
herein, include, without limitation, ligands, antibodies (e.g.,
monoclonal antibodies and bispecific antibodies), and multivalent
agents. In one embodiment, the PD-1 antagonist is a fusion protein,
e.g., an Fc fusion protein, such as AMP-244. In one embodiment, the
PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.
[0266] An exemplary anti-PD-1 antibody is OPDIVO.RTM./nivolumab
(BMS-936558) or an antibody that comprises the CDRs or variable
regions of one of antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4 and 4A11
described in WO 2006/121168. In certain embodiments, an anti-PD-1
antibody is MK-3475 (KEYTRUDA.RTM./pembrolizumab/formerly
lambrolizumab) described in WO 2012/145493; AMP-514/MEDI-0680
described in WO 2012/145493; and CT-011 (pidilizumab; previously
CT-AcTibody or BAT; see, e.g., Rosenblatt et al. (2011) J.
Immunotherapy 34:409). Further known PD-1 antibodies and other PD-1
inhibitors include those described in WO 2009/014708, WO 03/099196,
WO 2009/114335, WO 2011/066389, WO 2011/161699, WO 2012/145493,
U.S. Pat. Nos. 7,635,757 and 8,217,149, and U.S. Patent Publication
No. 2009/0317368. Any of the anti-PD-1 antibodies disclosed in WO
2013/173223 may also be used. An anti-PD-1 antibody that competes
for binding with, and/or binds to the same epitope on PD-1 as, as
one of these antibodies may also be used in combination
treatments.
[0267] In certain embodiments, the anti-PD-1 antibody binds to
human PD-1 with a K.sub.D of 5.times.10.sup.-8 M or less, binds to
human PD-1 with a K.sub.D of 1.times.10.sup.-8 M or less, binds to
human PD-1 with a K.sub.D of 5.times.10.sup.-9 M or less, or binds
to human PD-1 with a K.sub.D of between 1.times.10.sup.-8M and
1.times.10.sup.-10 M or less.
[0268] Provided herein are methods for treating a
hyperproliferative disease (e.g., cancer), comprising administering
an agonist anti-huCD40 antibody and an antagonist PD-L1 antibody to
a subject. In certain embodiments, the agonist anti-huCD40 antibody
is administered at a subtherapeutic dose, the anti-PD-L1 antibody
is administered at a subtherapeutic dose, or both are administered
at a subtherapeutic dose. Provided herein are methods for altering
an adverse event associated with treatment of a hyperproliferative
disease with an immunostimulatory agent, comprising administering
an agonist anti-huCD40 antibody and a subtherapeutic dose of
anti-PD-L1 antibody to a subject. In certain embodiments, the
subject is human. In certain embodiments, the anti-PD-L1 antibody
is a human sequence monoclonal antibody and the agonist anti-huCD40
antibody is a humanized monoclonal antibody, such as an antibody
comprising the CDRs or variable regions of the antibodies disclosed
herein.
[0269] In one embodiment, the anti-PD-L1 antibody is BMS-936559
(referred to as 12A4 in WO 2007/005874 and U.S. Pat. No.
7,943,743), MSB0010718C (WO 2013/79174), or an antibody that
comprises the CDRs or variable regions of 3G10, 12A4, 10A5, 5F8,
10H10, 1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT
Publication WO 07/005874 and U.S. Pat. No. 7,943,743. In certain
embodiment an anti-PD-L1 antibody is MEDI4736 (also known as
Anti-B7-H1) or MPDL3280A (also known as RG7446). Any of the
anti-PD-L1 antibodies disclosed in WO 2013/173223, WO 2011/066389,
WO 2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149 and U.S.
Publication No. 2009/145493 may also be used. Anti-PD-L1 antibodies
that compete with and/or bind to the same epitope as that of any of
these antibodies may also be used in combination treatments.
[0270] In yet further embodiment, the agonist anti-huCD40 antibody
of the present invention is combined with an antagonist of
PD-1/PD-L1 signaling, such as a PD-1 antagonist or a PD-L1
antagonist, in combination with a third immunotherapeutic agent. In
one embodiment the third immunotherapeutic agent is a GITR
antagonist or an OX-40 antagonist, such as the anti-GITR or
anti-OX40 antibodies disclosed herein.
[0271] In another aspect, the immuno-oncology agent is a GITR
agonist, such as an agonistic GITR antibody. Suitable GITR
antibodies include, for example, BMS-986153, BMS-986156, TRX-518
(WO 06/105021, WO 09/009116) and MK-4166 (WO 11/028683).
[0272] In another aspect, the immuno-oncology agent is an IDO
antagonist. Suitable IDO antagonists include, for example,
INCB-024360 (WO 2006/122150, WO 07/75598, WO 08/36653, WO
08/36642), indoximod, or NLG-919 (WO 09/73620, WO 09/1156652, WO
11/56652, WO 12/142237).
[0273] Provided herein are methods for treating a
hyperproliferative disease (e.g., cancer), comprising administering
an agonist anti-huCD40 antibody described herein and a CTLA-4
antagonist antibody to a subject. In certain embodiments, the
agonist anti-huCD40 antibody is administered at a subtherapeutic
dose, the anti-CTLA-4 antibody is administered at a subtherapeutic
dose, or both are administered at a subtherapeutic dose. Provided
herein are methods for altering an adverse event associated with
treatment of a hyperproliferative disease with an immunostimulatory
agent, comprising administering an agonist anti-huCD40 antibody and
a subtherapeutic dose of anti-CTLA-4 antibody to a subject. In
certain embodiments, the subject is human. In certain embodiments,
the anti-CTLA-4 antibody is an antibody selected from the group
consisting of: YERVOY.RTM. (ipilimumab or antibody 10D1, described
in PCT Publication WO 01/14424), tremelimumab (formerly
ticilimumab, CP-675,206), and the anti-CTLA-4 antibodies described
in the following publications: WO 98/42752; WO 00/37504; U.S. Pat.
No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. (USA)
95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology
22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al.
(1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodies
disclosed in WO 2013/173223 may also be used.
[0274] Provided herein are methods for treating a
hyperproliferative disease (e.g., cancer), comprising administering
an agonist anti-huCD40 antibody and an anti-LAG-3 antibody to a
subject. In further embodiments, the agonist anti-huCD40 antibody
is administered at a subtherapeutic dose, the anti-LAG-3 antibody
is administered at a subtherapeutic dose, or both are administered
at a subtherapeutic dose. Provided herein are methods for altering
an adverse event associated with treatment of a hyperproliferative
disease with an immunostimulatory agent, comprising administering
an agonist anti-huCD40 antibody and a subtherapeutic dose of
anti-LAG-3 antibody to a subject. In certain embodiments, the
subject is human. In certain embodiments, the anti-LAG-3 antibody
is a human sequence monoclonal antibody and the agonist anti-huCD40
antibody is a humanized monoclonal antibody, such as an antibody
comprising the CDRs or variable regions of the antibodies disclosed
herein. Examples of anti-LAG3 antibodies include antibodies
comprising the CDRs or variable regions of antibodies 25F7, 26H10,
25E3, 8B7, 11F2 or 17E5, which are described in U.S. Patent
Publication No. US 2011/0150892 and WO 2014/008218. In one
embodiment, an anti-LAG-3 antibody is BMS-986016. Other art
recognized anti-LAG-3 antibodies that can be used include IMP731
described in US 2011/007023. IMP-321 may also be used. Anti-LAG-3
antibodies that compete with and/or bind to the same epitope as
that of any of these antibodies may also be used in combination
treatments.
[0275] In certain embodiments, the anti-LAG-3 antibody binds to
human LAG-3 with a K.sub.D of 5.times.10.sup.-8 M or less, binds to
human LAG-3 with a K.sub.D of 1.times.10.sup.-8 M or less, binds to
human LAG-3 with a K.sub.D of 5.times.10.sup.-9 M or less, or binds
to human LAG-3 with a K.sub.D of between 1.times.10.sup.-8 M and
1.times.10.sup.-10 M or less.
[0276] Administration of agonist anti-huCD40 antibodies described
herein and antagonists, e.g., antagonist antibodies, to one or more
second target antigens such as LAG-3 and/or CTLA-4 and/or PD-1
and/or PD-L1 can enhance the immune response to cancerous cells in
the patient. Cancers whose growth may be inhibited using the
antibodies of the instant disclosure include cancers typically
responsive to immunotherapy. Representative examples of cancers for
treatment with the combination therapy of the instant disclosure
include those cancers specifically listed above in the discussion
of monotherapy with agonist anti-huCD40 antibodies.
[0277] In certain embodiments, the combination of therapeutic
antibodies discussed herein can be administered concurrently as a
single composition in a pharmaceutically acceptable carrier, or
concurrently as separate compositions with each antibody in a
pharmaceutically acceptable carrier. In another embodiment, the
combination of therapeutic antibodies can be administered
sequentially. For example, an anti-CTLA-4 antibody and an agonist
anti-huCD40 antibody can be administered sequentially, such as
anti-CTLA-4 antibody being administered first and agonist
anti-huCD40 antibody second, or agonist anti-huCD40 antibody being
administered first and anti-CTLA-4 antibody second. Additionally or
alternatively, an anti-PD-1 antibody and an agonist anti-huCD40
antibody can be administered sequentially, such as anti-PD-1
antibody being administered first and agonist anti-huCD40 antibody
second, or agonist anti-huCD40 antibody being administered first
and anti-PD-1 antibody second. Additionally or alternatively, an
anti-PD-L1 antibody and an agonist anti-huCD40 antibody can be
administered sequentially, such as anti-PD-L1 antibody being
administered first and agonist anti-huCD40 antibody second, or
agonist anti-huCD40 antibody being administered first and
anti-PD-L1 antibody second. Additionally or alternatively, an
anti-LAG-3 antibody and an agonist anti-huCD40 antibody can be
administered sequentially, such as anti-LAG-3 antibody being
administered first and agonist anti-huCD40 antibody second, or
agonist anti-huCD40 antibody being administered first and
anti-LAG-3 antibody second.
[0278] Furthermore, if more than one dose of the combination
therapy is administered sequentially, the order of the sequential
administration can be reversed or kept in the same order at each
time point of administration, sequential administrations can be
combined with concurrent administrations, or any combination
thereof. For example, the first administration of a combination
anti-CTLA-4 antibody and agonist anti-huCD40 antibody can be
concurrent, the second administration can be sequential with
anti-CTLA-4 antibody first and agonist anti-huCD40 antibody second,
and the third administration can be sequential with agonist
anti-huCD40 antibody first and anti-CTLA-4 antibody second, etc.
Additionally or alternatively, the first administration of a
combination anti-PD-1 antibody and agonist anti-huCD40 antibody can
be concurrent, the second administration can be sequential with
anti-PD-1 antibody first and agonist anti-huCD40 antibody second,
and the third administration can be sequential with agonist
anti-huCD40 antibody first and anti-PD-1 antibody second, etc.
Additionally or alternatively, the first administration of a
combination anti-PD-L1 antibody and agonist anti-huCD40 antibody
can be concurrent, the second administration can be sequential with
anti-PD-L1 antibody first and agonist anti-huCD40 antibody second,
and the third administration can be sequential with agonist
anti-huCD40 antibody first and anti-PD-L1 antibody second, etc.
Additionally or alternatively, the first administration of a
combination anti-LAG-3 antibody and agonist anti-huCD40 antibody
can be concurrent, the second administration can be sequential with
anti-LAG-3 antibody first and agonist anti-huCD40 antibody second,
and the third administration can be sequential with agonist
anti-huCD40 antibody first and anti-LAG-3 antibody second, etc.
Another representative dosing scheme can involve a first
administration that is sequential with agonist anti-huCD40 first
and anti-CTLA-4 antibody (and/or anti-PD-1 antibody and/or
anti-PD-L1 antibody and/or anti-LAG-3 antibody) second, and
subsequent administrations may be concurrent.
[0279] Optionally, an agonist anti-huCD40 as sole immunotherapeutic
agent, or the combination of an agonist anti-huCD40 antibody and
one or more additional immunotherapeutic antibodies (e.g.,
anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3
blockade) can be further combined with an immunogenic agent, such
as cancerous cells, purified tumor antigens (including recombinant
proteins, peptides, and carbohydrate molecules), cells, and cells
transfected with genes encoding immune stimulating cytokines (He et
al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor
vaccines that can be used include peptides of melanoma antigens,
such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or
tyrosinase, or tumor cells transfected to express the cytokine
GM-CSF (discussed further below). A CD40 agonist and one or more
additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/or
LAG-3 blockade) can also be further combined with standard cancer
treatments. For example, a CD40 agonist and one or more additional
antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3
blockade) can be effectively combined with chemotherapeutic
regimes. In these instances, it is possible to reduce the dose of
other chemotherapeutic reagent administered with the combination of
the instant disclosure (Mokyr et al. (1998) Cancer Research 58:
5301-5304). An example of such a combination is a combination of
CD40 agonist antibody with or without and an additional antibody,
such as anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or
anti-PD-L1 antibodies and/or anti-LAG-3 antibodies) further in
combination with decarbazine for the treatment of melanoma. Another
example is a combination of agonist anti-huCD40 antibody with or
without and anti-CTLA-4 antibodies and/or anti-PD-1 antibodies
and/or anti-PD-L1 antibodies and/or LAG-3 antibodies further in
combination with interleukin-2 (IL-2) for the treatment of
melanoma. The scientific rationale behind the combined use of CD40
agonism and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade
with chemotherapy is that cell death, which is a consequence of the
cytotoxic action of most chemotherapeutic compounds, should result
in increased levels of tumor antigen in the antigen presentation
pathway. Other combination therapies that may result in synergy
with a combined CD40 agonism with or without and CTLA-4 and/or PD-1
and/or PD-L1 and/or LAG-3 blockade through cell death include
radiation, surgery, or hormone deprivation. Each of these protocols
creates a source of tumor antigen in the host. Angiogenesis
inhibitors can also be combined with a combined CD40 agonism and
CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade. Inhibition
of angiogenesis leads to tumor cell death, which can be a source of
tumor antigen fed into host antigen presentation pathways.
[0280] An agonist anti-huCD40 antibody as sole immunotherapeutic
agent, or a combination of CD40 agonist and CTLA-4 and/or PD-1
and/or PD-L1 and/or LAG-3 blocking antibodies can also be used in
combination with bispecific antibodies that target Fc.alpha. or
Fc.gamma. receptor-expressing effector cells to tumor cells. See,
e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243. Bispecific antibodies
can be used to target two separate antigens. The T cell arm of
these responses would be augmented by the use of a combined CD40
agonism and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3
blockade.
[0281] In another example, an agonistic anti-CD40 antibody as sole
immunotherapeutic agent or a combination of an anti-CD40 antibody
and additional immunostimulating agent, e.g., anti-CTLA-4 antibody
and/or anti-PD-1 antibody and/or anti-PD-L1 antibody and/or LAG-3
agent, e.g., antibody, can be used in conjunction with an
anti-neoplastic antibody, such as RITUXAN.RTM. (rituximab),
HERCEPTIN.RTM. (trastuzumab), BEXXAR.RTM. (tositumomab),
ZEVALIN.RTM. (ibritumomab), CAMPATH.RTM. (alemtuzumab),
LYMPHOCIDE.RTM. (eprtuzumab), AVASTIN.RTM. (bevacizumab), and
TARCEVA.RTM. (erlotinib), and the like. By way of example and not
wishing to be bound by theory, treatment with an anti-cancer
antibody or an anti-cancer antibody conjugated to a toxin can lead
to cancer cell death (e.g., tumor cells) which would potentiate an
immune response mediated by the immunostimulating agent, e.g.,
CD40, TIGIT, CTLA-4, PD-1, PD-L1 or LAG-3 agent, e.g., antibody. In
an exemplary embodiment, a treatment of a hyperproliferative
disease (e.g., a cancer tumor) can include an anti-cancer agent,
e.g., antibody, in combination with an agonist anti-huCD40 antibody
and optionally an additional immunostimulating agent, e.g.,
anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3
agent, e.g., antibody, concurrently or sequentially or any
combination thereof, which can potentiate an anti-tumor immune
responses by the host.
[0282] Provided herein are methods for altering an adverse event
associated with treatment of a hyperproliferative disease (e.g.,
cancer) with an immunostimulatory agent, comprising administering
an agonist anti-huCD40 antibody with or without and a
subtherapeutic dose of anti-CTLA-4 and/or anti-PD-1 and/or
anti-PD-L1 and/or anti-LAG-3 agent, e.g., antibody, to a subject.
For example, the methods described herein provide for a method of
reducing the incidence of immunostimulatory therapeutic
antibody-induced colitis or diarrhea by administering a
non-absorbable steroid to the patient. As used herein, a
"non-absorbable steroid" is a glucocorticoid that exhibits
extensive first pass metabolism such that, following metabolism in
the liver, the bioavailability of the steroid is low, i.e., less
than about 20%. In one embodiment described herein, the
non-absorbable steroid is budesonide. Budesonide is a
locally-acting glucocorticosteroid, which is extensively
metabolized, primarily by the liver, following oral administration.
ENTOCORT EC.RTM. (ASTRA-ZENECA) is a pH- and time-dependent oral
formulation of budesonide developed to optimize drug delivery to
the ileum and throughout the colon. ENTOCORT EC.RTM. is approved in
the U.S. for the treatment of mild to moderate Crohn's disease
involving the ileum and/or ascending colon. The usual oral dosage
of ENTOCORT EC.RTM. for the treatment of Crohn's disease is 6 to 9
mg/day. ENTOCORT EC.RTM. is released in the intestines before being
absorbed and retained in the gut mucosa. Once it passes through the
gut mucosa target tissue, ENTOCORT EC.RTM. is extensively
metabolized by the cytochrome P450 system in the liver to
metabolites with negligible glucocorticoid activity. Therefore, the
bioavailability is low (about 10%). The low bioavailability of
budesonide results in an improved therapeutic ratio compared to
other glucocorticoids with less extensive first-pass metabolism.
Budesonide results in fewer adverse effects, including less
hypothalamic-pituitary suppression, than systemically-acting
corticosteroids. However, chronic administration of ENTOCORT
EC.RTM. can result in systemic glucocorticoid effects such as
hypercorticism and adrenal suppression. See PDR 58th ed. 2004;
608-610.
[0283] In still further embodiments, a CD40 agonist with or without
CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade (i.e.,
immunostimulatory therapeutic antibodies against CD40 and
optionally anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or
anti-LAG-3 antibodies) in conjunction with a non-absorbable steroid
can be further combined with a salicylate. Salicylates include
5-ASA agents such as, for example: sulfasalazine (AZULFIDINE.RTM.,
PHARMACIA &UPJOHN); olsalazine (DIPENTUM.RTM., PHARMACIA &
UPJOHN); balsalazide (COLAZAL.RTM., SALIX Pharmaceuticals, Inc.);
and mesalamine (ASACOL.RTM., PROCTER & GAMBLE Pharmaceuticals;
PENTASA.RTM., SHIRE US; CANASA.RTM., AXCAN SCANDIPHARM, Inc.;
ROWASA.RTM., SOLVAY).
[0284] In accordance with the methods described herein, a
salicylate administered in combination with agonist anti-huCD40
antibody with or without anti-CTLA-4 and/or anti-PD-1 and/or
anti-PD-L1 and/or LAG-3 antibodies and a non-absorbable steroid can
includes any overlapping or sequential administration of the
salicylate and the non-absorbable steroid for the purpose of
decreasing the incidence of colitis induced by the
immunostimulatory antibodies. Thus, for example, methods for
reducing the incidence of colitis induced by the immunostimulatory
antibodies described herein encompass administering a salicylate
and a non-absorbable concurrently or sequentially (e.g., a
salicylate is administered 6 hours after a non-absorbable steroid),
or any combination thereof. Further, a salicylate and a
non-absorbable steroid can be administered by the same route (e.g.,
both are administered orally) or by different routes (e.g., a
salicylate is administered orally and a non-absorbable steroid is
administered rectally), which may differ from the route(s) used to
administer the anti-huCD40 and anti-CTLA-4 and/or anti-PD-1 and/or
anti-PD-L1 and/or anti-LAG-3 antibodies.
[0285] The agonist anti-huCD40 antibodies and combination antibody
therapies described herein may also be used in conjunction with
other well known therapies that are selected for their particular
usefulness against the indication being treated (e.g., cancer).
Combinations of the agonist anti-huCD40 antibodies described herein
may be used sequentially with known pharmaceutically acceptable
agent(s).
[0286] For example, the agonist anti-huCD40 antibodies and
combination antibody therapies described herein can be used in
combination (e.g., simultaneously or separately) with an additional
treatment, such as irradiation, chemotherapy (e.g., using
camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin,
doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin,
paclitaxel, carboplatin-paclitaxel (TAXOL), doxorubicin, 5-fu, or
camptothecin+apo2l/TRAIL (a 6.times. combo)), one or more
proteasome inhibitors (e.g., bortezomib or MG132), one or more
Bcl-2 inhibitors (e.g., BH3I-2' (bcl-xl inhibitor), indoleamine
dioxygenase-1 (IDO1) inhibitor (e.g., INCB24360), AT-101
(R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070
(obatoclax), or MCL-1 (myeloid leukemia cell differentiation
protein-1) antagonists), iAP (inhibitor of apoptosis protein)
antagonists (e.g., smac7, smac4, small molecule smac mimetic,
synthetic smac peptides (see Fulda et al., Nat Med 2002; 8:808-15),
ISIS23722 (LY2181308), or AEG-35156 (GEM-640)), HDAC (histone
deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab),
angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agents
targeting VEGF and VEGFR (e.g., AVASTIN.RTM.), synthetic
triterpenoids (see Hyer et al., Cancer Research 2005; 65:4799-808),
c-FLIP (cellular FLICE-inhibitory protein) modulators (e.g.,
natural and synthetic ligands of PPAR.gamma. (peroxisome
proliferator-activated receptor .gamma.), 5809354 or 5569100),
kinase inhibitors (e.g., Sorafenib), trastuzumab, cetuximab,
Temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus,
Bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors,
Lenalildomide, GSK3.beta. inhibitors, IAP inhibitors and/or
genotoxic drugs.
[0287] The agonist anti-huCD40 antibodies and combination antibody
therapies described herein can further be used in combination with
one or more anti-proliferative cytotoxic agents. Classes of
compounds that may be used as anti-proliferative cytotoxic agents
include, but are not limited to, the following:
[0288] Alkylating agents (including, without limitation, nitrogen
mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas
and triazenes): Uracil mustard, Chlormethine, Cyclophosphamide
(CYTOXAN.TM.) fosfamide, Melphalan, Chlorambucil, Pipobroman,
Triethylenemelamine, Triethylenethiophosphoramine, Busulfan,
Carmustine, Lomustine, Streptozocin, Dacarbazine, and
Temozolomide.
[0289] Antimetabolites (including, without limitation, folic acid
antagonists, pyrimidine analogs, purine analogs and adenosine
deaminase inhibitors): Methotrexate, 5-Fluorouracil, Floxuridine,
Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,
Pentostatine, and Gemcitabine.
[0290] Suitable anti-proliferative agents for combining with
agonist anti-huCD40 antibodies, without limitation, taxanes,
paclitaxel (paclitaxel is commercially available as TAXOL.TM.),
docetaxel, discodermolide (DDM), dictyostatin (DCT), Peloruside A,
epothilones, epothilone A, epothilone B, epothilone C, epothilone
D, epothilone E, epothilone F, furanoepothilone D, desoxyepothilone
Bl, [17]-dehydrodesoxyepothilone B, [18]dehydrodesoxyepothilones B,
C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A,
trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D,
16-desmethylepothilone B, epothilone B10, discoderomolide,
patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A
(Discodermolide), TZT-1027 (soblidotin), ILX-651 (tasidotin
hydrochloride), Halichondrin B, Eribulin mesylate (E-7389),
Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703,
Maytansinoid immunoconjugates (DM-1), MKC-1, ABT-751, T1-38067,
T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312,
eleutherobin,
17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,
cyclostreptin, isolaulimalide, laulimalide,
4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, and
cryptothilone 1, in addition to other microtubuline stabilizing
agents known in the art.
[0291] In cases where it is desirable to render aberrantly
proliferative cells quiescent in conjunction with or prior to
treatment with agonist anti-huCD40 antibodies described herein,
hormones and steroids (including synthetic analogs), such as
17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone,
Fluoxymesterone, Dromostanolone propionate, Testolactone,
Megestrolacetate, Methylprednisolone, Methyl-testosterone,
Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,
Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate,
Leuprolide, Flutamide, Toremifene, ZOLADEX.TM., can also be
administered to the patient. When employing the methods or
compositions described herein, other agents used in the modulation
of tumor growth or metastasis in a clinical setting, such as
antimimetics, can also be administered as desired.
[0292] Methods for the safe and effective administration of
chemotherapeutic agents are known to those skilled in the art. In
addition, their administration is described in the standard
literature. For example, the administration of many of the
chemotherapeutic agents is described in the Physicians' Desk
Reference (PDR), e.g., 1996 edition (Medical Economics Company,
Montvale, N.J. 07645-1742, (USA); the disclosure of which is
incorporated herein by reference thereto.
[0293] The chemotherapeutic agent(s) and/or radiation therapy can
be administered according to therapeutic protocols well known in
the art. It will be apparent to those skilled in the art that the
administration of the chemotherapeutic agent(s) and/or radiation
therapy can be varied depending on the disease being treated and
the known effects of the chemotherapeutic agent(s) and/or radiation
therapy on that disease. Also, in accordance with the knowledge of
the skilled clinician, the therapeutic protocols (e.g., dosage
amounts and times of administration) can be varied in view of the
observed effects of the administered therapeutic agents on the
patient, and in view of the observed responses of the disease to
the administered therapeutic agents.
XI. Characterization of Specific Agonist Anti-CD40 Antibodies of
the Present Invention
[0294] Agonist anti-CD40 antibodies of the present invention were
obtained as described in Example 1. Variable domains and CDR
sequence regions of exemplary antibodies of the present invention
are provided in the Sequence Listing, and are summarized at Table
2. Variable domain and CDR region numbering is the same for all
antibodies derived from the same original clone, i.e. the humanized
variants provided herein do not include any insertions or
deletions.
TABLE-US-00002 TABLE 2 Antibody Variable Domains and CDRs Clone
Chain Variable Domain CDR1 CDR2 CDR3 12D6 Heavy chain 1-119 31-35
50-66 99-108 12D6 Light chain 1-112 24-39 55-61 94-102 5F11 Heavy
chain 1-117 31-35 50-66 99-106 5F11 Light chain 1-111 24-38 54-60
93-101 8E8 Heavy chain 1-122 31-35 50-66 99-111 8E8 Light chain
1-112 24-39 55-61 94-102 5G7 Heavy chain 1-113 31-35 50-66 99-102
5G7 Light chain 1-107 24-34 50-56 89-97 19G3 Heavy chain 1-112
31-35 50-66 99-101 19G3 Light chain 1-112 24-39 55-61 94-102
[0295] The invention also provides anti-huCD40 antibodies related
to those disclosed herein by sequence by being derived from the
same murine germline sequences, specifically the V and J region
gene segments. The murine germline sequences for each of the
antibodies disclosed herein are provided at Table 3.
TABLE-US-00003 TABLE 3 Mouse Germline Sequences for anti-huCD40
mAbs Clone Chain V region J region 12D6 Heavy chain VH1-39_01 IGHJ4
12D6 Light chain VK1-110_01 IGKJ1 5F11 Heavy chain VH1-4_02 IGHJ3
5F11 Light chain VK3-5_01 IGKJ5 8E8 Heavy chain VH1-80_01 IGHJ2 8E8
Light chain VK1-110_01 IGKJ2 5G7 Heavy chain VH1-18_01 IGHJ4 5G7
Light chain VK10-96_01 IGKJ2 19G3 Heavy chain VH5-9-4_01 IGHJ3 19G3
Light chain VK1-117_01 IGKJ2
[0296] The invention further provides humanized anti-huCD40
antibodies derived from the murine parental antibodies disclosed
herein by replacement of murine framework sequences with human
framework sequences, with or without additional mutations ("back
mutations") to restore antigen (human CD40) affinity that would
otherwise be lost in the humanization or to remove sequence
liabilities. See Example 3.
[0297] The invention also provides antibody constructs comprising
the novel variable domain sequences disclosed herein and constant
domains with modified Fc regions having enhanced affinity for
Fc.gamma.RIIb as compared with their affinity for other Fc
receptors, i.e. activating receptors. Such agonistic anti-huCD40
antibodies with enhanced Fc.gamma.RIIb-specificity are expected to
exhibit superior efficacy in treatment of cancer and chronic
infection. Li & Ravetch (2011) Science 333:1030; White et al.
(2011) J. Immunol. 187:1754. Without intending to be limited by
theory, such Fc.gamma.RIIb-specific agonistic anti-CD40 mAbs may
exhibit enhanced adjuvant effects by increasing the maturation of
dendritic cell promoting expansion and activation of cytotoxic CD8+
T cells, leading to enhanced anti-tumor response. Id. Without
intending to be limited by theory, FcR-mediated signal enhancement
of agonist CD40 antibodies due to increased receptor clustering, or
"cross-linking," of the present invention may be a major
contributor to therapeutic efficacy. Cross-linking of CD40 agonist
antibodies by FcR engagement by the Fc portion of the antibody may
increase signal strength and thereby enhance activation of
cells.
[0298] The relative binding affinity of antibodies for activating
(A) versus inhibitory (I) Fc receptors can be expressed as the
"A/I" ratio, and is typically a function of the structure of the Fc
region of an IgG antibody. See WO 2012/087928. Antibodies having
enhanced specificity for binding to inhibitory receptor
Fc.gamma.RIIb have lower A/I ratios. Preferred antibodies for the
agonistic anti-huCD40 antibodies of the present invention have, for
example, A/I ratios of less than 5, 4, 3, 2, 1, 0.5, 0.3, 0.1,
0.05, 0.03 or 0.01.
[0299] Human IgG1constant domains comprising mutations to enhance
Fc.gamma.RIIb specificity are also provided in the Sequence
Listing, and are summarized at Table 4 and illustrated at FIG. 1.
Sequence variants are defined with reference to human IgG1f
constant domain sequence provided at SEQ ID NO: 65. The
nomenclature regarding positions (numbering) of mutations in the Fc
region is according to the EU index as in Kabat et al. (1981)
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.),
which facilitates comparison of Fc sequences at equivalent
positions in antibodies with differing variable domain lengths. See
also Edelman et al. (1969) Proc. Nat'l Acad. Sci. (USA) 63:78; WO
2012/130831 (using the same numbering system). It does not match
the sequence numbering in the Sequence Listing. FIG. 1 provides a
graphical representation of the Fc sequence variants of Table 4,
from which one of skill in the art could readily recognize the
corresponding positions in the antibody sequences disclosed herein.
SE and SELF variants are described at Chu et al. (2008) Mol.
Immunol. 45:3926. P238D, V4, V7, V8, V9, V11 and V12 variants are
described at Mimoto et al. (2013) Protein Engineering Design &
Selection 26:589 (e.g. at table 1 therein).
TABLE-US-00004 TABLE 4 Fc Sequence Variants Designation SEQ ID:
Sequence Variants IgG1f 65 SE 66 S267E SELF 67 S267E L328F P238D 68
P238D V4 69 P238D P271G V4-D270E 70 P238D P271G D270E V7 71 E233D
P238D P271G A330R V8 72 G237D P238D H268D P271G V9 73 G237D P238D
P271G A330R V9-D270E 74 G237D P238D P271G A330R D270E V11 75 G237D
P238D H268D P271G A330R V12 76 E233D G237D P238D H268D P271G
A330R
[0300] Additional Fc sequence variants with enhanced affinity for
Fc.gamma.RIIb are disclosed at Yu et al. (2013) J. Am. Chem. Soc.
135:9723 (and WO 2014/184545), including V262E and V264E, e.g. for
use in combination with S267E and L328F.
[0301] Additional variants with D270E mutations were produced to
reduce the D270 isomeization that occurred in the V4 and V9 Fc
sequence variants, which otherwise exhibited enhanced isomerization
rates in accelerated degradation studies suggesting .about.12-16%
isomerization after two years at 4.degree. C. in PBS, compared with
.about.5-7% for other variants. See Table 5 (providing data from a
single experiment but replicate experiments showed comparable
values). Substitution of aspartic acid at position 270 with
glutamic acid eliminated the possibility of such DG isomerization,
resulting in antibodies that are more chemically stable and
antibody preparations that are more homogenous. Other D270
substitutions of V9, such as D270A, D270Q, D270S and D270T,
effectively eliminated binding to Fc.gamma.RIIa and Fc.gamma.RIIb
receptors (K.sub.D was greater than 5 .mu.M). Although the D270E
mutation reduced binding to Fc.gamma.RIIb receptor by an order of
magnitude, the V9-D270E variant maintained a favorable bias in
affinity for Fc.gamma.RIIb receptor compared with Fc.gamma.RIIa,
and acceptable absolute affinity for Fc.gamma.RIIb. See Example
8.
TABLE-US-00005 TABLE 5 Fc Sequence Variant Affinity for Fc.gamma.
Receptors (K.sub.D in nM) Fc.gamma.RIIa- Designation H131
Fc.gamma.RIIa-R131 Fc.gamma.RIIb IgG1f 530 850 3900 SE 520 22 98
SELF 1100 2 11 P238D >5000 >5000 950 V4 >5000 1900 150
V4-D270E >5000 >5000 1800 V7 >5000 1600 84 V8 >5000
1400 93 V9 >5000 420 15 V9-D270A,Q,S,T >5000 >5000
>5000 V9-D270E >5000 >5000 150 V11 -- 450 15 V12 >5000
490 21
[0302] Agonist activity of various anti-CD40 antibodies of the
present invention was measured. See Example 7, and FIGS. 3A, 3B and
4. Agonist activity was found to depend on both the variable domain
sequences (mAb clone number), which determine antigen (human CD40)
binding, and the sequence of the Fc region, which determines Fc
receptor (Fc.gamma.RIIb) binding.
[0303] The present disclosure is further illustrated by the
following examples, which should not be construed as further
limiting. The contents of all figures and all references, Genbank
sequences, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
EXAMPLES
Example 1
Generation of Mouse Monoclonal Antibodies Against Human CD40
[0304] Murine anti-human CD40 monoclonal antibodies were generated
using wild type Balb/c mice (Charles Rivers Labs) that express
mouse antibody genes, as follows.
[0305] Antigen
[0306] A huCD40-muFc soluble recombinant protein was used as the
antigen for immunizations. The soluble fusion protein has a MW of
91.6 K.sub.D and is composed of the extracellular portion of huCD40
linked to a mouse IgG2b Fc at its C-terminus. This fusion protein
is referred to herein as "huCD40-muFc fusion protein". The fusion
protein was generated by standard recombinant DNA methods and
expressed in transfected CHO cells, which secreted the soluble
fusion protein into the culture supernatant. The CHO host cells
used for transfection were obtained from INVITROGEN (Cat
#11619-012). The secreted soluble fusion protein was purified for
use as immunogen.
[0307] Immunization of Mice
[0308] To generate mouse monoclonal antibodies to human CD40,
Balb/c mice were immunized with purified huCD40-muFc fusion
protein. The mice were approximately 2-4 months of age upon the
first infusion of antigen. Purified recombinant huCD40-muFc antigen
preparation (10 .mu.g purified from transfected mammalian cells
expressing the fusion protein) was used to immunize the mice using
two immunization protocols (A and B). Protocol A consisted of four
weekly footpad (FP) immunizations and protocol B of seven weekly
subcutaneous (SC)/intraperitoneal (IP)/Hock immunizations. In both
cases, the immunogen was mixed 1:1 with RIBI adjuvant (SIGMA Cat #
M6536).
[0309] All mice were bled one week after the fourth immunizations
to assess antigen specific titers. Final bleeds were also obtained
from each mouse at time of sacrifice. The immune response was
monitored by retro orbital bleeds. The plasma was screened by ELISA
analysis using the recombinant protein used for immunizations. Mice
from protocol A received two final boosts intravenously (IV) and in
the footpads (FP) with soluble antigen on days -2 and -3 before
fusion. Mice from protocol B received two final boosts
intravenously as well as IP and in the hock with the soluble
antigen on days -2 and -3 before fusion.
[0310] All four mice from protocol A (IDs 291763, 291764, 291765,
291766) were sacrificed. Lymph node and spleen cells were extracted
and mixed for fusion (fusions 3582 and 3583). Only two mice from
protocol B (IDs 294286 and 294288) were sacrificed. Spleen and
lymph nodes from each mouse were mixed and fused (fusions 3716 and
3717).
[0311] Generation of Hybridomas Producing Monoclonal Antibodies to
Human CD40
[0312] Mouse splenocytes and lymph nodes isolated from high titer
Balb/c mice were fused with a mouse myeloma fusion partner using
the electric field based electrofusion Hybrimune instrument and a
large 0.9m1 fusion chamber (BTX Harvard Apparatus, Inc., Holliston,
Mass.). Single cell suspensions of lymphocytes from immunized mice
were fused to an equal number of P3X63 Ag8.6.53 (ATCC CRL 1580)
non-secreting mouse myeloma cells. Resulting cells were plated at
2.0.times.10.sup.4 cells/well in flat bottom microtiter plates in
selective CLONACELL-HY Medium E (catalog #03805; STEMCELL
Technologies Inc., Vancouver BC, Canada) with addition of
Aminopterin to select for hybridomas. After about 7 days, the
culture medium was replaced with Medium E (without
aminopterin).
[0313] After 10 to 12 days, individual wells were screened for the
presence of mouse IgG/mouse kappa light chain antibodies using a
homogenous HTRF assay. In this assay, supernatants from 96 well
fusion plates were mixed with custom labeled Terbium-Cryptate goat
anti-mouse IgG (Fey specific) (CISBIO US Inc. Bedford, Mass.) and
goat anti-mouse IgG Fab'2 labeled with AlexaFluor 647 (Jackson
ImmunoResearch; Catalog #109-605-098). Incubation was for 1 hour.
The plates were then read on a RUBYSTAR reader. Hybridoma cells
from wells positive for mouse IgG/mouse kappa light chain
antibodies were then screened either by FACS using CHO cells
transfected with human CD40 and CHO untransfected cells as control
(fusions 3582/3583) or by ELISA using recombinant protein followed
by FACS on Daudi B cells and Jurkat T cells as negative control
(fusions 3716/3717). FACS positive parental lines were transferred
to 24-well plates. A few days later, cell supernatants from
individual wells were rescreened by FACS to confirm IgG specificity
to human CD40.
[0314] A panel of antigen specific hybridomas were cloned by serial
dilution and rescreened by FACS using either CHO transfectants or
Daudi cells. Nineteen antibodies from fusions 3582/3583 and twenty
antibodies from fusions 3716/3717 were purified and tested for
functional activity. From this panel of antibodies, five strong
agonists were selected for second subcloning. These five
antibodies, namely 1802.3582.19G3.F10.E1, 1802.3583.5G7.F12.G3,
1802.3583.8E8.C10.G2, 1802.3716.12D6.B1.E3, and
1802.3717.5F11.A11.E7 were subsequently submitted to sequencing and
further analysis.
Example 2
Generation of Fully Human Anti-huCD40 Antibodies
[0315] Fully human anti-huCD40 monoclonal antibodies that bind to
the same epitope and/or cross-block the binding of the humanized
anti-CD40 antibodies disclosed herein may find use in methods of
the present invention. Such antibodies may be generated using
transgenic mice that express human antibody genes, as follows.
[0316] Antigen
[0317] A huCD40 soluble recombinant protein is used as the antigen
for immunization. The soluble fusion protein is composed of the
extracellular portion of huCD40 linked to a mouse IgG2a Fc at its
C-terminus. This fusion protein is referred to herein as
"huCD40-muFc fusion protein." The fusion protein is generated by
standard recombinant DNA methods and expressed in transfected CHO
cells, which secrete the soluble fusion protein into the culture
supernatant. The CHO host cells used for transfection are obtained
from INVITROGEN (Cat #11619-012). The secreted soluble fusion
protein is purified for use as immunogen. The sequence of full
length human CD40 including signal sequence is provided at SEQ ID
NO: 1.
[0318] Transgenic Mice
[0319] Fully human monoclonal antibodies to human CD40 are prepared
using mice from the CMD++; JKD++; KCo5(9272)+{circumflex over ( )};
SC20+ genotype (hereafter called KM.RTM. mice). Individual
transgene designations are in parentheses, followed by line numbers
for randomly integrated transgenes. The symbols ++ and + indicate
homozygous or hemizygous; however, because the mice are routinely
screened using a PCR-based assay that does not allow us to
distinguish between heterozygosity and homozygosity for the
randomly integrated human Ig transgenes, a + designation may be
given to mice that are actually homozygous for these elements. In
this strain, the endogenous mouse kappa light chain gene has been
homozygously disrupted as described in Chen et al. (1993) EMBO J.
12:811-820 and the endogenous mouse heavy chain gene has been
homozygously disrupted as described in example 1 of WO 2001/09187.
Furthermore, this mouse strain carries a human kappa light chain
transgene, KCo5, as described in Fishwild et al. (1996) Nature
Biotechnology 14:845-851, a yeast artificial chromosome (YAC)
carrying most of the human kappa light chain locus, as described in
WO 2000/026373.
[0320] Immunization of Mice
[0321] To generate fully human monoclonal antibodies to human CD40,
KM mice are immunized with purified huCD40-muFc fusion protein.
General immunization schemes are described in Lonberg et al. (1994)
Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature
Biotechnology 14: 845-851 and WO 98/24884. The mice are
approximately 4 months of age upon the first infusion of antigen.
Either purified recombinant huCD40-muFc antigen preparation (10
.mu.g purified from transfected mammalian cells expressing the
fusion protein) or 300-19 cells transfected with human CD40 are
used to immunize the mice intraperitoneally and subcutaneously. The
immunogens are mixed 1:1 with RIBI adjuvant (SIGMA Cat #
M6536).
[0322] The mice are immunized five times at 5-7 day intervals. The
first and second immunizations are performed with the recombinant
protein. The third immunization is with the cells, the fourth
immunization with the protein and the fifth immunization with the
cells. Mice are bled one week after the last immunizations to
assess antigen specific titers. The immune response is monitored by
retro orbital bleeds. The plasma is screened by FACS analysis using
the transfected 300-19 cells, and mice with highest titers for
anti-human CD40 human IgG are used for fusions. Mice receive a
final boost by intravenous (IV) and intraperitoneal (IP) injection
of soluble antigen two days and transfected cells three days before
sacrifice and removal of the spleen.
[0323] Generation of Hybridomas Producing Human Monoclonal
Antibodies to Human CD40
[0324] Mouse splenocytes isolated from high titer KM mice and a
mouse myeloma fusion partner are fused with an electric field based
electrofusion using a Cyto Pulse large chamber cell fusion
electroporator (Cyto Pulse Sciences, Inc., Glen Burnie, Md.).
Single cell suspensions of splenic lymphocytes from immunized mice
are fused to an equal number of P3X63 Ag8.6.53 (ATCC CRL 1580)
non-secreting mouse myeloma cells (fusion number: 2541). Resulting
cells are plated at 2.0.times.10.sup.4 cells/well in flat bottom
microtiter plates in selective DMEM medium containing high glucose
(CELLGRO #10-013-CM) and 10% fetal calf serum (HYCLONE #
SH30071.03), and supplemented with beta-mercaptoethanol
(1000.times., GIBCO #21985-023), 7 mM HEPES (CELLGRO 25-060-C1),
additional 2 mM L-glutamine (CELLGRO 25-005-C1), HAT (50.times.,
SIGMA # H-0262), 5% Hybridoma Cloning Factor (BIOVERIS #210001),
10% P388DI (ATCC # CRL TIB-63) conditioned medium and
Penicillin-Streptomycin (100.times., CELLGRO #30-002-CI). After
about 7 days, some of the medium containing HAT is replaced with
medium containing HT (CELLGRO #25-047-CI).
[0325] After 10 to 12 days, individual wells are screened for the
presence of human IgG/human kappa light chain antibodies using a
homogenous HTRF assay. In this assay, supernatants from 96 well
fusion plates are mixed with Europium-cryptate labeled goat
anti-human IgG (Fc fragment specific), biotinylated goat anti-human
kappa light chain (Bethyl # A80-115B), streptavidin-XLent and
incubated for 1 hour. The plates are then read on a RUBYSTAR
reader.
[0326] Hybridoma cells from wells positive for human IgG/human
kappa light chain or human IgG/human lambda light chain antibodies
are then screened by FACS using 300-19 cells transfected with human
CD40 and 300-19 untransfected cells as control. FACS positive
parental lines are transferred to 24-well plates. A few days later,
cell supernatants from individual wells are rescreened by FACS to
confirm IgG specificity to human CD40.
[0327] The hybridomas are cloned by serial dilution and re-screened
by FACS.
Example 3
Humanization of Anti-huCD40 Antibodies
[0328] Parental (murine) antibodies of the present invention were
humanized for potential use as human therapeutics. The closest
matching human germline region sequence was selected for each mouse
variable domain, and these human framework regions were used to
replace murine frameworks in the "CDR graft" versions of the
antibodies. Additional amino acid substitutions, such as framework
back-mutations, were then made as necessary to restore the binding
affinity of the humanized antibody. Further amino acid
substitutions were made to eliminate sequence liabilities. The
sequence relationships between the various forms of the antibodies
of the present invention are provided at Table 6. The binding of
various agonistic anti-huCD40 antibodies to soluble human CD40 was
determined by Biolayer Interferometry (BLI) analysis using a
FORTEBIO Octet RED (Rapid system--Extended Detection) label-free
interaction analysis instrument. All studies were performed in 10
mM Na.sub.xPO.sub.4, 130 mM NaCl, 0.05% Surfactant P20 (pH 7.1) at
25.degree. C. Briefly, anti-huCD40 antibody supernatants (diluted
to 10 .mu.g/ml or captured undiluted if supernatant concentration
was less than 10 .mu.g/ml) were captured on protein A coated
biosensors (PALL FORTEBIO #18-5010) using a loading time of 90s and
shake speed of 1000 rpm. Supernatants were first screened for
binding to 1 .mu.M recombinant human CD40 monomer (huCD40-monomer)
using 180s association and dissociation times, at 1000 rpm, with
two 15s conditioning steps using 10 mM glycine pH 1.5 in between
binding cycles. All supernatants that demonstrated a binding signal
in the 1 .mu.M huCD40-monomer experiment were then tested for
binding to seven different concentrations of huCD40-monomer in a
three-fold dilution series, where the highest concentration was
either 10 .mu.M huCD40-monomer or 1 .mu.M huCD40-monomer depending
on the strength of the binding signal in the 1 .mu.M screening
experiment. Results for selected antibodies and sequence variants
thereof are shown in Table 6.
TABLE-US-00006 TABLE 6 Binding Affinity of anti-CD40 Antibodies
K.sub.D Antibody Type V.sub.H V.sub.L (nM) 12D6 parental mouse
mouse 3.6 12D6-03 humanized VH1-18 CDR graft VKII O11 4.8 CDR graft
12D6-22 humanized VH1-18 CDR graft VKII O11 3.6 G29A 12D6-23
humanized VH1-18 M100bL VKII O11 4.3 CDR graft 12D6-24 humanized
VH1-18 M100bL VKII O11 5.6 G29A 5F11 parental mouse mouse 4.7
5F11-03 humanized VH1-e CDR graft VKIV B3 >5000 CDR graft
5F11-17 humanized VH1-e G27Y S30T V37I VKIV B3 8.4 R38K A40R M48I
R66K CDR graft V67T I69L 5F11-23 humanized VH1-e G27Y S30T V37I
VKIV B3 4.9 R38K A40R M48I R66K M4L V67T I69L N22S 5F11-45
humanized VH1-e G27Y S30T VKIV B3 12.3* R66K V67T I69L M4L 19G3
parental mouse mouse 870 19G3-03 humanized VH3-21 CDR graft VKII
O11 no CDR graft binding 19G3-11 humanized VH3-21 S94R VKII O11 850
CDR graft 19G3-22 humanized VH3-21 S94R VKII O11 99 N28Q 19G3-23
humanized VH3-21 S94R VKII O11 1300 G29A 5G7 parental mouse mouse
89 5G7-03 humanized VH1-18 CDR graft VKI A20 340 CDR graft 5G7-22
humanized VH1-18 M69L A93V VKI A20 50 CDR graft 5G7-25 humanized
VH1-18 M69L T71V VKI A20 69 A93V G55A CDR graft 5G7-28 humanized
VH1-18 M69L T71V VKI A20 230 A93V G55A M96L CDR graft 8E8 parental
mouse mouse 85 8E8-05 humanized VH1-e CDR graft VKII A19 no CDR
graft binding 8E8-56 humanized VH1-e L4F G27Y S30T VKII A19 82 R66K
V67A T68L I69L CDR graft 8E8-62 humanized VH1-e L4F G27Y S30T VKII
A19 31 R66K V67A T68L I69L CDR graft D99E 8E8-66 humanized VH1-e
L4F G27Y S30T VKII A19 82 R66K V67A T68L I69L G29A 8E8-67 humanized
VH1-e L4F G27Y S30T VKII A19 28 R66K V67A T68L I69L G29A D99E
8E8-70 humanized VH1-e L4F G27Y S30T VKII A19 low R66K V67A T68L
I69L CDR graft signal D99E G55S 8E8-71 humanized VH1-e L4F G27Y
S30T VKII A19 low R66K V67A T68L I69L G29A signal D99E G55S *= for
comparison purposes, the parental 5F11 exhibited a K.sub.D of 8.2
nM (rather than 4.7 nM) when run in the same assay in parallel with
the 5F11-45 antibody.
[0329] For humanized antibodies, V.sub.H and V.sub.L columns
provide the human variable domain germlines on which framework
regions are based. "CDR graft" refers to variable domains
comprising unmodified parental (mouse) CDRs grafted directly onto
the recited human framework sequences. Residue numbering for all
sequence variants in Table 6 is according to Kabat, and thus the
positions of sequence changes in Table 6 does not match the residue
numbering of the antibody sequences in the Sequence Listing.
Underlining is used to indicate sequence modifications that are
intended to correct potential sequence liabilities, i.e. residues
that are subject to chemical modification and potential
degradation, leading to product heterogeneity. Other sequence
modifications are intended to restore affinity, typically a
framework back mutation (reverting from the human germline
framework sequence to the original murine framework residue). The
M69L modification in the heavy chain of antibody 5G7 is both a
framework back mutation and a liability correction.
Example 4
Anti-CD40 Antibody Epitope Binning Experiments
[0330] Epitope binning experiments may be conducted to determine
which anti-human CD40 antibodies compete with which others for
binding to huCD40, and thus bind to similar epitopes. Pairwise
competition between anti-huCD40 antibodies is determined as
follows, in which a reference antibody is bound to the surface of a
sensor chip, a test antibody is pre-incubated with a huCD40
polypeptide construct in a mixture, and the pre-incubated mixture
is flowed over the sensor chip to determine the degree to which the
test antibody interferes with binding of the huCD40 polypeptide
construct to the reference antibody on the chip surface. Such
competition experiments may be performed using a BIACORE.RTM. SPR
instrument. Briefly, a reference anti-huCD40 antibody is
immobilized onto Sensor Chip CM5 chip (Series S, GE Healthcare CAT
# BR-1005-30) surfaces, flowcell2, flowcell3 & flowcell4 (5000
RUs), and flowcell1 is used as a negative control. A test antibody
is diluted to 120 .mu.g/mL (2.times.) at starting concentration. A
series of dilutions of the test antibody is made by diluting 1:3
concentration of antibody with buffer for seven different
concentrations and a control sample (with 0 .mu.g/ml) to obtain a
titration curve. Each antibody concentration series is divided into
two halves. In the first half of the concentration series, 40 nM
(2.times.) human CD40 antigen (e.g. huCD40/Fc) is added to make the
final concentration series (60 .mu.g/ml-0.0 .mu.g/ml) and 20 nM of
final antigen concentration in each well. In the second half of the
concentration series, in place of antigen, buffer is added to have
the antibody diluted to the same concentration, and this half is
treated as the blank. Complexes of the test anti-CD40 antibodies
and huCD40/Fc are incubated for 2 hours. 40 .mu.L complexes are
injected on the reference antibody-coated surface at a 30
.mu.L/min. A BIACORE.RTM. T200 surface plasmon resonance instrument
is used and the running buffer is HBE-EP, GE Healthcare CAT #
BR-1001-88, filtered, degassed, 0.01M HEPES, pH7.4, 0.15 NaCl, 3 mM
EDTA, 0.005% Surfactant P20. The surface is regenerated with 25 mM
NaOH (order code: BR-1003-58, GE Healthcare) at 100 .mu.L/min for 5
seconds. The data are analyzed using Microsoft Excel where the
concentration of test antibodies is plotted against the
corresponding response unit to obtain titration curves.
[0331] Results of such epitope binning experiments for the
anti-CD40 antibodies of the present invention are provided at FIG.
2. Antibodies 5F11 and 8E8 block ligand (CD40L) binding. The five
anti-CD40 antibodies of the present invention fall into three
epitope groups--12D6/5G7/19G3, 5F11/5G7/19G3, and 8E8.
Example 5
Epitope Mapping by HDX
[0332] The epitopes for anti-huCD40 antibodies 12D6, 5G7, 19G3 and
5F11 of the present invention were determined by hydrogen/deuterium
exchange mass spectrometry (HDX-MS). HDX-MS probes protein
conformation and conformational dynamics in solution by monitoring
the rate and extent of deuterium exchange of backbone amide
hydrogen atoms. Huang & Chen (2014) Anal. Bioanalytical Chem.
406:6541; Wei et al. (2014) Drug Disc. Today 19:95. The level of
HDX depends on the solvent accessibility of backbone amide hydrogen
atoms and the protein hydrogen bonds. The mass increase of the
protein upon HDX can be precisely measured by MS. When this
technique is paired with enzymatic digestion, structure features at
the peptide level can be resolved, enabling differentiation of
surface exposed peptides from those folded inside, or from those
sequestered at the interface of a protein-protein complex.
Typically, the deuterium labeling and subsequent quenching
experiments are performed, followed by enzymatic digestion, peptide
separation, and MS analysis.
[0333] Prior to epitope mapping experiments, non-deuteriated
experiments were carried out to generate a list of common peptides
for recombinant human CD40 monomer (10 .mu.M), and protein
complexes of CD40 with mAbs 12D6, 5G7, 19G3, and 5F11 (1:1 molar
ratio). In the HDX-MS experiment, 5 .mu.L of each sample (CD40 or
CD40 with mAbs 12D6, 5G7, 19G3, and 5F11 respectively) was diluted
into 55 .mu.L of D20 buffer (10 mM phosphate buffer, D20, pH7.0) to
start the labeling reactions. The reactions were carried out for
different periods of time: 20 sec, 1 min, 10 min and 240 min. By
the end of each labeling reaction period, the reaction was quenched
by adding quenching buffer (100 mM phosphate buffer with 4M GdnCl
and 0.4M TCEP, pH 2.5, 1:1, v/v) and 50 .mu.L of quenched sample
was injected into Waters HDX-MS system for analysis. The deuterium
uptake levels of common peptic peptides were monitored in the
absence/presence of CD40 mAbs. The obtained sequence coverage was
82%.
[0334] The HDX epitopes for anti-CD40 mAbs 12D6, 5G7, 19G3, and
5F11 are provided at Table 7. Antibody 12D6 protected two peptides,
one of which (residues 11-28) includes a portion of the signal
sequence and is thus unlikely to be physiologically relevant per
se, but indicates that 12D6 makes contacts in the region 21-28 that
are not made by mAbs 5G7 and 19G3.
TABLE-US-00007 TABLE 7 HDX Epitopes CD40 residues Clone (SEQ ID NO:
1) Sequence 12D6 11-28 WGCLLTAVHPEPPTACRE 12D6 21-35
EPPTACREKQYLINS 5G7 21-35 EPPTACREKQYLINS 19G3 21-35
EPPTACREKQYLINS 5F11 58-66 ECLPCGESE
Example 6
Epitope Mapping by Yeast Display
[0335] The epitopes for selected chimeric or humanized anti-huCD40
antibodies of the present invention are determined by displaying
randomly mutagenized huCD40 extracellular region variants on yeast,
and sorting these yeast based on their failure to bind to
particular antibodies. Selected yeast cells that fail to bind are
amplified and subjected to additional rounds of selection based on
their inability to bind to particular chimeric or humanized forms
of the antibodies of the present invention. See, e.g., Chao et al.
(2004) J. Mol. Biol. 342:539. Sequences for huCD40 variants are
determined for the resulting yeast and analyzed for the effects of
each residue on antibody binding. The binding epitope for the
antibodies of the present invention is determined as the loci
within the huCD40 sequence where single amino acid mutations
disrupt binding to the anti-huCD40 antibodies of the present
invention.
[0336] Briefly, error-prone PCR is used to clone human
CD40-encoding DNA into constructs allowing expression of the huCD40
variants as the amino-terminal portions of fusion proteins further
comprising a c-myc tag sequence and yeast cell wall protein Aga1p.
Such constructs, when expressed in yeast (Saccharomyces
cerevisiae), display the variant huCD40 polypeptides on the surface
of yeast cells, anchored to the cell surface by the Aga1p
polypeptide. The c-myc tag can optionally be used as a positive
control for display of huCD40 fusion proteins on a given yeast
cell. Yeast cells are sorted by FACS, and those that express as
properly folded huCD40-fusion proteins (as determined by binding of
a control mouse anti-huCD40 antibody detected by an allophycocyanin
(APC)-labeled goat anti-mouse IgG secondary), but do not bind to
the antibodies of the present invention (as determined by detection
with a phycoerythrin (PE) labeled goat anti-human IgG as a
secondary), are pooled, amplified, and used in subsequent rounds of
selection. The huCD40 sequence is determined for constructs from
yeast remaining after several rounds of selection. Control
experiments without anti-huCD40 antibody selection are performed to
confirm good mutant coverage at each position along the huCD40
sequence, and provide a baseline for normalizing the results
obtained with the selected libraries.
Example 7
Agonist Activity of Anti-CD40 Antibodies
[0337] The effect of Fc sequence variation on the agonist activity
of selected anti-CD40 antibodies of the present invention was
assessed by measuring activation of immature dendritic cells (DC).
Experiments were performed with anti-CD40 mAb 12D6-24 constructs
having human IgG1f (control), SE, SELF, P238D, V4, V8 and V12 Fc
sequences. Human Monocytes (CD14.+-.) were isolated from healthy
normal donors using plastic adherence or human CD14-micro beads
(MILTENYI Biotec). Monocytes were cultured with 100 ng/mL GM-CSF
(Miltenyi Biotec) and 100 ng/mL IL-4 (Miltenyi Biotec). Half of the
medium was removed and replenished on day 2 and day 5. Immature
dendritic cells were harvested at day 6-7. DC from two donors were
incubated with the indicated concentration of antibodies overnight
at 37.degree. C. Cell culture supernatants were collected and
assayed for human-IL-6 production (CISBIO). See FIGS. 3A and
3B.
[0338] Control human IgG1f antibodies did not induce IL-6 secretion
when used at up to 100 nM, and the P238D variant induced only
weakly. In contrast, SE, SELF, V9 and V12 variants all dramatically
enhanced IL-6 secretion, with V8 and V4 exhibiting intermediate
effects. These results roughly correlate with binding affinity for
Fc.gamma.RIIb, and confirm that use of the proper Fc sequence
variant can result in anti-CD40 antibodies with enhanced agonist
activity.
[0339] In addition, the differences in agonist activity between
antibodies having 8E8, 5G7, 12D6, 19G3 and 5F11 variable regions
were evaluated in experiments using chimeric anti-CD40 mAb
constructs having a common human IgG1f V12 constant domain.
Activation was measured on immature dendritic cells in vitro
(isolated as described in the preceding paragraph) by plating cells
in a 96 well plate, adding antibodies as indicated, and incubating
overnight at 37.degree. C. Cells were then harvested and stained
with a fluorescent anti-CD54 antibody, which was detected by
fluorescence activated cell sorting (FACS). See FIG. 4.
[0340] Control human IgG1f antibody did not significantly cause
CD54 upregulation, whereas 12D6 caused dramatic CD54 upregulation.
Antibodies 19G3 and 8G8 were somewhat less effective than 12D6, and
antibodies 5G7 and 5F11 were similar to each other and exhibited
relatively low stimulation. The activation results do not
necessarily correlate with binding affinity for CD40, since
antibody 5F11 has nearly the highest binding affinity and yet is a
weak agonist, whereas antibody 19G3 is the opposite. Regardless of
the reason for the differences, these results confirm that use of
the proper antigen binding domain sequence is important in
obtaining anti-CD40 antibodies with enhanced agonist activity.
Example 8
Remediation of DG Isomerization
[0341] Antibodies of the present invention having V4 and V9 Fc
variant sequences exhibit unacceptable levels of DG isomerization
at position D270. Such isomerization is undesirable because it
leads to product heterogeneity and potentially reduced efficacy. To
reduce such isomerization, the aspartic acid residue at position
270 of V4 Fc was changed to glutamic acid (D270E), and position 270
of V9 Fc was changed to alanine, glutamic acid, glutamine, serine
and threonine (D270A, D270E, D270Q, D270S, D270T). Supernatants
were obtained from cells expressing these antibodies and assayed
for binding to hCD32/Fc.gamma.RII. Selected purified antibodies
without mutations at position 270 were used as controls. These
experiments were performed on antibodies with variable domains from
mAbs 12D6-24 and 5F11-45. Results are provided at FIG. 5.
[0342] For both V4 and V9 Fc variants, the D270E substitution led
to a modest decrease in receptor binding. Other V9 Fc variants
tested (D270A, D270Q, D270S, D270T) led to a greater decrease in
receptor binding. Results were independent of whether the antibody
was 12D6 or 5F11. In all cases the relative (rank order) binding to
the three receptors tested (Fc.gamma.RIIa-H131, Fc.gamma.RIIa-R131,
and Fc.gamma.RIIb) remained similar, with roughly equivalent
binding to Fc.gamma.RIIa-R131 and Fc.gamma.RIIb, and significantly
weaker binding to Fc.gamma.RIIa-H131.
[0343] For both V4 and V9 variants, the D270E substitution retained
acceptably high Fc.gamma.R affinity, and maintained the specificity
for Fc.gamma.RIIb that is desired for enhancing the agonistic
activity of the anti-CD40 antibodies of the present invention.
Example 9
Anti-CD40 Activity in Human CD40/Human Fc.gamma.R Transgenic
Mice
[0344] Selected agonist anti-CD40 antibodies of the present
invention were administered to mice transgenic for human CD40 and
human Fc.gamma. receptors [CD40.sup.-/-hCD40.sup.+
Fcgra.sup.-/-Fcgr1.sup.-/-
hFCGRI.sup.+hFCGRIIA.sup.+hFCGRIIB.sup.+hFCGRIIIA.sup.+hFCGRIIIB.sup.+].
Mice transgenic for human Fc.gamma. receptors are described at
Smith et al. (2012) Proc. Nat'l Acad. Sci. (USA) 109(16):6181-6.
Such transgenic human CD40/Fc.gamma.R mice are well suited to study
potential human therapeutic anti-CD40 antibodies, which necessarily
bind to human CD40 and human Fc.gamma.Rs. Agonist activity was
measured in the "DEC-OVA" model described in Li & Ravetch
(2011) Science 333:1030, and platelet levels were also measured.
Results are shown in FIGS. 6A and 6B. Antibodies showing the
greatest agonist activity, such as 12D6-V11 and to a lesser degree
12D6-V4, also exhibited the greatest decrease in platelet count at
24 hours. The decrease in platelet count was a transient
phenomenon, with levels returning to normal by 7 days
post-injection (data not shown).
[0345] 5F11 antibody recognizes epitopes overlapping with the CD40L
binding epitope, unlike 12D6 antibody that binds hCD40 distinct
from its CD40L binding epitope, similar to CP-870,893. 12D6
antibody blocks CP-870,893 binding, but 5F11 antibody does not
(data not shown). In vitro DC activation assays have demonstrated
significant activity for the SE-Fc, and V11-Fc variants of 12D6
while 12D6 Fc variants exhibit enhanced activity compared to 5F11
variants. Although the in vitro activity of the 12D6-Fc variants is
greater than the wild type IgG1, the in vitro studies do not
distinguish between the activities of the different Fc variants. We
therefore evaluated the activity of these antibodies and their Fc
variants in the humanized CD40/human Fc.gamma.R mice. As was
observed for clone CP-870,893, the selective Fc.gamma.RIIB-enhanced
V11 Fc variant of these mAbs displayed superior activity in vivo
compared to its Fc.gamma.RIIA/IIB-enhanced SE variant and the wild
type IgG1 subclass. Moreover, the magnitude of the in vivo activity
of the newly developed 12D6-V11 is about 20-fold higher than the
activity of CP-870,893 (IgG2).
[0346] The anti tumor activity of the CD40 agonistic Abs, 12D6-24
and 5F11-45 was evaluated. Wild type IgG1, SE and V11 mutants of
each clone were tested for their activity in the MC38 tumor model.
The Fc.gamma.RIIB-selectively enhanced V11 variants of these
antibodies have superior anti tumor activity compared to the other
tested Fc variants, consistent with their in vivo T cell
stimulation activity and with the anti tumor activity hierarchy
observed for CP-870,893 derivatives. Results are shown in FIG. 6C.
Tumor-free mice from the 12D6-24 treated group were re-challenged
with MC38 tumor cells 30 days after their CD40 Ab treatment.
Results are shown in FIG. 6D. All mice in this group rejected the
tumors within two weeks of the re-challenge, demonstrating that
long-term protection is mediated by single injection of
12D6-24.
[0347] These data indicate that the antitumor activity of agonistic
human CD40 Abs can be enhanced by Fc engineering that provides
selective enhancement of Fc.gamma.RIIB-engagement and that the V11
Fc variant is particularly effective at enhancing agonism.
TABLE-US-00008 TABLE 8 Summary of Sequence Listing SEQ ID
Description 1 Human CD40 (NP_001241) 2 Human CD40L-gp39
(NP_000065.1) 3 12D6 Chimeric Heavy Chain 4 12D6 Chimeric Light
Chain 5 12D6-03 Heavy Chain 6 12D6-03 Light Chain 7 12D6-22 Heavy
Chain 8 12D6-22 V9 Heavy Chain 9 12D6-22/12D6-24 Light Chain 10
12D6-23 Heavy Chain 11 12D6-23 Light Chain 12 12D6-24 Heavy Chain
13 12D6-24 P238D Heavy Chain 14 12D6-24 SE Heavy Chain 15 12D6-24
SELF Heavy Chain 16 12D6-24 V4 Heavy Chain 17 12D6-24 V4 D270E
Heavy Chain 18 12D6-24 V8 Heavy Chain 19 12D6-24 V9 Heavy Chain 20
12D6-24 V9 D270E Heavy Chain 21 12D6-24 V11 Heavy Chain 22 12D6-24
V12 Heavy Chain 23 5F11 Chimeric Heavy Chain 24 5F11 Chimeric Light
Chain 25 5F11-17 Heavy Chain 26 5F11-17 Light Chain 27 5F11-23
Heavy Chain 28 5F11-23 Light Chain 29 5F11-45 Heavy Chain 30
5F11-45 Light Chain 31 5F11-45 SE Heavy Chain 32 5F11-45 SELF Heavy
Chain 33 5F11-45 V4 Heavy Chain 34 5F11-45 D270E V4 Heavy Chain 35
5F11-45 V8 Heavy Chain 36 5F11-45 V9 Heavy Chain 37 5F11-45 V9
D270E Heavy Chain 38 5F11-45 V11 Heavy Chain 39 5F11-45 V12 Heavy
Chain 40 8E8 Chimeric Heavy Chain 41 8E8 Chimeric Light Chain 42
8E8-56 Heavy Chain 43 8E8-56 Light Chain 44 8E8-62 Heavy Chain 45
8E8-62 Light Chain 46 8E8-67 Heavy Chain 47 8E8-67 Light Chain 48
8E8-70 Heavy Chain 49 8E8-70 Light Chain 50 8E8-71 Heavy Chain 51
8E8-71 Light Chain 52 5G7 Chimeric Heavy Chain 53 5G7 Chimeric
Light Chain 54 5G7-22 Heavy Chain 55 5G7-22 Light Chain 56 5G7-25
Heavy Chain 57 5G7-25 Light Chain 58 19G3 Chimeric Heavy Chain 59
19G3 Chimeric Light Chain 60 19G3-11 Heavy Chain 61 19G3-11 V9 62
19G3-11 Light Chain 63 19G3-22 Heavy Chain 64 19G3-22 Light Chain
65 Human Constant Domain IgG1f 66 SE Constant Domain 67 SELF
Constant Domain 68 P238D Constant Domain 69 V4 Constant Domain 70
V4 D270E Constant Domain 71 V7 Constant Domain 72 V8 Constant
Domain 73 V9 Constant Domain 74 V9 D270E Constant Domain 75 V11
Constant Domain 76 V12 Constant Domain 77 Light Chain Kappa
Constant Domain 78 Signal Sequence
[0348] The Sequence Listing provides the sequences of the mature
heavy and light chains (i.e., sequences do not include signal
peptides). A signal sequence for production of the antibodies of
the present invention, for example in human cells, is provided at
SEQ ID NO: 78.
EQUIVALENTS
[0349] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments disclosed herein. Such
equivalents are intended to be encompassed by the following claims.
Sequence CWU 1
1
781277PRTHomo
sapiensSIGNAL(1)..(20)PEPTIDE(21)..(277)DOMAIN(21)..(193)extracellular
domainDOMAIN(194)..(215)transmembrane
domainDOMAIN(216)..(277)intracellular domain 1Met Val Arg Leu Pro
Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr1 5 10 15Ala Val His Pro
Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30Ile Asn Ser
Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45Ser Asp
Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60Ser
Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His65 70 75
80Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr
85 90 95Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys
Thr 100 105 110Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys
Ser Pro Gly 115 120 125Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser
Asp Thr Ile Cys Glu 130 135 140Pro Cys Pro Val Gly Phe Phe Ser Asn
Val Ser Ser Ala Phe Glu Lys145 150 155 160Cys His Pro Trp Thr Ser
Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175Ala Gly Thr Asn
Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu 180 185 190Arg Ala
Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200
205Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn
210 215 220Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe
Pro Asp225 230 235 240Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val
Gln Glu Thr Leu His 245 250 255Gly Cys Gln Pro Val Thr Gln Glu Asp
Gly Lys Glu Ser Arg Ile Ser 260 265 270Val Gln Glu Arg Gln
2752261PRTHomo sapiens 2Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg
Ser Ala Ala Thr Gly1 5 10 15Leu Pro Ile Ser Met Lys Ile Phe Met Tyr
Leu Leu Thr Val Phe Leu 20 25 30Ile Thr Gln Met Ile Gly Ser Ala Leu
Phe Ala Val Tyr Leu His Arg 35 40 45Arg Leu Asp Lys Ile Glu Asp Glu
Arg Asn Leu His Glu Asp Phe Val 50 55 60Phe Met Lys Thr Ile Gln Arg
Cys Asn Thr Gly Glu Arg Ser Leu Ser65 70 75 80Leu Leu Asn Cys Glu
Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95Asp Ile Met Leu
Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110Met Gln
Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120
125Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
130 135 140Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
Lys Gln145 150 155 160Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
Tyr Ala Gln Val Thr 165 170 175Phe Cys Ser Asn Arg Glu Ala Ser Ser
Gln Ala Pro Phe Ile Ala Ser 180 185 190Leu Cys Leu Lys Ser Pro Gly
Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205Ala Asn Thr His Ser
Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220Leu Gly Gly
Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn225 230 235
240Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
245 250 255Gly Leu Leu Lys Leu 2603448PRTArtificial
Sequencechimeric antibody with mouse variable domain, human
constant domainDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 3Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu
Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr
Ser Phe Thr Gly Tyr 20 25 30Asn Met Asn Trp Val Lys Gln Ser Asn Gly
Lys Ser Leu Glu Trp Ile 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Leu Gln Leu Lys Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser 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 Arg 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 Glu Glu Met 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 4454219PRTArtificial
Sequencechimeric antibody with mouse variable domain and human
constant domainDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 4Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Asn Leu Leu Ile Tyr Lys Leu Thr
Asn Arg Phe Phe Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Ile His Val Pro Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 2155448PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 5Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Glu Glu Met 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 4456219PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 6Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Leu Thr
Asn Arg Phe Phe Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Ile His Val Pro Trp
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 2157448PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 7Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Glu Glu Met 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 4458448PRTArtificial Sequencehumanized antibody
with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 8Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Asp Asp225 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 270Gly 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 Arg 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 Glu Glu Met 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 4459219PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 9Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Leu Thr
Asn Arg Phe Phe Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Ile His Val Pro Trp
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21510448PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 10Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Glu Glu Met 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 44511219PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 11Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Leu Thr
Asn Arg Phe Phe Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Ile His Val Pro Trp
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21512448PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 12Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Glu Glu Met 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 44513448PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 13Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Asp225 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 Glu Glu Met 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 44514448PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework
and
constant regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 14Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Glu 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 Glu Glu Met 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 44515448PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 15Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Glu 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 Phe 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 Glu Glu Met 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 44516448PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 16Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Asp225 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 270Gly 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 Glu Glu Met 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 44517448PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regions 17Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg 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 Asp225 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 Glu
260 265 270Gly 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 Glu Glu Met 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 44518448PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 18Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Asp Asp225 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 Asp Glu Asp
260 265 270Gly 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 Glu Glu Met 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 44519448PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50
55 60Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu Gln Leu Tyr Ala Leu Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Asp Asp225 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 270Gly 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 Arg 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 Glu Glu Met 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 44520448PRTArtificial Sequencehumanized antibody with mouse
CDRs and human framework and constant regions 20Gln 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 30Asn Met Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asn
Ile Asp Pro Tyr Tyr Gly Asn Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Leu Gly Leu Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr 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 Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Asp Asp225 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 Glu 260 265 270Gly 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 Arg 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 Glu Glu Met 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
44521448PRTArtificial Sequencehumanized antibody with mouse CDRs
and human framework and constant regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 21Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Asp Asp225 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 Asp Glu Asp
260 265 270Gly 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 Arg 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 Glu Glu Met 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 44522448PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(119)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(108)CDRH3 22Gln 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 30Asn Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asn Ile Asp Pro Tyr Tyr Gly Asn
Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Leu
Gln Leu Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr 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 Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Asp Leu Leu Gly Asp Asp225 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 Asp Glu Asp
260 265 270Gly 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 Arg 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 Glu Glu Met 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 44523446PRTArtificial
Sequencechimeric antibody with mouse variable domain and human
constant domainDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 23Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala
Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Ile Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44524218PRTArtificial
Sequencechimeric antibody with mouse variable domain and human
constant domainDOMAIN(1)..(111)variable
domainMISC_FEATURE(24)..(38)CDRL1MISC_FEATURE(54)..(60)CDRL2MISC_FEATURE(-
93)..(101)CDRL3 24Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys
Asn Val Asp Ser Tyr 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln
Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn
Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Gly Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Asn65 70 75 80Pro Val Glu Ala Asp Asp
Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Leu Thr
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21525446PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 25Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Ile Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44526218PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(111)variable
domainMISC_FEATURE(24)..(38)CDRL1MISC_FEATURE(54)..(60)CDRL2MISC_FEATURE(-
93)..(101)CDRL3 26Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys
Asn Val Asp Ser Tyr 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln
Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn
Leu Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Leu Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 210 21527446PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 27Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Ile Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44528218PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(111)variable
domainMISC_FEATURE(24)..(38)CDRL1MISC_FEATURE(54)..(60)CDRL2MISC_FEATURE(-
93)..(101)CDRL3 28Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys
Asn Val Asp Ser Tyr 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln
Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn
Leu Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Leu Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 210 21529446PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 29Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44530218PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(111)variable
domainMISC_FEATURE(24)..(38)CDRL1MISC_FEATURE(54)..(60)CDRL2MISC_FEATURE(-
93)..(101)CDRL3 30Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys
Asn Val Asp Ser Tyr 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln
Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser Asn
Leu Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn 85 90 95Glu Asp Pro Leu Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135
140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 210 21531446PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 31Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170
175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Glu His Glu Asp Pro Glu 260 265 270Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44532446PRTArtificial Sequencehumanized antibody with mouse
CDRs and human framework and constant
regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 32Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Glu His Glu Asp Pro Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Phe Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44533446PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 33Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Gly Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44534446PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regions 34Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Gly Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44535446PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regions 35Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Asp Asp Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp Gly Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44536446PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 36Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Asp Asp Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Gly Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275
280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro Arg
Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 44537446PRTArtificial Sequencehumanized antibody
with mouse CDRs and human framework and constant regions 37Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Leu 20 25
30Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr Thr Ala Tyr Asn Gln Lys
Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu Gln Arg Gly Ala Tyr Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170
175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Asp Asp Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Glu Gly Glu 260 265 270Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Pro Arg Pro Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44538446PRTArtificial Sequencehumanized antibody with mouse
CDRs and human framework and constant
regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 38Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Asp Asp Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp Gly Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Arg Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44539446PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(117)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(106)CDRH3 39Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Leu 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Thr Pro Ser Ser Gly Tyr
Thr Ala Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Thr Thr Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ile Leu
Gln Arg Gly Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro
Cys Pro Ala Pro Asp Leu Leu Gly Asp Asp Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp Gly Glu
260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala
Leu Pro Arg Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 44540451PRTArtificial
Sequencechimeric antibody with mouse variable domain and human
constant domainDOMAIN(1)..(122)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(111)CDRH3 40Gln Val Gln Phe Gln Gln Ser Gly Ala Glu Leu Ala
Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Trp Met Gln Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Thr Ile Tyr Pro Gly Asp Gly Asp
Ser Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Leu Leu Thr Ala
Asp Lys Ser Ser Ser Ile Ala Tyr65 70 75 80Met Gln Leu Asn Ser Leu
Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Phe Ser Leu
Tyr Asp Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
45041219PRTArtificial Sequencechimeric antibody with mouse variable
domain and human constant domainDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 41Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Arg 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Arg Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Phe Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21542451PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(122)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(111)CDRH3 42Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Trp Met Gln Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ile Tyr Pro Gly Asp Gly Asp
Ser Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Leu Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe Ser Leu
Tyr Asp Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360
365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
45043219PRTArtificial Sequencehumanized antibody with mouse CDRs
and human framework and constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 43Asp 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
Ser Leu Val His Arg 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21544451PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(122)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(111)CDRH3 44Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Trp Met Gln Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ile Tyr Pro Gly Asp Gly Asp
Ser Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Leu Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe Ser Leu
Tyr Glu Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
45045219PRTArtificial Sequencehumanized antibody with mouse CDRs
and human framework and constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 45Asp 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
Ser Leu Val His Arg 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21546451PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(122)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(111)CDRH3 46Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Trp Met Gln Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ile Tyr Pro Gly Asp Gly Asp
Ser Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Leu Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe Ser Leu
Tyr Glu Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
45047219PRTArtificial Sequencehumanized antibody with mouse CDRs
and human framework and constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 47Asp 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
Ser Leu Val His Arg 20 25 30Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21548451PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(122)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(111)CDRH3 48Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Trp Met Gln Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ile Tyr Pro Gly Asp Ser Asp
Ser Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Leu Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe Ser Leu
Tyr Glu Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405
410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu 435 440 445Ser Pro Gly 45049219PRTArtificial
Sequencehumanized antibody with mouse CDRs and human framework and
constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 49Asp 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
Ser Leu Val His Arg 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21550451PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(122)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(111)CDRH3 50Gln Val Gln Phe Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Trp Met Gln Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Thr Ile Tyr Pro Gly Asp Ser Asp
Ser Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Leu Leu Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe Ser Leu
Tyr Glu Gly Tyr Pro Tyr Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
45051219PRTArtificial Sequencehumanized antibody with mouse CDRs
and human framework and constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 51Asp 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
Ser Leu Val His Arg 20 25 30Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21552442PRTArtificial Sequencechimeric
antibody with mouse variable domain and human constant
domainDOMAIN(1)..(113)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(102)CDRH3 52Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val
Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr
Lys Phe Thr Asp Tyr 20 25 30Asn Met Asp Trp Val Lys Gln Ser His Gly
Lys Ser Leu Glu Trp Ile 35 40 45Gly Asp Ile Asn Pro Lys Asn Gly Gly
Thr Ile Tyr Asn Leu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val
Asp Met Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Arg Met Asp
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 100 105 110Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135
140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250
255Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
260 265 270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 275 280 285Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val 290 295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser305 310 315 320Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375
380Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe385 390 395 400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 405 410 415Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr 420 425 430Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 44053214PRTArtificial Sequencechimeric antibody with
mouse variable domain and human constant
domainDOMAIN(1)..(107)variable
domainMISC_FEATURE(24)..(34)CDRL1MISC_FEATURE(50)..(56)CDRL2MISC_FEATURE(-
89)..(97)CDRL3 53Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser
Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
Thr Val Lys Leu Leu Ile 35 40 45Tyr Tyr Ile Ser Arg Leu His Ser Gly
Val Pro Ser Arg Phe Arg Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Ser
Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr
Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly
Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21054442PRTArtificial Sequencehumanized antibody with mouse CDRs
and human framework and constant regionsDOMAIN(1)..(113)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(102)CDRH3 54Gln 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 Asp Tyr 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Pro Lys Asn Gly Gly
Thr Ile Tyr Asn Leu Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Arg Met Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135
140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250
255Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
260 265 270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 275 280 285Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val 290 295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser305 310 315 320Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375
380Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe385 390 395 400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 405 410 415Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr 420 425 430Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 44055214PRTArtificial Sequencehumanized antibody with
mouse CDRs and human framework and constant
regionsDOMAIN(1)..(107)variable
domainMISC_FEATURE(24)..(34)CDRL1MISC_FEATURE(50)..(56)CDRL2MISC_FEATURE(-
89)..(97)CDRL3 55Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Val Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Ile Ser Arg Leu His Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly
Thr Lys 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
21056442PRTArtificial Sequencehumanized antibody with mouse CDRs
and human framework and constant
regionsDOMAIN(1)..(113)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(102)CDRH3 56Gln 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 Asp Tyr 20 25 30Asn Met Asp Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Asn Pro Lys Asn Ala Gly
Thr Ile Tyr Asn Leu Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Val
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Arg Met Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 100 105 110Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135
140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln 180 185 190Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro225 230 235 240Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250
255Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
260 265 270Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 275 280 285Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val 290 295 300Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser305 310 315 320Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375
380Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe385 390 395 400Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 405 410 415Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr 420 425 430Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 44057214PRTArtificial Sequencehumanized antibody with
mouse CDRs and human framework and constant
regionsDOMAIN(1)..(107)variable
domainMISC_FEATURE(24)..(34)CDRL1MISC_FEATURE(50)..(56)CDRL2MISC_FEATURE(-
89)..(97)CDRL3 57Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Val Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Ile Ser Arg Leu His Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly
Thr Lys 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
21058441PRTArtificial Sequencechimeric antibody with mouse variable
domain and human constant domainDOMAIN(1)..(112)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(101)CDRH3 58Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ser Pro Glu
Lys Arg Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Gly Gly Gly Ser Tyr
Thr Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Glu Met Ser Ser Leu
Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Ser Arg Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135
140Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser145 150 155 160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 165 170 175Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 180 185 190Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 195 200 205Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235 240Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250
255Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
260 265 270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn305 310 315 320Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375
380Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe385 390 395 400Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 44059219PRTArtificial Sequencechimeric antibody with mouse
variable domain and human constant domainDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 59Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Phe Glu Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21560441PRTArtificial Sequencehumanized
antibody with mouse CDRs and human framework and constant
regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(101)CDRH3 60Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile Ser Gly Gly Gly Ser Tyr
Thr Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Arg Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135
140Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser145 150 155 160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 165 170 175Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 180 185 190Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 195 200 205Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235 240Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250
255Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
260 265 270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn305 310 315 320Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375
380Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe385 390 395 400Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 44061441PRTArtificial Sequencehumanized antibody with mouse
CDRs and human framework and constant
regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(101)CDRH3 61Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile Ser Gly Gly Gly Ser Tyr
Thr Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Arg Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135
140Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser145 150 155 160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 165 170 175Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 180 185 190Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 195 200 205Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220Pro Ala Pro Glu
Leu Leu Gly Asp Asp Ser Val Phe Leu Phe Pro Pro225 230 235 240Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250
255Val Val Val Asp Val Ser His Glu Asp Gly Glu Val Lys Phe Asn Trp
260 265 270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn305 310 315 320Lys Ala Leu Pro Arg Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375
380Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe385 390 395 400Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 44062219PRTArtificial Sequencehumanized antibody with mouse
CDRs and human framework and constant
regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 62Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Phe Glu Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200
205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21563441PRTArtificial Sequencehumanized antibody with mouse CDRs
and human framework and constant regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(31)..(35)CDRH1MISC_FEATURE(50)..(66)CDRH2MISC_FEATURE(-
99)..(101)CDRH3 63Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile Ser Gly Gly Gly Ser Tyr
Thr Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Arg Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135
140Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser145 150 155 160Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 165 170 175Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 180 185 190Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 195 200 205Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro225 230 235 240Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250
255Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
260 265 270Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 275 280 285Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 290 295 300His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn305 310 315 320Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375
380Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe385 390 395 400Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 405 410 415Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr 420 425 430Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 44064219PRTArtificial Sequencehumanized antibody with mouse
CDRs and human framework and constant
regionsDOMAIN(1)..(112)variable
domainMISC_FEATURE(24)..(39)CDRL1MISC_FEATURE(55)..(61)CDRL2MISC_FEATURE(-
94)..(102)CDRL3 64Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 20 25 30Gln Gly Asn Thr Tyr Phe Glu Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 21565329PRTHomo sapiens 65Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32566329PRTArtificial
Sequencevariant of human IgG1f constant domain 66Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Glu His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32567329PRTArtificial
Sequencevariant of human IgG1f constant domain 67Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Glu His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32568329PRTArtificial
Sequencevariant of human IgG1f constant domain 68Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32569329PRTArtificial
Sequencevariant of human IgG1f constant domain 69Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Asp Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu
Asp Gly Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260
265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro
Gly 32570329PRTArtificial Sequencevariant of human IgG1f constant
domain 70Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Glu Gly Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32571329PRTArtificial Sequencevariant of human IgG1f constant
domain 71Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Asp Leu
Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Gly Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Arg Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32572329PRTArtificial Sequencevariant of human IgG1f constant
domain 72Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Asp Asp Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser Asp Glu Asp Gly Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32573329PRTArtificial Sequencevariant of human IgG1f constant
domain 73Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Asp Asp Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Gly Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Arg Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32574329PRTArtificial Sequencevariant of human IgG1f constant
domain 74Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Asp Asp Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile S