U.S. patent application number 15/577375 was filed with the patent office on 2018-08-23 for antibodies against ox40 and uses thereof.
The applicant listed for this patent is BRISTOL-MYERS SQUIBB COMPANY. Invention is credited to Praveen AANUR, Bryan C. BARNHART, Zhehong CAI, Indrani CHAKRABORTY, Han CHANG, Yan FENG, Marie-Michelle Navarro GARCIA, Jochem GOKEMEIJER, Patrick GUIRNALDA, Michelle Minhua HAN, Sandra V. HATCHER, Karla A. HENNING, Christine HUANG, Maria JURE-KUNKEL, Thomas D. KEMPE, Alan J. KORMAN, Alexander T. KOZHICH, Ming LEI, Hadia LEMAR, Nils LONBERG, Mark MAURER, Christina Maria MILBURN, Michael QUIGLEY, Arvind RAJPAL, Maria RODRIGUEZ, Liang SCHWEIZER, Mark J. SELBY, Xiang SHAO, Mohan SRINIVASAN, Brenda L. STEVENS, Kent THUDIUM, Xi-Tao WANG, Susan Chien-Szu WONG, Aaron P. YAMNIUK, Zheng YANG.
Application Number | 20180237534 15/577375 |
Document ID | / |
Family ID | 56121193 |
Filed Date | 2018-08-23 |
United States Patent
Application |
20180237534 |
Kind Code |
A1 |
CAI; Zhehong ; et
al. |
August 23, 2018 |
ANTIBODIES AGAINST OX40 AND USES THEREOF
Abstract
Provided herein are antibodies, or antigen binding portions
thereof, that bind to OX40. Also provided are uses of these
proteins in therapeutic applications, such as in the treatment of
cancer. Further provided are cells that produce the antibodies,
polynucleotides encoding the heavy and/or light chain variable
region of the antibodies, and vectors comprising the
polynucleotides encoding the heavy and/or light chain variable
region of the antibodies.
Inventors: |
CAI; Zhehong; (Sunnyvale,
CA) ; CHAKRABORTY; Indrani; (Fremont, CA) ;
GARCIA; Marie-Michelle Navarro; (San Jose, CA) ;
KEMPE; Thomas D.; (Sunnyvale, CA) ; KORMAN; Alan
J.; (Piedmont, CA) ; KOZHICH; Alexander T.;
(Princeton, NJ) ; LEMAR; Hadia; (Tracy, CA)
; MAURER; Mark; (Seattle, WA) ; MILBURN; Christina
Maria; (Santa Cruz, CA) ; QUIGLEY; Michael;
(Ambler, PA) ; RODRIGUEZ; Maria; (San Jose,
CA) ; SHAO; Xiang; (Milpitas, CA) ;
SRINIVASAN; Mohan; (Cupertino, CA) ; STEVENS; Brenda
L.; (Seattle, WA) ; THUDIUM; Kent; (Oakland,
CA) ; WONG; Susan Chien-Szu; (Fremont, CA) ;
GOKEMEIJER; Jochem; (Wayland, MA) ; WANG; Xi-Tao;
(Wellesley, MA) ; CHANG; Han; (West Windsor,
NJ) ; HUANG; Christine; (Princeton, NJ) ;
JURE-KUNKEL; Maria; (Plainsboro, NJ) ; YANG;
Zheng; (Plainsboro, NJ) ; FENG; Yan;
(Pittsburgh, PA) ; GUIRNALDA; Patrick;
(Framingham, MA) ; LONBERG; Nils; (Woodside,
CA) ; BARNHART; Bryan C.; (Philadelphia, PA) ;
YAMNIUK; Aaron P.; (Lawrenceville, NJ) ; HENNING;
Karla A.; (Milpitas, CA) ; HAN; Michelle Minhua;
(Piedmont, CA) ; LEI; Ming; (Princeton, NJ)
; SCHWEIZER; Liang; (Shanghai PR, CN) ; HATCHER;
Sandra V.; (Hillsborough, NJ) ; RAJPAL; Arvind;
(San Francisco, CA) ; AANUR; Praveen; (Windsor,
NJ) ; SELBY; Mark J.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRISTOL-MYERS SQUIBB COMPANY |
Princeton |
NJ |
US |
|
|
Family ID: |
56121193 |
Appl. No.: |
15/577375 |
Filed: |
May 26, 2016 |
PCT Filed: |
May 26, 2016 |
PCT NO: |
PCT/US2016/034470 |
371 Date: |
November 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62168377 |
May 29, 2015 |
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62239574 |
Oct 9, 2015 |
|
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62264691 |
Dec 8, 2015 |
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62327140 |
Apr 25, 2016 |
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62333556 |
May 9, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/30 20130101;
C07K 2317/21 20130101; A61K 39/3955 20130101; C07K 16/3069
20130101; C07K 2317/92 20130101; C07K 2317/94 20130101; C07K
2317/52 20130101; C07K 2317/73 20130101; C07K 2317/567 20130101;
C07K 16/2818 20130101; C07K 2317/34 20130101; A61K 2039/505
20130101; A61P 35/00 20180101; A61K 2039/507 20130101; C07K 2317/56
20130101; C07K 2317/565 20130101; C07K 16/2878 20130101; C07K
2317/24 20130101; C07K 2317/31 20130101; G01N 33/574 20130101; G01N
2333/70514 20130101; C07K 16/3038 20130101; C07K 2317/74 20130101;
C07K 2317/76 20130101; C07K 2317/33 20130101; C07K 16/3023
20130101; C07K 2317/75 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; A61K 39/395 20060101
A61K039/395; G01N 33/574 20060101 G01N033/574; A61P 35/00 20060101
A61P035/00 |
Claims
1-2. (canceled)
3. An isolated monoclonal antibody which binds to OX40, comprising:
(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID
NOs: 87, 317, and 89, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 90-92, respectively; (b)
heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:
11-13, respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 14-16, respectively; (c) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 19-21,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 22-24, respectively; (d) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 19-21,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 25-27, respectively; (e) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 31-33,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 34-36, respectively; (f) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 39-41,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 42-44, respectively; (g) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 39-41,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 45-47, respectively; (h) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 51-53,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 54-56, respectively; (i) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 59-61,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 62-64, respectively; (j) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 67-69,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 70-72, respectively; (k) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 75-77,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 78-80, respectively, wherein, optionally,
the Asp-Gly sequence in SEQ ID NO: 76 is replaced an amino acid
sequence that does not undergo isomerization; (l) heavy chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 75-77,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 81-83, respectively; or (m) heavy chain
CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 87-89,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 90-92, respectively.
4. The antibody of claim 3, wherein the antibody comprises heavy
and light chain variable region sequences, which are at least 90%
identical to the amino acid sequences selected from the group
consisting of: (a) SEQ ID NOs: 318 and 94; (b) SEQ ID NOs: 17 and
18; (c) SEQ ID NOs: 28 and 29; (d) SEQ ID NOs: 28 and 30; (e) SEQ
ID NOs: 37 and 38; (f) SEQ ID NOs: 48 and 49; (g) SEQ ID NOs: 48
and 50; (h) SEQ ID NOs: 57 and 58; (i) SEQ ID NOs: 65 and 66; (j)
SEQ ID NOs: 73 and 74; (k) SEQ ID NOs: 84 and 85; (l) SEQ ID NOs:
84 and 86; and (m) SEQ ID NOs: 93 and 94.
5. The antibody of claim 3, wherein the antibody comprises heavy
chain and light chain sequences, which are at least 80% identical
to the amino acid sequences selected from the group consisting of:
(a) SEQ ID NOs: 124 and 116, respectively; (b) SEQ ID NOs: 95 and
96, respectively; (c) SEQ ID NOs: 97 and 98, respectively; (d) SEQ
ID NOs: 99 and 100, respectively; (e) SEQ ID NOs: 101 and 102,
respectively; (f) SEQ ID NOs: 103 and 104, respectively; (g) SEQ ID
NOs: 105 and 106, respectively; (h) SEQ ID NOs: 107 and 108,
respectively; (i) SEQ ID NOs: 109 and 110, respectively; (j) SEQ ID
NOs: 111 and 112, respectively; (k) SEQ ID NOs: 113 and 114,
respectively; (l) SEQ ID NOs: 115 and 116, respectively; (m) SEQ ID
NOs: 117 and 118, respectively; (n) SEQ ID NOs: 119 and 120,
respectively; (o) SEQ ID NOs: 121 and 122, respectively; (p) SEQ ID
NOs: 123 and 116, respectively; and (q) SEQ ID NOs: 125 and 116,
respectively.
6. The antibody of claim 3, wherein the antibody binds to all or a
portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human
OX40 (SEQ ID NO: 2) or to all or a portion of the sequence
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of human OX40
(SEQ ID NO: 2).
7. The antibody of claim 3, wherein the antibody comprises an Fc
having enhanced binding to an activating Fc.gamma.R.
8. The antibody of claim 3, wherein the antibody is selected from
the group consisting of an IgG1, an IgG2, an IgG3, an IgG4, and a
variant thereof.
9. The antibody of claim 3, wherein the antibody is a human or
humanized antibody.
10. An antibody which binds to OX40 comprising a heavy chain
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 282-296.
11. A nucleic acid encoding the heavy and/or light chain variable
region of the antibody of claim 4.
12. An expression vector comprising the nucleic acid of claim
11.
13. A cell transformed with the expression vector of claim 12.
14. A composition comprising the antibody of claim 3, and a
carrier.
15. A kit comprising the antibody of claim 3, and instructions for
use.
16. A method of stimulating an antigen-specific T cell response
comprising contacting the T cell with the antibody of claim 3, such
that an antigen-specific T cell response is stimulated.
17. A method of treating cancer comprising administering to a
subject in need thereof a therapeutically effective amount of the
antibody of claim 3, to treat the cancer.
18. The method of claim 17 comprising administering to the subject
an effective amount of an anti-OX40 antibody comprising CDR1, CDR2
and CDR3 domains of the heavy chain variable region having the
sequence set forth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3
domains of the light chain variable region having the sequence set
forth in SEQ ID NO: 94, wherein the method comprises at least one
administration cycle, wherein the cycle is a period of two weeks,
wherein for each of the at least one cycles, one dose of the
anti-OX40 antibody is administered at a dose of 1 mg/kg body
weight; a fixed dose of 20, 40, 80, 160, or 320 mg; a dose of about
1 mg/kg body weight; or a fixed dose of about 20, 40, 80, 160, or
320 mg.
19. The method of claim 17, wherein the cancer is selected from the
group consisting of: cervical cancer, bladder cancer, colorectal
cancer, ovarian cancer, non-small cell lung cancer, and squamous
cell carcinoma of the head and neck.
20. The method of claim 17, further comprising administering one or
more additional therapeutic or treatment.
21. The method of claim 20, wherein the additional therapeutic is
an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-LAG-3
antibody, an anti-CTLA-4 antibody, or an anti-TGF.beta.
antibody.
22. The method of claim 20, wherein the additional treatment is
administration of a vaccine.
23. A method of reducing or depleting the number of T regulatory
cells in a tumor of a subject in need thereof comprising
administering to the subject a therapeutically effective amount of
the antibody of claim 3, such that the number of T regulatory cells
is reduced or depleted.
24. A method of inhibiting the growth of tumor cells in a subject
in need thereof comprising administering to the subject a
therapeutically effective amount of the antibody of claim 3, such
that the growth of tumor cells is inhibited.
25. A method of detecting the presence of OX40 in a sample
comprising (i) contacting the sample with the antibody of claim 3,
under conditions that allow for formation of a complex between the
antibody and OX40, and (ii) detecting the formation of the complex.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Nos. 62/333,556, 62/327,140, 62/264,691, 62/239,574 and
62/168,377, filed May 9, 2016, Apr. 25, 2016, Dec. 8, 2015, Oct. 9,
2015 and May 29, 2015, respectively. The contents of the
aforementioned application are hereby incorporated by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 26, 2016, is named MXI_543PC_Sequence_Listing.txt and is
417,528 bytes in size.
BACKGROUND
[0003] OX40 (TNFRSF4), also known as ACT35, IMD16, TXGP1L, and
CD134, is a 50-kD type I transmembrane glycoprotein in the TNFSFR
family of costimulatory receptors expressed on activated CD4+ T
cells. In the context of cancer, OX40-expressing activated T cells
are found in tumor infiltrating lymphocytes. OX40 and its ligand,
OX40-L, play a crucial role in inducing and maintaining T-cell
responses. Recent studies have demonstrated the utility of
enhancing anti-tumor T cell function to fight cancer, with key
components of an effective response including the activation of
CD4+ T cells and promoting survival signals through memory and
effector T cells. Given the ongoing need for improved strategies
for treating diseases such as cancer through, e.g., enhancing
immune responses such as T cell responses, novel agents that
modulate T cell responses, such as those that target OX40, as well
as therapies (e.g., combination therapies) that use such agents,
would be therapeutically beneficial.
SUMMARY
[0004] Provided herein are antibodies, such as human monoclonal
antibodies, that specifically bind OX40 and have desirable
functional properties. These properties include high affinity
binding to human OX40 and cynomolgus OX40 and the ability to
stimulate antigen-specific T cell responses, e.g., in tumor-bearing
subjects. Also provided herein are methods of detecting OX40 in a
sample.
[0005] In one aspect, provided herein are antibodies, or
antigen-binding portions thereof, which specifically bind to OX40
and exhibit at least one of the following properties: [0006] (1)
binding to soluble human OX40, e.g., with a K.sub.D of 10 nM or
less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore; [0007]
(2) binding to membrane bound human OX40, e.g., with an EC.sub.50
of 1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;
[0008] (3) binding to cynomolgus OX40, e.g., binding to membrane
bound cynomolgus OX40, e.g., with an EC.sub.50 of 10 nM or less
(e.g., 0.01 nM to 10 nM), e.g., as measured by FACS; [0009] (4)
inducing or enhancing T cell activation, as evidenced by (i)
increased IL-2 and/or IFN-.gamma. production in OX40-expressing T
cells and/or (ii) enhanced T cell proliferation; [0010] (5)
inhibiting the binding of OX40 ligand to OX40, e.g., with an
EC.sub.50 of 1 nM or less as measured by FACS, e.g., in an assay
with hOX40-293 cells; [0011] (6) binding to an epitope on the
extracellular portion of mature human OX40 (SEQ ID NO: 2), e.g., an
epitope within the region DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179); [0012] (7)
competing for binding to human OX40 with 3F4, 14B6-1, 14B6-2, 23H3,
18E9, 8B11, 20B3, and 20C1; [0013] (8) competing for binding to
human OX40 with 6E1-1, 6E1-2, 14A2-1, and 14A2-2.
[0014] In certain embodiments, the anti-OX40 antibodies, or antigen
binding portions thereof, described herein stimulate an anti-tumor
immune response, for example, an antigen-specific T cell response.
In certain embodiments, the antibodies, or antigen binding portions
thereof, increase cytokine production (e.g., IL-2 and/or
IFN-.gamma.) in OX40-expressing T cells and/or increase T cell
proliferation. In certain embodiments, the antibodies bind to the
C1q component of human complement. In certain embodiments, the
antibodies induce NK cell-mediated lysis of activated CD4+ T cells.
In certain embodiments, the antibody promotes macrophage-mediated
phagocytosis of OX40 expressing cells. In certain embodiments, the
antibody inhibits regulatory T cell-mediated suppression of CD4+ T
cell proliferation.
[0015] In certain embodiments, the anti-OX40 antibodies, or antigen
binding portions thereof, bind to Fc receptors, such as one or more
activating Fc.gamma.Rs. In certain embodiments, the antibodies, or
antigen binding portions thereof, induce or enhance T cell
activation through multivalent crosslinking.
[0016] Provided herein are isolated monoclonal antibodies, or
antigen binding portions thereof, which specifically bind to OX40
and comprise the three variable heavy chain CDRs and the three
variable light chain CDRs that are in the variable heavy chain and
variable light chain pairs selected from: SEQ ID NOs: 318 and 94;
SEQ ID NOs: 17 and 18; 28 and 29; 28 and 30; 37 and 38; 48 and 49;
48 and 50; 57 and 58; 65 and 66; 73 and 74; 84 and 85; 84 and 86;
93 and 94.
[0017] Also provided herein are monoclonal antibodies, or antigen
binding portions thereof, which bind to OX40 and comprise:
[0018] (a) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 87, 317, and 89, respectively, and light chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 90-92,
respectively;
[0019] (b) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 11-13, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 14-16, respectively;
[0020] (c) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 19-21, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 22-24, respectively;
[0021] (d) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 19-21, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 25-27, respectively;
[0022] (e) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 31-33, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 34-36, respectively;
[0023] (f) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 39-41, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 42-44, respectively;
[0024] (g) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 39-41, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 45-47, respectively;
[0025] (h) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 51-53, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 54-56, respectively;
[0026] (i) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 59-61, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 62-64, respectively;
[0027] (j) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 67-69, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 70-72, respectively;
[0028] (k) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 75-77, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 78-80, respectively;
[0029] (l) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 75-77, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 81-83, respectively; or
[0030] (m) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 87-89, respectively, and light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 90-92, respectively.
[0031] Provided herein are monoclonal antibodies, or antigen
binding portions thereof, which bind to OX40 and comprise heavy and
light chain variable regions, wherein the heavy chain variable
region comprises an amino acid sequence which is at least 90%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence
selected from the group consisting of SEQ ID NOs: 318, 17, 28, 37,
48, 57, 65, 73, 84, and 93.
[0032] Provided herein are isolated monoclonal antibodies, or
antigen binding portions thereof, which bind to OX40 and comprise
heavy and light chain variable regions, wherein the light chain
variable region comprises an amino acid sequence which is at least
90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid
sequence selected from the group consisting of SEQ ID NOs: 94, 18,
29, 30, 38, 49, 50, 58, 66, 74, 85, 86, and 94.
[0033] Provided herein are isolated monoclonal antibodies, or
antigen binding portions thereof, which bind to OX40 and comprise
heavy and light chain variable region sequences at least 85%
identical, for example, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
identical, to the amino acid sequences selected from the group
consisting of: SEQ ID NOs: 318 and 94; 17 and 18; 28 and 29; 28 and
30; 37 and 38; 48 and 49; 48 and 50; 57 and 58; 65 and 66; 73 and
74; 84 and 85; 84 and 86; 93 and 94.
[0034] Provided herein are isolated monoclonal antibodies, or
antigen binding portions thereof, which bind to OX40 and comprise
heavy chain and light chain sequences at least 80%, 85%, 90%, 95%,
96%, 97%, 98% 99%, or 100% identical to the amino acid sequences
selected from the group consisting of: SEQ ID NOs: 124 and 116; 95
and 96; 97 and 98; 99 and 100; 101 and 102; 103 and 104; 105 and
106; 107 and 108; 109 and 110; 111 and 112; 113 and 114; 115 and
116; 117 and 118; 119 and 120; 121 and 122; 123 and 116; 124 and
116; and 125 and 116.
[0035] In certain embodiments, the isolated monoclonal antibodies,
or antigen binding portions thereof, (a) bind to the same epitope
on OX40 as 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3, or 20C1, or
binds to the same epitope on OX40 as 6E1-1, 6E1-2, 14A2-1 or
14A2-2, and (b) inhibit binding of 3F4, 14B6-1, 14B6-2, 23H3,
6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and/or 20C1 to OX40
on activated T cells by at least 50%, 60%, 70%, 80% or 90% as
measured by, e.g., FACS.
[0036] In certain embodiments, the anti-OX40 antibodies, or antigen
binding portions thereof, bind within the regions DVVSSKPCKPCTWCNLR
(SEQ ID NO: 178) or DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID
NO: 179) of mature extracellular portion of human OX40 (SEQ ID NO:
2). In some embodiments, the anti-OX40 antibodies, or antigen
binding portions thereof, described herein, bind to both human and
cynomolgus OX40. In some embodiments, the anti-OX40 antibodies, or
antigen binding portions thereof, described herein, do not bind to
mouse and/or rat OX40.
[0037] In certain embodiments, the anti-OX40 antibodies, or
antigen-binding portions thereof, are IgG1, IgG2, IgG3, or IgG4
antibodies, or variants thereof. In certain embodiments, methionine
residues in the CDR regions of the anti-OX40 antibodies, or
antigen-binding portions thereof, are substituted for amino acid
residues that do not undergo oxidation. In certain embodiments, the
anti-OX40 antibodies, or antigen-binding portions thereof, are
human or humanized antibodies. In certain embodiments, the
anti-OX40 antibodies comprise an Fc having enhanced binding to an
activating Fc.gamma.R.
[0038] Provided herein are isolated monoclonal antibodies, or
antigen binding portions thereof, which bind to OX40 comprising a
modified heavy chain constant region that comprises an IgG2 hinge
and at least one of CH1, CH2 and CH3 that is not of an IgG2
isotype.
[0039] In certain embodiments, the heavy chain comprises an amino
acid sequence selected from the group consisting of SEQ ID NOs: 95,
97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,
124 and 125, or a heavy chain that differs therefrom in at most 10
amino acids or is at least 95%, 96%, 97%, 98% or 99% identical to
an amino acid sequence of SEQ ID NOs: 95, 97, 99, 101, 103, 105,
107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and 125.
[0040] In certain embodiments, the light chain comprises an amino
acid sequence selected from the group consisting of SEQ ID NOs: 96,
98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122,
or a light chain that differs therefrom in at most 10 amino acids
or is at least 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112,
114, 116, 118, 120, and 122.
[0041] In certain embodiments, the anti-OX40 antibodies, or
antigen-binding portions thereof, are not immunogenic.
[0042] In certain embodiments, the anti-OX40 antibodies, or
antigen-binding portions thereof, lack an amino acid sequence that
undergoes isomerization. For instance, if the amino acid sequence
Asp-Gly is present in the heavy and/or light chain CDR sequences of
the antibody, the sequence is substituted with an amino acid
sequence that does not undergo isomerization. In one embodiment,
the antibody comprises the heavy chain variable region CDR2
sequence set forth in SEQ ID NO: 76, but wherein the Asp or Gly in
the Asp-Gly sequence (LISYDGSRKHYADSVKG; SEQ ID NO: 76) is replaced
with an amino acid sequence that does not undergo isomerization,
for example, an Asp-Ser or a Ser-Gly sequence. In another
embodiment, the antibody comprises the heavy chain variable region
CDR2 sequence set forth in SEQ ID NO: 88, but wherein the Asp or
Gly in the Asp-Gly sequence (AIDTDGGTFYADSVRG; SEQ ID NO: 88) is
replaced with an amino acid sequence that does not undergo
isomerization, for example, a Ser-Gly, an Asp-Ala, or a Ser-Thr
sequence.
[0043] Provided herein are antibodies which bind to OX40 comprising
an amino acid selected from the group consisting of SEQ ID NOs:
282-296. In one embodiment, the antibodies comprise a heavy chain
consisting of an amino acid sequence selected from the group
consisting of SEQ ID NOs: 282-296.
[0044] Provided herein are bispecific molecules comprising an
anti-OX40 antibody linked to a molecule having a second binding
specificity.
[0045] Provided herein are nucleic acids encoding the heavy and/or
light chain variable regions of the anti-OX40 antibodies, or
antigen binding portions thereof, expression vectors comprising the
nucleic acid molecules, and cells transformed with the expression
vectors.
[0046] Provided herein are immunoconjugates comprising the
anti-OX40 antibodies described herein, linked to an agent.
[0047] Provided herein are compositions comprising anti-OX40
antibodies, or antigen binding portions thereof, and a carrier.
[0048] Provided herein are kits comprising the anti-OX40
antibodies, or antigen binding portions thereof, and instructions
for use. In certain embodiments, the kits further comprise an
anti-CTLA4 antibody, anti-PD-1, or anti-PD-L1 antibody.
[0049] Provided herein is a method of preparing the anti-OX40
antibodies, comprising expressing an anti-OX40 antibody in a cell
and isolating the antibody from the cell.
[0050] Provided herein is a method of stimulating an
antigen-specific T cell response comprising contacting the T cell
with an anti-OX40 antibody, or antigen binding portion thereof,
such that an antigen-specific T cell response is stimulated.
[0051] Provided herein is a method of activating or co-stimulating
a T cell, e.g., an effector T cell, comprising contacting a cell,
e.g., an effector T cell, with an anti-OX40 antibody, or antigen
binding portion thereof, and CD3, wherein the effector T cell is
activated or co-stimulated.
[0052] Provided herein is a method of increasing IL-2 and/or
IFN-.gamma. production in and/or proliferation of a T cell
comprising contacting the T cell with an effective amount of an
anti-OX40 antibody, or antigen binding portion thereof.
[0053] Provided herein is a method of increasing IL-2 and/or
IFN-.gamma. production in T cells in a subject comprising
administering an effective amount of an anti-OX40 antibody, or
antigen binding portion thereof, bispecific molecule or conjugate
comprising the anti-OX40 antibody, or composition comprising the
anti-OX40 antibody, to increase IL-2 and/or IFN-.gamma. production
from the T cells.
[0054] Provided herein is a method of reducing or depleting the
number of T regulatory cells in a tumor of a subject in need
thereof comprising administering an effective amount of an
anti-OX40 antibody, or antigen binding portion thereof, bispecific
molecule or conjugate wherein the antibody, or antigen binding
portion thereof, has effector or enhanced effector function, to
reduce the number of T regulatory cells in the tumor.
[0055] Provided herein is a method of stimulating an immune
response in a subject comprising administering an effective amount
of an anti-OX40 antibody, or antigen binding portion thereof,
bispecific molecule or conjugate to the subject such that an immune
response in the subject is stimulated. In certain embodiments, the
subject has a tumor and an immune response against the tumor is
stimulated.
[0056] Provided herein is a method of inhibiting the growth of
tumor cells in a subject comprising administering to the subject an
anti-OX40 antibody, or antigen binding portion thereof, bispecific
molecule or conjugate such that growth of the tumor is inhibited in
the subject.
[0057] Provided herein is a method of treating cancer, e.g., by
immunotherapy, comprising administering to a subject in need
thereof a therapeutically effective amount an anti-OX40 antibody,
or antigen binding portion thereof, bispecific molecule or
conjugate comprising the anti-OX40 antibody, or composition
comprising the anti-OX40 antibody, to treat the cancer. In certain
embodiments, 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. In certain embodiments, the cancer is a metastatic cancer,
refractory cancer, or recurrent cancer.
[0058] In certain embodiments, the methods described herein further
comprise administering one or more additional therapeutics with an
anti-OX40 antibody, for example, an anti-PD1 antibody, a LAG-3
antibody, a CTLA-4 antibody, and/or a PD-L1 antibody.
[0059] Provided herein is a method of detecting the presence of
OX40 in a sample comprising contacting the sample with an anti-OX40
antibody, or an antigen binding portion thereof, under conditions
that allow for formation of a complex between the antibody, or
antigen binding portion thereof, and OX40, and detecting the
formation of a complex.
[0060] Provided herein are uses of the anti-OX40 antibodies
described herein for treating cancer, stimulating an immune
response in a subject, stimulating an antigen-specific T cell
response, activating or co-stimulating a T cell, increasing the
production of cytokines, such as IL-2 and/or IFN-.gamma., in and/or
proliferation of a T cell, reducing or depleting the number of T
regulatory cells in a tumor, and/or inhibiting the growth of tumor
cells. Also provided herein are uses of the anti-OX40 antibodies
described herein for preparing a medicament for stimulating an
immune response in a subject, stimulating an antigen-specific T
cell response, activating or co-stimulating a T cell, increasing
IL-2 and/or IFN-.gamma. production in and/or proliferation of a T
cell, reducing or depleting the number of T regulatory cells in a
tumor, and/or inhibiting the growth of tumor cells.
[0061] Provided herein is a method of treating a solid tumor in a
human subject, the method comprising administering to the subject
an effective amount of an anti-OX40 antibody comprising CDR1, CDR2
and CDR3 domains of the heavy chain variable region having the
sequence set forth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3
domains of the light chain variable region having the sequence set
forth in SEQ ID NO: 94, wherein the method comprises at least one
administration cycle, wherein the cycle is a period of two weeks,
wherein for each of the at least one cycles, one dose of the
anti-OX40 antibody is administered at a dose of 1 mg/kg body
weight; a fixed dose of 20, 40, 80, 160, or 320 mg; a dose of about
1 mg/kg body weight; or a fixed dose of about 20, 40, 80, 160, or
320 mg.
[0062] In one embodiment, the method comprises further
administering an anti-PD-1 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 301, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 302, wherein the method comprises at least one administration
cycle, wherein the cycle is a period of two, three, or four weeks,
wherein for each of the at least one cycles, one dose of the
anti-OX40 antibody is administered at a dose of 1 mg/kg body
weight; a fixed dose of 20, 40, 80, 160, or 320 mg; a dose of about
1 mg/kg body weight; or a fixed dose of about 20, 40, 80, 160, or
320 mg, and one dose of the anti-PD-1 antibody is administered at a
dose of 240, 360, or 480 mg or a dose of about 240, 360, or 480
mg.
[0063] In another embodiment, the method comprises further
administering an anti-CTLA-4 antibody comprising CDR1, CDR2 and
CDR3 domains of the heavy chain variable region having the sequence
set forth in SEQ ID NO: 309, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 310, wherein the method comprises at least one administration
cycle, wherein the cycle is a period of three weeks, wherein for
each of the at least one cycles, one dose of the anti-OX40 antibody
is administered at a dose of 1 mg/kg body weight; a fixed dose of
20, 40, 80, 160, or 320 mg; a dose of about 1 mg/kg body weight; or
a fixed dose of about 20, 40, 80, 160, or 320 mg, and one dose of
the anti-CTLA-4 antibody is administered at a dose of 1 mg/kg or a
dose of about 1 mg/kg,
[0064] wherein the anti-OX40 antibody is administered together with
the anti-CTLA-4 antibody for at least one cycle, followed by
anti-OX40 antibody monotherapy for at least one cycle. In some
embodiments, the treatment consists of 8 cycles. In one embodiment,
the anti-OX40 antibody is administered together with the
anti-CTLA-4 antibody for the first 4 cycles, followed by anti-OX40
antibody monotherapy for the last 4 cycles.
[0065] In some embodiments, the anti-OX40 antibody comprises heavy
and light chain sequences set forth in SEQ ID NOs: 124 and 116,
respectively.
[0066] In certain embodiments, the anti-OX40 antibody, or anti-OX40
antibody and anti-PD-1 or anti-CTLA-4 antibody, are formulated for
intravenous administration. In some embodiments, the anti-OX40 and
anti-PD-1 or anti-CTLA-4 antibody are formulated together. In other
embodiments, the anti-OX40 and anti-PD-1 or anti-CTLA-4 antibody
are formulated separately.
[0067] In some embodiments, the treatment consists of 8 cycles. In
one embodiment, the anti-OX40 antibody, or anti-OX40 antibody and
anti-PD-1 or anti-CTLA-4 antibody, are administered on Day 1 of
each cycle.
[0068] In certain embodiments, the anti-OX40 antibody is
administered prior to administration of the anti-PD-1 or
anti-CTLA-4 antibody. In one embodiment, the anti-OX40 antibody is
administered within about 30 minutes prior to administration of the
anti-PD-1 or anti-CTLA-4 antibody. In other embodiments, the
anti-OX40 antibody is administered after administration of the
anti-PD-1 or anti-CTLA-4 antibody. In yet other embodiments, the
anti-OX40 antibody is administered concurrently with the anti-PD-1
or anti-CTLA-4 antibody.
[0069] In certain embodiments, the treatment produces at least one
therapeutic effect chosen from a reduction in size of a tumor,
reduction in number of metastatic lesions over time, complete
response, partial response, and stable disease. In some
embodiments, the tumor is associated with a cancer selected from
the group consisting of: cervical cancer, bladder cancer,
colorectal cancer, and ovarian cancer.
[0070] Other features and advantages of the instant disclosure will
be apparent from the following detailed description and examples,
which should not be construed as limiting.
BRIEF DESCRIPTION OF THE FIGURES
[0071] FIG. 1A shows the nucleotide sequence (SEQ ID NO: 126) and
amino acid sequence (SEQ ID NO: 17) of the heavy chain variable
region of the 3F4 human monoclonal antibody. The CDR1 (SEQ ID NO:
11), CDR2 (SEQ ID NO: 12) and CDR3 (SEQ ID NO: 13) regions are
delineated and the V, D and J germline derivations are
indicated.
[0072] FIG. 1B shows the nucleotide sequence (SEQ ID NO: 127) and
amino acid sequence (SEQ ID NO: 18) of the kappa light chain
variable region of the 3F4 human monoclonal antibody. The CDR1 (SEQ
ID NO: 14), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 16) regions
are delineated and the V and J germline derivations are
indicated.
[0073] FIG. 2A shows the nucleotide sequence (SEQ ID NO: 128) and
amino acid sequence (SEQ ID NO: 28) of the heavy chain variable
region of the 14B6 (14B6-1 and 14B6-2) human monoclonal antibody.
The CDR1 (SEQ ID NO: 19), CDR2 (SEQ ID NO: 20) and CDR3 (SEQ ID NO:
21) regions are delineated and the V, D and J germline derivations
are indicated.
[0074] FIG. 2B shows the nucleotide sequence (SEQ ID NO: 129) and
amino acid sequence (SEQ ID NO: 29) of the kappa light chain
variable region of the 14B6-1 human monoclonal antibody. The CDR1
(SEQ ID NO: 22), CDR2 (SEQ ID NO: 23) and CDR3 (SEQ ID NO: 24)
regions are delineated and the V and J germline derivations are
indicated.
[0075] FIG. 2C shows the nucleotide sequence (SEQ ID NO: 130) and
amino acid sequence (SEQ ID NO: 30) of the kappa light chain
variable region of the 14B6-2 human monoclonal antibody. The CDR1
(SEQ ID NO: 26), CDR2 (SEQ ID NO: 27) and CDR3 (SEQ ID NO: 28)
regions are delineated and the V and J germline derivations are
indicated.
[0076] FIG. 3A shows the nucleotide sequence (SEQ ID NO: 131) and
amino acid sequence (SEQ ID NO: 37) of the heavy chain variable
region of the 23H3 human monoclonal antibody. The CDR1 (SEQ ID NO:
31), CDR2 (SEQ ID NO: 32) and CDR3 (SEQ ID NO: 33) regions are
delineated and the V, D and J germline derivations are
indicated.
[0077] FIG. 3B shows the nucleotide sequence (SEQ ID NO: 132) and
amino acid sequence (SEQ ID NO: 38) of the kappa light chain
variable region of the 23H3 human monoclonal antibody. The CDR1
(SEQ ID NO: 34), CDR2 (SEQ ID NO: 35) and CDR3 (SEQ ID NO: 36)
regions are delineated and the V and J germline derivations are
indicated.
[0078] FIG. 4A shows the nucleotide sequence (SEQ ID NO: 133) and
amino acid sequence (SEQ ID NO: 48) of the heavy chain variable
region of the 6E1 (6E1-1 and 6E1-2) human monoclonal antibody. The
CDR1 (SEQ ID NO: 39), CDR2 (SEQ ID NO: 40) and CDR3 (SEQ ID NO: 41)
regions are delineated and the V, D and J germline derivations are
indicated.
[0079] FIG. 4B shows the nucleotide sequence (SEQ ID NO: 134) and
amino acid sequence (SEQ ID NO: 49) of the kappa light chain
variable region of the 6E1-1 human monoclonal antibody. The CDR1
(SEQ ID NO: 42), CDR2 (SEQ ID NO: 43) and CDR3 (SEQ ID NO: 44)
regions are delineated and the V and J germline derivations are
indicated.
[0080] FIG. 4C shows the nucleotide sequence (SEQ ID NO: 135) and
amino acid sequence (SEQ ID NO: 50) of the kappa light chain
variable region of the 6E1-2 human monoclonal antibody. The CDR1
(SEQ ID NO: 45), CDR2 (SEQ ID NO: 46) and CDR3 (SEQ ID NO: 47)
regions are delineated and the V and J germline derivations are
indicated.
[0081] FIG. 5A shows the nucleotide sequence (SEQ ID NO: 136) and
amino acid sequence (SEQ ID NO: 57) of the heavy chain variable
region of the 18E9 human monoclonal antibody. The CDR1 (SEQ ID NO:
51), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 53) regions are
delineated and the V, D and J germline derivations are
indicated.
[0082] FIG. 5B shows the nucleotide sequence (SEQ ID NO: 137) and
amino acid sequence (SEQ ID NO: 58) of the kappa light chain
variable region of the 18E9 human monoclonal antibody. The CDR1
(SEQ ID NO: 54), CDR2 (SEQ ID NO: 55) and CDR3 (SEQ ID NO: 56)
regions are delineated and the V and J germline derivations are
indicated.
[0083] FIG. 6A shows the nucleotide sequence (SEQ ID NO: 138) and
amino acid sequence (SEQ ID NO: 65) of the heavy chain variable
region of the 8B11 human monoclonal antibody. The CDR1 (SEQ ID NO:
59), CDR2 (SEQ ID NO: 60) and CDR3 (SEQ ID NO: 61) regions are
delineated and the V, D and J germline derivations are
indicated.
[0084] FIG. 6B shows the nucleotide sequence (SEQ ID NO: 139) and
amino acid sequence (SEQ ID NO: 66) of the kappa light chain
variable region of the 8B11 human monoclonal antibody. The CDR1
(SEQ ID NO: 62), CDR2 (SEQ ID NO: 63) and CDR3 (SEQ ID NO: 64)
regions are delineated and the V and J germline derivations are
indicated.
[0085] FIG. 7A shows the nucleotide sequence (SEQ ID NO: 140) and
amino acid sequence (SEQ ID NO: 73) of the heavy chain variable
region of the 20B3 human monoclonal antibody. The CDR1 (SEQ ID NO:
67), CDR2 (SEQ ID NO: 68) and CDR3 (SEQ ID NO: 69) regions are
delineated and the V, D and J germline derivations are
indicated.
[0086] FIG. 7B shows the nucleotide sequence (SEQ ID NO: 141) and
amino acid sequence (SEQ ID NO: 74) of the kappa light chain
variable region of the 20B3 human monoclonal antibody. The CDR1
(SEQ ID NO: 70), CDR2 (SEQ ID NO: 71) and CDR3 (SEQ ID NO: 72)
regions are delineated and the V and J germline derivations are
indicated.
[0087] FIG. 8A shows the nucleotide sequence (SEQ ID NO: 142) and
amino acid sequence (SEQ ID NO: 84) of the heavy chain variable
region of the 14A2 (14A2-1 and 14A2-2) human monoclonal antibody.
The CDR1 (SEQ ID NO: 75), CDR2 (SEQ ID NO: 76) and CDR3 (SEQ ID NO:
77) regions are delineated and the V, D and J germline derivations
are indicated.
[0088] FIG. 8B shows the nucleotide sequence (SEQ ID NO: 143) and
amino acid sequence (SEQ ID NO: 85) of the kappa light chain
variable region of the 14A2-1 human monoclonal antibody. The CDR1
(SEQ ID NO: 78), CDR2 (SEQ ID NO: 79) and CDR3 (SEQ ID NO: 80)
regions are delineated and the V and J germline derivations are
indicated.
[0089] FIG. 8C shows the nucleotide sequence (SEQ ID NO: 144) and
amino acid sequence (SEQ ID NO: 86) of the kappa light chain
variable region of the 14A2-2 human monoclonal antibody. The CDR1
(SEQ ID NO: 81), CDR2 (SEQ ID NO: 82) and CDR3 (SEQ ID NO: 83)
regions are delineated and the V and J germline derivations are
indicated.
[0090] FIG. 9A shows the nucleotide sequence (SEQ ID NO: 145) and
amino acid sequence (SEQ ID NO: 93) of the heavy chain variable
region of the 20C1 human monoclonal antibody. The CDR1 (SEQ ID NO:
87), CDR2 (SEQ ID NO: 88) and CDR3 (SEQ ID NO: 89) regions are
delineated and the V, D and J germline derivations are
indicated.
[0091] FIG. 9B shows the nucleotide sequence (SEQ ID NO: 146) and
amino acid sequence (SEQ ID NO: 94) of the kappa light chain
variable region of the 20C1 human monoclonal antibody. The CDR1
(SEQ ID NO: 90), CDR2 (SEQ ID NO: 91) and CDR3 (SEQ ID NO: 92)
regions are delineated and the V and J germline derivations are
indicated.
[0092] FIG. 10A shows the nucleotide sequence (SEQ ID NO: 176) and
amino acid sequence (SEQ ID NO: 124) of the heavy chain of the
OX40.21 human monoclonal antibody. The nucleotide sequence (SEQ ID
NO: 168) and amino acid sequence (SEQ ID NO: 116) of the light
chain is shown in FIG. 10B.
[0093] FIGS. 11A-11D show binding curves and EC.sub.50s (in nM) of
various anti-OX40 antibodies for activated human T cells, with
hIgG1 and secondary antibodies serving as controls, as assessed by
FACS.
[0094] FIGS. 12A-12C show binding curves and EC.sub.50s (in nM) of
various anti-OX40 antibodies for activated cynomolgus T cells, with
hIgG1 and secondary antibodies serving as controls, as assessed by
FACS.
[0095] FIGS. 13A-13C show binding curves and K.sub.Ds of the
anti-OX40 antibody, OX40.21, for activated human T cells, HEK293
cells overexpressing human OX40, and CHO cells overexpressing
cynomolgus monkey OX40, as assessed by Scatchard analysis.
[0096] FIG. 14A shows immunohistological staining of various
acetone-fixed frozen human tissue sections with the anti-OX40
antibodies OX40.8, OX40.6, and OX40.16. Images show representative
staining at an antibody concentration of 1 .mu.g/ml for hyperplasic
tonsil and 5 .mu.g/ml for other tissues, with the exception of
OX40.16 at 10 .mu.g/ml in the endocardium and valves. While all
three antibodies positively stained a small subset of lymphocytes
in the tonsil, OX40.8 also stained myofilament-like structures in
the heart and OX40.6 stained the cardiac muscles,
endothelium/subendothelium matrix in small arteries in the tonsil,
and endocardium and valves in the heart. GC, germinal center; MZ,
mantle zone.
[0097] FIG. 14B shows immunostaining of various acetone-fixed
frozen human tissue sections with the anti-OX40 antibody OX40.21 (a
variant of OX40.16). Images show representative staining at an
antibody concentration of 5 .mu.g/ml. The antibody positively
stained a small subset of lymphocytes in the tonsil and thymus. The
positive cells in the tonsil were distributed in the germinal
center, mantle zone, and inter-follicular region, while the
positive cells in the thymus were primarily localized in the
medulla. No specific staining was observed in the heart, liver,
kidney, and lung. GC, germinal center; Me, medulla; MZ, mantle
zone.
[0098] FIG. 15A shows the distribution of OX40+ tumor infiltrating
lymphocytes in hepatocellular carcinoma (HCC), colorectal carcinoma
(CRC), head and neck squamous cell carcinoma (HNSCC), and melanoma
(Mel). A manual score of 12 to 20 cases for each tumor type was
performed by estimation of number of positive cells under the
20.times. objective of a microscope. Minimum, <1 cells per
20.times. objective field; Mild, 1.about.<10 cells per 20.times.
objective field; Moderate, 10.about.<50 cells per 20.times.
objective field; Marked, 50.about.<200 cells per 20.times.
objective; Intense, >200 cells per 20.times. objective
field.
[0099] FIGS. 15B and 15C shows immunohistological staining for CD3,
FoxP3, and OX40 on adjacent FFPE sections from colorectal carcinoma
(CRC) and head and neck squamous cell carcinoma (HNSCC) samples.
FIG. 15B is a low power view, showing that both OX40+ and FoxP3+
TILs are a small fraction of CD3+ TILs and primarily distributed in
tumor stroma. FIG. 15C is a higher power view showing potential
partial co-localization of OX40+ and FoxP3+ (Treg) TILs in CRC.
[0100] FIG. 16 shows the ability of various anti-OX40 antibodies to
inhibit the binding of OX40 ligand (OX40-L) to human
OX40-transfected 293 cells ("hOX40-293 cells"), with hIgG1 as the
control.
[0101] FIG. 17 summarizes the OX40-L blocking relationships between
various anti-OX40 antibodies.
[0102] FIG. 18 shows the ability of various anti-OX40 antibodies to
inhibit the binding of the anti-OX40 antibody clone L106 to
hOX40-293 cells, as assessed by FACS, with PE-labeled L106 only,
PE-labeled mIgG1, and unstained cells as controls.
[0103] FIGS. 19A-19C show the ability of various anti-OX40
antibodies to inhibit the binding of allophycocyanin
(APC)-conjugated OX40.1 antibody to hOX40-293 (FIGS. 19A and 19B)
or hOX40-HT1080 cells (FIG. 19C), as assessed by FACS. hIgG1 and/or
hIgG4 were used as controls.
[0104] FIGS. 19D-19G show the ability of various anti-OX40
antibodies to inhibit the binding of biotin-conjugated OX40.4 or
OX40.5 antibody to hOX40-293 cells. hIgG1 and streptavidin-APC were
used as controls.
[0105] FIG. 19H summarize the epitope bins in relation to OX40.1
based on results shown in FIGS. 19A-19C.
[0106] FIG. 19I summarizes the epitope bins in relation to OX40.5
or OX40.4 based on results shown in FIGS. 19D-19G.
[0107] FIG. 20A shows that OX40.21 binds only to non-reduced human
OX40, regardless of the presence of N-linked sugars.
[0108] FIGS. 20B and 20C show two N-glycopeptides that were
identified by peptide mapping after deglycosylation: 60% occupancy
for both AspN118 (FIG. 20B) and AspN12 (FIG. 20C).
[0109] FIG. 20D depicts the regions in OX40 bound by OX40.16,
OX40.21, and OX40.8.
[0110] FIG. 20E shows the identification of the peptides recognized
by OX40.16, OX40.21, and OX40.8 by LC-MS.
[0111] FIGS. 21A-21D show the effects of various anti-OX40
antibodies on human primary CD4+ T cell proliferation when
co-cultured with CHO-CD3-CD32A cells. CHO cells only, T cells only,
CHO cells co-cultured with T cells only, and hIgG1 were used as
controls.
[0112] FIGS. 22A-22D show the effects of various anti-OX40
antibodies on interferon gamma (IFN-.gamma.) secretion from human
primary CD4+ T cells co-cultured with CHO-CD3-CD32A cells. CHO
cells only, T cells only, CHO cells co-cultured with T cells only,
and hIgG1 were used as controls.
[0113] FIGS. 23A-23F show the effects of various anti-OX40
antibodies on the stimulation of IL-2 secretion from primary T
cells in cultures of staphylococcus enterotoxin B (SEB)-activated
human peripheral blood mononuclear cells (PBMCs), which were
isolated from different donors.
[0114] FIG. 24 shows the effects of various anti-OX40 antibodies on
NK92 cell induced lysis of activated CD4+ cells.
[0115] FIGS. 25A and 25B show the effects of various anti-OX40
antibodies on primary NK cell-mediated lysis of activated CD4+ T
cells isolated from two donors by NK:target cell ratios.
[0116] FIG. 26 shows the effects of various anti-OX40 antibodies on
the phagocytosis of hOX40-293 cells by primary human
macrophages.
[0117] FIG. 27 shows the level of binding of the human complement
C1q component to OX40.21. IgG1 and IgG1.1 (effectorless) were used
as controls.
[0118] FIGS. 28A-28C show that OX40 is expressed in tumor
infiltrating lymphocytes, with a pattern generally limited to CD4+
cells with minimal expression on CD8+ cells.
[0119] FIG. 28D shows that OX40 is expressed by CD4+ T cells and
regulatory T cells in mouse Sa1N tumors.
[0120] FIG. 28E shows that OX40 is expressed by CD4+ T cells, CD8+
T cells, and regulatory T cells in mouse MC38 tumors.
[0121] FIG. 29 shows the ability of various anti-OX40 antibodies to
reverse regulatory T (Treg) cell-mediated suppression of human CD4+
T cells. In both the presence and absence of Treg cells, anti-OX40
antibodies increased the proliferation of T responder (Tresp) cells
compared to the IgG1 isotype control.
[0122] FIGS. 30A and 30B show the clearance of intravenously
administered OX40.6 antibody from monkeys. Two of the monkeys
showed accelerated clearance, which correlated with the formation
of anti-drug antibodies.
[0123] FIG. 31 is a graph depicting T-cell proliferation results
for percentage antigenicity for various anti-OX40 antibodies, as
well as quality control samples QC-1, QC-2, and QC-3.
[0124] FIGS. 32A-32C show the effects of different isotypes of the
chimeric OX86 antibody (an antibody having the rat variable regions
of OX86 and mouse constant region that does not block the
interaction between OX40 and OX40-L, i.e., a non-blocking antibody)
on anti-tumor activity measured by changes in tumor volumes in
individual mice treated with these isotypes (mIgG1 and mIgG2
isotypes) in a MC38 colon adenocarcinoma model: (FIG. 32A) control
mouse IgG1 antibody ("control"), (FIG. 32B) OX86 mIgG1 antibody
("OX-40 mIgG1"), (FIG. 32C) OX86 mIgG2 antibody ("OX-40
mIgG2a).
[0125] FIGS. 33A-33C show the effects of different isotypes (mIgG1
and mIgG2a) of the OX86 antibody on the number of CD4+ regulatory T
cells in tumors and the periphery, and on cell numbers in the
spleen.
[0126] FIGS. 34A-34C show the effects of chimeric OX86 antibodies
with a human IgG1 on anti-tumor activity measured by changes in
tumor volumes in individual mice treated with the indicated
antibodies in the MC38 colon adenocarcinoma model: (FIG. 34A) human
IgG1 isotype control ("Isotype"), (FIG. 34B) OX86-hIgG1 chimeric
antibody ("OX86-hG1"), (FIG. 34C) OX86-hIgG1-S267E antibody
("OX86-hG1-S267E"). The S267E substitution in hIgG1 increases its
effector function by increasing binding to FcRs (CD32A and CD32B).
OX86-hIgG1 exhibited potent anti-tumor activity.
[0127] FIGS. 35A-35D show the effects of chimeric OX86 hIgG1
antibody on regulatory T cell depletion.
[0128] FIGS. 36A-36E show the effects of a blocking (i.e., blocks
the interaction between OX40 and OX40 ligand) hamster anti-mouse
OX40 antibody (8E5) at different dosages on anti-tumor activity by
changes in tumor volumes in individual mice treated with the
indicated antibodies in the subcutaneous mouse CT-26 tumor model.
(FIG. 36A) hamster Ig control, (FIG. 36B) 8E5 at 10 mg/kg, (FIG.
36C) 8E5 at 3 mg/kg, (FIG. 36D) 8E5 at 1 mg/kg, (FIG. 36E) 8E5 at
0.3 mg/kg.
[0129] FIGS. 37A-37D show the effects of combination therapy with
the OX86-rG1 antibody and an anti-PD1 antibody on anti-tumor
activity measured by changes in tumor volumes in individual mice
treated with the indicated antibodies and combination in the MC38
colon adenocarcinoma model: (FIG. 37A) isotype control, (FIG. 37B)
anti-PD1 antibody, (FIG. 37C) OX86-rG1 ("anti-OX40"), (FIG. 36D)
OX86-rG1+anti-PD1 antibody ("anti-OX40+anti-PD1").
[0130] FIGS. 38A and 38B show the ex vivo recall response to KLH,
CD69+ expressing CD4+CD8- T cells at Days 22 and 41, respectively.
Animals were immunized with KLH on Study Day 1. Data points
represent individual animal (males and females) results as a
percentage of CD4+CD8- T cells. Horizontal bars represent group
means.
[0131] FIG. 39 shows antibody binding to anti-his Fab captured
Fc.gamma.R-his proteins. Binding responses are plotted as a
percentage of the theoretical Rmax assuming a 1:1 mAb:Fc.gamma.R
binding stoichiometry. The bars for each antibody are shown in the
order provided by the color legends at the bottom of the slide.
[0132] FIG. 40 shows antibody binding to anti-his Fab captured
FcgR-his proteins. Binding responses are plotted as a percentage of
the theoretical Rmax assuming a 1:1 mAb:Fc.gamma.R binding
stoichiometry. The bars for each antibody are shown in the order
provided by the color legends at the bottom of the slide.
[0133] FIGS. 41A-41D show the effects of combination therapy with
an agonistic anti-OX40 antibody (OX86-rG1) and an anti-CTLA-4
antibody (9D9-mG2b) on anti-tumor activity measured by changes in
tumor volumes in individual mice treated with the indicated
antibodies and combination in the CT26 colon adenocarcinoma model:
(FIG. 41A) isotype control, (FIG. 41B) anti-CTLA-4 antibody, (FIG.
41C) OX86-rG1, (FIG. 41D) OX86-rG1+anti-CTLA-4 antibody.
[0134] FIG. 42 is a schematic of the Phase 1/2a clinical trial
protocol.
[0135] FIG. 43 is a schematic of the study visit schedule for the
Phase 1/2a clinical trial.
DETAILED DESCRIPTION
[0136] Described herein are isolated antibodies, particularly
monoclonal antibodies, e.g., human monoclonal antibodies, which
specifically bind to OX40 and thereby activate downstream OX40
signaling ("agonist anti-OX40 antibodies"). In certain embodiments,
the antibodies described herein are characterized by particular
functional features and/or comprise particular structural features,
such as CDR regions comprising specific amino acid sequences.
Provided herein are isolated antibodies, methods of making such
anti-OX40 antibodies, immunoconjugates, and bispecific molecules
comprising such antibodies, and pharmaceutical compositions
formulated to contain the antibodies. 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 therapies. Accordingly, the anti-OX40
antibodies described herein may be used in a treatment in a wide
variety of therapeutic applications, including, for example,
inhibiting tumor growth and treating viral infections.
Definitions
[0137] 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.
[0138] The term "OX40" as used herein refers to a receptor that is
a member of the TNF-receptor superfamily, which binds to OX40
ligand (OX40-L). OX40 is also referred to as tumor necrosis factor
receptor superfamily, member 4 (TNFRSF4), ACT35, IMD16, TXGP1L, and
CD134. The term "OX40" includes any variants or isoforms of OX40
which are naturally expressed by cells. Accordingly, antibodies
described herein may cross-react with OX40 from species other than
human (e.g., cynomolgus OX40). Alternatively, the antibodies may be
specific for human OX40 and may not exhibit any cross-reactivity
with other species. OX40 or any variants and isoforms thereof, may
either be isolated from cells or tissues which naturally express
them or be recombinantly produced using well-known techniques in
the art and/or those described herein.
[0139] The amino acid sequence of human OX40 precursor (Accession
No. NP_003318.1) is set forth in SEQ ID NO: 1. The amino acid
sequence of the extracellular domain of mature human OX40 is set
forth in SEQ ID NO: 2. The amino acid sequence of cynomolgus OX40
is set forth in SEQ ID NO: 3. The amino acid sequence of human
OX40-L is set forth in SEQ ID NO: 4.
[0140] The terms "Programmed Death 1," "Programmed Cell Death 1,"
"Protein PD-1," "PD-1," PD1," "PDCD1," "hPD-1" and "hPD-I," as used
herein, are used interchangeably, and include variants, isoforms,
species homologs of human PD-1, and analogs having at least one
common epitope with PD-1. The complete PD-1 sequence can be found
under GenBank Accession No. U64863.
[0141] The term "cytotoxic T lymphocyte-associated antigen-4,"
"CTLA-4," "CTLA4," "CTLA-4 antigen" and "CD152" (see, e.g., Murata
(1999) Am. J. Pathol. 155:453-460) are used interchangeably, and
include variants, isoforms, species homologs of human CTLA-4, and
analogs having at least one common epitope with CTLA-4 (see, e.g.,
Balzano (1992) Int. J. Cancer Suppl. 7:28-32). A complete sequence
of human CTLA-4 is set forth in GenBank Accession No. Ll 5006.
[0142] 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 portion thereof. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as V.sub.H) and a heavy chain constant region. 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 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 (C1q) of the
classical complement system.
[0143] 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.-6 M 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 OX40 may cross-react with OX40 from certain non-human primate
species (e.g., cynomolgus monkey), but may not cross-react with
OX40 from other species (e.g., murine OX40), or with an antigen
other than OX40.
[0144] 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. In certain embodiments, the anti-OX40 antibodies
described herein are of the IgG1 or IgG2 subtype. Immunoglobulins,
e.g., IgG1, exist in several allotypes, which differ from each
other in at most a few amino acids. "Antibody" may include, by way
of example, both naturally occurring and non-naturally occurring
antibodies; monoclonal and polyclonal antibodies; chimeric and
humanized antibodies; human and nonhuman antibodies; wholly
synthetic antibodies; and single chain antibodies.
[0145] The term "antigen-binding portion" 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 OX40).
It has been shown that the antigen-binding function of an antibody
can be performed by fragments of a full-length antibody. Examples
of binding fragments encompassed within the term "antigen-binding
portion" of an antibody, e.g., an anti-OX40 antibody described
herein, include (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L, V.sub.H, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the V.sub.H and CH1 domains; (iv) a Fv
fragment consisting of the V.sub.L and V.sub.H domains of a single
arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a V.sub.H domain; and (vi) an
isolated complementarity determining region (CDR) or (vii) a
combination of two or more isolated CDRs which may optionally be
joined by a synthetic linker. Furthermore, although the two domains
of the Fv fragment, V.sub.L and V.sub.H, are coded for by separate
genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain Fv (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" 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 can be produced by
recombinant DNA techniques, or by enzymatic or chemical cleavage of
intact immunoglobulins.
[0146] A "bispecific" or "bifunctional antibody" is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.
Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992).
[0147] 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 animals (e.g., a transgenic mouse having a genome
comprising a human heavy chain transgene and a light chain), to an
immortalized cell.
[0148] 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).
[0149] 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. The
antibodies described herein 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 and are
used synonymously.
[0150] 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 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.
[0151] 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.
[0152] 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.
[0153] "Allotype" refers to naturally occurring variants within a
specific isotype group, which variants differ in a few amino acids
(see, e.g., Jefferis et al. (2009) mAbs 1:1). Antibodies described
herein may be of any allotype. As used herein, antibodies referred
to as "IgG1f" or "IgG1.1f" isotype are IgG1 and effectorless IgG1.1
antibodies, respectively, of the allotype "f," i.e., having 214R,
356E and 358M according to the EU index as in Kabat, as shown,
e.g., in SEQ ID NO: 5 (see underlined residues in SEQ ID NO: 5 of
Table 23).
[0154] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen."
[0155] An "isolated antibody," as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to OX40 is substantially free of
antibodies that specifically bind antigens other than OX40). An
isolated antibody that specifically binds to an epitope of OX40
may, however, have cross-reactivity to other OX40 proteins from
different species.
[0156] As used herein, an antibody that "inhibits binding of OX40-L
to OX40" is intended to refer to an antibody that inhibits the
binding of OX40-L to OX40, e.g., in binding assays using hOX40-293
cells, with an EC50 of about 1 .mu.g/mL or less, such as about 0.9
.mu.g/mL or less, about 0.85 .mu.g/mL or less, about 0.8 .mu.g/mL
or less, about 0.75 .mu.g/mL or less, about 0.7 .mu.g/mL or less,
about 0.65 .mu.g/mL or less, about 0.6 .mu.g/mL or less, about 0.55
.mu.g/mL or less, about 0.5 .mu.g/mL or less, about 0.45 .mu.g/mL
or less, about 0.4 .mu.g/mL or less, about 0.35 .mu.g/mL or less,
about 0.3 .mu.g/mL or less, about 0.25 .mu.g/mL or less, about 0.2
.mu.g/mL or less, about 0.15 .mu.g/mL or less, or about 0.1
.mu.g/mL or less, in art-recognized methods, e.g., the FACS-based
binding assays described herein.
[0157] An "effector function" refers to the interaction of an
antibody Fc region with an Fc receptor or ligand, or a biochemical
event that results therefrom. Exemplary "effector functions"
include C1q 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
downregulation of a cell surface receptor (e.g., the B cell
receptor; BCR). Such effector functions generally require the Fc
region to be combined with a binding domain (e.g., an antibody
variable domain).
[0158] 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) receptor. Various properties of human Fc.gamma.Rs
are summarized in Table 1. The majority of innate effector cell
types coexpress 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 Isotype Fc.gamma. variants human IgG preference
Cellular distribution Fc.gamma.RI None High (K.sub.D IgG1 = 3 >
4 >> 2 Monocytes, macrophages, described ~10 nM) activated
neutrophils, dentritic cells? Fc.gamma.RIIA H131 Low to IgG1 > 3
> 2 > 4 Neutrophils, monocytes, medium macrophages,
eosinophils, R131 Low IgG1 > 3 > 4 > 2 dentritic cells,
platelets Fc.gamma.RIIIA V158 Medium IgG1 = 3 >> 4 > 2 NK
cells, monocytes, F158 Low IgG1 = 3 >> 4 > 2 macrophages,
mast cells, eosinophils, dentritic cells? Fc.gamma.RIIB I232 Low
IgG1 = 3 = 4 > 2 B cells, monocytes, T232 Low IgG1 = 3 = 4 >
2 macrophages, dentritic cells, mast cells
[0159] 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 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. 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).
[0160] A "hinge", "hinge domain" or "hinge region" or "antibody
hinge region" refers to the domain of a heavy chain constant region
that joins the CH1 domain to the CH2 domain and includes the upper,
middle, and lower portions of the hinge (Roux et al. J. Immunol.
1998 161:4083). The hinge provides varying levels of flexibility
between the binding and effector regions of an antibody and also
provides sites for intermolecular, disulfide bonding between the
two heavy chain constant regions. As used herein, a hinge starts at
Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., 1998 J
Immunol 161:4083). The sequences of wildtype IgG1, IgG2, IgG3 and
IgG4 hinges are shown in Tables 2 and 23.
TABLE-US-00002 TABLE 2 Hinge region amino acids C-terminal Ig Type
C.sub.H1 * Upper Hinge Middle Hinge Lower Hinge IgG1 VDKRV
EPKSCDKTHT CPPCP APELLGG (SEQ ID NO: (SEQ ID NO: 188) (SEQ ID NO:
192) (SEQ ID NO: 186) 200) IgG2 VDKTV ERK CCVECPPCP APPVAG (SEQ ID
NO: (SEQ ID NO: 193) (SEQ ID NO: 187) 201) IgG3 (17-15- VDKRV
ELKTPLGDTTHT CPRCP APELLGG 15-15) (SEQ ID NO: (SEQ ID NO: 189)
(EPKSCDTPPPCPRCP).sub.3 (SEQ ID NO: 186) (SEQ ID NO: 194) 200) IgG3
(17-15- VDKRV ELKTPLGDTTHT CPRCP APELLGG 15) (SEQ ID NO: (SEQ ID
NO: 189) (EPKSCDTPPPCPRCP).sub.2 (SEQ ID NO: 186) (SEQ ID NO: 195)
200) IgG3 (17-15) VDKRV ELKTPLGDTTHT CPRCP APELLGG (SEQ ID NO: (SEQ
ID NO: 189) (EPKSCDTPPPCPRCP).sub.1 (SEQ ID NO: 186) (SEQ ID NO:
196) 200) IgG3 (15-15- VDKRV EPKS CDTPPPCPRCP APELLGG 15) (SEQ ID
NO: (SEQ ID NO: 190) (EPKSCDTPPPCPRCP).sub.2 (SEQ ID NO: 186) (SEQ
ID NO: 197) 200) IgG3 (15) VDKRV EPKS CDTPPPCPRCP APELLGG (SEQ ID
NO: (SEQ ID NO: 190) (SEQ ID NO: 198) (SEQ ID NO: 186) 200) IgG4
VDKRV ESKYGPP CPSCP APEFLGG (SEQ ID NO: (SEQ ID NO: 191) (SEQ ID
NO: 199) (SEQ ID NO: 186) 200) *C-terminal amino acid sequences of
the CH1 domains.
[0161] The term "hinge" includes wild-type hinges (such as those
set forth in Table 23), as well as variants thereof (e.g.,
non-naturally-occurring hinges or modified hinges). For example,
the term "IgG2 hinge" includes wildtype IgG2 hinge, as shown in
Table 23, and variants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or
at most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions
or additions. Exemplary IgG2 hinge variants include IgG2 hinges in
which 1, 2, 3 or all 4 cysteines (C219, C220, C226 and C229) are
changed to another amino acid. In a specific embodiment, an IgG2
comprises a C219S substitution. In certain embodiments, a hinge is
a hybrid hinge that comprises sequences from at least two isotypes.
For example, a hinge may comprise the upper, middle or lower hinge
from one isotype and the remainder of the hinge from one or more
other isotypes. For example, a hinge can be an IgG2/IgG1 hinge, and
may comprise, e.g., the upper and middle hinges of IgG2 and the
lower hinge of IgG1. A hinge may have effector function or be
deprived of effector function. For example, the lower hinge of
wildtype IgG1 provides effector function.
[0162] The term "CH1 domain" refers to the heavy chain constant
region linking the variable domain to the hinge in a heavy chain
constant domain. As used herein, a CH1 domain starts at A118 and
ends at V215. The term "CH1 domain" includes wildtype CH1 domains
(such as having SEQ ID NO: 202 for IgG1 and SEQ ID NO: 203 for
IgG2; Table 23), as well as variants thereof (e.g., non-naturally
occurring CH1 domains or modified CH1 domains). For example, the
term "CH1 domain" includes wildtype CH1 domains and variants having
1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1
mutations, e.g., substitutions, deletions or additions. Exemplary
CH1 domains include CH1 domains with mutations that modify a
biological activity of an antibody, such as ADCC, CDC or half-life.
Modifications to the CH1 domain that affect a biological activity
of an antibody are provided herein.
[0163] The term "CH2 domain" refers to the heavy chain constant
region linking the hinge to the CH3 domain in a heavy chain
constant domain. As used herein, a CH2 domain starts at P238 and
ends at K340. The term "CH2 domain" includes wildtype CH2 domains
(such as having SEQ ID NO: 204 for IgG1 and SEQ ID NO: 205 for
IgG2; Table 23), as well as variants thereof (e.g., non-naturally
occurring CH2 domains or modified CH2 domains). For example, the
term "CH2 domain" includes wildtype CH2 domains and variants having
1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1
mutations, e.g., substitutions, deletions or additions. Exemplary
CH2 domains include CH2 domains with mutations that modify a
biological activity of an antibody, such as ADCC, CDC or half-life.
In certain embodiments, a CH2 domain comprises the substitutions
A330S/P331S that reduce effector function. Other modifications to
the CH2 domain that affect a biological activity of an antibody are
provided herein.
[0164] The term "CH3 domain" refers to the heavy chain constant
region that is C-terminal to the CH2 domain in a heavy chain
constant domain. As used herein, a CH3 domain starts at G341 and
ends at K447. The term "CH3 domain" includes wildtype CH3 domains
(such as having SEQ ID NO: 206 for IgG1 and SEQ ID NO: 207 for
IgG2; Table 23), as well as variants thereof (e.g., non-naturally
occurring CH3 domains or modified CH3 domains). For example, the
term "CH3 domain" includes wildtype CH3 domains and variants having
1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1
mutations, e.g., substitutions, deletions or additions. Exemplary
CH3 domains include CH3 domains with mutations that modify a
biological activity of an antibody, such as ADCC, CDC or half-life.
Modifications to the CH3 domain that affect a biological activity
of an antibody are provided herein.
[0165] 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 Fcs include
the various allotypes of Fcs (see, e.g., Jefferis et al. (2009)
mAbs 1:1).
[0166] The term "epitope" or "antigenic determinant" refers to a
site on an antigen (e.g., OX40) 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. Methods for determining what epitopes are
bound by a given antibody (i.e., epitope mapping) are well known in
the art and include, for example, immunoblotting and
immunoprecipitation assays, wherein overlapping or contiguous
peptides (e.g., from OX40) are tested for reactivity with a given
antibody (e.g., an anti-OX40 antibody). Methods of determining
spatial conformation of epitopes include techniques in the art and
those described herein, for example, x-ray crystallography,
2-dimensional nuclear magnetic resonance and HDX-MS (see, e.g.,
Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,
G. E. Morris, Ed. (1996)).
[0167] The term "epitope mapping" refers to the process of
identification of the molecular determinants on the antigen
involved in antibody-antigen recognition.
[0168] The term "binds to the same epitope" with reference to two
or more antibodies means that the antibodies bind to the same group
of amino acid residues, as determined by a given method. Techniques
for determining whether antibodies bind to the "same epitope on
OX40" with the antibodies described herein include art-recognized
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 (e.g., alanine scanning
mutagenesis--Cunningham & Wells (1985) Science 244:1081). 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 CDR1, 2 and 3
sequences are expected to bind to the same epitope.
[0169] Antibodies that "compete with another antibody for binding
to a target" refer to antibodies that (partially or completely)
inhibit 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).
[0170] Other art-recognized 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., Methods in Enzymology 9:242 (1983));
solid phase direct biotin-avidin EIA (see Kirkland et al., J.
Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid
phase direct labeled sandwich assay (see Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988));
solid phase direct label RIA using I-125 label (see Morel et al.,
Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA
(Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.
(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).
[0171] 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 2000
surface plasmon resonance (SPR) instrument using the predetermined
antigen, e.g., recombinant human OX40, as the analyte and the
antibody as the ligand, 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 OX40"
refers to an antibody that binds to soluble or cell bound human
OX40 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 OX40" refers to an
antibody that binds to cynomolgus OX40 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. In certain embodiments, antibodies
that do not cross-react with OX40 from a non-human species (e.g.,
murine OX40) exhibit essentially undetectable binding against these
proteins in standard binding assays.
[0172] The term "k.sub.assoc" or "k.sub.a", as used herein, is
intended to refer to the association rate constant of a particular
antibody-antigen interaction, whereas the term "k.sub.dis" or
"k.sub.d," as used herein, is intended to refer to the dissociation
rate constant of a particular antibody-antigen interaction. The
term "K.sub.D", as used herein, is intended to refer 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 by using
surface plasmon resonance, preferably using a biosensor system such
as a Biacore.RTM. SPR system or flow cytometry and Scatchard
analysis.
[0173] As used herein, the term "high affinity" for an IgG antibody
refers to an antibody having a K.sub.D of 10.sup.-8M or less, more
preferably 10.sup.-9 M or less and even more preferably 10.sup.-10
M or less for a target antigen. However, "high affinity" binding
can vary for other antibody isotypes. For example, "high affinity"
binding for an IgM isotype refers to an antibody having a K.sub.D
of 10.sup.-7 M or less, more preferably 10.sup.-8 M or less.
[0174] 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 portion
thereof that induces a response that is 50% of the maximal
response, i.e., halfway between the maximal response and the
baseline.
[0175] The term "binds to immobilized OX40," refers to the ability
of an antibody described herein to bind to OX40, for example,
expressed on the surface of a cell or attached to a solid
support.
[0176] The term "cross-reacts," as used herein, refers to the
ability of an antibody described herein to bind to OX40 from a
different species. For example, an antibody described herein that
binds human OX40 may also bind OX40 from another species (e.g.,
cynomolgus OX40). 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 OX40. 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] Also provided are "conservative sequence modifications" of
the sequences set forth herein, e.g., in Table 23, i.e., nucleotide
and amino acid sequence modifications which do not abrogate the
binding of the antibody encoded by the nucleotide sequence or
containing the amino acid sequence, to the antigen. Such
conservative sequence modifications include conservative nucleotide
and amino acid substitutions, as well as, nucleotide and amino acid
additions and deletions. For example, modifications can be
introduced into a sequence in Table 23 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-OX40 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)). Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of an anti-OX40 antibody
coding sequence, such as by saturation mutagenesis, and the
resulting modified anti-OX40 antibodies can be screened for
improved binding activity.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] The percent identity between two nucleotide sequences can be
determined using the GAP program in the GCG software package
(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and
a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5, or 6. The percent identity between two nucleotide or amino
acid sequences can also be determined using the algorithm of E.
Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. In addition, the percent identity between two amino acid
sequences can be determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6.
[0185] 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.
See www.ncbi.nlm.nih.gov.
[0186] 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).
[0187] Nucleic acids, e.g., cDNA, may be mutated, in accordance
with standard techniques to provide gene sequences. For coding
sequences, these mutations, may affect amino acid sequence as
desired. In particular, DNA sequences substantially homologous to
or derived from native V, D, J, constant, switches and other such
sequences described herein are contemplated.
[0188] 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.
[0189] 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 maybe 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.
[0190] As used herein, the term "antigen" refers to any natural or
synthetic immunogenic substance, such as a protein, peptide, or
hapten. An antigen may be OX40 or a fragment thereof. An antigen
may also be a tumor antigen, against which protective or
therapeutic immune responses are desired, e.g., antigens expressed
by a tumor cell (e.g., for use as a tumor vaccine to be
administered in combination with an anti-OX40 antibody). Antigens
include tumor-associated antigens for the prevention or treatment
of cancers. Examples of tumor-associated antigens include, but are
not limited to, sequences comprising all or part of the sequences
of .beta.hCG, gp100 or Pmel17, HER2/neu, WT1, mesothelin, CEA,
gp100, MART1, TRP-2, melan-A, NY-ESO-1, NY-BR-1, NY-CO-58, MN
(gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3, Tyrosinase, Telomerase,
SSX2 and MUC-1 antigens, and germ cell derived tumor antigens.
Tumor associated antigens also include the blood group antigens,
for example, Le.sup.a, Le.sup.b, LeX, LeY, H-2, B-1, B-2 antigens.
Alternatively, more than one antigen can be included in a
construct. For example, a MAGE antigen can be combined with other
antigens such as melanin A, tyrosinase, and gp100 along with
adjuvants such as GM-CSF or IL-12, and linked to an anti-APC
antibody.
[0191] Sequences of the foregoing antigens are well known in the
art. For example, an example of a MAGE-3 cDNA sequence is provided
in U.S. Pat. No. 6,235,525 (Ludwig Institute for Cancer Research);
examples of NY-ESO-1 nucleic acid and protein sequences are
provided in U.S. Pat. No. 5,804,381 and U.S. Pat. No. 6,069,233
(Ludwig Institute for Cancer Research); examples of Melan-A nucleic
acid and protein sequences are provided in U.S. Pat. No. 5,620,886
and U.S. Pat. No. 5,854,203 (Ludwig Institute for Cancer Research);
examples of NY-BR-1 nucleic acid and protein sequences are provided
in U.S. Pat. No. 6,774,226 and U.S. Pat. No. 6,911,529 (Ludwig
Institute for Cancer Research) and examples of NY-CO-58 nucleic
acid and protein sequences are provided in WO 02/90986 (Ludwig
Institute for Cancer Research); an example of an amino acid
sequence for the HER-2/neu protein is available at GENBANK.RTM.
Accession No. AAA58637; and a nucleotide sequence (mRNA) for human
carcinoembryonic antigen-like 1 (CEA-1) is available at
GENBANK.RTM. Accession No. NM020219.
[0192] 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 of a Treg cell.
[0193] 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.
[0194] Immunomodulatory targets include, for example, receptors on
the surface of a cell ("immunomodulatory receptors") and receptor
ligands ("immunomodulatory ligands").
[0195] "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.
[0196] "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.
[0197] "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.
[0198] "T effector" ("T.sub.eff") 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). Anti-OX40 antibodies described herein activate T.sub.eff
cells, e.g., CD4+ and CD8+ T.sub.eff cells.
[0199] An increased ability to stimulate an immune response or the
immune system, can result from an enhanced agonist activity of T
cell costimulatory receptors and/or an enhanced antagonist activity
of inhibitory receptors. An increased ability to stimulate an
immune response or the immune system may be reflected by a fold
increase of the EC.sub.50 or maximal level of activity in an assay
that measures an immune response, e.g., an assay that measures
changes in cytokine or chemokine release, cytolytic activity
(determined directly on target cells or indirectly via detecting
CD107a or granzymes) and proliferation. The ability to stimulate an
immune response or the immune system activity may be enhanced by at
least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 fold or more.
[0200] 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.
[0201] 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.
[0202] As used herein, the term "T cell-mediated response" refers
to a response mediated by T cells, including effector T cells
(e.g., CD8.sup.+ cells) and helper T cells (e.g., CD4.sup.+ cells).
T cell mediated responses include, for example, T cell cytotoxicity
and proliferation.
[0203] 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.sup.+ T
cells.
[0204] As used herein, the terms "inhibits" or "blocks" (e.g.,
referring to inhibition/blocking of binding of OX40-L to OX40 on
cells) are used interchangeably and encompass both partial and
complete inhibition/blocking. In certain embodiments, the anti-OX40
antibody inhibits binding of OX40-L to OX40 by at least about 50%,
for example, about 60%, 70%, 80%, 90%, 95%, 99%, or 100%,
determined, e.g., as further described herein. In certain
embodiments, the anti-OX40 antibody inhibits binding of OX40-L to
OX40 by no more than 50%, for example, by about 40%, 30%, 20%, 10%,
5% or 1%, determined, e.g., as further described herein.
[0205] As used herein, the term "inhibits growth" of a tumor
includes any measurable decrease in the growth of a tumor, e.g.,
the inhibition of growth of a tumor by at least about 10%, for
example, at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 99%, or
100%.
[0206] 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.
[0207] 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.
[0208] A "hematological malignancy" includes a lymphoma, leukemia,
myeloma or a lymphoid malignancy, as well as a cancer of the spleen
and the lymph nodes. Exemplary lymphomas include both B cell
lymphomas (a B-cell hematological cancer) and T cell lymphomas.
B-cell lymphomas include both Hodgkin's lymphomas and most
non-Hodgkin's lymphomas. Non-limiting examples of B cell lymphomas
include diffuse large B-cell lymphoma, follicular lymphoma,
mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic
lymphoma (overlaps with chronic lymphocytic leukemia), mantle cell
lymphoma (MCL), Burkitt's lymphoma, mediastinal large B cell
lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell
lymphoma, splenic marginal zone lymphoma, intravascular large
B-cell lymphoma, primary effusion lymphoma, lymphomatoid
granulomatosis. Non-limiting examples of T cell lymphomas include
extranodal T cell lymphoma, cutaneous T cell lymphomas, anaplastic
large cell lymphoma, and angioimmunoblastic T cell lymphoma.
Hematological malignancies also include leukemia, such as, but not
limited to, secondary leukemia, chronic lymphocytic leukemia, acute
myelogenous leukemia, chronic myelogenous leukemia, and acute
lymphoblastic leukemia. Hematological malignancies further include
myelomas, such as, but not limited to, multiple myeloma and
smoldering multiple myeloma. Other hematological and/or B cell- or
T-cell-associated cancers are encompassed by the term hematological
malignancy.
[0209] 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.
[0210] In reference to solid tumors, an effective amount comprises
an amount sufficient to cause a tumor to shrink and/or to decrease
the growth rate of the tumor (such as to suppress tumor growth) or
to prevent or delay other unwanted cell proliferation. In certain
embodiments, an effective amount is an amount sufficient to delay
tumor development. In some embodiments, an effective amount is an
amount sufficient to prevent or delay tumor recurrence. An
effective amount can be administered in one or more
administrations. The effective amount of the drug or composition
may: (i) reduce the number of cancer cells; (ii) reduce tumor size;
(iii) inhibit, retard, slow to some extent and may stop cancer cell
infiltration into peripheral organs; (iv) inhibit, i.e., slow to
some extent and may stop, tumor metastasis; (v) inhibit tumor
growth; (vi) prevent or delay occurrence and/or recurrence of
tumor; and/or (vii) relieve to some extent one or more of the
symptoms associated with the cancer. In one example, an "effective
amount" is the amount of anti-OX40 antibody and the amount of
anti-PD-1 antibody (e.g., nivolumab) or anti-CTLA-4 antibody (e.g.,
ipilimumab), in combination, clinically proven to affect a
significant decrease in cancer or slowing of progression of cancer,
such as an advanced solid tumor. As used herein, the terms "fixed
dose", "flat dose" and "flat-fixed dose" are used interchangeably
and refer to a dose that is administered to a patient without
regard for the weight or body surface area (BSA) of the patient.
The fixed or flat dose is therefore not provided as a mg/kg dose,
but rather as an absolute amount of the agent.
[0211] As used herein, a "body surface area (BSA)-based dose"
refers to a dose that is adjusted to the body-surface area (BSA) of
the individual patient. A BSA-based dose may be provided as mg/kg
body weight. Various calculations have been published to arrive at
the BSA without direct measurement, the most widely used of which
is the Du Bois formula (see Du Bois D, Du Bois E F (June 1916)
Archives of Internal Medicine 17 (6): 863-71; and Verbraecken, J.
et al. (April 2006). Metabolism--Clinical and Experimental 55 (4):
515-24). Other exemplary BSA formulas include the Mosteller formula
(Mosteller R D. N Engl J Med., 1987; 317:1098), the Haycock formula
(Haycock G B, et al., J Pediatr 1978, 93:62-66), the Gehan and
George formula (Gehan E A, George S L, Cancer Chemother Rep 1970,
54:225-235), the Boyd formula (Current, J D (1998), The Internet
Journal of Anesthesiology 2 (2); and Boyd, Edith (1935), University
of Minnesota. The Institute of Child Welfare, Monograph Series, No.
x. London: Oxford University Press), the Fujimoto formula (Fujimoto
S, et al., Nippon Eiseigaku Zasshi 1968; 5:443-50), the Takahira
formula (Fujimoto S, et al., Nippon Eiseigaku Zasshi 1968;
5:443-50), and the Schlich formula (Schlich E, et al., Ernahrungs
Umschau 2010; 57:178-183).
[0212] 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.
[0213] 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.
[0214] 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. 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.
[0215] The terms "patient" and "subject" refer to any human or
non-human animal that receives either prophylactic or therapeutic
treatment. For example, the methods and compositions described
herein can be used to treat a subject or patient having cancer,
such as an advanced solid tumor. The term "non-human animal"
includes all vertebrates, e.g., mammals and non-mammals, such as
non-human primates, sheep, dog, cow, chickens, amphibians,
reptiles, etc.
[0216] Various aspects described herein are described in further
detail in the following subsections.
I. Anti-OX40 Antibodies
[0217] Described herein are antibodies, e.g., fully human
antibodies, which are characterized by particular functional
features or properties. For example, the antibodies specifically
bind human OX40. Additionally, antibodies may cross react with OX40
from one or more non-human primates, such as cynomolgus OX40. Such
antibodies are useful in the treatment of cancer when used as
monotherapy, or when used in combination with an immuno-oncology
agent, such as an anti-PD-1 antibody (e.g., nivolumab) or
anti-CTLA-4 antibody (e.g., ipilimumab).
[0218] Anti-OX40 antibodies described herein exhibit one or more or
all of the following functional properties: [0219] (1) binding to
soluble human OX40, e.g., with a K.sub.D of 10 nM or less (e.g.,
0.01 nM to 10 nM), e.g., as measured by BIACORE.RTM. SPR analysis;
[0220] (2) binding to membrane bound human OX40, e.g., with an
EC.sub.50 of 1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as
measured by FACS; [0221] (3) binding to cynomolgus OX40, e.g.,
binding to membrane bound cynomolgus OX40, e.g., with an EC.sub.50
of 10 nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by
FACS; [0222] (4) inducing or enhancing T cell activation, as
evidenced by (i) increased IL-2 and/or IFN-.gamma. production in
OX40-expressing T cells and/or (ii) enhanced T cell proliferation;
[0223] (5) inhibiting the binding of OX40 ligand to OX40, e.g.,
with an EC.sub.50 of 1 nM or less as measured by FACS, e.g., in an
assay with hOX40-293 cells; [0224] (6) binding to an epitope on the
extracellular portion of mature human OX40 (SEQ ID NO: 2), e.g., an
epitope within the region DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179); [0225] (7)
competing for binding to human OX40 with 3F4, 14B6-1, 14B6-2, 23H3,
18E9, 8B11, 20B3, and 20C1; [0226] (8) competing for binding to
human OX40 with 6E1-1, 6E1-2, 14A2-1, and 14A2-2.
[0227] Preferably, the antibodies bind to OX40 with high affinity,
for example, with a K.sub.D of 10.sup.-7 M or less, 10.sup.-8M or
less, 10.sup.-9 M or less, 10.sup.-10 M or less, 10.sup.-11 M or
less, 10.sup.-12 M or less, 10.sup.-12 M to 10.sup.-7 M, 10.sup.-11
M to 10.sup.-7 M, 10.sup.-10 M to 10.sup.-7 M, or 10.sup.-9 M to
10.sup.-7 M. In certain embodiments, an anti-OX40 antibody binds to
soluble human OX40, e.g., as determined by BIACORE.RTM. SPR
analysis, with a K.sub.D of 10.sup.-7 M or less, 10.sup.-8 M or
less, 10.sup.-9 M (1 nM) or less, 10.sup.-10 M or less, 10.sup.-12
M to 10.sup.-7 M, 10.sup.-11 M to 10.sup.-7 M, 10.sup.-10 M to
10.sup.-7 M, 10.sup.-9 M to 10.sup.-7 M, or 10.sup.-8M to 10.sup.-7
M. In certain embodiments, the anti-OX40 antibody binds to bound
(e.g., cell membrane bound) human OX40, such as on activated human
T cells, e.g., as determined by flow cytometry, with a K.sub.D of
10.sup.-7 M or less, 10.sup.-8M or less, 10.sup.-9 M (1 nM) or
less, 10.sup.-10 M or less, 10.sup.-12 M to 10.sup.-7 M, 10.sup.-11
M to 10.sup.-8 M, 10.sup.-10 M to 10.sup.-8M, 10.sup.-9M to
10.sup.-8M, 10.sup.-11 M to 10.sup.-9 M, or 10.sup.-10 M to
10.sup.-9 M. In certain embodiments, an anti-OX40 antibody binds to
bound (e.g., cell membrane bound) human OX40, such as on activated
human T cells, e.g., as determined by FACS, with an EC.sub.50 of
10.sup.-7 M or less, 10.sup.-8 M or less, 10.sup.-9 M (1 nM) or
less, 10.sup.-10 M or less, 10.sup.-12 M to 10.sup.-7 M, 10.sup.-11
M to 10.sup.-8 M, 10.sup.-10 M to 10.sup.-8 M, 10.sup.-9 M to
10.sup.-8 M, 10.sup.-11 M to 10.sup.-9 M, or 10.sup.-10 M to
10.sup.-9 M. In certain embodiments, the anti-OX40 antibody binds
to soluble human OX40 with a K.sub.D of 10.sup.-7 M or less,
10.sup.-8 M or less, 10.sup.-9 M (1 nM) or less, 10.sup.-10 M or
less, 10.sup.-12 M to 10.sup.-7 M, 10.sup.-11 M to 10.sup.-7 M,
10.sup.-10 M to 10.sup.-7 M, 10.sup.-9 M to 10.sup.-7 M, or
10.sup.-8 M to 10.sup.-7 M, and to cell membrane bound human OX40
with a K.sub.D or EC.sub.50 of 10.sup.-7 M or less, 10.sup.-8 M or
less, 10.sup.-9 M (1 nM) or less, 10.sup.-10 M or less, 10.sup.-12
M to 10.sup.-7 M, 10.sup.-11 M to 10.sup.-8 M, 10.sup.-10 M to
10.sup.-8 M, 10.sup.-9 M to 10.sup.-8 M, 10.sup.-11 M to 10.sup.-9
M, or 10.sup.-10 M to 10.sup.-9 M.
[0228] Anti-OX40 antibodies described herein may bind to cynomolgus
OX40, e.g., bind to membrane bound cynomolgus OX40, e.g., with an
EC.sub.50 of 100 nM or less, 10 nM or less, 100 nM to 0.01 nM, 100
nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, e.g., as measured
by FACS (e.g., as described in the Examples).
[0229] Anti-OX40 antibodies described herein may stimulate or
enhance an immune response, e.g., by activating T.sub.eff cells,
limiting the suppression of Teffector cells by Treg cells,
depleting and/or inhibiting tumor Treg cells and/or activating NK
cells, e.g., in the tumor. For example, the anti-OX40 antibodies
may activate or costimulate T.sub.eff cells as evidenced, e.g., by
enhanced cytokine (e.g., IL-2 and IFN-.gamma.) secretion and/or
enhanced proliferation. In certain embodiments, CD3 stimulation is
also provided. In certain embodiments, the OX40 antibody increases
IL-2 secretion by a factor of 50%, 100% (i.e., 2 fold), 3 fold, 4
fold, 5 fold or more, optionally with a maximum of up to 10 fold,
30 fold, 100 fold, as measured, e.g., on primary human T cells or T
cells expressing human OX40 (e.g., as further described in the
Examples). In certain embodiments, the OX40 antibody increases
IFN-.gamma. secretion by a factor of 50%, 100% (i.e., 2 fold), 3
fold, 4 fold, 5 fold or more, optionally with a maximum of up to 10
fold, 30 fold, 100 fold, as measured, e.g., on primary human T
cells or T cells expressing human OX40 (e.g., as further described
in the Examples).
[0230] Anti-OX40 antibodies described herein may inhibit binding of
human OX40L to human OX40 on cells, e.g., 293 cells expressing
human OX40 (i.e., hOX40-293 cells), e.g., with an EC.sub.50 of 10
nM or less, 1 nM or less, 0.01 nM to 10 nM, 0.1 nM to 10 nM, or 0.1
nM to 1 nM (see Example 6).
[0231] Anti-OX40 antibodies described herein bind to an epitope on
OX40, as determined, for example, by binding to fragments of human
OX40. For example, in certain embodiments, the antibody binds to
all or a portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178)
of human OX40 (SEQ ID NO: 2) as determined, for example, by HDX or
by binding of the antibodies to fragments of human OX40, followed
by enzymatic cleavage (see Example 11). In other embodiments, the
antibody binds to all or a portion of the sequence
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of human OX40
(SEQ ID NO: 2).
[0232] In certain embodiments, the anti-OX40 antibodies described
herein bind to all or a portion of the sequence
SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182).
[0233] In other embodiments, the anti-OX40 antibodies described
herein bind to all or a portion of the sequence PCKPCTWCNLR (SEQ ID
NO: 183).
[0234] In yet other embodiments, the anti-OX40 antibodies that bind
to all or a portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO:
178) further bind to all or a portion of the sequence QLCTATQDTVCR
(SEQ ID NO: 184).
[0235] In additional embodiments, the anti-OX40 antibodies
described herein bind to all or a portion of the sequence
SQNTVCRPCGPGFYN (SEQ ID NO: 185).
[0236] Anti-OX40 antibodies described herein may compete for
binding to OX40 with (or inhibit binding of) anti-OX40 antibodies
comprising CDRs or variable regions described herein, e.g., 3F4,
14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1,
14A2-2, and/or 20C1. In certain embodiments, anti-OX40 antibodies
inhibit binding of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9,
8B11, 20B3, 14A2-1, 14A2-2, and/or 20C1 to human OX40 by at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or by 100%. In certain
embodiments, 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11,
20B3, 14A2-1, 14A2-2, and 20C1 inhibit binding of anti-OX40
antibodies to human OX40 by at least 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or by 100%.
[0237] In certain embodiments, the antibodies induce or enhance T
cell activation with multivalent crosslinking through, e.g., FcR
binding. In certain embodiments, the antibodies are multivalent,
e.g., bivalent. In certain embodiments, the antibodies are not
monovalent.
[0238] In certain embodiments, the antibodies have 1, 2, 3, 4, 5,
or 6 of the following features: [0239] (1) binding to soluble human
OX40, e.g., with a K.sub.D of 10 nM or less (e.g., 0.01 nM to 10
nM), e.g., as measured by BIACORE.RTM. SPR analysis; [0240] (2)
binding to membrane bound human OX40, e.g., with an EC.sub.50 of 1
nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;
[0241] (3) binding to cynomolgus OX40, e.g., binding to membrane
bound cynomolgus OX40, e.g., with an EC.sub.50 of 10 nM or less
(e.g., 0.01 nM to 10 nM), e.g., as measured by FACS; [0242] (4)
inducing or enhancing T cell activation, as evidenced by (i)
increased IL-2 and/or IFN-.gamma. production in OX40-expressing T
cells and/or (ii) enhanced T cell proliferation; [0243] (5)
inhibiting the binding of OX40 ligand to OX40, e.g., with an
EC.sub.50 of 1 nM or less as measured by FACS, e.g., in an assay
with hOX40-293 cells; [0244] (6) binding to an epitope on the
extracellular portion of mature human OX40 (SEQ ID NO: 2), e.g., an
epitope within the region DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179); [0245] (7)
competing for binding to human OX40 with 3F4, 14B6-1, 14B6-2, 23H3,
18E9, 8B11, 20B3, and 20C1; [0246] (8) competing for binding to
human OX40 with 6E1-1, 6E1-2, 14A2-1, and 14A2-2.
[0247] Accordingly, an antibody that exhibits one or more of these
functional properties (e.g., biochemical, immunochemical, cellular,
physiological or other biological activities, or the like) as
determined according to methodologies known to the art and
described herein, will be understood to relate to a statistically
significant difference in the particular activity relative to that
seen in the absence of the antibody (e.g., or when a control
antibody of irrelevant specificity is present). Preferably, the
anti-OX40 antibody increases a measured parameter (e.g., T cell
proliferation, cytokine production) by at least 10% of the measured
parameter, more preferably by at least 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 100% (i.e, 2 fold), 3 fold, 5 fold, or 10 fold.
Conversely, the antibody may decrease a measured parameter (e.g.,
tumor volume, OX40-L binding to OX40, quantity of regulatory T
cells in tumors) by at least 10% of the measured parameter, more
preferably by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%,
95%, or 99%.
[0248] Standard assays to evaluate the binding ability of the
antibodies toward OX40 of various species are known in the art,
including for example, ELISAs, Western blots, and RIAs. Suitable
assays are described in detail in the Examples. The binding
kinetics (e.g., binding affinity) of the antibodies also can be
assessed by standard assays known in the art, such as by
BIACORE.RTM. SPR analysis. Assays to evaluate the effects of the
antibodies on functional properties of OX40 (e.g., ligand binding,
T cell proliferation, cytokine production) are described in further
detail infra and in the Examples.
[0249] In certain embodiments, the anti-OX40 antibodies are not
native antibodies or are not naturally-occurring antibodies, e.g.,
anti-OX40 antibodies with post-translational modifications that are
different from those of antibodies that are naturally occurring,
such as by having more, less, or a different type of
post-translational modification.
II. Exemplary Anti-OX40 Antibodies
[0250] Particular antibodies described herein are antibodies, e.g.,
monoclonal antibodies, having the CDR and/or variable region
sequences of antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2,
18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1, isolated and
structurally characterized as described in Example 1, as well as
antibodies having at least 80% identity (e.g., at least 85%, at
least 90%, at least 95%, or at least 99% identity) to the variable
region or CDR sequences of antibodies 3F4, 14B6-1, 14B6-2, 23H3,
6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1. The
V.sub.H amino acid sequences of 3F4, 14B6 (14B6-1 and 14B6-2),
23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and
14A2-2), and 20C1 are set forth in SEQ ID NOs: 17, 28, 37, 48, 57,
65, 73, 84, and 93, respectively. The V.sub.L amino acid sequences
of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3,
14A2-1, 14A2-2, and 20C1 are set forth in SEQ ID NOs: 18, 29, 30,
38, 49, 50, 58, 66, 74, 85, 86, and 94, respectively.
[0251] Accordingly, provided herein are antibodies, or antigen
binding portion thereof, comprising heavy and light chain variable
regions, wherein the heavy chain variable region comprises an amino
acid sequence selected from the group consisting of SEQ ID NOs: 17,
28, 37, 48, 57, 65, 73, 84, and 93.
[0252] Also provided are antibodies, or antigen binding portions
thereof, comprising heavy and light chain variable regions, wherein
the light chain variable region comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 18, 29, 30, 38,
49, 50, 58, 66, 74, 85, 86, and 94.
[0253] Provided herein are antibodies, or antigen-binding portion
thereof, comprising: heavy and light chain variable region
sequences comprising SEQ ID NOs: 17 and 18; 28 and 29; 28 and 30;
37 and 38; 48 and 49; 48 and 50; 57 and 58; 65 and 66; 73 and 74;
84 and 85; 84 and 86; 93 and 94.
[0254] Anti-OX40 antibodies described herein may comprise the heavy
and light chain CDR1s, CDR2s and CDR3s of 3F4, 14B6-1, 14B6-2,
23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1, or
combinations thereof. The amino acid sequences of the V.sub.H CDR1s
of 3F4, 14B6 (14B6-1 and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2),
18E9, 8B11, 20B3, 14A2 (14A2-1 and 14A2-2), and 20C1 are set forth
in SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87,
respectively. The amino acid sequences of the V.sub.H CDR2s of 3F4,
14B6 (14B6-1 and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11,
20B3, 14A2 (14A2-1 and 14A2-2), and 20C1 are set forth in SEQ ID
NOs: 12, 20, 32, 40, 52, 60, 68, 76, and 88, respectively. The
amino acid sequences of the V.sub.H CDR3s of 3F4, 14B6 (14B6-1 and
14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2
(14A2-1 and 14A2-2), and 20C1 are set forth in SEQ ID NOs: 13, 21,
33, 41, 53, 61, 69, 77, and 89. The amino acid sequences of the
V.sub.L CDR1s of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9,
8B11, 20B3, 14A2-1, 14A2-2, and 20C1 are set forth in SEQ ID NOs:
14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81, and 90, respectively.
The amino acid sequences of the V.sub.L CDR2s of 3F4, 14B6-1,
14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and
20C1 are set forth in SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63,
71, 79, 82, and 91, respectively. The amino acid sequences of the
V.sub.L CDR3s of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9,
8B11, 20B3, 14A2-1, 14A2-2, and 20C1 are set forth in SEQ ID NOs:
16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92, respectively.
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).
[0255] Given that each of these antibodies bind to OX40 and that
antigen-binding specificity is provided primarily by the CDR1, 2
and 3 regions, the V.sub.H CDR1, 2 and 3 sequences and V.sub.LCDR1,
2 and 3 sequences can be "mixed and matched" (i.e., CDRs from
different antibodies can be mixed and match, although each antibody
must contain a V.sub.H CDR1, 2 and 3 and a V.sub.L CDR1, 2 and 3)
to create other anti-OX40 binding antibodies. OX40 binding of such
"mixed and matched" antibodies can be tested using the binding
assays described above and in the Examples (e.g., ELISAs).
Preferably, when V.sub.H CDR sequences are mixed and matched, the
CDR1, CDR2 and/or CDR3 sequence from a particular V.sub.H sequence
is replaced with a structurally similar CDR sequence(s). Likewise,
when V.sub.L CDR sequences are mixed and matched, the CDR1, CDR2
and/or CDR3 sequence from a particular V.sub.L sequence preferably
is replaced with a structurally similar CDR sequence(s). It will be
readily apparent to the ordinarily skilled artisan that novel
V.sub.H and V.sub.L sequences can be created by substituting one or
more V.sub.H and/or V.sub.L CDR region sequences with structurally
similar sequences from the CDR sequences disclosed herein for
monoclonal antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2,
18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1. "Mixed and matched"
antibodies having binding affinity, bioactivity and/or other
properties equivalent or superior to the specific antibodies
disclosed herein may be selected for use in the methods of the
present invention.
[0256] Provided herein are isolated antibodies, or antigen binding
portion thereof comprising:
[0257] (a) a heavy chain variable region CDR1 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 11,
19, 31, 39, 51, 59, 67, 75, and 87;
[0258] (b) a heavy chain variable region CDR2 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 12,
20, 32, 40, 52, 60, 68, 76, and 88;
[0259] (c) a heavy chain variable region CDR3 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 13,
21, 33, 41, 53, 61, 69, 77, and 89;
[0260] (d) a light chain variable region CDR1 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 14,
22, 25, 34, 42, 45, 54, 62, 70, 78, 81, and 90;
[0261] (e) a light chain variable region CDR2 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 15,
23, 26, 35, 43, 46, 55, 63, 71, 79, 82, and 91; and
[0262] (f) a light chain variable region CDR3 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 16,
24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92;
[0263] wherein the antibody specifically binds to human OX40.
[0264] In one embodiment, the antibody comprises heavy and light
chain variable regions, wherein the heavy chain variable region
CDR1, CDR2, and CDR3 regions comprise: SEQ ID NOs: 11-13; 19-21;
31-33; 39-41; 51-53; 59-61; 67-69; 74-77; 87-89; and 87, 317, and
89, respectively;
[0265] wherein the antibody specifically binds to human OX40.
[0266] In another embodiment, the antibody comprises heavy and
light chain variable regions, wherein the light chain variable
region CDR1, CDR2, and CDR3 regions comprise: SEQ ID NOs: 14-16;
22-24; 25-27; 34-36; 42-44; 45-47; 54-56; 62-64; 70-72; 78-80;
81-83; and 90-92, respectively;
[0267] wherein the antibody specifically binds to human OX40.
[0268] In a particular embodiment, the antibody comprises heavy and
light chain variable regions, wherein the antibody comprises:
[0269] (a) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 87, 317, and 89, respectively, and/or light chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 90-92,
respectively;
[0270] (b) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 11-13, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 14-16, respectively;
[0271] (c) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 22-24, respectively;
[0272] (d) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 25-27, respectively;
[0273] (e) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 31-33, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 34-36, respectively;
[0274] (f) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 39-41, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 42-44, respectively;
[0275] (g) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 39-41, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 45-47, respectively;
[0276] (h) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 51-53, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 54-56, respectively;
[0277] (i) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 59-61, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 62-64, respectively;
[0278] (j) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 67-69, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 70-72, respectively;
[0279] (k) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 75-77, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 78-80, respectively;
[0280] (l) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 75-77, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 81-83, respectively; or
[0281] (m) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 87-89, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 90-92, respectively;
[0282] wherein the antibody specifically binds to human OX40.
[0283] In another embodiment, the antibody comprises heavy and
light chain variable regions, wherein the antibody comprises:
[0284] (a) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 87, 317, and 89, respectively, and/or light chain CDR1,
CDR2, and CDR3 sequences consisting of SEQ ID NOs: 90-92,
respectively;
[0285] (b) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 11-13, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 14-16, respectively;
[0286] (c) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 22-24, respectively;
[0287] (d) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 25-27, respectively;
[0288] (e) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 31-33, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 34-36, respectively;
[0289] (f) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 39-41, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 42-44, respectively;
[0290] (g) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 39-41, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 45-47, respectively;
[0291] (h) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 51-53, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 54-56, respectively;
[0292] (i) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 59-61, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 62-64, respectively;
[0293] (j) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 67-69, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 70-72, respectively;
[0294] (k) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 75-77, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 78-80, respectively;
[0295] (l) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 75-77, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 81-83, respectively;
or
[0296] (m) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 87-89, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 90-92, respectively.
[0297] A VH domain, or one or more CDRs thereof, described herein
may be linked to a constant domain for forming a heavy chain, e.g.,
a full length heavy chain. Similarly, a VL domain, or one or more
CDRs thereof, described herein may be linked to a constant domain
for forming a light chain, e.g., a full length light chain. A full
length heavy chain (with the exception of the C-terminal lysine (K)
or with the exception of the C-terminal glycine and lysine (GK),
which may be absent) and full length light chain combine to form a
full length antibody. N-terminal glutamine and glutamate residues
may also be converted to pyroglutamate residues on both light and
heavy chains.
[0298] A VH domain described herein may be fused to the constant
domain of a human IgG, e.g., IgG1, IgG2, IgG3 or IgG4, which are
either naturally-occurring or modified, e.g., as further described
herein. For example, a heavy chain may comprise the amino acid
sequence of any VH domain described herein fused to the human IgG1
amino acid sequence set forth in SEQ ID NO: 5.
[0299] The human IgG1 constant domain may also be that of an
allotypic variant. For example, an allotypic variant of IgG1
comprises an R107K, E189D and M191L (underlined above, with
numbering according to that in SEQ ID NO: 6). Within the full
length heavy region, these amino acid substitutions are numbered
R214K, E356D and M358L.
[0300] A VL domain described herein may be fused to the constant
domain of a human kappa or lambda light chain. For example, a light
chain may comprise the amino acid sequence of any VL domain
described herein fused to the human IgG1 kappa light chain amino
acid sequence set forth in SEQ ID NO: 7.
[0301] In certain embodiments, the heavy chain constant region
comprises a lysine or another amino acid at the C-terminus, e.g.,
it comprises the following last amino acids: LSPGK (SEQ ID NO: 8)
for the heavy chain. In certain embodiments, the heavy chain
constant region is lacking one or more amino acids at the
C-terminus, and has, e.g., the C-terminal sequence LSPG (SEQ ID NO:
9) or LSP.
[0302] The amino acid sequences of exemplary heavy and light chains
are set forth in Table 23 and correspond to SEQ ID NOs: 95, 97, 99,
101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and
125 for the heavy chains and SEQ ID NOs: 96, 98, 100, 102, 104,
106, 108, 110, 112, 114, 116, 118, 120, and 122 for the light
chains.
[0303] Heavy and light chains comprising an amino acid sequence
that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or 70%
identical to any of the heavy or light chains set forth in Table 23
(or their variable regions), e.g., SEQ ID NOs: 95 and 96; 97 and
98; 99 and 100; 101 and 102; 103 and 104; 105 and 106; 107 and 108;
109 and 110; 111 and 112; 113 and 114; 115 and 116; 117 and 118;
119 and 120; 121 and 122; 123 and 116; 124 and 116; and 125 and 116
may be used for forming anti-human OX40 antibodies having the
desired characteristics, e.g., those further described herein.
Exemplary variants are those comprising an allotypic variation,
e.g., in the constant domain, and/or a mutation in the variable or
constant regions, such as the mutations disclosed herein. Heavy and
light chains comprising an amino acid sequence that differs in at
most 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino
acid (by substitution, addition or deletion) from any of the heavy
or light chains set forth in Table 23 (or their variable regions)
may be used for forming anti-human OX40 antibodies having the
desired characteristics, e.g., those further described herein.
[0304] In various embodiments, the antibodies described above
exhibit one or more, two or more, three or more, four or more, five
or more, six, or all of the following functional properties: [0305]
(1) binding to soluble human OX40, e.g., with a K.sub.D of 10 nM or
less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore; [0306]
(2) binding to membrane bound human OX40, e.g., with an EC.sub.50
of 1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;
[0307] (3) binding to cynomolgus OX40, e.g., binding to membrane
bound cynomolgus OX40, e.g., with an EC.sub.50 of 10 nM or less
(e.g., 0.01 nM to 10 nM), e.g., as measured by FACS; [0308] (4)
inducing or enhancing T cell activation, as evidenced by (i)
increased IL-2 and/or IFN-.gamma. production in OX40-expressing T
cells and/or (ii) enhanced T cell proliferation; [0309] (5)
inhibiting the binding of OX40 ligand to OX40, e.g., with an
EC.sub.50 of 1 nM or less as measured by FACS, e.g., in an assay
with hOX40-293 cells; [0310] (6) binding to an epitope on the
extracellular portion of mature human OX40 (SEQ ID NO: 2), e.g., an
epitope within the region DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179); [0311] (7)
competing for binding to human OX40 with 3F4, 14B6-1, 14B6-2, 23H3,
18E9, 8B11, 20B3, and 20C1; [0312] (8) competing for binding to
human OX40 with 6E1-1, 6E1-2, 14A2-1, and 14A2-2.
[0313] Such antibodies include, for example, human antibodies,
humanized antibodies, or chimeric antibodies.
[0314] In certain embodiments, the anti-OX40 antibodies described
herein bind to amino acid residues within the following region of
mature human OX40 (SEQ ID NO: 2):
TABLE-US-00003 (SEQ ID NO: 178) DVVSSKPCKPCTWCNLR,
corresponding to amino acid residues 46-62 of mature human OX40
(SEQ ID NO: 2).
[0315] In certain embodiments, the anti-OX40 antibodies described
herein bind to amino acid residues within the following region of
mature human OX40 (SEQ ID NO: 2):
TABLE-US-00004 (SEQ ID NO: 179)
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK,
corresponding to amino acid residues 89-124 of mature human OX40
(SEQ ID NO: 2).
[0316] In certain embodiments, the anti-OX40 antibodies described
herein that bind to all or a portion of the sequence
DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) bind to all or a portion of the
sequence SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182).
[0317] In other embodiments, the anti-OX40 antibodies described
herein that bind to all or a portion of the sequence
DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) bind to all or a portion of the
sequence PCKPCTWCNLR (SEQ ID NO: 183).
[0318] In yet other embodiments, the anti-OX40 antibodies that bind
to all or a portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO:
178) further bind to all or a portion of the sequence QLCTATQDTVCR
(SEQ ID NO: 184).
[0319] In additional embodiments, the anti-OX40 antibodies
described herein that bind to all or a portion of the sequence
DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) further bind to all or a portion
of the sequence SQNTVCRPCGPGFYN (SEQ ID NO: 185).
Modified Heavy Chain Constant Domains
[0320] The heavy chain constant region of anti-OX40 antibodies
described herein may be of any isotype, e.g., IgG1, IgG2, IgG3 and
IgG4, or combinations thereof and/or modifications thereof. In
certain embodiments, anti-OX40 antibodies comprise a modified heavy
chain constant region that alters the properties of the
antibody.
[0321] As discussed further herein and in the Examples,
cross-linking of anti-OX40 antibodies with unmodified hIgG1
constant regions (hIgG1 isotype antibodies) induce OX40 signaling
and promote T cell activation, and specifically to promote T cell
proliferation, IFN-.gamma. secretion, and IL-2 secretion.
Cross-linking can occur by, e.g., binding to human CD32A Fc.gamma.
receptors (Fc.gamma.Rs) expressed on the surface of transfected CHO
cells in an assay utilizing co-cultures of CHO-CD3-CD32A cells and
human primary CD4 T cells. Cross-linking can also occur, e.g., by
adding a soluble polyclonal anti-human Fc.gamma. antibody in
cultures of staphylococcus enterotoxin B (SEB)-activated human
peripheral blood mononuclear cells (PBMCs).
[0322] Anti-OX40 antibodies with modified heavy chain constant
regions (e.g., IgG1 constant region wherein the CH1/hinge region is
replaced with an hIgG2 CH1/hinge region) may have the ability to
alter the activities of the antibodies relative to antibodies with
a fully IgG1 heavy chain constant region. Exemplary activities that
may be altered include, but are not limited to, (1) T cell
activation in the presence or absence of cross-linking, (2) T cell
proliferation in the presence or absence of cross-linking, and/or
(3) cytokine secretion (e.g., IFN-.gamma., IL-2) in the presence or
absence of cross-linking. The methods described in the Examples can
be used to determine whether the anti-OX40 antibodies with modified
heavy chain constant regions exhibit these altered activities (see,
e.g., Example 27). In preferred embodiments, these altered
activities do not markedly affect the antigen-binding properties of
the antibodies, which can be assessed using, e.g., FACS, SPR).
[0323] Accordingly, provided herein are methods of altering the
activity of anti-OX40 antibodies comprising providing an anti-OX40
antibody that has a non-IgG2 hinge, and replacing the non-IgG2
hinge with an IgG2 hinge. In certain embodiments, a modified heavy
chain constant region comprises a hinge of the IgG2 isotype (an
"IgG2 hinge") and a CH1, CH2 and CH3 domain. In certain
embodiments, a modified heavy chain constant region comprises an
IgG2 hinge and a CH1, CH2 and CH3 domain, wherein at least one of
the CH1, CH2 and CH3 domains is not of the IgG2 isotype. The IgG2
hinge may be a wildtype IgG2 hinge, e.g., a wildtype human IgG2
hinge (e.g., ERKCCVECPPCPAPPVAG; SEQ ID NO: 208) or a variant
thereof, provided that the IgG2 hinge retains the ability to confer
to the antibody an altered activity relative to the same antibody
that comprises a non-IgG2 hinge. In certain embodiments, an IgG2
hinge variant retains similar rigidity or stiffness to that of a
wildtype IgG2 hinge. The rigidity of a hinge can be determined,
e.g., by computer modeling, electron microscopy, spectroscopy such
as Nuclear Magnetic Resonance (NMR), X-ray crystallography
(B-factors), or Sedimentation Velocity Analytical
ultracentrifugation (AUC) to measure or compare the radius of
gyration of antibodies comprising the hinge. A hinge may have
similar or higher rigidity relative to another hinge if an antibody
comprising the hinge has a value obtained from one of the tests
described in the previous sentence that differs from the value of
the same antibody with a different hinge, e.g., an IgG1 hinge, in
less than 5%, 10%, 25%, 50%, 75%, or 100%. A person of skill in the
art would be able to determine from the tests whether a hinge has
at least similar rigidity to that of another hinge by interpreting
the results of these tests. An exemplary human IgG2 hinge variant
is an IgG2 hinge that comprises a substitution of one or more of
the four cysteine residues (i.e., C219, C220, C226 and C229). A
cysteine may be replaced by a serine. An exemplary IgG2 hinge is a
human IgG2 hinge comprising a C219S mutation (e.g.,
ERKSCVECPPCPAPPVAG; SEQ ID NO: 209). Other IgG2 hinge variants that
may be used include human IgG2 hinges comprising a C220, C226
and/or C229 substitution, e.g., a C220S, C226S or C229S mutation
(which may be combined with a C219S mutation). An IgG2 hinge may
also be an IgG2 hinge in which a portion of the hinge is that of
another isotype (i.e., it is a chimeric hinge), provided that the
rigidity of the chimeric hinge is at least similar to that of a
wildtype IgG2 hinge. For example, an IgG2 hinge may be an IgG2
hinge in which the lower hinge (as defined in Table 2) is of an
IgG1 isotype, and is, e.g., a wildtype IgG1 lower hinge. Additional
IgG2 hinge mutations that may be used in an IgG2 hinge include the
SE (S267E), SELF (S267E/L328F), SDIE (S239D/I332E), SEFF and
GASDALIE (G236A/S239D/A330L/I332E) mutations.
[0324] A "hybrid" or "chimeric" hinge is referred to as being of a
specific isotype if more than half of the consecutive amino acids
of the hinge are from that isotype. For example, a hinge having an
upper and middle hinge of IgG2 and the lower hinge of IgG1 is
considered to be an IgG2 hinge.
[0325] In certain embodiments, an anti-OX40 antibody comprises a
modified heavy chain constant region that comprises an IgG2 hinge
comprising one of the following sequences:
TABLE-US-00005 (SEQ ID NO: 208) ERKCCVECPPCPAPPVAG; (SEQ ID NO:
209) ERKSCVECPPCPAPPVAG; (SEQ ID NO: 210) ERKCSVECPPCPAPPVAG; (SEQ
ID NO: 211) ERKXCVECPPCPAPPVAG; (SEQ ID NO: 212)
ERKCXVECPPCPAPPVAG; (SEQ ID NO: 213) ERKCCVECPPCPAPPVAGX; (SEQ ID
NO: 214) ERKSCVECPPCPAPPVAGX; (SEQ ID NO: 215) ERKCSVECPPCPAPPVAGX;
(SEQ ID NO: 216) ERKXCVECPPCPAPPVAGX; (SEQ ID NO: 217)
ERKCXVECPPCPAPPVAGX; (SEQ ID NO: 218) ERKCCVECPPCPAPELLGG; (SEQ ID
NO: 219) ERKSCVECPPCPAPELLGG; (SEQ ID NO: 220)
ERKCCSVECPPCPAPELLGG; (SEQ ID NO: 221) ERKXCVECPPCPAPELLGG; (SEQ ID
NO: 222) ERKCXVECPPCPAPELLGG; (SEQ ID NO: 223) ERKCCVECPPCPAPELLG;
(SEQ ID NO: 224) ERKSCVECPPCPAPELLG; (SEQ ID NO: 225)
ERKCCSVECPPCPAPELLG; (SEQ ID NO: 226) ERKXCVECPPCPAPELLG; (SEQ ID
NO: 227) ERKCXVECPPCPAPELLG; (SEQ ID NO: 228) ERKCCVECPPCPAP; (SEQ
ID NO: 229) ERKSCVECPPCPAP; (SEQ ID NO: 230) ERKCSVECPPCPAP; (SEQ
ID NO: 231) ERKXCVECPPCPAP; or (SEQ ID NO: 232) ERKCXVECPPCPAP,
[0326] wherein X is any amino acid, except a cysteine,
or any of the above sequences, in which 1-5, 1-3, 1-2 or 1 amino
acid is inserted between amino acid residues CVE and CPP. In
certain embodiments, THT or GGG is inserted. In certain
embodiments, 1, 1-2, or 1-3 amino acids are inserted between the
hinge and CH2 domain. For example, a glycine may be inserted
between the hinge and CH2 domain.
[0327] In certain embodiments, the hinge comprises SEQ ID NO: 208,
209, 210, 211, or 212, wherein 1, 2, 3 or all 4 amino acids P233,
V234, A235 and G237 (corresponding to the C-terminal 4 amino acids
"PVAG" (SEQ ID NO: 233) are deleted or substituted with another
amino acid, e.g., the amino acids of the C-terminus of the IgG1
hinge (ELLG (SEQ ID NO: 234) or ELLGG (SEQ ID NO: 235). In certain
embodiments, the hinge comprises SEQ ID NO: 208, 209, 210, 211, or
212, wherein V234, A235 and G237 are deleted or substituted with
another amino acid. In certain embodiments, the hinge comprises SEQ
ID NO: 208, 209, 210, 211, or 212, wherein A235 and G237 are
deleted or substituted with another amino acid. In certain
embodiments, the hinge comprises SEQ ID NO: 208, 209, 210, 211, or
212, wherein G237 is deleted or substituted with another amino
acid. In certain embodiments, the hinge comprises SEQ ID NO: 447,
448, 449, 450, or 451, wherein V234 and A235 are deleted or
substituted with another amino acid. Substitution of PVAG (SEQ ID
NO: 233) in an IgG2 with the corresponding amino acids of an IgG1
hinge, i.e., (ELLG (SEQ ID NO: 234) or ELLGG (SEQ ID NO: 235)) to
obtain a hybrid hinge, e.g., shown above, that provides a hinge
having the advantages of an IgG2 hinge and the effector function of
IgG1 hinges.
[0328] In certain embodiments, a modified heavy chain constant
region comprises a hinge that consists of or consists essentially
of one of the sequences shown above, e.g., any one of SEQ ID NOs:
208-232, and in certain embodiments, does not comprise additional
hinge amino acid residues.
[0329] In certain embodiments, a modified heavy chain constant
region comprises a CH1 domain that is a wildtype CH1 domain of the
IgG1 or IgG2 isotype ("IgG1 CH1 domain" or "IgG2 CH1 domain,"
respectively). CH1 domains of the isotypes IgG3 and IgG4 ("IgG3 CH1
domain and "IgG2 CH1 domain," respectively) may also be used. A CH1
domain may also be a variant of a wildtype CH1 domain, e.g., a
variant of a wildtype IgG1, IgG2, IgG3 or IgG4 CH1 domain.
Exemplary variants of CH1 domains include A114C and T173C and/or
C131, e.g., C131S.
[0330] In certain embodiments, a modified heavy chain constant
region comprises a CH2 domain that is a wildtype CH2 domain of the
IgG1, IgG2, IgG3 or IgG4 isotype ("IgG1 CH2 domain," "IgG2 CH2
domain," "IgG3 CH2 domain," or "IgG4 CH2 domain," respectively). A
CH2 domain may also be a variant of a wildtype CH2 domain, e.g., a
variant of a wildtype IgG1, IgG2, IgG3 or IgG4 CH2 domain.
Exemplary variants of CH2 domains include variants that modulate a
biological activity of the Fc region of an antibody, such as ADCC
or CDC or modulate the half-life of the antibody or its stability.
In one embodiment, the CH2 domain is a human IgG1 CH2 domain with
an A330S and P331S mutation, wherein the CH2 domain has reduced
effector function relative to the same CH2 mutation without the
mutations. Other mutations are further set forth herein
elsewhere.
[0331] In certain embodiments, a modified heavy chain constant
region comprises a CH3 domain that is a wildtype CH3 domain of the
IgG1, IgG2, IgG3 or IgG4 isotype ("IgG1 CH3 domain," "IgG2 CH3
domain," "IgG3 CH3 domain," or "IgG4 CH3 domain," respectively). A
CH3 domain may also be a variant of a wildtype CH3 domain, e.g., a
variant of a wildtype IgG1, IgG2, IgG3 or IgG4 CH3 domain.
Exemplary variants of CH3 domains include variants that modulate a
biological activity of the Fc region of an antibody, such as ADCC
or CDC or modulate the half-life of the antibody or its
stability.
[0332] Generally, variants of the CH1, hinge, CH2 or CH3 domains
may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations,
and/or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation, or 1-10 or
1-5 mutations, or comprise an amino acid sequence that is at least
about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to
that of the corresponding wildtype domain (CH1, hinge, CH2, or CH3
domain, respectively), provided that the heavy chain constant
region comprising the specific variant retains the necessary
biological activity.
[0333] Table 3 sets forth exemplary human heavy chain constant
regions comprising a human CH1, hinge, CH2 and/or CH3 domains,
wherein each domain is either a wildtype domain or a variant
thereof that provides the desired biological activity to the heavy
chain constant region. An unfilled cell in Table 3 indicates that
the domain is present or not, and if present can be of any isotype,
e.g., IgG1, IgG2, IgG3 or IgG4. For example, an antibody comprising
the heavy chain constant region 1 in Table 3 is an antibody that
comprises a heavy chain constant region comprising at least an IgG2
hinge, and which may also comprise a CH1, CH2 and/or CH3 domain,
and if present, which CH1, CH2 and/or CH3 domain is of an IgG1,
IgG2, IgG3 or IgG4 isotype. As another example for understanding
Table 3, an antibody comprising a heavy chain constant region 8 is
an antibody comprising a heavy chain constant region comprising an
IgG1 CH1 domain, and IgG2 hinge, an IgG1 CH2 domain, and which may
or may not also comprise a CH3 domain, which if present, may be of
an IgG1, IgG2, IgG3 or IgG4 isotype.
TABLE-US-00006 TABLE 3 Exemplary configurations of human heavy
chain constant regions MHCCR* CH1 Hinge CH2 CH3 1 IgG2 2 IgG1 IgG2
3 IgG2 IgG2 4 IgG2 IgG1 5 IgG2 IgG2 6 IgG2 IgG1 7 IgG2 IgG2 8 IgG1
IgG2 IgG1 9 IgG1 IgG2 IgG2 10 IgG2 IgG2 IgG1 11 IgG2 IgG2 IgG2 12
IgG1 IgG2 IgG1 13 IgG1 IgG2 IgG2 14 IgG2 IgG2 IgG1 15 IgG2 IgG2
IgG2 16 IgG2 IgG1 IgG1 17 IgG2 IgG1 IgG2 18 IgG2 IgG2 IgG1 19 IgG2
IgG2 IgG2 20 IgG1 IgG2 IgG1 IgG1 21 IgG1 IgG2 IgG1 IgG2 22 IgG1
IgG2 IgG2 IgG1 23 IgG1 IgG2 IgG2 IgG2 24 IgG2 IgG2 IgG1 IgG1 25
IgG2 IgG2 IgG1 IgG2 26 IgG2 IgG2 IgG2 IgG1 27 IgG2 IgG2 IgG2 IgG2
*Modified heavy chain constant region
[0334] In certain embodiments, an anti-OX40 antibody comprises a
heavy chain constant region shown in Table 3 and may have altered
activity relative to the same antibody comprising a heavy chain
constant region that does not comprise that specific heavy chain
constant region. In certain embodiments, an antibody comprising a
heavy chain constant region shown in Table 3 or 4 may have an
altered activity relative to the same antibody comprising a heavy
chain constant region that does not comprise an IgG2 hinge or the
same IgG2 hinge. In certain embodiments, an antibody comprising a
heavy chain constant region shown in Table 3 or 4 may have an
altered activity relative to the same antibody comprising a heavy
chain constant region that comprises a non-IgG2 hinge, and
comprises, e.g., an IgG1, IgG3 or IgG4 hinge. In certain
embodiments, an antibody comprising a heavy chain constant region
shown in Table 3 or 4 may have an altered activity relative to the
same antibody comprising a heavy chain constant region that does
not comprise one or more of the same CH1, hinge, CH2 or CH3 domain.
For example, in certain embodiments, an antibody comprising a heavy
chain constant region shown in Table 3 or 4 may have an altered
activity relative to the same antibody comprising a heavy chain
constant region that does not comprise an IgG2 hinge and a CH1, CH2
and/or CH3 domain of the specific isotype. For example, an antibody
comprising a heavy chain constant region 22 shown in Table 3, may
have an altered activity relative to (i) the same antibody
comprising a heavy chain constant region that does not comprise an
IgG2 hinge, and comprises, e.g., a non-IgG2 hinge (e.g., an IgG1,
IgG3 or IgG4 hinge); (ii) the same antibody comprising a heavy
chain constant region that does not comprise an IgG2 hinge and an
IgG1 CH1, and comprises, e.g., a non-IgG2 hinge and/or a non-IgG1
CH1; (iii) the same antibody comprising a heavy chain constant
region that does not comprise an IgG2 hinge and an IgG2 CH2, and
comprises, e.g., a non-IgG2 hinge and/or a non-IgG2 CH2; (iv) the
same antibody comprising a heavy chain constant region that does
not comprise an IgG2 hinge and an IgG1 CH3, and comprises, e.g., a
non-IgG2 hinge and/or a non-IgG1 CH3; (v) the same antibody
comprising a heavy chain constant region that does not comprise an
IgG2 hinge, an IgG1 CH1 and an IgG2 CH2, and comprises, e.g., a
non-IgG2 hinge and/or a non-IgG1 CH1 and/or a non-IgG2 CH2; (vi)
the same antibody comprising a heavy chain constant region that
does not comprise an IgG2 hinge, an IgG1 CH1 and an IgG1 CH3, and
comprises, e.g., a non-IgG2 hinge and/or a non-IgG1 CH1 and/or a
non-IgG1 CH3; (vii) the same antibody comprising a heavy chain
constant region that does not comprise an IgG2 hinge, an IgG2 CH2
and an IgG1 CH3, and comprises, e.g., a non-IgG2 hinge and/or a
non-IgG2 CH and/or a non-IgG1 CH3; (viii) or the same antibody
comprising a heavy chain constant region that does not comprise an
IgG2 hinge, an IgG1 CH1, IgG2 CH2 and an IgG1 CH3, and comprises,
e.g., a non-IgG2 hinge and/or a non-IgG1 CH1 and/or a non-IgG2 CH2
and/or a non-IgG1 CH3.
[0335] Exemplary modified heavy chain constant regions that may be
linked to anti-OX40 variable regions, e.g., the variable regions
described herein, are provided in Table 4, which sets forth the
identity of each of the domains.
TABLE-US-00007 TABLE 4 Exemplary modified heavy chain constant
regions Modified SEQ ID heavy chain NO of constant whole region CH1
Hinge CH2 CH3 MHCCR IgG1-IgG2- IgG1 IgG2/IgG1 IgG1 IgG1 wildtype
SEQ ID IgG1f wildtype SEQ ID NO: 240 wildtype SEQ ID NO: 244 SEQ ID
NO: SEQ ID NO: 204 202 NO: 204 IgG1-IgG2- IgG1 IgG2 wildtype IgG1
IgG1 wildtype SEQ ID IgG1f2 wildtype SEQ ID NO: 238 wildtype SEQ ID
NO: 245 SEQ ID SEQ ID NO: 206 NO: 202 NO: 204 IgG1- IgG1
IgG2C219S/IgG1 IgG1 IgG1 wildtype SEQ ID IgG2CS- wildtype SEQ ID
NO: 241 wildtype SEQ ID NO: 246 IgG1f SEQ ID SEQ ID NO: 206 NO: 202
NO: 204 IgG1- IgG1 IgG2 C219S IgG1 IgG1 wildtype SEQ ID IgG2CS-
wildtype SEQ ID NO: 239 wildtype SEQ ID NO: 247 IgG1f2 SEQ ID SEQ
ID NO: 206 NO: 202 NO: 204 IgG2-IgG1f IgG2 IgG2/IgG1 IgG1 IgG1
wildtype SEQ ID wildtype SEQ ID NO: 240 wildtype SEQ ID NO: 248 SEQ
ID SEQ ID NO: 206 NO: 203 NO: 204 IgG2-IgG1f2 IgG2 IgG2 wildtype
IgG1 IgG1 wildtype SEQ ID wildtype SEQ ID NO: 238 wildtype SEQ ID
NO: 249 SEQ ID SEQ ID NO: 206 NO: 203 NO: 204 IgG2CS- IgG2
IgG2C219S/IgG1 IgG1 IgG1 wildtype SEQ ID IgG1f wildtype SEQ ID NO:
241 wildtype SEQ ID NO: 250 SEQ ID SEQ ID NO: 206 NO: 203 NO: 204
IgG2CS- IgG2 IgG2 C219S IgG1 IgG1 wildtype SEQ ID IgG1f2 wildtype
SEQ ID NO: 239 wildtype SEQ ID NO: 251 SEQ ID SEQ ID NO: 206 NO:
203 NO: 204 IgG1-IgG2- IgG1 IgG2 wildtype IgG1 IgG1 wildtype SEQ ID
IgG1.1f wildtype SEQ ID NO: 238 A330S/P331S SEQ ID NO: 252 SEQ ID
SEQ ID NO: 206 NO: 202 NO: 243 IgG1- IgG1 IgG2 C219S IgG1 IgG1
wildtype SEQ ID IgG2CS- wildtype SEQ ID NO: 239 A330S/P331S SEQ ID
NO: 253 IgG1.1f SEQ ID SEQ ID NO: 206 NO: 202 NO: 243 IgG2-IgG1.1f
IgG2 IgG2 wildtype IgG1 IgG1 wildtype SEQ ID wildtype SEQ ID NO:
238 A330S/P331S SEQ ID NO: 254 SEQ ID SEQ ID NO: 206 NO: 203 NO:
243 IgG2CS- IgG2 IgG2 C219S IgG1 IgG1 wildtype SEQ ID IgG1.1f
wildtype SEQ ID NO: 239 A330S/P331S SEQ ID NO: 255 SEQ ID SEQ ID
NO: 206 NO: 203 NO: 243
[0336] Additional exemplary modified heavy chain constant regions
are provided in Table 5.
TABLE-US-00008 TABLE 5 SEQ ID NO of constant Constructs region
Description IgG1f 256 wild type IgG1f IgG1.1f 257 standard inert
IgG1.1f IgG2.3 258 IgG2 A-form (C219S) IgG2.5 259 IgG2 B-form
(C131S) IgG2.3G1-KH 260 CH1, upper hinge and lower hinge/ upper CH2
of IgG2.3, all else IgG1f IgG2.5G1-KH 261 CH1, upper hinge and
lower hinge/ upper CH2 of IgG2.5, all else IgG1f IgG2.3G1-AY 262
CH1 and upper hinge of IgG2.3, all else IgG1f IgG2.5G1-AY 263 CH1
and upper hinge of IgG2.5, all else IgG1f IgG2.3G1.1f-KH 264 CH1,
upper hinge and lower hinge/ upper CH2 of IgG2.3, all else IgG1.1f
IgG2.5G1.1f-KH 265 CH1, upper hinge and lower hinge/ upper CH2 of
IgG2.5, all else IgG1.1f IgG2.5G1-V27 266 IgG2-B-form variant
IgG2.3G1-V27 297 hHC-IgG2-C219S/hHC-IgG1f - S267E
[0337] In certain embodiments, an anti-OX40 antibody comprises a
modified heavy chain constant region comprising an IgG2 hinge
comprising any one of SEQ ID NOs: 238, 239, 240, 241, and 208-232
or a variant thereof, such as an IgG2 hinge comprising an amino
acid sequence that (i) differs from any one of SEQ ID NOs: 238,
239, 240, 241, and 208-232 in 1, 2, 3, 4 or 5 amino acids
substitutions, additions or deletions; (ii) differs from any one of
SEQ ID NOs: 238, 239, 240, 241, and 208-232 in at most 5, 4, 3, 2,
or 1 amino acids substitutions, additions or deletions; (iii)
differs from any one of SEQ ID NOs: 238, 239, 240, 241, and 208-232
in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions
or deletions and/or (iv) comprises an amino acid sequence that is
at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to any one of SEQ ID NOs: 238, 239, 240, 241, and
208-232, wherein in any of (i)-(iv), an amino acid substitution may
be a conservative amino acid substitution or a non-conservative
amino acid substitution; and wherein the modified heavy chain
constant region provides an altered activity to an anti-OX40
antibody relative to another heavy chain constant region, e.g., a
heavy chain constant region that comprises a non-IgG2 hinge or
relative to the same modified heavy chain constant region that
comprises a non-IgG2 hinge.
[0338] In certain embodiments, an anti-OX40 antibody comprises a
modified heavy chain constant region comprising an IgG1 CH1 domain
comprising SEQ ID NO: 202 or an IgG2 CH1 domain comprising SEQ ID
NO: 203, or a variant of SEQ ID NO: 202 or 203, which variant (i)
differs from SEQ ID NO: 202 or 203 in 1, 2, 3, 4 or 5 amino acids
substitutions, additions or deletions; (ii) differs from SEQ ID NO:
202 or 203 in at most 5, 4, 3, 2, or 1 amino acids substitutions,
additions or deletions; (iii) differs from SEQ ID NO: 202 or 203 in
1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or
deletions and/or (iv) comprises an amino acid sequence that is at
least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical
to SEQ ID NO: 202 or 203, wherein in any of (i)-(iv), an amino acid
substitution may be a conservative amino acid substitution or a
non-conservative amino acid substitution; and wherein the anti-OX40
antibody comprising a modified heavy chain constant region may have
an altered activity relative to that of the anti-OX40 antibody but
with another heavy chain constant region, e.g., a heavy chain
constant region that comprises a non-IgG2 hinge or relative to the
same modified heavy chain constant region that comprises a non-IgG2
hinge.
[0339] In certain embodiments, an anti-OX40 antibody comprises a
modified heavy chain constant region comprising an IgG1 CH2 domain
comprising SEQ ID NO: 204 or 298, or a variant of SEQ ID NO: 204 or
298, which variant (i) differs from SEQ ID NO: 204 or 298 in 1, 2,
3, 4 or 5 amino acids substitutions, additions or deletions; (ii)
differs from SEQ ID NO: 204 or 298 in at most 5, 4, 3, 2, or 1
amino acids substitutions, additions or deletions; (iii) differs
from SEQ ID NO: 204 or 298 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids
substitutions, additions or deletions and/or (iv) comprises an
amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% identical to SEQ ID NO: 204 or 298, wherein in
any of (i)-(iv), an amino acid substitution may be a conservative
amino acid substitution or a non-conservative amino acid
substitution; and wherein the modified heavy chain constant region
may provide an altered activity to an anti-OX40 antibody relative
to that of another heavy chain constant region, e.g., a heavy chain
constant region that comprises a non-IgG2 hinge or relative to the
same modified heavy chain constant region that comprises a non-IgG2
hinge.
[0340] In certain embodiments, an anti-OX40 antibody comprises a
modified heavy chain constant region comprising an IgG1 CH3 domain
comprising SEQ ID NO: 206, or a variant of SEQ ID NO: 206, which
variant (i) differs from SEQ ID NO: 206 in 1, 2, 3, 4 or 5 amino
acids substitutions, additions or deletions; (ii) differs from SEQ
ID NO: 206 in at most 5, 4, 3, 2, or 1 amino acids substitutions,
additions or deletions; (iii) differs from SEQ ID NO: 206 in 1-5,
1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or
deletions and/or (iv) comprises an amino acid sequence that is at
least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical
to SEQ ID NO: 206, wherein in any of (i)-(iv), an amino acid
substitution may be a conservative amino acid substitution or a
non-conservative amino acid substitution; and wherein the modified
heavy chain constant region may provide an altered activity
relative to that of another heavy chain constant region, e.g., a
heavy chain constant region that comprises a non-IgG2 hinge or
relative to the same modified heavy chain constant region that
comprises a non-IgG2 hinge.
[0341] Modified heavy chain constant regions may also comprise a
combination of the CH1, hinge, CH2 and CH3 domains described
above.
[0342] In certain embodiments, an anti-OX40 antibody comprises a
modified heavy chain constant region comprising any one of SEQ ID
NOs: 244-281, or a variant of any one of SEQ ID NOs: 244-281, which
variant (i) differs from any one of SEQ ID NOs: 244-281 in 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more amino acids substitutions, additions
or deletions; (ii) differs from any one of SEQ ID NOs: 244-281 in
at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids substitutions,
additions or deletions; (iii) differs from any one of SEQ ID NOs:
244-281 in 1-5, 1-3, 1-2, 2-5, 3-5, 1-10, or 5-10 amino acids
substitutions, additions or deletions and/or (iv) comprises an
amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 244-281,
wherein in any of (i)-(iv), an amino acid substitution may be a
conservative amino acid substitution or a non-conservative amino
acid substitution; and wherein the modified heavy chain constant
region may provide an altered activity relative to that of another
heavy chain constant region, e.g., a heavy chain constant region
that comprises a non-IgG2 hinge or relative to the same modified
heavy chain constant region that comprises a non-IgG2 hinge.
[0343] Modified heavy chain constant regions may have (i) similar,
reduced or increased effector function (e.g., binding to an
Fc.gamma.R) relative to a wildtype heavy chain constant region and
or (ii) similar, reduced or increased half-life (or binding to the
FcRn receptor) relative to a wildtype heavy chain constant
region.
III. Antibodies Having Particular Germline Sequences
[0344] In certain embodiments, anti-OX40 antibodies described
herein comprise a heavy chain variable region from a particular
germline heavy chain immunoglobulin gene and/or a light chain
variable region from a particular germline light chain
immunoglobulin gene.
[0345] As discussed in the Examples of the present disclosure,
human antibodies specific for OX40 have been prepared that comprise
a heavy chain variable region that is the product of or derived
from human germline VH 1-08 gene, VH 6-6 gene, VH 5-51 gene, VH 3-9
gene, VH DP44 gene, VH 3-30.3 gene, VH 3-10 gene, and/or VH 3-13
gene. Accordingly, provided herein are isolated monoclonal
antibodies specific for human OX40, or antigen-binding portions
thereof, comprising a heavy chain variable region that is the
product of or derived from a human VH germline gene selected from
the group consisting of: VH 1-08 gene, VH 6-6 gene, VH 5-51 gene,
VH 3-9 gene, VH DP44 gene, VH 3-30.3 gene, VH 3-10 gene, and VH
3-13 gene.
[0346] Human antibodies specific for OX40 have been prepared that
comprise a light chain variable region that is the product of or
derived from human germline VK L5 gene, VK L6 gene, VK L15 gene, VK
A27 gene, and/or VK O14/O4 gene. Accordingly, provide herein are
isolated monoclonal antibodies, or antigen-binding portions
thereof, comprising a light chain variable region that is the
product of or derived from a human VK germline gene selected from
the group consisting of: VK L5 gene, VK L6 gene, VK L15 gene, VK
A27 gene, and VK O14/O4 gene.
[0347] Preferred antibodies described herein are those comprising a
heavy chain variable region that is the product of or derived from
one of the above-listed human germline VH genes and also comprising
a light chain variable region that is the product of or derived
from one of the above-listed human germline VK genes.
[0348] As used herein, a human antibody comprises heavy or light
chain variable regions that is "the product of" or "derived from" a
particular germline sequence if the variable regions of the
antibody are obtained from a system that uses human germline
immunoglobulin genes. Such systems include immunizing a transgenic
mouse carrying human immunoglobulin genes with the antigen of
interest or screening a human immunoglobulin gene library displayed
on phage with the antigen of interest. A human antibody that is
"the product of" or "derived from" a human germline immunoglobulin
sequence can be identified as such by comparing the amino acid
sequence of the human antibody to the amino acid sequences of human
germline immunoglobulins and selecting the human germline
immunoglobulin sequence that is closest in sequence (i.e., greatest
% identity) to the sequence of the human antibody. A human antibody
that is "the product of" or "derived from" a particular human
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 human antibody
typically is at least 90% identical in amino acids sequence to an
amino acid sequence encoded by a human germline immunoglobulin gene
and contains amino acid residues that identify the human antibody
as being human when compared to the germline immunoglobulin amino
acid sequences of other species (e.g., murine germline sequences).
In certain cases, a human 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.
Typically, a human antibody derived from a particular human
germline sequence will display no more than 10 amino acid
differences from the amino acid sequence encoded by the human
germline immunoglobulin gene. In certain cases, the human antibody
may display 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.
IV. Homologous Antibodies
[0349] Provided herein are antibodies having heavy and light chain
variable regions comprising amino acid sequences that are
homologous to the amino acid sequences of the preferred antibodies
described herein, and wherein the antibodies retain the desired
functional properties of the anti-OX40 antibodies described
herein.
[0350] For example, an isolated anti-OX40 antibody, or antigen
binding portion thereof, may comprise a heavy chain variable region
and a light chain variable region, wherein:
[0351] (a) the heavy chain variable region comprises an amino acid
sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 318, 17, 28, 37, 48, 57, 65, 73, 84, and
93, or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15,
1-20, 1-25, or 1-50 amino acid changes (i.e., amino acid
substitutions, additions or deletions) relative to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 318, 17,
28, 37, 48, 57, 65, 73, 84, and 93;
[0352] (b) the light chain variable region comprises an amino acid
sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 18, 29, 30, 38, 49, 50, 58, 66, 74, 85,
86, and 94, or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10,
1-15, 1-20, 1-25, or 1-50 amino acid changes (i.e., amino acid
substitutions, additions or deletions) relative to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 18, 29,
30, 38, 49, 50, 58, 66, 74, 85, 86, and 94;
[0353] (c) the antibody specifically binds to OX40, and
[0354] (d) the antibody exhibits 1, 2, 3, 4, 5, 6, or all of the
following functional properties:
[0355] (1) binding to soluble human OX40, e.g., with a K.sub.D of
10 nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by
Biacore;
[0356] (2) binding to membrane bound human OX40, e.g., with an
EC.sub.50 of 1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as
measured by FACS;
[0357] (3) binding to cynomolgus OX40, e.g., binding to membrane
bound cynomolgus OX40, e.g., with an EC.sub.50 of 10 nM or less
(e.g., 0.01 nM to 10 nM), e.g., as measured by FACS;
[0358] (4) inducing or enhancing T cell activation, as evidenced by
(i) increased IL-2 and/or IFN-.gamma. production in OX40-expressing
T cells and/or (ii) enhanced T cell proliferation;
[0359] (5) inhibiting the binding of OX40 ligand to OX40, e.g.,
with an EC.sub.50 of 1 nM or less as measured by FACS, e.g., in an
assay with hOX40-293 cells;
[0360] (6) binding to an epitope on the extracellular portion of
mature human OX40 (SEQ ID NO: 2), e.g., an epitope within the
region DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);
[0361] (7) competing for binding to human OX40 with 3F4, 14B6-1,
14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1;
[0362] (8) competing for binding to human OX40 with 6E1-1, 6E1-2,
14A2-1, and 14A2-2.
[0363] The antibody can be, for example, a human antibody, a
humanized antibody or a chimeric antibody.
[0364] In certain embodiments, the anti-OX40 antibody, or antigen
binding portion thereof, may comprise a heavy chain and a light
chain, wherein:
[0365] (a) the heavy chain comprises an amino acid sequence that is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,
121, 123, 124 and 125, or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4,
1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acid changes (i.e.,
amino acid substitutions, additions or deletions) relative to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,
121, 123, 124 and 125;
[0366] (b) the light chain comprises an amino acid sequence that is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,
and 122, or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10,
1-15, 1-20, 1-25, or 1-50 amino acid changes (i.e., amino acid
substitutions, additions or deletions) relative to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 96, 98,
100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, and 122;
[0367] (c) the antibody specifically binds to OX40, and
[0368] (d) the antibody exhibits 1, 2, 3, 4, 5, 6, or all of the
following functional properties: [0369] (1) binding to soluble
human OX40, e.g., with a K.sub.D of 10 nM or less (e.g., 0.01 nM to
10 nM), e.g., as measured by Biacore; [0370] (2) binding to
membrane bound human OX40, e.g., with an EC.sub.50 of 1 nM or less
(e.g., 0.01 nM to 1 nM), e.g., as measured by FACS; [0371] (3)
binding to cynomolgus OX40, e.g., binding to membrane bound
cynomolgus OX40, e.g., with an EC.sub.50 of 10 nM or less (e.g.,
0.01 nM to 10 nM), e.g., as measured by FACS; [0372] (4) inducing
or enhancing T cell activation, as evidenced by (i) increased IL-2
and/or IFN-.gamma. production in OX40-expressing T cells and/or
(ii) enhanced T cell proliferation; [0373] (5) inhibiting the
binding of OX40 ligand to OX40, e.g., with an EC.sub.50 of 1 nM or
less as measured by FACS, e.g., in an assay with hOX40-293 cells;
[0374] (6) binding to an epitope on the extracellular portion of
mature human OX40 (SEQ ID NO: 2), e.g., an epitope within the
region DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) or
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179); [0375] (7)
competing for binding to human OX40 with 3F4, 14B6-1, 14B6-2, 23H3,
18E9, 8B11, 20B3, and 20C1; [0376] (8) competing for binding to
human OX40 with 6E1-1, 6E1-2, 14A2-1, and 14A2-2.
[0377] Also provided are anti-OX40 antibodies comprising a VHCDR1,
VHCDR2, VHCDR3, VLCDR1, VLCDR2, and/or VLCDR3 that differs from the
corresponding CDR of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9,
8B11, 20B3, 14A2-1, 14A2-2, and/or 20C1, in 1, 2, 3, 4, 5, 1-2,
1-3, 1-4, or 1-5 amino acid changes (i.e., amino acid
substitutions, additions or deletions). In certain embodiments, the
antibody comprises 1-5 amino acid changes in each of 1, 2, 3, 4, 5
or 6 of the CDRs relative to the corresponding sequence in 3F4,
14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1,
14A2-2, and/or 20C1. In certain embodiments, the antibody comprises
at total of 1-5 amino acid changes across all CDRs relative to the
CDRs in 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3,
14A2-1, 14A2-2, and/or 20C1.
[0378] Antibodies having sequences with homology to those of 3F4,
14B6 (14B6-1 and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11,
20B3, 14A2 (14A2-1 and 14A2-2), and 20C1, e.g., the V.sub.H and
V.sub.L regions of SEQ ID NOs: 17 and 18; 28 and 29; 28 and 30; 37
and 38; 48 and 49; 48 and 50; 57 and 58; 65 and 66; 73 and 74; 84
and 85; 84 and 86; 93 and 94, respectively, or heavy and light
chains of SEQ ID NOs: 95 and 96; 97 and 98; 99 and 100; 101 and
102; 103 and 104; 105 and 106; 107 and 108; 109 and 110; 111 and
112; 113 and 114; 115 and 116; 117 and 118; 119 and 120; 121 and
122; 123 and 116; 124 and 116; and 125 and 116, respectively, or
CDRs can be obtained by mutagenesis (e.g., site-directed or
PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID
NOs: 17, 28, 37, 48, 57, 65, 73, 84, and 93 and/or SEQ ID NOs: 18,
29, 30, 38, 49, 50, 58, 66, 74, 85, 86, and 94 or SEQ ID NOs: 95,
97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,
124 and 125 and/or SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, 120, and 122, followed by testing of the
encoded altered antibody for retained function (i.e., the functions
set forth in (1) through (7) above) using the functional assays
described herein.
V. Antibodies with Conservative Modifications
[0379] Anti-OX40 antibodies provided herein may comprise a heavy
chain variable region comprising CDR1, CDR2 and CDR3 sequences and
a light chain variable region comprising CDR1, CDR2 and CDR3
sequences, wherein one or more of these CDR sequences comprise
specified amino acid sequences based on the antibodies described
herein (e.g., 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11,
20B3, 14A2-1, 14A2-2, and 20C1), or conservative modifications
thereof, and wherein the antibodies retain the desired functional
properties of the anti-OX40 antibodies described herein.
Accordingly, the anti-OX40 antibody, or antigen binding portion
thereof, may comprise a heavy chain variable region comprising
CDR1, CDR2, and CDR3 sequences and a light chain variable region
comprising CDR1, CDR2, and CDR3 sequences, wherein: [0380] (a) the
heavy chain variable region CDR3 sequence comprises an amino acid
sequence selected from the group consisting of amino acid sequences
of SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77, and 89, and
conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3,
1-4 or 1-5 conservative amino acid substitutions; [0381] (b) the
light chain variable region CDR3 sequence comprises an amino acid
sequence selected from the group consisting of amino acid sequence
of SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92,
and conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2,
1-3, 1-4 or 1-5 conservative amino acid substitutions; [0382] (c)
the antibody specifically binds to OX40, and [0383] (d) the
antibody exhibits 1, 2, 3, 4, 5, 6, or all of the following
functional properties: [0384] (1) binding to soluble human OX40,
e.g., with a K.sub.D of 10 nM or less (e.g., 0.01 nM to 10 nM),
e.g., as measured by Biacore; [0385] (2) binding to membrane bound
human OX40, e.g., with an EC.sub.50 of 1 nM or less (e.g., 0.01 nM
to 1 nM), e.g., as measured by FACS; [0386] (3) binding to
cynomolgus OX40, e.g., binding to membrane bound cynomolgus OX40,
e.g., with an EC.sub.50 of 10 nM or less (e.g., 0.01 nM to 10 nM),
e.g., as measured by FACS; [0387] (4) inducing or enhancing T cell
activation, as evidenced by (i) increased IL-2 and/or IFN-.gamma.
production in OX40-expressing T cells and/or (ii) enhanced T cell
proliferation; [0388] (5) inhibiting the binding of OX40 ligand to
OX40, e.g., with an EC.sub.50 of 1 nM or less as measured by FACS,
e.g., in an assay with hOX40-293 cells; [0389] (6) binding to an
epitope on the extracellular portion of mature human OX40 (SEQ ID
NO: 2), e.g., an epitope within the region
TABLE-US-00009 [0389] (SEQ ID NO: 178) DVVSSKPCKPCTWCNLR or (SEQ ID
NO: 179) DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK;
[0390] (7) competing for binding to human OX40 with 3F4, 14B6-1,
14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1; [0391] (8) competing for
binding to human OX40 with 6E1-1, 6E1-2, 14A2-1, and 14A2-2.
[0392] In a preferred embodiment, the heavy chain variable region
CDR2 sequence comprises an amino acid sequence selected from the
group consisting of amino acid sequences of SEQ ID NOs: 12, 20, 32,
40, 52, 60, 68, 76, 88, and 317, and conservative modifications
thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative
amino acid substitutions; and the light chain variable region CDR2
sequence comprises an amino acid sequence selected from the group
consisting of amino acid sequences of SEQ ID NOs: 15, 23, 26, 35,
43, 46, 55, 63, 71, 79, 82, and 91, and conservative modifications
thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative
amino acid substitutions. In another preferred embodiment, the
heavy chain variable region CDR1 sequence comprises an amino acid
sequence selected from the group consisting of amino acid sequences
of SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87, and
conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3,
1-4 or 1-5 conservative amino acid substitutions; and the light
chain variable region CDR1 sequence comprises an amino acid
sequence selected from the group consisting of amino acid sequences
of SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81, and 90,
and conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2,
1-3, 1-4 or 1-5 conservative amino acid substitutions.
[0393] In various embodiments, the antibodies can be, for example,
human antibodies, humanized antibodies or chimeric antibodies.
[0394] Conservative amino acid substitutions may also be made in
portions of the antibodies other than, or in addition to, the CDRs.
For example, conservative amino acid modifications may be made in a
framework region or in the Fc region. A variable region or a heavy
or light chain may comprise 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5,
1-10, 1-15, 1-20, 1-25, or 1-50 conservative amino acid
substitutions relative to the anti-OX40 antibody sequences provided
herein. In certain embodiments, the anti-OX40 antibody comprises a
combination of conservative and non-conservative amino acid
modification.
VI. Competing Antibodies and Same Epitope Binding Antibodies
[0395] Also provided herein are antibodies that compete for binding
to OX40 with the anti-OX40 antibodies described herein (e.g.,
antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11,
20B3, 14A2-1, 14A2-2, and 20C1). Such competing antibodies can be
identified based on their ability to competitively inhibit binding
to OX40 of one or more of monoclonal antibodies 3F4, 14B6-1,
14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and
20C1 in standard OX40 binding assays. For example, standard ELISA
assays or competitive ELISA assays can be used in which a
recombinant human OX40 protein is immobilized on a plate, various
concentrations of unlabeled first antibody are added, the plate is
washed, labeled second antibody is added, washed, and the amount of
bound label is measured. If the increasing concentration of the
unlabeled (first) antibody (also referred to as the "blocking
antibody") inhibits the binding of the labeled (second) antibody,
the first antibody is said to inhibit the binding of the second
antibody to the target on the plate, or is said to compete with the
binding of the second antibody. Additionally or alternatively,
BIACORE.RTM. SPR analysis can be used to assess the ability of the
antibodies to compete. The ability of a test antibody to inhibit
the binding of an anti-OX40 antibody described herein to OX40
demonstrates that the test antibody can compete with the antibody
for binding to OX40.
[0396] Accordingly, provided herein are anti-OX40 antibodies that
inhibit the binding of the anti-OX40 antibodies described herein to
OX40 on cells, e.g., activated T 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 100%, by using, e.g., FACS as
described in the Examples.
[0397] In other embodiments, provided herein are anti-OX40
antibodies which bind to the same epitope as one or more of the
anti-OX40 antibodies described herein (e.g., antibodies 3F4,
14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1,
14A2-2, and 20C1), as determined using art-recognized epitope
mapping techniques, such as those described below.
[0398] Art-recognized epitope mapping techniques include, for
example, structural methods, such as X-ray crystal structure
determination (e.g., WO2005/044853), molecular modeling and nuclear
magnetic resonance (NMR) spectroscopy, including NMR determination
of the H-D exchange rates of labile amide hydrogens in OX40 when
free and when bound in a complex with an antibody of interest
(Zinn-Justin et al. Biochemistry 1992; 31:11335-47; Zinn-Justin et
al. Biochemistry 1993; 32, 6884-91).
[0399] For X-ray crystallography, crystallization may be
accomplished using any known method in the art (e.g. Giege et al.
Acta Crystallogr 1994; D50:339-50; McPherson, Eur J Biochem 1990;
189:1-23), including microbatch (e.g. Chayen, Structure 19976;
5:1269-74), hanging-drop vapor diffusion (e.g. McPherson, J Biol
Chem 1976; 251:6300-3), 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 such 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.
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, Meth. Enzymol.
1985; 114 & 115, H. W. Wyckoff et al., eds., Academic Press;
U.S. Patent Application Publication No. 2004/0014194), and BUSTER
(Bricogne, Acta Cryst 1993; D49:37-60; Bricogne, Meth Enzymol 1997;
276A:361-423; Carter & Sweet, eds.; Roversi et al., Acta Cryst.
2000; D56:1313-23).
[0400] Other epitope mapping 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. One such method is alanine
scanning mutagenesis, as described, e.g., by Cunningham and Wells,
Science 1989; 244:1081-5. Another suitable method is deep
mutational scanning (see, e.g., Araya et al., Trends in
Biotechnology 2011; 29:435-42; Forsyth et al., mAbs 2013;
5:523-32).
[0401] Additionally or alternatively, computational combinatorial
methods for epitope mapping, including the mapping of
conformational discontinuous epitopes, can be used.
[0402] Additionally or alternatively, epitope mapping can be
achieved by testing binding of an antibody to peptides comprising
fragments of OX40, e.g., non-denatured or denatured fragments. A
series of overlapping peptides encompassing the sequence of OX40
(e.g., human OX40) 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 OX40
bound to a well of a microtiter plate) or on a chip. Other methods
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. The peptides are then regarded as leads for the
definition of the epitope recognized by the antibody used to screen
the peptide library.
[0403] Epitopes also can 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, for example, as described in the Examples
herein and by Wei et al., Drug Discovery Today 2014; 19:95. FPOP
may be conducted, for example, as described by Hambley et al. (J
American Soc Mass Spectrometry 2005; 16:2057).
[0404] Antibodies that compete for binding with the anti-OX40
antibodies described herein may be produced and identified using
art-known methods. For example, mice may be immunized with human
OX40 as described herein, hybridomas produced, and the resulting
monoclonal antibodies screened for the ability to compete with an
antibody described herein for binding to OX40 using the methods
described above.
[0405] Antibodies that bind to the same epitope as the anti-OX40
antibodies described herein may be produced by immunizing mice with
a smaller fragment of OX40 containing the epitope to which the
antibody binds. The epitope or region comprising the epitope can be
identified using the methods described above. Alternatively, the
method of Jespers et al. (Biotechnology 1994; 12:899) may be used
to guide the selection of antibodies recognizing the same epitope
and therefore exhibiting similar properties to the anti-OX40
antibodies described herein. For example, using phage display,
first the heavy chain of the anti-OX40 antibody is paired with a
repertoire of (preferably human) light chains to select a
OX40-binding antibody, and then the new light chain is paired with
a repertoire of (preferably human) heavy chains to select a
(preferably human) OX40-binding antibody recognizing the same
epitope or epitope region on OX40 as an anti-OX40 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.
[0406] In some embodiments, provided herein are antibodies which
bind to all or a portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID
NO: 178), corresponding to amino acid residues 46-62 of mature
human OX40 (SEQ ID NO: 2), as determined by the methods in the
Examples.
[0407] In certain embodiments, the anti-OX40 antibodies described
herein that bind to all or a portion of the sequence
SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182), as determined by
the methods in the Examples.
[0408] In other embodiments, the anti-OX40 antibodies described
herein that bind to all or a portion of the sequence PCKPCTWCNLR
(SEQ ID NO: 183), as determined by the methods in the Examples.
[0409] In yet other embodiments, the anti-OX40 antibodies that bind
to all or a portion of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO:
178) further bind to all or a portion of the sequence QLCTATQDTVCR
(SEQ ID NO: 184), as determined by the methods in the Examples.
[0410] In additional embodiments, the anti-OX40 antibodies
described herein that bind to all or a portion of the sequence
SQNTVCRPCGPGFYN (SEQ ID NO: 185), as determined by the methods in
the Examples.
[0411] In additional embodiments, the anti-OX40 antibody binds
within the region DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO:
179), corresponding to amino acid residues 89-124 of mature human
OX40 (SEQ ID NO: 2), as determined by the methods in the
Examples.
VII. Engineered and Modified Antibodies
VH and VL Regions
[0412] Also provided herein 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., V.sub.H and/or V.sub.L), 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.
[0413] One type of variable region engineering that can be
performed is CDR grafting. 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. U.S.A. 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.)
[0414] Accordingly, another embodiment pertains to a monoclonal
antibody, or antigen binding portion thereof, comprising a heavy
chain variable region comprising CDR1, CDR2, and CDR3 sequences
comprising an amino acid sequence selected from the group
consisting of SEQ ID SEQ ID NOs: 11-13; 19-21; 31-33; 39-41; 51-53;
59-61; 67-69; 74-77; and 87-89, respectively, and a light chain
variable region comprising CDR1, CDR2, and CDR3 sequences
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 14-16; 22-24; 25-27; 34-36; 42-44; 45-47;
54-56; 62-64; 70-72; 78-80; 81-83; and 90-92, respectively. Thus,
such antibodies contain the V.sub.H and V.sub.L CDR sequences of
monoclonal antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2,
18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1, yet may contain
different framework sequences.
[0415] 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 (available on the Internet at
www.mrc-cpe.cam.ac.uk/vbase), 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 V.sub.H Sequences Reveals about Fifty
Groups of V.sub.H 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 V.sub.H 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.
[0416] 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
V.sub.H CDR1, 2 and 3 sequences, and the V.sub.L 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).
[0417] 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. Typically 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.
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.
[0418] 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, can be evaluated
in in vitro or in vivo assays as described herein and provided in
the Examples. Preferably conservative modifications (as discussed
above) 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.
[0419] Accordingly, also provided herein are anti-OX40 monoclonal
antibodies, or antigen binding portions thereof, comprising a heavy
chain variable region comprising: (a) a V.sub.H CDR1 region
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87,
or an amino acid sequence having one, two, three, four or five
amino acid substitutions, deletions or additions as compared to SEQ
ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87; (b) a V.sub.H CDR2
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76, 88, and
317, or an amino acid sequence having one, two, three, four or five
amino acid substitutions, deletions or additions as compared to SEQ
ID NOs: 12, 20, 32, 40, 52, 60, 68, 76, and 88; (c) a V.sub.H CDR3
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77, and 89,
or an amino acid sequence having one, two, three, four or five
amino acid substitutions, deletions or additions as compared to SEQ
ID NOs: 13, 21, 33, 41, 53, 61, 69, 77, and 89; (d) a V.sub.L CDR1
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78,
81, and 90, or an amino acid sequence having one, two, three, four
or five amino acid substitutions, deletions or additions as
compared to SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81,
and 90; (e) a V.sub.L CDR2 region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 15, 23, 26, 35,
43, 46, 55, 63, 71, 79, 82, and 91, or an amino acid sequence
having one, two, three, four or five amino acid substitutions,
deletions or additions as compared to SEQ ID NOs: 15, 23, 26, 35,
43, 46, 55, 63, 71, 79, 82, and 91; and (f) a V.sub.L CDR3 region
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80,
83, and 92, or an amino acid sequence having one, two, three, four
or five amino acid substitutions, deletions or additions as
compared to SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83,
and 92.
[0420] Methionine residues in CDRs of antibodies can be oxidized,
resulting in potential chemical degradation and consequent
reduction in potency of the antibody. Accordingly, one or more
methionine residues in the heavy and/or light chain CDRs of the
anti-OX40 antibodies described herein may be replaced with amino
acid residues that do not undergo oxidative degradation.
[0421] Similarly, deamidation sites may be removed from the
antibodies, particularly in the CDRs.
[0422] 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
[0423] In various embodiments, the antibodies described herein are
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.
[0424] 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
[0425] In addition to the activity of a therapeutic antibody
arising from binding of the antigen binding domain to the antigen
(e.g. blocking of a cognate ligand or receptor protein in the case
of antagonist antibodies, or induced signaling in the case of
agonist antibodies), the Fc portion of the antibody interact with
the immune system generally in complex ways to elicit any number of
biological effects. Effector functions, such as the Fc region of an
immunoglobulin, are responsible for many important antibody
functions, such as antigen-dependent cellular cytotoxicity (ADCC),
complement dependent cytotoxicity (CDC), and antibody-dependent
cell-mediated phagocytosis (ADCP), result in killing of target
cells, albeit by different mechanisms. There are five major
classes, or isotypes, of heavy chain constant region (IgA, each
with characteristic effector functions. These isotypes can be
further subdivided into subclasses, for example, IgG is separated
into four subclasses known as IgG1, IgG2, IgG3, and IgG4. IgG
molecules interact with three classes of Fc.gamma. receptors
(Fc.gamma.R) specific for the IgG class of antibody, namely
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII. The important
sequences for the binding of IgG to the Fc.gamma.R receptors have
been reported to be located in the CH2 and CH3 domains. The serum
half-life of an antibody is influenced by the ability of that
antibody to bind to the neonatal Fc receptor (FcRn).
[0426] Anti-OX40 antibodies described herein 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 Fc.gamma. receptors (activating
receptors Fc.gamma.RI (CD64), Fc.gamma.RIIA, Fc.gamma.RIIC (CD32);
Fc.gamma.RIIIA and Fc.gamma.RIIIB (CD16) and inhibiting receptor
Fc.gamma.RIIB), and for the first component of complement (C1q).
Human IgG1 and IgG3 bind to all Fc.gamma. 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
Fc.gamma. 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.gtoreq.IgG3>>IgG4.gtoreq.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, or 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.
[0427] Accordingly, anti-OX40 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.
[0428] In certain embodiments, anti-OX40 variable regions described
herein are linked to an Fc that binds to one or more activating Fc
receptors (Fc.gamma.I/CD64, Fc.gamma.IIa/CD32 or
Fc.gamma.IIIa/CD16), and thereby stimulate ADCC and may cause T
cell depletion. In particular embodiments, anti-OX40 variable
regions described herein are linked to an Fc that causes depletion.
In other embodiments, anti-OX40 variable regions described herein
are linked to a human IgG1 or IgG3 Fc, i.e., the antibodies are of
the IgG1 or IgG3 isotype. In other embodiments, anti-OX40
antibodies are depleting antibodies. For example, they may deplete
T.sub.reg cells that are in the tumor microenvironment (and thereby
enhance anti-tumor activity), but not significantly deplete
T.sub.eff cells that are in the tumor microenvironment and mediate
the anti-tumor effect, and/or not significantly deplete T.sub.reg
and T.sub.eff cells that are outside of the tumor, e.g., in the
periphery. In other embodiments, anti-OX40 antibodies are of an
isotype, (either naturally occurring or non-naturally occurring
(e.g., including mutation(s)) isotype that stimulate T.sub.reg cell
depletion or elimination at the tumor site and concomitant
activation of T.sub.eff cells. In other embodiments, anti-OX40
antibodies create an elevated T.sub.eff to T.sub.reg ratio at the
tumor site, which is indicative of potent anti-tumor activity, and
preferably without significantly depleting T.sub.reg and T.sub.eff
cells that are outside of the tumor, e.g., in the periphery.
[0429] In certain embodiments, anti-OX40 antibodies block the
immunosuppressive activity of T.sub.regs. In other embodiments,
anti-OX40 antibodies have an Fc receptor with reduced or eliminated
FcR binding, e.g., reduced binding to activating FcRs. In certain
embodiments, anti-OX40 antibodies have an Fc that binds to or has
enhanced binding to FcRIIb, 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.
[0430] Anti-OX40 variable regions described herein may be linked to
a non-naturally occurring Fc region, e.g., an effectorless or
mostly effectorless Fc (e.g., human IgG2 or IgG4) or,
alternatively, an Fc with enhanced binding to one or more
activating Fc receptors (Fc.gamma.I, Fc.gamma.IIa or
Fc.gamma.IIIa), such as to enhance T.sub.reg depletion in the tumor
environment.
[0431] Variable regions described herein may be linked to an Fc
comprising one or more modification, typically to alter one or more
functional properties of the antibody, such as serum half-life,
complement fixation, Fc receptor binding, and/or antigen-dependent
cellular cytotoxicity. Furthermore, an antibody described herein
may be chemically modified (e.g., one or more chemical moieties can
be attached to the antibody) or it may be modified to alter its
glycosylation, to alter one or more functional properties of the
antibody. Each of these embodiments is described in further detail
below. The numbering of residues in the Fc region is that of the EU
index of Kabat. Sequence variants disclosed herein are provided
with reference to the residue number followed by the amino acid
that is substituted in place of the naturally occurring amino acid,
optionally preceded by the naturally occurring residue at that
position. Where multiple amino acids may be present at a given
position, e.g. if sequences differ between naturally occurring
isotypes, or if multiple mutations may be substituted at the
position, they are separated by slashes (e.g. "X/Y/Z").
[0432] For example, one may make modifications in the Fc region in
order to generate an Fc variant with (a) increased or decreased
antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased
or decreased complement mediated cytotoxicity (CDC). (c) increased
or decreased affinity for C1q and/or (d) increased or decreased
affinity for a Fc receptor relative to the parent Fe. Such Fc
region variants will generally comprise at least one amino acid
modification in the Fc region. Combining amino acid modifications
is thought to be particularly desirable. For example, the variant
Fc region may include two, three, four, five, etc substitutions
therein, e.g. of the specific Fc region positions identified
herein. Exemplary Fc sequence variants are disclosed herein, and
are also provided at U.S. Pat. Nos. 5,624,821; 6,277,375;
6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publications
WO 00/42072; WO 01/58957; WO 04/016750; WO 04/029207; WO 04/035752;
WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO
05/040217, WO 05/092925 and WO 06/020114.
Reducing Effector Function
[0433] ADCC activity may be reduced by modifying the Fc region. In
certain embodiments, sites that affect binding to Fc receptors may
be removed, preferably sites other than salvage receptor binding
sites. In other embodiments, an Fc region may be modified to remove
an ADCC site. ADCC sites are known in the art; see, for example,
Sarmay et al. (1992) Molec. Immunol. 29 (5): 633-9 with regard to
ADCC sites in IgG1. In one embodiment, the G236R and L328R variant
of human IgG1 effectively eliminates Fc .gamma. R binding. Horton
et al. (2011) J. Immunol. 186:4223 and Chu et al. (2008) Mol.
Immunol. 45:3926. In other embodiments, the Fc having reduced
binding to Fc.gamma.Rs comprised the amino acid substitutions
L234A, L235E and G237A. Gross et al. (2001) Immunity 15:289.
[0434] CDC activity may also be reduced by modifying the Fc region.
Mutations at IgG1 positions D270, K322, P329 and P331, specifically
alanine mutations D270A, K322A, P329A and P331A, significantly
reduce the ability of the corresponding antibody to bind C1q and
activate complement. Idusogie et al. (2000) J. Immunol. 164:4178;
WO 99/51642. Modification of position 331 of IgG1 (e.g. P331S) has
been shown to reduce complement binding. Tao et al. (1993) J. Exp.
Med. 178:661 and Canfield & Morrison (1991) J. Exp. Med.
173:1483. In another example, one or more amino acid residues
within amino acid positions 231 to 239 are altered to thereby
reduce the ability of the antibody to fix complement. WO
94/29351.
[0435] In some embodiments, the Fc with reduced complement fixation
has the amino acid substitutions A330S and P331S. Gross et al.
(2001) Immunity 15:289.
[0436] For uses where effector function is to be avoided
altogether, e.g. when antigen binding alone is sufficient to
generate the desired therapeutic benefit, and effector function
only leads to (or increases the risk of) undesired side effects,
IgG4 antibodies may be used, or antibodies or fragments lacking the
Fc region or a substantial portion thereof can be devised, or the
Fc may be mutated to eliminate glycosylation altogether (e.g.
N297A). Alternatively, a hybrid construct of human IgG2 (C.sub.H1
domain and hinge region) and human IgG4 (C.sub.H2 and C.sub.H3
domains) has been generated that is devoid of effector function,
lacking the ability to bind the Fc.gamma.Rs (like IgG2) and unable
to activate complement (like IgG4). Rother et al. (2007) Nat.
Biotechnol. 25:1256. See also Mueller et al. (1997) Mol. Immunol.
34:441; Labrijn et al. (2008) Curr. Op. Immunol. 20:479 (discussing
Fc modifications to reduce effector function generally).
[0437] In other embodiments, the Fc region is altered by replacing
at least one amino acid residue with a different amino acid residue
to reduce all effector function(s) of the antibody. For example,
one or more amino acids selected from amino acid residues 234, 235,
236, 237, 297, 318, 320 and 322 can be replaced with a different
amino acid residue such that the antibody has decreased affinity
for an effector ligand but retains the antigen-binding ability of
the parent antibody. The effector ligand to which affinity is
altered can be, for example, an Fc receptor (residues 234, 235,
236, 237, 297) or the C1 component of complement (residues 297,
318, 320, 322). U.S. Pat. Nos. 5,624,821 and 5,648,260, both by
Winter et al.
[0438] One early patent application proposed modifications in the
IgG Fc region to decrease binding to Fc.gamma.RI to decrease ADCC
(234A; 235E; 236A; G237A) or block binding to complement component
C1q to eliminate CDC (E318A or V/K320A and K322A/Q). WO 88/007089.
See also Duncan & Winter (1988) Nature 332:563; Chappel et al.
(1991) Proc. Nat'l Acad. Sci. (USA) 88:9036; and Sondermann et al.
(2000) Nature 406:267 (discussing the effects of these mutations on
Fc.gamma.RIII binding).
[0439] Fc modifications reducing effector function also include
substitutions, insertions, and deletions at positions 234, 235,
236, 237, 267, 269, 325, and 328, such as 234G, 235G, 236R, 237K,
267R, 269R, 325L, and 328R. An Fc variant may comprise 236R/328R.
Other modifications for reducing Fc.gamma.R and complement
interactions include substitutions 297A, 234A, 235A, 237A, 318A,
228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S, 229S, 238S, 233P,
and 234V. These and other modifications are reviewed in Strohl
(2009) Current Opinion in Biotechnology 20:685-691. Effector
functions (both ADCC and complement activation) can be reduced,
while maintaining neonatal FcR binding (maintaining half-life), by
mutating IgG residues at one or more of positions 233-236 and
327-331, such as E233P, L234V, L235A, optionally G2364, A327G,
A330S and P331S in IgG1; E233P, F234V, L235A, optionally G2364 in
IgG4; and A330S and P331S in IgG2. See Armour et al. (1999) Eur. J.
Immunol. 29:2613; WO 99/58572. Other mutations that reduce effector
function include L234A and L235A in IgG1 (Alegre et al. (1994)
Transplantation 57:1537); V234A and G237A in IgG2 (Cole et al.
(1997) J. Immunol. 159:3613; see also U.S. Pat. No. 5,834,597); and
S228P and L235E for IgG4 (Reddy et al. (2000) J. Immunol.
164:1925). Another combination of mutations for reducing effector
function in a human IgG1 include L234F, L235E and P331S. Oganesyan
et al. (2008) Acta Crystallogr. D. Biol. Crystallogr. 64:700. See
generally Labrijn et gal. (2008) Curr. Op. Immunol. 20:479.
Additional mutations found to decrease effector function in the
context of an Fc (IgG1) fusion protein (abatacept) are C226S, C229S
and P238S (EU residue numbering). Davis et al. (2007) J. Immunol.
34:2204.
[0440] Other Fc variants having reduced ADCC and/or CDC are
disclosed at Glaesner et al. (2010) Diabetes Metab. Res. Rev.
26:287 (F234A and L235A to decrease ADCC and ADCP in an IgG4);
Hutchins et al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:11980
(F234A, G237A and E318A in an IgG4); An et al. (2009) MAbs 1:572
and U.S. Pat. App. Pub. 2007/0148167 (H268Q, V309L, A330S and P331S
in an IgG2); McEarchern et al. (2007) Blood 109:1185 (C226S, C229S,
E233P, L234V, L235A in an IgG1); Vafa et al. (2014) Methods 65:114
(V234V, G237A, P238S, H268A, V309L, A330S, P331S in an IgG2).
[0441] In certain embodiments, an Fc is chosen that has essentially
no effector function, i.e., it has reduced binding to Fc.gamma.Rs
and reduced complement fixation. An exemplary Fc, e.g., IgG1 Fc,
that is effectorless comprises the following five mutations: L234A,
L235E, G237A, A330S and P331S. Gross et al. (2001) Immunity 15:289.
Exemplary heavy chains comprising these mutations are set forth in
the Sequence Listing, as detailed at Table 23 (e.g. SEQ ID NO: 11).
These five substitutions may be combined with N297A to eliminate
glycosylation as well.
Enhancing Effector Function
[0442] Alternatively. ADCC activity may be increased by modifying
the Fc region. With regard to ADCC activity, human
IgG1.gtoreq.IgG3>>IgG4.gtoreq.IgG2, so an IgG1 constant
domain, rather than an IgG2 or IgG4, might be chosen for use in a
drug where ADCC is desired. Alternatively, the Fc region may be
modified to increase antibody dependent cellular cytotoxicity
(ADCC) and/or to increase the affinity for an Fc.gamma. receptor by
modifying one or more amino acids at the following positions: 234,
235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252,
254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272,
276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296,
298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324,
325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340,
360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433,
434, 435, 436, 437, 438 or 439. See WO 2012/142515; see also WO
00/42072. Exemplary substitutions include 236A, 239D, 239E, 268D,
267E, 268E, 268F, 324T, 332, and 332E. Exemplary variants include
239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F,
267E/324T, and 267E/268F/324T. For example, human IgG1 Fcs
comprising the G236A variant, which can optionally be combined with
I332E, have been shown to increase the Fc.gamma.IIA/Fc.gamma.IIB
binding affinity ratio approximately 15-fold. Richards et al.
(2008) Mol. Cancer Therap. 7:2517; Moore et al. (2010) mAbs 2:181.
Other modifications for enhancing Fc.gamma.R and complement
interactions include but are not limited to substitutions 298A,
333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L,
292P, 300L, 396L, 305I, and 396L. These and other modifications are
reviewed in Strohl (2009) Current Opinion in Biotechnology
20:685-691. Specifically, both ADCC and CDC may be enhanced by
changes at position E333 of IgG1, e.g. E333A. Shields et al. (2001)
J. Biol. Chem. 276:6591. The use of P247I and A339D/Q mutations to
enhance effector function in an IgG1 is disclosed in WO2006/020114,
and D280H, K290S.+-.S298D/V is disclosed in WO2004/074455. The
K326A/W and E333A/S variants have been shown to increase effector
function in human IgG1, and E333S in IgG2. Idusogie et al. (2001)
J. Immunol. 166:2571.
[0443] Specifically, the binding sites on human IgG1 for
Fc.gamma.R1, Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped,
and variants with improved binding have been described. Shields et
al. (2001) J. Biol. Chem. 276:6591-6604. Specific mutations at
positions 256, 290, 298, 333, 334 and 339 were shown to improve
binding to Fc.gamma.RIII, including the combination mutants
T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A (having
enhanced Fc.gamma.RIIIa binding and ADCC activity). Other IgG1
variants with strongly enhanced binding to Fc.gamma.RIIIa have been
identified, including variants with S239D/I332E and
S239D/I332E/A330L mutations which showed the greatest increase in
affinity for Fc.gamma.RIIIa, a decrease in Fc.gamma.RIIb binding,
and strong cytotoxic activity in cynomolgus monkeys. Lazar et al.
(2006) Proc. Nat'l Acad Sci. (USA) 103:4005; Awan et al. (2010)
Blood 115:1204; Desjarlais & Lazar (2011) Exp. Cell Res.
317:1278. Introduction of the triple mutations into antibodies such
as alemtuzumab (CD52-specific), trastuzumab (HER2/neu-specific),
rituximab (CD20-specific), and cetuximab (EGFR-specific) translated
into greatly enhanced ADCC activity in vitro, and the S239D/I332E
variant showed an enhanced capacity to deplete B cells in monkeys.
Lazar et al. (2006) Proc. Nat'l Acad Sci. (USA) 103:4005. In
addition, IgG1 mutants containing L235V, F243L, R292P, Y300L, V305I
and P396L mutations which exhibited enhanced binding to
Fc.gamma.RIIIa and concomitantly enhanced ADCC activity in
transgenic mice expressing human Fc.gamma.RIIIa in models of B cell
malignancies and breast cancer have been identified. Stavenhagen et
al. (2007) Cancer Res. 67:8882; U.S. Pat. No. 8,652,466; Nordstrom
et al. (2011) Breast Cancer Res. 13:R123.
[0444] Different IgG isotypes also exhibit differential CDC
activity (IgG3>IgG1>>IgG2.apprxeq.IgG4). Dangl et al.
(1988) EMBO J. 7:1989. For uses in which enhanced CDC is desired,
it is also possible to introduce mutations that increase binding to
C1q. The ability to recruit complement (CDC) may be enhanced by
mutations at K326 and/or E333 in an IgG2, such as K326W (which
reduces ADCC activity) and E333S, to increase binding to C1q, the
first component of the complement cascade. Idusogie et al. (2001)
J. Immunol. 166:2571. Introduction of S267E/H268F/S324T (alone or
in any combination) into human IgG1 enhances C1q binding. Moore et
al. (2010) mAbs 2:181. The Fc region of the IgG1/IgG3 hybrid
isotype antibody "113F" of Natsume et al. (2008) Cancer Res.
68:3863 (FIG. 1 therein) also confers enhanced CDC. See also
Michaelsen et al. (2009) Scand. J. Immunol. 70:553 and Redpath et
al. (1998) Immunology 93:595.
[0445] Additional mutations that can increase or decrease effector
function are disclosed at Dall'Acqua et al. (2006) J. Immunol.
177:1129. See also Carter (2006) Nat. Rev. Immunol. 6:3439 Presta
(2008) Curr. Op. Immunol. 20:460.
[0446] Fc variants that enhance affinity for the inhibitory
receptor Fc.gamma.RIIb may also be used, e.g. to enhance
apoptosis-inducing or adjuvant activity. Li & Ravetch (2011)
Science 333:1030; 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.sup.+ 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. 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 FcyRIIb
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.sup.R131) may be
obtained by adding the P238D substitution. Mimoto et al. (2013)
Protein. Eng. Des. & Selection 26:589; WO 2012/115241.
[0447] 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
259I, 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, 311A, 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.
[0448] Modification of certain conserved residues in IgG Fc
(I253/H310/Q311/H433/N434), such as the N434A variant (Yeung et al.
(2009) J. Immunol. 182:7663), has 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.
[0449] 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.
[0450] 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.
[0451] 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, 422I, 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).
[0452] 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.
[0453] Alternatively, under some circumstances it may be desirable
to decrease the half-life of an antibody, 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
[0454] When using an IgG4 constant domain, it is 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.
[0455] 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.
[0456] 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 immunoabsorbent assay (ELBA), 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.
[0457] 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).
[0458] 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).
[0459] Additionally, an antibody with enhanced ADCC can be made by
altering glycosylation. For example, removal of fucose from heavy
chain Asn297-linked oligosaccharides has been shown to enhance
ADCC, based on improved binding to Fc.gamma.RIIIa. Shields et al.
(2002) JBC 277:26733; Niwa et al. (2005) J. Immunol. Methods 306:
151; Cardarelli et al. (2009) Clin. Cancer Res. 15:3376 (MDX-1401);
Cardarelli et al. (2010) Cancer Immunol. Immunotherap. 59:257
(MDX-1342). Such low fucose antibodies may be produced, e.g., in
knockout Chinese hamster ovary (CHO) cells lacking
fucosyltransferase (FUT8) (Yamane-Ohnuki et al. (2004) Biotechnol.
Bioeng. 87:614), or in other cells that generate afucosylated
antibodies. See, e.g., Zhang et al. (2011) mAbs 3:289 and Li et al.
(2006) Nat. Biotechnol. 24:210 (both describing antibody production
in glycoengineered Pichia pastoris.); Mossner et al. (2010) Blood
115:4393; Shields et al. (2002) J. Biol. Chem. 277:26733; Shinkawa
et al. (2003) J. Biol. Chem. 278:3466; EP 1176195B1. ADCC can also
be enhanced as described in PCT Publication WO 03/035835, which
discloses use of a variant CHO cell line, Lec13, with reduced
ability to attach fucose to Asn(297)-linked carbohydrates, also
resulting in hypofucosylation of antibodies expressed in that host
cell (see also Shields, R. L. et al. (2002) J. Biol. Chem.
277:26733-26740). Alternatively, fucose analogs may be added to
culture medium during antibody production to inhibit incorporation
of fucose into the carbohydrate on the antibody (see, e.g., WO
2009/135181).
[0460] Increasing bisecting GlcNac structures in antibody-linked
oligosaccharides also enhances ADCC. PCT Publication WO 99/54342 by
Umana et al. describes cell lines engineered to express
glycoprotein-modifying glycosyl transferases (e.g.,
beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures which results in increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech. 17:176-180).
[0461] Additional glycosylation variants have been developed that
are devoid of galactose, sialic acid, fucose and xylose residues
(so-called GNGN glycoforms), which exhibit enhanced ADCC and ADCP
but decreased CDC, as well as others that are devoid of sialic
acid, fucose and xylose (so-called G1/G2 glycoforms), which exhibit
enhanced ADCC, ADCP and CDC. U.S. Pat. App. Pub. No. 2013/0149300.
Antibodies having these glycosylation patterns are optionally
produced in genetically modified N. benthamiana plants in which the
endogenous xylosyl and fucosyl transferase genes have been
knocked-out.
[0462] 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
(see, e.g., WO 2013/095966).
VIII. Antibody Physical Properties
[0463] 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) Annu
Rev Biochem 41:673-702; Gala and Morrison (2004) J. Immunol
172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro
(2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature
316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).
Glycosylation has been known to occur at motifs containing an
N-X-S/T sequence. In some some embodiments, the anti-OX40 antibody
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.
[0464] 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). For instance, if the amino acid sequence Asp-Gly is
present in the heavy and/or light chain CDR sequences of the
antibody, the sequence is substituted with an amino acid sequence
that does not undergo isomerization. In one embodiment, the
antibody comprises the heavy chain variable region CDR2 sequence
set forth in SEQ ID NO: 76, but wherein the Asp or Gly in the
Asp-Gly sequence (LISYDGSRKHYADSVKG; SEQ ID NO: 76) is replaced
with an amino acid sequence that does not undergo isomerization,
for example, an Asp-Ser or a Ser-Gly sequence. In another
embodiment, the antibody comprises the heavy chain variable region
CDR2 sequence set forth in SEQ ID NO: 88, but wherein the Asp or
Gly in the Asp-Gly sequence (AIDTDGGTFYADSVRG; SEQ ID NO: 88) is
replaced with an amino acid sequence that does not undergo
isomerization, for example, a Ser-Gly, an Asp-Ala, or a Ser-Thr
sequence.
[0465] 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-OX40 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.
[0466] 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 T.sub.M1 (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). 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). In certain embodiments, the anti-OX40 antibodies
disclosed herein (e.g., the OX40.21 antibody) have increased
stability.
[0467] Accordingly, in other embodiments, 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.
IX. Methods of Engineering Antibodies
[0468] As discussed herein, anti-OX40 antibodies having V.sub.H and
V.sub.L sequences disclosed herein can be used to create new
anti-OX40 antibodies by modifying the VH and/or VL sequences, or
the constant region(s) of the antibodies. Thus, in another
embodiment, the structural features of anti-OX40 antibodies
described herein, e.g. 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2,
18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1, are used to create
structurally related anti-OX40 antibodies that retain at least one
functional property of the antibodies described herein, such as
binding to human OX40 and cynomolgus OX40. For example, one or more
CDR regions of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11,
20B3, 14A2-1, 14A2-2, and 20C1, or mutations thereof, can be
combined recombinantly with known framework regions and/or other
CDRs to create additional, recombinantly-engineered, anti-OX40
antibodies, as discussed above. Other types of modifications
include those described in the previous section. The starting
material for the engineering method is one or more of the V.sub.H
and/or V.sub.L sequences provided herein, or one or more CDR
regions thereof. To create the engineered antibody, it is not
necessary to actually prepare (i.e., express as a protein) an
antibody having one or more of the V.sub.H and/or V.sub.L sequences
provided herein, or one or more CDR regions thereof. Rather, the
information contained in the sequence(s) is used as the starting
material to create a "second generation" sequence(s) derived from
the original sequence(s) and then the "second generation"
sequence(s) is prepared and expressed as a protein.
[0469] Accordingly, provided herein are methods for generating
anti-OX40 antibodies comprising:
[0470] (a) providing: (i) a heavy chain variable region antibody
sequence comprising a CDR1 sequence selected from the group
consisting of SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87, a
CDR2 sequence selected from the group consisting of SEQ ID NOs: 12,
20, 32, 40, 52, 60, 68, 76, 88, and 317 and/or a CDR3 sequence
selected from the group consisting of SEQ ID NOs: 13, 21, 33, 41,
53, 61, 69, 77, and 89; and (ii) a light chain variable region
antibody sequence comprising a CDR1 sequence selected from the
group consisting of SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70,
78, 81, and 90, a CDR2 sequence selected from the group consisting
of SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82, and 91,
and/or a CDR3 sequence selected from the group consisting of SEQ ID
NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92;
[0471] (b) altering at least one amino acid residue within the
heavy chain variable region antibody sequence and/or the light
chain variable region antibody sequence to create at least one
altered antibody sequence; and
[0472] (c) expressing the altered antibody sequence as a
protein.
[0473] Standard molecular biology techniques can be used to prepare
and express the altered antibody sequence.
[0474] Preferably, the antibody encoded by the altered antibody
sequence(s) is one that retains one, some or all of the functional
properties of the anti-OX40 antibodies described herein, which
include, [0475] (1) binding to soluble human OX40, e.g., with a
K.sub.D of 10 nM or less (e.g., 0.01 nM to 10 nM), e.g., as
measured by Biacore; [0476] (2) binding to membrane bound human
OX40, e.g., with an EC.sub.50 of 1 nM or less (e.g., 0.01 nM to 1
nM), e.g., as measured by FACS; [0477] (3) binding to cynomolgus
OX40, e.g., binding to membrane bound cynomolgus OX40, e.g., with
an EC.sub.50 of 10 nM or less (e.g., 0.01 nM to 10 nM), e.g, as
measured by FACS; [0478] (4) inducing or enhancing T cell
activation, as evidenced by (i) increased IL-2 and/or IFN-.gamma.
production in OX40-expressing T cells and/or (ii) enhanced T cell
proliferation; [0479] (5) inhibiting the binding of OX40 ligand to
OX40, e.g., with an EC.sub.50 of 1 nM or less as measured by FACS,
e.g., in an assay with hOX40-293 cells; [0480] (6) binding to an
epitope on the extracellular portion of mature human OX40 (SEQ ID
NO: 2), e.g., an epitope within the region DVVSSKPCKPCTWCNLR (SEQ
ID NO: 178) or DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO:
179); [0481] (7) competing for binding to human OX40 with 3F4,
14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3, and 20C1; [0482] (8)
competing for binding to human OX40 with 6E1-1, 6E1-2, 14A2-1, and
14A2-2.
[0483] The altered antibody may exhibit one or more, two or more,
three or more, four or more, five or more, six, or all of the
functional properties set forth as (1) through (7) above. The
functional properties of the altered antibodies can be assessed
using standard assays available in the art and/or described herein,
such as those set forth in the Examples (e.g., ELISAs, FACS).
[0484] In certain embodiments of the methods of engineering
antibodies described herein, mutations can be introduced randomly
or selectively along all or part of an anti-OX40 antibody coding
sequence and the resulting modified anti-OX40 antibodies can be
screened for binding activity and/or other functional properties as
described herein. Mutational methods have been described in the
art. For example, PCT Publication WO 02/092780 by Short describes
methods for creating and screening antibody mutations using
saturation mutagenesis, synthetic ligation assembly, or a
combination thereof. Alternatively, PCT Publication WO 03/074679 by
Lazar et al. describes methods of using computational screening
methods to optimize physiochemical properties of antibodies.
X. Nucleic Acid Molecules
[0485] Also provided herein are 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 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.
[0486] Nucleic acids provided 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.
[0487] Preferred nucleic acids molecules are those encoding the VH
and VL sequences of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9,
8B11, 20B3, 14A2-1, 14A2-2, and 20C1. Exemplary DNA sequences
encoding the VH sequences of 3F4, 14B6, 23H3, 6E1, 18E9, 8B11,
20B3, 14A2, and 20C1 are set forth in SEQ ID NOs: 126, 128, 131,
133, 136, 138, 140, 142, and 145, respectively. Exemplary DNA
sequences encoding the VL sequences of 3F4, 14B6-1, 14B6-2, 23H3,
6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1 are set
forth in SEQ ID NOs: 127, 129, 130, 132, 134, 135, 137, 139, 141,
143, 144, and 146, respectively. Exemplary DNA sequences encoding
heavy chain sequences are set forth in SEQ ID NOs: 147, 149, 151,
153, 155, 157, 159, 161, 163, 165, 167, 169171, 173, 175, 176, and
177. Exemplary DNA sequences encoding light chain sequences are set
forth in SEQ ID NOs: 148, 150, 152, 154, 156, 158, 160, 162, 164,
166, 168, 170, 172, and 174.
[0488] Exemplary nucleotide sequences encoding the mature VH and VL
domains of an anti-OX40 antibody are set forth in SEQ ID NOs: 145
and 146, respectively. An exemplary nucleotide sequence encoding
the heavy chain of an anti-OX40 antibody is set forth in SEQ ID NO:
177. An exemplary nucleotide sequence encoding the light chain of
an OX40 antibody is set forth in SEQ ID NO: 168.
[0489] Methods for making the anti-OX40 antibodies provided herein
can include expressing the heavy chain and the light chains in a
cell line comprising the nucleotide sequences encoding the heavy
and light chains with a signal peptide. Host cells comprising these
nucleotide sequences are also provided herein.
[0490] 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, refers to the joining of the two DNA fragments
such that the amino acid sequences they encode remain in-frame.
[0491] 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., 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 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.
[0492] 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.
[0493] 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
(Gly.sub.4-Ser).sub.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).
[0494] Also provided herein are nucleic acid molecules encoding VH
and VL sequences that are homologous to those of the 3F4, 14B6-1,
14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and
20C1 monoclonal antibodies. Exemplary nucleic acid molecules encode
VH and VL sequences that are at least 70% identical, for example,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 99% identical, to nucleic acid molecules encoding
the VH and VL sequences of the 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1,
6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1 monoclonal
antibodies. Also provided herein are nucleic acid molecules with
conservative substitutions (i.e., substitutions that do not alter
the resulting amino acid sequence upon translation of nucleic acid
molecule), e.g., for codon optimization.
XI. Antibody Generation
[0495] Anti-OX40 antibodies described 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.
[0496] 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.
[0497] 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.).
[0498] In one embodiment, the antibodies described herein are human
monoclonal antibodies. Such human monoclonal antibodies directed
against OX40 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."
[0499] 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.
[0500] In certain embodiments, antibodies described herein are
raised using a mouse that carries human immunoglobulin sequences on
transgenes and transchomosomes, 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.
[0501] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-OX40 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.
[0502] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-OX40 antibodies described herein. For
example, mice carrying both a human heavy chain transchromosome and
a human light chain tranchromosome, 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-OX40 antibodies described herein.
[0503] Additional mouse systems described in the art for raising
human antibodies, e.g., human anti-OX40 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.
[0504] 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.
[0505] 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
[0506] To generate fully human antibodies to OX40, 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 OX40 antigen and/or cells expressing
OX40 or fragment thereof, 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 OX40 or fragment thereof. 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 OX40 antigen
can be used to immunize the HuMAb mice intraperitoneally. In the
event that immunizations using a purified or enriched preparation
of the OX40 antigen do not result in antibodies, mice can also be
immunized with cells expressing OX40, e.g., a cell line, to promote
immune responses. Exemplary cell lines include OX40-overexpressing
stable CHO and Raji cell lines.
[0507] 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-OX40 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
OX40
[0508] To generate hybridomas producing the 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.
[0509] To purify the 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
antibodies can then be aliquoted and stored at -80.degree. C.
Generation of Transfectomas Producing Monoclonal Antibodies to
OX40
[0510] 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).
[0511] 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.
[0512] 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). Exemplary signal sequences for use in
antibody heavy and light chains include the signal sequences
originally found in the anti-OX40 antibodies described herein.
[0513] 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).
[0514] 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).
[0515] 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). The antibodies disclosed
herein can also be produced in glycoengineered strains of the yeast
Pichia pastoris. Li et al. (2006) Nat. Biotechnol. 24:210.
[0516] 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.
[0517] 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.
XII. Assays
[0518] Anti-OX40 antibodies described herein can be tested for
binding to OX40 by, for example, standard ELISA. Briefly,
microtiter plates are coated with purified OX40 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
OX40-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,
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 OX40, but not
to a control cell line that does not express OX40. Briefly, the
binding of anti-OX40 antibodies is assessed by incubating OX40
expressing CHO cells with the anti-OX40 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 which develop the highest titers will be used for
fusions.
[0519] An ELISA assay as described above can be used to screen for
antibodies and, thus, hybridomas that produce antibodies that show
positive reactivity with the OX40 immunogen. Hybridomas that
produce antibodies that bind, preferably with high affinity, to
OX40 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.
[0520] To purify anti-OX40 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 OD.sub.280 using 1.43 extinction coefficient.
The monoclonal antibodies can be aliquoted and stored at
-80.degree. C.
[0521] To determine if the selected anti-OX40 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 OX40 coated-ELISA plates as described above.
[0522] 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.
[0523] To test the binding of antibodies to live cells expressing
OX40, flow cytometry can be used, as described in the Examples.
Briefly, cell lines expressing membrane-bound OX40 (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
Fluorescein-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.
[0524] Anti-OX40 antibodies can be further tested for reactivity
with the OX40 antigen by Western blotting. Briefly, cell extracts
from cells expressing OX40 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.).
[0525] Methods for analyzing binding affinity, cross-reactivity,
and binding kinetics include standard assays known in the art, for
example, BIACORE.RTM. SPR analysis using a BIACORE.RTM. 2000 SPR
instrument (Biacore AB, Uppsala, Sweden).
[0526] In certain embodiments, the anti-OX40 antibody specifically
binds to the extracellular region of human OX40. For example, the
antibody may specifically bind to a particular domain (e.g., a
functional domain) within the extracellular domain of OX40. In a
particular embodiment, the antibody specifically binds to the site
on OX40 to which OX40-L binds. In other embodiments, the antibody
specifically binds to the extracellular region of human OX40 and
the extracellular region of cynomolgus OX40.
XIII. Immunoconjugates, Antibody Derivatives and Diagnostics
[0527] Anti-OX40 antibodies described herein can be used for
diagnostic purposes, including sample testing and in vivo imaging,
and for this purpose the antibody (or binding fragment thereof) can
be conjugated to an appropriate detectable agent, to form an
immunoconjugate. For diagnostic purposes, appropriate agents are
detectable labels that include radioisotopes, for whole body
imaging, and radioisotopes, enzymes, fluorescent labels and other
suitable antibody tags for sample testing.
[0528] The detectable labels can be any of the various types used
currently in the field of in vitro diagnostics, including
particulate labels including metal sols such as colloidal gold,
isotopes such as I.sup.125 or Tc.sup.99 presented for instance with
a peptidic chelating agent of the N.sub.2S.sub.2, N.sub.3S or
N.sub.4 type, chromophores including fluorescent markers, biotin,
luminescent markers, phosphorescent markers and the like, as well
as enzyme labels that convert a given substrate to a detectable
marker, and polynucleotide tags that are revealed following
amplification such as by polymerase chain reaction. A biotinylated
antibody would then be detectable by avidin or streptavidin
binding. Suitable enzyme labels include horseradish peroxidase,
alkaline phosphatase and the like. For instance, the label can be
the enzyme alkaline phosphatase, detected by measuring the presence
or formation of chemiluminescence following conversion of 1,2
dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyl
dioxetane (AMPPD), disodium
3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1
3,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and
CDP-Star.RTM. or other luminescent substrates well-known to those
in the art, for example the chelates of suitable lanthanides such
as Terbium(III) and Europium(III). The detection means is
determined by the chosen label. Appearance of the label or its
reaction products can be achieved using the naked eye, in the case
where the label is particulate and accumulates at appropriate
levels, or using instruments such as a spectrophotometer, a
luminometer, a fluorimeter, and the like, all in accordance with
standard practice.
[0529] Preferably, conjugation methods result in linkages which are
substantially (or nearly) non-immunogenic, e.g., peptide- (i.e.
amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-,
and ether linkages. These linkages are nearly non-immunogenic and
show reasonable stability within serum (see e.g. Senter, P. D.,
Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009/059278; WO
95/17886).
[0530] Depending on the biochemical nature of the moiety and the
antibody, different conjugation strategies can be employed. In case
the moiety is naturally occurring or recombinant polypeptide of
between 50 to 500 amino acids, there are standard procedures in
text books describing the chemistry for synthesis of protein
conjugates, which can be easily followed by the skilled artisan
(see e.g. Hackenberger, C. P. R., and Schwarzer, D., Angew. Chem.
Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment the
reaction of a maleinimido moiety with a cysteine residue within the
antibody or the moiety is used. This is an especially suited
coupling chemistry in case e.g. a Fab or Fab'-fragment of an
antibody is used. Alternatively in one embodiment coupling to the
C-terminal end of the antibody or moiety is performed. C-terminal
modification of a protein, e.g. of a Fab-fragment can e.g. be
performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem.
7 (2009) 3361-3371).
[0531] In general, site specific reaction and covalent coupling is
based on transforming a natural amino acid into an amino acid with
a reactivity which is orthogonal to the reactivity of the other
functional groups present. For example, a specific cysteine within
a rare sequence context can be enzymatically converted in an
aldehyde (see Frese, M. A., and Dierks, T., ChemBioChem. 10 (2009)
425-427). It is also possible to obtain a desired amino acid
modification by utilizing the specific enzymatic reactivity of
certain enzymes with a natural amino acid in a given sequence
context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004)
119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; and
Protease-catalyzed formation of C--N bonds is described in Bordusa,
F., Highlights in Bioorganic Chemistry (2004) 389-403).
[0532] Site specific reaction and covalent coupling can also be
achieved by the selective reaction of terminal amino acids with
appropriate modifying reagents.
[0533] The reactivity of an N-terminal cysteine with benzonitrils
(see Ren et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662)
can be used to achieve a site-specific covalent coupling.
[0534] Native chemical ligation can also rely on C-terminal
cysteine residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids
and Molecular Biology (2009), 22 (Protein Engineering), 65-96).
[0535] EP 1 074 563 describes a conjugation method which is based
on the faster reaction of a cysteine within a stretch of negatively
charged amino acids than a cysteine located in a stretch of
positively charged amino acids.
[0536] The moiety may also be a synthetic peptide or peptide mimic.
In case a polypeptide is chemically synthesized, amino acids with
orthogonal chemical reactivity can be incorporated during such
synthesis (see e.g. de Graaf et al., Bioconjug. Chem. 20 (2009)
1281-1295). Since a great variety of orthogonal functional groups
is at stake and can be introduced into a synthetic peptide,
conjugation of such peptide to a linker is standard chemistry.
[0537] In order to obtain a mono-labeled polypeptide, the conjugate
with 1:1 stoichiometry may be separated by chromatography from
other conjugation side-products. This procedure can be facilitated
by using a dye labeled binding pair member and a charged linker. By
using this kind of labeled and highly negatively charged binding
pair member, mono conjugated polypeptides are easily separated from
non-labeled polypeptides and polypeptides which carry more than one
linker, since the difference in charge and molecular weight can be
used for separation. The fluorescent dye can be useful for
purifying the complex from un-bound components, like a labeled
monovalent binder.
[0538] In one embodiment, the moiety attached to the anti-OX40
antibody is selected from the group consisting of a binding moiety,
a labeling moiety, and a biologically active moiety.
[0539] Anti-OX40 antibodies described herein also may be conjugated
to a therapeutic agent to form an immunoconjugate such as an
antibody-drug conjugate (ADC). Suitable therapeutic agents include
antimetabolites, alkylating agents, DNA minor groove binders, DNA
intercalators, DNA crosslinkers, histone deacetylase inhibitors,
nuclear export inhibitors, proteasome inhibitors, topoisomerase I
or II inhibitors, heat shock protein inhibitors, tyrosine kinase
inhibitors, antibiotics, and anti-mitotic agents. In the ADC, the
antibody and therapeutic agent preferably are conjugated via a
linker cleavable such as a peptidyl, disulfide, or hydrazone
linker. More preferably, the linker is a peptidyl linker such as
Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID
NO: 180), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys,
Lys, Cit, Ser, or Glu. The ADCs can be prepared as described in
U.S. Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; PCT
Publications WO 02/096910; WO 07/038658; WO 07/051081; WO
07/059404; WO 08/083312; and WO 08/103693; U.S. Patent Publications
20060024317; 20060004081; and 20060247295; the disclosures of which
are incorporated herein by reference.
[0540] More specifically, in an ADC, the antibody is conjugated to
a drug, with the antibody functioning as a targeting agent for
directing the ADC to a target cell expressing its antigen, such as
a cancer cell. Preferably, the antigen is a tumor associated
antigen, i.e., one that is uniquely expressed or overexpressed by
the cancer cell. Once there, the drug is released, either inside
the target cell or in its vicinity, to act as a therapeutic agent.
For a review on the mechanism of action and use of ADCs in cancer
therapy, see Schrama et al., Nature Rev. Drug Disc. 2006, 5,
147.
[0541] For cancer treatment, the drug preferably is a cytotoxic
drug that causes death of the targeted cancer cell. Cytotoxic drugs
that can be used in ADCs include the following types of compounds
and their analogs and derivatives: [0542] (a) enediynes such as
calicheamicin (see, e.g., Lee et al., J. Am. Chem. Soc. 1987, 109,
3464 and 3466) and uncialamycin (see, e.g., Davies et al., WO
2007/038868 A2 (2007) and Chowdari et al., U.S. Pat. No. 8,709,431
B2 (2012)); [0543] (b) tubulysins (see, e.g., Domling et al., U.S.
Pat. No. 7,778,814 B2 (2010); Cheng et al., U.S. Pat. No. 8,394,922
B2 (2013); and Cong et al., U.S. Ser. No. 14/177,376, filed Feb.
11, 2014)); [0544] (c) CC-1065 and duocarmycin (see, e.g., Boger,
U.S. Pat. No. 6,5458,530 B1 (2003); Sufi et al., U.S. Pat. No.
8,461,117 B2 (2013); and Zhang et al., US 2012/0301490 A1 (2012));
[0545] (d) epothilones (see, e.g., Vite et al., US 2007/0275904 A1
(2007) and U.S. RE42930 E (2011)); [0546] (e) auristatins (see,
e.g., Senter et al., U.S. Pat. No. 6,844,869 B2 (2005) and Doronina
et al., U.S. Pat. No. 7,498,298 B2 (2009)); [0547] (f)
pyrrolobezodiazepine (PBD) dimers (see, e.g., Howard et al., US
2013/0059800 A1(2013); US 2013/0028919 A1 (2013); and WO
2013/041606 A1 (2013)); and [0548] (g) maytansinoids such as DM1
and DM4 (see, e.g., Chari et al., U.S. Pat. No. 5,208,020 (1993)
and Amphlett et al., U.S. Pat. No. 7,374,762 B2 (2008)).
[0549] In an ADC, a linker covalently connects the antibody and the
drug. Typically, there is one drug molecule attached to each
linker, but the linker can be branched, allowing the attachment of
plural drug molecules to increase the drug payload delivered per
ADC. Further, each antibody may have more than one linker attached.
The number drug molecules carried on an ADC is referred to as the
drug-antibody ratio (DAR). For instance, if each heavy chain of the
antibody has attached to it one linker that in turn has one drug
molecule attached, the DAR is 2. Preferably, the DAR is between 1
and 5, more preferably between 2 and 4. Those skilled in the art
will also appreciate that, while in each individual ADC the
antibody is conjugated to an integer number of drug molecules, as a
whole, a preparation of the ADC may analyze for a non-integer DAR,
reflecting a statistical average. In summary, the architecture of
an ADC may be represented by the following formula:
[Antibody]-[Linker-(Drug).sub.n].sub.m
where typically m is 1, 2, 3, 4, 5, or 6 (preferably 2, 3, or 4)
and n is 1, 2, or 3.
[0550] In some embodiments, the linker contains a cleavable group
that is cleaved inside or in the vicinity of the target cell, to
release the drug. In other embodiments, the linker does not contain
a cleavable group but, rather, the ADC relies on catabolism of the
antibody to release the drug.
[0551] One type of cleavable group is a pH sensitive group. The pH
in blood plasma is slightly above neutral, while the pH inside a
lysosome--where most ADCs end up after internalization inside a
target cell--is acidic, circa 5. Thus, a cleavable group whose
cleavage is acid catalyzed will cleave at a rate several orders of
magnitude faster inside a lysosome than in the blood plasma.
Examples of acid-sensitive groups include cis-aconityl amides and
hydrazones, as described in Shen et al., U.S. Pat. No. 4,631,190
(1986); Shen et al., U.S. Pat. No. 5,144,011 (1992); Shen et al.,
Biochem. Biophys. Res. Commun. 1981, 102, 1048; and Yang et al.,
Proc. Nat'l Acad. Sci (USA), 1988, 85, 1189; the disclosures of
which are incorporated herein by reference.
[0552] In another embodiment, the cleavable group is a disulfide.
Disulfides can be cleaved by a thiol-disulfide exchange mechanism,
at a rate dependent on the ambient thiol concentration. As the
intracellular concentration of glutathione and other thiols is
higher than their serum concentrations, the cleavage rate of a
disulfide will be higher intracellularly, i.e., after
internalization of the ADC. Further, the rate of thiol-disulfide
exchange can be modulated by adjustment of the steric and
electronic characteristics of the disulfide (e.g., an alkyl-aryl
disulfide versus an alkyl-alkyl disulfide; substitution on the aryl
ring, etc.), enabling the design of disulfide linkages that have
enhanced serum stability or a particular cleavage rate. For
additional disclosures relating to disulfide cleavable groups in
conjugates, see, e.g., Thorpe et al., Cancer Res. 1988, 48, 6396;
Santi et al., U.S. Pat. No. 7,541,530 B2 (2009); Ng et al., U.S.
Pat. No. 6,989,452 B2 (2006); Ng et al., WO 2002/096910 A1 (2002);
Boyd et al., U.S. Pat. No. 7,691,962 B2 (2010); and Sufi et al., US
2010/0145036 A1 (2010); the disclosures of which are incorporated
herein by reference.
[0553] A preferred cleavable group is a peptide that is cleaved
selectively by a protease inside the target cell, as opposed to by
a protease in the serum. Typically, a cleavable peptide group
comprises from 1 to 20 amino acids, preferably from 1 to 6 amino
acids, more preferably from 1 to 3 amino acids. The amino acid(s)
can be natural and/or non-natural .alpha.-amino acids. Natural
amino acids are those encoded by the genetic code, as well as amino
acids derived therefrom, e.g., hydroxyproline,
.gamma.-carboxyglutamate, citrulline, and O-phosphoserine. In this
context, the term "amino acid" also includes amino acid analogs and
mimetics. Analogs are compounds having the same general
H.sub.2N(R)CHCO.sub.2H structure of a natural amino acid, except
that the R group is not one found among the natural amino acids.
Examples of analogs include homoserine, norleucine,
methionine-sulfoxide, and methionine methyl sulfonium. An amino
acid mimetic is a compound that has a structure different from the
general chemical structure of an .alpha.-amino acid but functions
in a manner similar to one. The amino acid can be of the "L"
stereochemistry of the genetically encoded amino acids, as well as
of the enantiomeric "D" stereochemistry.
[0554] Preferably, a cleavable peptide group contains an amino acid
sequence that is a cleavage recognition sequence for a protease.
Many cleavage recognition sequences are known in the art. See,
e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.
Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175
(1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al.
Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244:
412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995); the
disclosures of which are incorporated herein by reference.
[0555] More preferably, a cleavable peptide group comprises an
amino acid sequence selected for cleavage by an endosomal or
lysosomal protease, especially the latter. Examples of such
proteases include cathepsins B, C, D, H, L and S, especially
cathepsin B. Cathepsin B preferentially cleaves peptides at a
sequence -AA.sup.2-AA.sup.1- where AA.sup.1 is a basic or strongly
hydrogen bonding amino acid (such as lysine, arginine, or
citrulline) and AA.sup.2 is a hydrophobic amino acid (such as
phenylalanine, valine, alanine, leucine, or isoleucine), for
example Val-Cit (where Cit denotes citrulline) or Val-Lys, written
in the N-to-C direction. For additional information regarding
cathepsin-cleavable groups, see Dubowchik et al., Biorg. Med. Chem.
Lett. 1998, 8, 3341; Dubowchik et al., Bioorg. Med. Chem. Lett.,
1998, 8, 3347; and Dubowchik et al., Bioconjugate Chem. 2002, 13,
855; the disclosures of which are incorporated by reference.
Another enzyme that can be utilized for cleaving peptidyl linkers
is legumain, a lysosomal cysteine protease that preferentially
cleaves at Ala-Ala-Asn.
[0556] In a preferred embodiment, the linker in ADCs comprises a
di- or tripeptide that is preferentially cleaved by a protease
located inside the target cell. Preferably, the di- or tripeptide
is cleavable by cathepsin B, more preferably a Val-Cit or Val-Lys
dipeptide.
[0557] Single amino acid cleavable peptide groups also can be used,
as disclosed in Chen et al., US 2010/0113476 A1 (2010), the
disclosure of which is incorporated herein by reference.
[0558] For conjugates that are not intended to be internalized by a
cell, the cleavable group can be chosen such that it is cleaved by
a protease present in the extracellular matrix in the vicinity of
the target cell, e.g., a protease released by nearby dying cells or
a tumor-associated protease. Exemplary extracellular
tumor-associated proteases are matrix metalloproteases (MMP),
plasmin, thimet oligopeptidase (TOP) and CD10. See, e.g., Trouet et
al., U.S. Pat. No. 5,962,216 (1999) and U.S. Pat. No. 7,402,556 B2
(2008); Dubois et al., U.S. Pat. No. 7,425,541 B2 (2008); and
Bebbington et al., U.S. Pat. No. 6,897,034 B2 (2005); the
disclosures of which are incorporated herein by reference.
[0559] The linker can perform other functions in addition to
covalently linking the antibody and the drug. For instance, the
linker can contain poly(ethylene glycol) (PEG) groups, which
enhance solubility either during the performance the conjugation
chemistry or in the final ADC product.
[0560] The linker can further include a self-immolating moiety
located adjacent to a cleavable peptide group. The self-immolating
group serves as a spacer that prevents the antibody and/or the drug
moiety from sterically interfering with the cleavage of the peptide
group by a protease but thereafter spontaneously releases itself
(i.e., self-immolates) so as to not interfere with the action of
the drug. See Carl et al., J. Med. Chem. 1981, 24 (3), 479; Carl et
al., WO 81/01145 (1981); Dubowchik et al., Pharmacology &
Therapeutics 1999, 83, 67; Firestone et al., U.S. Pat. No.
6,214,345 B1 (2001); Toki et al., J. Org. Chem. 2002, 67, 1866;
Doronina et al., Nature Biotechnology 2003, 21 (7), 778 (erratum,
p. 941); de Groot et al., Org. Chem. 2001, 66, 8815; Boyd et al.,
U.S. Pat. No. 7,691,962 B2 (2010); Boyd et al., US 2008/0279868 A1
(2008); Sufi et al., WO 2008/083312 A2 (2008); Feng, U.S. Pat. No.
7,375,078 B2 (2008); Jeffrey, U.S. Pat. No. 8,039,273 B2 (2011);
and Senter et al., US 2003/0096743 A1 (2003); the disclosures of
which are incorporated by reference.
[0561] A preferred self-immolating group is a p-aminobenzyl
oxycarbonyl (PABC) group, whose structure and mechanism of action
is depicted below:
##STR00001##
[0562] Thus, in a preferred embodiment, an ADC has a linker
comprising a di- or tripeptide that is preferentially cleaved by a
protease located inside the target cell and, adjacent to the di- or
tripeptide, a self-immolating group. Preferably, the di- or
tripeptide is cleavable by cathepsin B. Preferably, the
self-immolating group is a PABC group.
[0563] Numerous techniques can be used for conjugating the antibody
and the drug. In a preferred one, an .epsilon.-amino group in the
side chain of a lysine residue in the antibody is reacted with
2-iminothiolane to introduce a free thiol (--SH) group. The thiol
group can react with a maleimide or other nucleophile acceptor
group to effect conjugation, as illustrated below:
##STR00002##
[0564] Typically, a thiolation level of two to three thiols per
antibody is achieved. For a representative procedure, see Chowdari
et al., U.S. Pat. No. 8,709,431 B2 (2014), the disclosure of which
is incorporated herein by reference. Thus, in one embodiment, an
antibody of this invention has one or more lysine residues
(preferably two or three) modified by reaction with
iminothiolane.
[0565] An alternative conjugation technique employs copper-free
"click chemistry," in which an azide group adds across the strained
alkyne bond of a cyclooctyne to form an 1,2,3-triazole ring. See,
e.g., Agard et al., J. Amer. Chem. Soc. 2004, 126, 15046; Best,
Biochemistry 2009, 48, 6571, the disclosures of which are
incorporated herein by reference. The azide can be located on the
antibody and the cyclooctyne on the drug moiety, or vice-versa. A
preferred cyclooctyne group is dibenzocyclooctyne (DIBO). Various
reagents having a DIBO group are available from
Invitrogen/Molecular Probes, Eugene, Oreg. The reaction below
illustrates click chemistry conjugation for the instance in which
the DIBO group is attached to the antibody:
##STR00003##
In an ADC made by this technique, the linker comprises a
1,2,3-triazole ring.
[0566] Yet another conjugation technique involves introducing a
non-natural amino acid into an antibody, with the non-natural amino
acid providing a functionality for conjugation with a reactive
functional group in the drug moiety. For instance, the non-natural
amino acid p-acetylphenylalanine can be incorporated into an
antibody or other polypeptide, as taught in Tian et al., WO
2008/030612 A2 (2008). The ketone group in p-acetylphenylalanine
can be a conjugation site by the formation of an oxime with a
hydroxylamino group on the linker-drug moiety. Alternatively, the
non-natural amino acid p-azidophenylalanine can be incorporated
into an antibody to provide an azide functional group for
conjugation via click chemistry, as discussed above. Non-natural
amino acids can also be incorporated into an antibody or other
polypeptide using cell-free methods, as taught in Goerke et al., US
2010/0093024 A1 (2010) and Goerke et al., Biotechnol. Bioeng. 2009,
102 (2), 400-416. The foregoing disclosures are incorporated herein
by reference. Thus, in one embodiment, the antibody has one or more
amino acids replaced by a non-natural amino acid, which preferably
is p-acetylphenylalanine or p-azidophenylalanine, more preferably
p-acetylphenylalanine.
[0567] Still another conjugation technique uses the enzyme
transglutaminase (preferably bacterial transglutaminase or BTG), as
taught in Jeger et al., Angew. Chem. Int. Ed. 2010, 49, 9995. BTG
forms an amide bond between the side chain carboxamide of a
glutamine and an alkyleneamino group, which can be, for example,
the .epsilon.-amino group of a lysine or a 5-amino-n-pentyl group.
In a typical conjugation reaction, the glutamine residue is located
on the antibody, while the alkyleneamino group is located on the
linker-drug moiety, as shown below:
##STR00004##
[0568] The positioning of a glutamine residue on a polypeptide
chain has a large effect on its susceptibility to BTG mediated
transamidation. None of the glutamine residues on an antibody are
normally BTG substrates. However, if the antibody is
deglycosylated--the glycosylation site being asparagine 297
(N297)--nearby glutamine 295 (Q295) is rendered BTG susceptible.
Alternatively, an antibody can be synthesized glycoside free by
introducing an N297A mutation in the constant region, to eliminate
the N297 glycosylation site. Further, it has been shown that an
N297Q substitution in an antibody not only eliminates
glycosylation, but also introduces a second glutamine residue (at
position 297) that too is susceptible BTG-mediated transamidation.
Thus, in one embodiment, the anti-OX40 antibody is deglycosylated.
In another embodiment, the anti-OX40 antibody has an N297Q
substitution. Those skilled in the art will appreciate that
deglycosylation by post-synthesis modification or by introducing an
N297A mutation generates two BTG-reactive glutamine residues per
antibody (one per heavy chain, at position 295), while an antibody
with an N297Q substitution will have four BTG-reactive glutamine
residues (two per heavy chain, at positions 295 and 297).
[0569] Further, another conjugation technique uses the enzyme
Sortase A, as taught in Levary et al., PLoS One 2011, 6(4), e18342;
Proft, Biotechnol. Lett. 2010, 32, 1-10; Ploegh et al., WO
2010/087994 A2 (2010); and Mao et al., WO 2005/051976 A2 (2005),
the disclosures of which are incorporated herein by reference. The
Sortase A recognition motif (typically LPXTG (SEQ ID NO: 181),
where X is any natural amino acid) may be attached to the antibody
and the nucleophilic acceptor motif (typically GGG) may be located
on the drug moiety, or vice-versa.
[0570] Anti-OX40 antibodies described herein also can be used for
detecting OX40, such as human OX40, e.g., human OX40 in tissues or
tissue samples. The antibodies may be used, e.g., in an ELISA assay
or in flow cytometry. In certain embodiments, the anti-OX40
antibody is contacted with cells, e.g., cells in a tissue, for a
time appropriate for specific binding to occur, and then a reagent,
e.g., an antibody that detects the anti-OX40 antibody, is added.
Exemplary assays are provided in the Examples. The anti-OX40
antibody may be a fully human antibody, or it may be a chimeric
antibody, such as an antibody having human variable regions and
murine constant regions or a portion thereof. Exemplary methods for
detecting OX40, e.g., human OX40, in a sample (cell or tissue
sample) comprise (i) contacting a sample with an anti-OX40
antibody, for a time sufficient for allowing specific binding of
the anti-OX40 antibody to OX40 in the sample, and (2) contacting
the sample with a detection reagent, e.g., an antibody, that
specifically binds to the anti-OX40 antibody, such as to the Fc
region of the anti-OX40 antibody, to thereby detect OX40 bound by
the anti-OX40 antibody. Wash steps may be included after the
incubation with the antibody and/or detection reagent. Anti-OX40
antibodies for use in these methods do not have to be linked to a
label or detection agents, as a separate detection agent can be
used.
XIV. Bispecific Molecules
[0571] Anti-OX40 antibodies described herein may be used for
forming bispecific molecules. For example, the antibody, or
antigen-binding portions 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. In one embodiment, the anti-OX40
antibody may be linked to an antibody or scFv that binds
specifically to any protein that may be used as potential targets
for combination treatments, such as the proteins described herein
(e.g., antibodies to PD-1, PD-L1, or LAG-3). Alternatively, the
antibody may 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, the anti-OX40 antibody 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.
[0572] Accordingly, provided herein are bispecific molecules
comprising at least one first binding specificity for OX40 and a
second binding specificity for a second target epitope. In one
embodiment, the bispecific molecule is multispecific, e.g., the
molecule further includes a third binding specificity.
[0573] In certain embodiments, the bispecific molecules comprises
as a binding specificity at least one antibody, or an antibody
fragment thereof, including, e.g., an Fab, Fab', F(ab').sub.2, Fv,
or a single chain Fv (scFv). 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.
[0574] While human monoclonal antibodies are preferred, other
antibodies can be employed in the bispecific molecules described
herein, including, e.g., murine, chimeric and humanized
antibodies.
[0575] Bispecific molecules provided 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.).
[0576] 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.
[0577] 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, mAb.times.(scFv).sub.2,
Fab.times.F(ab').sub.2 or ligand.times.Fab fusion protein. A
bispecific antibody may comprise an antibody comprising an scFv at
the C-terminus of each heavy chain. 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. No. 5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No.
4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat. No. 5,091,513; U.S.
Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S. Pat. No.
5,258,498; and U.S. Pat. No. 5,482,858.
[0578] 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.
XV. Compositions
[0579] Further provided are compositions, e.g., a pharmaceutical
compositions, containing one or more anti-OX40 antibodies, alone or
in combination with antibodies to other targets, 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, the composition can comprise a
combination of antibodies (or immunoconjugates or bispecifics)
described herein that bind to different epitopes on OX40 or that
have complementary activities.
[0580] In certain embodiments, the composition comprises an
anti-OX40 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, 1-300 mg/ml,
or 100-300 mg/ml.
[0581] Pharmaceutical compositions described herein also can be
administered in combination therapies, i.e., combined with other
agents. For example, the combination therapy can include
administration of an anti-OX40 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.
[0582] In certain embodiments, therapeutic compositions disclosed
herein 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-PDL-1 antibody, an anti-GITR antibody, an anti-CD137 antibody,
or an anti-LAG-3 antibody.
[0583] As used herein, "pharmaceutically acceptable carriers"
include 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.
[0584] The pharmaceutical compositions 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.
[0585] The pharmaceutical compositions 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.
[0586] 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.
[0587] 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 which delay absorption
such as aluminum monostearate and gelatin.
[0588] 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. A
pharmaceutical composition may comprise a preservative or may be
devoid of a preservative. Supplementary active compounds can be
incorporated into the compositions.
[0589] 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.
[0590] 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 herein. 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.
[0591] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred 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.
[0592] 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.
[0593] For administration of the anti-OX40 antibody, the dosage
ranges from about 0.0001 to 100 mg/kg, about 0.01 to 5 mg/kg, about
0.01 to 10 mg/kg, about 0.1 to 1 mg/kg, about 0.1 to 0.5 mg/kg, or
about 0.5 to 0.8 mg/kg of the host body weight. For example,
dosages can be 0.2 mg/kg body weight, 0.3 mg/kg body weight, 0.5
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. In certain embodiments, the dosage is 0.2 mg/kg. In
some embodiments, the dosage is 0.25 mg/kg. In other embodiments,
the dosage is 0.5 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. Exemplary dosage regimens
for the antibodies described herein include 1 mg/kg body weight or
3 mg/kg body weight via intravenous administration, with the
antibody being given using one of the following dosing schedules:
(i) every four weeks for six dosages, then every three months; (ii)
every three weeks; (iii) 3 mg/kg body weight once followed by 1
mg/kg body weight every three weeks.
[0594] In certain embodiments, for combination treatment with an
anti-OX40 antibody and anti-PD-1 or anti-CTLA-4 antibody, the
antibodies are administered at a fixed dose. Accordingly, in some
embodiments, the anti-OX40 antibody is administered at a fixed dose
of about 25 to about 320 mg, for example, about 25 to about 160 mg,
about 25 to about 80 mg, about 25 to about 40 mg, about 40 to about
320 mg, about 40 to about 160 mg, about 40 to about 80 mg, about 80
to about 320 mg, about 30 to about 160 mg, or about 160 to about
320 mg. In one embodiment, the anti-OX40 antibody is administered
at a dose of 20 mg or about 20 mg. In another embodiment, the
anti-OX40 antibody is administered at a dose of 40 mg or about 40
mg. In another embodiment, the anti-OX40 antibody is administered
at a dose of 80 mg or about 80 mg. In another embodiment, the
anti-OX40 antibody is administered at a dose of 160 mg or about 160
mg. In another embodiment, the anti-OX40 antibody is administered
at a dose of 320 mg or about 320 mg.
[0595] In some embodiments, the anti-PD-1 antibody is administered
at a fixed dose of about 100 to 300 mg, For example, the dosage of
the immuno-oncology agent can be 240 mg or about 240 mg, 360 mg or
about 360 mg, or 480 mg or about 480 mg. In certain embodiments,
the dose of the anti-PD1 antibody 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 or about 0.3 mg/kg
body weight, 1 mg/kg body weight or about 1 mg/kg body weight, 3
mg/kg body weight or about 3 mg/kg body weight, 5 mg/kg body weight
or about 5 mg/kg body weight, or 10 mg/kg body weight or about 10
mg/kg body weight, or within the range of 1-10 mg/kg. In some
embodiments, the dosage of the anti-PD-1 antibody is 240 mg or
about 240 mg administered once every 2 weeks (Q2W). This dosage can
be adjusted proportionately (at 120 mg per week) for longer or
shorter periods, e.g., 360 mg administered once every 3 weeks (Q3W)
or 480 mg administered once every 4 weeks (Q4W).
[0596] In some embodiments, the anti-CTLA-4 antibody is
administered at a dose of about 0.1 mg/kg to about 10 mg/kg. For
example, dosages can be 1 mg/kg or about 1 mg/kg or 3 mg/kg or
about 3 mg/kg, of the host body weight.
[0597] Exemplary dosage regimens for combination treatment with an
anti-OX40 and anti-PD-1 or anti-CTLA-4 antibody are provided infra
under the section titled "Uses and Methods."
[0598] In certain embodiments, the anti-OX40 antibody is
administered to a patient with an infusion duration of about 15
minutes to about 60 minutes, for example, about 30 minutes.
[0599] In certain embodiments, the anti-PD-1 antibody (e.g.,
nivolumab) is administered to a patient with an infusion duration
of about 15 minutes to about 60 minutes, for example, about 30
minutes, when administered at a dose of 3 mg/kg (0.1 mg/kg/min). In
certain embodiments, the anti-PD-1 antibody is administered to a
patient with an infusion duration of about 45 minutes to 75
minutes, for example, about 60 minutes, when administered at a dose
of 10 mg/kg.
[0600] In certain embodiments, the anti-CTLA-4 antibody (e.g.,
ipilimumab) is administered to a patient with an infusion duration
of about 15 minutes to 120 minutes, for example, about 30 minutes
when administered at a dose of 3 mg/kg. In certain embodiments, the
anti-CTLA-4 antibody is administered to a patient with an infusion
duration of about 15 minutes to 120 minutes, for example, 90
minutes, when administered at a dose of 10 mg/kg.
[0601] In certain embodiments, when administered on the same day,
the anti-OX40 antibody is administered before the anti-PD-1 or
anti-CTLA-4 antibody. In certain embodiments, when administered on
the same day, the anti-OX40 antibody is administered after the
anti-PD-1 or anti-CTLA-4 antibody. In certain embodiments, when
administered on the same day, the anti-OX40 antibody is
administered simultaneously with the anti-PD-1 or anti-CTLA-4
antibody.
[0602] In certain embodiments, when administered on the same day,
the anti-OX40 antibody is administered about 15 to 45 minutes
(e.g., about 30 minutes) before the anti-PD-1 or anti-CTLA-4
antibody. In certain embodiments, when administered on the same
day, the anti-OX40 antibody is administered about 15 to 45 minutes
(e.g., about 30 minutes) after the anti-PD-1 or anti-CTLA-4
antibody.
[0603] Alternatively, anti-OX40 antibodies provided herein can be
administered at a flat dose (flat dose regimen).
[0604] In some cases, two or more monoclonal antibodies with
different binding specificities are administered simultaneously,
such that the dosage of each antibody administered falls within the
ranges above. In addition, the antibodies usually are 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.
[0605] Anti-OX40 antibodies described herein may be administered
with another antibody at the dosage regimen of the other antibody.
For example, the anti-OX40 antibody may be administered with an
anti-PD-1 antibody, such as nivolumab (OPDIVO), every two weeks as
an i.v. infusion over 60 minutes until disease progression or
unacceptable toxicity occurs. Alternatively, the anti-OX40 antibody
may be administered with pembrolizumab (KEYTRUDA) every 3 weeks as
an i.v. infusion over 30 minutes until disease progression or
unacceptable toxicity occurs.
[0606] Antibodies 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 be administered a prophylactic
regime.
[0607] 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 which 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.
[0608] "Therapeutically effective dosages" of the antibodies
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 results in increased
survival, and/or prevention of 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.
[0609] 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 OX40 levels), 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.
[0610] Antibodies and compositions 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.
[0611] Alternatively, the antibody 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.
[0612] 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.
[0613] Antibody compositions can be administered with medical
devices known in the art. For example, in one embodiment, the
composition is 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 in
administering the antibodies 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 medicants 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.
[0614] In certain embodiments, the anti-OX40 antibodies are
formulated to ensure proper distribution in vivo. For example, the
blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure the antibodies cross the BBB (if desired,
e.g., for brain cancers), 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 which 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); p120
(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.
Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion;
I. J. Fidler (1994) Immunomethods 4:273.
XVI. Uses and Methods
[0615] Anti-OX40 antibodies and compositions described herein have
numerous in vitro and in vivo applications involving, for example,
enhancement of immune response by activating OX40 signaling, or
detection of OX40. In a preferred embodiment, the antibodies are
human antibodies. For example, anti-OX40 antibodies described
herein can be contacted with cells in culture, in vitro or ex vivo,
or administered 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
portion thereof, described herein such that the immune response in
the subject is modified. Preferably, the response is enhanced,
stimulated or up-regulated.
[0616] 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., a T-cell mediated immune response, e.g., an antigen specific
T cell response). In a particular embodiment, the methods are
particularly suitable for treatment of cancer in vivo. To achieve
antigen-specific enhancement of immunity, anti-OX40 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
anti-OX40 antibodies are administered together with another agent,
the two can be administered separately or simultaneously.
[0617] Also encompassed are methods for detecting the presence of
human OX40 antigen in a sample, or measuring the amount of human
OX40 antigen, comprising contacting the sample, and a control
sample, with anti-OX40 antibodies (or antigen binding portions
thereof) described herein, under conditions that allow for
formation of a complex between the antibody and human OX40. 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 OX40 antigen in the sample. The
anti-OX40 antibodies described herein also can be used to purify
human OX40 via immunoaffinity purification.
[0618] Given the ability of anti-OX40 antibodies described herein
to stimulate or co-stimulate T cell responses, e.g.,
antigen-specific T cell responses, also provided herein are in
vitro and in vivo methods of using the antibodies to stimulate,
enhance or upregulate antigen-specific T cell responses, e.g.,
anti-tumor T cell responses. In certain embodiments, CD3
stimulation is also included (e.g., by coincubation with a cell
expressing membrane CD3), which stimulation can be provided at the
same time, before, or after stimulation with an anti-OX40 antibody.
In one embodiment, the method comprises contacting T cells with an
anti-OX40 antibody described herein, and optionally with an
anti-CD3 antibody, such that an antigen-specific T cell response is
stimulated. Any suitable indicator of an antigen-specific T cell
response can be used to measure the antigen-specific T cell
response. Non-limiting examples of such suitable indicators include
increased T cell proliferation in the presence of the antibody
and/or increase cytokine production in the presence of the
antibody. In a preferred embodiment, interleukin-2 and/or
interferon-.gamma. production by the antigen-specific T cell is
stimulated.
[0619] T cells that can be enhanced or co-stimulated with anti-OX40
antibodies include CD4+ T cells and CD8+ T cells. The T cells can
be T.sub.eff cells, e.g., CD4+ T.sub.eff cells, CD8+ T.sub.eff
cells, Thelper (T.sub.h) cells and T cytotoxic (T.sub.c) cells.
[0620] Also provided are methods of stimulating an immune response
(e.g., an antigen-specific T cell response) in a subject comprising
administering a therapeutically effective amount of an anti-OX40
antibody described herein to the subject such that an immune
response (e.g., an antigen-specific T cell response) in the subject
is stimulated. In a preferred embodiment, the subject is a
tumor-bearing subject and an immune response against the tumor is
stimulated. 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 stimulated.
[0621] Further provided are methods for inhibiting growth of tumor
cells in a subject comprising administering to the subject a
therapeutically effective amount of an anti-OX40 antibody described
herein such that growth of the tumor is inhibited in the subject.
Also provided are methods of treating viral infection in a subject
comprising administering to the subject an anti-OX40 antibody
described herein such that the viral infection is treated in the
subject.
[0622] Also encompassed herein are methods for depleting Treg cells
from the tumor microenvironment of a subject having a tumor, e.g.,
cancerous tumor, comprising administering to the subject a
therapeutically effective amount of an anti-OX40 antibody described
herein that comprises an Fc that stimulates depletion of T.sub.reg
cells in the tumor microenvironment. An Fc may, e.g., be an Fc with
effector function or enhanced effector function, such as binding or
having enhanced binding to one or more activating Fc receptors. In
a preferred embodiment, T.sub.reg depletion occurs without
significant depletion or inhibition of T.sub.eff in the tumor
microenvironment, and without significant depletion or inhibition
of T.sub.eff cells and T.sub.reg cells outside of the tumor
microenvironment, e.g., in the periphery. In certain embodiments,
the subject has higher levels of OX40 on T.sub.reg cells than on
T.sub.eff cells, e.g., in the tumor microenvironment.
[0623] In certain embodiments, the subject is treated with an
anti-OX40 antibody having an Fc that enhances agonism, e.g., binds
to or has enhanced binding to the inhibitory FcRIIb. Anti-OX40
antibodies may deplete Tregs in tumors and/or Tregs in tumor
infiltrating lymphocytes (TILs).
[0624] In certain embodiments, the anti-OX40 antibody is given to a
subject as an adjunctive therapy. Treatments of subjects having
cancer with the anti-OX40 antibody may lead to prolonged survival,
e.g., long-term durable response relative to the current standard
of care; long term survival of at least 3 months, 6 months, 9
months, 1, 2, 3, 4, 5, 10 or more years, or recurrence-free
survival of at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5,
or 10 or more years. In certain embodiments, treatment of a subject
having cancer with the anti-OX40 antibody prevents recurrence of
cancer or delays recurrence of cancer by, e.g., 3 months, 6 months,
9 months, 1, 2, 3, 4, 5, or 10 or more years. The anti-OX40
antibody treatment can be used as a first-, second-, or third-line
treatment.
[0625] In preferred embodiments, the anti-OX40 antibody is not
significantly toxic. For example, the antibody is not significantly
toxic to an organ of a human, e.g., one or more of the liver,
kidney, brain, lungs, and heart, as determined, e.g., in clinical
trials. In certain embodiments, the antibody does not significantly
trigger an undesirable immune response, e.g., autoimmunity or
inflammation.
[0626] In certain embodiments, treatment of a subject with the
anti-OX40 antibody does not result in overstimulation of the immune
system to the extent that the subject's immune system then attacks
the subject itself (e.g., autoimmune response) or results in, e.g.,
anaphylaxis. Thus, the antibodies preferably do not cause
anaphylaxis.
[0627] In certain embodiments, treatment of a subject with the
anti-OX40 antibody does not cause significant inflammatory
reactions, e.g., immune-mediated pneumonitis, immune-mediated
colitis, immune mediated hepatitis, immune-mediated nephritis or
renal dysfunction, immune-mediated hypophysitis, immune-mediated
hypothyroidism and hyperthyroidism, or other immune-mediated
adverse reactions.
[0628] In certain embodiments, the anti-OX40 antibody provides
synergistic anti-tumor effects in combination with another cancer
therapy, such as a compound that stimulates the immune system
(e.g., an immune-oncology agent), e.g., a compound described herein
or a compound modulating a target described herein.
[0629] These and other methods described herein are discussed in
further detail below.
Cancer
[0630] Activation of OX40 by anti-OX40 antibodies can enhance the
immune response to cancerous cells in the patient. Accordingly,
provided herein are methods for treating a subject having cancer,
comprising administering to the subject the anti-OX40 antibodies
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 and/or that prolonged survival is achieved. The
anti-OX40 antibody can be used alone to inhibit the growth of
cancerous tumors. Alternatively, the anti-OX40 antibody can be used
in conjunction with another agent, e.g., another immunogenic agent,
a standard cancer treatment, or another antibody, as described
below.
[0631] 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
anti-OX40 antibodies described herein. The antibody may be a human
antibody. Additionally or alternatively, the antibody can be a
chimeric or humanized antibody.
[0632] Also provided herein are combination therapies comprising
administration of an anti-OX40 antibody and an anti-PD-1 or
anti-CTLA-4 antibody to treat subjects having tumors (e.g.,
advanced solid tumors).
[0633] In certain embodiments, provided herein are methods of
treating cancer wherein an anti-OX40 antibody and an anti-PD-1
antibody or anti-CTLA-4 antibody are administered to a patient with
a tumor (e.g., advanced solid tumor) according to a defined
clinical dosage regimen. In certain embodiments, the anti-OX40
antibody is OX40.21. In certain embodiments, the anti-PD-1 antibody
is BMS-936558 (nivolumab). In certain embodiments, the anti-CTLA-4
antibody is ipilimumab (Yervoy.RTM.). In certain embodiments,
dosage regimens are adjusted to provide the optimum desired
response (e.g., an effective response).
[0634] As used herein, adjunctive or combined administration
(coadministration) includes simultaneous administration of the
compounds in the same or different dosage form, or separate
administration of the compounds (e.g., sequential administration).
Thus, the anti-OX40 and anti-PD-1 antibody or anti-CTLA-4 antibody
can be simultaneously administered in a single formulation.
Alternatively, the anti-OX40 and anti-PD-1 antibody or anti-CTLA-4
antibody can be formulated for separate administration and are
administered concurrently or sequentially (e.g., one antibody is
administered within about 30 minutes prior to administration of the
second antibody).
[0635] For example, the anti-PD1 antibody or anti-CTLA-4 antibody
can be administered first and followed by (e.g., immediately
followed by) the administration of the anti-OX40 antibody, or vice
versa. In certain embodiments, the anti-PD-1 antibody or
anti-CTLA-4 antibody is administered prior to administration of the
anti-OX40 antibody. In another embodiment, the anti-PD-1 antibody
or anti-CTLA-4 antibody is administered after administration of the
anti-OX40 antibody. In another embodiment, the anti-OX40 antibody
and anti-PD-1 antibody or anti-CTLA-4 antibody are administered
concurrently. Such concurrent or sequential administration
preferably results in both antibodies being simultaneously present
in treated patients.
[0636] Cancers whose growth may be inhibited with anti-OX40
antibodies, or combination therapy with an anti-OX40 and an
anti-PD-1 or anti-CTLA-4 antibody, include cancers typically
responsive to immunotherapy and those that are not typically
responsive to immunotherapy. Cancers may be cancers with solid
tumors or blood malignancies (liquid tumors). 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 squamous 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 cancer, 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
or cancers of viral origin (e.g., human papilloma virus
(HPV-related or -originating tumors)), 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 (M0), 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 hematologic malignancy, e.g., 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/leukaemia
(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, B cell 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,
unresectable and/or refractory cancers (e.g., cancers refractory to
previous immunotherapy, e.g., with a blocking CTLA-4 or PD-1
antibody), and recurrent cancers.
[0637] In certain embodiments, the patient being treated with the
anti-OX40 antibody, or combination of anti-OX40 antibody and
anti-PD-1 or anti-CTLA-4 antibody, has an advanced solid tumor. For
example, in one embodiment, the patient to be treated has cervical
cancer. In another embodiment, the patient to be treated has
colorectal (CRC) cancer. In another embodiment, the patient to be
treated has bladder cancer (e.g., unresectable locally advanced or
metastatic bladder cancer). In another embodiment, the patient to
be treated has ovarian cancer (e.g., unresectable locally advanced
or metastatic ovarian cancer).
[0638] In one embodiment, the patient being treated with the
anti-OX40 antibody, or combination of anti-OX40 antibody and
anti-PD-1 or anti-CTLA-4 antibody, has non-small cell lung cancer
(NSCLC). In another embodiment, the patient to be treated has
squamous cell carcinoma of the head and neck (SCCHN). In another
embodiment, the patient to be treated has B-cell non-Hodgkin's
lymphoma (B-NHL). In another embodiment, the patient to be treated
has myeloma. In another embodiment, the patient has melanoma. In
another embodiment, the patient to be treated has diffuse large
B-cell lymphoma (DLBCL).
[0639] In certain embodiments, the anti-OX40 antibody is
administered to patients having a cancer that exhibited an
inadequate response to a prior treatment, e.g., a prior treatment
with an immuno-oncology drug, or patients having a cancer that is
refractory or resistant, either intrinsically refractory or
resistant (e.g., refractory to a PD-1 pathway antagonist), or a
wherein the resistance or refractory state is acquired. For
example, subjects who are not responsive or not sufficiently
responsive to a first therapy or who see disease progression
following treatment, e.g., anti-PD-1 treatment, may be treated by
administration of the anti-OX40 antibody alone or in combination
with another therapy (e.g., with an anti-PD-1 therapy).
[0640] In certain embodiments, the anti-OX40 antibody is
administered to patients who have not previously received (i.e.,
been treated with) an immuno-oncology agent, e.g., a PD-1 pathway
antagonist.
[0641] In certain embodiments, the anti-OX40 antibody may be
administered with a standard of care treatment (e.g., surgery,
radiation, and chemotherapy). In other embodiments, the anti-OX40
antibody may be administered as a maintenance therapy, e.g., a
therapy that is intended to prevent the occurrence or recurrence of
tumors.
[0642] In certain embodiments, the anti-OX40 antibody may be
administered with another treatment, e.g., radiation, surgery, or
chemotherapy. For example, anti-OX40 antibody adjunctive therapy
may be administered when there is a risk that micrometastases may
be present and/or in order to reduce the risk of a relapse.
[0643] In certain embodiments, the anti-OX40 antibody can be
administered as a monotherapy, or as the only immunostimulating
therapy. In other embodiments, the anti-OX40 antibody can also be
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).
[0644] In humans, some tumors have been shown to be immunogenic
such as melanomas. By lowering the threshold of T cell activation
via OX40 activation, the tumor responses in the host can be
activated, allowing treatment of non-immunogenic tumors or those
having limited immunogenicity.
[0645] In some embodiments, the anti-OX40 antibody can be used in
conjunction with a vaccination protocol. 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 such strategy, 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 U.S.A. 90: 3539-43).
[0646] 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. OX40
activation 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.
[0647] 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 which can be used in
conjunction with OX40 activation 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).
[0648] 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 OX40
activation to activate more potent anti-tumor responses.
[0649] Anti-OX40 antibodies described herein can also be 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).
For example, the anti-OX40 antibody can be used in combination with
decarbazine to treat melanoma. In another example, the anti-OX40
antibody can be used in combination with interleukin-2 (IL-2) to
treat melanoma. The scientific rationale behind the combined use of
anti-OX40 antibodies and chemotherapy is that cell death, 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 anti-OX40 antibodies 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 used in combination with the
anti-OX40 antibody. Inhibition of angiogenesis leads to tumor cell
death which may feed tumor antigen into host antigen presentation
pathways.
[0650] Anti-OX40 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
the activation of OX40. Alternatively, antigen may be delivered
directly to DCs by the use of bispecific antibodies which bind to
tumor antigen and a dendritic cell specific cell surface
marker.
[0651] Tumors evade host immune surveillance by a large variety of
mechanisms. Many of these mechanisms may be overcome by the
inactivation of proteins which are expressed by the tumors and
which are immunosuppressive. 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-OX40 antibodies
to counteract the effects of the immunosuppressive agent and favor
tumor immune responses by the host.
[0652] Other antibodies which activate host immune responsiveness
can be used in combination with the anti-OX40 antibodies described
herein. These include molecules on the surface of dendritic cells
which activate DC function and antigen presentation. Anti-CD40
antibodies are able to substitute effectively for T cell helper
activity (Ridge et al. (1998) Nature 393: 474-478) and can be used
in conjunction with anti-OX40 antibodies. 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),
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-OX40
antibodies.
[0653] Bone marrow transplantation is currently being used to treat
a variety of tumors of hematopoietic origin. While graft versus
host disease is a consequence of this treatment, therapeutic
benefit may be obtained from graft vs. tumor responses. Anti-OX40
antibodies can be used to increase the effectiveness of the donor
engrafted tumor specific T cells.
[0654] 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-OX40 antibodies can
increase the frequency and activity of the adoptively transferred T
cells.
Infectious Diseases
[0655] Also provided herein are methods to treat patients who have
been exposed to particular toxins or pathogens. Accordingly,
provided herein are methods of treating an infectious disease in a
subject comprising administering to the subject anti-OX40
antibodies described herein, such that the subject is treated for
the infectious disease. In certain embodiments, the anti-OX40
antibody is a chimeric or humanized antibody.
[0656] Similar to its application to tumors as discussed above,
anti-OX40 antibodies can be used alone, or as an adjuvant, in
combination with vaccines, to stimulate 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. Anti-OX40 antibodies
may be 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-OX40 antibody administration, thus provoking a strong T cell
response.
[0657] Some examples of pathogenic viruses causing infections
treatable by the 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.
[0658] Some examples of pathogenic bacteria causing infections
treatable by the 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.
[0659] Some examples of pathogenic fungi causing infections
treatable by the 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.
[0660] Some examples of pathogenic parasites causing infections
treatable by the 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.
[0661] In all of the above methods, anti-OX40 antibodies 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).
Autoimmune Reactions
[0662] Anti-OX40 antibodies may provoke and amplify autoimmune
responses. Indeed, induction of anti-tumor responses using tumor
cell and peptide vaccines reveals that many anti-tumor responses
involve anti-self reactivities (van Elsas et al. (2001) J. Exp.
Med. 194:481-489; Overwijk, et al. (1999) Proc. Natl. Acad. Sci.
U.S.A. 96: 2982-2987; Hurwitz, (2000) supra; Rosenberg & White
(1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4).
Therefore, anti-OX40 antibodies can be used in conjunction with
various self proteins in order to devise vaccination protocols to
efficiently generate immune responses against these self proteins
for disease treatment. For example, Alzheimer's disease involves
inappropriate accumulation of A.beta. peptide in amyloid deposits
in the brain; antibody responses against amyloid are able to clear
these amyloid deposits (Schenk et al., (1999) Nature 400:
173-177).
[0663] Other self proteins can also be used as targets such as IgE
for the treatment of allergy and asthma, and TNFc for rheumatoid
arthritis. Finally, antibody responses to various hormones may be
induced by the use of anti-OX40 antibodies. Neutralizing antibody
responses to reproductive hormones can be used for contraception.
Neutralizing antibody response to hormones and other soluble
factors that are required for the growth of particular tumors can
also be considered as possible vaccination targets.
[0664] Analogous methods as described above for the use of
anti-OX40 antibodies can be used for induction of therapeutic
autoimmune responses to treat patients having an inappropriate
accumulation of other self-antigens, such as amyloid deposits,
including A.beta. in Alzheimer's disease, cytokines such as
TNF.alpha., and IgE.
Vaccines
[0665] The anti-OX40 antibodies described herein can be used to
stimulate antigen-specific immune responses by coadministration of
the antibodies 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-OX40 antibody such
that an immune response to the antigen in the subject is enhanced.
The antibody may be a human anti-OX40 antibody (such as any of the
human anti-OX40 antibodies described herein). In other embodiments,
the antibody can be a chimeric or humanized antibody. 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.
[0666] In certain embodiments, a peptide or fusion protein
comprising the epitope to which the anti-OX40 antibody binds is
used as a vaccine instead of, or in addition to, the anti-OX40
antibody.
[0667] 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.
[0668] As previously described, anti-OX40 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-OX40 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 address problems related to the
development of resistance to drugs or a change in the antigenicity
of the tumor cells which would render them unreactive with the
antibody.
[0669] Also provided herein are kits comprising the anti-OX40
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 which binds to an epitope in OX40 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
which otherwise accompanies the kit.
Treatment Protocols
[0670] Suitable protocols for treating a solid tumor (e.g., an
advanced solid tumor) in a human patient include, for example,
administering to the patient an effective amount of an anti-OX40
antibody comprising CDR1, CDR2 and CDR3 domains of the heavy chain
variable region having the sequence set forth in SEQ ID NO: 318,
and CDR1, CDR2 and CDR3 domains of the light chain variable region
having the sequence set forth in SEQ ID NO: 94, wherein the method
comprises at least one administration cycle, wherein the cycle is a
period of two weeks (Q2W), wherein for each of the at least one
cycles, at least one dose of the anti-OX40 antibody is administered
at a dose of 1 mg/kg body weight; a fixed dose of 20, 40, 80, 160,
or 320 mg; a dose of about 1 mg/kg body weight; or a fixed dose of
about 20, 40, 80, 160, or 320 mg.
[0671] Another suitable protocol for treating a solid tumor in a
human patient includes, for example, administering to the patient
an effective amount of each of:
[0672] (a) an anti-OX40 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 94, and
[0673] (b) an anti-PD-1 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 301, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 302,
[0674] wherein the method comprises at least one administration
cycle, wherein the cycle is a period of two weeks, wherein for each
of the at least one cycles, at least one dose of the anti-OX40
antibody is administered at a dose of 1 mg/kg body weight; a fixed
dose of 20, 40, 80, 160, or 320 mg; a dose of about 1 mg/kg body
weight; or a fixed dose of about 20, 40, 80, 160, or 320 mg, and at
least one dose of the anti-PD-1 antibody is administered at flat
dose of 240 mg or a flat dose of about 240 mg. In some embodiments,
the anti-PD-1 antibody is administered once every three weeks (q3w)
at a fixed dose of 360 mg, or once every four weeks (q4w) at a dose
of 480 mg.
[0675] Another suitable protocol for treating a solid tumor in a
human patient includes, for example, administering to the patient
an effective amount of each of:
[0676] (a) an anti-OX40 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 94, and
[0677] (b) an anti-CTLA-4 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 309, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 310,
[0678] wherein the method comprises at least one administration
cycle, wherein the cycle is a period of three weeks (q3w), wherein
for each of the at least one cycles, at least one dose of the
anti-OX40 antibody is administered at a dose of 1 mg/kg body
weight; a fixed dose of 20, 40, 80, 160, or 320 mg; a dose of about
1 mg/kg body weight; or a fixed dose of about 20, 40, 80, 160, or
320 mg, and at least one dose of the anti-CTLA-4 antibody is
administered at flat dose of 1 mg/kg body weight or a flat dose of
about 1 mg/kg body weight. In one embodiment, the anti-OX40
antibody is administered together with the anti-CTLA-4 antibody for
at least one cycle, followed by anti-OX40 antibody monotherapy for
at least one cycle. In certain embodiments, the anti-OX40 antibody
is administered together with ipilimumab for the initial four
cycles, followed by anti-OX40 antibody monotherapy for subsequent
cycles.
[0679] In some embodiments, the anti-OX40 antibody and anti-PD-1
antibody are administered at the following doses:
[0680] (a) 1 mg/kg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody;
[0681] (b) 20 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody;
[0682] (c) 40 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody;
[0683] (d) 80 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody;
[0684] (e) 160 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody; or
[0685] (f) 320 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody.
[0686] In some embodiments, the anti-OX40 antibody and anti-CTLA-4
antibody are administered at the following doses:
[0687] (a) 1 mg/kg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody;
[0688] (b) 20 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody;
[0689] (c) 40 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody;
[0690] (d) 80 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody;
[0691] (e) 160 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody; or
[0692] (f) 320 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody.
[0693] In one embodiment, the dose of the anti-OX40 and/or
anti-PD-1 or anti-CTLA-4 antibody is calculated per body weight,
e.g., mg/kg body weight. In another embodiment, the dose of the
anti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody is a flat-fixed
dose. In another embodiment, the dose of the anti-OX40 and/or
anti-PD-1 or anti-CTLA-4 antibody is varied over time. For example,
the anti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody may be
initially administered at a high dose and may be lowered over time.
In another embodiment, the anti-OX40 and/or anti-PD-1 or
anti-CTLA-4 antibody is initially administered at a low dose and
increased over time.
[0694] In another embodiment, the amount of the anti-OX40 and/or
anti-PD-1 or anti-CTLA-4 antibody administered is constant for each
dose. In another embodiment, the amount of antibody administered
varies with each dose. For example, the maintenance (or follow-on)
dose of the antibody can be higher or the same as the loading dose
which is first administered. In another embodiment, the maintenance
dose of the antibody can be lower or the same as the loading
dose.
[0695] In some embodiments, the anti-OX40 and/or anti-PD-1 or
anti-CTLA-4 antibody are formulated for intravenous administration.
In some embodiments, the anti-OX40 antibody, or anti-OX40 antibody
and anti-PD-1 or CTLA-4 antibody, are administered on Day 1 of each
cycle.
[0696] In some embodiments, the anti-OX40 and/or anti-PD-1 or
anti-CTLA-4 antibody are administered once per week, once every two
weeks, once every three weeks, or once every four weeks, or as long
as a clinical benefit is observed or until there is a complete
response, confirmed progressive disease or unmanageable
toxicity.
[0697] In one embodiment, a cycle of administration is two weeks,
which can be repeated, as necessary. In another embodiment, the
cycle is three weeks. In some embodiments, the treatment consists
of up to eight cycles. In other embodiments, the treatment consists
of up to 12 cycles.
[0698] In one embodiment, one dose each of an anti-OX40 antibody
and an anti-PD-1 antibody is administered per two week cycle. In
another embodiment, one dose each of the anti-PD-1 antibody and
anti-OX40 antibody is administered per three week cycle. In another
embodiment, one dose each of the anti-PD-1 antibody and anti-OX40
antibody is administered per four week cycle.
[0699] In one embodiment, one dose each of the anti-OX40 antibody
and anti-CTLA-4 antibody is administered per three week cycle. In
some embodiments, one dose each of the anti-OX40 antibody and
anti-CTLA-4 antibody is administered per three week cycle for the
first four cycles, followed by anti-OX40 antibody monotherapy for
the fifth through eighth cycles.
[0700] In another embodiment, the anti-OX40 antibody and anti-PD-1
or anti-CTLA-4 antibody are administered as a first line of
treatment (e.g., the initial or first treatment). In another
embodiment, the anti-OX40 antibody and anti-PD-1 or anti-CTLA-4
antibody are administered as a second line of treatment (e.g.,
after the initial or first treatment, including after relapse
and/or where the first treatment has failed).
[0701] In another aspect, the invention features any of the
aforementioned embodiments, wherein the anti-PD-1 antibody is
replaced by, or combined with, an anti-PD-L1 or anti-PD-L2
antibody.
[0702] In some embodiments, the human patient has a cancer selected
from the group consisting of cervical cancer, bladder cancer,
colorectal cancer, and ovarian cancer.
[0703] In certain embodiments, the anti-OX40 antibody comprises a
heavy chain variable region CDR1 comprising the sequence set forth
in SEQ ID NO: 87, a heavy chain variable region CDR2 comprising the
sequence set forth in SEQ ID NO: 317, a heavy chain variable region
CDR3 comprising the sequence set forth in SEQ ID NO: 89, a light
chain variable region CDR1 comprising the sequence set forth in SEQ
ID NO: 90, a light chain variable region CDR2 comprising the
sequence set forth in SEQ ID NO: 91, and a light chain variable
region CDR3 comprising the sequence set forth in SEQ ID NO: 92. In
certain embodiments, the anti-OX40 antibody comprises heavy and
light chain variable regions comprising the sequences set forth in
SEQ ID NOs: 318 and 94, respectively. In certain embodiments, the
anti-OX40 antibody comprises heavy and light chain sequences
comprising the sequences set forth in SEQ ID NOs: 124 and 116,
respectively.
[0704] In certain embodiments, the anti-PD-1 antibody comprises a
heavy chain variable region CDR1, CDR2, and CDR3 comprising the
sequences set forth in SEQ ID NOs: 303-305, respectively, and light
chain variable region CDR1, CDR2, and CDR3 comprising the sequences
set forth in SEQ ID NOs: 306-308, respectively. In certain
embodiments, the anti-PD-1 antibody comprises heavy and light chain
variable regions sequences set forth in SEQ ID NOs: 301 and 302,
respectively. In certain embodiments, the anti-PD-1 antibody
comprises heavy and light chain sequences set forth in SEQ ID NOs:
299 and 300, respectively.
[0705] In certain embodiments, the anti-CTLA-4 antibody comprises a
heavy chain variable region CDR1, CDR2, and CDR3 comprising the
sequences set forth in SEQ ID NOs: 311-313, respectively, and light
chain variable region CDR1, CDR2, and CDR3 comprising the sequences
set forth in SEQ ID NOs: 314-316, respectively. In certain
embodiments, the anti-CTLA-4 antibody comprises heavy and light
chain variable regions sequences set forth in SEQ ID NOs: 309 and
310, respectively.
Outcomes
[0706] With respect to target lesions, responses to therapy may
include:
TABLE-US-00010 Complete Response (CR) Disappearance of all target
lesions. Any (RECIST V1.1) pathological lymph nodes (whether target
or non-target) must have reduction in short axis to <10 mm.
Partial Response (PR) At least a 30% decrease in the sum of the
(RECIST V1.1) diameters of target lesions, taking as reference the
baseline sum diameters. Progressive Disease (PD) At least a 20%
increase in the sum of the (RECIST V1.1) diameters of target
lesions, taking as reference the smallest sum on study (this
includes the baseline sum if that is the smallest on study). In
addition to the relative increase of 20%, the sum must also
demonstrate an absolute increase of at least 5 mm. (Note: the
appearance of one or more new lesions is also considered
progression). Stable Disease (SD) Neither sufficient shrinkage to
qualify for (RECIST V1.1) PR nor sufficient increase to qualify for
PD, taking as reference the smallest sum diameters while on study.
Immune-related Complete Disappearance of all target lesions. Any
Response (irCR) pathological lymph nodes (whether target (irRECIST)
or non-target) must have reduction in short axis to <10 mm.
Immune-related Partial At least a 30% decrease in the sum of
Response (irPR) diameters of target lesions and all new (irRECIST)
measurable lesions (ie Percentage Change in Tumor Burden), taking
as reference the baseline sum diameters. Note: the appearance of
new measurable lesions is factored into the overall Tumor Burden,
but does not automatically qualify as progressive disease until the
sum of the diameters increases by .gtoreq.20% when compared to
nadir. Immune-related Progressive At least a 20% increase in Tumor
Burden Disease (irPD) (ie the sum of diameters of target lesions,
(irRECIST) and any new measurable lesions) taking as reference the
smallest sum on study (this includes the baseline sum if that is
the smallest on study). In addition to the relative increase of
20%, the sum must also demonstrate an absolute increase of at least
5 mm. Tumor assessments using immune- related criteria for
progressive disease incorporates the contribution of new measurable
lesions. Each net percentage change in tumor burden per assessment
accounts for the size and growth kinetics of both old and new
lesions as they appear. Immune-related Stable Neither sufficient
shrinkage to qualify for Disease (irSD) irPR nor sufficient
increase to qualify for (irRECIST) irPD, taking as reference the
smallest sum diameters while on study.
[0707] With respect to non-target lesions, responses to therapy may
include:
TABLE-US-00011 Complete Response (CR) Disappearance of all
non-target lesions. (RECIST V1.1) All lymph nodes must be
non-pathological in size (<10 mm short axis). Non-CR/Non-PD
Persistence of one or more non-target (RECIST V1.1) lesion(s).
Progressive Disease (PD) Unequivocal progression of existing non-
(RECIST V1.1) target lesions. The appearance of one or more new
lesions is also considered progression. Immune-related Complete
Disappearance of all non-target lesions. All Response (irCR) lymph
nodes must be non-pathological in (irRECIST) size (<10 mm short
axis). Immune-related Progressive Increases in number or size of
non-target Disease (irPD) lesion(s) does not constitute progressive
(irRECIST) disease unless/until Tumor Burden increases by 20% (ie
the sum of the diameters at nadir of target lesions and any new
measurable lesions increases by the required amount). Non-target
lesions are not considered in the definition of Stable Disease and
Partial Response.
[0708] Patients treated according to the methods disclosed herein
preferably experience improvement in at least one sign of cancer.
In one embodiment, improvement is measured by a reduction in the
quantity and/or size of measurable tumor lesions. In another
embodiment, lesions can be measured on chest x-rays or CT or MRI
films. In another embodiment, cytology or histology can be used to
evaluate responsiveness to a therapy.
[0709] In one embodiment, the patient treated exhibits a complete
response (CR), a partial response (PR), stable disease (SD),
immune-related complete disease (irCR), immune-related partial
response (irPR), or immune-related stable disease (irSD). In
another embodiment, the patient treated experiences tumor shrinkage
and/or decrease in growth rate, i.e., suppression of tumor growth.
In another embodiment, unwanted cell proliferation is reduced or
inhibited. In yet another embodiment, one or more of the following
can occur: the number of cancer cells can be reduced; tumor size
can be reduced; cancer cell infiltration into peripheral organs can
be inhibited, retarded, slowed, or stopped; tumor metastasis can be
slowed or inhibited; tumor growth can be inhibited; recurrence of
tumor can be prevented or delayed; one or more of the symptoms
associated with cancer can be relieved to some extent.
[0710] In other embodiments, administration of effective amounts of
the anti-OX40 antibody and anti-PD-1 or anti-CTLA-4 antibody
according to any of the methods provided herein produces at least
one therapeutic effect selected from the group consisting of
reduction in size of a tumor, reduction in number of metastatic
lesions appearing over time, complete remission, partial remission,
or stable disease. In still other embodiments, the methods of
treatment produce a comparable clinical benefit rate
(CBR=CR+PR+SD.gtoreq.6 months) better than that achieved by an
anti-OX40 antibody or anti-PD-1 or anti-CTLA-4 antibody alone. In
other embodiments, the improvement of clinical benefit rate is
about 20% 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to an
anti-OX40 antibody or anti-PD-1 or anti-CTLA-4 antibody alone.
Combination Therapies
[0711] In addition to the combinations therapies provided above,
anti-OX40 antibodies described herein can be used in combination
therapy, as described below.
[0712] Methods of combination therapy include those in which an
anti-OX40 antibody, or a combination of anti-OX40 antibody and
anti-PD-1 or anti-CTLA-4 antibody, is coadministered with one or
more additional agents, e.g., small molecule drugs, antibodies or
antigen binding portions thereof, and which are effective in
stimulating immune responses to thereby further enhance, stimulate
or upregulate immune responses in a subject. For instance, as shown
in the Examples, the administration of an anti-OX40 antibody and an
antagonist anti-PD-1 antibody to mice can result in a synergic
effect in inhibiting tumor growth.
[0713] The anti-OX40 antibody can be combined with (i) an agonist
of a stimulatory (e.g., co-stimulatory) molecule (e.g., receptor or
ligand) and/or (ii) an antagonist of an inhibitory signal or
molecule (e.g., receptor or ligand) on immune cells, such as T
cells, both of which result in amplifying immune responses, such as
antigen-specific T cell responses. In certain aspects, an
immuno-oncology agent is (i) an agonist of a stimulatory (including
a co-stimulatory) molecule (e.g., receptor or ligand) or (ii) an
antagonist of an inhibitory (including a co-inhibitory) molecule
(e.g., receptor or ligand) on cells involved in innate immunity,
e.g., NK cells, and wherein the immuno-oncology agent enhances
innate immunity. Such immuno-oncology agents are often referred to
as immune checkpoint regulators, e.g., immune checkpoint inhibitor
or immune checkpoint stimulator.
[0714] In certain embodiments, the anti-OX40 antibody is
administered with an agent that targets a stimulatory or inhibitory
molecule that is a member of the immunoglobulin super family
(IgSF). For example, the anti-OX40 antibody may be administered to
a subject with an agent that targets a member of the IgSF family to
increase an immune response. In other embodiments, the anti-OX40
antibody may be administered with an agent that targets (or binds
specifically to) a member of the B7 family of membrane-bound
ligands that 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 or a
co-stimulatory or co-inhibitory receptor binding specifically to a
B7 family member.
[0715] The anti-OX40 antibody may also be administered with an
agent that targets a member of the TNF and TNFR family of molecules
(ligands or receptors), such as CD40 and CD40L, GITR, GITR-L, CD70,
CD27L, CD30, CD30L, 4-1BBL, CD137, 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, EDA1, EDA2, TNFR1, Lymphotoxin
.alpha./TNF.beta., TNFR2, TNF.alpha., LT.beta.R, Lymphotoxin
.alpha.1.beta.2, FAS, FASL, RELT, DR6, TROY, and NGFR (see, e.g.,
Tansey (2009) Drug Discovery Today 00:1).
[0716] T cell responses can be stimulated by a combination of
anti-OX40 antibodies and one or more of the following agents:
[0717] (1) An antagonist (inhibitor or blocking agent) of a protein
that inhibits T cell activation (e.g., immune checkpoint
inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, as
described above, and any of the following proteins: TIM-3, Galectin
9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA,
B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4;
and/or [0718] (2) 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, GITR, GITR-L, CD70, CD27, CD40, DR3 and CD28H.
[0719] Exemplary agents that modulate one of the above proteins and
may be combined with the anti-OX40 antibody for treating cancer,
include: Yervoy.TM. (ipilimumab) or Tremelimumab (to CTLA-4),
galiximab (to B7.1), BMS-936558 (to PD-1), MK-3475 (to PD-1),
AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1),
MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3), IMP321 (to
LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137), CDX-1127 (to
CD27), Atacicept (to TACI), CP-870893 (to CD40), Lucatumumab (to
CD40), Dacetuzumab (to CD40), Muromonab-CD3 (to CD3), Ipilumumab
(to CTLA-4).
[0720] Anti-OX40 antibodies may also be administered with
pidilizumab (CT-011).
[0721] Other molecules that can be combined with the anti-OX40
antibody for the treatment of cancer include antagonists of
inhibitory receptors on NK cells or agonists of activating
receptors on NK cells. For example, the anti-OX40 antibody can be
combined with antagonists of KIR (e.g., lirilumab).
[0722] T cell activation is also regulated by soluble cytokines,
and anti-OX40 antibodies may be administered to a subject, e.g.,
having cancer, with antagonists of cytokines that inhibit T cell
activation or agonists of cytokines that stimulate T cell
activation.
[0723] In certain embodiments, anti-OX40 antibodies can be used in
combination with (i) antagonists (or inhibitors or blocking agents)
of proteins of the IgSF family or B7 family or the TNF family that
inhibit T cell activation or antagonists of cytokines that inhibit
T cell activation (e.g., IL-6, IL-10, TGF-.beta., VEGF;
"immunosuppressive cytokines") and/or (ii) agonists of stimulatory
receptors of the IgSF family, B7 family or the TNF family or of
cytokines that stimulate T cell activation, for stimulating an
immune response, e.g., for treating proliferative diseases, such as
cancer.
[0724] 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 (WO11/70024, WO11/107553, WO11/131407, WO13/87699,
WO13/119716, WO13/132044) or FPA-008 (WO11/140249; WO13169264;
WO14/036357).
[0725] The anti-OX40 antibodies may also be administered with
agents that inhibit TGF-.beta. signaling.
[0726] Additional agents that may be combined with the anti-OX40
antibodies described herein include agents that enhance tumor
antigen presentation, e.g., dendritic cell vaccines, GM-CSF
secreting cellular vaccines, CpG oligonucleotides, and imiquimod,
or therapies that enhance the immunogenicity of tumor cells (e.g.,
anthracyclines).
[0727] Yet other therapies that may be combined with the anti-OX40
antibodies include therapies that deplete or block Treg cells,
e.g., an agent that specifically binds to CD25.
[0728] Another therapy that may be combined with the anti-OX40
antibodies is a therapy that inhibits a metabolic enzyme such as
indoleamine dioxigenase (IDO), dioxigenase, arginase, or nitric
oxide synthetase.
[0729] Another class of agents that may be used with the anti-OX40
antibodies includes agents that inhibit the formation of adenosine
or inhibit the adenosine A2A receptor.
[0730] Other therapies that may be combined with anti-OX40
antibodies for treating cancer include therapies that
reverse/prevent T cell anergy or exhaustion and therapies that
trigger an innate immune activation and/or inflammation at a tumor
site.
[0731] The anti-OX40 antibody may be combined with more than one
immuno-oncology agent, and may be, e.g., combined with a
combinatorial approach that targets multiple elements of the immune
pathway, such as one or more of the following: a therapy that
enhances tumor antigen presentation (e.g., dendritic cell vaccine,
GM-CSF secreting cellular vaccines, CpG oligonucleotides,
imiquimod); a therapy that inhibits negative immune regulation
e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or
depleting or blocking Tregs or other immune suppressing cells; a
therapy that stimulates positive immune regulation, e.g., with
agonists that stimulate the CD-137 and/or GITR pathway and/or
stimulate T cell effector function; a therapy that increases
systemically the frequency of anti-tumor T cells; a therapy that
depletes or inhibits Tregs, such as Tregs in the tumor, e.g., using
an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25
bead depletion; a therapy that impacts the function of suppressor
myeloid cells in the tumor; a therapy that enhances immunogenicity
of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell
transfer including genetically modified cells, e.g., cells modified
by chimeric antigen receptors (CAR-T therapy); a therapy that
inhibits a metabolic enzyme such as indoleamine dioxigenase (IDO),
dioxigenase, arginase, or nitric oxide synthetase; a therapy that
reverses/prevents T cell anergy or exhaustion; a therapy that
triggers an innate immune activation and/or inflammation at a tumor
site; administration of immune stimulatory cytokines; or blocking
of immuno repressive cytokines.
[0732] Anti-OX40 antibodies can be used together with one or more
of agonistic agents that ligate positive costimulatory receptors,
blocking agents that attenuate signaling through inhibitory
receptors, antagonists, 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.
[0733] In certain embodiments, the anti-OX40 antibody is
administered to a subject together with a BRAF inhibitor if the
subject is BRAF V600 mutation positive.
[0734] In certain embodiments, the anti-OX40 antibody is
administered together with another immunostimulatory antibody.
[0735] Provided herein are methods for stimulating an immune
response in a subject comprising administering to the subject the
anti-OX40 antibody, and one or more additional immunostimulatory
antibodies, such as an anti-PD-1 antagonist, e.g., antagonist
antibody, an anti-PD-L1 antagonist, e.g., antagonist antibody, an
antagonist anti-CTLA-4 antagonist, e.g., antagonist antibody and/or
an anti-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 the anti-OX40 antibody and
an antagonist anti-PD-1 antibody. In one embodiment, the subject is
administered the anti-OX40 antibody and an antagonist anti-PD-L1
antibody. In one embodiment, the subject is administered the
anti-OX40 antibody and an antagonist anti-CTLA-4 antibody. In one
embodiment, the anti-OX40 antibody is a human antibody.
Alternatively, the anti-OX40 antibody can be, for example, a
chimeric or humanized 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).
[0736] Provided herein are methods for treating a
hyperproliferative disease (e.g., cancer), comprising administering
the anti-OX40 antibody with an antagonist PD-1 antibody, an
antagonist PD-L1 antibody, an anti-CTLA-4 antibody, or an anti-LAG3
antibody to a subject. In certain embodiments, one or both
antibodies 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 the anti-OX40 antibody and a
subtherapeutic dose of an anti-PD-1, anti-PD-L1, anti-CTLA-4, or
anti-LAG3 antibody to a subject (e.g., a human). In certain
embodiments, the anti-OX40 antibody comprises the CDRs or variable
regions of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11,
20B3, 14A2-1, 14A2-2, and 20C1, or is another agonist anti-OX40
antibody described herein.
[0737] 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.
[0738] An exemplary anti-PD-1 antibody is 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-PD1 antibody is
MK-3475 (Lambrolizumab) described in WO2012/145493; and AMP-514
described in WO 2012/145493. 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 WO2013/173223 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. Another approach to target the PD-1
receptor is the recombinant protein composed of the extracellular
domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called
AMP-224. In certain embodiments, the antibody has at least about
90% variable region amino acid sequence identity with the
above-mentioned antibodies.
[0739] In certain embodiments, the anti-OX40 antibody is used in
combination with nivolumab, which comprises heavy and light chains
comprising the sequences shown in SEQ ID NOs: 299 and 300,
respectively, or antigen binding fragments and variants thereof. In
certain embodiments, the antibody has heavy and light chain CDRs or
variable regions of nivolumab. Accordingly, in one embodiment, the
antibody comprises CDR1, CDR2, and CDR3 domains of the VH of
nivolumab having the sequence set forth in SEQ ID NO: 301, and
CDR1, CDR2 and CDR3 domains of the VL of nivolumab having the
sequence set forth in SEQ ID NO: 302. In certain embodiments, the
antibody comprises CDR1, CDR2 and CDR3 domains comprising the
sequences set forth in SEQ ID NOs: 303-305, respectively, and CDR1,
CDR2 and CDR3 domains comprising the sequences set forth in SEQ ID
NOs: 306-308, respectively. In certain embodiments, the antibody
comprises VH and/or VL regions comprising the amino acid sequences
set forth in SEQ ID NO: 301 and/or SEQ ID NO: 302, respectively. In
certain embodiments, the antibody has at least about 90%, e.g., at
least about 90%, 95%, or 99% variable region identity with SEQ ID
NO: 301 or SEQ ID NO: 302.
[0740] Exemplary anti-PD-L1 antibodies include BMS-936559 (referred
to as 12A4 in WO 2007/005874 and U.S. Pat. No. 7,943,743), 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 embodiments, the anti-PD-L1 antibody is
MEDI4736 (also known as Anti-B7-H1), MPDL3280A (also known as
RG7446), MSB0010718C (WO2013/79174), or rHigM12B7. Any of the
anti-PD-L1 antibodies disclosed in WO2013/173223, WO2011/066389,
WO2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149 and U.S.
Publication No. 2009/145493 may also be used.
[0741] Exemplary anti-CTLA-4 antibodies include Yervoy.TM.
(ipilimumab or antibody 10D1, described in PCT Publication WO
01/14424), tremelimumab (formerly ticilimumab, CP-675,206), or an
anti-CTLA-4 antibody described in any of 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 WO2013/173223 may also be used.
[0742] Exemplary anti-LAGS 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.
US2011/0150892, WO10/19570 and WO2014/008218. In one embodiment, an
anti-LAG-3 antibody is BMS-986016. Other art recognized anti-LAG-3
antibodies that can be used include IMP731 and IMP-321, described
in US 2011/007023, WO08/132601, and WO09/44273.
[0743] In certain embodiments, the anti-OX40 antibody is used in
combination with ipilimumab. In certain embodiments, the antibody
has heavy and light chain CDRs or variable regions of ipilimumab.
Accordingly, in one embodiment, the antibody comprises CDR1, CDR2,
and CDR3 domains of the VH of ipilimumab having the sequence set
forth in SEQ ID NO: 309, and CDR1, CDR2 and CDR3 domains of the VL
of ipilimumab having the sequence set forth in SEQ ID NO: 310. In
certain embodiments, the antibody comprises CDR1, CDR2 and CDR3
domains comprising the sequences set forth in SEQ ID NOs: 311-313,
respectively, and CDR1, CDR2 and CDR3 domains comprising the
sequences set forth in SEQ ID NOs: 314-316, respectively. In
certain embodiments, the antibody comprises VH and/or VL regions
comprising the amino acid sequences set forth in SEQ ID NO: 309
and/or SEQ ID NO: 310, respectively. In certain embodiments, the
antibody has at least about 90%, e.g., at least about 90%, 95%, or
99% variable region identity with SEQ ID NO: 309 or SEQ ID NO:
310.
[0744] Administration anti-OX40 antibodies 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 anti-OX40 antibodies include cancers
typically responsive to immunotherapy and those that are not
typically responsive to immunotherapy. Representative examples of
cancers for treatment with the combination therapy of the instant
disclosure include those cancers listed herein.
[0745] 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. 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, and sequential administrations can be
combined with concurrent administrations, or any combination
thereof. For example, the first administration of a combination of
anti-OX40 antibody and anti-PD1 antibody (and/or anti-CTLA-4
antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody) can
be concurrent, the second administration can be sequential with
anti-PD1 antibody first and the anti-OX40 antibody second, and the
third administration can be sequential with the anti-OX40 antibody
first and anti-PD1 antibody second, etc. Another representative
dosing scheme involves a first administration that is sequential
with the anti-OX40 first and anti-PD1 antibody (and/or anti-CTLA-4
antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody)
second, and subsequent administrations may be concurrent.
[0746] In certain embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
the anti-OX40 antibody and an immuno-oncology agent. Exemplary
immune-oncology agents include CD137 (4-1BB) agonists (e.g., an
agonistic CD137 antibody such as urelumab or PF-05082566
(WO12/32433)); GITR agonists (e.g., an agonistic anti-GITR
antibody), CD40 agonists (e.g., an agonistic CD40 antibody); CD40
antagonists (e.g., an antagonistic CD40 antibody such as
lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob
7/4); CD27 agonists (e.g., an agonistic CD27 antibody such as
varlilumab (CDX-1127)), MGA271 (to B7H3) (WO11/109400)); KIR
antagonists (e.g., lirilumab); IDO antagonists (e.g., INCB-024360
(WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod,
NLG-919 (WO09/73620, WO09/1156652, WO11/56652, WO12/142237) or
F001287); Toll-like receptor agonists (e.g., TLR2/4 agonists (e.g.,
Bacillus Calmette-Guerin); TLR7 agonists (e.g., Hiltonol or
Imiquimod); TLR7/8 agonists (e.g., Resiquimod); or TLR9 agonists
(e.g., CpG7909)); and TGF-.beta. inhibitors (e.g., GC1008,
LY2157299, TEW7197, or IMC-TR1).
[0747] In one embodiment, the anti-OX40 antibody is administered
prior to administration of a second agent, e.g., an immuno-oncology
agent. In another embodiment, the anti-OX40 antibody is
administered concurrently with the second agent, e.g., an
immunology-oncology agent. In yet another embodiment, the anti-OX40
antibody is administered after administration of the second agent.
The administration of the two agents may start at times that are,
e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6
hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7
days, or one or more weeks apart, or administration of the second
agent may start, e.g., 30 minutes, 60 minutes, 90 minutes, 120
minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours,
3 days, 5 days, 7 days, or one or more weeks after the first agent
has been administered.
[0748] In certain embodiments, the anti-OX40 antibody and a second
agent, e.g., an immuno-oncology agent, are administered
simultaneously, e.g., are infused simultaneously, e.g., over a
period of 30 or 60 minutes, to a patient. The anti-OX40 antibody
may be co-formulated with the second agent, e.g., an
immuno-oncology agent.
[0749] Optionally, the anti-OX40 antibody as sole immunotherapeutic
agent, or a combination of the anti-OX40 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 combination of the anti-OX40 antibody
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 combination of the
anti-OX40 antibody 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, the dose of other chemotherapeutic reagent administered
with the combination can be reduced (Mokyr et al. (1998) Cancer
Research 58: 5301-5304). For example, such a combination may
include the anti-OX40 antibody with or without and an additional
antibody (e.g., 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 or interleukin-2 (IL-2) for the
treatment of melanoma. The scientific rationale behind combining an
agonistic anti-OX40 antibody with 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 combination of the anti-OX40 antibody
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 combination of the anti-OX40 antibody 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.
[0750] In certain embodiments, the anti-OX40 antibody can be used
as the sole immunotherapeutic agent, or a combination of the
anti-OX40 antibody 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 combination of the
anti-OX40 antibody and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3
blockade.
[0751] In another example, the anti-OX40 antibody can be used as
the sole immunotherapeutic agent, or a combination of the anti-OX40
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.,
OX40, 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 the anti-OX40 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.
[0752] Tumors evade host immune surveillance by a large variety of
mechanisms. Many of these mechanisms may be overcome by the
inactivation of proteins, which are expressed by the tumors and
which are immunosuppressive. 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 further combined with the anti-OX40
antibody with or without an additional immunostimulating agent,
e.g., an anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or
anti-LAG-3 agent, such as antibody, to counteract the effects of
immunosuppressive agents and favor anti-tumor immune responses by
the host.
[0753] Other agents (e.g., antibodies) that can be used to activate
host immune responsiveness can be further used in combination with
the anti-OX40 antibody with or without an additional
immunostimulating agent, such as anti-CTLA-4 and/or anti-PD-1
and/or anti-PD-L1 and/or anti-LAG-3 antibody. These include
molecules on the surface of dendritic cells that activate DC
function and antigen presentation. Anti-CD40 antibodies (Ridge et
al., supra) can be used in conjunction with the anti-OX40 antibody
and optionally an additional immunostimulating agent, e.g., an
anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3
agent, e.g., antibody. Other activating antibodies to T cell
costimulatory molecules Weinberg et al., supra, Melero et al.
supra, Hutloff et al., supra, may also provide for increased levels
of T cell activation.
[0754] As discussed above, bone marrow transplantation is currently
being used to treat a variety of tumors of hematopoietic origin.
Anti-OX40 immunotherapy alone or combined with CTLA-4 and/or PD-1
and/or PD-L1 and/or LAG-3 blockade can be used to increase the
effectiveness of the donor engrafted tumor specific T cells.
[0755] Several experimental treatment protocols involve ex vivo
activation and expansion of antigen specific T cells and adoptive
transfer of these cells into recipients in order to
antigen-specific T cells against tumor (Greenberg & Riddell,
supra). These methods can also be used to activate T cell responses
to infectious agents such as CMV. Ex vivo activation in the
presence of the anti-OX40 antibody with or without an additional
immunostimulating therapy, e.g., anti-CTLA-4 and/or anti-PD-1
and/or anti-PD-L1 and/or anti-LAG-3 antibodies can be expected to
increase the frequency and activity of the adoptively transferred T
cells.
[0756] Provided herein are methods for altering an adverse event
associated with the treatment of a hyperproliferative disease
(e.g., cancer) with an immunostimulatory agent, comprising
administering the anti-OX40 antibody with or without an 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, 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 58.sup.th ed. 2004; 608-610.
[0757] In still further embodiments, the anti-OX40 antibody with or
without CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade
(i.e., anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-Ll 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).
[0758] In accordance with the methods described herein, a
salicylate administered in combination with the anti-OX40 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 include
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-OX40 antibody
and anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or
anti-LAG-3 antibodies.
[0759] Anti-OX40 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 with anti-OX40 antibodies may be used sequentially
with known pharmaceutically acceptable agent(s).
[0760] For example, anti-OX40 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+apo21/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-x1 inhibitor), indoleamine
dioxygenase-1 inhibitor (e.g., INCB24360, indoximod, NLG-919, or
F001287), 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),
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.
[0761] Anti-OX40 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:
[0762] 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.
[0763] 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.
[0764] Suitable anti-proliferative agents for combining with
anti-OX40 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
B1, [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.
[0765] In cases where it is desirable to render aberrantly
proliferative cells quiescent in conjunction with or prior to
treatment with the anti-OX40 antibody, 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.
[0766] In certain embodiments, the anti-OX40 antibody is
administered in combination (concurrently or separately) with
nivolumab to treat a patient with cancer, for example, colorectal
or bladder cancer.
[0767] In certain embodiments, the anti-OX40 antibody is
administered in combination (concurrently or separately) with
ipilimumab to treat a patient with cancer, for example, ovarian,
bladder, or prostate cancer.
[0768] 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. 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.
[0769] 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.
XVII. Kits and Unit Dosage Forms
[0770] Also provided herein are kits which include a pharmaceutical
composition containing an anti-OX40 antibody (e.g., OX40.21) and an
anti-PD-1 (e.g., nivolumab) or anti-CTLA-4 (ipilimumab) antibody,
and a pharmaceutically-acceptable carrier, in a therapeutically
effective amount adapted for use in the preceding methods. The kits
optionally also can include instructions, e.g., comprising
administration schedules, to allow a practitioner (e.g., a
physician, nurse, or patient) to administer the composition
contained therein to administer the composition to a patient having
cancer (e.g., a solid tumor). The kit also can include a
syringe.
[0771] Optionally, the kits include multiple packages of the
single-dose pharmaceutical compositions each containing an
effective amount of the anti-OX40 antibody or anti-PD-1 or
anti-CTLA-4 antibody for a single administration in accordance with
the methods provided above. Instruments or devices necessary for
administering the pharmaceutical composition(s) also may be
included in the kits. For instance, a kit may provide one or more
pre-filled syringes containing an amount of the anti-OX40 antibody
or anti-PD-1 or anti-CTLA-4 antibody.
[0772] In one embodiment, the present invention provides a kit for
treating a solid tumor in a human patient, the kit comprising a
dose of an anti-OX40 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 94, and instructions for use in the methods described herein.
In certain embodiments, the kit further comprises (a) a dose of an
anti-PD-1 antibody comprising CDR1, CDR2 and CDR3 domains of the
heavy chain variable region having the sequence set forth in SEQ ID
NO: 301, and CDR1, CDR2 and CDR3 domains of the light chain
variable region having the sequence set forth in SEQ ID NO: 302, or
(b) a dose of an anti-CTLA-4 antibody comprising CDR1, CDR2 and
CDR3 domains of the heavy chain variable region having the sequence
set forth in SEQ ID NO: 309, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 310.
Embodiments
[0773] 1. An isolated antibody, or antigen binding portion thereof,
which binds to human OX40 and exhibits the following
properties:
[0774] (a) binds to membrane-bound human OX40;
[0775] (b) binds to cynomolgus OX40;
[0776] (c) binds to soluble human OX40;
[0777] (d) induces or enhances T cell activation;
[0778] (e) inhibits the binding of OX40 ligand to OX40;
[0779] (f) competes for binding to human OX40 with one or more of
antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11,
20B3, 14A2-1, 14A2-2, and 20C1.
2. The antibody of embodiment 1, wherein the antibody does not bind
to mouse and/or rat OX40. 3. The antibody, or antigen binding
portion thereof, of embodiment 1 or 2, wherein the antibody
stimulates an anti-tumor immune response. 4. The antibody, or
antigen binding portion thereof, of any one of the preceding
embodiments, wherein the antibody stimulates an antigen-specific T
cell response. 5. The antibody, or antigen binding portion thereof,
of any one of the preceding embodiments, wherein the antibody
increases IL-2 and/or IFN-.gamma. production in OX40-expressing T
cells. 6. The antibody, or antigen binding portion thereof, of any
one of the preceding embodiments, wherein the antibody increases T
cell proliferation. 7. The antibody, or antigen binding portion
thereof, of any one of the preceding embodiments, wherein the
antibody binds to Fc receptors. 8. The antibody, or antigen binding
portion thereof, of embodiment 7, wherein the antibody binds to one
or more activating Fc.gamma.Rs. 9. The antibody, or antigen binding
portion thereof, of any one of the preceding embodiments, wherein
the antibody binds to soluble human OX40 with a K.sub.D of about 1
nM or less, such as 0.5 nM or less or 0.1 nM or less, as measured
by Biacore. 10. The antibody, or antigen binding portion thereof,
of any one of the preceding embodiments, wherein the antibody binds
to membrane bound human OX40 with an EC.sub.50 of 50 nM or less,
such as 10 nM or less or 1 nM or less, as measured by FACS. 11. The
antibody, or antigen binding portion thereof, of any one of the
preceding embodiments, wherein the antibody binds to membrane bound
cynomolgus OX40 with an EC.sub.50 of 50 nM or less, such as 10 nM
or less or 1 nM or less as measured by FACS. 12. The antibody, or
antigen binding portion thereof, of any one of the preceding
embodiments, wherein the antibody induces or enhances T cell
activation through multivalent cross-linking. 13. The antibody, or
antigen binding portion thereof, of any one of the preceding
embodiments, wherein the antibody binds the C1q component of human
complement. 14. The antibody, or antigen binding portion thereof,
of any one of the preceding embodiments, wherein the antibody
induces NK cell-mediated lysis of activated CD4+ T cells. 15. The
antibody, or antigen binding portion thereof, of any one of the
preceding embodiments, wherein the antibody promotes
macrophage-mediated phagocytosis of OX40 expressing cells. 16. The
antibody, or antigen binding portion thereof, of any one of the
preceding embodiments, wherein the antibody inhibits regulatory T
cell-mediated suppression of CD4+ T cell proliferation. 17. The
antibody, or antigen binding portion thereof, of any one of the
preceding embodiments, wherein the antibody binds to the sequence
DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ ID NO: 2).
18. The antibody, or antigen binding portion thereof, of any one of
embodiments 1-16, wherein the antibody binds to the sequence
DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of human OX40
(SEQ ID NO: 2). 19. An isolated monoclonal antibody, or antigen
binding portion thereof, which specifically binds to OX40 and
comprises the three variable heavy chain CDRs and the three
variable light chain CDRs that are in the variable heavy chain and
variable light chain pairs selected from the group consisting
of:
[0780] (a) SEQ ID NOs: 318 and 94;
[0781] (b) SEQ ID NOs: 17 and 18;
[0782] (c) SEQ ID NOs: 28 and 29;
[0783] (d) SEQ ID NOs: 28 and 30;
[0784] (e) SEQ ID NOs: 37 and 38;
[0785] (f) SEQ ID NOs: 48 and 49;
[0786] (g) SEQ ID NOs: 48 and 50;
[0787] (h) SEQ ID NOs: 57 and 58;
[0788] (i) SEQ ID NOs: 65 and 66;
[0789] (j) SEQ ID NOs: 73 and 74;
[0790] (k) SEQ ID NOs: 84 and 85;
[0791] (l) SEQ ID NOs: 84 and 86; and
[0792] (m) SEQ ID NOs: 93 and 94.
20. An isolated monoclonal antibody, or antigen binding portion
thereof, which binds to OX40, comprising:
[0793] (a) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 87, 317, and 89, respectively, and/or light chain CDR1,
CDR2, and CDR3 sequences comprising SEQ ID NOs: 90-92,
respectively;
[0794] (b) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 11-13, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 14-16, respectively;
[0795] (c) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 22-24, respectively;
[0796] (d) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 25-27, respectively;
[0797] (e) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 31-33, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 34-36, respectively;
[0798] (f) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 39-41, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 42-44, respectively;
[0799] (g) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 39-41, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 45-47, respectively;
[0800] (h) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 51-53, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 54-56, respectively;
[0801] (i) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 59-61, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 62-64, respectively;
[0802] (j) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 67-69, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 70-72, respectively;
[0803] (k) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 75-77, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 78-80, respectively;
[0804] (l) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 75-77, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 81-83, respectively; or
[0805] (m) heavy chain CDR1, CDR2, and CDR3 sequences comprising
SEQ ID NOs: 87-89, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences comprising SEQ ID NOs: 90-92, respectively.
21. An isolated monoclonal antibody, or antigen binding portion
thereof, which binds to OX40, comprising:
[0806] (a) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 87, 317, and 89, respectively, and/or light chain CDR1,
CDR2, and CDR3 sequences consisting of SEQ ID NOs: 90-92,
respectively;
[0807] (b) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 11-13, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 14-16, respectively;
[0808] (c) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 22-24, respectively;
[0809] (d) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 25-27, respectively;
[0810] (e) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 31-33, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 34-36, respectively;
[0811] (f) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 39-41, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 42-44, respectively;
[0812] (g) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 39-41, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 45-47, respectively;
[0813] (h) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 51-53, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 54-56, respectively;
[0814] (i) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 59-61, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 62-64, respectively;
[0815] (j) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 67-69, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 70-72, respectively;
[0816] (k) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 75-77, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 78-80, respectively;
[0817] (l) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 75-77, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 81-83, respectively;
or
[0818] (m) heavy chain CDR1, CDR2, and CDR3 sequences consisting of
SEQ ID NOs: 87-89, respectively, and/or light chain CDR1, CDR2, and
CDR3 sequences consisting of SEQ ID NOs: 90-92, respectively.
22. An isolated monoclonal antibody, or antigen binding portion
thereof, which binds to OX40 and comprises heavy and light chain
variable regions, wherein the heavy chain variable region comprises
an amino acid sequence which is at least 90% identical to the amino
acid sequence selected from the group consisting of SEQ ID NOs: 17,
28, 37, 48, 57, 65, 73, 84, and 93. 23. An isolated monoclonal
antibody, or antigen binding portion thereof, which binds to OX40
and comprises heavy and light chain variable regions, wherein the
light chain variable region comprises an amino acid sequence which
is at least 90% identical to the amino acid sequence selected from
the group consisting of SEQ ID NOs: 18, 29, 30, 38, 49, 50, 58, 66,
74, 85, 86, and 94. 24. An isolated monoclonal antibody, or antigen
binding portion thereof, which binds to OX40 and comprises heavy
and light chain variable region sequences at least 85% identical to
the amino acid sequences selected from the group consisting of:
[0819] (a) SEQ ID NOs: 318 and 94;
[0820] (b) SEQ ID NOs: 17 and 18;
[0821] (c) SEQ ID NOs: 28 and 29;
[0822] (d) SEQ ID NOs: 28 and 30;
[0823] (e) SEQ ID NOs: 37 and 38;
[0824] (f) SEQ ID NOs: 48 and 49;
[0825] (g) SEQ ID NOs: 48 and 50;
[0826] (h) SEQ ID NOs: 57 and 58;
[0827] (i) SEQ ID NOs: 65 and 66;
[0828] (j) SEQ ID NOs: 73 and 74;
[0829] (k) SEQ ID NOs: 84 and 85;
[0830] (l) SEQ ID NOs: 84 and 86; and
[0831] (m) SEQ ID NOs: 93 and 94.
25. The antibody, or antigen binding portion thereof, of embodiment
24, wherein the heavy and light chain variable regions comprise an
amino acid sequence at least 90% identical to the heavy and light
chain variable regions selected from the group consisting of
(a)-(l) of embodiment 24. 26. The antibody, or antigen binding
portion thereof, of embodiment 25, wherein the heavy and light
chain variable region comprises an amino acid sequence at least 95%
identical to the heavy and light chain variable regions selected
from the group consisting of (a)-(l) of embodiment 24. 27. The
antibody, or antigen binding portion thereof, of embodiment 26,
wherein the heavy and light chain variable region comprises the
heavy and light chain variable regions selected from the group
consisting of (a)-(l) of embodiment 24. 28. An isolated monoclonal
antibody, or antigen binding portion thereof, which binds to OX40
and comprises heavy chain and light chain sequences at least 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid
sequences selected from the group consisting of:
[0832] (a) SEQ ID NOs: 124 and 116, respectively;
[0833] (b) SEQ ID NOs: 95 and 96, respectively;
[0834] (c) SEQ ID NOs: 97 and 98, respectively;
[0835] (d) SEQ ID NOs: 99 and 100, respectively;
[0836] (e) SEQ ID NOs: 101 and 102, respectively;
[0837] (f) SEQ ID NOs: 103 and 104, respectively;
[0838] (g) SEQ ID NOs: 105 and 106, respectively;
[0839] (h) SEQ ID NOs: 107 and 108, respectively;
[0840] (i) SEQ ID NOs: 109 and 110, respectively;
[0841] (j) SEQ ID NOs: 111 and 112, respectively;
[0842] (k) SEQ ID NOs: 113 and 114, respectively;
[0843] (l) SEQ ID NOs: 115 and 116, respectively;
[0844] (m) SEQ ID NOs: 117 and 118, respectively;
[0845] (n) SEQ ID NOs: 119 and 120, respectively;
[0846] (o) SEQ ID NOs: 121 and 122, respectively;
[0847] (p) SEQ ID NOs: 123 and 116, respectively; and
[0848] (q) SEQ ID NOs: 125 and 116, respectively.
29. The antibody, or antigen binding portion thereof, of embodiment
28, wherein the heavy and light chains comprises the heavy and
light chains selected from the group consisting of (a)-(r) of
embodiment 28. 30. An isolated monoclonal antibody, or antigen
binding portion thereof, which (a) binds to the same epitope on
OX40 as the antibody of embodiment 27, and/or (b) inhibits binding
of the antibody of embodiment 27 to OX40 on activated T cells by at
least 95% as measured by FACS. 31. The antibody, or antigen binding
portion thereof, of any one of embodiments 19-30, wherein the
antibody binds to the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178)
of human OX40 (SEQ ID NO: 2). 32. The antibody, or antigen binding
portion thereof, of any one of embodiments 19-30, wherein the
antibody binds to the sequence DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK
(SEQ ID NO: 179) of human OX40 (SEQ ID NO: 2). 33. The antibody, or
antigen binding portion thereof, of any one of embodiments 19-31,
wherein the antibody binds to both human and cynomolgus OX40. 34.
The antibody, or antigen binding portion thereof, of any one of the
preceding embodiments, wherein the antibody is selected from the
group consisting of an IgG1, an IgG2, an IgG3, an IgG4, or a
variant thereof. 35. The antibody, or antigen binding portion
thereof, of embodiment 34, wherein the antibody is an IgG1
antibody. 36. The antibody of embodiment 35, wherein the antibody,
or antigen binding portion thereof, comprises an Fc having enhanced
binding to an activating Fc.gamma.R. 37. The antibody, or antigen
binding portion thereof, of any one of the preceding embodiments,
wherein one or more methionine residues in the CDR regions are
substituted for amino acid residues that do not undergo oxidation.
38. The antibody, or antigen binding portion thereof, of any one of
the preceding embodiments, wherein the antibody, or antigen binding
portion thereof, is a human or humanized antibody. 39. The
antibody, or antigen binding portion thereof, of any one of the
preceding embodiments, wherein the antibody is not immunogenic, as
assessed according to Example 21. 40. The antibody, or antigen
binding portion thereof, of any one of the preceding embodiments,
wherein the amino acid sequence Asp-Gly, if present in the heavy
and/or light chain CDR sequences, is substituted with an amino acid
sequence that does not undergo isomerization. 41. The antibody, or
antigen binding portion thereof, of embodiment 40, wherein the
antibody comprises the heavy chain variable region CDR2 sequence
set forth in SEQ ID NO: 76, but wherein the Asp-Gly sequence is
replaced an amino acid sequence that does not undergo
isomerization. 42. The antibody of embodiment 41, wherein the Asp
or Gly in the Asp-Gly sequence is replaced with Ser. 43. A
bispecific molecule comprising the antibody of any one of the
preceding embodiments linked to a molecule having a second binding
specificity. 44. A nucleic acid encoding the heavy and/or light
chain variable region of the antibody, or antigen binding portion
thereof, of any one of embodiments 1-42. 45. An expression vector
comprising the nucleic acid molecule of embodiment 44. 46. A cell
transformed with an expression vector of embodiment 45. 47. An
immunoconjugate comprising the antibody according to any one of
embodiments 1-42, linked to an agent. 48. A composition comprising
the antibody, or antigen binding portion thereof, bispecific
molecule or immunoconjugate, of any one of embodiments 1-43 and 47,
and a carrier. 49. A kit comprising the antibody, or antigen
binding portion thereof, or bispecific molecule, or immunoconjugate
of any one of embodiments 1-43 and 47 and instructions for use. 50.
A method of preparing an OX40 antibody, or antigen binding portion
thereof, comprising expressing the antibody, or antigen binding
portion thereof, in the cell of embodiment 46 and isolating the
antibody, or antigen binding portion thereof, from the cell. 51. A
method of stimulating an antigen-specific T cell response
comprising contacting the T cell with the antibody, or antigen
binding portion thereof, bispecific molecule or immunoconjugate, of
any one of embodiments 1-43 and 47 such that an antigen-specific T
cell response is stimulated. 52. A method of activating or
co-stimulating an effector T cell, comprising contacting an
effector T cell with an anti-OX40 antibody, or antigen binding
portion thereof, bispecific molecule or immunoconjugate, of any one
of embodiments 1-43 and 47 and CD3, wherein the effector T cell is
activated or co-stimulated. 53. A method of increasing IL-2 and/or
IFN-.gamma. production in a T cell comprising contacting the T cell
with an effective amount of the antibody, or antigen binding
portion thereof, bispecific molecule or immunoconjugate, of any one
of embodiments 1-43 and 47. 54. A method of increasing T cell
proliferation comprising contacting the cell with an effective
amount of the antibody, or antigen binding portion thereof,
bispecific molecule or immunoconjugate, of any one of embodiments
1-43 and 47. 55. A method of increasing IL-2 and/or IFN-.gamma.
production in T cells in a subject comprising administering an
effective amount of the antibody, or antigen binding portion
thereof, bispecific molecule or immunoconjugate, of any one of
embodiments 1-43 and 47, to increase IL-2 and/or IFN-.gamma.
production from the T cells. 56. A method of reducing or depleting
the number of T regulatory cells in a tumor of a subject in need
thereof comprising administering an effective amount of an
antibody, or antigen binding portion thereof, bispecific molecule
or immunoconjugate, of any one of embodiments 1-43 and 47, wherein
the antibody, or antigen binding portion thereof, has effector or
enhanced effector function, to reduce the number of T regulatory
cells in the tumor. 57. A method of stimulating an immune response
in a subject comprising administering the antibody, or antigen
binding portion thereof, bispecific molecule or immunoconjugate, of
any one of embodiments 1-43 and 47 to the subject such that an
immune response in the subject is stimulated. 58. The method of
embodiment 57, wherein the subject has a tumor and an immune
response against the tumor is stimulated. 59. A method for
inhibiting the growth of tumor cells in a subject comprising
administering to the subject the antibody, or antigen binding
portion thereof, bispecific molecule or immunoconjugate, of any one
of embodiments 1-43 and 47, such that growth of the tumor is
inhibited. 60. A method of treating cancer comprising administering
to a subject in need thereof a therapeutically effective amount of
the antibody, or antigen binding portion thereof, bispecific
molecule or immunoconjugate, of any one of embodiments 1-43 and 47,
to treat the cancer. 61. The method of embodiment 60, 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. 62. The method of
embodiment 60 or 61 wherein the cancer is a metastatic cancer,
refractory cancer, or recurrent cancer. 63. The method of any one
of embodiments 56-62, further comprising administering one or more
additional therapeutics. 64. The method of embodiment 63, wherein
the one or more additional therapeutics is an antibody or a small
molecule. 65. The method of embodiment 64, wherein the additional
therapy is an anti-PD1 antibody, a LAG-3 antibody, a CTLA-4
antibody, a PD-L1 antibody, or an anti-TGF.beta. antibody. 66. A
method of treating a solid tumor in a human subject, the method
comprising administering to the subject an effective amount of an
anti-OX40 antibody comprising CDR1, CDR2 and CDR3 domains of the
heavy chain variable region having the sequence set forth in SEQ ID
NO: 318, and CDR1, CDR2 and CDR3 domains of the light chain
variable region having the sequence set forth in SEQ ID NO: 94,
wherein the method comprises at least one administration cycle,
wherein the cycle is a period of two weeks, wherein for each of the
at least one cycles, one dose of the anti-OX40 antibody is
administered at a dose of 1 mg/kg body weight; a fixed dose of 20,
40, 80, 160, or 320 mg; a dose of about 1 mg/kg body weight; or a
fixed dose of about 20, 40, 80, 160, or 320 mg. 67. A method of
treating a solid tumor in a human subject, the method comprising
administering to the subject an effective amount of each of:
[0849] (a) an anti-OX40 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 94,
[0850] (b) an anti-PD-1 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 301, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 302,
[0851] wherein the method comprises at least one administration
cycle, wherein the cycle is a period of two, three, or four weeks,
wherein for each of the at least one cycles, one dose of the
anti-OX40 antibody is administered at a dose of 1 mg/kg body
weight; a fixed dose of 20, 40, 80, 160, or 320 mg; a dose of about
1 mg/kg body weight; or a fixed dose of about 20, 40, 80, 160, or
320 mg, and one dose of the anti-PD-1 antibody is administered at a
dose of 240, 360, or 480 mg or a dose of about 240, 360, or 480
mg.
68. The method of embodiment 67, wherein the anti-OX40 antibody and
anti-PD-1 antibody are administered at the following doses:
[0852] (a) 1 mg/kg body weight anti-OX40 antibody and 240 mg, 360
mg, or 480 mg of anti-PD-1 antibody;
[0853] (b) 20 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody;
[0854] (c) 40 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody;
[0855] (d) 80 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody;
[0856] (e) 160 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody; or
[0857] (f) 320 mg anti-OX40 antibody and 240 mg, 360 mg, or 480 mg
of anti-PD-1 antibody.
69. A method of treating a solid tumor in a human subject, the
method comprising administering to the subject an effective amount
of each of:
[0858] (a) an anti-OX40 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 318, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 94,
[0859] (b) an anti-CTLA-4 antibody comprising CDR1, CDR2 and CDR3
domains of the heavy chain variable region having the sequence set
forth in SEQ ID NO: 309, and CDR1, CDR2 and CDR3 domains of the
light chain variable region having the sequence set forth in SEQ ID
NO: 310,
[0860] wherein the method comprises at least one administration
cycle, wherein the cycle is a period of three weeks, wherein for
each of the at least one cycles, one dose of the anti-OX40 antibody
is administered at a dose of 1 mg/kg body weight; a fixed dose of
20, 40, 80, 160, or 320 mg; a dose of about 1 mg/kg body weight; or
a fixed dose of about 20, 40, 80, 160, or 320 mg, and one dose of
the anti-CTLA-4 antibody is administered at a dose of 1 mg/kg or a
dose of about 1 mg/kg,
[0861] wherein the anti-OX40 antibody is administered together with
the anti-CTLA-4 antibody for at least one cycle, followed by
anti-OX40 antibody monotherapy for at least one cycle.
70. The method of embodiment 67, wherein the anti-OX40 antibody and
anti-CTLA-4 antibody are administered at the following doses:
[0862] (a) 1 mg/kg body weight anti-OX40 antibody and 1 mg/kg
anti-CTLA-4 antibody;
[0863] (b) 20 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody;
[0864] (c) 40 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody;
[0865] (d) 80 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody;
[0866] (e) 160 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody; or
[0867] (f) 320 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4
antibody.
71. The method of any one of embodiments 66-70, wherein the
anti-OX40 antibody, or anti-OX40 antibody and anti-PD-1 or
anti-CTLA-4 antibody, are formulated for intravenous
administration. 72. The method of any one of embodiments 67-71,
wherein the anti-OX40 and anti-PD-1 or anti-CTLA-4 antibody are
formulated together. 73. The method of any one of embodiments
67-71, wherein the anti-OX40 and anti-PD-1 or anti-CTLA-4 antibody
are formulated separately. 74. The method of any one of embodiments
66-68, and 71-73, wherein the treatment consists of up to 12
cycles. 75. The method of any one of embodiments 69-73, wherein the
treatment consists of 8 cycles. 76. The method of embodiment 75,
wherein the anti-OX40 antibody is administered together with the
anti-CTLA-4 antibody for the first 4 cycles, followed by anti-OX40
antibody monotherapy for the last 4 cycles. 77. The method of any
one of embodiments 66-76, wherein the anti-OX40 antibody, or
anti-OX40 antibody and anti-PD-1 or anti-CTLA-4 antibody, are
administered on Day 1 of each cycle. 78. The method of any one of
embodiments 67-77, wherein the anti-OX40 antibody is administered
prior to administration of the anti-PD-1 or anti-CTLA-4 antibody.
79. The method of embodiment 78, wherein the anti-OX40 antibody is
administered within about 30 minutes prior to administration of the
anti-PD-1 or anti-CTLA-4 antibody. 80. The method of any one of
embodiments 67-77, wherein the anti-OX40 antibody is administered
after administration of the anti-PD-1 or anti-CTLA-4 antibody. 81.
The method of any one of embodiments 67-77, wherein the anti-OX40
antibody is administered concurrently with the anti-PD-1 or
anti-CTLA-4 antibody. 82. The method of any one of embodiments
66-81, wherein the treatment produces at least one therapeutic
effect chosen from a reduction in size of a tumor, reduction in
number of metastatic lesions over time, complete response, partial
response, and stable disease. 83. The method of any one of
embodiments 66-82, wherein the solid tumor is associated with a
cancer selected from the group consisting of: cervical cancer,
bladder cancer, colorectal cancer, and ovarian cancer. 84. The
method of any one of embodiments 66-83, wherein the anti-OX40
antibody comprises heavy chain and light chain variable region CDRs
comprising the amino acid sequences set forth in SEQ ID NOs: 87,
317 and 89, and 90-92, respectively. 85. The method of any one of
embodiments 66-84, wherein the anti-OX40 antibody comprises heavy
and light chain variable region sequences set forth in SEQ ID NOs:
318 and 94, respectively. 86. The method of any one of embodiments
66-85, wherein the anti-OX40 antibody comprises heavy and light
chain sequences set forth in SEQ ID NOs: 124 and 116, respectively.
87. The method of any one of embodiments 67, 68, and 71-86, wherein
the anti-PD-1 antibody comprises heavy chain and light chain
variable region CDRs comprising the amino acid sequences set forth
in SEQ ID NOs: 303-305 and 306-308, respectively. 88. The method of
any one of embodiments 67, 68, and 71-87, wherein the anti-PD-1
antibody comprises heavy and light chain variable region sequences
set forth in SEQ ID NOs: 301 and 302, respectively. 89. The method
of any one of embodiments 69-86, wherein the anti-CTLA-4 antibody
comprises heavy chain and light chain variable region CDRs
comprising the amino acid sequences set forth in SEQ ID NOs:
311-313 and 314-316, respectively. 90. The method of any one of
embodiments 69-86 and 89, wherein the anti-CTLA-4 antibody
comprises heavy and light chain variable region sequences set forth
in SEQ ID NOs: 309 and 310, respectively. 91. A kit for treating a
solid tumor in a human subject, the kit comprising a dose of an
anti-OX40 antibody comprising CDR1, CDR2 and CDR3 domains of the
heavy chain variable region having the sequence set forth in SEQ ID
NO: 318, and CDR1, CDR2 and CDR3 domains of the light chain
variable region having the sequence set forth in SEQ ID NO: 94, and
instructions for use. 92. The kit of embodiment 91, further
comprising (a) a dose of an anti-PD-1 antibody comprising CDR1,
CDR2 and CDR3 domains of the heavy chain variable region having the
sequence set forth in SEQ ID NO: 301, and CDR1, CDR2 and CDR3
domains of the light chain variable region having the sequence set
forth in SEQ ID NO: 302, or (b) a dose of an anti-CTLA-4 antibody
comprising CDR1, CDR2 and CDR3 domains of the heavy chain variable
region having the sequence set forth in SEQ ID NO: 309, and CDR1,
CDR2 and CDR3 domains of the light chain variable region having the
sequence set forth in SEQ ID NO: 310. 93. A method of detecting the
presence of OX40 in a sample comprising contacting the sample with
the antibody, or antigen binding portion thereof, of any one of
embodiments 1-38, under conditions that allow for formation of a
complex between the antibody, or antigen binding. 94. An antibody
which binds to OX40 comprising an amino acid sequence selected from
the group consisting of SEQ ID NOs: 282-296. 95. The antibody of
embodiment 94, wherein the antibody comprises a heavy chain
consisting of an amino acid sequence selected from the group
consisting of SEQ ID NOs: 282-296. 96. An isolated monoclonal
antibody which binds to OX40, comprising heavy chain CDR1, CDR2,
and CDR3 sequences comprising SEQ ID NOs: 87, 317, and 89,
respectively, and light chain CDR1, CDR2, and CDR3 sequences
comprising SEQ ID NOs: 90-92, respectively. 97. An isolated
monoclonal antibody which binds to OX40, comprising heavy and light
chain variable regions comprising the amino acid sequences of SEQ
ID NOs: 318 and 94, respectively. 98. An isolated monoclonal
antibody which binds to OX40, comprising heavy and light chains
comprising the amino acid sequences of SEQ ID NOs: 124 and 116,
respectively. 99. An isolated monoclonal antibody which binds to
OX40, wherein the antibody binds to all or a portion of the
sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ ID
NO: 2). 100. A composition comprising an isolated monoclonal
antibody according to any one of embodiments 96-99 and a carrier.
101. A nucleic acid encoding the heavy and/or light chain variable
region of the antibody of embodiment 96 or 97, or the heavy and/or
light chain of embodiment 98. 102. A kit comprising the antibody of
any one of embodiments 96-99. 103. A method of stimulating an
antigen-specific T cell response comprising contacting the T cell
with an antibody according to any one of embodiments 96-99. 104. A
method of treating cancer comprising administering to a subject in
need thereof a therapeutically effective amount of an antibody
according to any one of embodiments 96-99, to treat the cancer.
105. The method of embodiment 104, wherein the cancer or solid
tumor is selected from the group consisting of: cervical cancer,
bladder cancer, colorectal cancer, ovarian cancer, non-small cell
lung cancer, and squamous cell carcinoma of the head and neck.
EXAMPLES
Example 1: Generation of Anti-OX40 Antibodies
[0868] Human anti-OX40 monoclonal antibodies were generated in
Hco7, Hco12, Hco17, and Hco38 strains of HuMAb.RTM. transgenic mice
("HuMAb" is a Trade Mark of Medarex, Inc., Princeton, N.J.) and KM
mice (the KM Mouse.RTM. strain contains the SC20 transchromosome as
described in PCT Publication WO 02/43478) using recombinant
hexahistidine-OX40 antigen.
[0869] A total of 52 mice, including 5 genotypes of transgenic mice
(KM, Hco7, Hco12, Hco17, and Hco38), were immunized with different
immunization strategies. The immunogen was huOX40-6.times.his
prepared in-house and used at 2.0 mg/mL for a total dose of 20
.mu.g per mouse. Routes of administration included: base of tail
injection, Hock immunization, intraperitoneal (ip) and subcutaneous
(sc) injection, and adjuvant (Ribi, Cat#56322, Sigma). 27 fusions
from 30 mice were performed and screened. 541 ELISA antigen
positive antibodies were identified from these 27 fusions, and
further characterization led to the isolation of antibodies of
particular interest, including the antibodies designated as 3F4,
14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3 (also referred
to as OX40.17), 14A2-1, 14A2-2, and 20C1. Their variable region
amino acid sequences and isotype are set forth in FIGS. 1-9. The
heavy and light chain variable regions of 3F4 consist of amino acid
sequences SEQ ID NOs: 17 and 18. The heavy and light chain variable
regions of 14B6-1 consist of amino acid sequences SEQ ID NOs: 28
and 29. The heavy and light chain variable regions of 14B6-2
consist of amino acid sequences SEQ ID NOs: 28 and 30. The heavy
and light chain variable regions of 23H3 consist of amino acid
sequences SEQ ID NOs: 37 and 38. The heavy and light chain variable
regions of 6E1-1 consist of amino acid sequences SEQ ID NOs: 48 and
49. The heavy and light chain variable regions of 6E1-2 consist of
amino acid sequences SEQ ID NOs: 48 and 50. The heavy and light
chain variable regions of 18E9 consist of amino acid sequences SEQ
ID NOs: 57 and 58. The heavy and light chain variable regions of
8B11 consist of amino acid sequences SEQ ID NOs: 65 and 66. The
heavy and light chain variable regions of 20B3 consist of amino
acid sequences SEQ ID NOs: 73 and 74. The heavy and light chain
variable regions of 14A2-1 consist of amino acid sequences SEQ ID
NOs: 84 and 85. The heavy and light chain variable regions of
14A2-2 consist of amino acid sequences SEQ ID NOs: 84 and 86. The
heavy and light chain variable regions of 20C1 consist of amino
acid sequences SEQ ID NOs: 93 and 94.
[0870] cDNA sequencing identified one heavy and one light chain for
each of the antibodies 3F4, 23H3, 18E9, 8B11, 20B3 (also referred
to as OX40.17) and 20C1, and one heavy chain and two light chains
(light chain 1 or "L1" and light chain 2 or "L2") for each of the
antibodies 14B6, 14A2 and 6E1. By protein analysis, a single light
chain was identified for antibodies 14B6, 6E1 and 14A2, and
N-terminal sequencing and molecular weight determination indicated
that it was light chain L1 for 14B6 and 14A2 and light chain L2 for
6E1. Antibodies 14B6-1 and 14B6-2 correspond to antibody 14B6 with
a light chain L1 and L2, respectively. Antibodies 14A2-1 and 14A2-2
correspond to antibody 14A2 with a light chain L1 and L2,
respectively. Antibodies 6E1-1 and 6E1-2 correspond to antibody 6E1
with a light chain L1 and L2, respectively. The amino acid and
nucleotide sequences of each of the light chains of the 3
antibodies are provided in Table 23.
[0871] For some of the antibodies above, substitutions in the
parental antibody were made in HCDR2 in order to remove the
presence of an isomerization site (DG), and framework substitutions
(due to derivation from the DP44 germline) were introduced to make
the framework more like a commonly expressed antibody. For
20C1-based antibodies, three additional unusual framework residues
were reverted to germline (A2V, D24G, and G82bS). The G82bS
framework reversion also eliminates a deamidation site (NG). A
summary of the various substitutions introduced into the parental
hybrid clone sequences is provided in Table 6.
TABLE-US-00012 TABLE 6 Parental Name hybridoma clone Isotype
Variable region substitutions OX40.6 23H3 g1f Anti-OX40 23H3 with
VH-H13Q/M87T OX40.7 23H3 g1f Anti-OX40 23H3 with VH-M87T/M95Y
OX40.8 14A2 g1f Anti-OX40 14A2 with VH-G103W OX40.9 14A2 g1f
Anti-OX40 14A2 with VH-M97Y/G103W OX40.10 14A2 g1f Anti-OX40 14A2
with VH-D53S OX40.11 14A2 g1f Anti-OX40 14A2 with VH-G54S OX40.12
14A2 g1f Anti-OX40 14A2 with VH-D53S/G103W OX40.13 14A2 g1f
Anti-OX40 14A2 with VH-G54S/G103W OX40.14 14A2 g1f Anti-OX40 14A2
with VH-D53S/M97Y/G103W OX40.15 14A2 g1f Anti-OX40 14A2 with
VH-G54S/M97Y/G103W OX40.16 20C1 g1f Anti-OX40 20C1 with VH-
A2V/H13Q/D24G/M87T/G82bS OX40.17 20B3 g1f Anti-OX40 20B3 (no
substitutions) OX40.18 3F4 g1f Anti-OX40 3F4 with
VH-N27Y/N72D/P102Y OX40.19 14A2 g1f Anti-OX40 14A2 with
VH-M97L/G103W OX40.20 20C1 g1f Anti-OX40 20C1 with VH-
A2V/H13Q/D24G/D54S/M87T/G82bS OX40.21 20C1 g1f Anti-OX40 20C1 with
VH- A2V/H13Q/D24G/G55A/M87T/G82bS OX40.22 20C1 g1f Anti-OX40 20C1
with VH- A2V/H13Q/D24G/D54S/G55T/M87T/G82bS
*Depending on the germline, there may be a D53 and G54 or a D54 and
G55 present, as a potential isomerization site
Example 2: Binding of Anti-OX40 Antibodies to Activated Primary
Human T Cells
[0872] The human monoclonal anti-OX40 antibodies generated in
Example 1 were tested for the ability to bind to activated primary
human T cells.
[0873] Cells were activated for several days before the binding
assay in order to induce OX40 expression. Briefly, PBMCs were
cultured for three or four days with magnetic beads coated with
anti-human CD3 plus anti-human CD28, in the presence of recombinant
human IL-2. On the day of the assay, the beads were removed and the
cells stained with a titration of each anti-OX40 antibody. Bound
antibodies were detected with a fluorescently conjugated anti-human
IgG polyclonal secondary antibody, and the cells were co-stained
for CD4 and CD25 to detect activated CD4 T cells. The fluorescence
intensity of the staining was measured using a FACSCanto II flow
cytometer (Becton Dickinson). The geometric mean fluorescence
intensity (GMFI) or median fluorescence intensity (MedFI) of
anti-OX40 antibody staining was calculated for the CD4+CD25+
population (FACSDiva software). EC.sub.50s for antibody binding
were calculated using GraphPad Prism software.
[0874] As shown in FIG. 11A, the anti-OX40 antibodies bound to
activated primary human T cells with subnanomolar EC.sub.50s.
Notably, OX40.5 showed lowest binding of the anti-OX40 antibodies
tested. The same experiment was performed with anti-OX40 antibodies
with variable region substitutions. Initial experiments were
carried out using antibodies in the form of supernatants from
cultures of host cells transfected with recombinant antibody
expression vectors. As shown in FIG. 11B, certain substitutions
caused a significant loss of binding, namely for antibodies OX40.7,
OX40.9, OX40.14 and OX40.15. A set of antibodies with variable
region substitutions was analyzed further using purified antibody
material.
[0875] As shown in FIGS. 11C and 11D, all tested antibodies bound
with subnanomolar EC50s to OX40, except for OX40.18, which showed
lower binding than the 3F4 hybridoma parent clone. OX40.5 showed
the lowest binding among the panel of antibodies tested in FIG.
11B, whereas OX40.1 showed the lowest binding among the panel of
antibodies tested in FIG. 11C. A summary of the EC.sub.50 values is
presented in Table 7 below.
TABLE-US-00013 TABLE 7 EC.sub.50s for binding of OX40 antibodies to
activated human primary T cells. Human T Cell Binding EC50 (nM)
Name (mean .+-. SD) n 3F4 0.19 .+-. 0.15 3 8B11 0.14 .+-. 0.07 2
18E9 0.22 .+-. 0.12 2 20B3 0.34 .+-. 0.23 3 20C1 0.10 .+-. 0.06 3
23H3 0.15 .+-. 0.09 3 6E1 0.97 .+-. 0.05 2 14A2 0.33 .+-. 0.30 3
14B6 0.18 .+-. 0.16 2 OX40.6 0.13 .+-. 0.06 7 OX40.8 0.14 .+-. 0.07
7 OX40.16 0.06 .+-. 0.03 5 OX40.17 0.26 .+-. 0.17 3 OX40.18 3.15
.+-. 3.9 2 OX40.21 0.07 .+-. 0.02 5 OX40.1 0.35 .+-. 0.09 2 OX40.4
0.36 .+-. 0.06 2 OX40.5 3.20 .+-. 0.00 2
Example 3: Binding of Anti-OX-40 Antibodies to Activated Primary
Cynomolgus Macaque T Cells
[0876] The human monoclonal anti-OX-40 antibodies that were tested
for binding to activated primary human T cells in Example 1 were
tested for the ability to bind activated primary cynomolgus macaque
T cells.
[0877] Briefly, cells were activated for several days before the
binding assay in order to induce OX40 expression. Total leukocytes
were isolated from cynomolgus macaque peripheral blood by lysis of
red blood cells using an ammonium chloride buffer. The leukocytes
were then cultured for four to five days with in flasks pre-coated
with anti-human CD3 plus anti-human CD28 antibodies that
cross-react with cynomolgus macaque, in the presence of recombinant
human IL-2, in order to expand and activate T cells. On the day of
the assay, the cells were harvested and stained with a titration of
each anti-OX40 antibody. Bound antibodies were detected with a
fluorescently conjugated anti-human IgG polyclonal secondary
antibody, and the cells were co-stained for CD4 and CD25 to detect
activated CD4 T cells. The fluorescence intensity of the staining
was measured using a FACSCanto II flow cytometer (Becton
Dickinson). The geometric mean fluorescence intensity (GMFI) or
median fluorescence intensity (MedFI) of anti-OX40 antibody
staining was calculated for the CD4+CD25+ population (FACSDiva
software). Dose-response curves were generated and EC50s for
antibody binding were calculated using GraphPad Prism software.
[0878] As shown in FIGS. 12A and 12B, the anti-OX40 antibodies
tested bound with high potency to activated cynomolgus macaque CD4
T cells, with EC.sub.50s ranging from 0.068 nM (20C1) to 1.4 nM
(20B3). 18E9 and 20B3 bound with EC.sub.50s between 1 and 1.5 nM,
while the remaining antibodies bound with EC.sub.50s below 1 nM.
OX40.1 showed the lowest binding among the anti-OX40 antibodies
tested in FIG. 12A, and OX40.5 showed the lowest binding among the
anti-OX40 antibodies tested in FIG. 12B. The same experiment was
performed with anti-OX40 antibodies with variable region
substitutions. As shown in FIG. 12C, OX40.6, OX40.8 and OX40.21
antibodies showed the highest potency of binding, with EC.sub.50s
of 0.12 nM or lower. OX40.1 showed much lower binding to
cynomolgous macaque CD4 T cells. No binding of the OX40.21 antibody
was detected on activated mouse or rat CD4+ T cells. A summary of
the EC.sub.50 values is presented in Table 8 below.
TABLE-US-00014 TABLE 8 Cyno T Cell Binding EC50 (nM) Name (mean
.+-. SD) n 3F4 0.37 .+-. 0.13 4 8B11 0.20 .+-. 0.08 4 18E9 41.70
.+-. 37.78 4 20B3 1.18 .+-. 0.27 4 20C1 * 0.17 .+-. 0.07 4 23H3
0.18 .+-. 0.10 4 6E1 0.77 .+-. 0.14 4 14A2 0.27 .+-. 0.02 4 14B6
0.31 1 OX40.6 0.11 .+-. 0.01 5 OX40.8 0.10 .+-. 0.02 4 OX40.16 0.06
.+-. 0.01 3 OX40.17 0.52 .+-. 0.12 3 OX40.18 OX40.21 0.07 .+-. 0.01
5 OX40.1 23.40 .+-. 37.75 4 OX40.4 1.3 1 OX40.5 ~2.2e+012 1
Example 4: Scatchard Analysis of Binding of Anti-OX40 Antibodies to
Activated Primary T Cells and Cells Overexpressing Human and
Cynomolgus Monkey OX40
[0879] The binding of OX40.21 (IgG1 isotype) to activated human T
cells was further assessed using Scatchard analysis. Briefly,
OX40.21 was radioiodinated with .sup.125I--Na (1mCi; PerkinElmer
Catalog NEZ033H001 MC) using IODO-GEN.RTM. solid phase iodination
reagent (1,3,4,6-tetrachloro-3a-6a-diphenylglycouril; Pierce
Catalog 28601). Activated human CD4+ T cells were isolated from
peripheral blood mononuclear cells (PBMC), Donor W-326470,
purchased from the Stanford Blood Bank. CD4+ T cells were isolated
by negative selection (RosetteSep.TM. Human CD4+ T cell enrichment
cocktail, StemCell Technologies Catalog 15062) and frozen. The
isolated CD4+ T cells were activated for four days before the
binding assay in order to induce OX40 expression, as follows.
Thawed cells were cultured for four days with magnetic beads coated
with anti-human CD3 plus anti-human CD28 (human T-Expander CD3/CD28
Dynabeads, Invitrogen Catalog 111.41D), at a 1:1 bead cell ratio,
in the presence of 200 IU/mL recombinant human IL-2 (Peprotech
Catalog 200-02).
[0880] Radioiodinated OX40.21 IgG1 binding to activated human T
cells was demonstrated by incubating activated human T cells with a
titration of .sup.125I-OX40.21 IgG1. Nonspecific binding was
determined by binding in the presence of a titration of a 100 fold
molar excess of unlabeled antibody and was subtracted from total
CPM to calculate specific binding. A linear standard curve of
.sup.125I-OX40.21 IgG1 concentration versus CPM was used to
extrapolate specific activity, maximal nM bound .sup.125I-OX40.21
IgG1 and thereby calculate receptor number per cell.
[0881] As shown in Table 9 and FIG. 13A, saturable binding of
OX40.21 IgG1 was observed on activated human T cells endogenously
expressing OX40, with a K.sub.D of 0.05 nM for each of two T cell
donors.
TABLE-US-00015 TABLE 9 Binding of .sup.125I-OX40.21 to Activated
Human T Cells Non-Specific Total Binding Binding Specific Binding
.sup.125I-labeled .sup.125I-labeled .sup.125I-labeled Ab Conc. (nM)
Antibody (nM) Antibody (nM) Antibody (nM) 10 0.0317 0.0292 0.0120
0.0117 0.0197 0.0175 5 0.0283 0.0275 0.0073 0.0072 0.0210 0.0204
2.5 0.0230 0.0256 0.0036 0.0030 0.0195 0.0226 1.25 0.0205 0.0221
0.0029 0.0025 0.0176 0.0196 0.625 0.0197 0.0223 0.0014 0.0017
0.0183 0.0207 0.3125 0.0197 0.0229 0.0013 0.0012 0.0184 0.0217
0.15625 0.0177 0.0201 0.0012 0.0011 0.0165 0.0190 0.078125 0.0140
0.0152 0.0010 0.0011 0.0129 0.0141 0.039063 0.0083 0.0091 0.0007
0.0010 0.0076 0.0081 0.019531 0.0057 0.0057 0.0009 0.0010 0.0049
0.0047 0.009766 0.0024 0.0030 0.0007 0.0008 0.0017 0.0022
[0882] The same assay was performed using HEK293 cells
overexpressing human OX40 ("hOX40-293"). Briefly, radioiodinated
OX40.21 binding to overexpressed human OX40 was demonstrated by
incubating hOX40-293 cells with a titration of .sup.125I-OX40.21.
Nonspecific binding was determined by binding in the presence of a
titration of a 100 fold molar excess of unlabeled antibody and was
subtracted from total CPM to calculate specific binding. A linear
standard curve of .sup.125I-OX40.21 concentration versus CPM was
used to extrapolate maximal nM bound .sup.125I-OX40.21 and thereby
calculate receptor numbers per cell. As shown in FIG. 13B and Table
10, saturable binding of OX40.21 IgG1 was observed for binding to
OX40 expressed on hOX40-293 cells. The average K.sub.D for binding
from two test conditions using different numbers of hOX40-293 cells
per sample was 0.22 nM.
TABLE-US-00016 TABLE 10 OX40.21 Binding to hOX40-293 Cells
Non-Specific Total Binding Binding Specific Binding
.sup.125I-labeled .sup.125I-labeled .sup.125I-labeled Ab Conc. (nM)
Antibody (nM) Antibody (nM) Antibody (nM) 10 0.1866 0.1712 0.0129
0.0137 0.1737 0.1575 5 0.1800 0.1710 0.0080 0.0099 0.1720 0.1611
2.5 0.1799 0.1643 0.0065 0.0065 0.1734 0.1578 1.25 0.1722 0.1628
0.0057 0.0054 0.1665 0.1574 0.625 0.1583 0.1436 0.0067 0.0048
0.1515 0.1388 0.3125 0.0986 0.0936 0.0038 0.0044 0.0948 0.0891
0.15625 0.0624 0.0501 0.0035 0.0048 0.0589 0.0453 0.078125 0.0351
0.0289 0.0035 0.0033 0.0316 0.0255 0.039063 0.0211 0.0162 0.0038
0.0027 0.0173 0.0136 0.019531 0.0117 0.0091 0.0029 0.0028 0.0088
0.0063 0.009766 0.0075 0.0056 0.0027 0.0028 0.0048 0.0028
[0883] The same assay was performed using CHO cells overexpressing
cynomolgus monkey OX40 ("cynoOX40-CHO"). Briefly, radioiodinated
OX40.21 binding to cynomologus OX40 was demonstrated by incubating
cynoOX40-CHO cells with a titration of .sup.125I-OX40.21.
Nonspecific binding was determined by binding in the presence of a
titration of a 100 fold molar excess of unlabeled antibody and was
subtracted from total CPM to calculate specific binding. A linear
standard curve of .sup.125I-OX40.21 concentration versus CPM was
used to extrapolate maximal nM bound .sup.125I-OX40.21 and thereby
calculate receptor numbers per cell. As shown in FIG. 13C and Table
11, saturable binding of OX40.21 IgG1 was observed for binding to
cynomologus OX40 expressed on cynoOX40-CHO cells. The average
K.sub.D for binding from two test conditions using different
numbers of cells per sample was 0.63 nM.
TABLE-US-00017 TABLE 11 OX40.21 Binding to cynoOX40-CHO Cells
Non-Specific Total Binding Binding Specific Binding
.sup.125I-labeled .sup.125I-labeled .sup.125I-labeled Ab Conc. (nM)
antibody (nM) antibody (nM) antibody (nM) 20 0.1781 0.1814 0.0266
0.0414 0.1515 0.1400 10 0.1768 0.1651 0.0161 0.0197 0.1607 0.1454 5
0.1629 0.1820 0.0109 0.0171 0.1520 0.1649 2.5 0.1665 0.1659 0.0080
0.0092 0.1586 0.1567 1.25 0.1197 0.1839 0.0079 0.0084 0.1117 0.1755
0.625 0.0892 0.1197 0.0060 0.0074 0.0832 0.1123 0.3125 0.0630
0.0754 0.0057 0.0053 0.0573 0.0701 0.15625 0.0318 0.0437 0.0049
0.0050 0.0269 0.0386 0.078125 0.0158 0.0212 0.0030 0.0034 0.0128
0.0179 0.039063 0.0082 0.0110 0.0027 0.0029 0.0055 0.0082 0.019531
0.0058 0.0058 0.0026 0.0023 0.0032 0.0035 0.009766 0.0030 0.0032
0.0022 0.0021 0.0009 0.0011
Example 5: Specific Binding of Anti-OX40 Antibodies to
Lymphocytes
[0884] The specificity of various OX40 antibodies was tested on a
panel of 22 normal human tissue types, including spleen, tonsil,
thymus, cerebrum, cerebellum, heart, liver, lung, kidney, pancreas,
pituitary, peripheral nerves, stomach, colon, small intestine,
thyroid, skin, skeletal muscle, prostate, uterus, testes, and
placenta by immunohistochemistry.
[0885] Fresh, frozen and/or OCT-embedded human tissues were
purchased from multiple commercial tissue networks/vendors
(Asterand Inc. Detroit, Mich.; Cooperative Human Tissue Network,
Philadelphia, Pa.; ProteoGenex Inc, Culver City, Calif.). To detect
tissue binding, a series of anti-OX40 antibodies (OX40.6-FITC,
OX40.8-FITC, 6E1-FITC, OX40.16-FITC, OX40.17-FITC, OX40.20-FITC,
and OX40.21-FITC) were fluoresceinated and applied to acetone fixed
cryostat sections, followed by an anti-FITC bridging antibody and
visualization by the EnVision+ System. A nonspecific
fluoresceinated human IgG1 was used as isotype control antibody.
HT1080 cells stably expressing human OX40 (HT1080/huOX40) and
hyperplasic human tonsil tissue sections were used as positive
control cells and tissues. To determine if FITC conjugation has any
impact on binding properties, both FITC-conjugated and
un-conjugated anti-OX40 antibodies were compared in HT1080/huOX40
cells using anti-huIgG as bridging antibody. Stained slides were
evaluated under a light microscope.
[0886] Initial tests revealed that both un-conjugated and
FITC-conjugated anti-OX40 antibodies specifically stained the
cytoplasm and membrane of human OX40 transfected cells but not
parent HT1080 cells. There was no difference between unconjugated
and FITC-conjugated anti-OX40 antibodies. These results suggest
that the antibodies were suitable for immunohistochemistry
analyses, and that FITC conjugation has no impact on tissue binding
properties.
[0887] All anti-OX40 antibodies tested exhibited positive staining
in a small subset, either as scattered or small clusters, of
mononuclear cells (MNC) in lymphoid tissues (tonsil, spleen, and
thymus) and lymphoid-rich tissues (colon, stomach, and small
intestine), as well as a few scattered MNC in multiple tissues
(lung, skin, and thyroid). Based on morphology, these positive
cells are primarily lymphocytes.
[0888] In addition to staining a subset of lymphocytes, the OX40.6
antibody, a ligand blocker, displayed strong staining in subsets of
endothelium/subendothelial matrix and interstitial elements, more
often associated with small arteries and adventitia of vessel and
its surrounding connective tissues, in virtually all tissues
examined (FIG. 14A), as well as specialized interstitial tissue
elements such as sheath-like interstitium surrounding the
seminiferous tubule in the testis. The OX40.8 antibody, a ligand
non-blocker, positively labeled myofilament-like structures in
cardiac muscles of the heart (FIG. 14A) and mesangial-like cells in
glomerulus of the kidney. Staining with another ligand non-blocker,
i.e., the 6E1 antibody, also revealed staining in cardiac muscle
cells, as well as in neurons and neuropils of cerebrum and
cerebellum and a subset of tubule epithelial cells in kidney. In
general, the staining of non-lymphocytes was detected only when the
antibodies were used at relatively high concentrations (3 or 5
.mu.g/ml), but not at lower concentrations (1 .mu.g/ml), suggesting
low affinity binding or potential off-target binding.
[0889] Further testing of other ligand blocking antibodies, OX40.16
(FIG. 14A) and OX40.17, revealed clean staining of a subset of
lymphocyte, with no specific staining of other tissue elements for
all tissues examined. The OX40.21 antibody, a variant of the
OX40.16 antibody, had a similar binding pattern as the OX40.16
antibody (FIG. 14B). Immunohistochemistry in a similar panel of
normal cynomolgus tissues revealed very similar staining pattern to
human, demonstrating the utility of cynomolgus monkey as a relevant
preclinical species.
Example 6: Expression of OX40 in Cancers
[0890] FFPE (formalin fixed paraffin embedded) tumor tissue samples
were purchased from commercial tissue venders (n=12-20 for each
tumor type). To detect binding to tissues, an automated IHC assay
with a commercial anti-human OX40 antibody was developed using the
Leica BondRX platform. Briefly, heat-induced antigen retrieval
(HIER) was performed in pH9 ER2 buffer (Leica) for 20 min at
95.degree. C. The mouse anti-human OX40 monoclonal antibody clone
ACT35 (BD Pharmingen) was incubated at 5 .mu.g/ml for 60 minutes,
followed by Novolink Max polymer (Leica) for 30 minutes. Finally,
slides were reacted with DAB substrate-chromogen solution for 6
minutes, counterstained with Mayer's hematoxylin, dehydrated,
cleared, and coverslipped with Permount. Dako protein block was
used as diluent for the primary antibody.
[0891] To profile TILs, commercially available anti-CD3 (T cell
marker) and anti-FoxP3 (Treg marker) monoclonal antibodies were
used to stain adjacent sections. Commercial mouse IgG1 was used as
a negative control and hyperplasic human tonsil tissue was used as
a positive control. After immunostaining, slides were manually
evaluated and scored under a light microscope.
[0892] In the four tumor types examined, CD3+ TILs were present in
all samples examined, with the amount of TILs varying across
samples and the distribution within the same tissue heterogeneous.
In some cases, TILs were more heavily distributed in the tumor and
host interface, as expected. Most TILs were localized in the tumor
stroma in the vast majority tissue samples. However, they were
readily found in intratumoral nests in many cases. Positive OX40
staining was observed in a small fraction of TILs and primarily
distributed in the tumor stroma. In general, the abundance of OX40+
TILs was in proportion to that of CD3+ TILs. Among the four tumor
types examined, OX40+ TILs were more abundant in HCC and CRC (FIGS.
15A-15C).
Example 7: Human Monoclonal Anti-OX40 Antibodies that Block the
Binding of OX-40L to OX-40
[0893] Several anti-OX40 antibodies were tested for their ability
to block the binding of recombinant soluble OX40L to human
OX40-transfected 293 cells. Briefly, 293 cells stably transfected
with human OX40 were first pre-incubated with varying
concentrations of anti-OX40 antibodies. A fixed concentration (0.2
.mu.g/mL) of recombinant soluble his-tagged human OX40L (OX40L-His,
R & D Systems) was then added and the samples incubated
further. After washing the cells, bound OX40L-His was detected
using an in-house APC-labeled anti-His tag antibody. The
fluorescence intensity of the staining was measured using a
FACSCanto II flow cytometer (Becton Dickinson). The geometric mean
fluorescence intensity (GMFI) of APC-anti-His tag
antibody/OX40L-His staining for the cell population was calculated
(FACSDiva software). Dose-response curves were generated and EC50s
for antibody blocking of OX40L binding were calculated using
GraphPad Prism software; the EC50s are shown in Table 12.
TABLE-US-00018 TABLE 12 EC50 values for blocking of OX40L/OX40
interaction as measured by FACS. Antibody Clone EC50 (nM)
1466.C5.C8 1.0 3F4.G11.D2 0.54 8611.H9.C1 0.45 18E9.G5.H4 0.48
2063.G12.A2 0.93 20C1.F2.D1 0.34 6E1.A12.A2 no blocking 23H3.C6
0.38 OX40.4 no blocking OX40.5 ~1.2e+013
[0894] As shown in FIG. 16, most of the anti-OX40 antibodies tested
fully blocked the binding of soluble human OX40L to human OX40 on
the surface of transfected cells, with the exception of 6E1,
OX40.4, and OX40.5. The incomplete blocking by OX40.5 may be due to
a lower potency of binding to human OX40 than the other antibodies
tested or binding to an overlapping but different epitope. In
contrast, 6E1 and OX40.4 did not block the binding of human OX-40L
to OX40. This lack of blocking is likely due to binding to a
different epitope than the remaining antibodies.
Example 8: Antibody Competition/Binning
[0895] Antibody binning experiments were carried out as follows.
One or more anti-OX40 antibodies were coated directly onto a
Biacore CM5 chip using amine coupling chemistry. Anti-OX40
antibodies, serially diluted (1:3) from a starting concentration of
60 .mu.g/mL, were incubated with 20 nM of OX40-6.times.-His antigen
for at least 1 hour. The incubated complex was flowed over the
antibody coupled surfaces and observed for cross-blocking. The
exercise was repeated with several antibodies on the surface to
create the epitope map based on mutual cross-blocking of all the
antibodies. OX40L was also coated on the surface to identify and
bin the antibodies that were able block OX40-OX40L interaction.
Experiments were carried out on Biacore T200 or Biacore 3000 SPR
instruments.
[0896] As summarized in FIG. 17, antibodies 20C1, 20B3, 8B11, 23H3,
18E9, 14B6, OX40.1, and OX40.2 were ligand blockers; antibody 3F4
was a partial ligand blocker; and 14A2, 6E1, and OX40.5 were ligand
non-blockers.
Example 9: Biophysical Properties of OX40 Antibodies
[0897] The affinity of several OX40 antibodies for soluble human
OX40 was tested by SPR analysis. Briefly, affinity measurements
were carried out by capturing 1-10 .mu.g/mL of the respective
antibody on a CM5 chip coated with anti-human-CH1.
Human-OX40-6.times.HIS antigen in either a single concentration of
400 nM or a 1:2 serial dilution from 400 nM was used. Experiments
were carried out on BIACORE.RTM. T200 or BIACORE.RTM. 3000 SPR
instruments. Data was fit to a 1:1 model.
[0898] As shown in Table 13, the anti-OX40 antibodies tested had
dissociation constants (K.sub.Ds) in the range of 10.sup.-8 M to
10.sup.-9 M.
TABLE-US-00019 TABLE 13 K.sub.D values for OX-40 antibodies Clone
K.sub.D (M) k-on (1/Ms) k-off (1/s) 3F4 7.13e-9 5.31e4 3.79e-4 8B11
1.05e-8 4.8e4 5.1e-4 14B6 8.84e-9 7.4E 4 6.54e-4 6E1 1.1e-8 1.28e5
1.41e-3 14A2 1.51e-9 1.46e5 2.2e-4 18E9 2.04e-9 6.83E4 1.39e-4 20B3
3.71e-9 5.42e4 2.01e-4 23H3 3.6e-9 1.1E5 3.95e-4 20C1 3.22e-9
6.48E4 2.09e-4 OX40.21 1.49e-9 9.41e+5 0.0014
[0899] The thermal stability of the OX40.21 antibody was also
tested, with results summarized in Table 14. Thermal stabilities
were determined using GE Healthcare CAP-DSC. Samples were run at
250 .mu.g/mL concentration in PBS. The scan rate was 60.degree.
C./hr. Data was fit to a non-2-state model. The OX40.21 antibody
was determined to be one of the more stable antibodies tested when
considered together with other attributes (e.g., low off-target
effects, immunogenicity, etc).
TABLE-US-00020 TABLE 14 % Clone Tm1 Tm2 Tm3 reversibility 3F4 68 83
8B11 72.7 82.9 14B6 66.3 70.5 18E9 65.8 71.2 23H3 72.3 82.7 20C1
68.0 83.0 OX40.21 72.2 79.5 48% at 80.degree. C.
[0900] The pharmacokinetics of the OX40.21 antibody after single
intravenous dosing to cynomolgus monkeys also was tested. The
OX40.21 antibody exhibited acceptable pharmacokinetic (PK)
properties after single intravenous (IV) dosing to cynomolgus
monkeys with linear PK (0.4 to 4 mg/kg) and a long terminal
half-life (6 days).
TABLE-US-00021 TABLE 15 Pharmacokinetic parameters of OX40.1 after
intravenous administration in cynomolgus monkeys (N = 3) Dose
AUC(INF)* t.sub.1/2 CLT Vss (mg/kg) (.mu.g/mL .times. day) (day)
(mL/h/kg) (mL/kg) 0.4 86 .+-. 5 5.6 .+-. 0.5 0.20 .+-. 0.01 36 .+-.
2 4 785 .+-. 138 6.2 .+-. 0.6 0.22 .+-. 0.04 49 .+-. 12 PK
parameters were calculated by a non-compartmental method. Values
are mean .+-. SD. *PK parameters were calculated using plasma conc.
up to 10 days, except monkey 2 at 0.4 mg/kg up to 7 days *%
AUCextra ranged between 24% and 42%
[0901] The human PK parameters of OX40.21 were projected from
cynomolgus monkey PK data using allometric scaling (assuming power
exponent=0.85 for CLT and 1 for Vss). The projected human t.sub.1/2
was 10 days (Table 16). PK parameters were calculated by a
two-compartment method.
TABLE-US-00022 TABLE 16 Projected Human Pharmacokinetic Parameters
of OX40.21 Dose AUC(INF) t.sub.1/2 CLT Vss Ab (mg/kg) (.mu.g/mL
.times. day) (day) (mL/h/kg) (mL/kg) OX40.21 1 303 10 0.14 47
Example 10: FACS Cross-Blocking of PE-Labeled Anti-OX40 Antibody
Clone L106 by a Panel of Unlabeled Anti-OX40 Antibodies
[0902] Several anti-OX40 antibodies were tested for their ability
to block the binding of PE-labeled anti-OX40 antibody clone L106 to
human OX40-transfected 293 cells. Briefly, 293 cells stably
transfected with human OX40 were first incubated with varying
concentrations of unlabeled anti-OX40 antibodies. The cells were
then washed and incubated with a fixed concentration of 2.5
.mu.g/mL of PE-labeled L106 antibody (BD Biosciences). The
fluorescence intensity of the staining was measured using a
FACSCanto II flow cytometer (Becton Dickinson). The geometric mean
fluorescence intensity (GMFI) of PE-L106 antibody staining for the
cell population was calculated (FACSDiva software). Dose-response
curves for blocking of L106 binding were generated using GraphPad
Prism software.
[0903] As shown in FIG. 18, the 18E9 and OX40.1 antibodies fully
blocked L106 binding to human OX40-transfected cells, while 20B3
showed partial blocking. The remaining antibodies showed little or
no blocking, indicating that 18E9 and OX40.1 bind to a different
epitope on OX40 than the other antibodies tested.
Example 11: Cross-Block Analysis of Anti-OX40 Antibodies
[0904] This experiment was performed to test the cross-blocking
properties of various anti-OX40 antibodies to assess binding
specificities. In brief, the OX40 antibody OX40.1 was conjugated to
allophycocyanin (APC), and human OX40 antibodies OX40.4 and OX40.5
were biotinylated. A panel of unconjugated human OX40 antibodies
were applied in dose to engineered 293 or HT1080 cell lines that
over-express human OX40 protein on their surface and were permitted
to bind at 4.degree. C. for 30 min. Without washout of the
unconjugated Ab, APC-OX40.1 (1 .mu.g/mL), biotin-OX40.4 (0.4
.mu.g/mL), or biotin-OX40.5 (0.4 .mu.g/mL) was applied to the assay
wells and allowed to bind at 4.degree. C. for 30 min. Cells were
washed and if necessary further incubated in the presence of
streptavidin-APC conjugate under the same conditions. After the
final wash, cells were analyzed on a FACSCanto flow cytometer (BD
Bioscience, San Jose, Calif.). Mean fluorescence intensity (MFI)
signal was proportional to bound conjugated antibody.
[0905] As shown in FIGS. 19A-19C, binding of APC-OX40.1 to cells
overexpressing human OX-40 protein was blocked by OX40.2 and
OX40.5, but only modestly, if at all, by OX40.4. Binding of
APC-OX40.1 was blocked by 8B11.H9, 3F4.G11, 20B3.G2, and 14B6.C5,
but not by 6E1.A12 and 14A2.B9. A diagram of the observed binding
relationships between the antibodies evaluated in FIGS. 19A-19C is
shown in FIG. 19H.
[0906] FIGS. 19D-19E show that binding of biotin-OX40.4 was
strongly blocked by 20B3.G2, moderately blocked by 20C1.F2, weakly
blocked, if at all, by 3F4.G11 and 23H3.C6, and was not blocked by
14A2.B9. Binding of biotin-OX40.5 was strongly blocked by 20B3.G2,
23H3.C6, and 20C1.F2, moderately blocked by 3F4.G11, and weakly
blocked by 14A2.B9.
[0907] FIGS. 19F-19G show that binding of biotin-OX40.4 was not
blocked by OX40.1 or OX40.8, and was only weakly blocked, if at
all, by OX40.5 or OX40.6. Binding of biotin-OX40.5 was blocked by
OX40.1, moderately blocked by OX40.6, and was only very weakly
blocked, if at all, by OX40.4 or OX40.6.
[0908] A diagram of the observed binding relationships between the
Abs evaluated in FIGS. 19D-19G is shown in FIG. 19I.
Example 12: Anti-OX40 Antibodies Bind to a Conformation
Epitope/Epitope Mapping
[0909] This example shows that OX40.21 binds to non-denatured human
OX40, but not to denatured human OX40, and that binding is not
affected by N-glycosylation.
[0910] Binding of OX40.21 to native or denatured OX40 that has
N-linked glycosylation or not was determined as follows. Samples of
native (i.e., non-denatured) and denatured human OX40 were
incubated with or without the enzyme N-glycanase PNGase F to remove
N-glycosylation. Samples of native human OX40 with or without
N-linked glycosylation were subjected to SDS gel electrophoresis,
and samples of denatured human OX40 with or without N-linked
glycosylation were subjected to denaturing SDS gel
electrophoresis.
[0911] As shown in FIG. 20A, OX40.21 binds only to native OX40, and
not to the denatured form, and the presence or absence of
glycosylation does not affect binding to OX40. FIGS. 20B and 20C
show that two N-glycopeptides were identified by peptide mapping
after deglycosylation (60% occupancy for both AspN118 and
AspN12).
[0912] These data suggest that OX40.21 binds to an epitope that is
conformational and independent of N-linked glycosylation.
[0913] Epitope mapping studies were also conducted using mass
spectrometry. Peptide fragments of his-tagged human OX40 ("hOX40")
were generated by enzymatic digestion with endoproteinases. LC-MS
was performed using AB Sciex 5600 Triple-TOF.
[0914] As shown in FIGS. 20D and 20E, binding experiments from
native hOX40 by limited proteolysis revealed that OX40.16 and
OX40.21 bound predominantly to the peptide DVVSSKPCKPCTWCNLR (SEQ
ID NO: 178), which corresponds to amino acids 46-62 of the
extracellular portion of mature human OX-40 (SEQ ID NO: 2). OX40.8
bound to the peptide DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID
NO: 179), which corresponds to amino acids 89-124 of the
extracellular portion of mature human OX40 (SEQ ID NO: 2). The
location of the epitope bound by OX40.21 overlaps part of the
binding site of OX40 ligand as determined by crystal structure of
the human OX40/OX40L complex (Protein Data Bank (PDB) ID code
2HEV). Additional peptides identified by mass spectrometry for
OX40.21 are shown in the upper panel of FIG. 20E, and include
QLCTATQDTVCR (SEQ ID NO: 184), SQNTVCRPCGPGFYN (SEQ ID NO: 185),
SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182), and PCKPCTWCNLR
(SEQ ID NO: 183).
Example 13: Anti-OX-40 Antibodies Promote T Cell Proliferation and
Induce IFN-.gamma. and IL-2 Secretion from T Cells
[0915] Anti-OX-40 antibodies were tested for their ability to
induce T cell activity in vitro by measuring the proliferation of
and amount of IL-2 and IFN-.gamma. secreted by T cells incubated
with the antibodies.
[0916] A transfected CHO cell line was generated for use as
artificial antigen-presenting cells in a primary T cell activation
assay. The CHO-CD3-CD32A cell line expresses anti-human CD3
antibody in a single-chain Fv format, along with the human Fc
receptor CD32A to present anti-OX40 antibodies on the CHO cell
surface. Briefly, human primary CD4 T cells were isolated by
negative selection (RosetteSep.TM., StemCell Technologies) and
co-cultured with irradiated CHO-CD3-CD32A cells at an 8:1 T:CHO
ratio, in the presence of graded doses of anti-OX40 antibodies or
isotype control antibody. After 3 to 4 days in culture at
37.degree. C., supernatants were harvested for assessment of T cell
activation by means of measurement of secreted human IFN.gamma.
either by ELISA (BD Biosciences) or HTRF assay (Cisbio), following
the manufacturers' recommendations. Afterwards, tritiated thymidine
was added for the final approximately 18 hours of culture to
measure T proliferation by tritiated thymidine incorporation, as an
additional assessment of T cell activation.
[0917] As shown in FIGS. 21A-21D and 22A-22D (and summarized in
Table 17 below), most tested anti-OX40 antibodies strongly
potentiated human CD4 T cell activation stimulated by CHO-CD3-CD32
cells, in a dose-dependent manner, as measured by proliferation and
IFN.gamma. secretion. The panel of antibodies tested in this assay
co-stimulated T cell activation at least as well as, or better
than, OX40.1, OX40.4, and OX40.5.
TABLE-US-00023 TABLE 17 Proliferation EC.sub.50 (nM) IFN.gamma.
EC.sub.50 (nM) Name (mean .+-. SD) n (mean .+-. SD) n 3F4 0.016
.+-. 0.008 5 8B11 0.022 .+-. 0.027 3 18E9 0.010 .+-. 0.005 3 20B3
0.008 .+-. 0.003 3 20C1 0.008 .+-. 0.006 4 23H3 0.028 .+-. 0.017 3
6E1 0.014 .+-. 0.008 2 14A2 0.037 .+-. 0.044 4 14B6 0.012 .+-.
0.008 3 OX40.6 0.032 .+-. 0.028 2 0.033 .+-. 0.004 2 OX40.8 0.043
.+-. 0.037 2 0.024 1 OX40.16 0.017 1 0.044 1 OX40.17 0.009.+-. 1
0.044 1 OX40.18 0.230.+-. 1 0.490 1 OX40.21 0.011 .+-. 0.006 9
0.043 .+-. 0.023 9 OX40.1 0.024 .+-. 0.012 4 OX40.4 0.094 1
~2.3e+009 1 OX40.5 1.900 1 ~37 1
[0918] The anti-human OX40 antibodies were also tested for their
effects on stimulating primary T cells in cultures of
staphylococcus enterotoxin B (SEB)-activated human peripheral blood
mononuclear cells (PBMCs). Human whole blood was obtained from
AllCells, Inc. (Berkeley, Calif.) or from donors at Bristol-Myers
Squibb, Redwood City, Calif. under the auspices of an in-house
phlebotomy program. PBMCs were isolated by gradient purification on
a Ficoll-Hypaque cushion and cultured for 3 days in culture medium
supplemented with fixed, suboptimal (85 ng/mL) of superantigen
staphylococcus enterotoxin B (SEB; Toxin Technologies, Sarasota,
Fla.) in the presence of graded doses of OX40 antibodies or isotype
control antibody together with 2-5 .mu.g/mL of soluble
cross-linking antibody, F(ab')2 goat anti-human Fc.gamma.. After
culturing for 3 days at 37.degree. C., supernatants were harvested
for assessment of T cell activation by means of ELISA measurement
of secreted human IL-2. Briefly, culture supernatants were diluted
1:10 in sample diluent and tested for the presence of human IL-2 by
ELISA (BD Bioscience) per the manufacturer's recommended protocol.
Following the addition of TMB substrate, assay plates were read on
a Spectramax 340PC reader using Softmax operating software at a
wavelength of 650 nm. Measured optical densities of the chromogenic
substrate were proportional to bound detecting antibody.
[0919] Data from PBMCs isolated from different donors are shown in
FIGS. 23A-23F. In general, solubly-crosslinked clone 20C1.F2
elicited a more robust cytokine response (EC50 of 1.3-2.0 nM)
compared to solubly-crosslinked clones 23H3.C6, 8B11.H9, 3F4.G11,
18E9.G5, 6E1.A12, and 20B3.G2 (FIGS. 23A-23C). With respect to
antibodies with variable region mutations, OX-40.21, in general,
elicited a more robust cytokine response compared to
solubly-crosslinked antibodies OX-40.17, OX40.18, OX40.6, and
OX40.8 (FIGS. 23D-23F). Data from these donors together with data
from 8 additional donors, in which additional anti-OX40 antibodies
were tested, collectively demonstrate that on average, OX40.21
exhibited superior potency in enhancing T cell responses compared
to OX40.1, OX40.2, OX40.4, OX40.5, OX40.17, and OX40.18. These
results further demonstrate that OX40.21 elicits responses that are
comparable to those elicited by OX40.6 and OX40.8 (Table 18).
TABLE-US-00024 TABLE 18 Donor # OX40.21 OX40.17 OX40.18 OX40.6
OX40.8 OX40.1 OX40.2 OX40.4 OX40.5 BMS-009 EC50 (nM) 0.34 0.97 3.21
0.45 0.50 BMS-012 EC50 (nM) 0.29 1.74 ~1.67 0.66 0.09 BMS-016 EC50
(nM) 0.04 1.40 >100 0.29 0.09 WB10024 EC50 (nM) 0.82 1.06 ~3.17
~0.42 ~0.44 1.87 2.53 1.25 ~1.83 WB10025 EC50 (nM) 1.31 1.35 ~1.14
~0.85 0.79 2.08 ~3.26 1.08 3.07 WB10026 EC50 (nM) 0.79 1.68 3.02
0.28 0.63 2.24 3.16 1.14 2.59 WB10027 EC50 (nM) 1.08 1.15 ~3.27
0.45 ~0.46 1.26 2.26 0.93 ~3.09 WB10137 EC50 (nM) 0.56 0.92 >100
0.56 0.42 ~1.824 >100 1.37 >100 BMS-001 EC50 (nM) 0.41 0.41
~84.76 0.49 ~0.42 0.64 4.95 ~0.89 ~0.43 BMS-004 EC50 (nM) ~0.84
1.03 2.43 0.48 0.55 2.52 13.89 0.84 1.94 BMS-015 EC50 (nM) 0.76
1.16 >100 ~0.46 ~0.42 2.41 >100 0.91 1.92 Mean EC50 (nM) 0.64
1.17 2.89 0.46 0.44 1.86 5.36 1.07 2.38 *Each set of experiments
was performed on different days.
Example 14: Anti-OX40 Antibody Promotion of NK92-Mediated Cell
Lysis Using Cell Lines
[0920] Several anti-human OX40 antibodies were tested for their
ability to promote NK92 cell-mediated lysis of activated CD4.sup.+
T cells using calcein release as a read-out. Briefly, CD4.sup.+ T
cells for use as target cells were separated by negative selection
using magnetic beads and activated for 72 hours with beads coated
with anti-CD3 and anti-CD28. After three days, NK92 cells were
plated with calcein AM-labeled activated CD4.sup.+ cells at a ratio
of 5 to 1. A titration of each anti-OX40 antibody was added and
cells were incubated for two hours. Calcein release was measured by
reading the fluorescence intensity of the media using an Envision
plate reader (Perkin Elmer). The percentage of antibody-dependent
cell lysis was calculated based on mean fluorescence intensity
(MFI) with the following formula: [(test MFI-mean background)/(mean
maximum-mean background)].times.100.
[0921] As shown in FIG. 24, OX40.8 and OX40.16 induced the highest
amount of specific lysis of target cells (60% and 30%,
respectively). The EC.sub.50 of OX40.8 was 16 ng/mL and that for
OX40.16 was 4 ng/mL. All other antibodies tested induced ADCC at
levels too low for accurate quantitation.
Example 15: Anti-OX40 Antibody Promotion of NK-Mediated Cell Lysis
of Primary Human CD4+ T Cells
[0922] Several anti-human OX40 antibodies were tested for their
ability to promote primary NK cell-mediated lysis of activated
CD4.sup.+ T cells. Briefly, CD4.sup.+ T cells for use as target
cells were separated from PBMCs from two donors by magnetic
selection and activated for 72 hours with beads coated with
anti-CD3 and anti-CD28. NK cells, for use as effectors, were
separated from a separate donor by negative selection using
magnetic beads and activated with IL-2 for 24 hrs. Following the
activation period, NK effector cells were mixed with
calcein-labeled target T cells at 20:1, 10:1, or 5:1 ratios in the
presence of antibody at 1 .mu.g/ml for 2 hours. The level of
calcein released by lysed target cells was measured by reading the
fluorescence intensity of the media using an Envision plate reader
(Perkin Elmer). The percentage of antibody-dependent cell lysis was
calculated based on mean fluorescence intensity (MFI) with the
following formula: [(test MFI-mean background)/(mean maximum-mean
background)].times.100.
[0923] As shown in FIGS. 25A and 25B, activated CD4+ T cell targets
from two donors were lysed most effectively by OX40.8. Lower levels
of ADCC activity were seen with both OX40.21 and OX40.1.
Example 16: OX40 Antibody Promotion of Macrophage-Mediated Cell
Phagocytosis of OX40-Expressing HEK293 Cells
[0924] To determine the antibody-mediated phagocytic activity of
several OX40 antibodies, primary human macrophages were cultured
for four hours with CellTrace Violet-labeled HEK293/OX40 cells and
a titration of anti-OX40 antibodies. After four hours, cells were
harvested, stained with anti-CD64-APC, and run on a flow cytometer.
Cells that stained double positive for CD64 and CellTrace Violet
were considered to have been phagocytosed. The percentage of target
cells phagocytosed was calculated using the formula:
100.times.(Number of double positive cells/Total number of
CellTrace Violet positive cells).
[0925] As shown in FIG. 26, all tested anti-OX40 antibodies induced
the phagocytosis of OX40-expressing target cells in a
dose-dependent manner. OX40.8 had the highest overall level of
phagocytosis and the lowest EC.sub.50 concentration of 6.2 ng/mL.
This demonstrates that human IgG1 anti-OX40 antibodies induce
FcR-mediated phagocytosis in a dose-dependent manner.
Example 17: Anti-OX40 Antibodies Bind the C1q Component of Human
Complement
[0926] A colorimetric ELISA assay was developed to evaluate whether
the C1q component of human serum complement binds to the OX40.21
antibody. All tested antibodies were coated on a high binding
immunoassay plate at 10 .mu.g/mL. After blocking unoccupied protein
binding sites, graded doses of human C1q (3.125-200 .mu.M) were
added to the wells, including blocked empty wells that served as
controls for non-specific background C1q binding to the assay
plate. Binding of C1q to the immobilized antibodies was detected
using a combination of biotinylated mouse anti-C1q antibody and
streptavidin-poly-HRP, together with tetramethylbenzidine
substrate. The results are reported as the optical density read at
450 nm minus 630 nm.
[0927] As shown in FIG. 27, C1q bound to OX40.21 (solid squares)
and the human IgG1 isotype control (open circles) in a dose
dependent manner. The level of C1q binding to OX40.21 however, was
lower than to the human IgG1 isotype control antibody. As expected,
there was little background signal (gray circles) and no evident
C1q binding to an IgG1.1 isotype control (solid black circles). The
IgG1.1 antibody contains five mutations in the Fc portion designed
to eliminate C1q binding and FcR interaction. This result
demonstrates that the C1q component of human serum complement can
bind to OX40.21 and indicates that OX40.21 may induce complement
mediated lysis of OX40-expressing cells in vivo.
Example 18: OX40 is Expressed in Tumor Infiltrating Lymphocytes
[0928] OX-40 is expressed in tumor infiltrating lymphocytes, with a
pattern that is generally limited to CD4+ cells (FIG. 28A) with
minimal expression on CD8+ T cells (FIG. 28B) in colorectal, lung,
and ovarian cancer (FIG. 28C).
[0929] Similarly, OX40 is expressed by CD4+ T cells and Tregs in
mouse Sa1N tumors (FIG. 28D) and mouse MC38 tumors (FIG. 28E). To
test expression of mouse OX-40 in tumors, 2.times.10.sup.6 SA1N
sarcoma cells or 2.times.10.sup.6 MC38 cells were implanted
subcutaneously into AJ or B6 mice respectively. On day 15
post-implantation, tumors were harvested, dissociated into single
cell suspensions, and stained for flow cytometry. T cell
populations were identified based on their expression of CD8, CD4
and Foxp3. For Sa1N tumors, CD4+ Foxp3+ cells from the tumor are
shown in the red histogram, CD4+ Foxp4- cells are in the blue
histogram and CD8+ cells are in the orange histogram (FIG. 28D).
Isotype control stained cells are in the green histogram. For MC38
tumors, Tregs are shown in the blue histogram, CD4+ cells are shown
in the green histogram and CD8+ cells in the red histogram (FIG.
28E).
Example 19: Anti-OX40 Antibody Reversal of Treg Cell-Mediated
Suppression
[0930] Several anti-human OX40 antibodies were tested for their
ability to reverse regulatory T (Treg) cell-mediated suppression of
human CD4.sup.+ T cell proliferation. Briefly, Treg and T responder
(Tresp) cells were isolated by enriching PBMCs for CD4.sup.+ cells
by magnetic bead separation and then sorting
CD4.sup.+CD25.sup.hiCD127.sup.lo Treg and
CD4.sup.+CD25.sup.loCD127.sup.hiCD45RO.sup.+ Tresp cells. Tresp
cells were then labeled with proliferation dye and plated with
titrating numbers of Treg cells, beginning at a 1:1 ratio. Cultures
were stimulated with 3 .mu.g/mL plate-bound anti-CD3, 1 .mu.g/mL
soluble anti-CD28, and 2 .mu.g/mL plate-bound anti-OX40 or isotype
control. After 96 hours, Tresp cell proliferation was measured by
assessing dye dilution using flow cytometry.
[0931] As shown in FIG. 29, in both the presence and absence of
Treg cells, the anti-OX40 antibodies increased Tresp cell
proliferation compared to the isotype control. This suggests that
the anti-OX40 antibodies tested reversed the suppressive effects of
Treg cells on Tresp cell proliferation.
Example 20: Toxicity Studies
[0932] OX40.6 (2 mg/kg) was administered intravenously to monkeys
on Days 1 (FIG. 30A) and 29 (FIG. 30B) to evaluate any associated
toxicities. No evidence of tolerability issues or clinical
pathology abnormalities was observed. OX40.6 stimulated an enhanced
immune response to KLH, as characterized by a trend towards
enhanced CD69 expression in CD4+ T cells in an ex vivo KLH recall
assay. Two of 4 monkeys exhibited accelerated clearance, which
correlated with the formation of anti-drug antibodies.
[0933] The concentration of OX40.6 in cynomolgus monkey serum
samples for the experiment above was analyzed by a
chemiluminescence (CL) immunoassay. OX40. 6 antibody was used to
prepare calibrators and quality control (QC) samples.
Biotinylated-human-OX40-his was immobilized on streptavidin-coated
microplates (Greiner Bio-one) as a capture molecule for OX40.6.
Samples, standards, and quality control samples brought up to a
final matrix of 10% cyno serum were incubated on the plates.
Samples were analyzed at 10% minimum required dilution in 1%
BSA/PBS/0.05% Tween 20 (PTB) containing 2% mouse serum. The unbound
material was washed away and the captured OX40.6 antibody was
detected using an HRP-labeled mouse monoclonal anti-human IgG
antibody as the detection molecule. Following addition of
SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo
Scientific), the concentration of OX40.6 in cyno serum samples was
calculated from luminescence intensity as measured by a M5 plate
reader using a 4-parameter logistic (4-PL) calibration curve
generated from OX40.6 antibody calibrators. The range of the OX40.6
antibody calibration curve was from 5 to 5,000 ng/mL in cyno serum.
The upper and lower limits of quantification were 5,000 and 10
ng/mL, respectively (i.e., ULOQ 5000 ng/mL, LLOQ 10 ng/mL). Quality
control samples were prepared at 3750, 400, and 20 ng/mL in
cynomolgus monkey serum and analyzed on each plate to ensure
acceptable assay performance. Calibrators, QCs, and samples were
diluted 5-fold in PTB containing 2% mouse serum. Four streptavidin
plates were used to analyze the samples. Assay performance was
within an acceptable range: interplate % CV of standards was below
25%, and QC recovery was within .+-.30% nominal values.
[0934] The presence of anti-drug antibodies to OX40.6 in cynomolgus
monkey serum in the experiment described above was determined by
electrochemiluminescence (ECL) bridging immunoassay. Specifically,
mouse monoclonal anti-human IgG Fc antibody was used to prepare the
positive control. Biotinylated-anti-OX40: anti-hOX40-his was used
at 25 ng/mL as the capture molecule and
ruthenylated-anti-OX40:anti-hOX40-his was used at 25 ng/mL as the
detection molecule. Samples were analyzed at 100-fold dilution in
1% BSA/PBS/0.05% Tween 20 (PTB) containing capture and detection
molecules. After 2 hours of incubation in polypropylene plates, the
sample mix was transferred to streptavidin-coated MSD plates.
Following a one hour incubation, unbound material was washed away,
MSD read buffer was added, and ECL was measured with the MSD plate
reader S16000. The positive control (Mouse anti-human IgG Fc) was
prepared at 1000 (HPC), 100 (MPC), and 10 ng/mL (LPC) in cynomolgus
serum. Pooled cynomolgus serum was used as a negative control (NC).
The signal ratio for HPC, MPC, and LPC versus NC was 102, 10, and
2, respectively. One streptavidin plate was used to analyze the
samples. Assay performance was within the acceptable range: % CV of
the PC was below 10%, and the raw signal for the negative control
(54 RLU) was comparable to the raw signal for predose samples
(48-55 RLU).
Example 21: Immunogenicity Risk Assessment Study
[0935] In vitro T cell proliferation assays were conducted for
several of the anti-human OX40 antibodies to assess their human
immunogenicity potential. Briefly, peripheral blood mononuclear
cells (PBMC) from healthy volunteers were isolated by Ficoll (GE
Healthcare) and gradient centrifugation, and human lymphocyte
antigen (HLA) Class II was characterized by polymerase chain
reaction (PCR) amplification and hybridization with oligonucleotide
probes (ProImmune).
[0936] A panel of 40 PBMC donors having HLA Class II types closely
matching world population frequencies was used for an assay run.
PBMCs were labeled with CFSE (Invitrogen) to monitor proliferation
and plated on 96 well plates in 6 replicates at 200,000 cells per
well in RPMI (Lonzo) containing 10% human AB (Bioreclamation), non
essential amino acids (Gibco), and pen-strep (Gibco). Anti-human
OX40 antibodies, controls proteins, reference antibodies, and ConA
were cultured with PBMCs at 1 .mu.M for 7 days, after which media
was washed away and cells were labeled with an anti-human CD4 APC
(BD science) monoclonal antibody. After removal of unbound anti-CD4
antibody with a wash step, cells were fixed with 3.7% formalin
(Sigma) in PBS, and analyzed by flow cytometry to determine the
percentage of proliferating CD4+ cells.
[0937] The percentage of 40 donors that showed a positive response
(defined as a significant increase in proliferating CD4+ T cells
relative to media-incubated PBMCs) for the different anti-human
OX40 antibodies is shown in FIG. 31. All variants of the anti-human
OX40 antibodies showed low potential to activate CD4+ cells in this
assay, comparable to the low QC protein, with the exception of
OX40.16 and OX40.21, which did not show a positive CD4
proliferation response in any of the 40 donors. These results
suggest that these anti-human OX40 antibodies have low potential to
elicit an anti-drug antibody response in humans.
Example 22: Binding to Activated Fc Receptors Enhances Anti-mOX40
Activity in a Colon Carcinoma Model
[0938] To test the role of FcR binding in the activity of
anti-mouse OX40 antibodies in mouse tumor models, anti-OX40
antibodies of different isotypes were tested. C57BL/6 mice were
subcutaneously injected with 2 million MC38 tumor cells. After 7
days, tumor volumes were determined and mice were randomized into
treatment groups so as to have comparable mean tumor volumes.
Antibodies formulated in PBS were administered intraperitoneally on
days 7, 10, and 14 at 200 .mu.g per dose in a volume of 200
.mu.l.
[0939] In syngeneic mouse tumor models, anti-murine OX40 antibodies
(e.g., OX86, rat IgG1) exhibit anti-tumor activity. Since varying
the isotype of many antibodies specific for T cell surface
receptors (both co-stimulatory and co-inhibitory) can alter the
anti-tumor activity of these antibodies, mouse Fc isotype variants
of OX86, an antibody which does not block the OX40/OX40L
interaction, were generated. As shown in FIGS. 32A-32C, OX86
formatted as a mouse IgG2a Fc (FIG. 32C) results in superior
anti-tumor activity compared to OX86 formatted as a mouse IgG1
(FIG. 32B). This is likely due both to depletion of Treg cells at
the tumor site and to T effector cell expansion from
antibody-mediated agonism of OX40.
[0940] To confirm the effects of different antibody isotypes on
tumor infiltrating T cell populations, tumors from MC38 mice that
were treated with the different isotypes were assessed by flow
cytometry. Selected mice were sacrificed and tumors and spleens
were harvested for analysis on day 15 after tumor implantation.
Single cell suspensions were prepared by dissociating tumor and
lymph node with the back of a syringe in a 24 well plate. Cell
suspensions were passed through 70 .mu.m filters, pelleted,
resuspended, and counted. Cells were then plated in 96 well plates
with 1.times.10.sup.6 cells per well for staining. Samples were
then analyzed on a FACS Canto flow cytometer (BD). Analysis of the
spleens and tumors of tumor bearing mice treated with anti-OX40
antibodies show that the IgG2a isotype can deplete CD4+ Tregs in
tumors (FIG. 33A), and that IgG1 and IgG2a isotypes can activate T
cell expansion in the periphery (FIG. 33B) and result in increased
cell numbers in the spleen (FIG. 33C). These results suggest that
agonism of OX40 (but not necessarily blocking the OX40/OX40L
interaction) and Fc receptor binding of the OX86 antibody promotes
anti-tumor activity.
[0941] The role of human Fc and FcRs were tested using mice where
mouse FcRs have been knocked out and replaced with the human FcRs.
These experiments were performed using a bone marrow chimera system
where CD45.1 congenic hosts were irradiated then reconstituted with
human FcR transgenic bone marrow cells. These mice were then
allowed to reconstitute for 8 weeks before being inoculated with
2.times.10.sup.6 MC38 tumor cells. After 7 days, tumor volumes were
determined and mice were randomized into treatment groups so as to
have comparable mean tumor volumes. Antibodies formulated in PBS
were administered intraperitoneally on days 7, 10, and 14 at 200
.mu.g per dose in a volume of 200 .mu.l. Mice were treated with
either a control human IgG1 (FIG. 34A), a chimeric OX-86 human G1
hybrid Ab (FIG. 34B), or the OX-86 human G1 hybrid with a S267E
mutation (FIG. 34C). The results were similar to what was observed
with the mouse isotypes, i.e., the human IgG1 antibody had a
significant anti-tumor effect as it can bind to activating FcRs,
while the S267E mutation which increases binding to both CD32B and
CD32A had higher activity (FIGS. 34A-34C). This higher level of
activity is likely due to increased agonism on effector T cells as
well as increased depletion of Tregs at the tumor site.
[0942] T cell populations at the tumor site and spleens of tumor
bearing mice were examined as described earlier. Tregs were less
prevalent in mice treated with either the G1 or G1 S267E antibody,
with a larger effect seen with the S267E isotype (FIG. 35A).
Increases in the percentages of CD8+ T cells (FIG. 35B) and CD4+
effector (FIG. 35C) and at the tumor site were also evident, and
these increases were greater with the G1 S267E antibody. Increased
cellularity in the spleens of mice treated with the anti-OX-40
antibodies was also noted (FIG. 35D). These results suggest that
the OX86-hIgG1 antibody exhibited potent anti-tumor activity (FIGS.
34A-34C) and measurable Treg depletion (FIGS. 35A-35D).
Example 23: A Blocking Anti-OX40 Antibody Exhibits Anti-Tumor
Activity in a Mouse Tumor Model
[0943] The following experiment was conducted to determine whether
an antibody which blocks the interaction between OX40/OX40L
exhibits potent anti-tumor activity. To this end, a hamster
anti-mouse OX40 antibody which blocks the OX40/OX40L interaction
(hamster IgG1 8E5 antibody) was generated and tested for its
anti-tumor activity in a subcutaneous mouse CT-26 tumor model.
CT-26 is a mouse colon adenocarcinoma tumor cell line whose solid
tumor growth can be monitored in BALB/c mice when the cells are
transplanted subcutaneously.
[0944] Female BALB/c mice (Charles River Laboratories, Hollister,
Calif.) were acclimated for a minimum of three days prior to the
start of the studies. Mice were housed 5 animals per cage, and the
cages were placed in microisolator ventilated racks. Housing was at
18-26.degree. C. and 50+20% relative humidity with at least twelve
room air changes per hour. A 12 h light/dark cycle was maintained.
Animals were provided with sanitized laboratory rodent diet and
municipal water ad libitum.
[0945] CT-26 cells were maintained in RPMI-1640 medium (Hyclone,
Cat. No. SH30096.01) supplemented with 10% fetal bovine serum (FBS;
Hyclone, Cat. No. SH30071.03). Approximately twice a week, cells
contained in a single T175 flask were divided and expanded to four
T175 flasks at a 1:5 dilution until sufficient number of cells were
obtained for tumor implantation. The cells were harvested near 80%
confluence, washed and resuspended in PBS.
[0946] On Day 0, 1.times.10.sup.6 CT-26 cells were implanted into
the mice using a 1 cc syringe (Becton Dickinson, Franklin Lakes,
N.J.) and 27 gauge 5/8 inch needle. Tumors were then measured two
times weekly in 3 dimensions with an electronic caliper (Mitutoyo,
Aurora, Ill.) and recorded. Tumor volumes (mm.sup.3) were
calculated using the formula:
width.times.length.times.height.times.0.5. Following tumor volume
measurements on Day 6 post implantation, mice were staged according
to tumor volume. Mice with a mean tumor volume of 26 mm.sup.3 were
randomized into groups and treated as shown in Table 19.
[0947] The hamster isotype control antibody is an inert Armenian
hamster IgG monoclonal antibody (mAb) to GST (clone PIP, catalog #
BE0260; BioXcell, West Lebanon, N.H.). It was prepared in PBS
immediately prior to administration to provide doses of 10 mg/kg
per mouse via intraperitoneal (IP) injection on Days 6, 10 and 14
as shown in Table 19.
[0948] The monoclonal antibody against mouse OX40 (clone 8E5) was
prepared in PBS immediately prior to administration to provide
doses of 10, 3, 1 or 0.3 mg/kg per mouse via IP injection on Days
6, 10 and 13 as shown in Table 19.
TABLE-US-00025 TABLE 19 Treatment Treatment Dose N Route schedule
Hamster IgG Istoype Control 10 mg/kg 12 IP Days 6, 10, 14 mAb
Hamster anti-mouse OX40 10 mg/kg 12 IP Days 6, 10, 14 (clone 8E5)
mAb Hamster anti-mouse OX40 3 mg/kg 12 IP Days 6, 10, 14 (clone
8E5) mAb Hamster anti-mouse OX40 1 mg/kg 12 IP Days 6, 10, 14
(clone 8E5) mAb Hamster anti-mouse OX40 0.3 mg/kg 12 IP Days 6, 10,
14 (clone 8E5) mAb
[0949] Animals were checked daily for postural, grooming, and
respiratory changes, as well as lethargy. Animals were weighed two
times weekly and euthanized if weight loss was .gtoreq.20%. Mice
were checked for the presence and size of tumors twice weekly until
death or euthanasia. Tumors were measured in 3 dimensions with an
electronic caliper (Mitutoyo, Aurora, Ill.) and recorded. Response
to treatment compounds was measured as a function of tumor growth.
If the tumor reached a volume of .gtoreq.1500 mm.sup.3 or appeared
ulcerated, animals were euthanized.
[0950] As shown in FIGS. 36A-36E, the hamster anti-mouse OX40 mAb
(clone 8E5; FIGS. 36B-36E showing treatment with different doses of
8E5) demonstrated potent anti-tumor activity in the subcutaneous
CT-26 model as compared to the hamster IgG isotype control group
(FIG. 36A). The 8E5 antibody was administered at doses ranging from
0.3 to 10 mg/kg, and even at the lowest dose evaluated (0.3 mg/kg),
10 of 12 mice were tumor-free (TF) at the end of the study period
(Day 72). Although the number of tumor-free mice did not differ
significantly amongst each dose group, with each group having 9 or
10 tumor-free mice by the end of the study period, mice treated
with either of the two highest doses (3 or 10 mg/kg) showed more
tumor growth delays as compared to mice treated with the two lowest
doses (0.3 or 1 mg/kg). There were no tumor-free mice in the
isotype control-treated group; all mice in that group had been
sacrificed by Day 39 as a result of ulceration or tumor burden
(>1500 mm.sup.3).
[0951] These data indicate that an anti-OX40 antibody that blocks
the interaction between OX40 and OX40-L demonstrates potent
anti-tumor activity in a subcutaneous mouse CT-26 tumor model when
administered to mice with established tumors.
Example 24: OX40 Agonism Synergizes with PD-1 Blockage in a Murine
MC38 Colon Carcinoma Model
[0952] To test for synergy between anti-OX-40 antibody and
anti-PD-1 antibody treatments, combinations of these antibodies
were tested in the MC38 mouse tumor model. C57BL/6 mice were
subcutaneously injected with 2 million MC38 tumor cells. After 7
days, tumor volumes were determined and mice were randomized into
treatment groups so as to have comparable mean tumor volumes.
Antibodies formulated in PBS were administered intraperitoneally on
days 7, 10, and 14 at 200 .mu.g per dose in a volume of 200
.mu.l.
[0953] As shown in FIGS. 37A-37D, both the anti-PD-1 antibody (FIG.
37B) and anti-OX40 antibody (FIG. 37C) showed minimal activity when
used alone, but had significant anti-tumor activity when combined
(FIG. 37D), with 5 of 8 mice rendered tumor free.
Example 25: OX40 Agonism Enhances the Response to Vaccines in
Cynomolgus Monkey
[0954] Enhancement of immune responses to vaccines was measured to
evaluate the ability of the OX40.6 antibody to stimulate immune
responses in cynomolgus monkeys. This approach was selected because
the desired effect, i.e., enhancement of immune responses to
tumors, cannot be evaluated in healthy non-human primates, as they
lack tumors.
[0955] Monkeys were immunized with keyhole limpet hemocyanin (KLH)
on Day 1 (10 mg, intramuscularly) and with hepatitis B virus
surface antigen (HBsAg) (ENGERIX-B) (20 .mu.g intramuscularly on
Days 1 and 29). Following administration of the vaccines, the
monkeys were dosed intravenously with 0 or 2 mg/kg of OX40.6
antibody on Days 1 and 29. Immune responses were measured on Days
22 and 41 by ex vivo T cell response to KLH and by T-cell-dependent
antibody responses to KLH and HBsAg. As shown in FIGS. 38A and 38B,
OX40.6-related findings at 2 mg/kg at Days 22 (FIG. 38A) and 41
(FIG. 38B) included an increase in the ex vivo recall response to
KLH, characterized by increases in the mean percent of CD69+,
IFN-gamma+, and TNF-alpha+ expressing CD4+CD8- T cells.
Example 26: Fc Receptor Binding for Antibodies with Engineered
Constant Domains
[0956] This Example demonstrates that antibodies having modified
heavy chain constant regions comprising the CH1 and hinge of IgG2
bind to Fc.gamma.Rs when they contain CH2 and CH3 domains of
IgG1.
[0957] In addition to antigen binding by the variable domains,
antibodies can engage Fc-gamma receptors (FcgRs) through
interaction with the constant domains. These interactions mediate
effector functions such as antibody-dependent cellular cytotoxicity
(ADCC) and antibody-dependent cellular phagocytosis (ADCP).
Effector function activity is high for the IgG1 isotype, but very
low or absent for IgG2 and IgG4 due to these isotypes having lower
affinity for FcgRs. In addition, the effector function of IgG1 can
be modified through mutation of amino acid residues within the
constant regions to alter FcgR affinity and selectivity.
[0958] The binding of antibodies to Fc gamma receptors (Fc.gamma.Rs
or FcgRs) was studied using biosensor technologies including
Biacore surface plasmon resonance (SPR) and Fortebio Biolayer
Interferometry (BLI). SPR studies were performed on a Biacore T100
instrument (GE Healthcare) at 25.degree. C. The Fab fragment from a
murine anti-6.times.His antibody was immobilized on a CM5 sensor
chip using EDC/NHS to a density of .about.3000 RU. Various
his-tagged FcgRs (7 ug/ml) were captured via the C-terminal his-tag
using a contact time of 30 s at 10 ul/min, and the binding of 1.0
uM antibody was evaluated in a running buffer of 10 mM NaPO4, 130
mM NaCl, 0.05% p20 (PBS-T) pH 7.1. FcgRs used for these experiments
included CD64 (FcgRI), CD32a-H131 (FcgRIIa-H131), CD32a-R131
(FcgRIIa-R131), CD32b (FcgRIIb), CD16a-V158 (FcgRIIIa-V158),
CD16b-NA1 (FcgRIIIb-NA1), and CD16B-NA2 (FcgRIIIb-NA2). BLI
experiments were performed on a Fortebio Octet RED instrument
(Pall, Fortebio) at 25.degree. C. in 10 mM NaPO4, 130 mM NaCl,
0.05% p20 (PBS-T) pH 7.1. Antibodies were captured out of undiluted
expression supernatants on protein A coated sensors, followed by
the binding of 1 .mu.M hCD32a-H131, hCD32a-R131, hCD32b,
hCD16a-V158, or 0.1 .mu.M hCD64 analytes.
[0959] First, antibodies were made that contain modified IgG1 Fc
domains including the substitutions S267E (SE) and S267E/L328F
(SELF), as well as various combinations of the mutations P238D,
P271G, H268D, A330R, G237D, E233D, referred to as V4, V7, V8, V9
and V12. The binding of these antibodies was studied by Biacore SPR
with comparison to IgG1f, IgG2.3 (IgG2-C219S) and IgG4.1
(IgG4-S228P) antibodies, as well as an IgG1.1f antibody which has
been engineered to reduce binding to all FcgRs. The results, which
are shown in FIG. 70, demonstrate the expected FcgR binding
properties for IgG1f, IgG2.3 and IgG4.1 and the mutated IgG1
antibodies, including increased CD32a-H131, CD32a-R131 and CD32b
binding for SE and SELF, as well as increased selectivity of the
V4, V7, V8, V9 and V12 mutants for CD32b over CD32a-H131 and
CD32a-R131 (FIG. 39).
[0960] The next set of constructs was used to engineer effector
function into the otherwise effector function negative IgG2
isotype. For this study, the mutations described above were
introduced in the context of IgG2.3 constant region, or an
IgG2.3/IgG1f hybrid termed IgG2.3G1-AY (Table 20). Antibodies were
expressed at small scale as supernatants, and tested for binding to
FcgRs using Fortebio Octet BioLayer Interferometry biosensor
technology. Since the antibodies were present at low concentration
in the supernatants, the experiment was performed by capturing
antibodies out of the supernatants using protein A coated sensors,
followed by binding of FcgR analytes in solution. Purified and
supernatant control IgG1f including wild type IgG1, SE, P238D, V4
and V12 antibodies were also included for comparison, and each of
these control antibodies demonstrated expected FcgR binding
properties (FIG. 40). The IgG2.3 antibody also demonstrated the
expected binding profile, with appreciable binding to only
CD32a-H131. However, all mutations to introduce S267E, L328F,
P238D, P271G, H268D, A330R, G237D, or E233D mutations into IgG2.3
failed to recapitulate the FcgR affinity of the corresponding
engineered IgG1 mAbs (FIG. 40). In contrast, the IgG2.3G1-AY
construct was able to fully preserve the FcgR binding properties of
wild type IgG1, while retaining the CH1 and hinge regions of
IgG2.3. In addition, all IgG2.3G1-AY mutants containing S267E,
L328F, P238D, P271G, H268D, A330R, G237D, and E233D demonstrated
FcgR binding properties comparable to the IgG1 version mAbs
containing the same mutations (FIG. 40). This demonstrates the
successful engineering of antibodies with CH1 and hinge regions of
IgG2 combined with effector function of wild type or mutant
IgG1.
TABLE-US-00026 TABLE 20 Engineered IgG2 constructs Set ID Construct
Seq ID# 1 IgG2.3 hHC-IgG2-C219S 258 IgG2.3-V13 hHC-IgG2-C219S -
P238D 267 IgG2.3-V14 hHC-IgG2-C219S - P238D, P271G 268 IgG2.3-V15
hHC-IgG2-C219S - P238D, H268D, P271G 269 IgG2.3-V16 hHC-IgG2-C219S
- P238D, P271G, A330R 270 IgG2.3-V17 hHC-IgG2-C219S - P238D, H268D,
P271G, A330R 271 IgG2.3-V18 hHC-IgG2-C219S - S267E 272 IgG2.3-V19
hHC-IgG2-C219S - S267E, L328F 273 2 IgG2.3G1
hHC-IgG2-C219S/hHC-IgG1f 262 IgG2.3G1-AY-V20
hHC-IgG2-C219S/hHC-IgG1f - P238D 274 IgG2.3G1-AY-V21
hHC-IgG2-C219S/hHC-IgG1f - P238D, P271G 275 IgG2.3G1-AY-V22
hHC-IgG2-C219S/hHC-IgG1f - 276 P238D, H268D, P271G IgG2.3G1-AY-V23
hHC-IgG2-C219S/hHC-IgG1f - 277 P238D, P271G, A330R IgG2.3G1-AY-V24
hHC-IgG2-C219S/hHC-IgG1f - 278 P238D, H268D, P271G, A330R
IgG2.3G1-AY-V25 hHC-IgG2-C219S/hHC-IgG1f - 279 G237D, P238D, H268D,
P271G, A330R IgG2.3G1-AY-V26 hHC-IgG2-C219S/hHC-IgG1f - 280 E233D,
G237D, P238D, H268D, P271G, A330R IgG2.3G1-AY-V27
hHC-IgG2-C219S/hHC-IgG1f - S267E 266 IgG2.3G1-AY-V28
hHC-IgG2-C219S/hHC-IgG1f - S267E, L328F 281
[0961] This engineering strategy was further explored by producing
other antibodies formatted with IgG2.3G1-AY, IgG2.3G1-AY-S267E
(IgG2.3G1-AY-V27), as well as IgG2-B-form variants (IgG2.5G1-AY and
IgG2.5G1-AY-V27), and other hybrid antibodies containing different
combinations of IgG1 and IgG2 constant domains, and testing the
binding of these antibodies to anti-his Fab captured his-tagged
FcgRs using Biacore SPR technology. In agreement with the Octet
supernatant data, the SPR data showed that the IgG2.3G1-AY and
IgG2.3G1-AY-V27 antibodies had comparable FcgR binding properties
to IgG1f and IgG1f-S267E respectively, despite containing the CH1
and hinge regions of an A-form IgG2 antibody (IgG2.3) (Table 21).
Similar data was also obtained using IgG2.5G1-AY and
IgG2.5G1-AY-V27 antibodies, demonstrating the successful
engineering of B-form IgG2 antibodies (containing C131S mutation
termed IgG2.5) having IgG1f or modified IgG1f like effector
functions. Data for several other antibodies with IgG2.3G1-AY,
IgG2.3G1-AY-V27, IgG2.5G1-AY, or IgG2.5G1-AY-V27 constant regions
but different variable regions shows that this engineering strategy
is broadly applicable to other antibodies independent of the
variable domains (Table 21).
TABLE-US-00027 TABLE 21 % Rmax values for 1 uM antibodies binding
to anti-his Fab captured FcgR-his proteins hCD32a- hCD32a- hCD16a-
hCD16B- mAb hCD64 H131 R131 hCD32b V158 NA2 mAb8-IgG1f 80% 82% 51%
27% 51% 21% mAb9-IgG1f 70% 33% 19% 4% 28% 10% mAb11-IgG2.3 2% 44%
17% 5% 1% 0% mAb6-IgG2.3 3% 66% 14% 3% 1% 0% mAb4-IgG2.3 1% 39% 6%
1% 1% 0% mAb5-IgG2.3 6% 100% 30% 4% 3% 0% mAb12-IgG2.3 2% 39% 7% 1%
1% 0% mAb13-IgG2.3 2% 40% 7% 1% 1% 0% mAb11-IgG2.5 0% 40% 13% 3% 0%
-1% mAb7-IgG2.5 4% 72% 19% 2% 2% 0% mAb8-IgG2.5 3% 59% 14% 3% 2% 0%
mAb10-IgG2.5 1% 29% 5% 1% 1% 0% mAb6-IgG2.5 3% 75% 17% 4% 2% 0%
mAb4-IgG2.5 2% 46% 8% 1% 1% 0% mAb5-IgG2.5 6% 89% 26% 5% 4% 1%
mAb12-IgG2.5 1% 36% 6% 1% 1% 0% mAb13-IgG2.5 -2% 39% 4% -2% 0% -2%
mAb8-IgG2.3G1-AY 77% 61% 38% 10% 38% 13% mAb10-IgG2.3G1-AY 67% 23%
14% 4% 24% 8% mAb7-IgG2.5G1-AY 80% 73% 45% 12% 47% 19%
mAb8-IgG2.5G1-AY 77% 70% 45% 17% 48% 22% mAb7-IgG2.3G1-AY-V27 84%
68% 92% 76% 26% 7% mAb8-IgG2.3G1-AY-V27 78% 67% 80% 67% 24% 7%
mAb10-IgG2.3G1-AY-V27 69% 24% 57% 40% 12% 3% mAb7-IgG2.5G1-AY-V27
81% 74% 89% 84% 32% 9% mAb8-IgG2.5G1-AY-V27 77% 76% 79% 77% 33%
10%
Example 27: Effects of Anti-OX40 Antibodies with Modified Heavy
Chain Constant Regions on T Cell Proliferation and IFN-.gamma. and
IL-2 Secretion from T Cells with or without Cross-Linking
[0962] Anti-OX40 antibodies with modified IgG2 CH1/hinge regions
may have the ability to promote T cell activation in the absence of
cross-linking, and thus may be able to promote T cell activation in
vivo in the absence or low expression of cell types expressing
Fc.gamma.Rs, and possibly to promote anti-tumor activity in a wider
range of tumor types than IgG1 isotype antibodies.
[0963] Alternatively, modified CH1/hinge region antibodies may
still require cross-linking in order to promote T cell activation,
but may have increased agonist activity when bound to Fc.gamma.Rs
compared to IgG1 isotype antibodies, and thus be more potent in
promoting T cell activation and anti-tumor activity.
[0964] Anti-OX40 antibodies having modified heavy chain constant
regions comprising the sequences shown in Table 22 are generated
and tested for their effects on T cell proliferation and
IFN-.gamma. and IL-2 secretion from T cells with or without
cross-linking using the assays described below. The light chain
sequences for antibodies OX40.6, 40.8, 40.16, and 40.21 correspond
to SEQ ID NOs: 96, 110, and 116 (for both OX40.16 and 40.21),
respectively.
TABLE-US-00028 TABLE 22 Constructs SEQ ID NO OX40.6-Vh-hHC-IgG2.3
282 OX40.8-Vh-hHC-IgG2.3 283 OX40.16-Vh-hHC-IgG2.3 284
OX40.6-Vh-hHC-IgG2.3G1 285 OX40.8-Vh-hHC-IgG2.3G1 286
OX40.16-Vh-hHC-IgG2.3G1 287 OX40.6-Vh-hHC-IgG2.3G1-V27 288
OX40.8-Vh-hHC-IgG2.3G1-V27 289 OX40.16-Vh-hHC-IgG2.3G1-V27 290
OX40.6-Vh-hHC-IgG2.5 291 OX40.8-Vh-hHC-IgG2.5 292
OX40.16-Vh-hHC-IgG2.5 293 OX40.21-Vh-hHC-IgG2.5 294
OX40.21-Vh-hHC-IgG2.5G1 295 OX40.21-Vh-hHC-IgG2.5G1-V27 296
CHO-CD3+/-CD32 Assay
[0965] Anti-OX-40 antibodies with the sequences shown in Table 22
are tested for their ability to induce T cell activity in vitro by
measuring the proliferation of and amount of IL-2 and IFN-.gamma.
secreted by T cells incubated with the antibodies.
[0966] Transfected CHO cell lines are generated for use as
artificial antigen-presenting cells in a primary T cell activation
assay. The CHO-CD3-CD32A cell line expresses anti-human CD3
antibody in a single-chain Fv format, along with the human Fc
receptor (FcR) CD32A to present anti-OX40 antibodies on the CHO
cell surface. The CHO-CD3 cell line expresses anti-human CD3
antibody in a single-chain Fv format without FcR.
[0967] Briefly, human primary CD4 T cells are isolated by negative
selection (RosetteSep.TM., StemCell Technologies) and co-cultured
with either irradiated CHO-CD3-CD32A cells, or irradiated CHO-CD3
cells, at an 8:1 T:CHO ratio, in the presence of graded doses of
anti-OX40 antibodies or isotype control antibody. After 3 to 4 days
in culture at 37.degree. C., supernatants are harvested for
assessment of T cell activation by means of measurement of secreted
human IFN-.gamma. either by ELISA (BD Biosciences) or HTRF assay
(Cisbio), following the manufacturers' recommendations. Afterwards,
tritiated thymidine is added for the final approximately 18 hours
of culture to measure T proliferation by tritiated thymidine
incorporation, as an additional assessment of T cell
activation.
SEB PBMC Assay
[0968] Anti-OX-40 antibodies with the sequences shown in Table 22
are tested for their effects on stimulating primary T cells in
cultures of staphylococcus enterotoxin B (SEB)-activated human
peripheral blood mononuclear cells (PBMCs). Human whole blood
samples are obtained from AllCells, Inc. (Berkeley, Calif.) or from
donors at Bristol-Myers Squibb, Redwood City, Calif. under the
auspices of an in-house phlebotomy program. PBMCs are isolated by
gradient purification on a Ficoll-Hypaque cushion and cultured for
3 days in culture medium supplemented with fixed, suboptimal (85
ng/mL) of superantigen staphylococcus enterotoxin B (SEB; Toxin
Technologies, Sarasota, Fla.) in the presence of graded doses of
OX40 antibodies or isotype control antibody. In some cases, 2-5
.mu.g/mL of soluble cross-linking antibody, F(ab')2 goat anti-human
Fc.gamma., is also added to the cultures. After culturing for 3
days at 37.degree. C., supernatants are harvested for assessment of
T cell activation by means of ELISA measurement of secreted human
IL-2. Briefly, culture supernatants are diluted 1:10 in sample
diluent and tested for the presence of human IL-2 by ELISA (BD
Bioscience) per the manufacturer's recommended protocol. Following
the addition of TMB substrate, assay plates are read on a
Spectramax 340PC reader using Softmax operating software at a
wavelength of 650 nm. Measured optical densities of the chromogenic
substrate were proportional to bound detecting antibody.
MLR Assay
[0969] Anti-OX-40 antibodies with the sequences shown in Table 22
tested for their ability to potentiate primary human T cell
proliferation and IFN-.gamma.secretion in a T cell: Dendritic Cell
Allogeneic Mixed Lymphocyte Reaction (T:DC AlloMLR). Total T cells
are isolated from peripheral blood from healthy human donors by
negative selection (RosetteSep, Stemcell Technologies). Monocytes
are isolated from peripheral blood from healthy human donors using
CD14 microbeads (Miltenyi), and cultured for six days in the
presence of GM-CSF and IL-4 to derive immature dendritic cells
(DCs). DCs and T cells are co-cultured in the presence of graded
doses of OX40 antibodies or isotype control antibody. In some
cases, 2-5 .mu.g/mL of soluble cross-linking antibody, such as
F(ab')2 goat anti-human Fc.gamma., is also added to the cultures.
Supernatant from each sample is harvested between Day 4 and Day 7
for measurement of secreted IFN-.gamma. by ELISA (BD Biosciences)
or HTRF assay (Cisbio), following the manufacturers'
recommendations. After supernatant harvest, the cell cultures are
pulsed with 1 .mu.Ci/well of .sup.3[H]-thymidine for the last 16-18
hours of the culture. The cells are harvested onto filter plates,
and .sup.3[H] counts per minute of cell-incorporated
.sup.3[H]-thymidine are read as a measure of T cell
proliferation.
Example 28: Anti-Tumor Activity of OX40 Agonist mAb with CTLA-4
Blockade in a CT26 Model
[0970] To test for synergy between anti-OX-40 antibody and
anti-CTLA-4 antibody treatments, combinations of these antibodies
were tested in the CT26 mouse tumor model. Mice were inoculated
with CT26 tumors and mAb dosing was initiated at Day +3 following
inoculation (dosed on Days 3, 7, and 10) with 200 .mu.g/mouse of
the antibodies indicated in FIGS. 41A-41D.
[0971] As shown in FIGS. 41A-41D, both the anti-CTLA-4 antibody
(FIG. 41B) and anti-OX40 antibody (FIG. 41C) showed minimal
activity when used alone (1 of 8 mice tumor free for both
treatments), but had significant anti-tumor activity when combined
(FIG. 41D), with 4 of 8 mice rendered tumor free.
Example 29: Phase 112a Trial in Subject Having Solid Tumors
[0972] A Phase 1/2a study of OX40.21 administered alone or in
combination with nivolumab or ipilimumab is conducted in subjects
having advanced solid tumors to demonstrate the efficacy of
administering OX40.21 alone or in combination with nivolumab or
ipilimumab.
1. Objective
[0973] The primary objective of the study is to assess the safety,
tolerability, dose-limiting toxicities (DLTs), and maximum
tolerated dose (MTD)/recommended phase 2 dose (RP2D) of OX40.21
administered alone or in combination with nivolumab or ipilimumab
in subjects with advanced malignant tumors.
[0974] Secondary objectives include investigating the preliminary
anti-tumor activity of OX40.21 administered alone or in combination
with nivolumab or ipilimumab in subjects with advanced malignant
tumors; characterizing the PK of OX40.21 administered alone and in
combination with nivolumab or ipilimumab; and characterizing the
immunogenicity of OX40.21 administered alone or in combination with
nivolumab or ipilimumab and the immunogenicity of nivolumab or
ipilimumab administered with OX40.21. Additional exploratory
objectives include exploring potential associations between
anti-tumor activity and select biomarker measures in tumor biopsy
specimens and peripheral blood prior to treatment and following
administration of OX40.21 alone or in combination with nivolumab or
ipilimumab; assessing the potential effect of OX40.21 monotherapy
and combination therapy on QTc interval; characterizing nivolumab
PK in subjects receiving the combination of nivolumab and OX40.21;
characterizing ipilimumab PK in subjects receiving the combination
of ipilimumab and OX40.21; assessing the overall survival (OS) in
subjects treated with OX40.21 alone and in combination with
nivolumab or ipilimumab; and exploring potential relationships
between dose/exposure and anti-tumor activity, pharmacodynamic (PD)
effects (selected biomarkers in the peripheral blood and tumor
biopsy specimens), and key safety measures in subjects treated with
OX40.21 alone and in combination with nivolumab or ipilimumab.
2. Study Design and Duration
[0975] This is a Phase 1/2a, open label study of OX40.21 in
subjects with advanced solid tumors that integrates initial OX40.21
monotherapy with subsequent nivolumab or ipilimumab combination
therapy.
[0976] Study sections (dose escalation and dose expansion) proceed
in a phased approach based on study-emergent safety, PK, and PD
data. The first section of the study begins with OX40.21
monotherapy dose escalation cohorts. Clinical data from the first 3
monotherapy dose cohorts serve as a foundation for initiating dose
escalation of OX40.21 in combination with nivolumab. Clinical data
from the first 3 monotherapy dose cohorts in addition to the
clinical data from the first cohort of OX40.21 in combination with
nivolumab serve as a foundation for initiating dose escalation of
OX40.21 in combination with ipilimumab. After establishment of a
tolerable and pharmacologically active RP2D of OX40.21 in the dose
escalation section, dose expansion in specific tumor cohorts is
initiated.
3. Dose Escalation
[0977] A schematic of the study design for Part 1A is shown in FIG.
42.
[0978] The dose escalation phase of the study evaluates the safety
and tolerability of OX40.21, alone or in combination with nivolumab
or ipilimumab, in subjects with advanced solid tumors.
[0979] The initial dose level of OX40.21 is 20 mg. Dose escalation
decisions for subsequent doses are based on DLTs using a BLRM model
(for OX40.21 monotherapy) or a BLRM (-Copula) model (for OX40.21 in
combination with nivolumab or ipilimumab). The DLT period is 28
days for both monotherapy and combination therapy dose escalation
parts. The DLT rate is determined based on the incidence, severity,
and duration of AEs that occur within the DLT period and for which
no alternative cause can be identified. Dose selection for the next
monotherapy cohort/dose level takes into account the BLRM (-Copula)
recommendation in conjunction with all available PK, PD, and
clinical and laboratory safety data from all treated subjects.
Starting dose selection of OX40.21 for Part 2A is determined using
data available from Part 1A, including clinical and laboratory
safety assessments, PK/PD data, and modeling recommendation within
Bayesian hierarchical modeling framework by incorporating
single-agent toxicity profiles of both OX40.21 (Part 1A) and
nivolumab (CA209-003). Starting dose selection of OX40.21 for Part
3A is determined using data available from Parts 1A and 2A,
including clinical and laboratory safety assessments, PK/PD data,
and modeling recommendation within Bayesian modeling framework by
incorporating single-agent toxicity profiles of both OX40.21 (Part
1A) and ipilimumab (CA184-022). Actual doses can be modified per
the BLRM (-Copula), but do not exceed doubling of the previously
tested dose.
[0980] During dose escalation for all dose cohorts, the initial
subject (sentinel subject) is observed for 5 days before additional
subjects in that cohort are treated with study drug.
[0981] Approximately 30 subjects are enrolled in each dose
escalation part. The number of subjects in each dose escalation
cohort varies depending on BLRM (-Copula) recommendations.
Initially, approximately 3 subjects are treated at the starting
dose levels of OX40.21 or OX40.21 in combination with nivolumab or
ipilimumab. Additional cohorts of approximately 3 evaluable
subjects are treated at recommended dose levels per BLRM (-Copula)
during the dose escalation phase. At least 6 DLT-evaluable subjects
are treated at the MTD.
[0982] Part 1A: Enrollment begins in Part 1A, OX40.21 monotherapy
dose escalation. The initial dose of OX40.21 for Part 1A is 20 mg,
with expected subsequent doses of 40, 80, 160, and 320 mg. Actual
doses can be modified per the BLRM but do not exceed doubling of
the previously tested dose.
[0983] Part 2A: Part 2A is the combination arm of OX40.21 with
nivolumab that is initiated after at least 3 dose levels in the
monotherapy dose escalation are found to be tolerated or an MTD has
been determined in the monotherapy dose escalation (Part 1A). The
starting dose of OX40.21 in Part 2A is at least 1 dose level below
a dose demonstrated to be tolerated in Part 1A to ensure further
safety of the combination. At no time does the dose for OX40.21 in
Part 2A exceed the highest tolerated dose in Part 1A. Nivolumab is
administered at a flat dose of 240 mg. Each treatment cycle is 2
weeks in length and study drugs are administered every 2 weeks
starting on Day 1 of each cycle for up to 12 cycles.
[0984] Part 3A: Part 3A is the combination arm of OX40.21 with
ipilimumab that is initiated only after at least 3 dose levels in
the monotherapy dose escalation are found to be tolerated or an MTD
is determined in the monotherapy dose escalation (Part 1A) and at
least 1 dose cohort is found to be tolerated in the OX40.21 with
nivolumab dose escalation part. The starting dose of OX40.21 in
Part 3A is at least 1 dose level below a dose demonstrated to be
tolerated in Part 1A. At no time does the dose for OX40.21 in Part
3A exceed the highest tolerated dose in Part 1A to further ensure
safety of the combination doses in treated subjects. Ipilimumab is
administered at a dose of 1 mg/kg. Each treatment cycle is 3 weeks
in length. OX40.21 is administered every 3 weeks starting on Cycle
1 Day 1, up to and including 8 cycles, and ipilimumab is
administered every 3 weeks starting on Day 1 for 4 cycles. Only
OX40.21 is administered in the last 4 cycles.
Dose Expansion:
[0985] Treatment in the dose expansion cohorts is initiated when
the MTD/RP2D has been determined based on the evaluation of
totality of available clinical safety (DLTs, significant AEs
occurring after the DLT period), PK, PD, and modeling data from the
dose escalation (Parts 1A, 2A, and 3A). Approximately 110 subjects
are treated in all dose expansion cohorts.
[0986] Part 1B is the OX40.21 monotherapy dose expansion cohort in
subjects with cervical cancer at the MTD/RP2D determined in Part
1A. Dosing of OX40.21 begins on Day 1 of each cycle and is
administered every 2 weeks for up to 12 cycles. Approximately 12
subjects are treated in this expansion cohort.
[0987] Parts 2B is the combination therapy (OX40.21 with nivolumab)
dose expansion part in subjects with CRC at the MTD/RP2D determined
in Part 2A. Nivolumab is administered at a flat dose of 240 mg.
Each treatment cycle is 2 weeks in length and study drugs are
administered every 2 weeks starting on Day 1 of each cycle for up
to 12 cycles. Approximately 35 subjects are treated in this
expansion cohort.
[0988] Part 2C is the combination therapy (OX40.21 with nivolumab)
dose expansion part in subjects with BC at the MTD/RP2D determined
in Part 2A. Each treatment cycle is 2 weeks in length and study
drugs are administered every 2 weeks starting on Day 1 of each
cycle for up to 12 cycles. Approximately 27 subjects are treated in
this expansion cohort.
[0989] Part 3B is the combination therapy (OX40.21 with ipilimumab)
dose expansion part in subjects with OC at the MTD/RP2D determined
in Part 3A. Each treatment cycle is 3 weeks in length. Ipilimumab
is administered in the initial 4 cycles in combination with
OX40.21. Then the subject continues on OX40.21 monotherapy for up
to an additional 4 cycles for a total of up to 24 weeks (8 cycles)
of treatment. Approximately 35 subjects with OC are treated in this
expansion cohort.
Summary of Study Periods:
[0990] Subjects complete up to 5 periods in the study: Screening
(up to 28 days), Treatment (up to 24 weeks), Safety Follow-up
(minimum 100 days), Response Follow-up, and Survival Long-term
Follow-up (up to approximately 2 years from the first dose) as
described below. The study visit schematic is presented in FIG.
43.
Screening Period:
[0991] The Screening period lasts for up to 28 days. The screening
period begins by establishing the subject's initial eligibility and
signing of the informed consent form. Subjects are enrolled using
an Interactive Response Technology (IRT).
Treatment Period:
[0992] The Treatment period consists of up to 24 weeks of dosing.
Following each treatment cycle, the decision to treat a subject
with the next cycle of study therapy, up to 24 weeks of treatment,
is based on risk/benefit and tumor assessments. Tumor assessments
are performed every 8 weeks for every 2-week (q2w) dosing regimen
and every 9 weeks for every 3-week (q3w) dosing regimen.
Assessments of partial response (PR) and complete response (CR)
mare confirmed at least 4 weeks following initial assessment. Tumor
progression or response endpoints are assessed using Response
Evaluation Criteria In Solid Tumors (RECIST) v1.1.
[0993] Subjects with a response of stable disease (SD), PR, or CR
at the end of a given cycle continue to the next treatment cycle.
Subjects are generally allowed to continue study therapy until the
first occurrence of one of the following: 1) completion of the
maximum number of cycles; 2) progressive disease; 3) clinical
deterioration suggesting that no further benefit from treatment is
likely; 4) intolerability to therapy; or 5) meeting the criteria
for discontinuation of study therapy.
Safety Follow-Up:
[0994] Upon completion of study therapy, subjects enter the Safety
Follow-up period. After the end of treatment (EOT) visit, subjects
are evaluated for any new adverse events (AEs) for at least 100
days after the last dose of therapy. Follow-up visits occur at Days
30, 60 and 100 after the last dose or the date of discontinuation.
Subjects (except those who withdraw consent for study
participation) complete 3 clinical Safety Follow-up visits
regardless of whether they start new anti-cancer therapy.
Survival Follow-Up:
[0995] After completion of the Safety Follow-up period, subjects
enter the Survival Follow-up period. Subjects are followed
approximately every 3 months (12 weeks) until death, lost to
follow-up, withdrawal of consent, or conclusion of the study,
whichever comes first. The duration of this phase is up to 2 years
following the first dose of study drug.
Response Follow-Up:
[0996] After completion of the Safety Follow-up period, all
subjects with ongoing SD, PR, or CR at the EOT visit enter the
Response Follow-up period, which occurs simultaneously with the
Survival Follow-up period. These subjects continue to have
radiological and clinical tumor assessments every 3 months (12
weeks) during the Response Follow-up period or until disease
progression or withdrawal of study consent. Radiological tumor
assessments for subjects who have ongoing clinical benefit continue
to be collected after subjects complete the survival phase of the
study. Subjects who have disease progression following initial
course of study therapy are not evaluated for response beyond the
EOT visit and are allowed to receive other tumor directed therapy
as required.
Duration of Study:
[0997] The total duration of study time for any individual subject
is approximately 2 years. The study ends when the last subject
completes their last study visit, which is approximately 4 years
after the start of the study.
Number of Subjects:
[0998] Approximately 225 subjects will be enrolled, and
approximately 200 subjects will be treated in the study.
Study Population:
[0999] Subjects are at least 18 years old and have histologic or
cytologic confirmation of a malignancy that is advanced
(metastatic, recurrent, refractory and/or unresectable) with
measurable disease per RECIST v1.1.
Dose Escalation and Stopping Rules
[1000] In Parts 1A, 2A, and 3A, the BLRM and BLRM (-Copula) models
are utilized for dose escalation recommendations after DLT
information becomes available for each cohort of subjects. OX40.21
dose selection for the next cohort/dose level takes into account
the BLRM (-Copula) recommendation in conjunction with clinical
recommendation and all available PK, PD, and clinical and
laboratory safety data from all treated subjects.
Dose-Limiting Toxicities
[1001] To guide dose escalation, DLTs are defined based on the
incidence, intensity, and duration of AEs for which no clear
alternative cause is identified. The DLT period is 28 days of
initiation of the study drug(s). For subject management, an AE that
meets DLT criteria, regardless of the cycle in which it occurs,
leads to discontinuation of study drug. Subjects who withdraw from
the study during the DLT evaluation interval for reasons other than
a DLT may be replaced with a new subject at the same dose level.
The incidence of DLT(s) during the DLT evaluation period is used in
dose escalation decisions and to define the MTD. AEs occurring
after the DLT period are considered for the purposes of defining
the MTD, if they are determined to have no clear alternative cause
and are not related to disease progression. Subjects experiencing a
DLT are not retreated with study drug and enter the safety
follow-up period of the study. AEs are graded according to the
National Cancer Institute (NCI) Common Terminology Criteria for
Adverse Events (CTCAE) v4.03.
Non-Hematologic DLT:
[1002] A. Hepatic DLT [1003] Any .gtoreq.Grade 3 elevation of AST,
ALT, or total bilirubin [1004] AST, ALT, or total bilirubin, [1005]
Grade 2 AST or ALT with symptomatic liver inflammation (e.g., right
upper quadrant tenderness, jaundice, pruritis) [1006] AST or
ALT>3.times.ULN and concurrent total bilirubin >2.times.ULN
without initial findings of cholestasis (elevated serum alkaline
phosphatase [ALP]) (e.g., findings consistent with Hy's law or FDA
definition of potential drug-induced liver injury or pDILI)
[1007] B. Non-Hepatic DLT [1008] Grade 2 or greater uveitis,
episcleritis, or iritis [1009] Any other Grade 2 eye pain or
blurred vision that does not respond to topical therapy and does
not improve to Grade 1 severity within 2 weeks OR requires systemic
treatment [1010] Grade 3 or greater pneumonitis, bronchospasm,
neurologic toxicity, hypersensitivity reaction, or infusion
reaction [1011] Any Grade 3 or greater non-dermatologic,
non-hepatic toxicity will be considered a DLT with the following
specific exceptions: [1012] Grade 3 or Grade 4 electrolyte
abnormalities that are not complicated by associated clinical
adverse experiences, last less than 72 hours, and either resolve
spontaneously or respond to conventional medical intervention
[1013] Grade 3 nausea, vomiting, or diarrhea that lasts less than
72 hours, and either resolves spontaneously or responds to
conventional medical intervention [1014] Grade 3 or 4 elevation of
amylase or lipase not associated with clinical or radiographic
evidence of pancreatitis [1015] Isolated Grade 3 fever not
associated with hemodynamic compromise (e.g., hypotension,
clinical, or laboratory evidence of impaired end-organ perfusion)
[1016] Grade 3 endocrinopathy that is well controlled by hormone
replacement [1017] Grade 3 tumor flare (defined as pain,
irritation, or rash that localizes to sites of known or suspected
tumor) [1018] Grade 3 fatigue for less than 7 days [1019] Grade 3
infusion reaction that returns to Grade 1 in less than 6 hours
[1020] Dermatologic DLT [1021] Grade 4 rash [1022] Grade 3 rash if
no improvement (i.e., resolution to .ltoreq.Grade 1) after a 1- to
2-week infusion delay. Subjects who have not experienced a Grade 3
skin AE may resume treatment in the presence of Grade 2 skin
toxicity.
[1023] Hematologic DLT [1024] Grade 4 neutropenia .gtoreq.5 days in
duration [1025] Grade 4 thrombocytopenia or Grade 3
thrombocytopenia with clinically significant bleeding, or any
requirement for platelet transfusion [1026] Grade 4 anemia not
explained by underlying disease [1027] Grade 4 febrile neutropenia
[1028] Grade 3 febrile neutropenia that lasts >48 hours [1029]
Grade .gtoreq.3 hemolysis (i.e., requiring transfusion or medical
intervention such as steroids) Treatment with Additional Cycles
Beyond 24 Weeks
[1030] Subjects are treated for 24 weeks unless criteria for study
drug discontinuation are met earlier. Subjects completing
approximately 24 weeks of treatment with ongoing disease control
(CR, PR, or SD) are eligible for an additional 24 weeks of study
therapy in monotherapy (Part 1) and combination therapy (Parts 2
and 3) beyond the initial 24 weeks when the risk/benefit assessment
favors continued administration of study therapy. Upon completion
of the additional 24 weeks of study therapy, subjects enter the
Safety Follow-up period.
Treatment Beyond Progression
[1031] Treatment beyond progression is allowed in select subjects
with initial RECIST v1.1-defined progressive disease after
determining that the benefit/risk assessment favors continued
administration of study therapy (e.g., subjects are continuing to
experience clinical benefit, tolerating treatment, and meeting
other criteria).
Retreatment
[1032] Retreatment is allowed if confirmed disease progression
occurs during the response follow-up period. Subjects completing
approximately 24 weeks (or additional cycles of treatment, if
appropriate) of therapy who enter the response follow-up period
with ongoing disease control (CR, PR, or SD) without any
significant toxicity are eligible for retreatment. Such subjects
are eligible for retreatment on a case-by-case basis after
evaluation and determining whether the risk/benefit ratio supports
administration of further study therapy, and the subject continues
to meet eligibility criteria for treatment with study therapy.
Subjects meeting criteria for retreatment are treated with the
originally assigned monotherapy or combination therapy regimen
(e.g., the same dose and dose schedule as administered during the
first 24 weeks), unless that dose and schedule were subsequently
found to exceed the MTD, in which case the subject is treated at
the next lower dose deemed tolerable/safe.
Inclusion Criteria
[1033] 1) Signed Written Informed Consent [1034] a) The subject
must sign the informed consent form prior to the performance of any
study-related procedures that are not considered part of SOC.
[1035] b) Consent for tumor biopsy samples (mandatory pre- and
on-treatment biopsies are required for the dose expansion cohorts,
and for additional subjects added to any of the previously
completed dose escalation cohorts and optional for dose escalation
cohorts).
[1036] 2) Target Population [1037] Subjects must be at least 18
years old and have histologic or cytologic confirmation of a
malignancy that is advanced (metastatic, recurrent, refractory,
and/or unresectable) with measurable disease per RECIST v1.1.
[1038] A. Dose Escalation: [1039] Subjects must have received, and
then progressed, or have been refractory or intolerant to, at least
1 standard treatment regimen in the advanced or metastatic setting,
if such a therapy exists. Subjects who are ineligible for any
standard therapy are allowed to enroll provided their ineligibility
is documented in medical records. The following tumor histologies
are permitted except for subjects with primary central nervous
system (CNS) tumors, or with CNS metastases as the only site of
active disease. [1040] (i) Melanoma: BRAF mutation status must be
documented if known. [1041] (ii) NSCLC: EGFR, ALK, KRAS, and ROS1
mutational status must be documented if known [1042] (iii) Head and
neck cancer restricted to squamous cell carcinoma. HPV status must
be documented if known [1043] (iv) Transitional cell carcinoma of
the genitourinary tract [1044] (v) Renal cell carcinoma [1045] (vi)
Pancreatic adenocarcinoma [1046] (vii) CRC: MSI, KRAS, and BRAF
status must be documented if known. [1047] (viii) Cervical cancer:
HPV status must be documented if known. [1048] (ix) Triple negative
breast cancer HER2, ER and PR status must be documented [1049] (x)
Adenocarcinoma of the endometrium [1050] (xi) Ovarian cancer [1051]
(xii) Prostate adenocarcinoma [1052] (xiii) Hepatocellular
cancer-Child Pugh A only [1053] (xiv) Small cell lung cancer [1054]
(xv) Gastric and gastric esophageal junction cancer: HER2 Status
must be documented if known.
[1055] B. Dose Expansion: [1056] Parts 1B, 2B, 2C, and 3B [1057]
The following tumor types will be permitted: [1058] (a) Cervical
Cancer--Part 1B [1059] (i) Histologically confirmed cervical cancer
that is unresectable, metastatic, or recurrent with documented
disease progression [1060] (ii) Document tumor HPV status if known.
If unknown, subjects must consent to allow their submitted archived
tumor tissue sample (block or unstained slides) to be tested.
[1061] (iii) Prior therapy requirement: [1062] 1. Must have
received and then progressed or have been intolerant or refractory
to at least 1 standard systemic therapy, for metastatic and/or
unresectable disease (e.g., paclitaxel/cisplatin,
paclitaxel/cisplatin/bevacizumab). Concurrent chemotherapy
administered with primary radiation and adjuvant chemotherapy given
following completion of radiation therapy do not count as systemic
chemotherapy regimens. [1063] (b) Colorectal Cancer--Part 2B [1064]
(i) Histologically confirmed CRC that is metastatic or recurrent
with documented disease progression [1065] (ii) Document MSI, MMR,
KRAS, and BRAF status if known. If unknown, subjects must consent
to allow their submitted archived tumor tissue sample (block or
unstained slides) to be tested. [1066] (iii) Prior therapy
requirement: [1067] Subjects must have received and then progressed
or have been intolerant or refractory to at least 1 standard
systemic therapy, for metastatic and/or unresectable disease (or
have progressed within 6 months of adjuvant therapy). [1068] (c)
Bladder Cancer--Part 2C [1069] (i) Histologically or cytologically
confirmed urothelial carcinoma (including mixed histologies of
urothelial carcinoma with elements of other subtypes) of the renal
pelvis, ureter, bladder, or urethra with progression or refractory
disease [1070] (ii) Prior therapy requirement: [1071] Subjects must
have received and then progressed or have been intolerant or
refractory to at least 1 standard systemic therapy (e.g., platinum
based chemotherapy) regimen for the treatment of metastatic (Stage
IV) or locally advanced unresectable disease. [1072] (d)
Ovarian--Part 3B [1073] (i) Histologically or cytologically
confirmed ovarian carcinoma (including epithelial OC, primary
peritoneal, or fallopian tube carcinoma) with documented disease
progression [1074] (ii) Documented germline BRCA mutation status,
if known. If unknown, subjects must consent to allow their
submitted archived tumor tissue sample (block or unstained slides)
to be tested. [1075] (iii) Prior therapy requirement: [1076]
Subjects must have received and then progressed or have been
intolerant or refractory to at least 1 standard systemic therapy
(e.g., platinum-based chemotherapy), for metastatic and/or
unresectable disease.
[1077] 3) Eastern Cooperative Oncology Group (ECOG) performance
status of .ltoreq.1.
[1078] 4) Presence of at least 1 lesion with measurable disease as
defined by RECIST v1.1 for response assessment. Subjects with
lesions in a previously irradiated field as the sole site of
measurable disease are permitted to enroll provided the lesion(s)
have demonstrated clear progression and can be measured
accurately.
[1079] 5) For subjects requiring fresh tumor biopsy, subjects must
have at least one lesion accessible for pre- and on-treatment
biopsy, in addition to the minimum one RECIST v1.1 measurable
lesion required for response assessment. This lesion needs to be
distinct from index lesion(s) being evaluated for radiological
response.
[1080] 6) Subjects with prior exposure to therapy with any agent
specifically targeting checkpoint pathway inhibition (such as
anti-PD-1, anti-PD-L1, anti-PD-L2, anti-LAG-3, and anti-CTLA-4
antibody) are permitted after a washout period of any time greater
than 4 weeks from the last treatment
[1081] Note: [1082] (i) Subjects who experienced prior Grade 1 to 2
checkpoint therapy-related immune-mediated AEs must have confirmed
recovery from these events at the time of study entry, other than
endocrinopathies treated with supplementation, as documented by
resolution of all related clinical symptoms, abnormal findings on
physical examination, and/or associated laboratory abnormalities.
Where applicable, these subjects must also have completed steroid
tapers for treatment of these AEs by a minimum of 14 days prior to
commencing treatment with study therapy. (ii) Eligibility of
subjects with prior .gtoreq.Grade 3 checkpoint therapy-related
immune AEs, will be considered on a case-by-case basis after
discussion with the Medical Monitor (e.g., asymptomatic isolated
Grade 3 lipase elevations without clinical or radiological features
of pancreatitis are permitted to enroll).
[1083] 7) Subjects with prior therapy with any agent specifically
targeting T-cell co-stimulation pathways except anti-OX40 antibody,
anti-CD137, anti-GITR antibody, and anti-CD27 are permitted after a
washout period of any time greater than 4 weeks from the last
treatment.
[1084] 8) Prior palliative radiotherapy must have been completed at
least 2 weeks prior to first dose of study drug. Subjects with
symptomatic tumor lesions at baseline that may require palliative
radiotherapy within 4 weeks of first dose of study drug are
strongly encouraged to receive palliative radiotherapy prior to
enrollment.
[1085] 9) Subjects enrolled into dose escalation and expansion
cohorts must consent to the acquisition of existing formalin-fixed,
paraffin-embedded (FFPE) tumor tissue, either a block or a minimum
of 15 unstained slides (25 slides preferred), for performance of
correlative studies. If an archived sample is not available,
subject must consent to a pre-treatment tumor biopsy. Subjects
unable to provide an archived tumor sample and who either do not
consent to a pre-treatment tumor biopsy or do not have accessible
lesions are not eligible. (However, subjects whose pre-treatment
biopsy yields inadequate tissue quantity or quality will not be
ineligible on this basis alone). For any additional subjects added
to any of the previously completed dose escalation cohorts,
mandatory pre- and on-treatment biopsies are required.
[1086] 10) Subjects enrolled into dose expansion, or added to any
previously completed dose escalation cohort, are required to
undergo mandatory pre- and on treatment biopsies at acceptable
clinical risk. (a) The solid tumor tissue specimen must be a core
needle, excisional, or incisional biopsy. Fine needle biopsies,
drainage of pleural effusions with cytospins, or punch biopsies are
not considered adequate for biomarker review. Biopsies of bone
lesions that do not have a soft tissue component or decalcified
bone tumor samples are also not acceptable. (b) Biopsied lesions
should be distinct from index lesion(s) being evaluated for
radiological response
[1087] 11) Adequate organ function for subjects as defined by the
following: [1088] (a) Neutrophils .gtoreq.1500/.mu.L (stable off
any growth factor within 4 weeks of first study drug
administration) [1089] (b) Platelets .gtoreq.80.times.103/.mu.L
(transfusion to achieve this level is not permitted within 2 weeks
of first study drug administration) [1090] (c) Hemoglobin .gtoreq.8
g/dL (transfusion to achieve this level is not permitted within 2
weeks of first study drug administration) [1091] (d) ALT and AST
.ltoreq.3.times. upper limit of normal (ULN) [1092] (e) Total
bilirubin .ltoreq.1.5.times.ULN (except subjects with Gilbert's
Syndrome who must have normal direct bilirubin) [1093] (f) Normal
thyroid function or stable on hormone supplementation per
investigator assessment [1094] (g) Albumin .gtoreq.2 mg/dl [1095]
(h) Serum creatinine .ltoreq.1.5.times.ULN or creatinine clearance
(CrCl).gtoreq.40 ml/min (measured using the Cockcroft-Gault formula
below):
[1095] Female CrCl=(140-age in years).times.weight in kg.times.0.85
72.times.serum creatinine in mg/dL
Male CrCl=(140-age in years).times.weight in kg.times.1.00
72.times.serum creatinine in mg/dL
[1096] 12) Ability to comply with treatment, PK and PD sample
collection, and required study follow-up
Age and Reproductive Status
[1097] a) Men and women, ages .gtoreq.18 years at the time of
informed consent.
[1098] b) Women of childbearing potential (WOCBP) must have a
negative serum or urine pregnancy test (minimum sensitivity 25 IU/L
or equivalent units of human chorionic gonadotrophin [hCG]) within
24 hours prior to the start of study drug.
[1099] c) Women must not be breastfeeding.
[1100] d) WOCBP must agree to follow instructions for method(s) of
contraception for the duration of treatment with study drug OX40.21
plus 5 half-lives of study drug plus 30 days. This duration should
be 12 weeks for Parts 1 and 3 subjects (50 days plus 30 days) or 23
weeks for Part 2 subjects (130 days plus 30 days [duration of
ovulatory cycle]), for a total of up to 160 days post-treatment
completion.
[1101] e) Men who are sexually active with WOCBP must agree to
follow instructions for method(s) of contraception for the duration
of treatment with study drug OX40.21 plus 5 half-lives of the study
drug plus 90 days. The duration should be 20 weeks for Parts 1 and
3 subjects (50 days plus 90 days) or 31 weeks for Part 2 subjects
(130 days completion. In addition, male subjects must be willing to
refrain from sperm donation during this time.
[1102] f) Azoospermic males are exempt from contraceptive
requirements. WOCBP who are continuously not heterosexually active
are also exempt from contraceptive requirements, but still undergo
pregnancy testing.
Exclusion Criteria
1) Target Disease Exceptions
[1103] a) Subjects with known or suspected CNS metastases or
untreated CNS metastases, or with the CNS as the only site of
disease, are excluded. However, subjects with controlled brain
metastases are allowed to enroll. Controlled brain metastases are
defined as no radiographic progression for at least 4 weeks
following radiation and/or surgical treatment (or 4 weeks of
observation if no intervention is clinically indicated), and off of
steroids for at least 2 weeks, and no new or progressive
neurological signs and symptoms.
[1104] b) Subjects with carcinomatous meningitis
[1105] c) For ovarian cancer: [1106] i) ovarian cancer subjects
with history of bowel obstruction in the prior 6 months or with
Tenckhoff catheter are excluded. [1107] ii) up to 4 prior
anti-cancer treatments are permitted (i.e, chemotherapy,
radiotherapy, hormonal, or immunotherapy). Restarting the same
regimen after a drug holiday may be considered one regimen; however
it would be counted as two regimens if there was any other regimen
used in between.
2) Medical History and Concurrent Diseases
[1108] a) Subjects with a prior malignancy, different from the one
used for enrollment in this study, diagnosed within less than 2
years prior to study entry are excluded (except non-melanoma skin
cancers and in situ cancers such as bladder, colon,
cervical/dysplasia, melanoma, or breast). In addition, subjects
with other second malignancies diagnosed more than 2 years ago who
have received therapy with curative intent with no evidence of
disease during the interval who are considered to present a low
risk for recurrence are eligible.
[1109] b) Other active malignancy requiring concurrent
intervention
[1110] c) Prior organ allograft
[1111] d) Previous treatment: [1112] i) Prior anti-cancer
treatments are permitted (i.e, chemotherapy, radiotherapy,
hormonal, or immunotherapy) [1113] ii) Toxicity (except for
alopecia) related to prior anti-cancer therapy and/or surgery must
either have resolved, returned to baseline or Grade 1 or have been
deemed irreversible [1114] iii) For cytotoxic agents at least 4
weeks must have elapsed between the last dose of prior to
anti-cancer therapy and initiation of study therapy [1115] iv) For
non-cytotoxic agents at least 4 weeks or 5 half-lives (whichever is
shorter) must have elapsed from last dose of prior anti-cancer
therapy and the initiation of study therapy.
[1116] e) Prior therapy with anti-OX40 antibody
[1117] f) Subjects with active, known, or suspected autoimmune
disease are excluded. Subjects with vitiligo, type 1 diabetes
mellitus, residual hypothyroidism due to autoimmune condition only
requiring hormone replacement, euthyroid subjects with a history of
Grave's disease (subjects with suspected autoimmune thyroid
disorders must be negative for thyroglobulin and thyroid peroxidase
antibodies and thyroid stimulating immunoglobulin prior to first
dose of study drug), psoriasis not requiring systemic treatment, or
conditions not expected to recur in the absence of an external
trigger are permitted to enroll. Subjects with well controlled
asthma and/or mild allergic rhinitis (seasonal allergies) are
eligible.
[1118] g) Subjects with history of life-threatening toxicity
related to prior immune therapy (e.g., anti-CTLA-4 or
anti-PD-1/PD-L1 treatment or any other antibody or drug
specifically targeting T-cell co-stimulation or immune checkpoint
pathways) except those that are unlikely to re-occur with standard
countermeasures (e.g., hormone replacement after adrenal
crisis)
[1119] h) Subjects with interstitial lung disease that is
symptomatic or that may interfere with the detection or management
of suspected drug-related pulmonary toxicity
[1120] i) Chronic obstructive pulmonary disease requiring recurrent
steroid bursts or chronic steroids at doses greater than 10 mg/day
of prednisone or the equivalent
[1121] j) Subjects with a condition requiring systemic treatment
with either corticosteroids (>10 mg daily prednisone
equivalents) or other immunosuppressive medications within 14 days
of study drug administration except for adrenal replacement steroid
doses >10 mg daily prednisone equivalent in the absence of
active autoimmune disease. Note: Treatment with a short course of
steroids (<5 days) up to 7 days prior to initiating study drug
is permitted.
[1122] k) Uncontrolled or significant cardiovascular disease,
including but not limited to any of the following: [1123] i)
Myocardial infarction or stroke/transient ischemic attack within
the past 6 months [1124] ii) Uncontrolled angina within the past 3
months [1125] iii) Any history of clinically significant
arrhythmias (such as ventricular tachycardia, ventricular
fibrillation, or torsades de pointes) [1126] iv) History of other
clinically significant heart disease (e.g., cardiomyopathy,
congestive heart failure with New York Heart Association functional
classification III-IV, pericarditis, significant pericardial
effusion) [1127] v) Cardiovascular disease-related requirement for
daily supplemental oxygen therapy [1128] vi) QT interval corrected
for heart rate using Fridericia's formula (QTcF) prolongation
>480 msec
[1129] l) History of any chronic hepatitis as evidenced by the
following: [1130] i) Positive test for hepatitis B surface antigen
[1131] ii) Positive test for qualitative hepatitis C viral load (by
PCR) [1132] Note: Subjects with positive hepatitis C antibody and
negative quantitative hepatitis C by PCR are eligible. History of
resolved hepatitis A virus infection is not an exclusion criterion.
Additional testing or substitute testing per institutional
guidelines to rule out infection is permitted.
[1133] m) Evidence of active infection that requires systemic
antibacterial, antiviral, or antifungal therapy .ltoreq.7 days
prior to initiation of study drug therapy (does not apply to viral
infections that are presumed to be associated with the underlying
tumor type required for study entry)
[1134] n) Known history of testing positive for HIV or known
acquired immunodeficiency syndrome.
[1135] o) Evidence or history of active or latent tuberculosis
infection including PPD recently converted to positive; chest x-ray
with evidence of infectious infiltrate; recent unexplained changes
in fever/chill patterns.
[1136] p) Any major surgery within 4 weeks of study drug
administration. Subjects must have recovered from the effects of
major surgery or significant traumatic injury at least 14 days
before the first dose of study drug.
[1137] q) Use of non-oncology vaccines containing live virus for
prevention of infectious diseases within 4 weeks prior to study
drug. The use of inactivated seasonal influenza vaccines, e.g.,
Fluzone.RTM., is permitted.
[1138] r) Use of pRBC or platelet transfusion within 2 weeks prior
to the first dose of study drug
[1139] s) A known or underlying medical or psychiatric condition
and/or social reason that could make the administration of study
drug hazardous to the subjects or could adversely affect the
ability of the subject to comply with or tolerate the study.
3) Allergies and Adverse Drug Reaction
[1140] a) History of allergy to nivolumab or ipilimumab (Parts 2
and 3 only, respectively)
[1141] b) History of any significant drug allergy (such as
anaphylaxis or hepatotoxicity) to prior anti-cancer immune
modulating therapies (e.g., checkpoint inhibitors, T-cell
co-stimulatory antibodies)
Study Assessments:
[1142] Physical examinations, vital sign measurements, 12-lead
electrocardiograms (ECGs), and clinical laboratory evaluations are
performed at selected times throughout the dosing interval.
Subjects are closely monitored for AEs throughout the study. [1143]
Safety Assessments: AEs are assessed during the study and for 100
days after the last treatment. AEs are evaluated according to NCI
CTCAE v4.03. Subjects are followed until all treatment-related AEs
have recovered to baseline or are deemed irreversible. [1144]
Efficacy Assessments: Disease assessment with CT and/or MRI as
appropriate are performed at baseline and every 8 weeks (.+-.1
week) for q2w dosing regimens and every 9 weeks (.+-.1 week) for
q3w dosing regimens, then every 12 weeks during the treatment and
response follow-up phases until discontinuation of treatment or
withdrawal from study. Tumor assessments at other time points are
performed if there are concerns about tumor progression. Assessment
of tumor response is made according to RECIST v1.1 for subjects
with malignant tumors. [1145] Pharmacokinetic and Immunogenicity
Assessments: Samples for PK and immunogenicity assessments are
collected for subjects receiving OX40.21 alone or in combination
with nivolumab or ipilimumab. The PK of OX40.21 is characterized by
non-compartmental analysis (NCA) method. Immunogenicity samples are
analyzed for anti-OX40.21 antibodies and/or anti-nivolumab
antibodies and/or anti-ipilimumab antibodies by validated
immunoassays. [1146] Exploratory Biomarker Assessments: To explore
potential predictive markers for clinical response to OX40.21 in
relation to dose and PK, 3 types of specimens are obtained from all
subjects for biomarker testing: (i) whole blood, (ii) serum/plasma,
and (iii) tumor tissue.
Statistical Considerations
Sample Size Determination
Dose Escalation:
[1147] As a Phase 1 dose escalation trial, the sample size for each
dose escalation cohort depends on observed toxicity and posterior
inference. Approximately 30 subjects are treated during each dose
escalation part (OX40.21 monotherapy [Part 1A], OX40.21 in
combination with nivolumab [Part 2A], and OX40.21 in combination
with ipilimumab [Part 3A]) for a combined total of about 90
subjects in Parts 1A, 2A, and 3A. Initially, approximately 3
subjects are treated at the starting dose levels of OX40.21 or
OX40.21 in combination with nivolumab or ipilimumab. Additional
cohorts of approximately 3 evaluable subjects are treated at
recommended dose levels per BLRM (-Copula) recommendations during
the dose escalation phase. At least 6 DLT-evaluable subjects are
treated at the MTD.
Dose Expansion:
[1148] In general terms, the expansion phase sizing is based on
target response rates (target overall response rate) and the
ability to identify a signal for such clinical response that is
above the standard of care (historical overall response rate).
[1149] Approximately 12 subjects are treated in the Part 1B dose
expansion cohort. Approximately 35 subjects are treated in the Part
2B dose expansion cohort. Approximately 27 subjects are treated in
the Part 2C dose expansion cohort. Approximately 35 subjects are
treated in the Part 3B dose expansion cohort.
Endpoints
Primary Endpoints
[1150] The assessment of safety is based on the incidence of AEs,
serious AEs, AEs leading to discontinuation, and deaths. In
addition, clinical laboratory test abnormalities are examined.
Secondary Endpoints
[1151] Efficacy: The anti-tumor activity of OX40.21 alone and
OX40.21 in combination with nivolumab or ipilimumab is measured by
ORR, duration of response, and progression free survival rate
(PFSR) at 24 weeks based on RECIST v1.1. The above are determined
based on tumor measurements occurring at baseline, every 8 weeks
(.+-.1 week) for q2w dosing regimens and every 9 weeks (.+-.1 week)
for q3w dosing regimens during the treatment period, and every 3
months (12 weeks) during the survival follow-up period. [1152] Best
overall response (BOR) is assessed per RECIST 1.1 criteria. [1153]
ORR is the proportion of all treated subjects whose BOR is either
CR or PR. [1154] Duration of response, computed for all treated
subjects with a BOR of CR or PR, is the time between the date of
first response and the date of disease progression or death,
whichever occurs first. [1155] PFSR at 24 weeks is defined as the
proportion of treated subjects remaining progression free and
surviving at 24 weeks. The proportion is calculated by the
Kaplan-Meier estimate, which takes into account censored data.
Pharmacokinetics
[1156] Selected parameters, such as Cmax, Tmax, AUC(0-t), and
AUC(TAU), are assessed in 2 cycles depending on the schedule for
monotherapy or in combination with nivolumab or ipilimumab.
Parameters such as Ctau, CLT, Css-avg, accumulation index (AI), and
effective elimination half-life (T-HALFeff) are assessed in the
second cycle when intensive PK is collected.
Immunogenicity
[1157] The secondary objective of immunogenicity is assessed by the
frequency of positive ADA to OX40.21 or nivolumab or
ipilimumab.
Exploratory Endpoints
[1158] Exploratory objectives related to OS are assessed by OS rate
at a certain time point (e.g., 2 years). OS rate is the proportion
of subjects alive at that time point. OS for a subject is defined
the time from the date of first dose of study medication to the
date of death from any cause. Exploratory objectives related to
biomarkers are assessed by the change from baseline or baseline
level biomarker measurements in peripheral blood (e.g., soluble
factors including, but not limited to, cytokine and chemokines) or
tumor tissue (e.g., tumor-infiltrating lymphocytes).
[1159] For subjects with multiple ECG measurements, the following
parameters are optionally assessed: changes in the ECG intervals
QT, QTc, QRS, and P-R interval from baseline.
Analyses
[1160] Safety analyses: All recorded AEs are listed and tabulated
by system organ class, preferred term, and treatment. Vital signs
and clinical laboratory test results are listed and summarized by
treatment. Any significant physical examination findings and
clinical laboratory results are also noted. ECG readings are
evaluated, and abnormalities, if present, are noted.
[1161] Efficacy analyses: Listing of tumor measurements are
provided by subject and study day in each arm and dose level.
Individual subject's BOR is listed based on RECIST 1.1. To describe
the anti-tumor activity of OX40.21 alone or in combination with
nivolumab or ipilimumab, ORR is calculated. ORR and corresponding
2-sided 95% CI by the Clopper-Pearson method are provided by
treatment and/or dose level and tumor type. Median duration of
response and corresponding 2-sided 95% CI are reported by treatment
and/or dose level and tumor type. Duration of response is analyzed
using the Kaplan-Meier method. In addition, PFSR, the probability
of a subject remaining progression free or surviving to 24 weeks,
is estimated by the Kaplan-Meier methodology by treatment, tumor
type, and dose level. The corresponding 95% CI is derived based on
Greenwood formula. OS is plotted using the Kaplan-Meier method.
Median OS and corresponding 2-sided 95% CI are reported.
[1162] Pharmacokinetic analyses: All individual PK parameters are
listed for each analyte, including any exclusions and reasons for
exclusion from summaries. Summary statistics are tabulated for each
PK parameter by treatment. Geometric means and coefficients of
variation are presented for Cmax, AUC(0-t), AUC(TAU), Ctau, CLT,
Cssavg, and AI. Medians and ranges are presented for Tmax. Means
and standard deviations are presented for all other PK parameters
(e.g., T-HALFeff).
[1163] OX40.21 dose dependency is assessed in dose escalation
monotherapy. To describe the dependency on dose of OX40.21, scatter
plots of Cmax, AUC(0-t), and AUC(TAU) versus dose are provided for
each day measured. An exploratory assessment of dose
proportionality based on a power model and a CI around the power
coefficient is performed. Nivolumab and ipilimumab end of infusion
and trough (Ctrough) concentrations and OX40.21 trough
concentration are tabulated by treatment and study day using
summary statistics. These data may also be pooled with other
datasets for population PK analysis.
[1164] Immunogenicity analysis: All available immunogenicity data
are provided by treatment, dose, and immunogenicity status. The
frequency of subjects with positive ADA assessment of OX40.21,
nivolumab, and ipilimumab are determined.
[1165] Exploratory biomarker analyses: Summary statistics for
biomarkers and their corresponding changes (or percent changes)
from baseline are tabulated by planned study day and dose in each
arm. The time course of biomarker measures are represented
graphically. If there is indication of meaningful pattern over
time, further analysis (e.g., by linear mixed model) is performed
to characterize the relationship. Methods such as, but not limited
to, logistic regression are used to explore possible associations
between biomarker measures from peripheral blood or tumor biopsy
and clinical outcomes.
TABLE-US-00029 TABLE 23 SUMMARY OF SEQUENCES SEQ ID Description
Sequence 1 Human OX40 precursor
MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVS
RCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCR
CRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSS
DAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVA
AILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADA HSTLAKI 2
Extracellular domain of
LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCK mature human
OX40 PCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPG
DNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQ
PTEAWPRTSQGPSTRPVEVPGGRAVAA 3 Cynomolgus OX40
MCVGARRLGRGPCAALLLLGLGLSTTAKLHCVGDTYPSNDRCCQECRPGNGMVS
RCNRSQNTVCRPCGPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCR
CRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSS
DAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPAVA
AILGLGLALGLLGPLAMLLALLLLRRDQRLPPDAPKAPGGGSFRTPIQEEQADA HSALAKI 4
Human OX40-L MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSTL
QVSHRYPRIQ SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS
QEVNISLHYQ KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL
ILIHQNPGEF CVL* 5 human IgG1 constant
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL domain
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 6 human IgG1
constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL domain
(allotypic variant) TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 7 human IgG1
kappa light RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA chain
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 8
heavy chain constant LSPGK region alternative C-terminus 9 heavy
chain constant LSPG region alternative C-terminus 10 Human IgG1
kappa light RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS chain
constant region (CL)
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 11 3F4
VH CDR1 SYDVN 12 3F4 VH CDR2 WMNPNSGNTGYAPKFQG 13 3F4 VH CDR3
IYSSSYNWFDP 14 3F4 VL CDR1 RASQSVSSYLA 15 3F4 VL CDR2 DASNRAT 16
3F4 VL CDR3 QQRSNWPLT 17 3F4 VH
QVQLVQSGAEVKKPGASVKVSCKASGNTFTSYDVNWVRQATGQGLEWMG
WMNPNSGNTGYAPKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR
IYSSSYNWFDPWGQGTLVTVSS 18 3F4 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTF GGGTKVEIK 19 14B6
VH CDR1 SNWIG 20 14B6 VH CDR2 FIYPGDSDTRYSPSFQG 21 14B6 VH CDR3
YGDDWYFDL 22 14B6 VL1 CDR1 RASQSVSSYLA 23 14B6 VL1 CDR2 DASNRAT 24
14B6 VL1 CDR3 QQRGDWPIT 25 14B6 VL2 CDR1 RASQGISSWLA 26 14B6 VL2
CDR2 AASSLQS 27 14B6 VL2 CDR3 QQYNSYPRIT 28 14B6 VH
EVQLEQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMG
FIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDIAMYYCAR
YGDDWYFDLWGRGTLVTVSS 29 14B6 VL1
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWFQQRPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFSLTISSLEPEDFAVYYCQQRGDWPITF GQGTRLEIK 30 14B6
VL2 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRIT FGQGTRLEIK 31
23H3 VH CDR1 NYAMY 32 23H3 VH CDR2 AIGIGGDTFYTDSVKG 33 23H3 VH CDR3
MGTGYFFDY 34 23H3 VL CDR1 RASQSVSSYLA 35 23H3 VL CDR2 DASNRAT 36
23H3 VL CDR3 QQRSNWPLT 37 23H3 VH
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVS
AIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDMAVYYCARM
GTGYFFDYWGQGTLVTVSS 38 23H3 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTF GPGTKVDIK 39 6E1
VH CDR1 SFAMH 40 6E1 VH CDR2 VISYDGSIKYYTDSVKG 41 6E1 VH CDR3
DGNYGSARYFQH 42 6E1 VL1 CDR1 RASQGISSWLA 43 6E1 VL1 CDR2 AASSLQS 44
6E1 VL1 CDR3 QQYNSYPRT 45 6E1 VL2 CDR1 RASQSVSSYLA 46 6E1 VL2 CDR2
DASNRAT 47 6E1 VL2 CDR3 QQRSNWPYT 48 6E1 VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFAMHWVRQAPGKGLEWVT
VISYDGSIKYYTDSVKGRFTFSRDNSKNTLYLQMNSLRAEDTAVYYCTR
DGNYGSARYFQHWGQGTLVTVSS 49 6E1 VL1
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTF GQGTKVEIK 50 6E1
VL2 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPYTF GQGTKLEIK 51 18E9
VH CDR1 SSAMH 52 18E9 VH CDR2 AIGTGGDTYYADSVKG 53 18E9 VH CDR3
DFYDILTGIFDY 54 18E9 VL CDR1 RASQGISSWLA 55 18E9 VL CDR2 AASSLQS 56
18E9 VL CDR3 QQANSFPST 57 18E9 VH
EVQLVQSGGGLVHPGGSLRLSCAHSGFTFTSSAMHWVRQAPGKGLEWIS
AIGTGGDTYYADSVKGRFTISRDNAKNSLYLQINSLRAEDMAVYYCARD
FYDILTGIFDYWGQGTLVTVSS 58 18E9 VL
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQHKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPSTF GQGTKVEIK 59 8B11
VH CDR1 SDAMY 60 8B11 VH CDR2 AIGIGGDTYYTDSVMG 61 8B11 VH CDR3
LGMGYYFDY 62 8B11 VL CDR1 RASQSVSSYLA 63 8B11 VL CDR2 DASNRAT 64
8B11 VL CDR3 QQRSNWPPT 65 8B11 VH
MEFVLSWVFLVAILKGVQCEIQLVQSGGGLVHPGGSLRLSCAGSGFTFS
SDAMYWVRQAPGKGLEWVSAIGIGGDTYYTDSVMGRFTISRDNAKNSLY
LQMNSLRAEDMAVYYCARLGMGYYFDYWGQGTLVTVSS 66 8B11 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTF GQGTKVEIK 67 20B3
VH CDR1 SYDMH 68 20B3 VH CDR2 VIGTAGDTYYPGSVKG 69 20B3 VH CDR3
GGMGNYFDY 70 20B3 VL CDR1 RASQSVSSYLA 71 20B3 VL CDR2 DASNRAT 72
20B3 VL CDR3 QQRSNWPLT 73 20B3 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQTTGKGLEWVS
VIGTAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARG
GMGNYFDYWGQGTLVTVSS 74 20B3 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTF GGGTKVEIK 75 14A2
VH CDR1 NYALH 76 14A2 VH CDR2 LISYDGSRKHYADSVKG 77 14A2 VH CDR3
LTMVREGG 78 14A2 VL1 CDR1 RASQSVSSSYLA 79 14A2 VL1 CDR2 GASSRAT 80
14A2 VL1 CDR3 QQYGSSPFT 81 14A2 VL2 CDR1 RVSQGISSYLN 82 14A2 VL2
CDR2 SASNLQS 83 14A2 VL2 CDR3 QRTYNAPYT 84 14A2 VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVA
LISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAS LTMVREGGQGTLVTVSS
85 14A2 VL1 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLI
YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFT FGPGTKVDIK 86
14A2 VL2 DIQLTQSPSSLSASVGDRVTITCRVSQGISSYLNWYRQKPGKVPKLLIY
SASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYGQRTYNAPYTF GGGTKVEIK 87 20C1
VH CDR1 SYAMY 88 20C1 VH CDR2 AIDTDGGTFYADSVRG 89 20C1 VH CDR3
LGEGYFFDY 90 20C1 VL CDR1 RASQSVSSYLA 91 20C1 VL CDR2 DASNRAT 92
20C1 VL CDR3 QQRSNWPPT 93 20C1 VH
EAQLVQSGGGLVHPGGSLRLSCADSGFTFSSYAMYWVRQAPGKGLEWVS
AIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNGLRAEDMAVYFCARL
GEGYFFDYWGQGTLVTVSS 94 20C1 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTF GGGTKVEIK 95
OX40.6 heavy chain EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLE
WVSAIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAV
YYCARMGTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 96 OX40.6 light chain
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL
LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS
NWPLTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 97 OX40.7 heavy chain
EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLE
WVSAIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAV
YYCARYGTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 98 OX40.7 light chain
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL
LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS
NWPLTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 99 OX40.8 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 100 OX40.8 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 101 OX40.9 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTYVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 102 OX40.9 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 103 OX40.10 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYSGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTMVREGGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 104 OX40.10 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 105 OX40.11 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYDSSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTMVREGGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 106 OX40.11 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 107 OX40.12 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYSGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 108 OX40.12 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 109 OX40.13 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYDSSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTMVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 110 OX40.13 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 111 OX40.14 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYSGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTYVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 112 OX40.14 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 113 OX40.15 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYDSSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTYVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 114 OX40.15 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 115 OX40.16 heavy chain
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLE
WVSAIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAV
YFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 116 OX40.16 light chain
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL (shared by OX40.20,
LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS OX40.21, OX40.22)
NWPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 117 OX40.17 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQTTGKGLE
WVSVIGTAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAV
YYCARGGMGNYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 118 OX40.17 light chain
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL
LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS
NWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 119 OX40.18 heavy chain
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVNWVRQATGQGLE
WMGWMNPNSGNTGYAPKFQGRVTMTRDTSISTAYMELSSLRSEDTA
VYYCARIYSSSYNWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 120 OX40.18 light chain
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL
LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS
NWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 121 OX40.19 heavy chain
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLE
WVALISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTA
VYYCASLTLVREWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 122 OX40.19 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR
LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY
GSSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 123 OX40.20 heavy chain
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLE
WVSAIDTSGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAV
YFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG -- OX40.20 light chain SEQ ID NO:
116 124 OX40.21 heavy chain
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLE
WVSAIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAV
YFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG -- OX40.21 light chain SEQ ID NO:
116 125 OX40.22 heavy chain
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLE
WVSAIDTSTGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAV
YFCARLGEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG
TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG -- OX40.22 light chain SEQ ID NO:
116 126 3F4 VH (nucleotide
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT sequence)
CAGTGAAGGTCTCCTGCAAGGCTTCTGGAAACACCTTCACCAGTTATGA
TGTCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGGATGGGA
TGGATGAACCCTAACAGTGGTAACACAGGCTATGCACCGAAGTTCCAGG
GCAGAGTCACCATGACCAGGAACACCTCCATAAGCACAGCCTACATGGA
GCTGAGCAGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGA
ATATATAGCAGCTCGTACAACTGGTTCGACCCCTGGGGCCAGGGAACCC TGGTCACCGTCTCCTCA
127 3F4 VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT
AGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA 128 14B6 VH
GAGGTGCAGCTGGAGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGT
CTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCAACTG
GATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGG
TTCATCTATCCTGGTGACTCTGATACCAGGTACAGCCCGTCCTTCCAAG
GCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCA
GTGGAGCAGCCTCAAGGCCTCGGACATCGCCATGTATTACTGTGCGAGA
TATGGGGATGACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCA CTGTCTCCTCA 129
14B6 VL1 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT
AGCCTGGTTCCAACAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCTCTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTGGCGACTGGCCCATCACCTTC
GGCCAAGGGACACGACTGGAGATTAAA 130 14B6 VL2
GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTT
AGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTAT
GCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGA
TTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCGGATCACC
TTCGGCCAAGGGACACGACTGGAGATTAAA 131 23H3 VH
GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACATCCTGGGGGGT
CCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAACTATGC
TATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCA
GCCATTGGTATTGGTGGTGACACATTCTATACAGACTCCGTGAAGGGCC
GATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTCTCTTCAAAT
GAACAGCCTGAGAGCCGAGGACATGGCTGTGTATTACTGTGCAAGAATG
GGAACTGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA 132 23H3
VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT
AGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTC
GGCCCTGGGACCAAAGTGGATATCAAA 133 6E1 VH
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGT
CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTTTGC
TATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGACA
GTTATTTCATATGATGGAAGCATTAAATACTACACAGACTCCGTGAAGG
GCCGATTCACCTTCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCA
AATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTACGAGA
GATGGAAACTATGGTTCGGCGAGATACTTCCAGCACTGGGGCCAGGGCA
CCCTGGTCACCGTCTCCTCA 134 6E1 VL1
GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTT
AGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTAT
GCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGA
TTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCGGACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAA 135 6E1 VL2
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT
AGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGTACACTTTT
GGCCAGGGGACCAAGCTGGAGATCAAA 136 18E9 VH
GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCATCCTGGGGGGT
CCCTGAGACTCTCCTGTGCACACTCTGGATTCACCTTCACTAGCTCTGC
TATGCACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAATGGATATCA
GCTATTGGTACTGGTGGTGACACATACTATGCAGACTCCGTGAAGGGCC
GATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAAT
AAACAGCCTGAGAGCCGAGGACATGGCTGTATATTACTGTGCAAGAGAC
TTTTACGATATTTTGACTGGTATCTTTGACTACTGGGGCCAGGGAACCC TGGTCACCGTCTCCTCA
137 18E9 VL GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTT
AGCCTGGTATCAGCATAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT
GCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGA
TTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCTTCGACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAA 138 8B11 VH
ATGGAGTTTGTGCTGAGCTGGGTTTTCCTTGTTGCTATATTAAAAGGTG
TCCAGTGTGAAATTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACATCC
TGGGGGGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGT
AGCGATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGT
GGGTATCAGCTATTGGTATTGGTGGTGACACATACTATACAGACTCCGT
GATGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTAT
CTTCAAATGAACAGCCTGAGAGCCGAGGACATGGCTGTGTATTACTGTG
CAAGGCTGGGGATGGGGTACTACTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCCTCA
139 8B11 VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT
AGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAA 140 20B3 VH
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGT
CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACGA
CATGCACTGGGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGGTCTCA
GTTATTGGTACTGCTGGTGACACATACTATCCAGGCTCCGTGAAGGGCC
GATTCACCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTCAAAT
GAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTGCAAGAGGG
GGGATGGGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA 141 20B3
VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT
AGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA 142 14A2 VH
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGT
CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGC
TCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCA
CTTATATCATATGATGGAAGCAGGAAACACTACGCAGACTCCGTGAAGG
GCCGATTCAGTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCA
AATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGT
CTTACTATGGTTCGGGAGGGGGGCCAGGGAACCCTGGTCACCGTCTCCT CA 143 14A2 VL1
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTA
CTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC
TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCA
GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGA
AGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCATTCACT
TTCGGCCCTGGGACCAAAGTGGATATCAAA 144 14A2 VL2
GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG
ACAGAGTCACCATCACTTGCCGGGTGAGTCAGGGCATTAGCAGTTATTT
AAATTGGTATCGGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTAT
AGTGCATCCAATTTGCAATCTGGAGTCCCATCTCGGTTCAGTGGCAGTG
GATCTGGGACAGATTTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGA
TGTTGCAACTTATTACGGTCAACGGACTTACAATGCCCCTTACACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA 145 20C1 VH
GAGGCTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCATCCTGGGGGGT
CCCTGAGACTCTCCTGTGCAGACTCTGGATTCACCTTCAGTAGCTATGC
TATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCA
GCTATTGATACTGATGGTGGCACATTCTATGCAGACTCCGTGCGGGGCC
GATTCACCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAAT
GAACGGCCTGAGAGCCGAGGACATGGCTGTGTATTTCTGTGCAAGACTT
GGGGAAGGGTACTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA 146 20C1
VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT
AGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAA 147 OX40.6 heavy chain
GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAA
CTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAG
TGGGTATCAGCCATTGGTATTGGTGGTGACACATTCTATACAGACT
CCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTC
CTTGTCTCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTG
TATTACTGTGCAAGAATGGGAACTGGGTACTTCTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGT 148 OX40.6
light chain GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTC
CTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGT
TCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAG
CCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGC
AACTGGCCTCTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
GTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG
GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 149 OX40.7 heavy chain
GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACATCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAA
CTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAG
TGGGTATCAGCCATTGGTATTGGTGGTGACACATTCTATACAGACT
CCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTC
CTTGTCTCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTG
TATTACTGTGCAAGATATGGAACTGGGTACTTCTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGT 150 OX40.7
light chain GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTC
CTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGT
TCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAG
CCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGC
AACTGGCCTCTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
GTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG
GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 151 OX40.8 heavy chain
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATGATGGAAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 152 OX40.8 light chain
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 153 OX40.9 heavy
chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATGATGGAAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 154 OX40.9 light chain
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 155 OX40.10 heavy
chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATAGTGGAAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGGGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 156 OX40.10 light
chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 157 OX40.11 heavy
chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATGATAGTAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGGGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 158 OX40.11 light
chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 159 OX40.12 heavy
chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATAGTGGAAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 160 OX40.12 light
chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 161 OX40.13 heavy
chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATGATAGTAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 162 OX40.13 light
chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 163 OX40.14 heavy
chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATAGTGGAAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 164 OX40.14 light
chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 165 OX40.15 heavy
chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATGATAGTAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 166 OX40.15 light
chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 167 OX40.16 heavy
chain GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCAGCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAG
CTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAG
TGGGTATCAGCTATTGATACTGATGGTGGCACATTCTATGCAGACT
CCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTC
CTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTG
TATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA 168 OX40.16
light chain GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG
(shared by OX40.20,
AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTT OX40.21, OX40.22)
AGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGA
TTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCCACTTTC
GGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTG
TCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC
TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG
TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC
ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTAG 169
OX40.17 heavy chain GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAG
CTACGACATGCACTGGGTCCGCCAAACTACAGGAAAAGGTCTGGAG
TGGGTCTCAGTTATTGGTACTGCTGGTGACACATACTATCCAGGCT
CCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATGCCAAGAACTC
CTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTG
TATTACTGTGCAAGAGGGGGGATGGGGAACTACTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA 170 OX40.17
light chain GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTC
CTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGT
TCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAG
CCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGC
AACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
GTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG
GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA G 171 OX40.18 heavy
chain CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGG
CCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAG
TTATGATGTCAACTGGGTGCGACAGGCCACTGGACAAGGGCTTGAG
TGGATGGGATGGATGAACCCTAACAGTGGTAACACAGGCTATGCAC
CGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACCTCCATAAG
CACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTTTATTACTGTGCGAGAATATATAGCAGCTCGTACAACTGGTTCG
ACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCAC
CAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC
TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC
CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC
CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA
CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGA
CAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCT
TCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA
GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA
CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG
CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC
CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTG
ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT
GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG
TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCCCCGGGTTGA 172
OX40.18 light chain GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTC
CTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGT
TCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAG
CCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGC
AACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
GTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG
GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA G 173 OX40.19 heavy
chain CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA
GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAA
CTATGCTCTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG
TGGGTGGCACTTATATCATATGATGGAAGCAGGAAACACTACGCAG
ACTCCGTGAAGGGCCGATTCAGTATCTCCAGAGACAATTCCAAGAA
CACACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCT
GTGTATTACTGTGCGAGTCTTACTCTGGTTCGGGAGTGGGGCCAGG
GAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC
GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG
AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA
CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGTAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 174 OX40.19 light
chain GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG
GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG
CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACA
GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAG
CAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAT
GGTAGCTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 175 OX40.20 heavy
chain GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCAGCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAG
CTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAG
TGGGTATCAGCTATTGATACTAGTGGTGGCACATTCTATGCAGACT
CCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTC
CTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTG
TATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA -- OX40.20
light chain SEQ ID NO: 168 176 OX40.21 heavy chain
GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCAGCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAG
CTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAG
TGGGTATCAGCTATTGATACTGATGCTGGCACATTCTATGCAGACT
CCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTC
CTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTG
TATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA -- OX40.21
light chain SEQ ID NO: 168 177 OX40.22 heavy chain
GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTTCAGCCTGGGG
GGTCCCTGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCAGTAG
CTATGCTATGTACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAG
TGGGTATCAGCTATTGATACTAGTACTGGCACATTCTATGCAGACT
CCGTGCGGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTC
CTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTG
TATTTCTGTGCAAGACTTGGGGAAGGGTACTTCTTTGACTACTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC
ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCC GGGTTGA -- OX40.22
light chain SEQ ID NO: 168 178 hOX40 epitope DVVSSKPCKPCTWCNLR 179
hOX40 epitope DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK 180 peptide
linker PVGVV 181 sortase A recognition motif LPXTG, wherein X is
any amino acid 182 hOX40 epitope QNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR
183 hOX40 epitope PCKPCTWCNLR 184 hOX40 epitope QLCTATQDTVCR 185
hOX40 epitope SQNTVCRPCGPGFYN 186 IgG1 C-termianl C.sub.H1 VDKRV
(same for IgG3 (17-15-15- 15), igG3 (17-15-15), IgG3 (17-15), IgG3
(15-15-15), IgG3 (15), and IgG4 187 IgG2 C-terminal C.sub.H1 VDKTV
188 IgG1 upper hinge EPKSCDKTHT 189 IgG3 (17-15-15-15) upper
ELKTPLGDTTHT hinge (same for IgG3 (17-
15-15) and IgG3 (17-15)) 190 IgG3 (15-15-15) upper EPKS hinge (same
for IgG3(15)) 191 IgG4 upper hinge ESKYGPP 192 IgG1 middle hinge
CPPCP 193 IgG2 middle hinge CCVECPPCP 194 IgG3 (17-15-15-15) middle
CPRCP(EPKSCDTPPPCPRCP).sub.3 hinge 195 IgG3 (17-15-15) middle
CPRCP(EPKSCDTPPPCPRCP).sub.2 hinge 196 IgG3 (17-15) middle hinge
CPRCP(EPKSCDTPPPCPRCP).sub.1 197 IgG3 (15-15-15) middle
CDTPPPCPRCP(EPKSCDTPPPCPRCF).sub.2 hinge 198 IgG3 (15) middle hinge
CDTPPPCPRCP 199 IgG4 middle hinge CPSCP 200 IgG1 lower hinge (same
APELLGG for IgG3 (17-15-15-15), IgG3 (17-15-15), IgG3 (17-15), IgG3
(15-15-15), IgG3 (15), and IgG4) 201 IgG2 lower hinge APPVAG 202
Wildtype human IgG1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG CH1
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV 203 Wildtype
human IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG CH1
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV 204 Wildtype
human IgG1 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN CH2
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAK 205
Wildtype human IgG2
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN CH2
AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTK 206
Wildtype human IgG1
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN CH3
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG 207
Wildtype human IgG2
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN CH3
NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 208
Alternative hinge ERKCCVECPPCPAPPVAG 209 Alternative hinge
ERKSCVECPPCPAPPVAG 210 Alternative hinge ERKCSVECPPCPAPPVAG 211
Alternative hinge ERKXCVECPPCPAPPVAG 212 Alternative hinge
ERKCXVECPPCPAPPVAG 213 Alternative hinge ERKCCVECPPCPAPPVAGX 214
Alternative hinge ERKSCVECPPCPAPPVAGX 215 Alternative hinge
ERKCSVECPPCPAPPVAGX 216 Alternative hinge ERKXCVECPPCPAPPVAGX 217
Alternative hinge ERKCXVECPPCPAPPVAGX 218 Alternative hinge
ERKCCVECPPCPAPELLGG 219 Alternative hinge ERKSCVECPPCPAPELLGG 220
Alternative hinge ERKCCSVECPPCPAPELLGG 221 Alternative hinge
ERKXCVECPPCPAPELLGG 222 Alternative hinge ERKCXVECPPCPAPELLGG 223
Alternative hinge ERKCCVECPPCPAPELLG 224 Alternative hinge
ERKSCVECPPCPAPELLG 225 Alternative hinge ERKCCSVECPPCPAPELLG 226
Alternative hinge ERKXCVECPPCPAPELLG 227 Alternative hinge
ERKCXVECPPCPAPELLG 228 Alternative hinge ERKCCVECPPCPAP 229
Alternative hinge ERKSCVECPPCPAP 230 Alternative hinge
ERKCSVECPPCPAP 231 Alternative hinge ERKXCVECPPCPAP 232 Alternative
hinge ERKCXVECPPCPAP 233 Portion of hinge PVAG 234 Portion of hinge
ELLG 235 Portion of hinge ELLGG 236 Portion of hinge SCDKTHT 237
Portion of hinge CCVE 238 WT human IgG2 hinge ERKCCVECPPCPAPPVAG
239 Human IgG2 hinge with ERKSCVECPPCPAPPVAG C219S 240 IgG2/IgG1
hinge ERKCCVECPPCPAPELLGG 241 IgG2 (C219S)/IgG1 hinge
ERKSCVECPPCPAPELLGG 242 Wild type human IgG1 EPKSCDKTHTCPPCPAPELLGG
hinge 243 Human IgG1 CH2 with
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN A330S/P331S
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAK 244
IgG1-IgG2-IgG1f ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
ERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 245 IgG1-IgG2-IgG1f2
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 246 IgG1-IgG2CS-IgG1f
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
ERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 247 IgG1-IgG2CS-IgG1f2
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
ERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 248 IgG2-IgG1f
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 249 IgG2-IgG1f2
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 250 IgG2C5-IgG1f
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 251 IgG2CS-IgG1f2
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 252 IgG1-IgG2-IgG1.1f
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRREMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 253 IgG1-IgG2CS-IgG1.1f
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
ERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 254 IgG2-IgG1.1f
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 255 IgG2CS-IgG1.1f
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 256 IgG1f
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 257 IgG1.1f
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV
EPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 258 IgG2.3
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT
KVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV
LTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL
SPGK 259 IgG2.5 ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT
KVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV
LTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 260 IgG2.3G1-KH
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT
KVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 261 IgG2.5G1-KH
ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT
KVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 262 IgG2.3G1-AY
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT
KVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 263
IgG2.5G1-AY ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT
KVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 264
IgG2.3G1.1f-KH ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT
KVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 265
IgG2.5G1.1f-KH ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT
KVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 266
IgG2.5G1-V27 ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKCCVECPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 267
IgG2.3-V13 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 268 IgG2.3-V14
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 269 IgG2.3-V15
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSD
EDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 270 IgG2.3-V16
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDDEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPRPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 271 IgG2.3-V17
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSD
EDGEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPRPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 272 IgG2.3-V18
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 273 IgG2.3-V19
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 274 IgG2.3G1-AY-V20
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 275 IgG2.3G1-AY-V21
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 276 IgG2.3G1-AY-V22
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 277 IgG2.3G1-AY-V23
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 278 IgG2.3G1-AY-V24
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 279 IgG2.3G1-AY-V25
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 280 IgG2.3G1-AY-V26
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 281 IgG2.3G1-AY-V28
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPPVAGPSVFLFPPKPDKTLMISRTPEVTCVVVDVEH
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 282 OX40.6-Vh-hHC-IgG2.3
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVS
AIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARM
GTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 283
OX40.8-Vh-hHC-IgG2.3
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVA
LISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAS
LTMVREWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY
TCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF
RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 284
OX40.16-Vh-hHC-IgG2.3
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS
AIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARL
GEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 285
OX40.6-Vh-hHC- EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVS
IgG2.3G1 AIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARM
GTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 286
OX40.8-Vh-hHC- QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVA
IgG2.3G1 LISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAS
LTMVREWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY
TCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
287 OX40.16-Vh-hHC-
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS IgG2.3G1
AIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARL
GEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 288
OX40.6-Vh-hHC- EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVS
IgG2.3G1-V27 AIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARM
GTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 289
OX40.8-Vh-hHC- QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVA
IgG2.3G1-V27 LISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAS
LTMVREWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY
TCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 290
OX40.16-Vh-hHC- EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS
IgG2.3G1-V27 AIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARL
GEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 291
OX40.6-Vh-hHC-IgG2.5
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYWVRQAPGKGLEWVS
AIGIGGDTFYTDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARM
GTGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 292
OX40.8-Vh-hHC-IgG2.5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHWVRQAPGKGLEWVA
LISYDGSRKHYADSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAS
LTMVREWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY
TCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF
RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 293
OX40.16-Vh-hHC-IgG2.5
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS
AIDTDGGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARL
GEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 294
OX40.21-Vh-hHC-IgG2.5
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS
AIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARL
GEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 295
OX40.21-Vh-hHC- EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS
IgG2.5G1 AIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARL
GEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 296
OX40.21-Vh-hHC- EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLEWVS
IgG2.5G1-V27 AIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCARL
GEGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ
TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 297
IgG2.3G1-V27 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVE
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 298 Human IgG1 CH2 with
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY A330S/P331S
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAK 299
Heavy chain-nivolumab QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGL
EWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDT
AVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG
LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGK 300 Light chain-nivolumab
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI
YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPR
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK
VYACEVTHQGLSSPVTKSFNRGEC 301 Heavy chain variable
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVA region-nivolumab
VIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT NDDYWGQGTLVTVSS
302 Light chain variable region-
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY nivolumab
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTF GQGTKVEIK 303
HCDR1-nivolumab NSGMH 304 HCDR2-nivolumab VIWYDGSKRYYADSVKG 305
HCDR3-nivolumab NDDY 306 LCDR1-nivolumab RASQSVSSYLA 307
LCDR2-nivolumab DASNRAT 308 LCDR3-nivolumab QQSSNWPRT 309 Heavy
chain variable QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVT
region-ipilimumab (from
FISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAR WO01/014424)
TGWLGPFDYWGQGTLVTVSS 310 Light chain variable region-
EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLI ipilimumab (from
YGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT WO01/014424)
FGQGTKVEIK 311 HCDR1-ipilimumab SYTMH (from WO01/014424) 312
HCDR2-ipilimumab FISYDGNNKYYADSVKG (from WO01/014424) 313
HCDR3-ipilimumab TGWLGPFDY (from WO01/014424) 314 LCDR1-ipilimumab
RASQSVGSSYLA (from WO01/014424) 315 LCDR2-ipilimumab GAFSRAT (from
WO01/014424) 316 LCDR3-ipilimumab QQYGSSPWT (from WO01/014424) 317
HCDR2 of OX40.21 AIDTDAGTFYADSVRG 318 VH of OX40.21
EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYWVRQAPGKGLE
WVSAIDTDAGTFYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAV
YFCARLGEGYFFDYWGQGTLVTVSS
[1166] Table 23 provides the sequences of the mature variable
regions and heavy and light chains and where indicated, sequences
with signal peptides.
EQUIVALENTS
[1167] 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
3181277PRTHomo sapiensmisc_feature(1)..(277)Human OX40 precursor
1Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1
5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys
Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu
Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser
Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn
Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys
Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala
Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro
Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro
Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135
140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn
145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala
Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile
Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly
Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val
Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly
Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg
Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255
Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260
265 270 Thr Leu Ala Lys Ile 275 2189PRTHomo
sapiensmisc_feature(1)..(189)Extracellular domain of mature human
OX40 2Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys
His 1 5 10 15 Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser
Arg Ser Gln 20 25 30 Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe
Tyr Asn Asp Val Val 35 40 45 Ser Ser Lys Pro Cys Lys Pro Cys Thr
Trp Cys Asn Leu Arg Ser Gly 50 55 60 Ser Glu Arg Lys Gln Leu Cys
Thr Ala Thr Gln Asp Thr Val Cys Arg 65 70 75 80 Cys Arg Ala Gly Thr
Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp 85 90 95 Cys Ala Pro
Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala 100 105 110 Cys
Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln 115 120
125 Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro
130 135 140 Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro
Ile Thr 145 150 155 160 Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser
Gln Gly Pro Ser Thr 165 170 175 Arg Pro Val Glu Val Pro Gly Gly Arg
Ala Val Ala Ala 180 185 3277PRTMacaca
fascicularismisc_feature(1)..(277)Cynomolgus OX40 3Met Cys Val Gly
Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu
Leu Gly Leu Gly Leu Ser Thr Thr Ala Lys Leu His Cys Val 20 25 30
Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys Gln Glu Cys Arg Pro 35
40 45 Gly Asn Gly Met Val Ser Arg Cys Asn Arg Ser Gln Asn Thr Val
Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser
Ala Lys Pro 65 70 75 80 Cys Lys Ala Cys Thr Trp Cys Asn Leu Arg Ser
Gly Ser Glu Arg Lys 85 90 95 Gln Pro Cys Thr Ala Thr Gln Asp Thr
Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr
Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe
Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys
Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160
Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Pro Thr Gln Pro 165
170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Thr Thr Val Gln Pro
Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Arg Pro Ser Thr Arg
Pro Val Glu 195 200 205 Val Pro Arg Gly Pro Ala Val Ala Ala Ile Leu
Gly Leu Gly Leu Ala 210 215 220 Leu Gly Leu Leu Gly Pro Leu Ala Met
Leu Leu Ala Leu Leu Leu Leu 225 230 235 240 Arg Arg Asp Gln Arg Leu
Pro Pro Asp Ala Pro Lys Ala Pro Gly Gly 245 250 255 Gly Ser Phe Arg
Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270 Ala Leu
Ala Lys Ile 275 4183PRTHomo sapiensmisc_feature(1)..(183)Human
OX40-L 4Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala
Arg 1 5 10 15 Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser
Val Ile Gln 20 25 30 Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile
Cys Leu His Phe Ser 35 40 45 Thr Leu Gln Val Ser His Arg Tyr Pro
Arg Ile Gln Ser Ile Lys Val 50 55 60 Gln Phe Thr Glu Tyr Lys Lys
Glu Lys Gly Phe Ile Leu Thr Ser Gln 65 70 75 80 Lys Glu Asp Glu Ile
Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn 85 90 95 Cys Asp Gly
Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu 100 105 110 Val
Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln 115 120
125 Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr
130 135 140 Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr
Ser Leu 145 150 155 160 Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile
Leu Ile His Gln Asn 165 170 175 Pro Gly Glu Phe Cys Val Leu 180
5329PRTHomo sapiensmisc_feature(1)..(329)human IgG1 constant domain
5Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly 325 6329PRTHomo sapiensmisc_feature(1)..(329)human IgG1
constant domain (allotypic variant) 6Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310
315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 7107PRTHomo
sapiensmisc_feature(1)..(107)human IgG1 kappa light chain 7Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20
25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 105 85PRTArtificial SequenceSynthetic heavy
chain constant region alternative C-terminus 8Leu Ser Pro Gly Lys 1
5 94PRTArtificial SequenceSynthetic heavy chain constant region
alternative C-terminus 9Leu Ser Pro Gly 1 10107PRTHomo
sapiensmisc_feature(1)..(107)Human IgG1 kappa light chain constant
region (CL) 10Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 115PRTArtificial
SequenceSynthetic 3F4 VH CDR1 11Ser Tyr Asp Val Asn 1 5
1217PRTArtificial SequenceSynthetic 3F4 VH CDR2 12Trp Met Asn Pro
Asn Ser Gly Asn Thr Gly Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly
1311PRTArtificial SequenceSynthetic 3F4 VH CDR3 13Ile Tyr Ser Ser
Ser Tyr Asn Trp Phe Asp Pro 1 5 10 1411PRTArtificial
SequenceSynthetic 3F4 VL CDR1 14Arg Ala Ser Gln Ser Val Ser Ser Tyr
Leu Ala 1 5 10 157PRTArtificial SequenceSynthetic 3F4 VL CDR2 15Asp
Ala Ser Asn Arg Ala Thr 1 5 169PRTArtificial SequenceSynthetic 3F4
VL CDR3 16Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5
17120PRTArtificial SequenceSynthetic 3F4 VH 17Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Asn Thr Phe Thr Ser Tyr 20 25 30 Asp
Val Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Pro Lys Phe
50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Ser Ser Ser Tyr Asn Trp
Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 18107PRTArtificial SequenceSynthetic 3F4 VL 18Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 195PRTArtificial SequenceSynthetic 14B6 VH CDR1
19Ser Asn Trp Ile Gly 1 5 2017PRTArtificial SequenceSynthetic 14B6
VH CDR2 20Phe Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser
Phe Gln 1 5 10 15 Gly 219PRTArtificial SequenceSynthetic 14B6 VH
CDR3 21Tyr Gly Asp Asp Trp Tyr Phe Asp Leu 1 5
2211PRTArtificial SequenceSynthetic 14B6 VL1 CDR1 22Arg Ala Ser Gln
Ser Val Ser Ser Tyr Leu Ala 1 5 10 237PRTArtificial
SequenceSynthetic 14B6 VL1 CDR2 23Asp Ala Ser Asn Arg Ala Thr 1 5
249PRTArtificial SequenceSynthetic 14B6 VL1 CDR3 24Gln Gln Arg Gly
Asp Trp Pro Ile Thr 1 5 2511PRTArtificial SequenceSynthetic 14B6
VL2 CDR1 25Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10
267PRTArtificial SequenceSynthetic 14B6 VL2 CDR2 26Ala Ala Ser Ser
Leu Gln Ser 1 5 2710PRTArtificial SequenceSynthetic 14B6 VL2 CDR3
27Gln Gln Tyr Asn Ser Tyr Pro Arg Ile Thr 1 5 10 28118PRTArtificial
SequenceSynthetic 14B6 VH 28Glu Val Gln Leu Glu Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly
Ser Gly Tyr Ser Phe Thr Ser Asn 20 25 30 Trp Ile Gly Trp Val Arg
Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Phe Ile Tyr
Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Ile Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Tyr Gly Asp Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly
Thr 100 105 110 Leu Val Thr Val Ser Ser 115 29107PRTArtificial
SequenceSynthetic 14B6 VL1 29Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Phe Gln
Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala
Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Glu Pro 65 70
75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Gly Asp Trp Pro
Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
30108PRTArtificial SequenceSynthetic 14B6 VL2 30Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40
45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn
Ser Tyr Pro Arg 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys 100 105 315PRTArtificial SequenceSynthetic 23H3 VH CDR1
31Asn Tyr Ala Met Tyr 1 5 3216PRTArtificial SequenceSynthetic 23H3
VH CDR2 32Ala Ile Gly Ile Gly Gly Asp Thr Phe Tyr Thr Asp Ser Val
Lys Gly 1 5 10 15 339PRTArtificial SequenceSynthetic 23H3 VH CDR3
33Met Gly Thr Gly Tyr Phe Phe Asp Tyr 1 5 3411PRTArtificial
SequenceSynthetic 23H3 VL CDR1 34Arg Ala Ser Gln Ser Val Ser Ser
Tyr Leu Ala 1 5 10 357PRTArtificial SequenceSynthetic 23H3 VL CDR2
35Asp Ala Ser Asn Arg Ala Thr 1 5 369PRTArtificial
SequenceSynthetic 23H3 VL CDR3 36Gln Gln Arg Ser Asn Trp Pro Leu
Thr 1 5 37117PRTArtificial SequenceSynthetic 23H3 VH 37Glu Val Gln
Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Asn Tyr 20 25
30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ala Ile Gly Ile Gly Gly Asp Thr Phe Tyr Thr Asp Ser
Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Ser Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met
Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Gly Thr Gly Tyr Phe Phe
Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
38107PRTArtificial SequenceSynthetic 23H3 VL 38Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys 100 105 395PRTArtificial SequenceSynthetic 6E1 VH CDR1 39Ser
Phe Ala Met His 1 5 4017PRTArtificial SequenceSynthetic 6E1 VH CDR2
40Val Ile Ser Tyr Asp Gly Ser Ile Lys Tyr Tyr Thr Asp Ser Val Lys 1
5 10 15 Gly 4112PRTArtificial SequenceSynthetic 6E1 VH CDR3 41Asp
Gly Asn Tyr Gly Ser Ala Arg Tyr Phe Gln His 1 5 10
4211PRTArtificial SequenceSynthetic 6E1 VL1 CDR1 42Arg Ala Ser Gln
Gly Ile Ser Ser Trp Leu Ala 1 5 10 437PRTArtificial
SequenceSynthetic 6E1 VL1 CDR2 43Ala Ala Ser Ser Leu Gln Ser 1 5
449PRTArtificial SequenceSynthetic 6E1 VL1 CDR3 44Gln Gln Tyr Asn
Ser Tyr Pro Arg Thr 1 5 4511PRTArtificial SequenceSynthetic 6E1 VL2
CDR1 45Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10
467PRTArtificial SequenceSynthetic 6E1 VL2 CDR2 46Asp Ala Ser Asn
Arg Ala Thr 1 5 479PRTArtificial SequenceSynthetic 6E1 VL2 CDR3
47Gln Gln Arg Ser Asn Trp Pro Tyr Thr 1 5 48121PRTArtificial
SequenceSynthetic 6E1 VH 48Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Val Ile Ser
Tyr Asp Gly Ser Ile Lys Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Phe Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Thr Arg Asp Gly Asn Tyr Gly Ser Ala Arg Tyr Phe Gln His Trp
Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
49107PRTArtificial SequenceSynthetic 6E1 VL1 49Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40
45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn
Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 50107PRTArtificial SequenceSynthetic 6E1 VL2 50Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Arg Ser Asn Trp Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 515PRTArtificial SequenceSynthetic 18E9 VH
CDR1 51Ser Ser Ala Met His 1 5 5216PRTArtificial SequenceSynthetic
18E9 VH CDR2 52Ala Ile Gly Thr Gly Gly Asp Thr Tyr Tyr Ala Asp Ser
Val Lys Gly 1 5 10 15 5312PRTArtificial SequenceSynthetic 18E9 VH
CDR3 53Asp Phe Tyr Asp Ile Leu Thr Gly Ile Phe Asp Tyr 1 5 10
5411PRTArtificial SequenceSynthetic 18E9 VL CDR1 54Arg Ala Ser Gln
Gly Ile Ser Ser Trp Leu Ala 1 5 10 557PRTArtificial
SequenceSynthetic 18E9 VL CDR2 55Ala Ala Ser Ser Leu Gln Ser 1 5
569PRTArtificial SequenceSynthetic 18E9 VL CDR3 56Gln Gln Ala Asn
Ser Phe Pro Ser Thr 1 5 57120PRTArtificial SequenceSynthetic 18E9
VH 57Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala His Ser Gly Phe Thr Phe
Thr Ser Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45 Ser Ala Ile Gly Thr Gly Gly Asp Thr
Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Ile Asn Ser Leu
Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Phe
Tyr Asp Ile Leu Thr Gly Ile Phe Asp Tyr Trp Gly Gln 100 105 110 Gly
Thr Leu Val Thr Val Ser Ser 115 120 58107PRTArtificial
SequenceSynthetic 18E9 VL 58Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln His
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ser 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
595PRTArtificial SequenceSynthetic 8B11 VH CDR1 59Ser Asp Ala Met
Tyr 1 5 6016PRTArtificial SequenceSynthetic 8B11 VH CDR2 60Ala Ile
Gly Ile Gly Gly Asp Thr Tyr Tyr Thr Asp Ser Val Met Gly 1 5 10 15
619PRTArtificial SequenceSynthetic 8B11 VH CDR3 61Leu Gly Met Gly
Tyr Tyr Phe Asp Tyr 1 5 6211PRTArtificial SequenceSynthetic 8B11 VL
CDR1 62Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10
637PRTArtificial SequenceSynthetic 8B11 VL CDR2 63Asp Ala Ser Asn
Arg Ala Thr 1 5 649PRTArtificial SequenceSynthetic 8B11 VL CDR3
64Gln Gln Arg Ser Asn Trp Pro Pro Thr 1 5 65136PRTArtificial
SequenceSynthetic 8B11 VH 65Met Glu Phe Val Leu Ser Trp Val Phe Leu
Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Ile Gln Leu Val
Gln Ser Gly Gly Gly Leu Val His 20 25 30 Pro Gly Gly Ser Leu Arg
Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe 35 40 45 Ser Ser Asp Ala
Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp
Val Ser Ala Ile Gly Ile Gly Gly Asp Thr Tyr Tyr Thr Asp 65 70 75 80
Ser Val Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 85
90 95 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val
Tyr 100 105 110 Tyr Cys Ala Arg Leu Gly Met Gly Tyr Tyr Phe Asp Tyr
Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser 130 135
66107PRTArtificial SequenceSynthetic 8B11 VL 66Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 675PRTArtificial SequenceSynthetic 20B3 VH CDR1 67Ser
Tyr Asp Met His 1 5 6816PRTArtificial SequenceSynthetic 20B3 VH
CDR2 68Val Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys
Gly 1 5 10 15 699PRTArtificial SequenceSynthetic 20B3 VH CDR3 69Gly
Gly Met Gly Asn Tyr Phe Asp Tyr 1 5 7011PRTArtificial
SequenceSynthetic 20B3 VL CDR1 70Arg Ala Ser Gln Ser Val Ser Ser
Tyr Leu Ala 1 5 10 717PRTArtificial SequenceSynthetic 20B3 VL CDR2
71Asp Ala Ser Asn Arg Ala Thr 1 5 729PRTArtificial
SequenceSynthetic 20B3 VL CDR3 72Gln Gln Arg Ser Asn Trp Pro Leu
Thr 1 5 73117PRTArtificial SequenceSynthetic 20B3 VH 73Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Asp Met His Trp Val Arg Gln Thr Thr Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Val Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser
Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn
Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Met Gly Asn Tyr Phe
Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
74107PRTArtificial SequenceSynthetic 20B3 VL 74Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
755PRTArtificial SequenceSynthetic 14A2 VH CDR1 75Asn Tyr Ala Leu
His 1 5 7617PRTArtificial SequenceSynthetic 14A2 VH CDR2 76Leu Ile
Ser Tyr Asp Gly Ser Arg Lys His Tyr Ala Asp Ser Val Lys 1 5 10 15
Gly 778PRTArtificial SequenceSynthetic 14A2 VH CDR3 77Leu Thr Met
Val Arg Glu Gly Gly 1 5 7812PRTArtificial SequenceSynthetic 14A2
VL1 CDR1 78Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10
797PRTArtificial SequenceSynthetic 14A2 VL1 CDR2 79Gly Ala Ser Ser
Arg Ala Thr 1 5 809PRTArtificial SequenceSynthetic 14A2 VL1 CDR3
80Gln Gln Tyr Gly Ser Ser Pro Phe Thr 1 5 8111PRTArtificial
SequenceSynthetic 14A2 VL2 CDR1 81Arg Val Ser Gln Gly Ile Ser Ser
Tyr Leu Asn 1 5 10 827PRTArtificial SequenceSynthetic 14A2 VL2 CDR2
82Ser Ala Ser Asn Leu Gln Ser 1 5 839PRTArtificial
SequenceSynthetic 14A2 VL2 CDR3 83Gln Arg Thr Tyr Asn Ala Pro Tyr
Thr 1 5 84115PRTArtificial SequenceSynthetic 14A2 VH 84Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25
30 Ala Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Leu Ile Ser Tyr Asp Gly Ser Arg Lys His Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr Met Val Arg Glu
Gly Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
85108PRTArtificial SequenceSynthetic 14A2 VL1 85Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Gly Ser Ser Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys 100 105 86107PRTArtificial SequenceSynthetic 14A2 VL2 86Asp
Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Val Ser Gln Gly Ile Ser Ser Tyr
20 25 30 Leu Asn Trp Tyr Arg Gln Lys Pro Gly Lys Val Pro Lys Leu
Leu Ile 35 40 45 Tyr Ser Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Gly
Gln Arg Thr Tyr Asn Ala Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 875PRTArtificial SequenceSynthetic 20C1
VH CDR1 87Ser Tyr Ala Met Tyr 1 5 8816PRTArtificial
SequenceSynthetic 20C1 VH CDR2 88Ala Ile Asp Thr Asp Gly Gly Thr
Phe Tyr Ala Asp Ser Val Arg Gly 1 5 10 15 899PRTArtificial
SequenceSynthetic 20C1 VH CDR3 89Leu Gly Glu Gly Tyr Phe Phe Asp
Tyr 1 5 9011PRTArtificial SequenceSynthetic 20C1 VL CDR1 90Arg Ala
Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 917PRTArtificial
SequenceSynthetic 20C1 VL CDR2 91Asp Ala Ser Asn Arg Ala Thr 1 5
929PRTArtificial SequenceSynthetic 20C1 VL CDR3 92Gln Gln Arg Ser
Asn Trp Pro Pro Thr 1 5 93117PRTArtificial SequenceSynthetic 20C1
VH 93Glu Ala Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly
Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Asp Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Thr Asp Gly Gly Thr
Phe Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Gly Leu
Arg Ala Glu Asp Met Ala Val Tyr Phe Cys Ala 85 90 95 Arg Leu Gly
Glu Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val
Thr Val Ser Ser 115 94107PRTArtificial SequenceSynthetic 20C1 VL
94Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 95446PRTArtificial
SequenceSynthetic OX40.6 heavy chain 95Glu Val Gln Leu Val Gln Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Gly Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Tyr
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ala Ile Gly Ile Gly Gly Asp Thr Phe Tyr Thr Asp Ser Val Lys 50 55
60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser Leu
65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Met Gly Thr Gly Tyr Phe Phe Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185
190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310
315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
96214PRTArtificial SequenceSynthetic OX40.6 light chain 96Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150
155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
97446PRTArtificial SequenceSynthetic OX40.7 heavy chain 97Glu Val
Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Ala Ile Gly Ile Gly Gly Asp Thr Phe Tyr Thr Asp
Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Ser Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Gly Thr Gly Tyr Phe
Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150
155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275
280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395
400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 445 98214PRTArtificial SequenceSynthetic OX40.7
light chain 98Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr
Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 99444PRTArtificial SequenceSynthetic OX40.8 heavy
chain 99Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly
Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asn Tyr 20 25 30 Ala Leu His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser Tyr Asp Gly Ser Arg
Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu
Thr Met Val Arg Glu Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165
170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290
295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410
415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
100215PRTArtificial SequenceSynthetic OX40.8 light chain 100Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150
155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
101444PRTArtificial SequenceSynthetic OX40.9 heavy chain 101Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Ala Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Leu Ile Ser Tyr Asp Gly Ser Arg Lys His Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr Tyr Val Arg
Glu Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150
155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275
280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395
400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 102215PRTArtificial SequenceSynthetic OX40.9 light chain
102Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Phe Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130
135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu
Cys 210 215 103444PRTArtificial SequenceSynthetic OX40.10 heavy
chain 103Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Tyr 20 25 30 Ala Leu His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser Tyr Ser Gly Ser
Arg Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser
Leu Thr Met Val Arg Glu Gly Gly Gln Gly Thr Leu Val Thr 100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115
120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235
240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 104215PRTArtificial SequenceSynthetic
OX40.10 light chain 104Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90
95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala
100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser
Phe Asn Arg Gly Glu Cys 210 215 105444PRTArtificial
SequenceSynthetic OX40.11 heavy chain 105Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Leu Ile Ser Tyr Asp Ser Ser Arg Lys His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr Met Val Arg Glu Gly Gly Gln
Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180
185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys 195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305
310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln 370
375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 106215PRTArtificial SequenceSynthetic
OX40.11 light chain 106Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90
95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala
100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser
Phe Asn Arg Gly Glu Cys 210 215 107444PRTArtificial
SequenceSynthetic OX40.12 heavy chain 107Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Leu Ile Ser Tyr Ser Gly Ser Arg Lys His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr Met Val Arg Glu Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180
185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys 195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305
310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425
430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
108215PRTArtificial SequenceSynthetic OX40.12 light chain 108Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Phe Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145
150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210
215 109444PRTArtificial SequenceSynthetic OX40.13 heavy chain
109Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30 Ala Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser Tyr Asp Ser Ser Arg Lys
His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr
Met Val Arg Glu Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130
135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250
255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375
380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 435 440 110215PRTArtificial SequenceSynthetic OX40.13
light chain 110Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Phe
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100 105
110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn
Arg Gly Glu Cys 210 215 111444PRTArtificial SequenceSynthetic
OX40.14 heavy chain 111Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser Tyr
Ser Gly Ser Arg Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Ser Leu Thr Tyr Val Arg Glu Trp Gly Gln Gly Thr Leu Val Thr
100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val
Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215
220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340
345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440 112215PRTArtificial
SequenceSynthetic OX40.14 light chain 112Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly
Ser Ser Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180
185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys 195 200
205 Ser Phe Asn Arg Gly Glu Cys 210 215 113444PRTArtificial
SequenceSynthetic OX40.15 heavy chain 113Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Leu Ile Ser Tyr Asp Ser Ser Arg Lys His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr Tyr Val Arg Glu Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180
185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys 195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305
310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425
430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
114215PRTArtificial SequenceSynthetic OX40.15 light chain 114Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Phe Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145
150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210
215 115446PRTArtificial SequenceSynthetic OX40.16 heavy chain
115Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Thr Asp Gly Gly Thr Phe
Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Leu Gly Glu
Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 445 116214PRTArtificial
SequenceSynthetic OX40.16 light chain (shared by OX40.20, OX40.21,
OX40.22) 116Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly
Glu Cys 210 117446PRTArtificial SequenceSynthetic OX40.17 heavy
chain 117Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Asp Met His Trp Val Arg Gln Thr Thr Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Thr Ala Gly Asp
Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser
Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser
Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly
Gly Met Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp
Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235
240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360
365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 435 440 445 118214PRTArtificial
SequenceSynthetic OX40.17 light chain 118Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 119449PRTArtificial
SequenceSynthetic OX40.18 heavy chain 119Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Val
Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Pro Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Ser Ser Ser Tyr Asn Trp Phe
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile 245 250
255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly 120214PRTArtificial
SequenceSynthetic OX40.18 light chain 120Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 121444PRTArtificial
SequenceSynthetic OX40.19 heavy chain 121Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Leu Ile Ser Tyr Asp Gly Ser Arg Lys His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr Leu Val Arg Glu Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180
185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys 195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305
310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425
430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
122215PRTArtificial SequenceSynthetic OX40.19 light chain 122Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Phe Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145
150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210
215 123446PRTArtificial SequenceSynthetic OX40.20 heavy chain
123Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Thr Ser Gly Gly Thr Phe
Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Leu Gly Glu
Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 445 124446PRTArtificial
SequenceSynthetic OX40.21 heavy chain 124Glu Val Gln Leu Val Gln
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met
Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Asp Thr Asp Ala Gly Thr Phe Tyr Ala Asp Ser Val Arg 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Phe Cys Ala 85 90 95 Arg Leu Gly Glu Gly Tyr Phe Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180
185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305
310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 125446PRTArtificial SequenceSynthetic OX40.22 heavy chain
125Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Thr Ser Thr Gly Thr Phe
Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Leu Gly Glu
Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295
300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420
425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 126360DNAArtificial SequenceSynthetic 3F4 VH (nucleotide
sequence) 126caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggaaa caccttcacc agttatgatg tcaactgggt
gcgacaggcc 120actggacaag ggcttgagtg gatgggatgg atgaacccta
acagtggtaa cacaggctat 180gcaccgaagt tccagggcag agtcaccatg
accaggaaca cctccataag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgttt attactgtgc gagaatatat 300agcagctcgt
acaactggtt cgacccctgg ggccagggaa ccctggtcac cgtctcctca
360127321DNAArtificial SequenceSynthetic 3F4 VL 127gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggcctctcac tttcggcgga 300gggaccaagg tggagatcaa a
321128354DNAArtificial SequenceSynthetic 14B6 VH 128gaggtgcagc
tggagcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60tcctgtaagg
gttctggata cagctttacc agcaactgga tcggctgggt gcgccagatg
120cccgggaaag gcctggagtg gatggggttc atctatcctg gtgactctga
taccaggtac 180agcccgtcct tccaaggcca ggtcaccatc tcagccgaca
agtccatcag caccgcctac 240ctgcagtgga gcagcctcaa ggcctcggac
atcgccatgt attactgtgc gagatatggg 300gatgactggt acttcgatct
ctggggccgt ggcaccctgg tcactgtctc ctca 354129321DNAArtificial
SequenceSynthetic 14B6 VL1 129gaaattgtgt tgacacagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
agctacttag cctggttcca acagagacct 120ggccaggctc ccaggctcct
catctatgat gcatccaaca gggccactgg catcccagcc 180aggttcagtg
gcagtgggtc tgggacagac ttctctctca ccatcagcag cctagagcct
240gaagattttg cagtttatta ctgtcagcag cgtggcgact ggcccatcac
cttcggccaa 300gggacacgac tggagattaa a 321130324DNAArtificial
SequenceSynthetic 14B6 VL2 130gacatccaga tgacccagtc tccatcctca
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc
agctggttag cctggtatca gcagaaacca 120gagaaagccc ctaagtccct
gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagattttg caacttatta ctgccaacag tataatagtt accctcggat
caccttcggc 300caagggacac gactggagat taaa 324131351DNAArtificial
SequenceSynthetic 23H3 VH 131gaggttcagc tggtgcagtc tgggggaggc
ttggtacatc ctggggggtc cctgagactc 60tcctgtgcag gctctggatt caccttcagt
aactatgcta tgtactgggt tcgccaggct 120ccaggaaaag gtctggagtg
ggtatcagcc attggtattg gtggtgacac attctataca 180gactccgtga
agggccgatt caccatctcc agagacaatg ccaagaactc cttgtctctt
240caaatgaaca gcctgagagc cgaggacatg gctgtgtatt actgtgcaag
aatgggaact 300gggtacttct ttgactactg gggccaggga accctggtca
ccgtctcctc a 351132321DNAArtificial SequenceSynthetic 23H3 VL
132gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca
acagaaacct 120ggccaggctc ccaggctcct catctatgat gcatccaaca
gggccactgg catcccagcc 180aggttcagtg gcagtgggtc tgggacagac
ttcactctca ccatcagcag cctagagcct 240gaagattttg cagtttatta
ctgtcagcag cgtagcaact ggcctctcac tttcggccct 300gggaccaaag
tggatatcaa a 321133363DNAArtificial SequenceSynthetic 6E1 VH
133caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctttgcta tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtgacagtt atttcatatg
atggaagcat taaatactac 180acagactccg tgaagggccg attcaccttc
tccagagaca attccaagaa cactctgtat 240ctgcaaatga acagcctgag
agctgaggac acggctgtgt attactgtac gagagatgga 300aactatggtt
cggcgagata cttccagcac tggggccagg gcaccctggt caccgtctcc 360tca
363134321DNAArtificial SequenceSynthetic 6E1 VL1 134gacatccaga
tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca
120gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag
tataatagtt accctcggac gttcggccaa 300gggaccaagg tggaaatcaa a
321135321DNAArtificial SequenceSynthetic 6E1 VL2 135gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggccgtacac ttttggccag 300gggaccaagc tggagatcaa a
321136360DNAArtificial SequenceSynthetic 18E9 VH 136gaggttcagc
tggtgcagtc tgggggaggc ttggttcatc ctggggggtc cctgagactc 60tcctgtgcac
actctggatt caccttcact agctctgcta tgcactgggt tcgccaggct
120ccaggaaaag gtctggaatg gatatcagct attggtactg gtggtgacac
atactatgca 180gactccgtga agggccgatt caccatctcc agagacaatg
ccaagaactc cttgtatctt 240caaataaaca gcctgagagc cgaggacatg
gctgtatatt actgtgcaag agacttttac 300gatattttga ctggtatctt
tgactactgg ggccagggaa ccctggtcac cgtctcctca 360137321DNAArtificial
SequenceSynthetic 18E9 VL 137gacatccaga tgacccagtc tccatcttcc
gtgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc
agctggttag cctggtatca gcataaacca 120gggaaagccc ctaagctcct
gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct
240gaagattttg caacttacta ttgtcaacag gctaatagtt tcccttcgac
gttcggccaa 300gggaccaagg tggaaatcaa a 321138408DNAArtificial
SequenceSynthetic 8B11 VH 138atggagtttg tgctgagctg ggttttcctt
gttgctatat taaaaggtgt ccagtgtgaa 60attcagctgg tgcagtctgg gggaggcttg
gtacatcctg gggggtccct gagactctcc 120tgtgcaggct ctggattcac
cttcagtagc gatgctatgt actgggttcg ccaggctcca 180ggaaaaggtc
tggagtgggt atcagctatt ggtattggtg gtgacacata ctatacagac
240tccgtgatgg gccgattcac catctccaga gacaatgcca agaactcctt
gtatcttcaa 300atgaacagcc tgagagccga ggacatggct gtgtattact
gtgcaaggct ggggatgggg 360tactactttg actactgggg ccagggaacc
ctggtcaccg tctcctca 408139321DNAArtificial SequenceSynthetic 8B11
VL 139gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca
acagaaacct 120ggccaggctc ccaggctcct catctatgat gcatccaaca
gggccactgg catcccagcc 180aggttcagtg gcagtgggtc tgggacagac
ttcactctca ccatcagcag cctagagcct 240gaagattttg cagtttatta
ctgtcagcag cgtagcaact ggcctccgac gttcggccaa 300gggaccaagg
tggaaatcaa a 321140351DNAArtificial SequenceSynthetic 20B3 VH
140gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctacgaca tgcactgggt
ccgccaaact 120acaggaaaag gtctggagtg ggtctcagtt attggtactg
ctggtgacac atactatcca 180ggctccgtga agggccgatt caccatctcc
agagaaaatg ccaagaactc cttgtatctt 240caaatgaaca gcctgagagc
cggggacacg gctgtgtatt actgtgcaag aggggggatg 300gggaactact
ttgactactg gggccaggga accctggtca ccgtctcctc a
351141321DNAArtificial SequenceSynthetic 20B3 VL 141gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa a
321142345DNAArtificial SequenceSynthetic 14A2 VH 142caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cctctggatt caccttcagt aactatgctc tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcactt atatcatatg atggaagcag
gaaacactac 180gcagactccg tgaagggccg attcagtatc tccagagaca
attccaagaa cacactgtat 240ctgcaaatga acagcctgag agctgaggac
acggctgtgt attactgtgc gagtcttact 300atggttcggg aggggggcca
gggaaccctg gtcaccgtct cctca 345143324DNAArtificial
SequenceSynthetic 14A2 VL1 143gaaattgtgt tgacgcagtc tccaggcacc
ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
agcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct
cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag
240cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccatt
cactttcggc 300cctgggacca aagtggatat caaa 324144321DNAArtificial
SequenceSynthetic 14A2 VL2 144gacatccagt tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggtgagtca gggcattagc
agttatttaa attggtatcg gcagaaacca 120gggaaagttc ctaagctcct
gatctatagt gcatccaatt tgcaatctgg agtcccatct 180cggttcagtg
gcagtggatc tgggacagat ttcactctca ctatcagcag cctgcagcct
240gaagatgttg caacttatta cggtcaacgg acttacaatg ccccttacac
tttcggcgga 300gggaccaagg tggagatcaa a 321145351DNAArtificial
SequenceSynthetic 20C1 VH 145gaggctcagc tggtgcagtc tgggggaggc
ttggttcatc ctggggggtc cctgagactc 60tcctgtgcag actctggatt caccttcagt
agctatgcta tgtactgggt tcgccaggct 120ccaggaaaag gtctggagtg
ggtatcagct attgatactg atggtggcac attctatgca 180gactccgtgc
ggggccgatt caccatctcc agagacaatg ccaagaactc cttgtatctt
240caaatgaacg gcctgagagc cgaggacatg gctgtgtatt tctgtgcaag
acttggggaa 300gggtacttct ttgactactg gggccaggga accctggtca
ccgtctcctc a 351146321DNAArtificial SequenceSynthetic 20C1 VL
146gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca
acagaaacct 120ggccaggctc ccaggctcct catctatgat gcatccaaca
gggccactgg catcccagcc 180aggttcagtg gcagtgggtc tgggacagac
ttcactctca ccatcagcag cctagagcct 240gaagattttg cagtttatta
ctgtcagcag cgtagcaact ggcctcccac tttcggcgga 300gggaccaagg
tggagatcaa a 3211471338DNAArtificial SequenceSynthetic OX40.6 heavy
chain 147gaggttcagc tggtgcagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag gctctggatt caccttcagt aactatgcta tgtactgggt
tcgccaggct 120ccaggaaaag gtctggagtg ggtatcagcc attggtattg
gtggtgacac attctataca 180gactccgtga agggccgatt caccatctcc
agagacaatg ccaagaactc cttgtctctt 240caaatgaaca gcctgagagc
cgaggacacg gctgtgtatt actgtgcaag aatgggaact 300gggtacttct
ttgactactg gggccaggga accctggtca ccgtctcctc agctagcacc
360aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg
gggcacagcg 420gccctgggct gcctggtcaa ggactacttc cccgaaccgg
tgacggtgtc gtggaactca 480ggcgccctga ccagcggcgt gcacaccttc
ccggctgtcc tacagtcctc aggactctac 540tccctcagca gcgtggtgac
cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600aacgtgaatc
acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt
660gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg
accgtcagtc 720ttcctcttcc ccccaaaacc caaggacacc ctcatgatct
cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag ccacgaagac
cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1080aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200gacggctcct
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg
1260aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1320ctctccctgt ccccgggt 1338148642DNAArtificial
SequenceSynthetic OX40.6 light chain 148gaaattgtgt tgacacagtc
tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc agctacttag cctggtacca acagaaacct 120ggccaggctc
ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc
180aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag
cctagagcct 240gaagattttg cagtttatta ctgtcagcag cgtagcaact
ggcctctcac tttcggccct 300gggaccaaag tggatatcaa acgtacggtg
gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gt 6421491338DNAArtificial SequenceSynthetic OX40.7
heavy chain 149gaggttcagc tggtgcagtc tgggggaggc ttggtacatc
ctggggggtc cctgagactc 60tcctgtgcag gctctggatt caccttcagt aactatgcta
tgtactgggt tcgccaggct 120ccaggaaaag gtctggagtg ggtatcagcc
attggtattg gtggtgacac attctataca 180gactccgtga agggccgatt
caccatctcc agagacaatg ccaagaactc cttgtctctt 240caaatgaaca
gcctgagagc cgaggacacg gctgtgtatt actgtgcaag atatggaact
300gggtacttct ttgactactg gggccaggga accctggtca ccgtctcctc
agctagcacc 360aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 420gccctgggct gcctggtcaa ggactacttc
cccgaaccgg tgacggtgtc gtggaactca 480ggcgccctga ccagcggcgt
gcacaccttc ccggctgtcc tacagtcctc aggactctac 540tccctcagca
gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
600aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc
caaatcttgt 660gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 720ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
900cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 960tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagccaaa 1020gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggagga gatgaccaag 1080aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
1200gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg
gcagcagggg 1260aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1320ctctccctgt ccccgggt
1338150642DNAArtificial SequenceSynthetic OX40.7 light chain
150gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca
acagaaacct 120ggccaggctc ccaggctcct catctatgat gcatccaaca
gggccactgg catcccagcc 180aggttcagtg gcagtgggtc tgggacagac
ttcactctca ccatcagcag cctagagcct 240gaagattttg cagtttatta
ctgtcagcag cgtagcaact ggcctctcac tttcggccct 300gggaccaaag
tggatatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca
360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gt 6421511332DNAArtificial
SequenceSynthetic OX40.8 heavy chain 151caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatatg atggaagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300atggttcggg agtggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag
1140agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg gacaagagca
ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332152645DNAArtificial SequenceSynthetic OX40.8 light chain
152gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451531332DNAArtificial
SequenceSynthetic OX40.9 heavy chain 153caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatatg atggaagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300tacgttcggg agtggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332154645DNAArtificial SequenceSynthetic OX40.9 light chain
154gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451551332DNAArtificial
SequenceSynthetic OX40.10 heavy chain 155caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatata gtggaagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300atggttcggg aggggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332156645DNAArtificial SequenceSynthetic OX40.10 light chain
156gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451571332DNAArtificial
SequenceSynthetic OX40.11 heavy chain 157caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatatg atagtagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300atggttcggg aggggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332158645DNAArtificial SequenceSynthetic OX40.11 light chain
158gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451591332DNAArtificial
SequenceSynthetic OX40.12 heavy chain 159caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatata gtggaagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300atggttcggg agtggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332160645DNAArtificial SequenceSynthetic OX40.12 light chain
160gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451611332DNAArtificial
SequenceSynthetic OX40.13 heavy chain 161caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatatg atagtagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300atggttcggg agtggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332162645DNAArtificial SequenceSynthetic OX40.13 light chain
162gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451631332DNAArtificial
SequenceSynthetic OX40.14 heavy chain 163caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatata gtggaagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300tacgttcggg agtggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332164645DNAArtificial SequenceSynthetic OX40.14 light chain
164gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451651332DNAArtificial
SequenceSynthetic OX40.15 heavy chain 165caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatatg atagtagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300tacgttcggg agtggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332166645DNAArtificial SequenceSynthetic OX40.15 light chain
166gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451671341DNAArtificial
SequenceSynthetic OX40.16 heavy chain 167gaggttcagc tggtgcagtc
tgggggaggc ttggttcagc ctggggggtc cctgagactc 60tcctgtgcag gctctggatt
caccttcagt agctatgcta tgtactgggt tcgccaggct 120ccaggaaaag
gtctggagtg ggtatcagct attgatactg atggtggcac attctatgca
180gactccgtgc ggggccgatt caccatctcc agagacaatg ccaagaactc
cttgtatctt 240caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
tctgtgcaag acttggggaa 300gggtacttct ttgactactg gggccaggga
accctggtca ccgtctcctc agctagcacc 360aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420gccctgggct
gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
480ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc
aggactctac 540tccctcagca gcgtggtgac cgtgccctcc agcagcttgg
gcacccagac ctacatctgc 600aacgtgaatc acaagcccag caacaccaag
gtggacaaga gagttgagcc caaatcttgt 660gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg accgtcagtc 720ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
780tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 840ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 900cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 960tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag
1080aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 1140tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 1200gacggctcct tcttcctcta tagcaagctc
accgtggaca agagcaggtg gcagcagggg 1260aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1320ctctccctgt
ccccgggttg a 1341168645DNAArtificial SequenceSynthetic OX40.16
light chain (shared by OX40.20, OX40.21, OX40.22) 168gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggcctcccac tttcggcgga 300gggaccaagg tggagatcaa
acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gttag 6451691341DNAArtificial
SequenceSynthetic OX40.17 heavy chain 169gaggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt agctacgaca tgcactgggt ccgccaaact 120acaggaaaag
gtctggagtg ggtctcagtt attggtactg ctggtgacac atactatcca
180ggctccgtga agggccgatt caccatctcc agagaaaatg ccaagaactc
cttgtatctt 240caaatgaaca gcctgagagc cggggacacg gctgtgtatt
actgtgcaag aggggggatg 300gggaactact ttgactactg gggccaggga
accctggtca ccgtctcctc agctagcacc 360aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420gccctgggct
gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
480ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc
aggactctac 540tccctcagca gcgtggtgac cgtgccctcc agcagcttgg
gcacccagac ctacatctgc 600aacgtgaatc acaagcccag caacaccaag
gtggacaaga gagttgagcc caaatcttgt 660gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg accgtcagtc 720ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
780tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 840ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 900cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 960tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag
1080aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 1140tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 1200gacggctcct tcttcctcta tagcaagctc
accgtggaca agagcaggtg gcagcagggg 1260aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1320ctctccctgt
ccccgggttg a 1341170645DNAArtificial SequenceSynthetic OX40.17
light chain 170gaaattgtgt tgacacagtc tccagccacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag
cctggtacca acagaaacct 120ggccaggctc ccaggctcct catctatgat
gcatccaaca gggccactgg catcccagcc 180aggttcagtg gcagtgggtc
tgggacagac ttcactctca ccatcagcag cctagagcct 240gaagattttg
cagtttatta ctgtcagcag cgtagcaact ggccgctcac tttcggcgga
300gggaccaagg tggagatcaa acgtacggtg gctgcaccat ctgtcttcat
cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420cccagagagg ccaaagtaca gtggaaggtg
gataacgccc tccaatcggg taactcccag 480gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacg 540ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc
600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag
6451711350DNAArtificial SequenceSynthetic OX40.18 heavy chain
171caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc agttatgatg tcaactgggt
gcgacaggcc 120actggacaag ggcttgagtg gatgggatgg atgaacccta
acagtggtaa cacaggctat 180gcaccgaagt tccagggcag agtcaccatg
accagggaca cctccataag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgttt attactgtgc gagaatatat 300agcagctcgt
acaactggtt cgactactgg ggccagggaa ccctggtcac cgtctcctca
360gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag
cacctctggg 420ggcacagcgg ccctgggctg cctggtcaag gactacttcc
ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg
cacaccttcc cggctgtcct acagtcctca 540ggactctact ccctcagcag
cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600tacatctgca
acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagccc
660aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact
cctgggggga 720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc
tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt ggacgtgagc
cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg gcgtggaggt
gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa
aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc
tgcccccatc ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc
ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa
tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200ctggactccg
acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg
1260cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccactacacg 1320cagaagagcc tctccctgtc cccgggttga
1350172645DNAArtificial SequenceSynthetic OX40.18 light chain
172gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca
acagaaacct 120ggccaggctc ccaggctcct catctatgat gcatccaaca
gggccactgg catcccagcc 180aggttcagtg gcagtgggtc tgggacagac
ttcactctca ccatcagcag cctagagcct 240gaagattttg cagtttatta
ctgtcagcag cgtagcaact ggcctctcac tttcggcgga 300gggaccaagg
tggagatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca
360tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 420cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 480gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 540ctgagcaaag cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gttag 6451731332DNAArtificial
SequenceSynthetic OX40.19 heavy chain 173caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt aactatgctc tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcactt atatcatatg atggaagcag gaaacactac
180gcagactccg tgaagggccg attcagtatc tccagagaca attccaagaa
cacactgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagtcttact 300ctggttcggg agtggggcca gggaaccctg
gtcaccgtct cctcagctag caccaagggc 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaactcctgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgtgtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctatagcaa gctcaccgtg
gacaagagta ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtccccgg gt
1332174645DNAArtificial SequenceSynthetic OX40.19 light chain
174gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta gctcaccatt cactttcggc 300cctgggacca
aagtggatat caaacgtacg gtggctgcac catctgtctt catcttcccg
360ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct
gaataacttc 420tatcccagag aggccaaagt acagtggaag gtggataacg
ccctccaatc gggtaactcc 480caggagagtg tcacagagca ggacagcaag
gacagcacct acagcctcag cagcaccctg 540acgctgagca aagcagacta
cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct
cgcccgtcac aaagagcttc aacaggggag agtgt 6451751341DNAArtificial
SequenceSynthetic OX40.20 heavy chain 175gaggttcagc tggtgcagtc
tgggggaggc ttggttcagc ctggggggtc cctgagactc 60tcctgtgcag gctctggatt
caccttcagt agctatgcta tgtactgggt tcgccaggct 120ccaggaaaag
gtctggagtg ggtatcagct attgatacta gtggtggcac attctatgca
180gactccgtgc ggggccgatt caccatctcc agagacaatg ccaagaactc
cttgtatctt 240caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
tctgtgcaag acttggggaa 300gggtacttct ttgactactg gggccaggga
accctggtca ccgtctcctc agctagcacc 360aagggcccat cggtcttccc
cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420gccctgggct
gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca
480ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc
aggactctac 540tccctcagca gcgtggtgac cgtgccctcc agcagcttgg
gcacccagac ctacatctgc 600aacgtgaatc acaagcccag caacaccaag
gtggacaaga gagttgagcc caaatcttgt 660gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg accgtcagtc 720ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca
780tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg
gtacgtggac 840ggcgtggagg tgcataatgc caagacaaag ccgcgggagg
agcagtacaa cagcacgtac 900cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaatggcaa ggagtacaag 960tgcaaggtct ccaacaaagc
cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag
1080aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 1140tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 1200gacggctcct tcttcctcta tagcaagctc
accgtggaca agagcaggtg gcagcagggg 1260aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac gcagaagagc 1320ctctccctgt
ccccgggttg a 13411761341DNAArtificial SequenceSynthetic OX40.21
heavy chain 176gaggttcagc tggtgcagtc tgggggaggc ttggttcagc
ctggggggtc cctgagactc 60tcctgtgcag gctctggatt caccttcagt agctatgcta
tgtactgggt tcgccaggct 120ccaggaaaag gtctggagtg ggtatcagct
attgatactg atgctggcac attctatgca 180gactccgtgc ggggccgatt
caccatctcc agagacaatg ccaagaactc cttgtatctt 240caaatgaaca
gcctgagagc cgaggacacg gctgtgtatt tctgtgcaag acttggggaa
300gggtacttct ttgactactg gggccaggga accctggtca ccgtctcctc
agctagcacc 360aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 420gccctgggct gcctggtcaa ggactacttc
cccgaaccgg tgacggtgtc gtggaactca 480ggcgccctga ccagcggcgt
gcacaccttc ccggctgtcc tacagtcctc aggactctac 540tccctcagca
gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc
600aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc
caaatcttgt 660gacaaaactc acacatgccc accgtgccca gcacctgaac
tcctgggggg accgtcagtc 720ttcctcttcc ccccaaaacc caaggacacc
ctcatgatct cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag
ccacgaagac cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg
tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac
900cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa
ggagtacaag 960tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga
aaaccatctc caaagccaaa 1020gggcagcccc gagaaccaca ggtgtacacc
ctgcccccat cccgggagga gatgaccaag 1080aaccaggtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca
atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc
1200gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg
gcagcagggg 1260aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac gcagaagagc 1320ctctccctgt ccccgggttg a
13411771341DNAArtificial SequenceSynthetic OX40.22 heavy chain
177gaggttcagc tggtgcagtc tgggggaggc ttggttcagc ctggggggtc
cctgagactc 60tcctgtgcag gctctggatt caccttcagt agctatgcta tgtactgggt
tcgccaggct 120ccaggaaaag gtctggagtg ggtatcagct attgatacta
gtactggcac attctatgca 180gactccgtgc ggggccgatt caccatctcc
agagacaatg ccaagaactc cttgtatctt 240caaatgaaca gcctgagagc
cgaggacacg gctgtgtatt tctgtgcaag acttggggaa 300gggtacttct
ttgactactg gggccaggga accctggtca ccgtctcctc agctagcacc
360aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg
gggcacagcg 420gccctgggct gcctggtcaa ggactacttc cccgaaccgg
tgacggtgtc gtggaactca 480ggcgccctga ccagcggcgt gcacaccttc
ccggctgtcc tacagtcctc aggactctac 540tccctcagca gcgtggtgac
cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600aacgtgaatc
acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt
660gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg
accgtcagtc 720ttcctcttcc ccccaaaacc caaggacacc ctcatgatct
cccggacccc tgaggtcaca 780tgcgtggtgg tggacgtgag ccacgaagac
cctgaggtca agttcaactg gtacgtggac 840ggcgtggagg tgcataatgc
caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag
960tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc
caaagccaaa 1020gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga gatgaccaag 1080aaccaggtca gcctgacctg cctggtcaaa
ggcttctatc ccagcgacat cgccgtggag 1140tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200gacggctcct
tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg
1260aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
gcagaagagc 1320ctctccctgt ccccgggttg a 134117817PRTArtificial
SequenceSynthetic hOX40 epitope 178Asp Val Val Ser Ser Lys Pro Cys
Lys Pro Cys Thr Trp Cys Asn Leu 1 5 10 15 Arg 17936PRTArtificial
SequenceSynthetic hOX40 epitope 179Asp Ser Tyr Lys Pro Gly Val Asp
Cys Ala Pro Cys Pro Pro Gly His 1 5 10 15 Phe Ser Pro Gly Asp Asn
Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr 20 25 30 Leu Ala Gly Lys 35
1805PRTArtificial SequenceSynthetic peptide linker 180Pro Val Gly
Val Val 1 5 1815PRTArtificial SequenceSynthetic sortase A
recognition motifmisc_feature(3)..(3)Xaa can be any naturally
occurring amino acid 181Leu Pro Xaa Thr Gly 1 5 18231PRTArtificial
SequenceSynthetic hOX40 epitope 182Gln Asn Thr Val Cys Arg Pro Cys
Gly Pro Gly Phe Tyr Asn Asp Val 1 5 10 15 Val Ser Ser Lys Pro Cys
Lys Pro Cys Thr Trp Cys Asn Leu Arg 20 25 30 18311PRTArtificial
SequenceSynthetic hOX40 epitope 183Pro Cys Lys Pro Cys Thr Trp Cys
Asn Leu Arg 1 5 10
18412PRTArtificial SequenceSynthetic hOX40 epitope 184Gln Leu Cys
Thr Ala Thr Gln Asp Thr Val Cys Arg 1 5 10 18515PRTArtificial
SequenceSynthetic hOX40 epitope 185Ser Gln Asn Thr Val Cys Arg Pro
Cys Gly Pro Gly Phe Tyr Asn 1 5 10 15 1865PRTArtificial
SequenceSynthetic IgG1 C-termianl CH1 (same for IgG3 (17-15-15-15),
igG3 (17-15-15), IgG3 (17-15), IgG3 (15-15-15), IgG3 (15), and IgG4
186Val Asp Lys Arg Val 1 5 1875PRTArtificial SequenceSynthetic IgG2
C-terminal CH1 187Val Asp Lys Thr Val 1 5 18810PRTArtificial
SequenceSynthetic IgG1 upper hinge 188Glu Pro Lys Ser Cys Asp Lys
Thr His Thr 1 5 10 18912PRTArtificial SequenceSynthetic IgG3
(17-15-15-15) upper hinge (same for IgG3 (17-15-15) and IgG3
(17-15)) 189Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr 1 5 10
1904PRTArtificial SequenceSynthetic IgG3 (15-15-15) upper hinge
(same for IgG3(15)) 190Glu Pro Lys Ser 1 1917PRTArtificial
SequenceSynthetic IgG4 upper hinge 191Glu Ser Lys Tyr Gly Pro Pro 1
5 1925PRTArtificial SequenceSynthetic IgG1 middle hinge 192Cys Pro
Pro Cys Pro 1 5 1939PRTArtificial SequenceSynthetic IgG2 middle
hinge 193Cys Cys Val Glu Cys Pro Pro Cys Pro 1 5 19450PRTArtificial
SequenceSynthetic IgG3 (17-15-15-15) middle hinge 194Cys Pro Arg
Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys 1 5 10 15 Pro
Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro 20 25
30 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
35 40 45 Cys Pro 50 19535PRTArtificial SequenceSynthetic IgG3
(17-15-15) middle hinge 195Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys 1 5 10 15 Pro Arg Cys Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro 20 25 30 Arg Cys Pro 35
19620PRTArtificial SequenceSynthetic IgG3 (17-15) middle hinge
196Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys
1 5 10 15 Pro Arg Cys Pro 20 19741PRTArtificial SequenceSynthetic
IgG3 (15-15-15) middle hinge 197Cys Asp Thr Pro Pro Pro Cys Pro Arg
Cys Pro Glu Pro Lys Ser Cys 1 5 10 15 Asp Thr Pro Pro Pro Cys Pro
Arg Cys Pro Glu Pro Lys Ser Cys Asp 20 25 30 Thr Pro Pro Pro Cys
Pro Arg Cys Pro 35 40 19811PRTArtificial SequenceSynthetic IgG3
(15) middle hinge 198Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 1
5 10 1995PRTArtificial SequenceSynthetic IgG4 middle hinge 199Cys
Pro Ser Cys Pro 1 5 2007PRTArtificial SequenceSynthetic IgG1 lower
hinge (same for IgG3 (17-15-15-15), IgG3 (17-15-15), IgG3 (17-15),
IgG3 (15-15-15), IgG3 (15), and IgG4) 200Ala Pro Glu Leu Leu Gly
Gly 1 5 2016PRTArtificial SequenceSynthetic IgG2 lower hinge 201Ala
Pro Pro Val Ala Gly 1 5 20298PRTHomo
sapiensmisc_feature(1)..(98)Wildtype human IgG1 CH1 202Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Lys Val 20398PRTHomo
sapiensmisc_feature(1)..(98)Wildtype human IgG2 CH1 203Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Thr Val 204103PRTHomo
sapiensmisc_feature(1)..(103)Wildtype human IgG1 CH2 204Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 1 5 10 15 Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 20 25
30 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
35 40 45 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 50 55 60 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 65 70 75 80 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 85 90 95 Lys Thr Ile Ser Lys Ala Lys 100
205103PRTHomo sapiensmisc_feature(1)..(103)Wildtype human IgG2 CH2
205Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
1 5 10 15 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 20 25 30 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 35 40 45 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe Arg 50 55 60 Val Val Ser Val Leu Thr Val Val
His Gln Asp Trp Leu Asn Gly Lys 65 70 75 80 Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu 85 90 95 Lys Thr Ile Ser
Lys Thr Lys 100 206106PRTHomo sapiensmisc_feature(1)..(106)Wildtype
human IgG1 CH3 206Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100 105 207107PRTHomo
sapiensmisc_feature(1)..(107)Wildtype human IgG2 CH3 207Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 1 5 10 15 Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25
30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 100 105 20818PRTArtificial SequenceSynthetic
Alternative hinge 208Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro Val 1 5 10 15 Ala Gly 20918PRTArtificial
SequenceSynthetic Alternative hinge 209Glu Arg Lys Ser Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly
21018PRTArtificial SequenceSynthetic Alternative hinge 210Glu Arg
Lys Cys Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15
Ala Gly 21118PRTArtificial SequenceSynthetic Alternative
hingemisc_feature(4)..(4)Xaa can be any naturally occurring amino
acid 211Glu Arg Lys Xaa Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
Val 1 5 10 15 Ala Gly 21218PRTArtificial SequenceSynthetic
Alternative hingemisc_feature(5)..(5)Xaa can be any naturally
occurring amino acid 212Glu Arg Lys Cys Xaa Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro Val 1 5 10 15 Ala Gly 21319PRTArtificial
SequenceSynthetic Alternative hingemisc_feature(19)..(19)Xaa can be
any naturally occurring amino acid 213Glu Arg Lys Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Xaa
21419PRTArtificial SequenceSynthetic Alternative
hingemisc_feature(19)..(19)Xaa can be any naturally occurring amino
acid 214Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
Val 1 5 10 15 Ala Gly Xaa 21519PRTArtificial SequenceSynthetic
Alternative hingemisc_feature(19)..(19)Xaa can be any naturally
occurring amino acid 215Glu Arg Lys Cys Ser Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Xaa 21619PRTArtificial
SequenceSynthetic Alternative hingemisc_feature(4)..(4)Xaa can be
any naturally occurring amino acidmisc_feature(19)..(19)Xaa can be
any naturally occurring amino acid 216Glu Arg Lys Xaa Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Xaa
21719PRTArtificial SequenceSynthetic Alternative
hingemisc_feature(5)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(19)..(19)Xaa can be any naturally occurring amino
acid 217Glu Arg Lys Cys Xaa Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
Val 1 5 10 15 Ala Gly Xaa 21819PRTArtificial SequenceSynthetic
Alternative hinge 218Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly Gly 21919PRTArtificial
SequenceSynthetic Alternative hinge 219Glu Arg Lys Ser Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly Gly
22020PRTArtificial SequenceSynthetic Alternative hinge 220Glu Arg
Lys Cys Cys Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Glu 1 5 10 15
Leu Leu Gly Gly 20 22119PRTArtificial SequenceSynthetic Alternative
hingemisc_feature(4)..(4)Xaa can be any naturally occurring amino
acid 221Glu Arg Lys Xaa Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Glu
Leu 1 5 10 15 Leu Gly Gly 22219PRTArtificial SequenceSynthetic
Alternative hingemisc_feature(5)..(5)Xaa can be any naturally
occurring amino acid 222Glu Arg Lys Cys Xaa Val Glu Cys Pro Pro Cys
Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly Gly 22318PRTArtificial
SequenceSynthetic Alternative hinge 223Glu Arg Lys Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly
22418PRTArtificial SequenceSynthetic Alternative hinge 224Glu Arg
Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15
Leu Gly 22519PRTArtificial SequenceSynthetic Alternative hinge
225Glu Arg Lys Cys Cys Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15 Leu Leu Gly 22618PRTArtificial SequenceSynthetic
Alternative hingemisc_feature(4)..(4)Xaa can be any naturally
occurring amino acid 226Glu Arg Lys Xaa Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly 22718PRTArtificial
SequenceSynthetic Alternative hingemisc_feature(5)..(5)Xaa can be
any naturally occurring amino acid 227Glu Arg Lys Cys Xaa Val Glu
Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly
22814PRTArtificial SequenceSynthetic Alternative hinge 228Glu Arg
Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 1 5 10
22914PRTArtificial SequenceSynthetic Alternative hinge 229Glu Arg
Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 1 5 10
23014PRTArtificial SequenceSynthetic Alternative hinge 230Glu Arg
Lys Cys Ser Val Glu Cys Pro Pro Cys Pro Ala Pro 1 5 10
23114PRTArtificial SequenceSynthetic Alternative
hingemisc_feature(4)..(4)Xaa can be any naturally occurring amino
acid 231Glu Arg Lys Xaa Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 1 5
10 23214PRTArtificial SequenceSynthetic Alternative
hingemisc_feature(5)..(5)Xaa can be any naturally occurring amino
acid 232Glu Arg Lys Cys Xaa Val Glu Cys Pro Pro Cys Pro Ala Pro 1 5
10 2334PRTArtificial SequenceSynthetic Portion of hinge 233Pro Val
Ala Gly 1 2344PRTArtificial SequenceSynthetic Portion of hinge
234Glu Leu Leu Gly 1 2355PRTArtificial SequenceSynthetic Portion of
hinge 235Glu Leu Leu Gly Gly 1 5 2367PRTArtificial
SequenceSynthetic Portion of hinge 236Ser Cys Asp Lys Thr His Thr 1
5 2374PRTArtificial SequenceSynthetic Portion of hinge 237Cys Cys
Val Glu 1 23818PRTHomo sapiensmisc_feature(1)..(18)WT human IgG2
hinge 238Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
Pro Val 1 5 10 15 Ala Gly 23918PRTHomo
sapiensmisc_feature(1)..(18)Human IgG2 hinge with C219S 239Glu Arg
Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15
Ala Gly 24019PRTArtificial SequenceSynthetic IgG2/IgG1 hinge 240Glu
Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Glu Leu 1 5 10
15 Leu Gly Gly 24119PRTArtificial SequenceSynthetic IgG2
(C219S)/IgG1 hinge 241Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro Glu Leu 1 5 10 15 Leu Gly Gly 24222PRTHomo
sapiensmisc_feature(1)..(22)Wild type human IgG1 hinge 242Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15
Pro Glu Leu Leu Gly Gly 20 243103PRTHomo
sapiensmisc_feature(1)..(103)Human IgG1 CH2 with A330S/P331S 243Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 1 5 10
15 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
20 25 30 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 35 40 45 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 50 55 60 Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys 65 70 75 80 Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ser Ser Ile Glu 85 90 95 Lys Thr Ile Ser Lys Ala
Lys 100 244326PRTArtificial SequenceSynthetic IgG1-IgG2-IgG1f
244Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Lys Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro 100 105 110 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 195 200 205
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210
215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu
Ser Leu Ser Pro Gly 325 245325PRTArtificial SequenceSynthetic
IgG1-IgG2-1gG1f2 245Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95
Lys Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100
105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 195 200 205 Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225
230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser
Pro Gly 325 246325PRTArtificial SequenceSynthetic IgG1-IgG2CS-IgG1f
246Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Arg
Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130
135 140 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250
255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly 325
247325PRTArtificial SequenceSynthetic IgG1-IgG2CS-IgG1f2 247Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Arg Lys Ser Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 145 150
155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275
280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly 325
248326PRTArtificial SequenceSynthetic IgG2-IgG1f 248Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly
Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165
170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290
295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly 325 249325PRTArtificial
SequenceSynthetic IgG2-IgG1f2 249Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 165 170 175 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185
190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310
315 320 Ser Leu Ser Pro Gly 325 250326PRTArtificial
SequenceSynthetic IgG2CS-IgG1f 250Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Pro Gly 325 251325PRTArtificial
SequenceSynthetic IgG2CS-IgG1f2 251Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr
Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105
110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 195 200 205 Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230
235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro
Gly 325 252325PRTArtificial SequenceSynthetic IgG1-IgG2-IgG1.1f
252Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Arg
Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130
135 140 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Arg Glu Met Thr Lys Asn 225 230 235 240 Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250
255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly 325
253325PRTArtificial SequenceSynthetic IgG1-IgG2CS-IgG1.1f 253Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Arg Lys Ser
Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 145
150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265
270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly 325
254325PRTArtificial SequenceSynthetic IgG2-IgG1.1f 254Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 145 150 155
160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280
285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly 325 255326PRTArtificial
SequenceSynthetic IgG2CS-IgG1.1f 255Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Pro Gly 325 256330PRTArtificial
SequenceSynthetic IgG1f 256Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 257330PRTArtificial
SequenceSynthetic IgG1.1f 257Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val
Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195
200 205 Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315
320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
258326PRTArtificial SequenceSynthetic IgG2.3 258Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln
Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro
Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180
185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305
310 315 320 Ser Leu Ser Pro Gly Lys 325 259326PRTArtificial
SequenceSynthetic IgG2.5 259Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210
215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser
Leu Ser Pro Gly Lys 325 260326PRTArtificial SequenceSynthetic
IgG2.3G1-KH 260Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr
Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105
110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 195 200 205 Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230
235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro
Gly Lys 325 261326PRTArtificial SequenceSynthetic IgG2.5G1-KH
261Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Arg
1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro
Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg
Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130
135 140 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250
255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325
262327PRTArtificial SequenceSynthetic IgG2.3G1-AY 262Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155
160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280
285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325
263327PRTArtificial SequenceSynthetic IgG2.5G1-AY 263Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Arg 1 5 10 15 Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155
160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280
285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325
264326PRTArtificial SequenceSynthetic IgG2.3G1.1f-KH 264Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 145 150 155
160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280
285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325
265326PRTArtificial SequenceSynthetic IgG2.5G1.1f-KH 265Ala Ser Thr
Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 165 170 175 Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 195
200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315
320 Ser Leu Ser Pro Gly Lys 325 266327PRTArtificial
SequenceSynthetic IgG2.5G1-V27 266Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Glu His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Pro Gly Lys 325 267326PRTArtificial
SequenceSynthetic IgG2.3-V13 267Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro 100 105 110 Pro Val Ala Gly Asp Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195
200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315
320 Ser Leu Ser Pro Gly Lys 325 268326PRTArtificial
SequenceSynthetic IgG2.3-V14 268Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro 100 105 110 Pro Val Ala Gly Asp Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Gly Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195
200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315
320 Ser Leu Ser Pro Gly Lys 325 269326PRTArtificial
SequenceSynthetic IgG2.3-V15 269Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro 100 105 110 Pro Val Ala Gly Asp Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 130 135 140 Val Ser Asp Glu Asp Gly Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195
200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315
320 Ser Leu Ser Pro Gly Lys 325 270326PRTArtificial
SequenceSynthetic IgG2.3-V16 270Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro 100 105 110 Pro Val Ala Gly Asp Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Gly Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195
200 205 Arg Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315
320 Ser Leu Ser Pro Gly Lys 325 271326PRTArtificial
SequenceSynthetic IgG2.3-V17 271Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro 100 105 110 Pro Val Ala Gly Asp Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 130 135 140 Val Ser Asp Glu Asp Gly Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195
200 205 Arg Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305
310 315 320 Ser Leu Ser Pro Gly Lys 325 272326PRTArtificial
SequenceSynthetic IgG2.3-V18 272Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 130 135 140 Val Glu His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195
200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315
320 Ser Leu Ser Pro Gly Lys 325 273326PRTArtificial
SequenceSynthetic IgG2.3-V19 273Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 130 135 140 Val Glu His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Phe Pro 195
200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315
320 Ser Leu Ser Pro Gly Lys 325 274327PRTArtificial
SequenceSynthetic IgG2.3G1-AY-V20 274Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Asp Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Pro Gly Lys 325 275327PRTArtificial
SequenceSynthetic IgG2.3G1-AY-V21 275Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Asp Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp
Gly Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Pro Gly Lys 325 276327PRTArtificial
SequenceSynthetic IgG2.3G1-AY-V22 276Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Asp Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Asp Glu Asp
Gly Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Pro Gly Lys 325 277327PRTArtificial
SequenceSynthetic IgG2.3G1-AY-V23 277Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Asp Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser His Glu Asp
Gly Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205 Pro Arg Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 290 295 300 Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310
315 320 Leu Ser Leu Ser Pro Gly Lys 325 278327PRTArtificial
SequenceSynthetic IgG2.3G1-AY-V24 278Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Asp Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Asp Glu Asp
Gly Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 165 170 175 Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185
190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
195 200 205 Pro Arg Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser 275 280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325
279327PRTArtificial SequenceSynthetic IgG2.3G1-AY-V25 279Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn
Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Asp
Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp
Val Ser Asp Glu Asp Gly Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150
155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 195 200 205 Pro Arg Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275
280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325
280327PRTArtificial SequenceSynthetic IgG2.3G1-AY-V26 280Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn
Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Asp Leu Leu Gly Asp
Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp
Val Ser Asp Glu Asp Gly Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150
155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 195 200 205 Pro Arg Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275
280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325
281327PRTArtificial SequenceSynthetic IgG2.3G1-AY-V28 281Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn
Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp
Val Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150
155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Phe 195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275
280 285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325
282443PRTArtificial SequenceSynthetic OX40.6-Vh-hHC-IgG2.3 282Glu
Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ala Ile Gly Ile Gly Gly Asp Thr Phe Tyr Thr
Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Ser Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Gly Thr Gly Tyr
Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Ser Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 260 265
270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn
Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390
395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 283441PRTArtificial SequenceSynthetic OX40.8-Vh-hHC-IgG2.3
283Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30 Ala Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser Tyr Asp Gly Ser Arg Lys
His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr
Met Val Arg Glu Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130
135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Asn Phe Gly 180 185 190 Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Thr Val Glu
Arg Lys Ser Cys Val Glu Cys Pro Pro Cys 210 215 220 Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 225 230 235 240 Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250
255 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
260 265 270 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 275 280 285 Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val Val His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 305 310 315 320 Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln 325 330 335 Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 340 345 350 Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365 Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375
380 Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
385 390 395 400 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 284443PRTArtificial SequenceSynthetic OX40.16-Vh-hHC-IgG2.3
284Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Thr Asp Gly Gly Thr Phe
Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Leu Gly Glu
Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr
Val Glu Arg Lys Ser Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala
Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250
255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn
260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser
Val Leu Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Gln Pro Arg Glu
Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405
410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
285444PRTArtificial SequenceSynthetic OX40.6-Vh-hHC-IgG2.3G1 285Glu
Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ala Ile Gly Ile Gly Gly Asp Thr Phe Tyr Thr
Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Ser Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Gly Thr Gly Tyr
Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Ser Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 260 265
270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390
395 400 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 286442PRTArtificial SequenceSynthetic
OX40.8-Vh-hHC-IgG2.3G1 286Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser
Tyr Asp Gly Ser Arg Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Ser Leu Thr Met Val Arg Glu Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 180 185 190 Thr Gln
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205
Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys 210
215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330
335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 287444PRTArtificial
SequenceSynthetic OX40.16-Vh-hHC-IgG2.3G1 287Glu Val Gln Leu Val
Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala
Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ala Ile Asp Thr Asp Gly Gly Thr Phe Tyr Ala Asp Ser Val Arg
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Phe Cys Ala 85 90 95 Arg Leu Gly Glu Gly Tyr Phe Phe Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170
175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn
180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys
Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 260 265 270 Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295
300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420
425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
288444PRTArtificial SequenceSynthetic OX40.6-Vh-hHC-IgG2.3G1-V27
288Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Ile Gly Gly Asp Thr Phe
Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Ser Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Met Gly Thr
Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr
Val Glu Arg Lys Ser Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250
255 Thr Cys Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe
260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro 275 280 285 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375
380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 289442PRTArtificial SequenceSynthetic
OX40.8-Vh-hHC-IgG2.3G1-V27 289Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile
Ser Tyr Asp Gly Ser Arg Lys His Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Ser Leu Thr Met Val Arg Glu Trp Gly Gln Gly Thr
Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 180 185 190
Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195
200 205 Val Asp Lys Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro
Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Glu His Glu
Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315
320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420
425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
290444PRTArtificial SequenceSynthetic OX40.16-Vh-hHC-IgG2.3G1-V27
290Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Thr Asp Gly Gly Thr Phe
Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Leu Gly Glu
Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr
Val Glu Arg Lys Ser Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250
255 Thr Cys Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe
260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro 275 280 285 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375
380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 291443PRTArtificial SequenceSynthetic
OX40.6-Vh-hHC-IgG2.5 291Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser
Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Ile
Gly Gly Asp Thr Phe Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser Leu 65 70 75 80 Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Met Gly Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125 Ala Pro Ser Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr
Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro 210 215
220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val 290 295 300 Val His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys
Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340
345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 292441PRTArtificial
SequenceSynthetic OX40.8-Vh-hHC-IgG2.5 292Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Leu
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Leu Ile Ser Tyr Asp Gly Ser Arg Lys His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Ser Leu Thr Met Val Arg Glu Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 180
185 190 Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys 195 200 205 Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys 210 215 220 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser His
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 290 295 300
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305
310 315 320 Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
Gly Gln 325 330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425
430 Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 293443PRTArtificial
SequenceSynthetic OX40.16-Vh-hHC-IgG2.5 293Glu Val Gln Leu Val Gln
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met
Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Asp Thr Asp Gly Gly Thr Phe Tyr Ala Asp Ser Val Arg 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Phe Cys Ala 85 90 95 Arg Leu Gly Glu Gly Tyr Phe Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180
185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val
Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 290 295 300
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305
310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425
430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
294443PRTArtificial SequenceSynthetic OX40.21-Vh-hHC-IgG2.5 294Glu
Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ala Ile Asp Thr Asp Ala Gly Thr Phe Tyr Ala
Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Leu Gly Glu Gly Tyr
Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro
Ser Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Cys Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 260 265
270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn
Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390
395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435
440 295444PRTArtificial SequenceSynthetic OX40.21-Vh-hHC-IgG2.5G1
295Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ala Ile Asp Thr Asp Ala Gly Thr Phe
Tyr Ala Asp Ser Val Arg 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95 Arg Leu Gly Glu
Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Ser Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr
Val Glu Arg Lys Cys Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250
255 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro 275 280 285 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375
380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 296444PRTArtificial SequenceSynthetic
OX40.21-Vh-hHC-IgG2.5G1-V27 296Glu Val Gln Leu Val Gln Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Tyr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile
Asp Thr Asp Ala Gly Thr Phe Tyr Ala Asp Ser Val Arg 50 55 60 Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70
75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
Ala 85 90 95 Arg Leu Gly Glu Gly Tyr Phe Phe Asp Tyr Trp Gly Gln
Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180 185 190
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195
200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
Pro 210 215 220 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Glu
His Glu Asp Pro Glu Val Lys Phe 260 265 270 Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315
320 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415 Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
297327PRTArtificial SequenceSynthetic IgG2.3G1-V27 297Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Ser Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val
Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 145 150 155
160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 195 200 205 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280
285 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 305 310 315 320 Leu Ser Leu Ser Pro Gly Lys 325
298103PRTArtificial SequenceSynthetic Human IgG1 CH2 with
A330S/P331S 298Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile 1 5 10 15 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu 20 25 30 Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His 35 40 45 Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 50 55 60 Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 65 70 75 80 Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu 85 90 95 Lys
Thr Ile Ser Lys Ala Lys 100 299440PRTArtificial SequenceSynthetic
Heavy chain nivolumab 299Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala
Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210
215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330
335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser
Leu Ser Leu Gly Lys 435 440 300214PRTArtificial SequenceSynthetic
Light chain nivolumab 300Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser
Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210 301113PRTArtificial SequenceSynthetic
Heavy chain variable region nivolumab 301Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser 100 105 110 Ser 302107PRTArtificial
SequenceSynthetic Light chain variable region nivolumab 302Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 3035PRTArtificial SequenceSynthetic HCDR1
nivolumab 303Asn Ser Gly Met His 1 5 30417PRTArtificial
SequenceSynthetic HCDR2 nivolumab 304Val Ile Trp Tyr Asp Gly Ser
Lys Arg Tyr Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 3054PRTArtificial SequenceSynthetic HCDR3 nivolumab 305Asn
Asp Asp Tyr 1 30611PRTArtificial SequenceSynthetic LCDR1 nivolumab
306Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10
3077PRTArtificial SequenceSynthetic LCDR2 nivolumab 307Asp Ala Ser
Asn Arg Ala Thr 1 5 3089PRTArtificial SequenceSynthetic LCDR3
nivolumab 308Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5
309118PRTArtificial SequenceSynthetic Heavy chain variable region
ipilimumab (from WO01/014424) 309Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe
Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr
Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
310108PRTArtificial SequenceSynthetic Light chain variable region
ipilimumab (from WO01/014424) 310Glu Ile Val Leu Thr Gln Ser Pro
Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr
Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser
Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 3115PRTArtificial SequenceSynthetic HCDR1 ipilimumab (from
WO01/014424) 311Ser Tyr Thr Met His 1 5 31217PRTArtificial
SequenceSynthetic HCDR2 ipilimumab (from WO01/014424) 312Phe Ile
Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15
Gly 3139PRTArtificial SequenceSynthetic HCDR3 ipilimumab (from
WO01/014424) 313Thr Gly Trp Leu Gly Pro Phe Asp Tyr 1 5
31412PRTArtificial SequenceSynthetic LCDR1 ipilimumab (from
WO01/014424) 314Arg Ala Ser Gln Ser Val Gly Ser Ser Tyr Leu Ala 1 5
10 3157PRTArtificial SequenceSynthetic LCDR2 ipilimumab (from
WO01/014424) 315Gly Ala Phe Ser Arg Ala Thr 1 5 3169PRTArtificial
SequenceSynthetic LCDR3 ipilimumab (from WO01/014424) 316Gln Gln
Tyr Gly Ser Ser Pro Trp Thr 1 5 31716PRTArtificial
SequenceSynthetic HCDR2 of OX40.21 317Ala Ile Asp Thr Asp Ala Gly
Thr Phe Tyr Ala Asp Ser Val Arg Gly 1 5 10 15 318117PRTArtificial
SequenceSynthetic VH of OX40.21 318Glu Val Gln Leu Val Gln Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala
Ile Asp Thr Asp Ala Gly Thr Phe Tyr Ala Asp Ser Val Arg 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe
Cys Ala 85 90 95 Arg Leu Gly Glu Gly Tyr Phe Phe Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
* * * * *
References