U.S. patent application number 17/563502 was filed with the patent office on 2022-04-21 for anti-pd-l1 antibodies and uses thereof.
The applicant listed for this patent is I-Mab Biopharma US Limited. Invention is credited to Lei Fang, Bingshi Guo, Yongqiang Wang, Zhengyi Wang, Jingwu Zang.
Application Number | 20220119531 17/563502 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-21 |
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United States Patent
Application |
20220119531 |
Kind Code |
A1 |
Fang; Lei ; et al. |
April 21, 2022 |
ANTI-PD-L1 ANTIBODIES AND USES THEREOF
Abstract
Provided are anti-PD-L1 antibodies or fragments thereof. The
antibodies or fragments thereof specifically bind to the
immunoglobulin C domain of the PD-L1 protein. In various example,
the antibodies or fragments thereof include a VH CDR1 of SEQ ID NO:
1, a VH CDR2 of SEQ ID NO: 116, a VH CDR3 of SEQ ID NO: 117, a VL
CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of
SEQ ID NO: 6, or variants of each thereof. Methods of using the
antibodies or fragments thereof for treating and diagnosing
diseases such as cancer and infectious diseases are also
provided.
Inventors: |
Fang; Lei; (Shanghai,
CN) ; Wang; Yongqiang; (Shanghai, CN) ; Wang;
Zhengyi; (Shanghai, CN) ; Guo; Bingshi;
(Shanghai, CN) ; Zang; Jingwu; (Shanghai,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
I-Mab Biopharma US Limited |
Gaithersburg |
MD |
US |
|
|
Appl. No.: |
17/563502 |
Filed: |
December 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16610071 |
Oct 31, 2019 |
11220546 |
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PCT/CN2019/080458 |
Mar 29, 2019 |
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17563502 |
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International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2018 |
CN |
PCT/CN2018/081079 |
Claims
1. An antibody or fragment thereof, wherein the antibody or
fragment thereof has specificity to a human PD-L1 protein and
comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ
ID NO: 1 or a variant of SEQ ID NO: 1 having one, two or three
substitution, deletion or insertion as compared to SEQ ID NO: 1;
(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 116
or a variant of SEQ ID NO: 116 having one, two or three
substitution, deletion or insertion as compared to SEQ ID NO: 116,
wherein amino acid residue 12 of the VH CDR3 is Arg; (c) a VH CDR3
comprising the amino acid sequence of SEQ ID NO: 3 or a variant of
SEQ ID NO: 3 having one, two or three substitution, deletion or
insertion as compared to SEQ ID NO: 3; (d) a VL CDR1 comprising the
amino acid sequence of SEQ ID NO: 4 or a variant of SEQ ID NO: 4
having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 4; (e) a VL CDR2 comprising the amino acid
sequence of SEQ ID NO: 5 or a variant of SEQ ID NO: 5 having one,
two or three substitution, deletion or insertion as compared to SEQ
ID NO: 5; and (f) a VL CDR3 comprising the amino acid sequence of
SEQ ID NO: 6 or a variant of SEQ ID NO: 6 having one, two or three
substitution, deletion or insertion as compared to SEQ ID NO:
6.
2. The antibody or fragment thereof of claim 1, wherein the variant
of SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO:
61-67.
3. The antibody or fragment thereof of claim 1, wherein the variant
of SEQ ID NO: 116 is selected from the group consisting of SEQ ID
NO: 118-127.
4. The antibody or fragment thereof of claim 1, wherein the variant
of SEQ ID NO: 3 is selected from the group consisting of SEQ ID NO:
117 and 128-139.
5. The antibody or fragment thereof of claim 1, wherein the variant
of SEQ ID NO: 4 is selected from the group consisting of SEQ ID NO:
91-92.
6. The antibody or fragment thereof of claim 1, wherein the variant
of SEQ ID NO: 5 is selected from the group consisting of SEQ ID NO:
93-105.
7. The antibody or fragment thereof of claim 1, wherein the variant
of SEQ ID NO: 6 is selected from the group consisting of SEQ ID NO:
140 and 106-111.
8. The antibody or fragment thereof of claim 1, wherein the VH CDR1
comprises the amino acid sequence of SEQ ID NO: 1, the VH CDR2
comprises the amino acid sequence of SEQ ID NO: 116, the VH CDR3
comprises the amino acid sequence of SEQ ID NO: 3, the VL CDR1
comprises the amino acid sequence of SEQ ID NO: 4, the VL CDR2
comprises the amino acid sequence of SEQ ID NO: 5, and the VL CDR3
comprises the amino acid sequence of SEQ ID NO: 6.
9. The antibody or fragment thereof of claim 8, comprising a heavy
chain variable region comprising the amino acid sequence of SEQ IN
NO: 141 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 142.
10. The antibody or fragment thereof of claim 1, wherein amino acid
residues 4, 5 and 6 of the VL CDR3 are Ser, Asp and Ala,
respectively.
11. The antibody or fragment thereof of claim 10, wherein the VL
CDR3 comprises the amino acid sequence of SEQ ID NO: 140.
12. The antibody or fragment thereof of claim 11, wherein the VH
CDR1 comprises the amino acid sequence of SEQ ID NO: 1, the VH CDR2
comprises the amino acid sequence of SEQ ID NO: 116, the VH CDR3
comprises the amino acid sequence of SEQ ID NO: 3, the VL CDR1
comprises the amino acid sequence of SEQ ID NO: 4, the VL CDR2
comprises the amino acid sequence of SEQ ID NO: 5, and the VL CDR3
comprises the amino acid sequence of SEQ ID NO: 140.
13. The antibody or fragment thereof of claim 12, comprising a
heavy chain variable region comprising the amino acid sequence of
SEQ IN NO: 159 and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 160.
14. The antibody or fragment thereof of claim 1, wherein the second
amino acid residue of the VH CDR3 is Leu.
15. The antibody or fragment thereof of claim 14, wherein the VH
CDR3 comprises the amino acid sequence of SEQ ID NO: 117.
16. The antibody or fragment thereof of claim 15, wherein the VH
CDR1 comprises the amino acid sequence of SEQ ID NO: 1, the VH CDR2
comprises the amino acid sequence of SEQ ID NO: 116, the VH CDR3
comprises the amino acid sequence of SEQ ID NO: 117, the VL CDR1
comprises the amino acid sequence of SEQ ID NO: 4, the VL CDR2
comprises the amino acid sequence of SEQ ID NO: 5, and the VL CDR3
comprises the amino acid sequence of SEQ ID NO: 6.
17. The antibody or fragment thereof of claim 16, comprising a
heavy chain variable region comprising the amino acid sequence of
SEQ IN NO: 149 and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 150.
18. One or more polynucleotide encoding the antibody or fragment
thereof of claim 1.
19. A method of treating cancer or infection in a patient in need
thereof, comprising administering to the patient an effective
amount of the antibody or fragment thereof of claim 1.
20. The method of claim 19, which is for treating cancer
characterized with expression of PD-L1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 16/610,071, filed Oct. 31, 2019, which is the
U.S. national stage application of International Application
PCT/CN2019/080458, filed Mar. 29, 2019, which claims priority to
Chinese Patent Application PCT/CN2018/081079, filed Mar. 29, 2018,
the contents of each of which are incorporated herein by reference
in their entireties in the present disclosure.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 22, 2021, is named 54LW-269085-US2_ST25.txt and is 87,662
bytes in size.
BACKGROUND
[0003] Programmed death-ligand 1 (PD-L1), also known as cluster of
differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa
type 1 transmembrane protein believed to play a major role in
suppressing the immune system during particular events such as
pregnancy, tissue allografts, autoimmune disease and other disease
states such as hepatitis. The binding of PD-L1 to PD-1 or B7.1
transmits an inhibitory signal which reduces the proliferation of
CD8+ T cells at the lymph nodes and supplementary to that PD-1 is
also able to control the accumulation of foreign antigen specific T
cells in the lymph nodes through apoptosis which is further
mediated by a lower regulation of the gene Bcl-2.
[0004] It has been shown that upregulation of PD-L1 may allow
cancers to evade the host immune system. An analysis of tumor
specimens from patients with renal cell carcinoma found that high
tumor expression of PD-L1 was associated with increased tumor
aggressiveness and an increased risk of death. Many PD-L1
inhibitors are in development as immuno-oncology therapies and are
showing good results in clinical trials.
[0005] In addition to treatment of cancers, PD-L1 inhibition has
also shown promises in treating infectious diseases. In a mouse
model of intracellular infection, L. monocytogenes induced PD-L1
protein expression in T cells, NK cells, and macrophages. PD-L1
blockade (e.g., using blocking antibodies) resulted in increased
mortality for infected mice. Blockade reduced TNF.alpha. and nitric
oxide production by macrophages, reduced granzyme B production by
NK cells, and decreased proliferation of L. monocytogenes
antigen-specific CD8 T cells (but not CD4 T cells). This evidence
suggests that PD-L1 acts as a positive costimulatory molecule in
intracellular infection.
SUMMARY
[0006] The present disclosure provides anti-PD-L1 antibodies having
high binding affinity to human PD-L1 proteins and can effectively
block the interaction between PD-L1 and its receptor PD-1. Also
importantly, the examples demonstrate that these anti-PD-L1
antibodies promote T cell immune response and inhibit tumor growth.
Different from known anti-PD-L1 antibodies that bind to the
immunoglobulin V domain of the extracellular portion of the PD-L1
protein, these antibodies bind to the immunoglobulin C domain, in
particular amino acid residues Y134, K162, and N183. These
anti-PD-L1 antibodies are useful for therapeutic purposes such as
treating various types of cancer, as well as infections, and can
also be used for diagnostic and prognostic purposes.
[0007] One embodiment of the present disclosure provides an
anti-PD-L1 antibody or fragment thereof, which antibody or fragment
thereof can specifically bind to an immunoglobulin C (Ig C) domain
of a human Programmed death-ligand 1 (PD-L1) protein. In some
embodiments, the Ig C domain consists of amino acid residues
133-225. In some embodiments, the antibody or fragment thereof can
bind to at least one of amino acid residues Y134, K162, or N183 of
the PD-L1 protein. In some embodiments, the antibody or fragment
thereof can bind to at least one of amino acid residues Y134, K162,
and N183 of the PD-L1 protein. In some embodiments, the antibody or
fragment thereof does not bind to an immunoglobulin V (Ig V) domain
of the PD-L1 protein, wherein the Ig V domain consists of amino
acid residues 19-127.
[0008] One embodiment of the present disclosure provides an
anti-PD-L1 antibody or fragment thereof, wherein the antibody or
fragment thereof has specificity to a human Programmed death-ligand
1 (PD-L1) protein and comprises a VH CDR1 of SEQ ID NO: 1, a VH
CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ
ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6.
In some embodiments, the antibody or fragment thereof further
comprises a heavy chain constant region, a light chain constant
region, an Fc region, or the combination thereof. In some
embodiments, the light chain constant region is a kappa or lambda
chain constant region. In some embodiments, the antibody or
fragment thereof is of an isotype of IgG, IgM, IgA, IgE or IgD. In
some embodiments, the isotype is IgG1, IgG2, IgG3 or IgG4. Without
limitation, the antibody or fragment thereof is a chimeric
antibody, a humanized antibody, or a fully human antibody. In one
aspect, antibody or fragment thereof is a humanized antibody.
[0009] Through mutagenesis, the present disclosure has further
identified mutation hotspot residues in the VH CDR3 (see, e.g.,
antibodies A1, A2, C3, C4, C6, B1 and B6 in Examples 13-17) and VL
CDR3 (see, e.g., antibodies B3, C4 and A3 in Examples 13-17).
Therefore, the present disclosure also provides antibodies that
incorporate one or more of mutations at these hotspots.
[0010] In some embodiments, provided is an antibody or fragment
thereof, wherein the antibody or fragment thereof has specificity
to a human PD-L1 protein and comprises (a) a VH CDR1 comprising the
amino acid sequence of SEQ ID NO: 1 or a variant of SEQ ID NO: 1
having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 1; (b) a VH CDR2 comprising the amino acid
sequence of SEQ ID NO: 116 or a variant of SEQ ID NO: 116 having
one, two or three substitution, deletion or insertion as compared
to SEQ ID NO: 116; (c) a VH CDR3 comprising the amino acid sequence
of SEQ ID NO: 117 or a variant of SEQ ID NO: 117 having one, two or
three substitution, deletion or insertion as compared to SEQ ID NO:
117, wherein the second amino acid residue of the VH CDR3 is Leu;
(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or
a variant of SEQ ID NO: 4 having one, two or three substitution,
deletion or insertion as compared to SEQ ID NO: 4; (e) a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 5 or a variant of
SEQ ID NO: 5 having one, two or three substitution, deletion or
insertion as compared to SEQ ID NO: 5; and (f) a VL CDR3 comprising
the amino acid sequence of SEQ ID NO: 6 or a variant of SEQ ID NO:
6 having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 6.
[0011] In one embodiment, the VH CDR1 comprises the amino acid
sequence of SEQ ID NO: 1, the VH CDR2 comprises the amino acid
sequence of SEQ ID NO: 116, the VH CDR3 comprises the amino acid
sequence of SEQ ID NO: 117, the VL CDR1 comprises the amino acid
sequence of SEQ ID NO: 4, the VL CDR2 comprises the amino acid
sequence of SEQ ID NO: 5, and the VL CDR3 comprises the amino acid
sequence of SEQ ID NO: 6.
[0012] In one embodiment, the antibody or fragment thereof
comprises a heavy chain variable region comprising the amino acid
sequence of SEQ IN NO: 149 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 150.
[0013] Also provided, in one embodiment, is an antibody or fragment
thereof, wherein the antibody or fragment thereof has specificity
to a human PD-L1 protein and comprises: (a) a VH CDR1 comprising
the amino acid sequence of SEQ ID NO: 1 or a variant of SEQ ID NO:
1 having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 1; (b) a VH CDR2 comprising the amino acid
sequence of SEQ ID NO: 116 or a variant of SEQ ID NO: 116 having
one, two or three substitution, deletion or insertion as compared
to SEQ ID NO: 116; (c) a VH CDR3 comprising the amino acid sequence
of SEQ ID NO: 3 or a variant of SEQ ID NO: 3 having one, two or
three substitution, deletion or insertion as compared to SEQ ID NO:
3; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4
or a variant of SEQ ID NO: 4 having one, two or three substitution,
deletion or insertion as compared to SEQ ID NO: 4; (e) a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 5 or a variant of
SEQ ID NO: 5 having one, two or three substitution, deletion or
insertion as compared to SEQ ID NO: 5; and (f) a VL CDR3 comprising
the amino acid sequence of SEQ ID NO: 140 or a variant of SEQ ID
NO: 140 having one, two or three substitution, deletion or
insertion as compared to SEQ ID NO: 140, wherein at least (i) amino
acid residue 4 of the VL CDR3 is Ser, (ii) amino acid residue 5 of
the VL CDR3 is Asp, or (iii) amino acid residue 6 of the VL CDR3 is
Ala.
[0014] In one embodiment, the VH CDR1 comprises the amino acid
sequence of SEQ ID NO: 1, the VH CDR2 comprises the amino acid
sequence of SEQ ID NO: 116, the VH CDR3 comprises the amino acid
sequence of SEQ ID NO: 3, the VL CDR1 comprises the amino acid
sequence of SEQ ID NO: 4, the VL CDR2 comprises the amino acid
sequence of SEQ ID NO: 5, and the VL CDR3 comprises the amino acid
sequence of SEQ ID NO: 140.
[0015] In one embodiment, the antibody or fragment thereof
comprises a heavy chain variable region comprising the amino acid
sequence of SEQ IN NO: 159 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 160.
[0016] One embodiment provides an antibody or fragment thereof,
wherein the antibody or fragment thereof has specificity to a human
PD-L1 protein and comprises: (a) a VH CDR1 comprising the amino
acid sequence of SEQ ID NO: 1 or a variant of SEQ ID NO: 1 having
one, two or three substitution, deletion or insertion as compared
to SEQ ID NO: 1; (b) a VH CDR2 comprising the amino acid sequence
of SEQ ID NO: 116 or a variant of SEQ ID NO: 116 having one, two or
three substitution, deletion or insertion as compared to SEQ ID NO:
116; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:
3 or a variant of SEQ ID NO: 3 having one, two or three
substitution, deletion or insertion as compared to SEQ ID NO: 3;
(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or
a variant of SEQ ID NO: 4 having one, two or three substitution,
deletion or insertion as compared to SEQ ID NO: 4; (e) a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 5 or a variant of
SEQ ID NO: 5 having one, two or three substitution, deletion or
insertion as compared to SEQ ID NO: 5; and (f) a VL CDR3 comprising
the amino acid sequence of SEQ ID NO: 6 or a variant of SEQ ID NO:
6 having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 6.
[0017] In some embodiments, the VH CDR1 comprises the amino acid
sequence of SEQ ID NO: 1, the VH CDR2 comprises the amino acid
sequence of SEQ ID NO: 116, the VH CDR3 comprises the amino acid
sequence of SEQ ID NO: 3, the VL CDR1 comprises the amino acid
sequence of SEQ ID NO: 4, the VL CDR2 comprises the amino acid
sequence of SEQ ID NO: 5, and the VL CDR3 comprises the amino acid
sequence of SEQ ID NO: 6.
[0018] In some embodiments, the antibody or fragment thereof
comprises a heavy chain variable region comprising the amino acid
sequence of SEQ IN NO: 141 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 142.
[0019] Also provided, in some embodiments, is a composition
comprising the antibody or fragment thereof of the present
disclosure and a pharmaceutically acceptable carrier. Still also
provided, in some embodiments, is an isolated cell comprising one
or more polynucleotide encoding the antibody or fragment thereof of
the present disclosure.
[0020] Treatment methods and uses are also provided. In one
embodiment, a method of treating cancer or infection in a patient
in need thereof is provided, comprising administering to the
patient an effective amount of the antibody or fragment thereof of
the present disclosure. In some embodiments, the cancer is a solid
tumor. In some embodiments, the cancer is selected from the group
consisting of bladder cancer, liver cancer, colon cancer, rectal
cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer,
small cell lung cancer, non-small cell lung cancer, breast cancer,
urethral cancer, head and neck cancer, gastrointestinal cancer,
stomach cancer, oesophageal cancer, ovarian cancer, renal cancer,
melanoma, prostate cancer and thyroid cancer. In some embodiments,
the cancer is selected from the group consisting of bladder cancer,
liver cancer, pancreatic cancer, non-small cell lung cancer, breast
cancer, urethral cancer, colorectal cancer, head and neck cancer,
squamous cell cancer, Merkel cell carcinoma, gastrointestinal
cancer, stomach cancer, oesophageal cancer, ovarian cancer, renal
cancer, and small cell lung cancer. In some embodiments, the method
further comprises administering to the patient a second cancer
therapeutic agent. In some embodiments, the infection is viral
infection, bacterial infection, fungal infection or infection by a
parasite.
[0021] In another embodiment, a method of treating cancer or
infection in a patient in need thereof is provided, comprising: (a)
treating a cell, in vitro, with the antibody or fragment thereof of
the present disclosure; and (b) administering the treated cell to
the patient. In some embodiments, the method further comprises,
prior to step (a), isolating the cell from an individual. In some
embodiments, the cell is isolated from the patient. In some
embodiments, the cell is isolated from a donor individual different
from the patient. In some embodiments, the cell is a T cell,
non-limiting examples of which include a tumor-infiltrating T
lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination
thereof.
[0022] Diagnostic methods and uses are also provided. In one
embodiment, a method of detecting expression of PD-L1 in a sample
is provided, comprising contacting the sample with an antibody or
fragment thereof under conditions for the antibody or fragment
thereof to bind to the PD-L1, and detecting the binding which
indicates expression of PD-L1 in the sample. In some embodiments,
the sample comprises a tumor cell, a tumor tissue, an infected
tissue, or a blood sample.
[0023] Antibodies and fragment of the present disclosure can be
used to prepare bispecific antibodies. In one embodiment, a
bispecific antibody is provided, comprising a fragment of the
present disclosure and a second antigen-binding fragment having
specificity to a molecular on an immune cell. In some embodiments,
the molecule is selected from the group consisting of PD-1, CTLA-4,
LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT,
ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or
BTLA, CD47 and CD73. In some embodiments, the fragment and the
second fragment each is independently selected from a Fab fragment,
a single-chain variable fragment (scFv), or a single-domain
antibody. In some embodiments, the bispecific antibody further
comprises a Fc fragment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows that HL1210-3 can bind to human PD-L1 with high
affinity.
[0025] FIG. 2 shows that HL1210-3 can efficiently inhibit the
binding of human PD-L1 to human PD1.
[0026] FIG. 3 shows the HL1210-3 antibody can highly efficiently
inhibit the binding of PD-1 on PD-L1 expressed on mammalian
cells.
[0027] FIG. 4 shows that the tested anti-PD-L1 antibodies can
promote human T cell response.
[0028] FIG. 5 shows the binding kinetics of HL1210-3 to recombinant
PD-L1.
[0029] FIG. 6 shows that all tested humanized antibodies had
comparable binding efficacy to human PD-L1 in contact to chimeric
antibody.
[0030] FIG. 7 shows that all tested humanized antibodies can high
efficiently bind to PD-L1 expressed on mammalian cells, comparable
with chimeric antibody.
[0031] FIG. 8 shows that humanized antibody Hu1210-41 can bind to
rhesus PD-L1 with lower affinity and cannot bind to rat and mouse
PD-L1.
[0032] FIG. 9 shows that Hu1210-41 antibody can only specifically
binding to B7-H1 (PD-L1), not B7-DC, B7-1, B7-2, B7-H2, PD-1, CD28,
CTLA4, ICOS and BTLA.
[0033] FIG. 10 shows that Hu1210-41 can efficiently inhibit the
binding of human PD-L1 to human PD1 and B7-1.
[0034] FIG. 11 shows that Hu1210-41 can efficiently inhibit the
binding of human PD-L1 to human PD1 and B7-1.
[0035] FIG. 12 shows that the Hu1210-8, Hu1210-9, Hu1210-16,
Hu1210-17, Hu1210-21 and Hu1210-36 humanized antibodies can dose
dependently promote the IFN.gamma. and IL-2 production in mix
lymphocyte reaction.
[0036] FIG. 13 shows that the Hu1210-40, Hu1210-41 and Hu1210-17
humanized antibodies can dose dependently promote the IFN.gamma.
production in CMV recall assay.
[0037] FIG. 14 shows that Hu1210-31 can inhibit the tumor growth by
30% at 5 mg/kg in HCC827-NSG-xenograft model.
[0038] FIG. 15 shows that Hu1210-41 antibody can dose-dependently
inhibit the tumor growth in HCC827-NSG-xenograft model, while the
tumor weight was also dose-dependently suppressed by Hu1210-41
antibody.
[0039] FIG. 16 plots, for each PD-L1 mutant, the mean binding value
as a function of expression (control anti-PD-L1 mAb
reactivity).
[0040] FIG. 17 illustrates the locations of Y134, K162, and N183,
the residues (spheres) involved in binding to the anti-PD-L1
Hu1210-41 antibody.
[0041] FIG. 18 compares the S60R mutant to parental antibody
Hu1210-41 in terms of binding efficiency to PD-L1 expressed on
mammalian cells.
[0042] FIG. 19 shows the results of a binding assay (to human
PD-L1) for the derived antibodies.
[0043] FIG. 20 shows that antibody B6 more highly efficiently bound
to PD-L1 expressed on mammalian cells, as compared to the parental
antibody and Tecentriq.TM. (atezolizumab).
[0044] FIG. 21 shows the antibodies' effect on IL2 production in
Jurkat cells in which B6 also exhibited higher potency.
[0045] FIG. 22 shows the antibodies' in vitro activity to promote
IFN.gamma. production in a mixed lymphocyte setting.
[0046] FIG. 23 shows the antibodies' in vivo activity to inhibit
tumor growth.
DETAILED DESCRIPTION
Definitions
[0047] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity; for example, "an antibody," is
understood to represent one or more antibodies. As such, the terms
"a" (or "an"), "one or more," and "at least one" can be used
interchangeably herein.
[0048] As used herein, the term "polypeptide" is intended to
encompass a singular "polypeptide" as well as plural
"polypeptides," and refers to a molecule composed of monomers
(amino acids) linearly linked by amide bonds (also known as peptide
bonds). The term "polypeptide" refers to any chain or chains of two
or more amino acids, and does not refer to a specific length of the
product. Thus, peptides, dipeptides, tripeptides, oligopeptides,
"protein," "amino acid chain," or any other term used to refer to a
chain or chains of two or more amino acids, are included within the
definition of "polypeptide," and the term "polypeptide" may be used
instead of, or interchangeably with any of these terms. The term
"polypeptide" is also intended to refer to the products of
post-expression modifications of the polypeptide, including without
limitation glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, or modification by non-naturally occurring amino acids. A
polypeptide may be derived from a natural biological source or
produced by recombinant technology, but is not necessarily
translated from a designated nucleic acid sequence. It may be
generated in any manner, including by chemical synthesis.
[0049] The term "isolated" as used herein with respect to cells,
nucleic acids, such as DNA or RNA, refers to molecules separated
from other DNAs or RNAs, respectively, that are present in the
natural source of the macromolecule. The term "isolated" as used
herein also refers to a nucleic acid or peptide that is
substantially free of cellular material, viral material, or culture
medium when produced by recombinant DNA techniques, or chemical
precursors or other chemicals when chemically synthesized.
Moreover, an "isolated nucleic acid" is meant to include nucleic
acid fragments which are not naturally occurring as fragments and
would not be found in the natural state. The term "isolated" is
also used herein to refer to cells or polypeptides which are
isolated from other cellular proteins or tissues. Isolated
polypeptides is meant to encompass both purified and recombinant
polypeptides.
[0050] As used herein, the term "recombinant" as it pertains to
polypeptides or polynucleotides intends a form of the polypeptide
or polynucleotide that does not exist naturally, a non-limiting
example of which can be created by combining polynucleotides or
polypeptides that would not normally occur together.
[0051] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are homologous at that position. A
degree of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences. An
"unrelated" or "non-homologous" sequence shares less than 40%
identity, though preferably less than 25% identity, with one of the
sequences of the present disclosure.
[0052] A polynucleotide or polynucleotide region (or a polypeptide
or polypeptide region) has a certain percentage (for example, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence
identity" to another sequence means that, when aligned, that
percentage of bases (or amino acids) are the same in comparing the
two sequences. This alignment and the percent homology or sequence
identity can be determined using software programs known in the
art, for example those described in Ausubel et al. eds. (2007)
Current Protocols in Molecular Biology. Preferably, default
parameters are used for alignment. One alignment program is BLAST,
using default parameters. In particular, programs are BLASTN and
BLASTP, using the following default parameters: Genetic
code=standard; filter=none; strand=both; cutoff=60; expect=10;
Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR. Biologically equivalent
polynucleotides are those having the above-noted specified percent
homology and encoding a polypeptide having the same or similar
biological activity.
[0053] The term "an equivalent nucleic acid or polynucleotide"
refers to a nucleic acid having a nucleotide sequence having a
certain degree of homology, or sequence identity, with the
nucleotide sequence of the nucleic acid or complement thereof. A
homolog of a double stranded nucleic acid is intended to include
nucleic acids having a nucleotide sequence which has a certain
degree of homology with or with the complement thereof. In one
aspect, homologs of nucleic acids are capable of hybridizing to the
nucleic acid or complement thereof. Likewise, "an equivalent
polypeptide" refers to a polypeptide having a certain degree of
homology, or sequence identity, with the amino acid sequence of a
reference polypeptide. In some aspects, the sequence identity is at
least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some
aspects, the equivalent polypeptide or polynucleotide has one, two,
three, four or five addition, deletion, substitution and their
combinations thereof as compared to the reference polypeptide or
polynucleotide. In some aspects, the equivalent sequence retains
the activity (e.g., epitope-binding) or structure (e.g.,
salt-bridge) of the reference sequence.
[0054] Hybridization reactions can be performed under conditions of
different "stringency". In general, a low stringency hybridization
reaction is carried out at about 40.degree. C. in about
10.times.SSC or a solution of equivalent ionic
strength/temperature. A moderate stringency hybridization is
typically performed at about 50.degree. C. in about 6.times.SSC,
and a high stringency hybridization reaction is generally performed
at about 60.degree. C. in about 1.times.SSC. Hybridization
reactions can also be performed under "physiological conditions"
which is well known to one of skill in the art. A non-limiting
example of a physiological condition is the temperature, ionic
strength, pH and concentration of Mg.sup.2+ normally found in a
cell.
[0055] A polynucleotide is composed of a specific sequence of four
nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine
(T); and uracil (U) for thymine when the polynucleotide is RNA.
Thus, the term "polynucleotide sequence" is the alphabetical
representation of a polynucleotide molecule. This alphabetical
representation can be input into databases in a computer having a
central processing unit and used for bioinformatics applications
such as functional genomics and homology searching. The term
"polymorphism" refers to the coexistence of more than one form of a
gene or portion thereof. A portion of a gene of which there are at
least two different forms, i.e., two different nucleotide
sequences, is referred to as a "polymorphic region of a gene". A
polymorphic region can be a single nucleotide, the identity of
which differs in different alleles.
[0056] The terms "polynucleotide" and "oligonucleotide" are used
interchangeably and refer to a polymeric form of nucleotides of any
length, either deoxyribonucleotides or ribonucleotides or analogs
thereof. Polynucleotides can have any three-dimensional structure
and may perform any function, known or unknown. The following are
non-limiting examples of polynucleotides: a gene or gene fragment
(for example, a probe, primer, EST or SAGE tag), exons, introns,
messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,
dsRNA, siRNA, miRNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, nucleic acid probes and primers. A
polynucleotide can comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure can be imparted before or
after assembly of the polynucleotide. The sequence of nucleotides
can be interrupted by non-nucleotide components. A polynucleotide
can be further modified after polymerization, such as by
conjugation with a labeling component. The term also refers to both
double- and single-stranded molecules. Unless otherwise specified
or required, any embodiment of this disclosure that is a
polynucleotide encompasses both the double-stranded form and each
of two complementary single-stranded forms known or predicted to
make up the double-stranded form.
[0057] The term "encode" as it is applied to polynucleotides refers
to a polynucleotide which is said to "encode" a polypeptide if, in
its native state or when manipulated by methods well known to those
skilled in the art, it can be transcribed and/or translated to
produce the mRNA for the polypeptide and/or a fragment thereof. The
antisense strand is the complement of such a nucleic acid, and the
encoding sequence can be deduced therefrom.
[0058] As used herein, an "antibody" or "antigen-binding
polypeptide" refers to a polypeptide or a polypeptide complex that
specifically recognizes and binds to an antigen. An antibody can be
a whole antibody and any antigen binding fragment or a single chain
thereof. Thus the term "antibody" includes any protein or peptide
containing molecule that comprises at least a portion of an
immunoglobulin molecule having biological activity of binding to
the antigen. Examples of such include, but are not limited to a
complementarity determining region (CDR) of a heavy or light chain
or a ligand binding portion thereof, a heavy chain or light chain
variable region, a heavy chain or light chain constant region, a
framework (FR) region, or any portion thereof, or at least one
portion of a binding protein.
[0059] The terms "antibody fragment" or "antigen-binding fragment",
as used herein, is a portion of an antibody such as F(ab').sub.2,
F(ab).sub.2, Fab', Fab, Fv, scFv and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. The term "antibody fragment"
includes aptamers, spiegelmers, and diabodies. The term "antibody
fragment" also includes any synthetic or genetically engineered
protein that acts like an antibody by binding to a specific antigen
to form a complex.
[0060] A "single-chain variable fragment" or "scFv" refers to a
fusion protein of the variable regions of the heavy (V.sub.H) and
light chains (V.sub.L) of immunoglobulins. In some aspects, the
regions are connected with a short linker peptide of ten to about
25 amino acids. The linker can be rich in glycine for flexibility,
as well as serine or threonine for solubility, and can either
connect the N-terminus of the V.sub.H with the C-terminus of the
V.sub.L, or vice versa. This protein retains the specificity of the
original immunoglobulin, despite removal of the constant regions
and the introduction of the linker. ScFv molecules are known in the
art and are described, e.g., in U.S. Pat. No. 5,892,019.
[0061] The term antibody encompasses various broad classes of
polypeptides that can be distinguished biochemically. Those skilled
in the art will appreciate that heavy chains are classified as
gamma, mu, alpha, delta, or epsilon (.gamma., .mu., .alpha.,
.delta., .epsilon.) with some subclasses among them (e.g.,
.gamma.1-.gamma.4). It is the nature of this chain that determines
the "class" of the antibody as IgG, IgM, IgA IgG, or IgE,
respectively. The immunoglobulin subclasses (isotypes) e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG4, IgG5, etc. are well
characterized and are known to confer functional specialization.
Modified versions of each of these classes and isotypes are readily
discernable to the skilled artisan in view of the instant
disclosure and, accordingly, are within the scope of the instant
disclosure. All immunoglobulin classes are clearly within the scope
of the present disclosure, the following discussion will generally
be directed to the IgG class of immunoglobulin molecules. With
regard to IgG, a standard immunoglobulin molecule comprises two
identical light chain polypeptides of molecular weight
approximately 23,000 Daltons, and two identical heavy chain
polypeptides of molecular weight 53,000-70,000. The four chains are
typically joined by disulfide bonds in a "Y" configuration wherein
the light chains bracket the heavy chains starting at the mouth of
the "Y" and continuing through the variable region.
[0062] Antibodies, antigen-binding polypeptides, variants, or
derivatives thereof of the disclosure include, but are not limited
to, polyclonal, monoclonal, multispecific, human, humanized,
primatized, or chimeric antibodies, single chain antibodies,
epitope-binding fragments, e.g., Fab, Fab' and F(ab').sub.2, Fd,
Fvs, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH
domain, fragments produced by a Fab expression library, and
anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to LIGHT antibodies disclosed herein). Immunoglobulin or
antibody molecules of the disclosure can be of any type (e.g., IgG,
IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2) or subclass of immunoglobulin molecule.
[0063] Light chains are classified as either kappa or lambda (K,
.delta.). Each heavy chain class may be bound with either a kappa
or lambda light chain. In general, the light and heavy chains are
covalently bonded to each other, and the "tail" portions of the two
heavy chains are bonded to each other by covalent disulfide
linkages or non-covalent linkages when the immunoglobulins are
generated either by hybridomas, B cells or genetically engineered
host cells. In the heavy chain, the amino acid sequences run from
an N-terminus at the forked ends of the Y configuration to the
C-terminus at the bottom of each chain.
[0064] Both the light and heavy chains are divided into regions of
structural and functional homology. The terms "constant" and
"variable" are used functionally. In this regard, it will be
appreciated that the variable domains of both the light (VK) and
heavy (VH) chain portions determine antigen recognition and
specificity. Conversely, the constant domains of the light chain
(CK) and the heavy chain (CH1, CH2 or CH3) confer important
biological properties such as secretion, transplacental mobility,
Fc receptor binding, complement binding, and the like. By
convention the numbering of the constant region domains increases
as they become more distal from the antigen-binding site or
amino-terminus of the antibody. The N-terminal portion is a
variable region and at the C-terminal portion is a constant region;
the CH3 and CK domains actually comprise the carboxy-terminus of
the heavy and light chain, respectively.
[0065] As indicated above, the variable region allows the antibody
to selectively recognize and specifically bind epitopes on
antigens. That is, the VK domain and VH domain, or subset of the
complementarity determining regions (CDRs), of an antibody combine
to form the variable region that defines a three dimensional
antigen-binding site. This quaternary antibody structure forms the
antigen-binding site present at the end of each arm of the Y. More
specifically, the antigen-binding site is defined by three CDRs on
each of the VH and VK chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1,
CDR-L2 and CDR-L3). In some instances, e.g., certain immunoglobulin
molecules derived from camelid species or engineered based on
camelid immunoglobulins, a complete immunoglobulin molecule may
consist of heavy chains only, with no light chains. See, e.g.,
Hamers-Casterman et al., Nature 363:446-448 (1993).
[0066] In naturally occurring antibodies, the six "complementarity
determining regions" or "CDRs" present in each antigen-binding
domain are short, non-contiguous sequences of amino acids that are
specifically positioned to form the antigen-binding domain as the
antibody assumes its three dimensional configuration in an aqueous
environment. The remainder of the amino acids in the
antigen-binding domains, referred to as "framework" regions, show
less inter-molecular variability. The framework regions largely
adopt a .beta.-sheet conformation and the CDRs form loops which
connect, and in some cases form part of, the .beta.-sheet
structure. Thus, framework regions act to form a scaffold that
provides for positioning the CDRs in correct orientation by
inter-chain, non-covalent interactions. The antigen-binding domain
formed by the positioned CDRs defines a surface complementary to
the epitope on the immunoreactive antigen. This complementary
surface promotes the non-covalent binding of the antibody to its
cognate epitope. The amino acids comprising the CDRs and the
framework regions, respectively, can be readily identified for any
given heavy or light chain variable region by one of ordinary skill
in the art, since they have been precisely defined (see "Sequences
of Proteins of Immunological Interest," Kabat, E., et al., U.S.
Department of Health and Human Services, (1983); and Chothia and
Lesk, J. Mol. Biol., 196:901-917 (1987)).
[0067] In the case where there are two or more definitions of a
term which is used and/or accepted within the art, the definition
of the term as used herein is intended to include all such meanings
unless explicitly stated to the contrary. A specific example is the
use of the term "complementarity determining region" ("CDR") to
describe the non-contiguous antigen combining sites found within
the variable region of both heavy and light chain polypeptides.
This particular region has been described by Kabat et al., U.S.
Dept. of Health and Human Services, "Sequences of Proteins of
Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol.
196:901-917 (1987), which are incorporated herein by reference in
their entireties. The CDR definitions according to Kabat and
Chothia include overlapping or subsets of amino acid residues when
compared against each other. Nevertheless, application of either
definition to refer to a CDR of an antibody or variants thereof is
intended to be within the scope of the term as defined and used
herein. The appropriate amino acid residues which encompass the
CDRs as defined by each of the above cited references are set forth
in the table below as a comparison. The exact residue numbers which
encompass a particular CDR will vary depending on the sequence and
size of the CDR. Those skilled in the art can routinely determine
which residues comprise a particular CDR given the variable region
amino acid sequence of the antibody.
TABLE-US-00001 Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58
CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3
89-97 91-96
[0068] Kabat et al. also defined a numbering system for variable
domain sequences that is applicable to any antibody. One of
ordinary skill in the art can unambiguously assign this system of
"Kabat numbering" to any variable domain sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983).
[0069] In addition to table above, the Kabat number system
describes the CDR regions as follows: CDR-H1 begins at
approximately amino acid 31 (i.e., approximately 9 residues after
the first cysteine residue), includes approximately 5-7 amino
acids, and ends at the next tryptophan residue. CDR-H2 begins at
the fifteenth residue after the end of CDR-H1, includes
approximately 16-19 amino acids, and ends at the next arginine or
lysine residue. CDR-H3 begins at approximately the thirty third
amino acid residue after the end of CDR-H2; includes 3-25 amino
acids; and ends at the sequence W-G-X-G, where X is any amino acid.
CDR-L1 begins at approximately residue 24 (i.e., following a
cysteine residue); includes approximately 10-17 residues; and ends
at the next tryptophan residue. CDR-L2 begins at approximately the
sixteenth residue after the end of CDR-L1 and includes
approximately 7 residues. CDR-L3 begins at approximately the thirty
third residue after the end of CDR-L2 (i.e., following a cysteine
residue); includes approximately 7-11 residues and ends at the
sequence F or W-G-X-G, where X is any amino acid.
[0070] Antibodies disclosed herein may be from any animal origin
including birds and mammals. Preferably, the antibodies are human,
murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or
chicken antibodies. In another embodiment, the variable region may
be condricthoid in origin (e.g., from sharks).
[0071] As used herein, the term "heavy chain constant region"
includes amino acid sequences derived from an immunoglobulin heavy
chain. A polypeptide comprising a heavy chain constant region
comprises at least one of: a CH1 domain, a hinge (e.g., upper,
middle, and/or lower hinge region) domain, a CH2 domain, a CH3
domain, or a variant or fragment thereof. For example, an
antigen-binding polypeptide for use in the disclosure may comprise
a polypeptide chain comprising a CH1 domain; a polypeptide chain
comprising a CH1 domain, at least a portion of a hinge domain, and
a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3
domain; a polypeptide chain comprising a CH1 domain, at least a
portion of a hinge domain, and a CH3 domain, or a polypeptide chain
comprising a CH1 domain, at least a portion of a hinge domain, a
CH2 domain, and a CH3 domain. In another embodiment, a polypeptide
of the disclosure comprises a polypeptide chain comprising a CH3
domain. Further, an antibody for use in the disclosure may lack at
least a portion of a CH2 domain (e.g., all or part of a CH2
domain). As set forth above, it will be understood by one of
ordinary skill in the art that the heavy chain constant region may
be modified such that they vary in amino acid sequence from the
naturally occurring immunoglobulin molecule.
[0072] The heavy chain constant region of an antibody disclosed
herein may be derived from different immunoglobulin molecules. For
example, a heavy chain constant region of a polypeptide may
comprise a CH1 domain derived from an IgG1 molecule and a hinge
region derived from an IgG3 molecule. In another example, a heavy
chain constant region can comprise a hinge region derived, in part,
from an IgG1 molecule and, in part, from an IgG3 molecule. In
another example, a heavy chain portion can comprise a chimeric
hinge derived, in part, from an IgG1 molecule and, in part, from an
IgG4 molecule.
[0073] As used herein, the term "light chain constant region"
includes amino acid sequences derived from antibody light chain.
Preferably, the light chain constant region comprises at least one
of a constant kappa domain or constant lambda domain.
[0074] A "light chain-heavy chain pair" refers to the collection of
a light chain and heavy chain that can form a dimer through a
disulfide bond between the CL domain of the light chain and the CH1
domain of the heavy chain.
[0075] As previously indicated, the subunit structures and three
dimensional configuration of the constant regions of the various
immunoglobulin classes are well known. As used herein, the term "VH
domain" includes the amino terminal variable domain of an
immunoglobulin heavy chain and the term "CH1 domain" includes the
first (most amino terminal) constant region domain of an
immunoglobulin heavy chain. The CH1 domain is adjacent to the VH
domain and is amino terminal to the hinge region of an
immunoglobulin heavy chain molecule.
[0076] As used herein the term "CH2 domain" includes the portion of
a heavy chain molecule that extends, e.g., from about residue 244
to residue 360 of an antibody using conventional numbering schemes
(residues 244 to 360, Kabat numbering system; and residues 231-340,
EU numbering system; see Kabat et al., U.S. Dept. of Health and
Human Services, "Sequences of Proteins of Immunological Interest"
(1983). The CH2 domain is unique in that it is not closely paired
with another domain. Rather, two N-linked branched carbohydrate
chains are interposed between the two CH2 domains of an intact
native IgG molecule. It is also well documented that the CH3 domain
extends from the CH2 domain to the C-terminal of the IgG molecule
and comprises approximately 108 residues.
[0077] As used herein, the term "hinge region" includes the portion
of a heavy chain molecule that joins the CH1 domain to the CH2
domain. This hinge region comprises approximately 25 residues and
is flexible, thus allowing the two N-terminal antigen-binding
regions to move independently. Hinge regions can be subdivided into
three distinct domains: upper, middle, and lower hinge domains
(Roux et al., J. Immunol 161:4083 (1998)).
[0078] As used herein the term "disulfide bond" includes the
covalent bond formed between two sulfur atoms. The amino acid
cysteine comprises a thiol group that can form a disulfide bond or
bridge with a second thiol group. In most naturally occurring IgG
molecules, the CH1 and CK regions are linked by a disulfide bond
and the two heavy chains are linked by two disulfide bonds at
positions corresponding to 239 and 242 using the Kabat numbering
system (position 226 or 229, EU numbering system).
[0079] As used herein, the term "chimeric antibody" will be held to
mean any antibody wherein the immunoreactive region or site is
obtained or derived from a first species and the constant region
(which may be intact, partial or modified in accordance with the
instant disclosure) is obtained from a second species. In certain
embodiments the target binding region or site will be from a
non-human source (e.g. mouse or primate) and the constant region is
human.
[0080] As used herein, "percent humanization" is calculated by
determining the number of framework amino acid differences (i.e.,
non-CDR difference) between the humanized domain and the germline
domain, subtracting that number from the total number of amino
acids, and then dividing that by the total number of amino acids
and multiplying by 100.
[0081] By "specifically binds" or "has specificity to," it is
generally meant that an antibody binds to an epitope via its
antigen-binding domain, and that the binding entails some
complementarity between the antigen-binding domain and the epitope.
According to this definition, an antibody is said to "specifically
bind" to an epitope when it binds to that epitope, via its
antigen-binding domain more readily than it would bind to a random,
unrelated epitope. The term "specificity" is used herein to qualify
the relative affinity by which a certain antibody binds to a
certain epitope. For example, antibody "A" may be deemed to have a
higher specificity for a given epitope than antibody "B," or
antibody "A" may be said to bind to epitope "C" with a higher
specificity than it has for related epitope "D."
[0082] As used herein, the terms "treat" or "treatment" refer to
both therapeutic treatment and prophylactic or preventative
measures, wherein the object is to prevent or slow down (lessen) an
undesired physiological change or disorder, such as the progression
of cancer. Beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" can also mean
prolonging survival as compared to expected survival if not
receiving treatment. Those in need of treatment include those
already with the condition or disorder as well as those prone to
have the condition or disorder or those in which the condition or
disorder is to be prevented.
[0083] By "subject" or "individual" or "animal" or "patient" or
"mammal," is meant any subject, particularly a mammalian subject,
for whom diagnosis, prognosis, or therapy is desired. Mammalian
subjects include humans, domestic animals, farm animals, and zoo,
sport, or pet animals such as dogs, cats, guinea pigs, rabbits,
rats, mice, horses, cattle, cows, and so on.
[0084] As used herein, phrases such as "to a patient in need of
treatment" or "a subject in need of treatment" includes subjects,
such as mammalian subjects, that would benefit from administration
of an antibody or composition of the present disclosure used, e.g.,
for detection, for a diagnostic procedure and/or for treatment.
Anti-PD-L1 Antibodies
[0085] The present disclosure provides anti-PD-L1 antibodies with
high affinity to the human PD-L1 protein. The tested antibodies
exhibited potent binding and inhibitory activities and are useful
for therapeutic and diagnostics uses.
[0086] The PD-L1 protein is a 40 kDa type 1 transmembrane protein.
Its extracellular portion includes an N-terminal immunoglobulin V
(IgV) domain (amino acids 19-127) and a C-terminal immunoglobulin C
(IgC) domain (amino acids 133-225). PD-1 and PD-L1 interact through
the conserved front and side of their IgV domains, as do the IgV
domains of antibodies and T cell receptors. Not surprisingly, the
current anti-PD-L1 antibodies all bind to the IgV domain which can
disrupt the binding between PD-1 and PD-L1 . It is therefore a
surprising and unexpected finding of the present disclosure that
antibodies, such as many disclosed herein, that bind to the IgC
domain of the PD-L1 protein can still effectively, and perhaps even
more so, inhibit PD-L1, leading to even further improved
therapeutic effects.
[0087] One embodiment of the present disclosure, therefore,
provides an anti-PD-L1 antibody or fragment thereof, which antibody
or fragment thereof can specifically bind to an immunoglobulin C
(Ig C) domain of a human Programmed death-ligand 1 (PD-L1) protein.
In some embodiments, the Ig C domain consists of amino acid
residues 133-225.
[0088] In some embodiments, the antibody or fragment thereof can
bind to at least one of amino acid residues Y134, K162, or N183 of
the PD-L1 protein. In some embodiments, the antibody or fragment
thereof can bind to at least two of amino acid residues Y134, K162,
or N183 of the PD-L1 protein. In some embodiments, the antibody or
fragment thereof can bind to at least one of amino acid residues
Y134, K162, and N183 of the PD-L1 protein. In some embodiments, the
antibody or fragment thereof does not bind to an immunoglobulin V
(Ig V) domain of the PD-L1 protein, wherein the Ig V domain
consists of amino acid residues 19-127.
[0089] In accordance with one embodiment of the present disclosure,
provided is an antibody that includes the heavy chain and light
chain variable domains with the CDR regions as defined in SEQ ID
NO: 1-6.
TABLE-US-00002 TABLE 1 Sequences of the CDR regions Name Sequence
SEQ ID NO: VH CDR1 SYDMS 1 VH CDR2 TISDGGGYIYYSDSVKG 2 VH CDR3
EFGKRYALDY 3 VL CDR1 KASQDVTPAVA 4 VL CDR2 STSSRYT 5 VL CDR3
QQHYTTPLT 6
[0090] As demonstrated in the experimental examples, the antibodies
that contained these CDR regions, whether mouse, humanized or
chimeric, had potent PD-L1 binding and inhibitory activities.
Further computer modeling indicated that certain residues within
the CDR can be modified to retain or improve the property of the
antibodies. Such residues are referred to as "hot spots" which are
underlined in Table 1. In some embodiments, an anti-PD-L1 antibody
of the present disclosure includes the VH and VL CDR as listed in
Table 1, with one, two or three further modifications. Such
modifications can be addition, deletion or substation of amino
acids.
[0091] In some embodiments, the modification is substitution at no
more than one hot spot position from each of the CDRs. In some
embodiments, the modification is substitution at one, two or three
such hot spot positions. In one embodiment, the modification is
substitution at one of the hot spot positions. Such substitutions,
in some embodiments, are conservative substitutions.
[0092] A "conservative amino acid substitution" is one 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, including 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), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a nonessential amino acid residue in an
immunoglobulin polypeptide is preferably replaced with another
amino acid residue from the same side chain family. In another
embodiment, a string of amino acids can be replaced with a
structurally similar string that differs in order and/or
composition of side chain family members.
[0093] Non-limiting examples of conservative amino acid
substitutions are provided in the table below, where a similarity
score of 0 or higher indicates conservative substitution between
the two amino acids.
TABLE-US-00003 TABLE 2 Amino Acid Similarity Matrix C G P S A T D E
N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6
-4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1
7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3
-3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2
-2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1
-1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6
K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1
0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1
-1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C
12
TABLE-US-00004 TABLE 3 Conservative Amino Acid Substitutions For
Amino Acid Substitution With Alanine D-Ala, Gly, Aib, .beta.-Ala,
L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine
D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D-Gln Aspartic Acid D-Asp,
D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine D-Cys, S-Me-Cys, Met,
D-Met, Thr, D-Thr, L-Ser, D-Ser Glutamine D-Gln, Asn, D-Asn, Glu,
D-Glu, Asp, D-Asp Glutamic Acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gln,
D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, .beta.-Ala Isoleucine
D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met,
D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-Orn
Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val
Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline
D-Pro Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr,
Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile,
D-Ile, Met, D-Met
[0094] Specific examples of CDRs with suitable substitutions are
provided in SEQ ID NO: 61-111 of Example 11. In some embodiments,
therefore, an antibody of the present disclosure includes a VH CDR1
of SEQ ID NO: 1 or any one of 61-67. In some embodiments, an
antibody of the present disclosure includes a VH CDR2 of SEQ ID NO:
2 or any one of 68-77. In some embodiments, an antibody of the
present disclosure includes a VH CDR3 of SEQ ID NO: 1 or any one of
78-90. In some embodiments, an antibody of the present disclosure
includes a VL CDR1 of SEQ ID NO: 4 or any one of 91-92. In some
embodiments, an antibody of the present disclosure includes a VL
CDR2 of SEQ ID NO: 5 or any one of 93-105. In some embodiments, an
antibody of the present disclosure includes a VL CDR3 of SEQ ID NO:
6 or any one of 106-110.
[0095] In some embodiments, an antibody or fragment thereof
includes no more than one, no more than two, or no more than three
of the above substitutions. In some embodiments, the antibody or
fragment thereof includes a VH CDR1 of SEQ ID NO: 1 or any one of
SEQ ID NO: 61-67, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID
NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a
VL CDR3 of SEQ ID NO: 6.
[0096] In some embodiments, the antibody or fragment thereof
includes a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2 or
any one of SEQ ID NO: 68-77, a VH CDR3 of SEQ ID NO: 3, a VL CDR1
of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID
NO: 6.
[0097] In some embodiments, the antibody or fragment thereof
includes a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH
CDR3 of SEQ ID NO: 3 or any one of SEQ ID NO: 78-90, a VL CDR1 of
SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID
NO: 6.
[0098] In some embodiments, the antibody or fragment thereof
includes a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH
CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4 or any one of SEQ
ID NO: 91-92, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID
NO: 6.
[0099] In some embodiments, the antibody or fragment thereof
includes a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH
CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ
ID NO: 5 or any one of SEQ ID NO: 93-105, and a VL CDR3 of SEQ ID
NO: 6.
[0100] In some embodiments, the antibody or fragment thereof
includes a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH
CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ
ID NO: 5, and a VL CDR3 of SEQ ID NO: 6 or any one of SEQ ID NO:
106-111.
[0101] Non-limiting examples of VH are provided in SEQ ID NO: 7-26
and 113, out of which SEQ ID NO: 113 is the mouse VH, and SEQ ID
NO: 7-26 are humanized ones. Further, among the humanized VH, SEQ
ID NO: 9-15, 17-21 and 23-26 include one or more back-mutations to
the mouse version. Likewise, non-limiting examples of VL (VK) are
provided in SEQ ID NO: 27-33. SEQ ID NO: 28 and 30 are the
originally derived, CDR-grafted, humanized sequences as shown in
the examples. SEQ ID NO: 29 and 31-33 are humanized VL with
back-mutations.
[0102] The back-mutations are shown to be useful for retaining
certain characteristics of the anti-PD-L1 antibodies. Accordingly,
in some embodiments, the anti-PD-L1 antibodies of the present
disclosure, in particular the human or humanized ones, include one
or more of the back-mutations. In some embodiments, the VH
back-mutation (i.e., included amino acid at the specified position)
is one or more selected from (a) Ser at position 44, (b) Ala at
position 49, (c) Ala at position 53, (d) Ile at position 91, (e)
Glu at position 1, (f) Val at position 37, (g) Thr at position 40
(h) Val at position 53, (i) Glu at position 54, (j) Asn at position
77, (k) Arg at position 94, and (1) Thr at position 108, according
to Kabat numbering, and combinations thereof In some embodiments,
the back-mutations are selected from (a) Ser at position 44, (b)
Ala at position 49, (c) Ala at position 53, and/or (d) Ile at
position 91, according to Kabat numbering, and combinations
thereof.
[0103] In some embodiments, the VL back-mutation is one or more
selected from (a) Ser at position 22, (b) Gln at position 42, (c)
Ser at position 43, (d) Asp at position 60, and (e) Thr at position
63, according to Kabat numbering, and combinations thereof.
[0104] In some embodiments, the anti-PD-L1 antibody of the present
disclosure includes a VH of SEQ ID NO: 7-26, a VL of SEQ ID NO:
27-33, or their respective biological equivalents. A biological
equivalent of a VH or VL is a sequence that includes the designated
amino acids while having an overall 80%, 85%, 90%, 95%, 98% or 99%
sequence identity. A biological equivalent of SEQ ID NO: 20, for
instance, can be a VH that has an overall 80%, 85%, 90%, 95%, 98%
or 99% sequence identity to SEQ ID NO: 20 but retains the CDRs (SEQ
ID NO: 1-6 or their variants), and optionally retains one or more,
or all of the back-mutations. In one embodiment, the VH has the
amino acid sequence of SEQ ID NO: 20 and the VL has the amino acid
sequence of SEQ ID NO: 28.
Further Improved PD-L1 Antibodies
[0105] Through random mutagenesis with controlled mutation rates,
Examples 13-17 were able to identify a number of hotspot residues
in particular in the CDR3 of both the heavy chain (e.g., B6, C3,
C6, and Al) and the light chain (e.g., A3) variable regions (see
Tables 14 and 15). The mutagenesis was performed on a template
antibody derived from Hu1210-41 (as noted in the footnote of Table
14, the template antibody WT has a S60R (Kabat numbering)
substitution in the heavy chain CDR2). Also, compared to the
chimeric antibody, Hu1210-41 included a G53A substitution (see SEQ
ID NO:20) in VH CDR2. Among the tested mutant antibodies, antibody
B6 exhibited greatly improved binding affinity to human PD-L1 and
biological activities.
[0106] In one embodiment, therefore, provided are antibodies and
antigen-binding fragment that include the following CDRs (from the
S60R mutant) and their variants.
TABLE-US-00005 Name Sequence SEQ ID NO: VH CDR1 SYDMS 1 VH CDR2
TISDAGGYIYYRDSVKG 116 VH CDR3 EFGKRYALDY 3 VL CDR1 KASQDVTPAVA 4 VL
CDR2 STSSRYT 5 VL CDR3 QQHYTTPLT 6
[0107] In one embodiment, therefore, provided are antibodies and
antigen-binding fragment that include the following CDRs (from B6)
and their variants.
TABLE-US-00006 Name Sequence SEQ ID NO: VH CDR1 SYDMS 1 VH CDR2
TISDAGGYIYYRDSVKG 116 VH CDR3 ELPWRYALDY 117 VL CDR1 KASQDVTPAVA 4
VL CDR2 STSSRYT 5 VL CDR3 QQHYTTPLT 6
[0108] In one embodiment, provided is an antibody or fragment
thereof, wherein the antibody or fragment thereof has specificity
to a human PD-L1 protein and comprises: (a) a VH CDR1 comprising
the amino acid sequence of SEQ ID NO: 1 or a variant of SEQ ID NO:
1 having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 1; (b) a VH CDR2 comprising the amino acid
sequence of SEQ ID NO: 116 or a variant of SEQ ID NO: 116 having
one, two or three substitution, deletion or insertion as compared
to SEQ ID NO: 116; (c) a VH CDR3 comprising the amino acid sequence
of SEQ ID NO: 3 or a variant of SEQ ID NO: 3 having one, two or
three substitution, deletion or insertion as compared to SEQ ID NO:
3, wherein the second amino acid residue of the VH CDR3 is Leu; (d)
a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or a
variant of SEQ ID NO: 4 having one, two or three substitution,
deletion or insertion as compared to SEQ ID NO: 4; (e) a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 5 or a variant of
SEQ ID NO: 5 having one, two or three substitution, deletion or
insertion as compared to SEQ ID NO: 5; and (f) a VL CDR3 comprising
the amino acid sequence of SEQ ID NO: 6 or a variant of SEQ ID NO:
6 having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 6.
[0109] In one embodiment, provided is an antibody or fragment
thereof, wherein the antibody or fragment thereof has specificity
to a human PD-L1 protein and comprises: (a) a VH CDR1 comprising
the amino acid sequence of SEQ ID NO: 1 or a variant of SEQ ID NO:
1 having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 1; (b) a VH CDR2 comprising the amino acid
sequence of SEQ ID NO: 116 or a variant of SEQ ID NO: 116 having
one, two or three substitution, deletion or insertion as compared
to SEQ ID NO: 116; (c) a VH CDR3 comprising the amino acid sequence
of SEQ ID NO: 117 or a variant of SEQ ID NO: 117 having one, two or
three substitution, deletion or insertion as compared to SEQ ID NO:
117, wherein the second amino acid residue of the VH CDR3 is Leu;
(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or
a variant of SEQ ID NO: 4 having one, two or three substitution,
deletion or insertion as compared to SEQ ID NO: 4; (e) a VL CDR2
comprising the amino acid sequence of SEQ ID NO: 5 or a variant of
SEQ ID NO: 5 having one, two or three substitution, deletion or
insertion as compared to SEQ ID NO: 5; and (f) a VL CDR3 comprising
the amino acid sequence of SEQ ID NO: 6 or a variant of SEQ ID NO:
6 having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 6.
[0110] Example variants of SEQ ID NO: 1 have one amino acid
substitution at one of amino acid residues 1, 2 and 5, such as SEQ
ID NO: 61-67:
TABLE-US-00007 Name Sequence SEQ ID NO: VH CDR1 SYDMS 1 TYDMS 61
CYDMS 62 SFDMS 63 SHDMS 64 SWDMS 65 SYDMT 66 SYDMC 67
[0111] Example variants of SEQ ID NO: 116 have one or more amino
acid substitutions, such as SEQ ID NO: 118-127, 2 and 68-77. In
some embodiments, the variants are SEQ ID NO: 118-127.
TABLE-US-00008 Name Sequence SEQ ID NO: VH CDR2 TISDAGGYIYYRDSVKG
116 TISDAGAYIYYRDSVKG 118 TISDAGPYIYYRDSVKG 119 TISDAGGFIYYRDSVKG
120 TISDAGGHIYYRDSVKG 121 TISDAGGWIYYRDSVKG 122 TISDAGGYIYYRDTVKG
123 TISDAGGYIYYRDCVKG 124 TISDAGGYIYYRDSLKG 125 TISDAGGYIYYRDSIKG
126 TISDAGGYIYYRDSMKG 127 TISDGGGYIYYSDSVKG 2 TISDGGAYIYYSDSVKG 68
TISDGGPYIYYSDSVKG 69 TISDGGGFIYYSDSVKG 70 TISDGGGHIYYSDSVKG 71
TISDGGGWIYYSDSVKG 72 TISDGGGYIYYSDTVKG 73 TISDGGGYIYYSDCVKG 74
TISDGGGYIYYSDSLKG 75 TISDGGGYIYYSDSIKG 76 TISDGGGYIYYSDSMKG 77
[0112] In some embodiments, the third amino acid residue of the VH
CDR3 variant is Pro. In some embodiments, the fourth amino acid
residue of the VH CDR3 variant is Trp.
[0113] Example variants of SEQ ID NO: 3 have one or more amino acid
substitution at amino acid residues 1-6, such as SEQ ID NO:
78-90:
TABLE-US-00009 Name Sequence SEQ ID NO: VH CDR3 EFGKRYALDY 3
QFGKRYALDY 78 DFGKRYALDY 79 NFGKRYALDY 80 EYGKRYALDY 81 EHGKRYALDY
82 EWGKRYALDY 83 EFAKRYALDY 84 EFPKRYALDY 85 EFGRRYALDY 86
EFGKKYALDY 87 EFGKRFALDY 88 EFGKRHALDY 89 EFGKRWALDY 90
[0114] Example variants of SEQ ID NO: 117 have one or more amino
acid substitution at amino acid residues 1, 5 and 6, such as SEQ ID
NO: 128-139:
TABLE-US-00010 Name Sequence SEQ ID NO: VH CDR3 ELPWRYALDY (B6) 117
ELFNRYALDY (B1) 128 ELHFRYALDY (C3) 129 ELYFRYALDY (C6) 130
ELLHRYALDY (A1) 131 ELRGRYALDY (A2) 132 QLPWRYALDY 133 DLPWRYALDY
134 NLPWRYALDY 135 ELPWKYALDY 136 ELPWRFALDY 137 ELPWRHALDY 138
ELPWRWALDY 139
[0115] In some embodiments, the variant of SEQ ID NO: 4 has one
amino acid substitution at amino acid residue 3, such as SEQ ID NO:
91-92:
TABLE-US-00011 Name Sequence SEQ ID NO: VL CDR1 KASQDVTPAVA 4
KATQDVTPAVA 91 KACQDVTPAVA 92
[0116] In some embodiments, the variant of SEQ ID NO: 5 has one
amino acid substitution at one of amino acid residues 1-6, such as
SEQ ID NO: 93-105:
TABLE-US-00012 Name Sequence SEQ ID NO: VL CDR2 STSSRYT 5 TTSSRYT
93 CTSSRYT 94 SSSSRYT 95 SMSSRYT 96 SVSSRYT 97 STTSRYT 98 STCSRYT
99 STSTRYT 100 STSCRYT 101 STSSKYT 102 STSSRFT 103 STSSRHT 104
STSSRWT 105
[0117] Example variants of SEQ ID NO: 6 have one amino acid
substitution at one of amino acid residues 1 and 2, such as SEQ ID
NO: 106-111. Another example variant is SEQ ID NO: 140.
TABLE-US-00013 Name Sequence SEQ ID NO: VL CDR3 QQHYTTPLT 6
EQHYTTPLT 106 DQHYTTPLT 107 NQHYTTPLT 108 QEHYTTPLT 109 QDHYTTPLT
110 QNHYTTPLT 111 QQHSDAPLT (A3) 140
[0118] Mutant A3, which has substitutions at three residues in the
VL CDR3, also exhibited excellent binding affinity to human PD-L1.
In one embodiment, therefore, provided are antibodies and
antigen-binding fragment that include the following CDRs and their
variants:
TABLE-US-00014 Name Sequence SEQ ID NO: VH CDR1 SYDMS 1 VH CDR2
TISDAGGYIYYRDSVKG 116 VH CDR3 EFGKRYALDY 3 VL CDR1 KASQDVTPAVA 4 VL
CDR2 STSSRYT 5 VL CDR3 QQHSDAPLT 140
[0119] In one embodiment, therefore, provided is an antibody or
fragment thereof, wherein the antibody or fragment thereof has
specificity to a human PD-L1 protein and comprises: (a) a VH CDR1
comprising the amino acid sequence of SEQ ID NO: 1 or a variant of
SEQ ID NO: 1 having one, two or three substitution, deletion or
insertion as compared to SEQ ID NO: 1; (b) a VH CDR2 comprising the
amino acid sequence of SEQ ID NO: 116 or a variant of SEQ ID NO:
116 having one, two or three substitution, deletion or insertion as
compared to SEQ ID NO: 116; (c) a VH CDR3 comprising the amino acid
sequence of SEQ ID NO: 3 or a variant of SEQ ID NO: 3 having one,
two or three substitution, deletion or insertion as compared to SEQ
ID NO: 3; (d) a VL CDR1 comprising the amino acid sequence of SEQ
ID NO: 4 or a variant of SEQ ID NO: 4 having one, two or three
substitution, deletion or insertion as compared to SEQ ID NO: 4;
(e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or
a variant of SEQ ID NO: 5 having one, two or three substitution,
deletion or insertion as compared to SEQ ID NO: 5; and (f) a VL
CDR3 comprising the amino acid sequence of SEQ ID NO: 140 or a
variant of SEQ ID NO: 140 having one, two or three substitution,
deletion or insertion as compared to SEQ ID NO: 140, wherein at
least (i) amino acid residue 4 of the VL CDR3 is Ser, (ii) amino
acid residue 5 of the VL CDR3 is Asp, or (iii) amino acid residue 6
of the VL CDR3 is Ala.
[0120] Example variants of SEQ ID NO: 1 have one amino acid
substitution at one of amino acid residues 1, 2 and 5, such as SEQ
ID NO: 61-67.
[0121] Example variants of SEQ ID NO: 116 have one or more amino
acid substitutions, such as SEQ ID NO: 118-127, 2 and 68-77.
[0122] Example variants of SEQ ID NO: 3 have one or more amino acid
substitutions such as SEQ ID NO: 117 and 128-139.
TABLE-US-00015 Name Sequence SEQ ID NO: VH CDR3 ELPWRYALDY (B6) 117
ELFNRYALDY (B1) 128 ELHFRYALDY (C3) 129 ELYFRYALDY (C6) 130
ELLHRYALDY (A1) 131 ELRGRYALDY (A2) 132 QLPWRYALDY 133 DLPWRYALDY
134 NLPWRYALDY 135 ELPWKYALDY 136 ELPWRFALDY 137 ELPWRHALDY 138
ELPWRWALDY 139
[0123] Example variants of SEQ ID NO: 4 have one amino acid
substitution at amino acid residue 3, such as SEQ ID NO: 91-92.
[0124] Example variants of SEQ ID NO: 5 has one amino acid
substitution at one of amino acid residues 1-6, such as SEQ ID NO:
93-105.
[0125] In some embodiments, amino acid residue 4 of the VL CDR3
variant is Ser. In some embodiments, amino acid residue 5 of the VL
CDR3 variant is Asp. In some embodiments, amino acid residue 6 of
the VL CDR3 variant is Ala. Example variants of SEQ ID NO: 140 has
one amino acid substitution at one of amino acid residues 1 and 2,
such as SEQ ID NO: 161-166.
TABLE-US-00016 Name Sequence SEQ ID NO: VL CDR3 QQHSDAPLT 140
EQHSDAPLT 161 DQHSDAPLT 162 NQHSDAPLT 163 QEHSDAPLT 164 QDHSDAPLT
165 QNHSDAPLT 166
[0126] Examples of antibodies derived from the mutagenesis study or
their antigen-binding fragments include those having the heavy
chain and light chain variable regions provided in Table 15. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 141
and the light chain variable region includes SEQ ID NO: 142. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 143
and the light chain variable region includes SEQ ID NO: 144. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 145
and the light chain variable region includes SEQ ID NO: 146. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 147
and the light chain variable region includes SEQ ID NO: 148. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 149
and the light chain variable region includes SEQ ID NO: 150. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 151
and the light chain variable region includes SEQ ID NO: 152. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 153
and the light chain variable region includes SEQ ID NO: 154. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 155
and the light chain variable region includes SEQ ID NO: 156. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 157
and the light chain variable region includes SEQ ID NO: 158. In one
embodiment, the heavy chain variable region includes SEQ ID NO: 159
and the light chain variable region includes SEQ ID NO: 160.
[0127] It will also be understood by one of ordinary skill in the
art that antibodies as disclosed herein may be modified such that
they vary in amino acid sequence from the naturally occurring
binding polypeptide from which they were derived. For example, a
polypeptide or amino acid sequence derived from a designated
protein may be similar, e.g., have a certain percent identity to
the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, or 99% identical to the starting sequence.
[0128] In certain embodiments, the antibody comprises an amino acid
sequence or one or more moieties not normally associated with an
antibody. Exemplary modifications are described in more detail
below. For example, an antibody of the disclosure may comprise a
flexible linker sequence, or may be modified to add a functional
moiety (e.g., PEG, a drug, a toxin, or a label).
[0129] Antibodies, variants, or derivatives thereof of the
disclosure include derivatives that are modified, i.e., by the
covalent attachment of any type of molecule to the antibody such
that covalent attachment does not prevent the antibody from binding
to the epitope. For example, but not by way of limitation, the
antibodies can be modified, e.g., by glycosylation, acetylation,
pegylation, phosphorylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. Any
of numerous chemical modifications may be carried out by known
techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the antibodies may contain one or
more non-classical amino acids.
[0130] In some embodiments, the antibodies may be conjugated to
therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses,
lipids, biological response modifiers, pharmaceutical agents, or
PEG.
[0131] The antibodies may be conjugated or fused to a therapeutic
agent, which may include detectable labels such as radioactive
labels, an immunomodulator, a hormone, an enzyme, an
oligonucleotide, a photoactive therapeutic or diagnostic agent, a
cytotoxic agent, which may be a drug or a toxin, an ultrasound
enhancing agent, a non-radioactive label, a combination thereof and
other such agents known in the art.
[0132] The antibodies can be detectably labeled by coupling it to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged antigen-binding polypeptide is then
determined by detecting the presence of luminescence that arises
during the course of a chemical reaction. Examples of particularly
useful chemiluminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
ester.
[0133] The antibodies can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques
for conjugating various moieties to an antibody are well known,
see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting
Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer
Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc.
(1985); Hellstrom et al., "Antibodies For Drug Delivery", in
Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel
Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal
Antibodies '84: Biological And Clinical Applications, Pinchera et
al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future
Prospective Of The Therapeutic Use Of Radiolabeled Antibody In
Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And
Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985),
and Thorpe et al., "The Preparation And Cytotoxic Properties Of
Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)).
Bi-Functional Molecules
[0134] PD-L1 is an immune checkpoint molecule and is also a tumor
antigen. As a tumor antigen targeting molecule, an antibody or
antigen-binding fragment specific to PD-L1 can be combined with a
second antigen-binding fragment specific to an immune cell to
generate a bispecific antibody.
[0135] In some embodiments, the immune cell is selected from the
group consisting of a T cell, a B cell, a monocyte, a macrophage, a
neutrophil, a dendritic cell, a phagocyte, a natural killer cell,
an eosinophil, a basophil, and a mast cell. Molecules on the immune
cell which can be targeted include, for example, CD3, CD16, CD19,
CD28, and CD64. Other examples include PD-1, CTLA-4, LAG-3 (also
known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3,
OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL,
TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272),
killer-cell immunoglobulin-like receptors (KIRs), and CD47.
Specific examples of bispecificity include, without limitation,
PD-L1/PD-1, PD-L1/LAG3, PD-L1/TIGIT, and PD-L1/CD47.
[0136] As an immune checkpoint inhibitor, an antibody or
antigen-binding fragment specific to PD-L1 can be combined with a
second antigen-binding fragment specific to a tumor antigen to
generate a bispecific antibody. A "tumor antigen" is an antigenic
substance produced in tumor cells, i.e., it triggers an immune
response in the host. Tumor antigens are useful in identifying
tumor cells and are potential candidates for use in cancer therapy.
Normal proteins in the body are not antigenic. Certain proteins,
however, are produced or overexpressed during tumorigenesis and
thus appear "foreign" to the body. This may include normal proteins
that are well sequestered from the immune system, proteins that are
normally produced in extremely small quantities, proteins that are
normally produced only in certain stages of development, or
proteins whose structure is modified due to mutation.
[0137] An abundance of tumor antigens are known in the art and new
tumor antigens can be readily identified by screening. Non-limiting
examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30,
CD33, CD47, CD52, CD133, CD73, CEA, gpA33, Mucins, TAG-72, CIX,
PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin,
.alpha.V.beta.3, .alpha.5.beta.1, ERBB2, ERBB3, MET, IGF1R, EPHA3,
TRAILR1, TRAILR2, RANKL, FAP and Tenascin.
[0138] In some aspects, the monovalent unit has specificity to a
protein that is overexpressed on a tumor cell as compared to a
corresponding non-tumor cell. A "corresponding non-tumor cell" as
used here, refers to a non-tumor cell that is of the same cell type
as the origin of the tumor cell. It is noted that such proteins are
not necessarily different from tumor antigens. Non-limiting
examples include carcinoembryonic antigen (CEA), which is
overexpressed in most colon, rectum, breast, lung, pancreas and
gastrointestinal tract carcinomas; heregulin receptors (HER-2, neu
or c-erbB-2), which is frequently overexpressed in breast, ovarian,
colon, lung, prostate and cervical cancers; epidermal growth factor
receptor (EGFR), which is highly expressed in a range of solid
tumors including those of the breast, head and neck, non-small cell
lung and prostate; asialoglycoprotein receptor; transferrin
receptor; serpin enzyme complex receptor, which is expressed on
hepatocytes; fibroblast growth factor receptor (FGFR), which is
overexpressed on pancreatic ductal adenocarcinoma cells; vascular
endothelial growth factor receptor (VEGFR), for anti-angiogenesis
gene therapy; folate receptor, which is selectively overexpressed
in 90% of nonmucinous ovarian carcinomas; cell surface glycocalyx;
carbohydrate receptors; and polymeric immunoglobulin receptor,
which is useful for gene delivery to respiratory epithelial cells
and attractive for treatment of lung diseases such as Cystic
Fibrosis. Non-limiting examples of bispecificity in this respect
include PD-L1/EGFR, PD-L1/Her2, PD-L1/CD33, PD-L1/CD133, PD-L1/CEA
and PD-L1/VEGF.
[0139] Different format of bispecific antibodies are also provided.
In some embodiments, each of the anti-PD-L1 fragment and the second
fragment each is independently selected from a Fab fragment, a
single-chain variable fragment (scFv), or a single-domain antibody.
In some embodiments, the bispecific antibody further includes a Fc
fragment.
[0140] Bifunctional molecules that include not just antibody or
antigen binding fragment are also provided. As a tumor antigen
targeting molecule, an antibody or antigen-binding fragment
specific to PD-L1, such as those described here, can be combined
with an immune cytokine or ligand optionally through a peptide
linker. The linked immune cytokines or ligands include, but not
limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12,
IL-13, IL-15, GM-CSF, TNF-.alpha., CD40L, OX40L, CD27L, CD30L,
4-1BBL, LIGHT and GITRL. Such bi-functional molecules can combine
the immune checkpoint blocking effect with tumor site local immune
modulation.
Polynucleotides Encoding the Antibodies and Methods of Preparing
the Antibodies
[0141] The present disclosure also provides isolated
polynucleotides or nucleic acid molecules (e.g., SEQ ID NO: 34-60,
112, and 114) encoding the antibodies, variants or derivatives
thereof of the disclosure. The polynucleotides of the present
disclosure may encode the entire heavy and light chain variable
regions of the antigen-binding polypeptides, variants or
derivatives thereof on the same polynucleotide molecule or on
separate polynucleotide molecules. Additionally, the
polynucleotides of the present disclosure may encode portions of
the heavy and light chain variable regions of the antigen-binding
polypeptides, variants or derivatives thereof on the same
polynucleotide molecule or on separate polynucleotide
molecules.
[0142] Methods of making antibodies are well known in the art and
described herein. In certain embodiments, both the variable and
constant regions of the antigen-binding polypeptides of the present
disclosure are fully human. Fully human antibodies can be made
using techniques described in the art and as described herein. For
example, fully human antibodies against a specific antigen can be
prepared by administering the antigen to a transgenic animal which
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled.
Exemplary techniques that can be used to make such antibodies are
described in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140 which
are incorporated by reference in their entireties.
[0143] In certain embodiments, the prepared antibodies will not
elicit a deleterious immune response in the animal to be treated,
e.g., in a human. In one embodiment, antigen-binding polypeptides,
variants, or derivatives thereof of the disclosure are modified to
reduce their immunogenicity using art-recognized techniques. For
example, antibodies can be humanized, primatized, deimmunized, or
chimeric antibodies can be made. These types of antibodies are
derived from a non-human antibody, typically a murine or primate
antibody, that retains or substantially retains the antigen-binding
properties of the parent antibody, but which is less immunogenic in
humans. This may be achieved by various methods, including (a)
grafting the entire non-human variable domains onto human constant
regions to generate chimeric antibodies; (b) grafting at least a
part of one or more of the non-human complementarity determining
regions (CDRs) into a human framework and constant regions with or
without retention of critical framework residues; or (c)
transplanting the entire non-human variable domains, but "cloaking"
them with a human-like section by replacement of surface residues.
Such methods are disclosed in Morrison et al., Proc. Natl. Acad.
Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol.
44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988);
Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun.
31:169-217 (1994), and U.S. Pat. Nos. 5,585,089, 5,693,761,
5,693,762, and 6,190,370, all of which are hereby incorporated by
reference in their entirety.
[0144] De-immunization can also be used to decrease the
immunogenicity of an antibody. As used herein, the term
"de-immunization" includes alteration of an antibody to modify
T-cell epitopes (see, e.g., International Application Publication
Nos. WO/9852976 A1 and WO/0034317 A2). For example, variable heavy
chain and variable light chain sequences from the starting antibody
are analyzed and a human T-cell epitope "map" from each V region
showing the location of epitopes in relation to
complementarity-determining regions (CDRs) and other key residues
within the sequence is created. Individual T-cell epitopes from the
T-cell epitope map are analyzed in order to identify alternative
amino acid substitutions with a low risk of altering activity of
the final antibody. A range of alternative variable heavy and
variable light sequences are designed comprising combinations of
amino acid substitutions and these sequences are subsequently
incorporated into a range of binding polypeptides. Typically,
between 12 and 24 variant antibodies are generated and tested for
binding and/or function. Complete heavy and light chain genes
comprising modified variable and human constant regions are then
cloned into expression vectors and the subsequent plasmids
introduced into cell lines for the production of whole antibody.
The antibodies are then compared in appropriate biochemical and
biological assays, and the optimal variant is identified.
[0145] The binding specificity of antigen-binding polypeptides of
the present disclosure can be determined by in vitro assays such as
immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked
immunoabsorbent assay (ELISA).
[0146] Alternatively, techniques described for the production of
single-chain units (U.S. Pat. No. 4,694,778; Bird, Science
242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
55:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989))
can be adapted to produce single-chain units of the present
disclosure. Single-chain units are formed by linking the heavy and
light chain fragments of the Fv region via an amino acid bridge,
resulting in a single-chain fusion peptide. Techniques for the
assembly of functional Fv fragments in E. coli may also be used
(Skerra et al., Science 242: 1038-1041 (1988)).
[0147] Examples of techniques which can be used to produce
single-chain Fvs (scFvs) and antibodies include those described in
U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA
90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040
(1988). For some uses, including in vivo use of antibodies in
humans and in vitro detection assays, it may be preferable to use
chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule in which different portions of the antibody are derived
from different animal species, such as antibodies having a variable
region derived from a murine monoclonal antibody and a human
immunoglobulin constant region. Methods for producing chimeric
antibodies are known in the art. See, e.g., Morrison, Science
229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et
al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816397, which are incorporated herein
by reference in their entireties.
[0148] Humanized antibodies are antibody molecules derived from a
non-human species antibody that bind the desired antigen having one
or more complementarity determining regions (CDRs) from the
non-human species and framework regions from a human immunoglobulin
molecule. Often, framework residues in the human framework regions
will be substituted with the corresponding residue from the CDR
donor antibody to alter, preferably improve, antigen-binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for
antigen-binding and sequence comparison to identify unusual
framework residues at particular positions. (See, e.g., Queen et
al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323
(1988), which are incorporated herein by reference in their
entireties.) Antibodies can be humanized using a variety of
techniques known in the art including, for example, CDR-grafting
(EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991);
Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska.
et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain
shuffling (U.S. Pat. No. 5,565,332, which is incorporated by
reference in its entirety).
[0149] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods using antibody libraries derived from human
immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and
4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;
each of which is incorporated herein by reference in its
entirety.
[0150] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring that express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a desired target polypeptide. Monoclonal
antibodies directed against the antigen can be obtained from the
immunized, transgenic mice using conventional hybridoma technology.
The human immunoglobulin transgenes harbored by the transgenic mice
rearrange during B-cell differentiation, and subsequently undergo
class switching and somatic mutation. Thus, using such a technique,
it is possible to produce therapeutically useful IgG, IgA, IgM and
IgE antibodies. For an overview of this technology for producing
human antibodies, see Lonberg and Huszar Int. Rev. Immunol.
73:65-93 (1995). For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., PCT
publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos.
5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;
5,814,318; and 5,939,598, which are incorporated by reference
herein in their entirety. In addition, companies such as Abgenix,
Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0151] Completely human antibodies which recognize a selected
epitope can also be generated using a technique referred to as
"guided selection." In this approach a selected non-human
monoclonal antibody, e.g., a mouse antibody, is used to guide the
selection of a completely human antibody recognizing the same
epitope. (Jespers et al., Bio/Technology 72:899-903 (1988). See
also, U.S. Pat. No. 5,565,332, which is incorporated by reference
in its entirety.)
[0152] In another embodiment, DNA encoding desired monoclonal
antibodies may be readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light
chains of murine antibodies). The isolated and subcloned hybridoma
cells serve as a preferred source of such DNA. Once isolated, the
DNA may be placed into expression vectors, which are then
transfected into prokaryotic or eukaryotic host cells such as E.
coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or
myeloma cells that do not otherwise produce immunoglobulins. More
particularly, the isolated DNA (which may be synthetic as described
herein) may be used to clone constant and variable region sequences
for the manufacture antibodies as described in Newman et al., U.S.
Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated by
reference herein. Essentially, this entails extraction of RNA from
the selected cells, conversion to cDNA, and amplification by PCR
using Ig specific primers. Suitable primers for this purpose are
also described in U.S. Pat. No. 5,658,570. As will be discussed in
more detail below, transformed cells expressing the desired
antibody may be grown up in relatively large quantities to provide
clinical and commercial supplies of the immunoglobulin.
[0153] Additionally, using routine recombinant DNA techniques, one
or more of the CDRs of the antigen-binding polypeptides of the
present disclosure, may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody. The
framework regions may be naturally occurring or consensus framework
regions, and preferably human framework regions (see, e.g., Chothia
et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human
framework regions). Preferably, the polynucleotide generated by the
combination of the framework regions and CDRs encodes an antibody
that specifically binds to at least one epitope of a desired
polypeptide, e.g., LIGHT. Preferably, one or more amino acid
substitutions may be made within the framework regions, and,
preferably, the amino acid substitutions improve binding of the
antibody to its antigen. Additionally, such methods may be used to
make amino acid substitutions or deletions of one or more variable
region cysteine residues participating in an intrachain disulfide
bond to generate antibody molecules lacking one or more intrachain
disulfide bonds. Other alterations to the polynucleotide are
encompassed by the present disclosure and within the skill of the
art.
[0154] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
USA:851-855 (1984); Neuberger et al., Nature 372:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule, of appropriate antigen specificity,
together with genes from a human antibody molecule of appropriate
biological activity can be used. As used herein, a chimeric
antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region
derived from a murine monoclonal antibody and a human
immunoglobulin constant region.
[0155] Yet another highly efficient means for generating
recombinant antibodies is disclosed by Newman, Biotechnology 10:
1455-1460 (1992). Specifically, this technique results in the
generation of primatized antibodies that contain monkey variable
domains and human constant sequences. This reference is
incorporated by reference in its entirety herein. Moreover, this
technique is also described in commonly assigned U.S. Pat. Nos.
5,658,570, 5,693,780 and 5,756,096 each of which is incorporated
herein by reference.
[0156] Alternatively, antibody-producing cell lines may be selected
and cultured using techniques well known to the skilled artisan.
Such techniques are described in a variety of laboratory manuals
and primary publications. In this respect, techniques suitable for
use in the disclosure as described below are described in Current
Protocols in Immunology, Coligan et al., Eds., Green Publishing
Associates and Wiley-Interscience, John Wiley and Sons, New York
(1991) which is herein incorporated by reference in its entirety,
including supplements.
[0157] Additionally, standard techniques known to those of skill in
the art can be used to introduce mutations in the nucleotide
sequence encoding an antibody of the present disclosure, including,
but not limited to, site-directed mutagenesis and PCR-mediated
mutagenesis which result in amino acid substitutions. Preferably,
the variants (including derivatives) encode less than 50 amino acid
substitutions, less than 40 amino acid substitutions, less than 30
amino acid substitutions, less than 25 amino acid substitutions,
less than 20 amino acid substitutions, less than 15 amino acid
substitutions, less than 10 amino acid substitutions, less than 5
amino acid substitutions, less than 4 amino acid substitutions,
less than 3 amino acid substitutions, or less than 2 amino acid
substitutions relative to the reference variable heavy chain
region, CDR-H1, CDR-H2, CDR-H3, variable light chain region,
CDR-L1, CDR-L2, or CDR-L3. Alternatively, mutations can be
introduced randomly along all or part of the coding sequence, such
as by saturation mutagenesis, and the resultant mutants can be
screened for biological activity to identify mutants that retain
activity.
Cancer Treatment
[0158] As described herein, the antibodies, variants or derivatives
of the present disclosure may be used in certain treatment and
diagnostic methods.
[0159] The present disclosure is further directed to antibody-based
therapies which involve administering the antibodies of the
disclosure to a patient such as an animal, a mammal, and a human
for treating one or more of the disorders or conditions described
herein. Therapeutic compounds of the disclosure include, but are
not limited to, antibodies of the disclosure (including variants
and derivatives thereof as described herein) and nucleic acids or
polynucleotides encoding antibodies of the disclosure (including
variants and derivatives thereof as described herein).
[0160] The antibodies of the disclosure can also be used to treat
or inhibit cancer. PD-L1 can be overexpressed in tumor cells.
Tumor-derived PD-L1 can bind to PD-1 on immune cells thereby
limiting antitumor T-cell immunity. Results with small molecule
inhibitors, or monoclonal antibodies targeting PD-L1 in murine
tumor models, indicate that targeted PD-L1 therapy is an important
alternative and realistic approach to effective control of tumor
growth. As demonstrated in the experimental examples, the
anti-PD-L1 antibodies activated the adaptive immune response
machinery, which can lead to improved survival in cancer
patients.
[0161] Accordingly, in some embodiments, provided are methods for
treating a cancer in a patient in need thereof. The method, in one
embodiment, entails administering to the patient an effective
amount of an antibody of the present disclosure. In some
embodiments, at least one of the cancer cells (e.g., stromal cells)
in the patient expresses, over-express, or is induced to express
PD-L1. Induction of PD-L1 expression, for instance, can be done by
administration of a tumor vaccine or radiotherapy.
[0162] Tumors that express the PD-L1 protein include those of
bladder cancer, non-small cell lung cancer, renal cancer, breast
cancer, urethral cancer, colorectal cancer, head and neck cancer,
squamous cell cancer, Merkel cell carcinoma, gastrointestinal
cancer, stomach cancer, oesophageal cancer, ovarian cancer, renal
cancer, and small cell lung cancer. Accordingly, the presently
disclosed antibodies can be used for treating any one or more such
cancers.
[0163] Cellular therapies, such as chimeric antigen receptor (CAR)
T-cell therapies, are also provided in the present disclosure. A
suitable cell can be used, that is put in contact with an
anti-PD-L1 antibody of the present disclosure (or alternatively
engineered to express an anti-PD-L1 antibody of the present
disclosure). Upon such contact or engineering, the cell can then be
introduced to a cancer patient in need of a treatment. The cancer
patient may have a cancer of any of the types as disclosed herein.
The cell (e.g., T cell) can be, for instance, a tumor-infiltrating
T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination
thereof, without limitation.
[0164] In some embodiments, the cell was isolated from the cancer
patient him- or her-self. In some embodiments, the cell was
provided by a donor or from a cell bank. When the cell is isolated
from the cancer patient, undesired immune reactions can be
minimized.
[0165] Additional diseases or conditions associated with increased
cell survival, that may be treated, prevented, diagnosed and/or
prognosed with the antibodies or variants, or derivatives thereof
of the disclosure include, but are not limited to, progression,
and/or metastases of malignancies and related disorders such as
leukemia (including acute leukemias (e.g., acute lymphocytic
leukemia, acute myelocytic leukemia (including myeloblastic,
promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and
chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia
and chronic lymphocytic leukemia)), polycythemia vera, lymphomas
(e.g., Hodgkin's disease and non-Hodgkin's disease), multiple
myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and
solid tumors including, but not limited to, sarcomas and carcinomas
such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyo sarcoma,
colon carcinoma, pancreatic cancer, breast cancer, thyroid cancer,
endometrial cancer, melanoma, prostate cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma and
retinoblastoma.
Combination Therapies
[0166] In a further embodiment, the compositions of the disclosure
are administered in combination with an antineoplastic agent, an
antiviral agent, antibacterial or antibiotic agent or antifungal
agents. Any of these agents known in the art may be administered in
the compositions of the current disclosure.
[0167] In another embodiment, compositions of the disclosure are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions of the disclosure include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen,
chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and combinations (e.g., bethamethasone sodium phosphate);
and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
[0168] In an additional embodiment, the compositions of the
disclosure are administered in combination with cytokines.
Cytokines that may be administered with the compositions of the
disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and
TNF-.alpha..
[0169] In additional embodiments, the compositions of the
disclosure are administered in combination with other therapeutic
or prophylactic regimens, such as, for example, radiation
therapy.
[0170] Combination therapies are also provided, which includes the
use of one or more of the anti-PD-L1 antibody of the present
disclosure along with a second anticancer (chemotherapeutic) agent.
Chemotherapeutic agents may be categorized by their mechanism of
action into, for example, the following groups:
[0171] anti-metabolites/anti-cancer agents such as pyrimidine
analogs floxuridine, capecitabine, and cytarabine;
[0172] purine analogs, folate antagonists, and related
inhibitors;
[0173] antiproliferative/antimitotic agents including natural
products such as vinca alkaloid (vinblastine, vincristine) and
microtubule such as taxane (paclitaxel, docetaxel), vinblastin,
nocodazole, epothilones, vinorelbine (NAVELBINE.RTM.), and
epipodophyllotoxins (etoposide, teniposide);
[0174] DNA damaging agents such as actinomycin, amsacrine,
busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide
(CYTOXAN.RTM.), dactinomycin, daunorubicin, doxorubicin,
epirubicin, iphosphamide, melphalan, merchlorethamine, mitomycin,
mitoxantrone, nitrosourea, procarbazine, taxol, taxotere,
teniposide, etoposide, and triethylenethiophosphoramide;
[0175] antibiotics such as dactinomycin, daunorubicin, doxorubicin,
idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin
(mithramycin), and mitomycin;
[0176] enzymes such as L-asparaginase which systemically
metabolizes L-asparagine and deprives cells which do not have the
capacity to synthesize their own asparagine;
[0177] antiplatelet agents;
[0178] antiproliferative/antimitotic alkylating agents such as
nitrogen mustards cyclophosphamide and analogs (melphalan,
chlorambucil, hexamethylmelamine, and thiotepa), alkyl nitrosoureas
(carmustine) and analogs, streptozocin, and triazenes
(dacarbazine);
[0179] antiproliferative/antimitotic antimetabolites such as folic
acid analogs (methotrexate);
[0180] platinum coordination complexes (cisplatin, oxiloplatinim,
and carboplatin), procarbazine, hydroxyurea, mitotane, and
aminoglutethimide;
[0181] hormones, hormone analogs (estrogen, tamoxifen, goserelin,
bicalutamide, and nilutamide), and aromatase inhibitors (letrozole
and anastrozole);
[0182] anticoagulants such as heparin, synthetic heparin salts, and
other inhibitors of thrombin;
[0183] fibrinolytic agents such as tissue plasminogen activator,
streptokinase, urokinase, aspirin, dipyridamole, ticlopidine, and
clopidogrel;
[0184] antimigratory agents;
[0185] antisecretory agents (breveldin);
[0186] immunosuppressives tacrolimus, sirolimus, azathioprine, and
mycophenolate;
[0187] compounds (TNP-470, genistein) and growth factor inhibitors
(vascular endothelial growth factor inhibitors and fibroblast
growth factor inhibitors);
[0188] angiotensin receptor blockers, nitric oxide donors;
[0189] anti-sense oligonucleotides;
[0190] antibodies such as trastuzumab and rituximab;
[0191] cell cycle inhibitors and differentiation inducers such as
tretinoin;
[0192] inhibitors, topoisomerase inhibitors (doxorubicin,
daunorubicin, dactinomycin, eniposide, epirubicin, etoposide,
idarubicin, irinotecan, mitoxantrone, topotecan, and irinotecan),
and corticosteroids (cortisone, dexamethasone, hydrocortisone,
methylprednisolone, prednisone, and prednisolone);
[0193] growth factor signal transduction kinase inhibitors;
[0194] dysfunction inducers;
[0195] toxins such as Cholera toxin, ricin, Pseudomonas exotoxin,
Bordetella pertussis adenylate cyclase toxin, diphtheria toxin, and
caspase activators;
[0196] and chromatin.
[0197] Further examples of chemotherapeutic agents include:
[0198] alkylating agents such as thiotepa and cyclophosphamide
(CYTOXAN.RTM.);
[0199] alkyl sulfonates such as busulfan, improsulfan, and
piposulfan;
[0200] aziridines such as benzodopa, carboquone, meturedopa, and
uredopa;
[0201] emylerumines and memylamelamines including alfretamine,
triemylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide, and trimemylolomelamine;
[0202] acetogenins, especially bullatacin and bullatacinone;
[0203] a camptothecin, including synthetic analog topotecan;
[0204] bryostatin;
[0205] callystatin;
[0206] CC-1065, including its adozelesin, carzelesin, and bizelesin
synthetic analogs; cryptophycins, particularly cryptophycin 1 and
cryptophycin 8;
[0207] dolastatin;
[0208] duocarmycin, including the synthetic analogs KW-2189 and
CBI-TMI;
[0209] eleutherobin;
[0210] pancratistatin;
[0211] a sarcodictyin;
[0212] spongistatin;
[0213] nitrogen mustards such as chlorambucil, chlornaphazine,
cyclophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, and uracil mustard;
[0214] nitrosoureas such as carmustine, chlorozotocin, foremustine,
lomustine, nimustine, and ranimustine;
[0215] antibiotics such as the enediyne antibiotics (e.g.,
calicheamicin, especially calicheamicin gammaII and calicheamicin
phiI1), dynemicin including dynemicin A, bisphosphonates such as
clodronate, an esperamicin, neocarzinostatin chromophore and
related chromoprotein enediyne antibiotic chromomophores,
aclacinomycins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, carabicin, carrninomycin, carzinophilin,
chromomycins, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin;
[0216] anti-metabolites such as methotrexate and 5-fluorouracil
(5-FU);
[0217] folic acid analogs such as demopterin, methotrexate,
pteropterin, and trimetrexate;
[0218] purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, and thioguanine;
[0219] pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, and floxuridine;
[0220] androgens such as calusterone, dromostanolone propionate,
epitiostanol, mepitiostane, and testolactone;
[0221] anti-adrenals such as aminoglutethimide, mitotane, and
trilostane;
[0222] folic acid replinishers such as frolinic acid;
[0223] trichothecenes, especially T-2 toxin, verracurin A, roridin
A, and anguidine;
[0224] taxoids such as paclitaxel (TAXOL.RTM.) and docetaxel
(TAXOTERE.RTM.);
[0225] platinum analogs such as cisplatin and carboplatin;
[0226] aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; hestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformthine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; leucovorin; lonidamine; maytansinoids such as maytansine
and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine;
pentostatin; phenamet; pirarubicin; losoxantrone; fluoropyrimidine;
folinic acid; podophyllinic acid; 2-ethylhydrazide; procarbazine;
polysaccharide-K (PSK); razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-tricUorotriemylamine; urethane; vindesine; dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); cyclophosphamide; thiopeta; chlorambucil;
gemcitabine (GEMZAR.RTM.); 6-thioguanine; mercaptopurine;
methotrexate; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitroxantrone; vancristine; vinorelbine (NAVELBINE.RTM.);
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DFMO); retinoids such as retinoic acid;
capecitabine; FOLFIRI (fluorouracil, leucovorin, and
irinotecan);
[0227] and pharmaceutically acceptable salts, acids, or derivatives
of any of the above.
[0228] Also included in the definition of "chemotherapeutic agent"
are anti-hormonal agents such as anti-estrogens and selective
estrogen receptor modulators (SERMs), inhibitors of the enzyme
aromatase, anti-androgens, and pharmaceutically acceptable salts,
acids or derivatives of any of the above that act to regulate or
inhibit hormone action on tumors.
[0229] Examples of anti-estrogens and SERMs include, for example,
tamoxifen (including NOLVADEX.TM.), raloxifene, droloxifene,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and toremifene (FARESTON.RTM.)).
[0230] Inhibitors of the enzyme aromatase regulate estrogen
production in the adrenal glands. Examples include 4(5)-imidazoles,
aminoglutethimide, megestrol acetate (MEGACE.RTM.), exemestane,
formestane, fadrozole, vorozole (RIVISOR.RTM.)), letrozole
(FEMARA.RTM.), and anastrozole)(ARIMIDEX.RTM.).
[0231] Examples of anti-androgens include flutamide, nilutamide,
bicalutamide, leuprohde, and goserelin.
[0232] Examples of chemotherapeutic agents also include
anti-angiogenic agents including, but are not limited to, retinoid
acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN.RTM.,
ENDOSTATIN.RTM., suramin, squalamine, tissue inhibitor of
metalloproteinase-1, tissue inhibitor of metalloproteinase-2,
plasminogen activator inhibitor-1, plasminogen activator
inbibitor-2, cartilage-derived inhibitor, paclitaxel
(nab-paclitaxel), platelet factor 4, protamine sulphate (clupeine),
sulphated chitin derivatives (prepared from queen crab shells),
sulphated polysaccharide peptidoglycan complex (sp-pg),
staurosporine, modulators of matrix metabolism including proline
analogs ((1-azetidine-2-carboxylic acid (LACA)), cishydroxyproline,
d,I-3,4-dehydroproline, thiaproline, .alpha.,.alpha.'-dipyridyl,
beta-aminopropionitrile fumarate,
4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone, methotrexate,
mitoxantrone, heparin, interferons, 2 macroglobulin-serum, chicken
inhibitor of metalloproteinase-3 (ChIMP-3), chymostatin,
beta-cyclodextrin tetradecasulfate, eponemycin, fumagillin, gold
sodium thiomalate, d-penicillamine, beta-1-anticollagenase-serum,
alpha-2-antiplasmin, bisantrene, lobenzarit disodium,
n-2-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA",
thalidomide, angiostatic steroid, carboxy aminoimidazole, and
metalloproteinase inhibitors such as BB-94. Other anti-angiogenesis
agents include antibodies, preferably monoclonal antibodies against
these angiogenic growth factors: beta-FGF, alpha-FGF, FGF-5, VEGF
isoforms, VEGF-C, HGF/SF, and Ang-1/Ang-2.
[0233] Examples of chemotherapeutic agents also include
anti-fibrotic agents including, but are not limited to, the
compounds such as beta-aminoproprionitrile (BAPN), as well as the
compounds disclosed in U.S. Pat. No. 4,965,288 (Palfreyman, et al.)
relating to inhibitors of lysyl oxidase and their use in the
treatment of diseases and conditions associated with the abnormal
deposition of collagen and U.S. Pat. No. 4,997,854 (Kagan et al.)
relating to compounds which inhibit LOX for the treatment of
various pathological fibrotic states, which are herein incorporated
by reference. Further exemplary inhibitors are described in U.S.
Pat. No. 4,943,593 (Palfreyman et al.) relating to compounds such
as 2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine, U.S. Pat.
No. 5,021,456 (Palfreyman et al.), U.S. Pat. No. 5,059,714
(Palfreyman et al.), U.S. Pat. No. 5,120,764 (Mccarthy et al.),
U.S. Pat. No. 5,182,297 (Palfreyman et al.), U.S. Pat. No.
5,252,608 (Palfreyman et al.) relating to
2-(1-naphthyloxymemyl)-3-fluoroallylamine, and U.S. Pub. No.
2004/0248871 (Farjanel et al.), which are herein incorporated by
reference.
[0234] Exemplary anti-fibrotic agents also include the primary
amines reacting with the carbonyl group of the active site of the
lysyl oxidases, and more particularly those which produce, after
binding with the carbonyl, a product stabilized by resonance, such
as the following primary amines: emylenemamine, hydrazine,
phenylhydrazine, and their derivatives; semicarbazide and urea
derivatives; aminonitriles such as BAPN or 2-nitroethylamine;
unsaturated or saturated haloamines such as 2-bromo-ethylamine,
2-chloroethylamine, 2-trifluoroethyl amine, 3-bromopropylamine, and
p-halobenzylamines; and selenohomocysteine lactone.
[0235] Other anti-fibrotic agents are copper chelating agents
penetrating or not penetrating the cells. Exemplary compounds
include indirect inhibitors which block the aldehyde derivatives
originating from the oxidative deamination of the lysyl and
hydroxylysyl residues by the lysyl oxidases. Examples include the
thiolamines, particularly D-penicillamine, and its analogs such as
2-amino-5-mercapto-5-methylhexanoic acid,
D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio)butanoic acid,
p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid,
sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane
sulphurate, 2-acetamidoethyl-2-acetamidoethanethiol sulphanate, and
sodium-4-mercaptobutanesulphinate trihydrate.
[0236] Examples of chemotherapeutic agents also include
immunotherapeutic agents including and are not limited to
therapeutic antibodies suitable for treating patients. Some
examples of therapeutic antibodies include simtuzumab, abagovomab,
adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab,
anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab,
bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab,
cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab,
daratumumab, drozitumab, duligotumab, dusigitumab, detumomab,
dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab,
ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab,
flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab,
glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab,
inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab,
lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab,
matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab,
narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab,
ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab,
oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab,
pertuzumab, pintumomab, pritumumab, racotumomab, radretumab,
rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab,
siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab,
teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab,
ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab,
CC49, and 3F8. Rituximab can be used for treating indolent B-cell
cancers, including marginal-zone lymphoma, WM, CLL and small
lymphocytic lymphoma. A combination of Rituximab and chemotherapy
agents is especially effective.
[0237] The exemplified therapeutic antibodies may be further
labeled or combined with a radioisotope particle such as
indium-111, yttrium-90, or iodine-131.
[0238] In a one embodiment, the additional therapeutic agent is a
nitrogen mustard alkylating agent. Nonlimiting examples of nitrogen
mustard alkylating agents include chlorambucil.
[0239] In one embodiment, the compounds and compositions described
herein may be used or combined with one or more additional
therapeutic agents. The one or more therapeutic agents include, but
are not limited to, an inhibitor of Abl, activated CDC kinase
(ACK), adenosine A2B receptor (A2B), apoptosis signal-regulating
kinase (ASK), Auroa kinase, Bruton's tyrosine kinase (BTK),
BET-bromodomain (BRD) such as BRD4, c-Kit, c-Met, CDK-activating
kinase (CAK), calmodulin-dependent protein kinase (CaMK),
cyclin-dependent kinase (CDK), casein kinase (CK), discoidin domain
receptor (DDR), epidermal growth factor receptors (EGFR), focal
adhesion kinase (FAK), Flt-3, FYN, glycogen synthase kinase (GSK),
HCK, histone deacetylase (HDAC), IKK such as IKK.beta..epsilon.,
isocitrate dehydrogenase (IDH) such as IDH1, Janus kinase (JAK),
KDR, lymphocyte-specific protein tyrosine kinase (LCK), lysyl
oxidase protein, lysyl oxidase-like protein (LOXL), LYN, matrix
metalloprotease (MMP), MEK, mitogen-activated protein kinase
(MAPK), NEK9, NPM-ALK, p38 kinase, platelet-derived growth factor
(PDGF), phosphorylase kinase (PK), polo-like kinase (PLK),
phosphatidylinositol 3-kinase (PI3K), protein kinase (PK) such as
protein kinase A, B, and/or C, PYK, spleen tyrosine kinase (SYK),
serine/threonine kinase TPL2, serine/threonine kinase STK, signal
transduction and transcription (STAT), SRC,
serine/threonine-protein kinase (TBK) such as TBK1, TIE, tyrosine
kinase (TK), vascular endothelial growth factor receptor (VEGFR),
YES, or any combination thereof
[0240] ASK inhibitors include ASK1 inhibitors. Examples of ASK1
inhibitors include, but are not limited to, those described in WO
2011/008709 (Gilead Sciences) and WO 2013/112741 (Gilead
Sciences).
[0241] Examples of BTK inhibitors include, but are not limited to,
ibrutinib, HM71224, ONO-4059, and CC-292.
[0242] DDR inhibitors include inhibitors of DDR1 and/or DDR2.
Examples of DDR inhibitors include, but are not limited to, those
disclosed in WO 2014/047624 (Gilead Sciences), US 2009/0142345
(Takeda Pharmaceutical), US 2011/0287011 (Oncomed Pharmaceuticals),
WO 2013/027802 (Chugai Pharmaceutical), and WO 2013/034933
(Imperial Innovations).
[0243] Examples of HDAC inhibitors include, but are not limited to,
pracinostat and panobinostat.
[0244] JAK inhibitors inhibit JAK1, JAK2, and/or JAK3. Examples of
JAK inhibitors include, but are not limited to, filgotinib,
ruxolitinib, fedratinib, tofacitinib, baricitinib, lestaurtinib,
pacritinib, XL019, AZD1480, INCB039110, LY2784544, BMS911543, and
NS018.
[0245] LOXL inhibitors include inhibitors of LOXL1, LOXL2, LOXL3,
LOXL4, and/or LOXLS. Examples of LOXL inhibitors include, but are
not limited to, the antibodies described in WO 2009/017833 (Arresto
Biosciences).
[0246] Examples of LOXL2 inhibitors include, but are not limited
to, the antibodies described in WO 2009/017833 (Arresto
Biosciences), WO 2009/035791 (Arresto Biosciences), and WO
2011/097513 (Gilead Biologics).
[0247] MMP inhibitors include inhibitors of MMP1 through 10.
Examples of MMP9 inhibitors include, but are not limited to,
marimastat (BB-2516), cipemastat (Ro 32-3555), and those described
in WO 2012/027721 (Gilead Biologics).
[0248] PI3K inhibitors include inhibitors of PI3K.gamma.,
PI3K.delta., PI3K.beta., PI3K.alpha., and/or pan-PI3K. Examples of
PI3K inhibitors include, but are not limited to, wortmannin,
BKM120, CH5132799, XL756, and GDC-0980.
[0249] Examples of PI3K.gamma. inhibitors include, but are not
limited to, ZSTK474, AS252424, LY294002, and TG100115.
[0250] Examples of PI3K.delta. inhibitors include, but are not
limited to, PI3K II, TGR-1202, AMG-319, GSK2269557, X-339, X-414,
RP5090, KAR4141, XL499, OXY111A, IPI-145, IPI-443, and the
compounds described in WO 2005/113556 (ICOS), WO 2013/052699
(Gilead Calistoga), WO 2013/116562 (Gilead Calistoga), WO
2014/100765 (Gilead Calistoga), WO 2014/100767 (Gilead Calistoga),
and WO 2014/201409 (Gilead Sciences).
[0251] Examples of PI3K.beta. inhibitors include, but are not
limited to, GSK2636771, BAY 10824391, and TGX221.
[0252] Examples of PI3K.alpha. inhibitors include, but are not
limited to, buparlisib, BAY 80-6946, BYL719, PX-866, RG7604,
MLN1117, WX-037, AEZA-129, and PA799.
[0253] Examples of pan-PI3K inhibitors include, but are not limited
to, LY294002, BEZ235, XL147 (SAR245408), and GDC-0941.
[0254] Examples of SYK inhibitors include, but are not limited to,
tamatinib (R406), fostamatinib (R788), PRT062607, BAY-61-3606,
NVP-QAB 205 AA, R112, R343, and those described in U.S. Pat. No.
8,450,321 (Gilead Connecticut).
[0255] TKIs may target epidermal growth factor receptors (EGFRs)
and receptors for fibroblast growth factor (FGF), platelet-derived
growth factor (PDGF), and vascular endothelial growth factor
(VEGF). Examples of TKIs that target EGFR include, but are not
limited to, gefitinib and erlotinib. Sunitinib is a non-limiting
example of a TKI that targets receptors for FGF, PDGF, and
VEGF.
[0256] The anti-PD-L1 antibodies of the present disclosure can be
used, in some embodiments, together with an immune checkpoint
inhibitor. Immune checkpoints are molecules in the immune system
that either turn up a signal (co-stimulatory molecules) or turn
down a signal (co-inhibitory molecules). Many cancers protect
themselves from the immune system by inhibiting the T cell signal
through agonist for co-inhibitory molecules or antagonist for
co-stimulatory molecules. An immune checkpoint agonist or
antagonist can help stop such a protective mechanism by the cell
cells. An immune checkpoint agonist or antagonist may target any
one or more of the following checkpoint molecules, PD-1, CTLA-4,
LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as
CD137), TIM3, OX-40/OX40L, CD40/CD40L, LIGHT, ICOS/ICOSL,
GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also
known as CD272).
[0257] Programmed T cell death 1 (PD-1) is a trans-membrane protein
found on the surface of T cells, which, when bound to programmed T
cell death ligand 1 (PD-L1) on tumor cells, results in suppression
of T cell activity and reduction of T cell-mediated cytotoxicity.
Thus, PD-1 and PD-L1 are immune down-regulators or immune
checkpoint "off switches". Example PD-1 inhibitor include, without
limitation, nivolumab, (Opdivo) (BMS-936558), pembrolizumab
(Keytruda), pidilizumab, AMP-224, MEDI0680 (AMP-514), PDR001,
MPDL3280A, MEDI4736, BMS-936559 and MSB0010718C.
[0258] CTLA-4 is a protein receptor that downregulates the immune
system. Non-limiting examples of CTLA-4 inhibitors include
ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX-101)
and tremelimumab (formerly ticilimumab, CP-675,206).
[0259] Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint
receptor on the cell surface works to suppress an immune response
by action to Tregs as well as direct effects on CD8+ T cells. LAG-3
inhibitors include, without limitation, LAG525 and BMS-986016.
[0260] CD28 is constitutively expressed on almost all human CD4+ T
cells and on around half of all CD8 T cells. prompts T cell
expansion. Non-limiting examples of CD28 inhibitors include
TGN1412.
[0261] CD122 increases the proliferation of CD8+ effector T cells.
Non-limiting examples include NKTR-214.
[0262] 4-1BB (also known as CD137) is involved in T-cell
proliferation. CD137-mediated signaling is also known to protect T
cells, and in particular, CD8+ T cells from activation-induced cell
death. PF-05082566, Urelumab (BMS-663513) and lipocalin are example
CD137 inhibitors.
[0263] For any of the above combination treatments, the anti-PD-L1
antibody can be administered concurrently or separately from the
other anticancer agent. When administered separately, the
anti-PD-L1 antibody can be administered before or after the other
anticancer agent.
Treatment of Infections
[0264] As demonstrated in the experimental examples, the antibodies
of the present disclosure can activate immune response which can
then be useful for treating infections.
[0265] Infection is the invasion of an organism's body tissues by
disease-causing agents, their multiplication, and the reaction of
host tissues to these organisms and the toxins they produce. An
infection can be caused by infectious agents such as viruses,
viroids, prions, bacteria, nematodes such as parasitic roundworms
and pinworms, arthropods such as ticks, mites, fleas, and lice,
fungi such as ringworm, and other macroparasites such as tapeworms
and other helminths. In one aspect, the infectious agent is a
bacterium, such as Gram negative bacterium. In one aspect, the
infectious agent is virus, such as DNA viruses, RNA viruses, and
reverse transcribing viruses. Non-limiting examples of viruses
include Adenovirus, Coxsackievirus, Epstein-Barr virus, Hepatitis A
virus, Hepatitis B virus, Hepatitis C virus, Herpes simplex virus,
type 1, Herpes simplex virus, type 2, Cytomegalovirus, Human
herpesvirus, type 8, HIV, Influenza virus, Measles virus, Mumps
virus, Human papillomavirus, Parainfluenza virus, Poliovirus,
Rabies virus, Respiratory syncytial virus, Rubella virus,
Varicella-zoster virus.
[0266] The antibodies of the present disclosure can also be used to
treat an infectious disease caused by a microorganism, or kill a
microorganism, by targeting the microorganism and an immune cell to
effect elimination of the microorganism. In one aspect, the
microorganism is a virus including RNA and DNA viruses, a Gram
positive bacterium, a Gram negative bacterium, a protozoa or a
fungus. Non-limiting examples of infectious diseases and related
microorganisms are provided in Table 4 below.
TABLE-US-00017 TABLE 4 Infectious diseases and related
microorganism sources. Infectious Disease Microorganism Source
Acinetobacter infections Acinetobacter baumannii Actinomycosis
Actinomyces israelii, Actinomyces gerencseriae and
Propionibacterium propionicus African sleeping sickness Trypanosoma
brucei (African trypanosomiasis) AIDS (Acquired immunodeficiency
syndrome) HIV (Human immunodeficiency virus) Amebiasis Entamoeba
histolytica Anaplasmosis Anaplasma genus Anthrax Bacillus anthracis
Arcanobacterium haemolyticum infection Arcanobacterium haemolyticum
Argentine hemorrhagic fever Junin virus Ascariasis Ascaris
lumbricoides Aspergillosis Aspergillus genus Astrovirus infection
Astroviridae family Babesiosis Babesia genus Bacillus cereus
infection Bacillus cereus Bacterial pneumonia multiple bacteria
Bacterial vaginosis (BV) multiple bacteria Bacteroides infection
Bacteroides genus Balantidiasis Balantidium coli Baylisascaris
infection Baylisascaris genus BK virus infection BK virus Black
piedra Piedraia hortae Blastocystis hominis infection Blastocystis
hominis Blastomycosis Blastomyces dermatitidis Bolivian hemorrhagic
fever Machupo virus Borrelia infection Borrelia genus Botulism (and
Infant botulism) Clostridium botulinum Brazilian hemorrhagic fever
Sabia Brucellosis Brucella genus Burkholderia infection usually
Burkholderia cepacia and other Burkholderia species Buruli ulcer
Mycobacterium ulcerans Calicivirus infection Caliciviridae family
(Norovirus and Sapovirus) Campylobacteriosis Campylobacter genus
Candidiasis (Moniliasis; Thrush) usually Candida albicans and other
Candida species Cat-scratch disease Bartonella henselae Cellulitis
usually Group A Streptococcus and Staphylococcus Chagas Disease
(American trypanosomiasis) Trypanosoma cruzi Chancroid Haemophilus
ducreyi Chickenpox Varicella zoster virus (VZV) Chlamydia Chlamydia
trachomatis Chlamydophila pneumoniae infection Chlamydophila
pneumoniae Cholera Vibrio cholerae Chromoblastomycosis usually
Fonsecaea pedrosoi Clonorchiasis Clonorchis sinensis Clostridium
difficile infection Clostridium difficile Coccidioidomycosis
Coccidioides immitis and Coccidioides posadasii Colorado tick fever
(CTF) Colorado tick fever virus (CTFV) Common cold (Acute viral
rhinopharyngitis; usually rhinoviruses and coronaviruses. Acute
coryza) Creutzfeldt-Jakob disease (CJD) CJD prion Crimean-Congo
hemorrhagic fever Crimean-Congo hemorrhagic fever virus (CCHF)
Cryptococcosis Cryptococcus neoformans Cryptosporidiosis
Cryptosporidium genus Cutaneous larva migrans (CLM) usually
Ancylostoma braziliense; multiple other parasites Cyclosporiasis
Cyclospora cayetanensis Cysticercosis Taenia solium Cytomegalovirus
infection Cytomegalovirus Dengue fever Dengue viruses (DEN-1,
DEN-2, DEN-3 and DEN-4)- Flaviviruses Dientamoebiasis Dientamoeba
fragilis Diphtheria Corynebacterium diphtheriae Diphyllobothriasis
Diphyllobothrium Dracunculiasis Dracunculus medinensis Ebola
hemorrhagic fever Ebolavirus (EBOV) Echinococcosis Echinococcus
genus Ehrlichiosis Ehrlichia genus Enterobiasis (Pinworm infection)
Enterobius vermicularis Enterococcus infection Enterococcus genus
Enterovirus infection Enterovirus genus Epidemic typhus Rickettsia
prowazekii Erythema infectiosum (Fifth disease) Parvovirus B19
Exanthem subitum (Sixth disease) Human herpesvirus 6 (HHV-6) and
Human herpesvirus 7 (HHV-7) Fasciolopsiasis Fasciolopsis buski
Fasciolosis Fasciola hepatica and Fasciola gigantica Fatal familial
insomnia (FFI) FFI prion Filariasis Filarioidea superfamily Food
poisoning by Clostridium perfringens Clostridium perfringens
Free-living amebic infection multiple Fusobacterium infection
Fusobacterium genus Gas gangrene (Clostridial myonecrosis) usually
Clostridium perfringens; other Clostridium species Geotrichosis
Geotrichum candidum Gerstmann-Straussler-Scheinker GSS prion
syndrome (GSS) Giardiasis Giardia intestinalis Glanders
Burkholderia mallei Gnathostomiasis Gnathostoma spinigerum and
Gnathostoma hispidum Gonorrhea Neisseria gonorrhoeae Granuloma
inguinale (Donovanosis) Klebsiella granulomatis Group A
streptococcal infection Streptococcus pyogenes Group B
streptococcal infection Streptococcus agalactiae Haemophilus
influenzae infection Haemophilus influenzae Hand, foot and mouth
disease Enteroviruses, mainly Coxsackie A virus and Enterovirus
(HFMD) 71 (EV71) Hantavirus Pulmonary Syndrome (HPS) Sin Nombre
virus Helicobacter pylori infection Helicobacter pylori
Hemolytic-uremic syndrome (HUS) Escherichia coli O157:H7, O111 and
O104:H4 Hemorrhagic fever with renal Bunyaviridae family syndrome
(HFRS) Hepatitis A Hepatitis A Virus Hepatitis B Hepatitis B Virus
Hepatitis C Hepatitis C Virus Hepatitis D Hepatitis D Virus
Hepatitis E Hepatitis E Virus Herpes simplex Herpes simplex virus 1
and 2 (HSV-1 and HSV-2) Histoplasmosis Histoplasma capsulatum
Hookworm infection Ancylostoma duodenale and Necator americanus
Human bocavirus infection Human bocavirus (HBoV) Human ewingii
ehrlichiosis Ehrlichia ewingii Human granulocytic anaplasmosis
Anaplasma phagocytophilum (HGA) Human metapneumovirus infection
Human metapneumovirus (hMPV) Human monocytic ehrlichiosis Ehrlichia
chaffeensis Human papillomavirus (HPV) Human papillomavirus (HPV)
infection Human parainfluenza virus infection Human parainfluenza
viruses (HPIV) Hymenolepiasis Hymenolepis nana and Hymenolepis
diminuta Epstein-Barr Virus Infectious Epstein-Barr Virus (EBV)
Mononucleosis (Mono) Influenza (flu) Orthomyxoviridae family
Isosporiasis Isospora belli Kawasaki disease unknown; evidence
supports that it is infectious Keratitis multiple Kingella kingae
infection Kingella kingae Kuru Kuru prion Lassa fever Lassa virus
Legionellosis (Legionnaires' disease) Legionella pneumophila
Legionellosis (Pontiac fever) Legionella pneumophila Leishmaniasis
Leishmania genus Leprosy Mycobacterium leprae and Mycobacterium
lepromatosis Leptospirosis Leptospira genus Listeriosis Listeria
monocytogenes Lyme disease (Lyme borreliosis) usually Borrelia
burgdorferi and other Borrelia species Lymphatic filariasis
(Elephantiasis) Wuchereria bancrofti and Brugia malayi Lymphocytic
choriomeningitis Lymphocytic choriomeningitis virus (LCMV) Malaria
Plasmodium genus Marburg hemorrhagic fever (MHF) Marburg virus
Measles Measles virus Melioidosis (Whitmore's disease) Burkholderia
pseudomallei Meningitis multiple Meningococcal disease Neisseria
meningitidis Metagonimiasis usually Metagonimus yokagawai
Microsporidiosis Microsporidia phylum Molluscum contagiosum (MC)
Molluscum contagiosum virus (MCV) Mumps Mumps virus Murine typhus
(Endemic typhus) Rickettsia typhi Mycoplasma pneumonia Mycoplasma
pneumoniae Mycetoma numerous species of bacteria (Actinomycetoma)
and fungi (Eumycetoma) Myiasis parasitic dipterous fly larvae
Neonatal conjunctivitis most commonly Chlamydia trachomatis and
Neisseria (Ophthalmia neonatorum) gonorrhoeae (New) Variant
Creutzfeldt-Jakob vCID prion disease (vCJD, nvCJD) Nocardiosis
usually Nocardia asteroides and other Nocardia species
Onchocerciasis (River blindness) Onchocerca volvulus
Paracoccidioidomycosis Paracoccidioides brasiliensis (South
American blastomycosis) Paragonimiasis usually Paragonimus
westermani and other Paragonimus species Pasteurellosis Pasteurella
genus Pediculosis capitis (Head lice) Pediculus humanus capitis
Pediculosis corporis (Body lice) Pediculus humanus corporis
Pediculosis pubis (Pubic lice, Crab lice) Phthirus pubis Pelvic
inflammatory disease (PID) multiple Pertussis (Whooping cough)
Bordetella pertussis Plague Yersinia pestis Pneumococcal infection
Streptococcus pneumoniae Pneumocystis pneumonia (PCP) Pneumocystis
jirovecii Pneumonia multiple Poliomyelitis Poliovirus Prevotella
infection Prevotella genus Primary amoebic usually Naegleria
fowleri meningoencephalitis (PAM) Progressive multifocal JC virus
leukoencephalopathy Psittacosis Chlamydophila psittaci Q fever
Coxiella burnetii Rabies Rabies virus Rat-bite fever
Streptobacillus moniliformis and Spirillum minus Respiratory
syncytial virus infection Respiratory syncytial virus (RSV)
Rhinosporidiosis Rhinosporidium seeberi Rhinovirus infection
Rhinovirus Rickettsial infection Rickettsia genus Rickettsialpox
Rickettsia akari Rift Valley fever (RVF) Rift Valley fever virus
Rocky mountain spotted fever (RMSF) Rickettsia rickettsii Rotavirus
infection Rotavirus Rubella Rubella virus Salmonellosis Salmonella
genus SARS (Severe Acute Respiratory Syndrome) SARS coronavirus
Scabies Sarcoptes scabiei Schistosomiasis Schistosoma genus Sepsis
multiple Shigellosis (Bacillary dysentery) Shigella genus Shingles
(Herpes zoster) Varicella zoster virus (VZV) Smallpox (Variola)
Variola major or Variola minor Sporotrichosis Sporothrix schenckii
Staphylococcal food poisoning Staphylococcus genus Staphylococcal
infection Staphylococcus genus Strongyloidiasis Strongyloides
stercoralis Syphilis Treponema pallidum Taeniasis Taenia genus
Tetanus (Lockjaw) Clostridium tetani Tinea barbae (Barber's itch)
usually Trichophyton genus Tinea capitis (Ringworm of the Scalp)
usually Trichophyton tonsurans Tinea corporis (Ringworm of the
Body) usually Trichophyton genus Tinea cruris (Jock itch) usually
Epidermophytonfloccosum, Trichophyton rubrum, and Trichophyton
mentagrophytes Tinea manuum (Ringworm of the Hand) Trichophyton
rubrum Tinea nigra usually Hortaea werneckii Tinea pedis (Athlete's
foot) usually Trichophyton genus Tinea unguium (Onychomycosis)
usually Trichophyton genus Tinea versicolor (Pityriasis versicolor)
Malassezia genus Toxocariasis (Ocular Larva Migrans (OLM)) Toxocara
canis or Toxocara cati Toxocariasis (Visceral Larva Migrans (VLM))
Toxocara canis or Toxocara cati Toxoplasmosis Toxoplasma gondii
Trichinellosis Trichinella spiralis Trichomoniasis Trichomonas
vaginalis Trichuriasis (Whipworm infection) Trichuris trichiura
Tuberculosis usually Mycobacterium tuberculosis Tularemia
Francisella tularensis Ureaplasma urealyticum infection Ureaplasma
urealyticum Venezuelan equine encephalitis Venezuelan equine
encephalitis virus Venezuelan hemorrhagic fever Guanarito virus
Viral pneumonia multiple viruses West Nile Fever West Nile virus
White piedra (Tinea blanca) Trichosporon beigelii
Yersinia pseudotuberculosis infection Yersinia pseudotuberculosis
Yersiniosis Yersinia enterocolitica Yellow fever Yellow fever virus
Zygomycosis Mucorales order (Mucormycosis) and Entomophthorales
order (Entomophthoramycosis)
[0267] A specific dosage and treatment regimen for any particular
patient will depend upon a variety of factors, including the
particular antibodies, variant or derivative thereof used, the
patient's age, body weight, general health, sex, and diet, and the
time of administration, rate of excretion, drug combination, and
the severity of the particular disease being treated. Judgment of
such factors by medical caregivers is within the ordinary skill in
the art. The amount will also depend on the individual patient to
be treated, the route of administration, the type of formulation,
the characteristics of the compound used, the severity of the
disease, and the desired effect. The amount used can be determined
by pharmacological and pharmacokinetic principles well known in the
art.
[0268] Methods of administration of the antibodies, variants or
include but are not limited to intradermal, intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, epidural,
and oral routes. The antigen-binding polypeptides or compositions
may be administered by any convenient route, for example by
infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.) and may be administered together with other
biologically active agents. Thus, pharmaceutical compositions
containing the antigen-binding polypeptides of the disclosure may
be administered orally, rectally, parenterally, intracistemally,
intravaginally, intraperitoneally, topically (as by powders,
ointments, drops or transdermal patch), bucally, or as an oral or
nasal spray.
[0269] The term "parenteral" as used herein refers to modes of
administration which include intravenous, intramuscular,
intraperitoneal, intrasternal, subcutaneous and intra-articular
injection and infusion.
[0270] Administration can be systemic or local. In addition, it may
be desirable to introduce the antibodies of the disclosure into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0271] It may be desirable to administer the antibodies
polypeptides or compositions of the disclosure locally to the area
in need of treatment; this may be achieved by, for example, and not
by way of limitation, local infusion during surgery, topical
application, e.g., in conjunction, with a wound dressing after
surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the disclosure,
care must be taken to use materials to which the protein does not
absorb.
[0272] In another embodiment, the antibodies or composition can be
delivered in a vesicle, in particular a liposome (see Langer, 1990,
Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0273] In yet another embodiment, the antigen-binding polypeptide
or composition can be delivered in a controlled release system. In
one embodiment, a pump may be used (see Sefton, 1987, CRC Crit.
Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507;
Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
N.Y. (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev.
Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190;
During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.
Neurosurg. 71:105). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)). Other controlled release
systems are discussed in the review by Langer (1990, Science
249:1527-1533).
[0274] In a specific embodiment where the composition of the
disclosure comprises a nucleic acid or polynucleotide encoding a
protein, the nucleic acid can be administered in vivo to promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic acid expression vector and administering it so
that it becomes intracellular, e.g., by use of a retroviral vector
(see U .S . Pat. No. 4,980,286), or by direct injection, or by use
of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont),
or coating with lipids or cell-surface receptors or transfecting
agents, or by administering it in linkage to a homeobox-like
peptide which is known to enter the nucleus (see, e.g., Joliot et
al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.
Alternatively, a nucleic acid can be introduced intracellularly and
incorporated within host cell DNA for expression, by homologous
recombination.
[0275] The amount of the antibodies of the disclosure which will be
effective in the treatment, inhibition and prevention of an
inflammatory, immune or malignant disease, disorder or condition
can be determined by standard clinical techniques. In addition, in
vitro assays may optionally be employed to help identify optimal
dosage ranges. The precise dose to be employed in the formulation
will also depend on the route of administration, and the
seriousness of the disease, disorder or condition, and should be
decided according to the judgment of the practitioner and each
patient's circumstances. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0276] As a general proposition, the dosage administered to a
patient of the antigen-binding polypeptides of the present
disclosure is typically 0.1 mg/kg to 100 mg/kg of the patient's
body weight, between 0.1 mg/kg and 20 mg/kg of the patient's body
weight, or 1 mg/kg to 10 mg/kg of the patient's body weight.
Generally, human antibodies have a longer half-life within the
human body than antibodies from other species due to the immune
response to the foreign polypeptides. Thus, lower dosages of human
antibodies and less frequent administration is often possible.
Further, the dosage and frequency of administration of antibodies
of the disclosure may be reduced by enhancing uptake and tissue
penetration (e.g., into the brain) of the antibodies by
modifications such as, for example, lipidation.
[0277] The methods for treating an infectious or malignant disease,
condition or disorder comprising administration of an antibody,
variant, or derivative thereof of the disclosure are typically
tested in vitro, and then in vivo in an acceptable animal model,
for the desired therapeutic or prophylactic activity, prior to use
in humans. Suitable animal models, including transgenic animals,
are well known to those of ordinary skill in the art. For example,
in vitro assays to demonstrate the therapeutic utility of
antigen-binding polypeptide described herein include the effect of
an antigen-binding polypeptide on a cell line or a patient tissue
sample. The effect of the antigen-binding polypeptide on the cell
line and/or tissue sample can be determined utilizing techniques
known to those of skill in the art, such as the assays disclosed
elsewhere herein. In accordance with the disclosure, in vitro
assays which can be used to determine whether administration of a
specific antigen-binding polypeptide is indicated, include in vitro
cell culture assays in which a patient tissue sample is grown in
culture, and exposed to or otherwise administered a compound, and
the effect of such compound upon the tissue sample is observed.
[0278] Various delivery systems are known and can be used to
administer an antibody of the disclosure or a polynucleotide
encoding an antibody of the disclosure, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Diagnostic Methods
[0279] Over-expression of PD-L1 is observed in certain tumor
samples, and patients having PD-L1-over-expressing cells are likely
responsive to treatments with the anti-PD-L1 antibodies of the
present disclosure. Accordingly, the antibodies of the present
disclosure can also be used for diagnostic and prognostic
purposes.
[0280] A sample that preferably includes a cell can be obtained
from a patient, which can be a cancer patient or a patient desiring
diagnosis. The cell be a cell of a tumor tissue or a tumor block, a
blood sample, a urine sample or any sample from the patient. Upon
optional pre-treatment of the sample, the sample can be incubated
with an antibody of the present disclosure under conditions
allowing the antibody to interact with a PD-L1 protein potentially
present in the sample. Methods such as ELISA can be used, taking
advantage of the anti-PD-L1 antibody, to detect the presence of the
PD-L1 protein in the sample.
[0281] Presence of the PD-L1 protein in the sample (optionally with
the amount or concentration) can be used for diagnosis of cancer,
as an indication that the patient is suitable for a treatment with
the antibody, or as an indication that the patient has (or has not)
responded to a cancer treatment. For a prognostic method, the
detection can be done at once, twice or more, at certain stages,
upon initiation of a cancer treatment to indicate the progress of
the treatment.
Compositions
[0282] The present disclosure also provides pharmaceutical
compositions. Such compositions comprise an effective amount of an
antibody, and an acceptable carrier. In some embodiments, the
composition further includes a second anticancer agent (e.g., an
immune checkpoint inhibitor).
[0283] In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. Further, a "pharmaceutically acceptable
carrier" will generally be a non-toxic solid, semisolid or liquid
filler, diluent, encapsulating material or formulation auxiliary of
any type.
[0284] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the therapeutic is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents such as acetates,
citrates or phosphates. Antibacterial agents such as benzyl alcohol
or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; and agents for the adjustment of tonicity such as sodium
chloride or dextrose are also envisioned. These compositions can
take the form of solutions, suspensions, emulsion, tablets, pills,
capsules, powders, sustained-release formulations and the like. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical carriers are described in Remington's Pharmaceutical
Sciences by E. W. Martin, incorporated herein by reference. Such
compositions will contain a therapeutically effective amount of the
antigen-binding polypeptide, preferably in purified form, together
with a suitable amount of carrier so as to provide the form for
proper administration to the patient. The formulation should suit
the mode of administration. The parental preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0285] In an embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0286] The compounds of the disclosure can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
EXAMPLES
Example 1
Generation of Human Monoclonal Antibodies Against Human PD-L1
[0287] Anti-human-PD-L1 mouse monoclonal antibodies were generated
using the hybridoma technology.
[0288] Antigen: human PDL1-Fc protein and human PD-L1 highly
expressed CHOK1 cell line (PDL1-CHOK1 cell line).
[0289] Immunization: To generate mouse monoclonal antibodies to
human PD-L1, 6-8 week female BALB/c mice were firstly immunized
with 1.5.times.10.sup.7 PDL1-CHOK1 cells. Day 14 and 33 post first
immunization, the immunized mice were re-immunized with
1.5.times.10.sup.7 PDL1-CHOK1 cells respectively. To select mice
producing antibodies that bond PD-L1 protein, sera from immunized
mice were tested by ELISA. Briefly, microtiter plates were coated
with human PD-L1 protein at 1 .mu.g/ml in PBS, 100 .mu.l/well at
room temperature (RT) overnight, then blocked with 100 .mu.l/well
of 5% BSA. Dilutions of plasma from immunized mice were added to
each well and incubated for 1-2 hours at RT. The plates were washed
with PBS/Tween and then incubate with anti-mouse IgG antibody
conjugated with Horse Radish Peroxidase (HRP) for 1 hour at RT.
After washing, the plates were developed with ABTS substrate and
analyzed by spectrophotometer at OD 405 nm. Mice with sufficient
titers of anti-PDL1 IgG were boosted with 50 .mu.g human PDL1-Fc
protein at Day 54 post-immunization. The resulting mice were used
for fusions. The hybridoma supernatants were tested for anti-PD-L1
IgGs by ELISA.
[0290] Hybridoma clones HL1210-3, HL1207-3, HL1207-9 and HL1120-3
were selected for further analysis. The amino acid and
polynucleotide sequences of the variable regions of HL1210-3 are
provided in Table 5 below.
TABLE-US-00018 TABLE 5 HL1210-3 variable sequences Name Sequence
SEQ ID NO: HL1210-3 VH GAAGTGAAACTGGTGGAGTCTGGGGGAGACTTAGTGAAGC 112
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATT
CACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGACT
CCGGAGAAGAGTCTGGAGTGGGTCGCAACCATTAGTGATG
GTGGTGGTTACATCTACTATTCAGACAGTGTGAAGGGGCG
ATTTACCATCTCCAGAGACAATGCCAAGAACAACCTGTAC
CTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGT
ATATTTGTGCAAGAGAATTTGGTAAGCGCTATGCTTTGGA
CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA HL1210-3 VH
EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYDMSWVRQT 113
PEKSLEWVATISDGGGYIYYSDSVKGRFTISRDNAKNNLY
LQMSSLRSEDTALYICAREFGKRYALDYWGQGTSVT HL1210-3 VL
GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACAT 114
CGGTAGGAGACAGGGTCAGCATCTCCTGCAAGGCCAGTCA
GGATGTGACTCCTGCTGTCGCCTGGTATCAACAGAAGCCA
GGACAATCTCCTAAACTACTGATTTACTCCACATCCTCCC
GGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATC
TGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCT
GAAGACCTGGCAGTTTATTACTGTCAGCAACATTATACTA
CTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAA A HL1210-3 VL
DIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAWYQQKP 115
GQSPKLLIYSTSSRYTGVPDRFTGSGSGTDFTFTISSVQA
EDLAVYYCQQHYTTPLTFGAGTKLELK
Example 2
HL1210-3 Mouse Monoclonal Antibody's Binding Activity for Human
PD-L1
[0291] To evaluate the binding activity of hybridoma clone
HL1210-3, the purified mAb from this clone were subjected to ELISA
test. Briefly, microtiter plates were coated with human PD-L1-Fc
protein at 0.1 .mu.g/ml in PBS, 100 .mu.l/well at 4.degree. C.
overnight, then blocked with 100 .mu.l/well of 5% BSA. Three-fold
dilutions of HL1210-3 antibodies starting from 0.2 .mu.g/ml were
added to each well and incubated for 1-2 hours at RT. The plates
were washed with PBS/Tween and then incubate with goat-anti-mouse
IgG antibody conjugated with Horse Radish Peroxidase (HRP) for 1
hour at RT. After washing, the plates were developed with TMB
substrate and analyzed by spectrophotometer at OD 450-630 nm. As
shown in FIG. 1, HL1210-3 can bind to human PD-L1 with high
activity (EC.sub.50=5.539 ng/ml).
Example 3
HL1210-3 Mouse mAb Blocked Human PD-L1 Binding to its Receptor
PD-1
Receptor Blocking Assay by Using Recombinant Human PD-L1
[0292] To evaluate the blocking effect of HL1210-3 mouse mAb on
recombinant human PD-L1 to bind to its receptor PD-1, the ELISA
based receptor blocking assay was employed. Briefly, microtiter
plates were coated with human PD-L1-Fc protein at 1 .mu.g/ml in
PBS, 100 .mu.l/well at 4.degree. C. overnight, then blocked with
100 .mu.l/well of 5% BSA. 50 .mu.l biotin-labeled human PD-1-Fc
protein and 3-fold dilutions of HL1210-3 antibodies starting from 2
.mu.g/ml at 50 .mu.l were added to each well and incubated for 1
hour at 37.degree. C. The plates were washed with PBS/Tween and
then incubated with Streptavidin-HRP for 1 hour at 37.degree. C.
After washing, the plates were developed with TMB substrate and
analyzed by spectrophotometer at OD 450-630 nm. As shown in FIG. 2,
HL1210-3 can efficiently inhibit the binding of human PD-L1 to
human PD1 at IC.sub.50=0.7835 nM.
Receptor Blocking Assay by Using Mammalian Cell Expressed Human
PD-L1
[0293] To evaluate the blocking effect of HL1210-3 mouse mAb on
human PD-L1 expressed on mammalian cells to bind to its receptor
PD-1, the FACS-based receptor blocking assay was used. Briefly,
PDL1-CHOK1 cells were firstly incubated with 3-fold serious diluted
HL1210-3 mouse mAb starting at 20 .mu.g/ml at RT for 1 hour. After
wash by FACS buffer (PBS with 2% FBS), the biotin-labeled huPD-1
were added to each well and incubated at RT for 1 hour. Then, the
Streptavidin-PE were added to each well for 0.5 hour post twice
wash with FACS buffer. The mean florescence intensity (MFI)of PE
were evaluated by FACSAriaIII. As shown in FIG. 3, the HL1210-3
antibody can highly efficiently inhibit the binding of PD-1 on
PD-L1 expressed on mammalian cells at IC50 of 2.56 nM with 92.6%
top inhibition rate.
% .times. .times. of .times. .times. inhibition = ( 1 - MFI .times.
.times. of .times. .times. testing .times. .times. antibody MFI
.times. .times. of .times. .times. vehicle .times. .times. contorl
) .times. 1 .times. 0 .times. 0 .times. % ##EQU00001##
Example 4
HL1210-3 Mouse mAb Promoted Human T Cell Immune Response
[0294] To evaluate the effect of HL1210-3 mouse mAb, the response
of human T cells assessed in a mixed lymphocyte reaction setting.
Human DCs were differentiated from CD14+ monocytes in the presence
of GM-CSF and IL-4 for 7 days. CD4+ T cells isolated from another
donor were then co-cultured with the DCs and serial dilutions of
anti-PD-L1 blocking antibody. At day 5 post-inoculation, the
culture supernatant was assayed for IFN.gamma. production. The
results indicated that the HL1210-3 antibodies can dose-dependently
promote IFN.gamma. production, suggesting anti-PD-L1 antibody can
promote human T cell response (FIG. 4).
Example 5
The Binding Affinity of HL1210-3 Mouse mAb
[0295] The binding of the HL1210-3 antibodies to recombinant PD-L1
protein (human PD-L1-his taq) was tested with BIACORE.TM. using a
capture method. The HL1210-3 mouse mAb was captured using
anti-mouse Fc antibody coated on a CM5 chip. A series dilution of
human PD-L1-his taq protein was injected over captured antibody for
3 mins at a flow rate of 25 .mu.g/ml. The antigen was allowed to
dissociate for 900s. All the experiment were carried out on a
Biacore T200. Data analysis was carried out using Biacore T200
evaluation software. The result are shown in FIG. 5 and Table 6
below.
TABLE-US-00019 TABLE 6 Binding Kinetics of HL1210-3 to recombinant
human PD-L1 ka kd KD Antibody (1/Ms) (1/s) (M) HL1210-3 1.61E+05
4.69E-05 2.93E-10
Example 6
Humanization of the HL1210-3 Mouse mAb
[0296] The mAb HL1210-3 variable region genes were employed to
create a humanized MAb. In the first step of this process, the
amino acid sequences of the VH and VK of MAb HL1210-3 were compared
against the available database of human Ig gene sequences to find
the overall best-matching human germline Ig gene sequences. For the
light chain, the closest human match was the O18/Jk2 and
KV1-39*01/KJ2*04 gene, and for the heavy chain the closest human
match was the VH3-21 gene. VH3-11, VH3-23, VH3-7*01 and VH3-48
genes were also selected due to their close matches.
[0297] Humanized variable domain sequences were then designed where
the CDR1 (SEQ ID NO.4), 2 (SEQ ID NO.5) and 3 (SEQ ID NO.6) of the
HL1210-3 light chain were grafted onto framework sequences of the
O18/Jk2 and KV1-39*01/KJ2*04 gene, and the CDR1 (SEQ ID NO.1), 2
(SEQ ID NO.2), and 3 (SEQ ID NO.3) sequences of the HL1210-3 VH
were grafted onto framework sequences of the VH3-21, VH3-11,
VH3-23, VH3-48 or VH3-7*01 gene. A 3D model was then generated to
determine if there were any framework positions where replacing the
mouse amino acid to the human amino acid could affect binding
and/or CDR conformation. In the case of the light chain, 22S, 43S,
60D, 63T and 42Q (Kabat numbering, see Table 7) in framework were
identified. In the case of the heavy chain, 1E, 37V, 40T, 44S, 49A,
77N, 91I, 94R and 108T in the framework was involved in
back-mutations.
TABLE-US-00020 TABLE 7 Humanization Design Construct Mutation VH
Design I: VH3-21/JH6 Hu1210 VH Chimera Hu1210 VH.1 CDR-grafted
Hu1210 VH.1a S49A Hu1210 VH.1b S49A, G44S, Y91I VH Design II:
VH3-11/JH6 Hu1210 VH.2 CDR-grafted, Q1E Hu1210 VH.2a Q1E, S49A
Hu1210 VH.2b Q1E, I37V, S49A, G44S, Y91I VH Design III: VH3-23/JH6
Hu1210 VH.3 CDR-grafted, K94R Hu1210 VH.3a G44S, S49A, Y91I, K94R
VH Design IV: VH3-48/JH6 Hu1210 VH.4 CDR-grafted Hu1210 VH.4a S49A
Hu1210 VH.4b S49A, G44S, Y91I Hu1210 VH.4c D52E, S49A, G44S, Y91I
Hu1210 VH.4d G53A, S49A, G44S, Y91I Hu1210 VH.4e G53V, S49A, G44S,
Y91I VH Design V: VH3-7*01/ HJ1*01 Hu1210 VH.5 CDR-grafted Hu1210
VH.5a H91I Hu1210 VH.5b H91I, H108T Hu1210 VH.5c H91I, H77N Hu1210
VH.5d H91I, H77N, H40T VK Design I: 018/Jk2 Construct Mutation
Hu1210 Vk Chimera Hu1210 Vk.1 CDR-grafted Hu1210 Vk.1a A43 S VK
Design II: KV1-39*01/KJ2*04 Hu1210 Vk.2 CDR-grafted Hu1210 Vk.2a
L60D, L63T Hu1210 Vk.2b L60D, L63T, L42Q, L43S Hu1210 Vk.2c L60D,
L63T, L42Q, L43S, T22S
[0298] The amino acid and nucleotide sequences of some of the
humanized antibody are listed in Table 8 below.
TABLE-US-00021 TABLE 8 Humanized antibody sequences (bold indicates
CDR) SEQ ID Name Sequence NO: HL1210-VH
EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYDMSWVRQTPEKSLEWVAT 7
ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYICAREF
GKRYALDYWGQGTSVTVSS Hu1210 VH.1
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST 8
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.1a
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 9
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.1b
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 10
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.2
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWIRQAPGKGLEWVST 11
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.2a
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWIRQAPGKGLEWVAT 12
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.2b
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 13
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.3
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST 14
ISDGGGYIYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.3a
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 15
ISDGGGYIYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYICAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.4
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST 16
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.4a
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 17
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.4b
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 18
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.4c
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 19
ISEGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.4d
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 20
ISDAGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.4e
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 21
ISDVGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF
GKRYALDYWGQGTTVTVSS Hu1210 VH.5
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 22
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF
GKRYALDYWGQGTLVTVSS HU1210 VH.5a
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 23
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF
GKRYALDYWGQGTLVTVSS HU1210 VH.5b
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 24
ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF
GKRYALDYWGQGTTVTVSS HU1210 VH.5C
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 25
ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMNSLRAEDTAVYICAREF
GKRYALDYWGQGTLVTVSS HU1210 VH.5d
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQTPEKSLEWVAT 26
ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMNSLRAEDTAVYICAREF
GKRYALDYWGQGTLVTVSS HL1210-VK
DIVMTQSNKFMSTSVGDRVSISCKASQDVTPAVAWYQQKPGQSPKLLIYS 27
TSSRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYTTPLTFGA GTKLELK Hu1210
VK.1 DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 28
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK Hu1210
VK.1a DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKSPKLLIYS 29
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK Hu1210
Vk.2 DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 30
TSSRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR Hu1210
Vk.2a DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 31
TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR Hu1210
Vk.2b DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGQSPKLLIYS 32
TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR Hu1210
Vk.2c DIQMTQSPSSLSASVGDRVTISCKASQDVTPAVAWYQQKPGQSPKLLIYS 33
TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR HL1210
VH GAGGTGAAGCTGGTGGAGAGCGGCGGAGATCTGGTGAAGCCTGGCGGCAGCCTGAAGCTG 34
AGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGGCAGACC
CCCGAGAAGAGCCTGGAGTGGGTGGCCACCATCAGCGATGGCGGCGGCTACATCTACTAC
AGCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAACCTGTAC
CTGCAGATGAGCAGCCTGAGGAGCGAGGACACCGCCCTGTACATCTGCGCCAGGGAGTTC
GGCAAGAGGTACGCCCTGGACTACTGGGGACAGGGCACCAGCGTGACCGTGAGCAGC Hu1210
VH.1 GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG
35 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.1a GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG
36 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.1b GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG
37 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.2 GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG
38 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGATCAGACAGGCC
CCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.2a GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG
39 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGATCAGACAGGCC
CCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.2b GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG
40 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.3 GAGGTGCAGCTGCTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG
41 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTAC
CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.3a GAGGTGCAGCTGCTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG
42 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTAC
CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.4 GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG
43 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.4a GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG
44 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.4b GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG
45 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.4c GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG
46 AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGAAGGCGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC
Hu1210_VH.4d
GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 47
AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGCGGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC
Hu1210_VH.4e
GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 48
AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCC
CCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGTTGGCGGCTACATCTATTAC
TCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTAC
CTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTC
GGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210
VH.5 GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG
49 TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCT
CCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC
TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTAC
CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATTACTGCGCCAGGGAGTTT
GGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210
VH.5a GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG
50 TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCT
CCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC
TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTAC
CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTT
GGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210
VH.5b
GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG
51 TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCT
CCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC
TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACAACCTGTAC
CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTT
GGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210
VH.5c GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG
52 TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGACC
CCTGAGAAGAGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC
TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACAACCTGTAC
CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTT
GGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC
Hu1210_VH.5d
GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG 53
TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCT
CCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTAC
TCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTAC
CTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTT
GGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACAACCGTGACAGTGAGCTCC HL1210 VK
GACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGATAGGGTGAGC 54
ATCAGCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCC
GGCCAGAGCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCGAC
AGGTTCACAGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCGTGCAGGCC
GAGGACCTGGCCGTGTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCGCC
GGCACCAAGCTGGAGCTGAAG Hu1210 VK.1
GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACC 55
ATCACCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCC
GGCAAGGCCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCAGC
AGGTTTAGCGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCC
GAGGACATCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCCAG
GGCACCAAGCTGGAGATCAAG Hu1210 VK.1a
GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACC 56
ATCACCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCC
GGCAAGTCCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCAGC
AGGTTTAGCGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCC
GAGGACATCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCCAG
GGCACCAAGCTGGAGATCAAG Hu1210 VK.2
GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 57
ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCT
GGCAAGGCTCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCTCC
AGGTTTAGCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCC
GAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAG
GGCACCAAGCTGGAGATCAAGCGG Hu1210 VK.2a
GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 58
ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCT
GGCAAGGCTCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGAC
AGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCC
GAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAG
GGCACCAAGCTGGAGATCAAGCGG Hu1210 VK.2b
GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 59
ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCT
GGCCAGAGCCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGAC
AGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCC
GAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAG
GGCACCAAGCTGGAGATCAAGCGG Hu1210 VK.2c
GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 60
ATCAGCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCT
GGCCAGAGCCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGAC
AGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCC
GAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAG
GGCACCAAGCTGGAGATCAAGCGG
[0299] The humanized VH and VK genes were produced synthetically
and then respectively cloned into vectors containing the human
gamma 1 and human kappa constant domains. The pairing of the human
VH and the human VK created the 40 humanized antibodies (see Table
9).
TABLE-US-00022 TABLE 9 Humanized antibodies with their VH am VL
regions VH Hu1210 Hu1210 Hu1210 Hu1210 Hu1210 Hu1210 Hu1210 Vk VH.1
VH.1a VH.1b VH.2 VH.2a VH 2.b VH Hu1210 Vk.1a Hu1210-1 Hu1210-2
Hu1210-3 Hu1210-4 Hu1210-5 Hu1210 Vk.1a Hu1210-7 Hu1210-8 Hu1210-9
Hu1210-10 Hu1210-11 Hu1210 Vk H1210 chimera VH Hu1210 Hu1210 Hu1210
Hu1210 Hu1210 Vk VH.3 VH.3a VH.4 VH.4a VH.4b Hu1210 Vk.1 Hu1210-13
Hu1210-14 Hu1210-15 Hu1210-16 Hu1210-17 Hu1210 Vk.1a Hu1210-18
Hu1210-19 Hu1210-20 Hu1210-21 Hu1210-22 VH Hu1210 HU1210 HU1210
HU1210 HU1210 VK VH.5 VH.5a VH.5b VH.5c VH.5d Hu1210 Vk.2 Hu1210-23
Hu1210-27 Hu1210-31 Hu1210-32 Hu1210-36 Hu1210 Vk.2a Hu1210-24
Hu1210-28 Hu1210-33 Hu1210-37 Hu1210 Vk.2b Hu1210-25 Hu1210-29
Hu1210-34 Hu1210-38 Hu1210 Vk.2c Hu1210-26 Hu1210-30 Hu1210-35
Hu1210-39 VH Hu1210 Hu1210 Hu1210 Vk VH.4c VH.4d VH.4e Hu1210 Vk.1
Hu1210-40 Hu1210-41 Hu1210-42
Example 7
The Antigen Binding Properties of Humanized PD-L1 Antibodies
Binding Property to Recombinant Human PD-L1
[0300] To evaluate the antigen binding activity, the humanized
antibodies were subjected to ELISA test. Briefly, microtiter plates
were coated with human PD-L1-Fc protein at 0.1 .mu.g/ml in PBS, 100
.mu.l/well at 4.degree. C. overnight, then blocked with 100
.mu.l/well of 5% BSA. Five-fold dilutions of humanized antibodies
starting from 10 .mu.g/ml were added to each well and incubated for
1-2 hours at RT. The plates were washed with PBS/Tween and then
incubate with goat-anti-mouse IgG antibody conjugated with Horse
Radish Peroxidase (HRP) for 1 hour at RT. After washing, the plates
were developed with TMB substrate and analyzed by spectrophotometer
at OD 450-630 nm. As shown in FIG. 6, all the humanized antibodies
show comparable binding efficacy to human PD-L1 in contact to
chimeric antibody.
Binding Property to Mammalian Expressed Human PD-L1
[0301] To evaluate the antigen binding property, the humanized
antibodies were analyzed for its binding to mammalian expressed
PD-L1 by FACS. Briefly, PDL1-CHOK1 cells were firstly incubated
with 5-fold serious diluted humanized antibodies starting at 2
.mu.g/ml at RT for 1 hour. After wash by FACS buffer (PBS with 2%
FBS), the alexa 488-anti-human IgG antibody was added to each well
and incubated at RT for 1 hour. The MFI of Alexa 488 were evaluated
by FACSAriaIII. As shown in the FIG. 7, all the humanized
antibodies can high efficiently bind to PD-L1 expressed on
mammalian cells, which was comparable with chimeric antibody.
[0302] To explore the binding kinetics of the humanized antibody,
this example performed the affinity ranking by using Octet Red 96.
As shown in Table 10, hu1210-3, hu1210-8, hu1210-9, hu1210-14,
hu1210-17, hu1210-1 and Hu1210-22 show better affinity, which is
comparable with chimeric antibody.
TABLE-US-00023 TABLE 10 Affinity ranking of humanized antibodies
Antibody KD (M) kon(1/Ms) kdis(1/s) HU1210 7.16E-09 3.94E+05
2.83E-03 (mIgG) H1210 1.07E-09 1.62E+05 1.73E-04 chimera HU1210-1
4.25E-09 7.10E+04 3.02E-04 HU1210-2 3.23E-09 7.78E+04 2.51E-04
HU1210-3 2.64E-09 8.62E+04 2.28E-04 HU1210-4 7.68E-09 7.12E+04
5.46E-04 HU1210-5 4.83E-09 7.93E+04 3.83E-04 HU1210-7 4.78E-09
8.45E+04 4.04E-04 HU1210-8 1.64E-09 7.72E+04 1.27E-04 HU1210-9
2.33E-09 8.37E+04 1.95E-04 HU1210-10 7.03E-09 8.59E+04 6.04E-04
HU1210-11 4.18E-09 7.54E+04 3.15E-04 HU1210-13 4.36E-09 8.38E+04
3.66E-04 HU1210-14 2.34E-09 8.41E+04 1.97E-04 HU1210-15 4.45E-09
7.87E+04 3.50E-04 HU1210-16 3.14E-09 8.41E+04 2.64E-04 HU1210-17
2.20E-09 8.17E+04 1.80E-04 HU1210-18 4.50E-09 7.92E+04 3.57E-04
HU1210-19 2.50E-09 9.03E+04 2.25E-04 HU1210-20 4.51E-09 8.87E+04
4.00E-04 HU1210-21 3.12E-09 9.39E+04 2.93E-04 HU1210-22 2.56E-09
9.00E+04 2.30E-04
Full Kinetic Affinity of Humanized Antibodies by Biacore.RTM.
[0303] The binding of the humanized antibodies to recombinant PD-L1
protein (human PD-L1-his taq) was tested by BIACORE.TM. using a
capture method. The HL1210-3 mouse mAb were captured using
anti-mouse Fc antibody coated on a CM5 chip. A series dilution of
human PD-L1-his taq protein was injected over captured antibody for
3 mins at a flow rate of 25 .mu.g/ml. The antigen was allowed to
dissociate for 900 s. All the experiment were carried out on a
Biacore T200. Data analysis was carried out using Biacore T200
evaluation software and is shown in Table 11 below.
TABLE-US-00024 TABLE 11 Affinity by Biacore Antibody ka (1/Ms) kd
(1/s) KD (M) Hu1210-8 9.346E+4 7.169E-5 7.671E-10 Hu1210-9 9.856E+4
4.528E-5 4.594E-10 Hu1210-14 1.216E+5 5.293E-5 4.352E-10 Hu1210-16
9.978E+4 6.704E-5 6.720E-10 Hu1210-17 1.101E+5 2.128E-5 1.933E-10
Hu1210-28 1.289E+5 1.080E-4 8.378E-10 Hu1210-31 1.486E+5 1.168E-4
7.862E-10 Hu1210-36 1.461E+5 7.852E-5 5.376E-10 Hu1210-40 8.77E+04
1.31E-04 1.49E-09 Hu1210-41 9.17E+04 3.46E-05 3.78E-10 Hu1210-42
8.68E+04 7.53E-05 8.67E-10 1210 Chimera 1.236E+5 3.265E-5
2.642E-10
Cross Species Activity
[0304] To evaluate the binding of humanized antibodies to huPD-L1,
Mouse PD-L1, Rat PD-L1, Rhesus PD-L1, the antibodies were performed
for the ELISA testing. Briefly, microtiter plates were coated with
human, mouse, rat and rhesus PD-L1-Fc protein at 1 .mu.g/ml in PBS,
100 .mu.l/well at 4.degree. C. overnight, then blocked with 100
.mu.l/well of 5% BSA. Three-fold dilutions of humanized antibodies
starting from 1 .mu.g/ml were added to each well and incubated for
1-2 hours at RT. The plates were washed with PBS/Tween and then
incubate with goat-anti-mouse IgG antibody conjugated with Horse
Radish Peroxidase (HRP) for 1 hour at RT. After washing, the plates
were developed with TMB substrate and analyzed by spectrophotometer
at OD 450-630 nm. The Hu1210-41 antibody can bind to rhesus PD-L1
with lower affinity and cannot bind to rat and mouse PD-L1 (FIG.
8).
TABLE-US-00025 Human Rhesus Rat Mouse EC50 0.215 nM 0.628 nM No
binding No binding
Family Member Specificity
[0305] To evaluate the binding of humanized anti-PD-L1 antibody to
human B7 family and other immune checkpoint, the antibody was
evaluate for its binding to B7-H1 (PD-L1), B7-DC, B7-1, B7-2,
B7-H2, PD-1, CD28, CTLA4, ICOS and BTLA by ELISA. As shown in FIG.
9, the Hu1210-41 antibody can only specifically binding to B7-H1
(PD-L1).
Example 8
Humanized Antibodies Blocked Activity of Human PD-L1 to PD-1
Cell Based Receptor Blocking Assay
[0306] To evaluate the blocking effect of humanized antibodies on
human PD-L1 expressed on mammalian cells to bind to its receptor
PD-1, the FACS-based receptor blocking assay was employed. Briefly,
PDL1-CHOK1 cells were firstly incubated with 3-fold serious diluted
HL1210-3 mouse mAb starting at 20 .mu.g/ml at RT for 1 hour. After
wash by FACS buffer (PBS with 2% FBS), the biotin-labeled huPD-1
were added to each well and incubated at RT for 1 hour. Then, the
Streptavidin-PE were added to each well for 0.5 hour post twice
wash with FACS buffer. The mean florescence intensity (MFI)of PE
were evaluated by FACSAriaIII.
% .times. .times. of .times. .times. inhibition = ( 1 - MFI .times.
.times. of .times. .times. testing .times. .times. antibody MFI
.times. .times. of .times. .times. vehicle .times. .times. contorl
) .times. 1 .times. 00 .times. % ##EQU00002##
[0307] As shown in Table 12 below, Hu1210-3, Hu1210-9, Hu1210-8,
Hu1210-14, Hu1210-17, Hu1210-19 and Hu1210-22 antibodies show
comparable efficacy with chimeric antibody to blocking the binding
of PD-L1 to PD-1.
TABLE-US-00026 TABLE 12 PD-1 receptor blocking assay Bio-PD1 (30
.mu.g/ml) TOP EC50 H1210 chimera 87.16 3.961 Hu1210-8 86.35 4.194
Hu1210-9 85.7 4.038 Hu1210-16 88.02 5.436 Hu1210-17 80.88 4.424
Hu1210-3 84.28 3.693 Hu1210-14 79.56 3.572 Hu1210-19 87.45 4.52
Hu1210-22 85.83 4.505 Hu1210-27 103.9 11.48 Hu1210-31 92.91 6.179
Hu1210-36 91.75 8.175
Receptor Blocking Assay by Using Recombinant Human PD-L1
[0308] There are two receptors i.e. PD-1 and B7-1 for human PD-L1.
To explore the blocking property of humanized PD-L1 antibody to
these two proteins, the protein based receptor blocking assay was
employed here. Briefly, microtiter plates were coated with human
PD-L1-Fc protein at 1 .mu.g/ml in PBS, 100 .mu.l/well at 4.degree.
C. overnight, then blocked with 200 .mu.l/well of 5% BSA at
37.degree. C. for 2 hr. 50 .mu.l biotin-labeled human PD-1-Fc or
B7-1vprotein and 5-fold dilutions of PD-L1 antibodies starting from
100 nM at 50 .mu.l were added to each well and incubated for 1 hour
at 37.degree. C. The plates were washed with PBS/Tween and then
incubate with Streptavidin-HRP for 1 hour at 37.degree. C. After
washing, the plates were developed with TMB substrate and analyzed
by spectrophotometer at OD 450 nm. As shown in FIGS. 10 and 11,
Hu1210-41 can efficiently inhibit the binding of human PD-L1 to
human PD1 and B7-1.
Example 9
Humanized Antibody Promoted Human T Cell Immune Response
Mixed Lymphocyte Reaction Assay
[0309] To evaluate the in vitro function of humanized antibodies,
the response of human T cells assessed in a mixed lymphocyte
reaction setting. Human DCs were differentiated from CD14+
monocytes in the presence of GM-CSF and IL-4 for 7 days. CD4+ T
cells isolated from another donor were then co-cultured with the
DCs and serial dilutions of anti-PD-L1 blocking antibody. At day 5
post-inoculation, the culture supernatant was assayed for IL-2 and
IFN.gamma. production. The results indicated that the Hu1210-8,
Hu1210-9, Hu1210-16 and Hu1210-17 antibodies can dose-dependently
promote IL-2 and IFN.gamma. production, suggesting anti-PD-L1
antibodies can promote human T cell response.
CMV Recall Assay
[0310] To evaluate the in vitro function of humanized antibodies,
the response of human T cells assessed in CMV recall assay. Human
PBMCs were stimulated with 1 .mu.g/ml CMV antigen in the presence
of serious diluted humanized antibodies. As shown in FIGS. 12 and
13 the Hu1210-40, Hu1210-41 and Hu1210-17 can dose dependently
promote the IFN.gamma. production.
Example 10
Tumor Growth Inhibition by Anti-PD-L1 mAb
[0311] Cells from the human lung adenocarcinoma cell line HCC827
will be grafted into NOD scid gamma (NSG) mice. NSG mice are NOD
scid gamma deficient and the most immunodeficient mice making them
ideal recipients for human tumor cell and PBMC grafting. 10 days
post-graft, human PBMCs will be transplanted into the tumor-bearing
mice. Approximately 20 days post-graft, once the tumor volume has
reached 100-150 mm.sup.3, PD-L1 antibody will be administered to
the mice every other day at 5 mg/kg. Tumor volume will be monitored
every other day in conjunction with antibody administration. As
shown in FIG. 14, Hu1210-31 can inhibit the tumor growth by 30% at
5 mg/kg. Hu1210-41 antibody can dose-dependently inhibit the tumor
growth, while the tumor weight was also dose-dependently suppressed
by Hu1210-41 antibody (FIG. 15).
Example 11
Computer Simulation of Further Variation and Optimization of the
Humanized Antibodies
[0312] It was contemplated that certain amino acid residues within
the CDR regions or the framework regions could be changed to
further improve or retain the activity and/or stability of the
antibodies. Variants were tested, with a computational tool
(VectorNTI, available at www.ebi.ac.uk/tools/msa/clustalo/), with
respect to their structural, conformational and functional
properties, and those (within the CDR regions) that showed promises
are listed in the tables blow.
TABLE-US-00027 TABLE 13 VH and VL CDRs and their variants suitable
for inclusion in humanized antibodies Name Sequence SEQ ID NO: VH
CDR1 SYDMS 1 TYDMS 61 CYDMS 62 SFDMS 63 SHDMS 64 SWDMS 65 SYDMT 66
SYDMC 67 VH CDR2 TISDGGGYIYYSDSVKG 2 TISDGGAYIYYSDSVKG 68
TISDGGPYIYYSDSVKG 69 TISDGGGFIYYSDSVKG 70 TISDGGGHIYYSDSVKG 71
TISDGGGWIYYSDSVKG 72 TISDGGGYIYYSDTVKG 73 TISDGGGYIYYSDCVKG 74
TISDGGGYIYYSDSLKG 75 TISDGGGYIYYSDSIKG 76 TISDGGGYIYYSDSMKG 77 VH
CDR3 EFGKRYALDY 3 QFGKRYALDY 78 DFGKRYALDY 79 NFGKRYALDY 80
EYGKRYALDY 81 EHGKRYALDY 82 EWGKRYALDY 83 EFAKRYALDY 84 EFPKRYALDY
85 EFGRRYALDY 86 EFGKKYALDY 87 EFGKRFALDY 88 EFGKRHALDY 89
EFGKRWALDY 90 VL CDR1 KASQDVTPAVA 4 KATQDVTPAVA 91 KACQDVTPAVA 92
VL CDR2 STSSRYT 5 TTSSRYT 93 CTSSRYT 94 SSSSRYT 95 SMSSRYT 96
SVSSRY 97 STTSRYT 98 STCSRYT 99 STSTRYT 100 STSCRYT 101 STSSKYT 102
STSSRFT 103 STSSRHT 104 STSSRWT 105 VL CDR3 QQHYTTPLT 6 EQHYTTPLT
106 DQHYTTPLT 107 NQHYTTPLT 108 QEHYTTPLT 109 QDHYTTPLT 110
QNHYTTPLT 111 Underline: hotspot mutation residues and their
substitutes
Example 12
Identification of PD-L1 Epitope
[0313] This study was conducted to identify amino acid residues
involved in the binding of PD-L1 to the antibodies of the present
disclosure.
[0314] An alanine-scan library of PD-L1 was constructed. Briefly,
217 mutant clones of PD-L1 were generated on Integral Molecular's
protein engineering platform. Binding of Hu1210-41 Fab to each
variant in the PD-L1 mutation library was determined, in duplicate,
by high-throughput flow cytometry. Each raw data point had
background fluorescence subtracted and was normalized to reactivity
with PD-L1 wild-type (WT). For each PD-L1 variant, the mean binding
value was plotted as a function of expression (control anti-PD-L1
mAb reactivity). To identify preliminary critical clones (circles
with crosses), thresholds (dashed lines) of >70% WT binding to
control MAb and <30% WT reactivity to Hu1210-41 Fab were applied
(FIG. 16). Y134, K162, and N183 of PDL1 were identified as required
residues for Hu1210-41 binding. The low reactivity of N183A clone
with Hu1210-41 Fab suggests that it is the major energetic
contributor to Hu1210-41 binding, with lesser contributions by Y134
and K162.
[0315] The critical residues (spheres) were identified on a 3D
PD-L1 structure (PDB ID #5JDR, Zhang et al., 2017), illustrated in
FIG. 17. These residues, Y134, K162, and N183, therefore,
constitute an epitope of PD-L1 responsible for binding to
antibodies of various embodiments of the present disclosure.
[0316] It is interesting to note that Y134, K162, and N183 are all
located within the IgC domain of the PD-L1 protein. Both PD-1 and
PD-L1's extracellular portions have an IgV domain and an IgC
domain. It is commonly known that PD-L1 binds to PD-1 through
bindings between their IgV domains. Unlike such conventional
antibodies, however, Hu1210-41 binds to the IgC domain, which would
have been expected to be ineffective in inhibiting PD-1/PD-L1
binding. This different epitope of Hu1210-41, surprisingly, likely
contributes to the excellent activities of Hu1210-41.
Example 13
Antibody Engineering of Anti-PDL1 Antibody
[0317] Examples 13-17 attempted to identify further improved
antibodies based on Hu1210-41 using mutagenesis.
[0318] A fusion protein of activation-induced cytidine deaminase
(AID) with nuclease-inactive clustered regularly interspaced short
palindromic repeats (CRISPR)-associated protein 9 (dCas9) was used
for high-throughput screening of functional variants in 293 T
cells. Briefly, single guide (sg)RNAs recognizing 6 CDRs of
antibody can guide dCas9-AID fusion protein to 6 CDRs of antibody
and induce mutations in CDR region. The mutated antibodies were
displayed in the cell surface of 293 cells. The resulting cells
showed better binding potency than the non-mutated counterpart and
were FACS sorted out for subsequence sequencing. A mutation of S60
to R in the CDRH2 was identified as potentially having positive
effect on the antibody.
[0319] To evaluate the antigen binding property of the S6OR (CDRH2)
mutant, the antibodies were analyzed for their binding to mammalian
expressed PD-L1 by FACS. Briefly, PD-L1 Raji cells were first
incubated with 5-fold serially diluted humanized antibodies
starting at 2 .mu.g/ml at RT for 1 hour. After wash by FACS buffer
(PBS with 2% FBS), the Alexa 488-anti-human IgG antibody was added
to each well and incubated at RT for 1 hour. The MFI of Alexa 488
were evaluated by FACSAriaIII. As shown in the FIG. 18, the S60R
mutant highly efficiently bound to PD-L1 expressed on mammalian
cells, which was more potent than the parental antibody Hu1210-41.
This S60R mutant was then used as the parental antibody for further
mutation analyses below, and was referred to as "WT".
[0320] Four sub-libraries were constructed for antibody engineering
of anti-PD-L1 monoclonal antibody, using either of the following
strategies. In strategy 1, mutagenesis of heavy chain variable
domain VH CDR3 or VL-CDR3 was perform by highly random mutation. In
strategy 2, two CDR combination libraries composed of (VH-CDR3,
VL-CDR3 and VL-CDR1) or (VH-CDR1, VH-CDR2 and VL-CDR2) were
generated by CDR walking with controlled mutation rates.
[0321] Bio-Panning: the phage panning methods were adapted by
shortening the incubation/binding time prior to the harsh washing
condition. Briefly, 100 .mu.l magnetic streptavidin beads
(Invitrogen, USA) were blocked with 1 ml of MPBS for 1 hr at room
temperature. In another tube, library phage was pre-incubated
(5.times.10{circumflex over ( )}11.about.12 for each round) with
100 .mu.l magnetic streptavidin beads in 1 ml of MPBS to remove
unwanted binders. Magnet particle concentrator was used to separate
the phage and beads. The biotinylated PD-L1 protein was added to
the phage and incubated 2 h at room temperature, and gently mixed
using an over-head shaker. Beads carrying phage from the solution
were separated in the magnetic particle concentrator and the
supernatant was discarded. The beads were washed with fresh wash
buffer, ten times with PBST and ten times with PBS (pH7.4). 0.8ml,
0.25% Trypsin in PBS (Sigma, USA) was added and incubated for 20
min at 37.degree. C. to elute the phage. The output phage was
titrated and rescued for next round panning, decreasing antigen
concentration round by round.
ELISA Screening and On/Off Rate Ranking
[0322] Clones were picked and induced from the desired panning
output; phage ELISA was conducted for primary screening; positive
clones were analyzed by sequencing; unique hotspots were found.
Table 14 shows the mutations identified. As shown below, the FGK
residues in the CDRH3 are hotpot residues producing improved
antibodies.
TABLE-US-00028 TABLE 14 Mutations in the CDRs CDR-H1 CDR-H2 CDR-H3
CDR-L1 CDR-L2 CDR-L3 WT* SYDMS TISDAGGYIYYRDSVKG EFGKRYALDY
KASQDVTPAVA STSSRYT QQHYTTPLT SEQ ID NO: 1 116 3 4 5 6 B3 -----
----------------- ---------- --K-------- ------- M-------- C4 -----
----------------- ---------S ------W---- ------- ---S----- B1 -----
----------------- -IFN------ ----------- ------- --------- B6 -----
----------------- -LPW------ ----------- ------- --------- C3 -----
----------------- -LHF------ ----------- ------- --------- C6 -----
----------------- -LYF------ ----------- ------- --------- A1 -----
----------------- -LLH------ ----------- ------- --------- A2 -----
----------------- -LRG------ ----------- ------- --------- A3 -----
----------------- ---------- ----------- ------- ---SDA--- *WT
differs from Hu1210-41 by a S60R (Kabat numbering) substitution in
the heavy chain to improve affinity.
[0323] The amino acid sequences of the variable regions of these
antibodies are shown in Table 15 below.
TABLE-US-00029 TABLE 15 Antibody sequences Name Sequence SEQ ID NO:
WT-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 141
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF
GKRYALDYWGQGTTVTVSS WT-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 142
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK B3-VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF 143
GKRYALDYWGQGTTVTVSS B3-Vk
DIQMTQSPSSLSASVGDRVTITCKAKQDVTPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCMQHYTTPLTFGQ 144 GTKLEIK
C4-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF 145
GKRYALDSWGQGTTVTVSS C4-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVWPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPLTFGQ 146 GTKLEIK
B1-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREI 147
FNRYALDYWGQGTTVTVSS B1-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ 148 GTKLEIK
B6-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL 149
PWRYALDYWGQGTTVTVSS B6-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ 150 GTKLEIK
C3-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL 151
HFRYALDYWGQGTTVTVSS C3-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ 152 GTKLEIK
C6-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL 153
YFRYALDYWGQGTTVTVSS C6-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ 154 GTKLEIK
A1-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL 155
LHRYALDYWGQGTTVTVSS A1-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ 156 GTKLEIK
A2-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL 157
RGRYALDYWGQGTTVTVSS A2-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ 158 GTKLEIK
A3-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT
ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF 159
GKRYALDYWGQGTTVTVSS A3-Vk
DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS
TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSDAPLTFGQ 160 GTKLEIK
Example 14
Antigen Binding Properties of the PD-L1 Antibodies
[0324] As shown in Tables 14 and 15, totally 9 unique clones were
characterized and converted into full-length IgG.
Binding Property to Recombinant Human PD-L1
[0325] To evaluate the antigen binding activity, the antibodies
were subjected to ELISA test. Briefly, microtiter plates were
coated with human PD-L1-Fc protein at 2 .mu.g/ml in PBS, 100
.mu.l/well at 4.degree. C. overnight, then blocked with 100
.mu.l/well of 5% BSA. 4-fold dilutions of humanized antibodies
starting from 10 pg/ml were added to each well and incubated for
1-2 hours at RT. The plates were washed with PBS/Tween and then
incubate with goat-anti-mouse IgG antibody conjugated with Horse
Radish Peroxidase (HRP) for 1 hour at RT. After washing, the plates
were developed with TMB substrate and analyzed by spectrophotometer
at OD 450-630 nm. As shown in FIG. 19, all the humanized antibodies
showed excellent binding efficacy to human PD-L1, and B6 and C3
behaved better than the parental clone WT.
Binding Property to Mammalian Expressed Human PD-L1
[0326] To evaluate the antigen binding property, the antibodies
were analyzed for its binding to mammalian expressed PD-L1 by FACS.
Briefly, PDL1-Raji cells were firstly incubated with 5-fold serious
diluted humanized antibodies starting at 2 .mu.g/ml at RT for 1
hour. After wash by FACS buffer (PBS with 2% FBS), the Alexa
488-anti-human IgG antibody was added to each well and incubated at
RT for 1 hour. The MFI of Alexa 488 were evaluated by FACSAriaIII.
As shown in the FIG. 20, B6 highly efficiently bound to PD-L1
expressed on mammalian cells, which was more potent than the
parental antibody WT.
Affinity Ranking of Humanized Antibodies by Biacore
[0327] To explore the binding kinetics of the humanized antibody,
this example performed the affinity ranking using Biacore. As shown
Table 16, B6, C3, C6, A1 and A3 showed better affinity than the
parent antibody WT.
TABLE-US-00030 TABLE 16 Affinity ranking Antibody ka (1/Ms) kd
(1/s) KD (M) WT 1.77E+05 4.64E-04 2.63E-09 B3 1.19E+05 2.96E-04
2.49E-09 C4 1.13E+05 5.06E-04 4.50E-09 B1 1.63E+05 2.61E-04
1.60E-09 B6 2.42E+05 2.46E-04 1.02E-09 C3 2.18E+05 2.99E-04
1.37E-09 C6 2.06E+05 3.34E-04 1.63E-09 A1 2.03E+05 2.76E-04
1.36E-09 A2 1.87E+05 4.75E-04 2.55E-09 A3 2.18E+05 3.24E-04
1.49E-09
Example 15
Anti-PDL1 Antibody Cell Based Function
[0328] To test the ability of anti-PDL1 antibodies to stimulate T
cell response, hPD-1-expressed Jurkat cells were used. Briefly,
Jurkat is human T cell leukemia cell line that can produce IL2 upon
TCR stimulation. In this assay, Jurkat cells transfected with human
PD-1 gene by lentivirus were used as the responder cells. The
Raji-PDL1 cells was used as the antigen presenting cells (APC).
Staphylococcal Enterotoxins (SE) are used to stimulate TCR signal.
In this system, ectopically expressed huPDL1 can suppress SE
stimulated IL-2 production by Jurkat cells, while anti-PDL1
antibodies can reverse IL-2 production. In short, APCs
(2.5.times.10.sup.4) were co-cultured with PD-1 expressing Jurkat T
cells (1.times.10.sup.5) in the presence of SE stimulation.
Anti-PDL1 antibodies (starting from 100 nM and 1:4 serially diluted
for 8 dose) were added at the beginning of the culture. 48hr later,
culture supernatant was evaluated for IL2 production by ELISA. As
shown in FIG. 21, the B6 monoclonal antibodies were more potent
than parental antibody WT.
Example 16
Mixed Lymphocyte Reaction
[0329] To evaluate the in vitro function of PDL1 antibodies, the
response of human T cells was assessed in a mixed lymphocyte
reaction setting. Briefly, human DCs were differentiated from CD14+
monocytes in the presence of GM-CSF and IL-4 for 7 days. CD4+ T
cells isolated from another donor were then co-cultured with the
DCs and serial dilutions of anti-PD-L1 blocking antibody. At day 5
post-inoculation, the culture supernatant was assayed for
IFN.gamma. production. The results (FIG. 22) indicated that the B6
antibody was more potent than parental antibody WT in promoting
IFN.gamma. production.
Example 17
In Vivo Efficacy of PDL1 Antibody in MC38 Syngeneic Model
[0330] To evaluate the effect of PDL1 on tumor growth, the PDL1
humanized MC38 syngeneic tumor model was applied. In this model,
the human PDL1 gene was expressed in mouse MC38 cells, while the
extracellular domain of mouse PDL1 gene was replaced by human PDL1
gene. In this regard, the efficacy of human PDL1 antibody on tumor
growth could be evaluated in this PDL1 gene humanized MC38
syngeneic model. The huPDL1 MC38 cells were inoculated
subcutaneously into PDL1 humanized mice. When tumor reached the
volume of 100-150m.sup.3, the parental antibody WT and B6
antibodies were administrated intraperitoneally at 3 mg/kg twice
weekly for 6 doses. The result (FIG. 23) showed that B6 antibody
was more potent than the parental antibody WT from day 19 to
26.
[0331] The present disclosure is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the disclosure, and any
compositions or methods which are functionally equivalent are
within the scope of this disclosure. It will be apparent to those
skilled in the art that various modifications and variations can be
made in the methods and compositions of the present disclosure
without departing from the spirit or scope of the disclosure. Thus,
it is intended that the present disclosure cover the modifications
and variations of this disclosure provided they come within the
scope of the appended claims and their equivalents.
[0332] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference
Sequence CWU 1
1
16615PRTArtificial SequenceSynthetic 1Ser Tyr Asp Met Ser1
5217PRTArtificial SequenceSynthetic 2Thr Ile Ser Asp Gly Gly Gly
Tyr Ile Tyr Tyr Ser Asp Ser Val Lys1 5 10 15Gly310PRTArtificial
SequenceSynthetic 3Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr1 5
10411PRTArtificial SequenceSynthetic 4Lys Ala Ser Gln Asp Val Thr
Pro Ala Val Ala1 5 1057PRTArtificial SequenceSynthetic 5Ser Thr Ser
Ser Arg Tyr Thr1 569PRTArtificial SequenceSynthetic 6Gln Gln His
Tyr Thr Thr Pro Leu Thr1 57119PRTArtificial SequenceSynthetic 7Glu
Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Ser Leu Glu Trp
Val 35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Asn Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr
Ala Leu Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser
1158119PRTArtificial SequenceSynthetic 8Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile Ser
Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly
100 105 110Thr Thr Val Thr Val Ser Ser 1159119PRTArtificial
SequenceSynthetic 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr
Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Gly
Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val
Thr Val Ser Ser 11510119PRTArtificial SequenceSynthetic 10Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11511119PRTArtificial SequenceSynthetic 11Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile
Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 11512119PRTArtificial
SequenceSynthetic 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Ile Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr
Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Gly
Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val
Thr Val Ser Ser 11513119PRTArtificial SequenceSynthetic 13Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11514119PRTArtificial SequenceSynthetic 14Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile
Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 11515119PRTArtificial
SequenceSynthetic 15Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr
Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly
Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val
Thr Val Ser Ser 11516119PRTArtificial SequenceSynthetic 16Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Ile Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11517119PRTArtificial SequenceSynthetic 17Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 11518119PRTArtificial
SequenceSynthetic 18Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr
Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly
Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val
Thr Val Ser Ser 11519119PRTArtificial SequenceSynthetic 19Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Glu Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala
Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11520119PRTArtificial SequenceSynthetic 20Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys
85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 11521119PRTArtificial
SequenceSynthetic 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Val Gly Gly Tyr
Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly
Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val
Thr Val Ser Ser 11522119PRTArtificial SequenceSynthetic 22Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11523119PRTArtificial SequenceSynthetic 23Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Ile Cys
85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11524119PRTArtificial
SequenceSynthetic 24Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr
Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly
Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val
Thr Val Ser Ser 11525119PRTArtificial SequenceSynthetic 25Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11526119PRTArtificial SequenceSynthetic 26Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Val Arg Gln Thr Pro Glu Lys Ser Leu Glu Trp Val 35
40 45Ala Thr Ile Ser Asp Gly Gly Gly Tyr Ile Tyr Tyr Ser Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11527107PRTArtificial SequenceSynthetic 27Asp Ile Val Met Thr Gln
Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile
Ser Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75
80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
10528107PRTArtificial SequenceSynthetic 28Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10529107PRTArtificial SequenceSynthetic 29Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10530108PRTArtificial SequenceSynthetic 30Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
10531108PRTArtificial SequenceSynthetic 31Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
10532108PRTArtificial SequenceSynthetic 32Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
10533108PRTArtificial SequenceSynthetic 33Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Ser Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
10534357DNAArtificial SequenceSynthetic 34gaggtgaagc tggtggagag
cggcggagat ctggtgaagc ctggcggcag cctgaagctg 60agctgtgccg ccagcggctt
caccttcagc agctacgaca tgagctgggt gaggcagacc 120cccgagaaga
gcctggagtg ggtggccacc atcagcgatg gcggcggcta catctactac
180agcgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa
caacctgtac 240ctgcagatga gcagcctgag gagcgaggac accgccctgt
acatctgcgc cagggagttc 300ggcaagaggt acgccctgga ctactgggga
cagggcacca gcgtgaccgt gagcagc 35735357DNAArtificial
SequenceSynthetic 35gaggtgcagc tggtggagag cggaggagga ctggtgaagc
ccggaggcag cctgagactg 60agctgcgctg ccagcggctt caccttcagc agctacgaca
tgagctgggt gagacaggcc 120cctggcaaag gcctggagtg ggtgagcacc
atctccgatg gcggcggcta catctattac 180tccgacagcg tgaagggcag
gttcaccatc agcagggaca acgccaagaa cagcctgtac 240ctgcagatga
acagcctgag ggccgaggac accgccgtgt actactgcgc cagggagttc
300ggcaaaaggt acgccctgga ctactggggc cagggcacaa ccgtgaccgt gagcagc
35736357DNAArtificial SequenceSynthetic 36gaggtgcagc tggtggagag
cggaggagga ctggtgaagc ccggaggcag cctgagactg 60agctgcgctg ccagcggctt
caccttcagc agctacgaca tgagctgggt gagacaggcc 120cctggcaaag
gcctggagtg ggtggccacc atctccgatg gcggcggcta catctattac
180tccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa
cagcctgtac 240ctgcagatga acagcctgag ggccgaggac accgccgtgt
actactgcgc cagggagttc 300ggcaaaaggt acgccctgga ctactggggc
cagggcacaa ccgtgaccgt gagcagc 35737357DNAArtificial
SequenceSynthetic 37gaggtgcagc tggtggagag cggaggagga ctggtgaagc
ccggaggcag cctgagactg 60agctgcgctg ccagcggctt caccttcagc agctacgaca
tgagctgggt gagacaggcc 120cctggcaaaa gcctggagtg ggtggccacc
atctccgatg gcggcggcta catctattac 180tccgacagcg tgaagggcag
gttcaccatc agcagggaca acgccaagaa cagcctgtac 240ctgcagatga
acagcctgag ggccgaggac accgccgtgt acatctgcgc cagggagttc
300ggcaaaaggt acgccctgga ctactggggc cagggcacaa ccgtgaccgt gagcagc
35738357DNAArtificial SequenceSynthetic 38gaggtgcagc tggtggagag
cggaggagga ctggtgaagc ccggaggcag cctgagactg 60agctgcgctg ccagcggctt
caccttcagc agctacgaca tgagctggat cagacaggcc 120cctggcaaag
gcctggagtg ggtgagcacc atctccgatg gcggcggcta catctattac
180tccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa
cagcctgtac 240ctgcagatga acagcctgag ggccgaggac accgccgtgt
actactgcgc cagggagttc 300ggcaaaaggt acgccctgga ctactggggc
cagggcacaa ccgtgaccgt gagcagc 35739357DNAArtificial
SequenceSynthetic 39gaggtgcagc tggtggagag cggaggagga ctggtgaagc
ccggaggcag cctgagactg 60agctgcgctg ccagcggctt caccttcagc agctacgaca
tgagctggat cagacaggcc 120cctggcaaag gcctggagtg ggtggccacc
atctccgatg gcggcggcta catctattac 180tccgacagcg tgaagggcag
gttcaccatc agcagggaca acgccaagaa cagcctgtac 240ctgcagatga
acagcctgag ggccgaggac accgccgtgt actactgcgc cagggagttc
300ggcaaaaggt acgccctgga ctactggggc cagggcacaa ccgtgaccgt gagcagc
35740357DNAArtificial SequenceSynthetic 40gaggtgcagc tggtggagag
cggaggagga ctggtgaagc ccggaggcag cctgagactg 60agctgcgctg ccagcggctt
caccttcagc agctacgaca tgagctgggt gagacaggcc 120cctggcaaaa
gcctggagtg ggtggccacc atctccgatg gcggcggcta catctattac
180tccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa
cagcctgtac 240ctgcagatga acagcctgag ggccgaggac accgccgtgt
acatctgcgc cagggagttc 300ggcaaaaggt acgccctgga ctactggggc
cagggcacaa ccgtgaccgt gagcagc 35741357DNAArtificial
SequenceSynthetic 41gaggtgcagc tgctggagag cggaggagga ctggtgcaac
ccggaggcag cctgagactg 60agctgcgctg ccagcggctt caccttcagc agctacgaca
tgagctgggt gagacaggcc 120cctggcaaag gcctggagtg ggtgagcacc
atctccgatg gcggcggcta catctattac 180tccgacagcg tgaagggcag
gttcaccatc agcagggaca acagcaagaa caccctgtac 240ctgcagatga
acagcctgag ggccgaggac accgccgtgt actactgcgc cagggagttc
300ggcaaaaggt acgccctgga ctactggggc cagggcacaa ccgtgaccgt gagcagc
35742357DNAArtificial SequenceSynthetic 42gaggtgcagc tgctggagag
cggaggagga ctggtgcaac ccggaggcag cctgagactg 60agctgcgctg ccagcggctt
caccttcagc agctacgaca tgagctgggt gagacaggcc 120cctggcaaaa
gcctggagtg ggtggccacc atctccgatg gcggcggcta catctattac
180tccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa
caccctgtac 240ctgcagatga acagcctgag ggccgaggac accgccgtgt
acatctgcgc cagggagttc 300ggcaaaaggt acgccctgga ctactggggc
cagggcacaa ccgtgaccgt gagcagc 35743357DNAArtificial
SequenceSynthetic 43gaggtgcagc tggtggagag cggaggagga ctggtgcaac
ccggaggcag cctgagactg 60agctgcgctg ccagcggctt caccttcagc agctacgaca
tgagctgggt gagacaggcc 120cctggcaaag gcctggagtg ggtgagcacc
atctccgatg gcggcggcta catctattac 180tccgacagcg tgaagggcag
gttcaccatc agcagggaca acgccaagaa cagcctgtac 240ctgcagatga
acagcctgag ggatgaggac accgccgtgt actactgcgc cagggagttc
300ggcaaaaggt acgccctgga ctactggggc cagggcacaa ccgtgaccgt gagcagc
35744357DNAArtificial SequenceSynthetic 44gaggtgcagc tggtggagag
cggaggagga ctggtgcaac ccggaggcag cctgagactg 60agctgcgctg ccagcggctt
caccttcagc agctacgaca tgagctgggt gagacaggcc 120cctggcaaag
gcctggagtg ggtggccacc atctccgatg gcggcggcta catctattac
180tccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa
cagcctgtac 240ctgcagatga acagcctgag ggatgaggac accgccgtgt
actactgcgc cagggagttc 300ggcaaaaggt acgccctgga ctactggggc
cagggcacaa ccgtgaccgt gagcagc 35745357DNAArtificial
SequenceSynthetic 45gaggtgcagc tggtggagag cggaggagga ctggtgcaac
ccggaggcag cctgagactg 60agctgcgctg ccagcggctt caccttcagc agctacgaca
tgagctgggt gagacaggcc 120cctggcaaaa gcctggagtg ggtggccacc
atctccgatg gcggcggcta catctattac 180tccgacagcg tgaagggcag
gttcaccatc agcagggaca acgccaagaa cagcctgtac 240ctgcagatga
acagcctgag ggatgaggac accgccgtgt acatctgcgc cagggagttc
300ggcaaaaggt acgccctgga ctactggggc cagggcacaa ccgtgaccgt gagcagc
35746357DNAArtificial SequenceSynthetic 46gaggtgcagc tggtggagag
cggaggagga ctggtgcaac ccggaggcag cctgagactg 60agctgcgctg ccagcggctt
caccttcagc agctacgaca tgagctgggt gagacaggcc 120cctggcaaaa
gcctggagtg ggtggccacc atctccgaag gcggcggcta catctattac
180tccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa
cagcctgtac 240ctgcagatga acagcctgag ggatgaggac accgccgtgt
acatctgcgc cagggagttc 300ggcaaaaggt acgccctgga ctactggggc
cagggcacaa ccgtgaccgt gagcagc 35747357DNAArtificial
SequenceSynthetic 47gaggtgcagc tggtggagag cggaggagga ctggtgcaac
ccggaggcag cctgagactg 60agctgcgctg ccagcggctt caccttcagc agctacgaca
tgagctgggt gagacaggcc 120cctggcaaaa gcctggagtg ggtggccacc
atctccgatg cgggcggcta catctattac 180tccgacagcg tgaagggcag
gttcaccatc agcagggaca acgccaagaa cagcctgtac 240ctgcagatga
acagcctgag ggatgaggac accgccgtgt acatctgcgc cagggagttc
300ggcaaaaggt acgccctgga ctactggggc cagggcacaa ccgtgaccgt gagcagc
35748357DNAArtificial SequenceSynthetic 48gaggtgcagc tggtggagag
cggaggagga ctggtgcaac ccggaggcag cctgagactg 60agctgcgctg ccagcggctt
caccttcagc agctacgaca tgagctgggt gagacaggcc 120cctggcaaaa
gcctggagtg ggtggccacc atctccgatg ttggcggcta catctattac
180tccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa
cagcctgtac 240ctgcagatga acagcctgag ggatgaggac accgccgtgt
acatctgcgc cagggagttc 300ggcaaaaggt acgccctgga ctactggggc
cagggcacaa ccgtgaccgt gagcagc 35749357DNAArtificial
SequenceSynthetic 49gaggtgcagc tggtggagtc cggaggaggc ctggtgcaac
ctggaggctc cctgaggctg 60tcctgtgccg cttccggctt caccttcagc tcctacgata
tgagctgggt gaggcaggct 120cctggaaagg gcctggagtg ggtggccacc
atctccgacg gaggcggcta catctactac 180tccgactccg tgaagggcag
gttcaccatc tcccgggaca acgccaagaa ctccctgtac 240ctgcagatga
actctctcag ggctgaggac accgccgtgt attactgcgc cagggagttt
300ggcaagaggt acgccctgga ttactggggc cagggcacac tggtgacagt gagctcc
35750357DNAArtificial SequenceSynthetic 50gaggtgcagc tggtggagtc
cggaggaggc ctggtgcaac ctggaggctc cctgaggctg 60tcctgtgccg cttccggctt
caccttcagc tcctacgata tgagctgggt gaggcaggct 120cctggaaagg
gcctggagtg ggtggccacc atctccgacg gaggcggcta catctactac
180tccgactccg tgaagggcag gttcaccatc tcccgggaca acgccaagaa
ctccctgtac 240ctgcagatga actctctcag ggctgaggac accgccgtgt
atatctgcgc cagggagttt 300ggcaagaggt acgccctgga ttactggggc
cagggcacac tggtgacagt gagctcc 35751357DNAArtificial
SequenceSynthetic 51gaggtgcagc tggtggagtc cggaggaggc ctggtgcaac
ctggaggctc cctgaggctg 60tcctgtgccg cttccggctt caccttcagc tcctacgata
tgagctgggt gaggcaggct 120cctggaaagg gcctggagtg ggtggccacc
atctccgacg gaggcggcta catctactac 180tccgactccg tgaagggcag
gttcaccatc tcccgggaca acgccaagaa caacctgtac 240ctgcagatga
actctctcag ggctgaggac accgccgtgt atatctgcgc cagggagttt
300ggcaagaggt acgccctgga ttactggggc cagggcacac tggtgacagt gagctcc
35752357DNAArtificial SequenceSynthetic 52gaggtgcagc tggtggagtc
cggaggaggc ctggtgcaac ctggaggctc cctgaggctg 60tcctgtgccg cttccggctt
caccttcagc tcctacgata tgagctgggt gaggcagacc 120cctgagaaga
gcctggagtg ggtggccacc atctccgacg gaggcggcta catctactac
180tccgactccg tgaagggcag gttcaccatc tcccgggaca acgccaagaa
caacctgtac 240ctgcagatga actctctcag ggctgaggac accgccgtgt
atatctgcgc cagggagttt 300ggcaagaggt acgccctgga ttactggggc
cagggcacac tggtgacagt gagctcc 35753357DNAArtificial
SequenceSynthetic 53gaggtgcagc tggtggagtc cggaggaggc ctggtgcaac
ctggaggctc cctgaggctg 60tcctgtgccg cttccggctt caccttcagc tcctacgata
tgagctgggt gaggcaggct 120cctggaaagg gcctggagtg ggtggccacc
atctccgacg gaggcggcta catctactac 180tccgactccg tgaagggcag
gttcaccatc tcccgggaca acgccaagaa ctccctgtac 240ctgcagatga
actctctcag ggctgaggac accgccgtgt atatctgcgc cagggagttt
300ggcaagaggt acgccctgga ttactggggc cagggcacaa ccgtgacagt gagctcc
35754321DNAArtificial SequenceSynthetic 54gacatcgtga tgacccagag
ccacaagttc atgagcacca gcgtgggcga tagggtgagc 60atcagctgca aggccagcca
ggatgtgacc cctgccgtgg cctggtacca gcagaagccc 120ggccagagcc
ccaagctgct gatctacagc accagcagca ggtacaccgg cgtgcccgac
180aggttcacag gaagcggcag cggcaccgac ttcaccttca ccatcagcag
cgtgcaggcc 240gaggacctgg ccgtgtacta ctgccagcag cactacacca
cccctctgac cttcggcgcc 300ggcaccaagc tggagctgaa g
32155321DNAArtificial SequenceSynthetic 55gacatccaga tgacccagag
ccctagcagc ctgagcgcta gcgtgggcga cagggtgacc 60atcacctgca aggccagcca
ggatgtgacc cctgccgtgg cctggtacca gcagaagccc 120ggcaaggccc
ccaagctgct gatctacagc accagcagca ggtacaccgg cgtgcccagc
180aggtttagcg gaagcggcag cggcaccgac ttcaccttca ccatcagcag
cctgcagccc 240gaggacatcg ccacctacta ctgccagcag cactacacca
cccctctgac cttcggccag 300ggcaccaagc tggagatcaa g
32156321DNAArtificial SequenceSynthetic 56gacatccaga tgacccagag
ccctagcagc ctgagcgcta gcgtgggcga cagggtgacc 60atcacctgca aggccagcca
ggatgtgacc cctgccgtgg cctggtacca gcagaagccc 120ggcaagtccc
ccaagctgct gatctacagc accagcagca ggtacaccgg cgtgcccagc
180aggtttagcg gaagcggcag cggcaccgac ttcaccttca ccatcagcag
cctgcagccc 240gaggacatcg ccacctacta ctgccagcag cactacacca
cccctctgac cttcggccag 300ggcaccaagc tggagatcaa g
32157324DNAArtificial SequenceSynthetic 57gacattcaga tgacccagtc
ccctagcagc ctgtccgctt ccgtgggcga cagggtgacc 60atcacctgca aggccagcca
ggacgtgaca cctgctgtgg cctggtatca acagaagcct 120ggcaaggctc
ctaagctcct gatctacagc acatcctccc ggtacaccgg agtgccctcc
180aggtttagcg gcagcggctc cggcaccgat ttcaccctga ccatttcctc
cctgcagccc 240gaggacttcg ccacctacta ctgccagcag cactacacca
cacccctgac cttcggccag 300ggcaccaagc tggagatcaa gcgg
32458324DNAArtificial SequenceSynthetic 58gacattcaga tgacccagtc
ccctagcagc ctgtccgctt ccgtgggcga cagggtgacc 60atcacctgca aggccagcca
ggacgtgaca cctgctgtgg cctggtatca acagaagcct 120ggcaaggctc
ctaagctcct gatctacagc acatcctccc ggtacaccgg agtgcccgac
180aggtttaccg gcagcggctc
cggcaccgat ttcaccctga ccatttcctc cctgcagccc 240gaggacttcg
ccacctacta ctgccagcag cactacacca cacccctgac cttcggccag
300ggcaccaagc tggagatcaa gcgg 32459324DNAArtificial
SequenceSynthetic 59gacattcaga tgacccagtc ccctagcagc ctgtccgctt
ccgtgggcga cagggtgacc 60atcacctgca aggccagcca ggacgtgaca cctgctgtgg
cctggtatca acagaagcct 120ggccagagcc ctaagctcct gatctacagc
acatcctccc ggtacaccgg agtgcccgac 180aggtttaccg gcagcggctc
cggcaccgat ttcaccctga ccatttcctc cctgcagccc 240gaggacttcg
ccacctacta ctgccagcag cactacacca cacccctgac cttcggccag
300ggcaccaagc tggagatcaa gcgg 32460324DNAArtificial
SequenceSynthetic 60gacattcaga tgacccagtc ccctagcagc ctgtccgctt
ccgtgggcga cagggtgacc 60atcagctgca aggccagcca ggacgtgaca cctgctgtgg
cctggtatca acagaagcct 120ggccagagcc ctaagctcct gatctacagc
acatcctccc ggtacaccgg agtgcccgac 180aggtttaccg gcagcggctc
cggcaccgat ttcaccctga ccatttcctc cctgcagccc 240gaggacttcg
ccacctacta ctgccagcag cactacacca cacccctgac cttcggccag
300ggcaccaagc tggagatcaa gcgg 324615PRTArtificial SequenceSynthetic
61Thr Tyr Asp Met Ser1 5625PRTArtificial SequenceSynthetic 62Cys
Tyr Asp Met Ser1 5635PRTArtificial SequenceSynthetic 63Ser Phe Asp
Met Ser1 5645PRTArtificial SequenceSynthetic 64Ser His Asp Met Ser1
5655PRTArtificial SequenceSynthetic 65Ser Trp Asp Met Ser1
5665PRTArtificial SequenceSynthetic 66Ser Tyr Asp Met Thr1
5675PRTArtificial SequenceSynthetic 67Ser Tyr Asp Met Cys1
56817PRTArtificial SequenceSynthetic 68Thr Ile Ser Asp Gly Gly Ala
Tyr Ile Tyr Tyr Ser Asp Ser Val Lys1 5 10 15Gly6917PRTArtificial
SequenceSynthetic 69Thr Ile Ser Asp Gly Gly Pro Tyr Ile Tyr Tyr Ser
Asp Ser Val Lys1 5 10 15Gly7017PRTArtificial SequenceSynthetic
70Thr Ile Ser Asp Gly Gly Gly Phe Ile Tyr Tyr Ser Asp Ser Val Lys1
5 10 15Gly7117PRTArtificial SequenceSynthetic 71Thr Ile Ser Asp Gly
Gly Gly His Ile Tyr Tyr Ser Asp Ser Val Lys1 5 10
15Gly7217PRTArtificial SequenceSynthetic 72Thr Ile Ser Asp Gly Gly
Gly Trp Ile Tyr Tyr Ser Asp Ser Val Lys1 5 10
15Gly7317PRTArtificial SequenceSynthetic 73Thr Ile Ser Asp Gly Gly
Gly Tyr Ile Tyr Tyr Ser Asp Thr Val Lys1 5 10
15Gly7417PRTArtificial SequenceSynthetic 74Thr Ile Ser Asp Gly Gly
Gly Tyr Ile Tyr Tyr Ser Asp Cys Val Lys1 5 10
15Gly7517PRTArtificial SequenceSynthetic 75Thr Ile Ser Asp Gly Gly
Gly Tyr Ile Tyr Tyr Ser Asp Ser Leu Lys1 5 10
15Gly7617PRTArtificial SequenceSynthetic 76Thr Ile Ser Asp Gly Gly
Gly Tyr Ile Tyr Tyr Ser Asp Ser Ile Lys1 5 10
15Gly7717PRTArtificial SequenceSynthetic 77Thr Ile Ser Asp Gly Gly
Gly Tyr Ile Tyr Tyr Ser Asp Ser Met Lys1 5 10
15Gly7810PRTArtificial SequenceSynthetic 78Gln Phe Gly Lys Arg Tyr
Ala Leu Asp Tyr1 5 107910PRTArtificial SequenceSynthetic 79Asp Phe
Gly Lys Arg Tyr Ala Leu Asp Tyr1 5 108010PRTArtificial
SequenceSynthetic 80Asn Phe Gly Lys Arg Tyr Ala Leu Asp Tyr1 5
108110PRTArtificial SequenceSynthetic 81Glu Tyr Gly Lys Arg Tyr Ala
Leu Asp Tyr1 5 108210PRTArtificial SequenceSynthetic 82Glu His Gly
Lys Arg Tyr Ala Leu Asp Tyr1 5 108310PRTArtificial
SequenceSynthetic 83Glu Trp Gly Lys Arg Tyr Ala Leu Asp Tyr1 5
108410PRTArtificial SequenceSynthetic 84Glu Phe Ala Lys Arg Tyr Ala
Leu Asp Tyr1 5 108510PRTArtificial SequenceSynthetic 85Glu Phe Pro
Lys Arg Tyr Ala Leu Asp Tyr1 5 108610PRTArtificial
SequenceSynthetic 86Glu Phe Gly Arg Arg Tyr Ala Leu Asp Tyr1 5
108710PRTArtificial SequenceSynthetic 87Glu Phe Gly Lys Lys Tyr Ala
Leu Asp Tyr1 5 108810PRTArtificial SequenceSynthetic 88Glu Phe Gly
Lys Arg Phe Ala Leu Asp Tyr1 5 108910PRTArtificial
SequenceSynthetic 89Glu Phe Gly Lys Arg His Ala Leu Asp Tyr1 5
109010PRTArtificial SequenceSynthetic 90Glu Phe Gly Lys Arg Trp Ala
Leu Asp Tyr1 5 109111PRTArtificial SequenceSynthetic 91Lys Ala Thr
Gln Asp Val Thr Pro Ala Val Ala1 5 109211PRTArtificial
SequenceSynthetic 92Lys Ala Cys Gln Asp Val Thr Pro Ala Val Ala1 5
10937PRTArtificial SequenceSynthetic 93Thr Thr Ser Ser Arg Tyr Thr1
5947PRTArtificial SequenceSynthetic 94Cys Thr Ser Ser Arg Tyr Thr1
5957PRTArtificial SequenceSynthetic 95Ser Ser Ser Ser Arg Tyr Thr1
5967PRTArtificial SequenceSynthetic 96Ser Met Ser Ser Arg Tyr Thr1
5977PRTArtificial SequenceSynthetic 97Ser Val Ser Ser Arg Tyr Thr1
5987PRTArtificial SequenceSynthetic 98Ser Thr Thr Ser Arg Tyr Thr1
5997PRTArtificial SequenceSynthetic 99Ser Thr Cys Ser Arg Tyr Thr1
51007PRTArtificial SequenceSynthetic 100Ser Thr Ser Thr Arg Tyr
Thr1 51017PRTArtificial SequenceSynthetic 101Ser Thr Ser Cys Arg
Tyr Thr1 51027PRTArtificial SequenceSynthetic 102Ser Thr Ser Ser
Lys Tyr Thr1 51037PRTArtificial SequenceSynthetic 103Ser Thr Ser
Ser Arg Phe Thr1 51047PRTArtificial SequenceSynthetic 104Ser Thr
Ser Ser Arg His Thr1 51057PRTArtificial SequenceSynthetic 105Ser
Thr Ser Ser Arg Trp Thr1 51069PRTArtificial SequenceSynthetic
106Glu Gln His Tyr Thr Thr Pro Leu Thr1 51079PRTArtificial
SequenceSynthetic 107Asp Gln His Tyr Thr Thr Pro Leu Thr1
51089PRTArtificial SequenceSynthetic 108Asn Gln His Tyr Thr Thr Pro
Leu Thr1 51099PRTArtificial SequenceSynthetic 109Gln Glu His Tyr
Thr Thr Pro Leu Thr1 51109PRTArtificial SequenceSynthetic 110Gln
Asp His Tyr Thr Thr Pro Leu Thr1 51119PRTArtificial
SequenceSynthetic 111Gln Asn His Tyr Thr Thr Pro Leu Thr1
5112357DNAArtificial SequenceSynthetic 112gaagtgaaac tggtggagtc
tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt
cactttcagt agctatgaca tgtcttgggt tcgccagact 120ccggagaaga
gtctggagtg ggtcgcaacc attagtgatg gtggtggtta catctactat
180tcagacagtg tgaaggggcg atttaccatc tccagagaca atgccaagaa
caacctgtac 240ctgcaaatga gcagtctgag gtctgaggac acggccttgt
atatttgtgc aagagaattt 300ggtaagcgct atgctttgga ctactggggt
caaggaacct cagtcaccgt ctcctca 357113116PRTArtificial
SequenceSynthetic 113Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu
Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Thr Pro
Glu Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Gly Gly Gly
Tyr Ile Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Asn Leu Tyr65 70 75 80Leu Gln Met Ser Ser
Leu Arg Ser Glu Asp Thr Ala Leu Tyr Ile Cys 85 90 95Ala Arg Glu Phe
Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser
Val Thr 115114321DNAArtificial SequenceSynthetic 114gacattgtga
tgacccagtc tcacaaattc atgtccacat cggtaggaga cagggtcagc 60atctcctgca
aggccagtca ggatgtgact cctgctgtcg cctggtatca acagaagcca
120ggacaatctc ctaaactact gatttactcc acatcctccc ggtacactgg
agtccctgat 180cgcttcactg gcagtggatc tgggacggat ttcactttca
ccatcagcag tgtgcaggct 240gaagacctgg cagtttatta ctgtcagcaa
cattatacta ctccgctcac gttcggtgct 300gggaccaagc tggagctgaa a
321115107PRTArtificial SequenceSynthetic 115Asp Ile Val Met Thr Gln
Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile
Ser Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75
80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
10511617PRTArtificial SequenceSynthetic 116Thr Ile Ser Asp Ala Gly
Gly Tyr Ile Tyr Tyr Arg Asp Ser Val Lys1 5 10
15Gly11710PRTArtificial SequenceSynthetic 117Glu Leu Pro Trp Arg
Tyr Ala Leu Asp Tyr1 5 1011817PRTArtificial SequenceSynthetic
118Thr Ile Ser Asp Ala Gly Ala Tyr Ile Tyr Tyr Arg Asp Ser Val Lys1
5 10 15Gly11917PRTArtificial SequenceSynthetic 119Thr Ile Ser Asp
Ala Gly Pro Tyr Ile Tyr Tyr Arg Asp Ser Val Lys1 5 10
15Gly12017PRTArtificial SequenceSynthetic 120Thr Ile Ser Asp Ala
Gly Gly Phe Ile Tyr Tyr Arg Asp Ser Val Lys1 5 10
15Gly12117PRTArtificial SequenceSynthetic 121Thr Ile Ser Asp Ala
Gly Gly His Ile Tyr Tyr Arg Asp Ser Val Lys1 5 10
15Gly12217PRTArtificial SequenceSynthetic 122Thr Ile Ser Asp Ala
Gly Gly Trp Ile Tyr Tyr Arg Asp Ser Val Lys1 5 10
15Gly12317PRTArtificial SequenceSynthetic 123Thr Ile Ser Asp Ala
Gly Gly Tyr Ile Tyr Tyr Arg Asp Thr Val Lys1 5 10
15Gly12417PRTArtificial SequenceSynthetic 124Thr Ile Ser Asp Ala
Gly Gly Tyr Ile Tyr Tyr Arg Asp Cys Val Lys1 5 10
15Gly12517PRTArtificial SequenceSynthetic 125Thr Ile Ser Asp Ala
Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Leu Lys1 5 10
15Gly12617PRTArtificial SequenceSynthetic 126Thr Ile Ser Asp Ala
Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Ile Lys1 5 10
15Gly12717PRTArtificial SequenceSynthetic 127Thr Ile Ser Asp Ala
Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Met Lys1 5 10
15Gly12810PRTArtificial SequenceSynthetic 128Glu Leu Phe Asn Arg
Tyr Ala Leu Asp Tyr1 5 1012910PRTArtificial SequenceSynthetic
129Glu Leu His Phe Arg Tyr Ala Leu Asp Tyr1 5 1013010PRTArtificial
SequenceSynthetic 130Glu Leu Tyr Phe Arg Tyr Ala Leu Asp Tyr1 5
1013110PRTArtificial SequenceSynthetic 131Glu Leu Leu His Arg Tyr
Ala Leu Asp Tyr1 5 1013210PRTArtificial SequenceSynthetic 132Glu
Leu Arg Gly Arg Tyr Ala Leu Asp Tyr1 5 1013310PRTArtificial
SequenceSynthetic 133Gln Leu Pro Trp Arg Tyr Ala Leu Asp Tyr1 5
1013410PRTArtificial SequenceSynthetic 134Asp Leu Pro Trp Arg Tyr
Ala Leu Asp Tyr1 5 1013510PRTArtificial SequenceSynthetic 135Asn
Leu Pro Trp Arg Tyr Ala Leu Asp Tyr1 5 1013610PRTArtificial
SequenceSynthetic 136Glu Leu Pro Trp Lys Tyr Ala Leu Asp Tyr1 5
1013710PRTArtificial SequenceSynthetic 137Glu Leu Pro Trp Arg Phe
Ala Leu Asp Tyr1 5 1013810PRTArtificial SequenceSynthetic 138Glu
Leu Pro Trp Arg His Ala Leu Asp Tyr1 5 1013910PRTArtificial
SequenceSynthetic 139Glu Leu Pro Trp Arg Trp Ala Leu Asp Tyr1 5
101409PRTArtificial SequenceSynthetic 140Gln Gln His Ser Asp Ala
Pro Leu Thr1 5141119PRTArtificial SequenceSynthetic 141Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40
45Ala Thr Ile Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val
Tyr Ile Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
115142107PRTArtificial SequenceSynthetic 142Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105143119PRTArtificial SequenceSynthetic 143Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys
85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 115144107PRTArtificial
SequenceSynthetic 144Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Lys
Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr Ser Ser Arg Tyr Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Met Gln His Tyr Thr Thr Pro Leu 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105145119PRTArtificial
SequenceSynthetic 145Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Ala Gly Gly
Tyr Ile Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys 85 90 95Ala Arg Glu Phe
Gly Lys Arg Tyr Ala Leu Asp Ser Trp Gly Gln Gly 100 105 110Thr Thr
Val Thr Val Ser Ser 115146107PRTArtificial SequenceSynthetic 146Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Trp Pro Ala
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Thr Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His
Ser Thr Thr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105147119PRTArtificial SequenceSynthetic
147Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp
Thr Ala Val Tyr Ile Cys 85 90 95Ala Arg Glu Ile Phe Asn Arg Tyr Ala
Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
115148107PRTArtificial SequenceSynthetic 148Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105149119PRTArtificial SequenceSynthetic 149Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys
85 90 95Ala Arg Glu Leu Pro Trp Arg Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 115150107PRTArtificial
SequenceSynthetic 150Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr Ser Ser Arg Tyr Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105151119PRTArtificial
SequenceSynthetic 151Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Ala Gly Gly
Tyr Ile Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys 85 90 95Ala Arg Glu Leu
His Phe Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr
Val Thr Val Ser Ser 115152107PRTArtificial SequenceSynthetic 152Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Thr Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105153119PRTArtificial SequenceSynthetic 153Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45Ala
Thr Ile Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile
Cys 85 90 95Ala Arg Glu Leu Tyr Phe Arg Tyr Ala Leu Asp Tyr Trp Gly
Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
115154107PRTArtificial SequenceSynthetic 154Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105155119PRTArtificial SequenceSynthetic 155Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys
85 90 95Ala Arg Glu Leu Leu His Arg Tyr Ala Leu Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 115156107PRTArtificial
SequenceSynthetic 156Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr Ser Ser Arg Tyr Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105157119PRTArtificial
SequenceSynthetic 157Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Ser Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Asp Ala Gly Gly
Tyr Ile Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile Cys 85 90 95Ala Arg Glu Leu
Arg Gly Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr
Val Thr Val Ser Ser 115158107PRTArtificial SequenceSynthetic 158Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Thr Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105159119PRTArtificial SequenceSynthetic 159Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Ser Leu Glu Trp Val 35 40 45Ala
Thr Ile Ser Asp Ala Gly Gly Tyr Ile Tyr Tyr Arg Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Ile
Cys 85 90 95Ala Arg Glu Phe Gly Lys Arg Tyr Ala Leu Asp Tyr Trp Gly
Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
115160107PRTArtificial SequenceSynthetic 160Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Thr Pro Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Thr
Ser Ser Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Ser Asp Ala Pro Leu
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
1051619PRTArtificial SequenceSynthetic 161Glu Gln His Ser Asp Ala
Pro Leu Thr1 51629PRTArtificial SequenceSynthetic 162Asp Gln His
Ser Asp Ala Pro Leu Thr1 51639PRTArtificial SequenceSynthetic
163Asn Gln His Ser Asp Ala Pro Leu Thr1 51649PRTArtificial
SequenceSynthetic 164Gln Glu His Ser Asp Ala Pro Leu Thr1
51659PRTArtificial SequenceSynthetic 165Gln Asp His Ser Asp Ala Pro
Leu Thr1 51669PRTArtificial SequenceSynthetic 166Gln Asn His Ser
Asp Ala Pro Leu Thr1 5
* * * * *
References