U.S. patent application number 16/907794 was filed with the patent office on 2020-10-08 for anti-pd-1 antibodies and their uses.
The applicant listed for this patent is AbbVie Biotherapeutics Inc.. Invention is credited to Daniel E.H. AFAR, Fiona A. HARDING, Josue SAMAYOA.
Application Number | 20200317808 16/907794 |
Document ID | / |
Family ID | 1000004901918 |
Filed Date | 2020-10-08 |
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United States Patent
Application |
20200317808 |
Kind Code |
A1 |
AFAR; Daniel E.H. ; et
al. |
October 8, 2020 |
ANTI-PD-1 ANTIBODIES AND THEIR USES
Abstract
The present disclosure provides novel anti-PD-1 antibodies,
compositions including the new antibodies, nucleic acids encoding
the antibodies, and methods of making and using the same.
Inventors: |
AFAR; Daniel E.H.; (Los
Altos Hills, CA) ; HARDING; Fiona A.; (Mountain View,
CA) ; SAMAYOA; Josue; (San Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Biotherapeutics Inc. |
Redwood City |
CA |
US |
|
|
Family ID: |
1000004901918 |
Appl. No.: |
16/907794 |
Filed: |
June 22, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16660634 |
Oct 22, 2019 |
10730953 |
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16907794 |
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15875970 |
Jan 19, 2018 |
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16660634 |
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15704296 |
Sep 14, 2017 |
9914783 |
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15875970 |
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62394314 |
Sep 14, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/75 20130101;
C07K 2317/92 20130101; C07K 2317/76 20130101; C07K 16/30 20130101;
C07K 16/2896 20130101; C07K 2317/73 20130101; C07K 2317/565
20130101; A61K 39/395 20130101; C07K 2317/33 20130101; C07K 2317/24
20130101; C07K 2319/30 20130101; C07K 16/2818 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 39/395 20060101 A61K039/395; C07K 16/28 20060101
C07K016/28 |
Claims
1. An anti-PD-1 binding protein which comprises (i) a V.sub.H chain
comprising three CDRs; and (ii) a V.sub.L chain comprising three
CDRs, wherein: TABLE-US-00015 V.sub.H CDR#1 is (SEQ ID NO: 11)
GYTFTHYGMN; V.sub.H CDR#2 is (SEQ ID NO: 12) WVNTYTGEPTYADDFKG;
V.sub.H CDR#3 is (SEQ ID NO: 13) EGEGLGFGD; V.sub.L CDR#1 is (SEQ
ID NO: 14) RSSQSIVHSHGDTYLE; V.sub.L CDR#2 is (SEQ ID NO: 15)
KVSNRFS; and V.sub.L CDR#3 is (SEQ ID NO: 16) FQGSHIPVT.
2. The anti-PD-1 binding protein of claim 1, which is
humanized.
3. The anti-PD-1 binding protein of claim 2, which comprises a
V.sub.H chain corresponding in sequence to SEQ ID NO:36; and a
V.sub.L chain corresponding in sequence to SEQ ID NO:42.
4. The anti-PD-1 binding protein of claim 3, which is an IgG.
5. The anti-PD-1 binding protein of claim 4, which is an
IgG.sub.1.
6. The anti-PD-1 binding protein of claim 5 comprising a variant
CH2 domain having amino acid substitutions L234A and L235A.
7. The anti-PD-1 binding protein of claim 4, which is an
IgG.sub.4.
8. The anti-PD-1 binding protein of claim 7 comprising a variant Fc
region having an amino acid substitution S228P.
9. The anti-PD-1 binding protein of claim 3 comprising a kappa
light constant region.
10. The anti-PD-1 binding protein of claim 3 which comprises a
heavy chain corresponding in sequence to SEQ ID NO:51 or SEQ ID
NO:52, and a light chain corresponding in sequence to SEQ ID
NO:61.
11. The anti-PD-1 binding protein of claim 1 which has a K.sub.D of
less than about 100 nM.
12. The anti-PD-1 binding protein of claim 11 which has a K.sub.D
of less than about 10 nM.
13. A pharmaceutical composition comprising the anti-PD-1 binding
protein of claim 1, and a pharmaceutically acceptable carrier.
14. A nucleic acid comprising a nucleotide sequence encoding an
anti-PD-1 binding protein, wherein the binding protein comprises
(i) a V.sub.H chain comprising three CDRs; and (ii) a V.sub.L chain
comprising three CDRs, wherein: TABLE-US-00016 V.sub.H CDR#1 is
(SEQ ID NO: 11) GYTFTHYGMN; V.sub.H CDR#2 is (SEQ ID NO: 12)
WVNTYTGEPTYADDFKG; V.sub.H CDR#3 is (SEQ ID NO: 13) EGEGLGFGD;
V.sub.L CDR#1 is (SEQ ID NO: 14) RSSQSIVHSHGDTYLE; V.sub.L CDR#2 is
(SEQ ID NO: 15) KVSNRFS; and V.sub.L CDR#3 is (SEQ ID NO: 16)
FQGSHIPVT.
15. A vector comprising the nucleic acid of claim 14.
16. A prokaryotic host cell transformed with the vector of claim
15.
17. A eukaryotic host cell transformed with the vector of claim
15.
18. A eukaryotic host cell engineered to express the nucleic acid
of claim 14.
19. The eukaryotic host cell of claim 18 which is a mammalian host
cell.
20. A method of producing an anti-PD-1 binding protein thereof,
comprising: (a) culturing the host cell of claim 19 and (b)
recovering the anti-PD-1 binding protein.
21. A method of activating the immune system, comprising
administering to a patient in need thereof an effective amount of
an anti-PD-1 binding protein expressed by the host cell of claim
18.
22. A method of treating a cancer, comprising administering to a
patient in need thereof a therapeutically effective amount of an
anti-PD-1 binding protein, wherein the binding protein comprises
(i) a V.sub.H chain comprising three CDRs; and (ii) a V.sub.L chain
comprising three CDRs, wherein: TABLE-US-00017 V.sub.H CDR#1 is
(SEQ ID NO: 11) GYTFTHYGMN; V.sub.H CDR#2 is (SEQ ID NO: 12)
WVNTYTGEPTYADDFKG; V.sub.H CDR#3 is (SEQ ID NO: 13) EGEGLGFGD;
V.sub.L CDR#1 is (SEQ ID NO: 14) RSSQSIVHSHGDTYLE; V.sub.L CDR#2 is
(SEQ ID NO: 15) KVSNRFS; and V.sub.L CDR#3 is (SEQ ID NO: 16)
FQGSHIPVT.
23. The method of claim 22, wherein the cancer is selected from
bladder cancer, breast cancer, head and neck cancer, kidney cancer,
lung cancer, lymphoma, melanoma, and stomach cancer.
24. The method of claim 23, wherein the lung cancer is non-small
cell lung cancer.
25. The method of claim 21, in which the anti-PD-1 binding protein
is administered intravenously.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Application No. 62/394,314, filed Sep.
14, 2016, the contents of which are incorporated herein in its
entirety by reference thereto.
2. SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 10, 2017, is named 381493-327US_SL.txt and is 32,733 bytes
in size.
3. TECHNICAL FIELD
[0003] The present application pertains to, among other things,
novel anti-PD-1 antibodies, compositions including the new
antibodies, nucleic acids encoding the antibodies, and methods of
making and using the same.
4. BACKGROUND
[0004] Cancer therapies comprise a wide range of therapeutic
approaches, including surgery, radiation, and chemotherapy. While
the various approaches allow a broad selection of treatments to be
available to the medical practitioner to treat the cancer, existing
therapeutics suffer from a number of disadvantages, such as a lack
of selectivity of targeting cancer cells over normal, healthy
cells, and the development of resistance by the cancer to the
treatment.
[0005] Recent approaches based on targeted therapeutics, which
interfere with cellular processes of cancer cells preferentially
over normal cells, have led to chemotherapeutic regimens with fewer
side effects as compared to non-targeted therapies such as
radiation treatment.
[0006] Cancer immunotherapy has emerged as a promising therapeutic
approach to complement existing standards of care. See, e.g.,
Miller, et al. Cancer Cell, 27, 439-449 (2015). Such immunotherapy
approaches include the development of antibodies used to modulate
the immune system to kill cancer cells.
[0007] For example, interaction of PD-1, a type I cell surface
receptor, with either of its two ligands, PD-L1 or PD-L2, results
in a dominant negative checkpoint signal that limits subsequent
antigen receptor-driven cellular activation. The ligands for PD-1
are differentially expressed on various tissues and cell types,
including antigen-presenting cells of the immune system, and are
upregulated on many types of tumor cells. Upregulation of PD-L1
within the tumor microenvironment is a proposed mechanism of tumors
to subvert protective anti-tumor immune responses by the host.
Antibodies directed at PD-1 that block the interaction of the
receptor with either of its ligands result in inhibition of
negative signaling. In vitro inhibition of the PD-1 mediated
checkpoint signal has been demonstrated to result in prolonged
antigen-specific T cell activation. In vivo PD-1 blockade has been
shown to enhance anti-tumor immune responses in both syngeneic
mouse tumor models and in human clinical trials.
[0008] Anti-tumor immune responses in patients with solid tumors
have been enhanced by anti-PD-1 treatment. There are two approved
and marketed antagonistic anti-PD-1 monoclonal antibodies:
nivolumab (OPDIVO.RTM.) and pembrolizumab (KEYTRUDA.RTM.), with
approvals in the US and the European Union to treat diseases such
as unresectable or metastatic melanoma and metastatic non-small
cell lung cancer. Treatment of patients with these agents has
resulted in anti-tumor responses as measured by improvement in
either progression free survival and/or overall survival.
[0009] The recent failure of OPDIVO.RTM. to slow progression of
advanced lung cancer in a treatment-naive patient population in a
clinical trial comparing OPDIVO.RTM. with conventional chemotherapy
highlights the need for alternative approaches and additional
cancer treatments to complement existing therapeutic standards of
care.
5. SUMMARY
[0010] The present disclosure provides anti-PD-1 antibodies and
binding fragments thereof that specifically bind to PD-1. The amino
acid sequences of exemplary CDRs, as well as the amino acid
sequence of the V.sub.H and V.sub.L regions of the heavy and light
chains of exemplary anti-PD-1 antibodies are provided in the
Detailed Description below. Antibodies provided herein interfere
with the interaction of the PD-1 receptor with either of its
ligands (PD-L1, SEQ ID NO:3; PD-L2, SEQ ID NO:4), resulting in
inhibition of negative signaling and upregulation of the adaptive
immune response.
[0011] The anti-PD-1 antibodies may include modifications and/or
mutations that alter the properties of the antibodies, such as
increase half-life, increase or decrease ADCC, etc., as is known in
the art.
[0012] Nucleic acids comprising nucleotide sequences encoding the
anti-PD-1 antibodies of the disclosure are provided herein, as are
vectors comprising nucleic acids. Additionally, prokaryotic and
eukaryotic host cells transformed with a vector comprising a
nucleotide sequence encoding a disclosed anti-PD-1 antibody are
provided herein, as well as eukaryotic (such as mammalian) host
cells engineered to express the nucleotide sequences. Methods of
producing antibodies, by culturing host cells and recovering the
antibodies are also provided, and discussed further in the Detailed
Description below.
[0013] In another aspect, the present disclosure provides
compositions including the anti-PD-1 antibodies described herein.
The compositions generally comprise one or more anti-PD-1 antibody
as described herein, and/or salts thereof, and one or more
excipients, carriers or diluents.
[0014] The present disclosure provides methods of treating
subjects, such as human subjects, diagnosed with a solid tumor or a
blood malignancy with an anti-PD-1 antibody. The method generally
involves administering to the subject an amount of an anti-PD-1
antibody described herein effective to provide therapeutic benefit.
The subject may be diagnosed with any one of a number of solid
tumors or blood malignancies that may be newly diagnosed, relapsed,
or relapsed and refractory. An anti-PD-1 antibody is typically
administered as an intravenous infusion twice a week, once a week,
once every two weeks, once every three weeks, once every four
weeks, once every five weeks, once every six weeks, once every
seven weeks, or once every eight weeks.
[0015] The anti-PD-1 antibodies may be administered as single
therapeutic agents (monotherapy) or adjunctive to or with other
therapeutic agents typically, but not necessarily, those used for
the treatment of a solid tumor or blood malignancy. Therapeutic
agents typically will be used at their approved dose, route of
administration, and frequency of administration, but may be used at
lower dosages.
[0016] The anti-PD-1 antibodies may be administered via a variety
of routes or modes of administration, including but not limited to,
intravenous infusion and/or injection, intratumoral injection, and
subcutaneous injection. The amount administered will depend upon
the route of administration, the dosing schedule, the type of
cancer being treated, the stage of the cancer being treated, and
other parameters such as the age and weight of the patient, as is
well known in the art. Specific exemplary dosing schedules expected
to provide therapeutic benefit are provided in the Detailed
Description.
6. BRIEF DESCRIPTION OF THE FIGURES
[0017] FIGS. 1A-1B show the amino acid sequences of human PD-1 (SEQ
ID NO:1), murine PD-1 (SEQ ID NO:2), human PD-L1 (SEQ ID NO:3), and
human PD-L2 (SEQ ID NO:4). FIG. 1A depicts the sequences of human
and murine PD-1; FIG. 1B depicts the sequences of human PD-L1 and
PD-L2.
[0018] FIG. 2 provides amino acid sequences of V.sub.H and V.sub.L
regions in exemplary anti-PD-1 antibodies of the disclosure.
[0019] FIGS. 3A-3C show the effects of oxidative and variable
temperature conditions on exemplary humanized antibody Hu12A11.2b1
and its point mutations at Kabat 99 in CDR-H3. FIG. 3A depicts
binding to PD-1 in Jurkat cells of Hu12A11.2b1 stored at -80, 5,
25, or 40.degree. C. for 30 days or after being subjected to
oxidative conditions (1% hydrogen peroxide, "1% HP"; or 1%
tert-butyl hydroperoxide, "1% TBHP"); FIG. 3B depicts binding of
anti-PD-1 antibodies Hu12A11.2b1, Hu12A11.2b2, Hu12A11.2b3, and
Hu12A11.2b4; FIG. 3C depicts the binding of Hu12A11.2b4 to PD-1 in
Jurkat cells after incubation at -80, 5, 25, or 40.degree. C. for
30 days.
[0020] FIGS. 4A-4B show the biological activity of nivolumab and
Hu12A11.2b4 in mixed leukocyte response (MLR) and tetanus toxoid
antigen recall assays. FIG. 4A shows enhancement in IL-2 or
interferon-gamma (IFN-.gamma.) levels after treatment with 10
.mu.g/mL of nivolumab, Hu12A11.2b4, or isotype control in mixed
leukocyte cultures. FIG. 4B shows dose-dependent response of
nivolumab, Hu12A11.2b4, or isotype control on IFN-.gamma.
enhancement in tetanus toxoid response assay.
7. DETAILED DESCRIPTION
[0021] The present disclosure concerns antibodies and fragments
that specifically bind PD-1, compositions comprising the
antibodies, polynucleotides encoding anti-PD-1 antibodies, host
cells capable of producing the antibodies, methods and compositions
useful for making the antibodies and binding fragments, and various
methods of using the same.
[0022] As will be appreciated by skilled artisans, antibodies are
"modular" in nature. Throughout the disclosure, various specific
embodiments of the various "modules" composing the antibodies are
described. As specific non-limiting examples, various specific
embodiments of V.sub.H CDRs, V.sub.H chains, V.sub.L CDRs and
V.sub.L chains are described. It is intended that all of the
specific embodiments may be combined with each other as though each
specific combination were explicitly described individually.
7.1. Abbreviations
[0023] The antibodies, binding fragments, and polynucleotides
described herein are, in many embodiments, described by way of
their respective polypeptide or polynucleotide sequences. Unless
indicated otherwise, polypeptide sequences are provided in
N.fwdarw.C orientation; polynucleotide sequences in 5'.fwdarw.3'
orientation. For polypeptide sequences, the conventional three or
one-letter abbreviations for the genetically encoded amino acids
may be used, as noted in TABLE 1, below.
TABLE-US-00001 TABLE 1 Encoded Amino Acid Abbreviations Amino Acid
Three Letter Abbreviation One-Letter Abbreviation Alanine Ala A
Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C
Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H
Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0024] Certain sequences are defined by structural formulae
specifying amino acid residues belonging to certain classes (e.g.,
aliphatic, hydrophobic, etc.). The various classes to which the
genetically encoded amino acids belong as used herein are noted in
TABLE 2, below. Some amino acids may belong to more than one class.
Cysteine, which contains a sulfhydryl group, and proline, which is
conformationally constrained, are not assigned classes.
TABLE-US-00002 TABLE 2 Encoded Amino Acid Classes Class Amino Acids
Aliphatic A, I, L,V Aromatic F, Y, W Non-Polar M, A, I, L, V Polar
N, Q, S, T Basic H, K, R Acidic D, E Small A, G
[0025] The abbreviations used for the various exemplary antibodies
disclosed herein are provided in TABLE 3, below:
TABLE-US-00003 TABLE 3 Antibody Abbreviations Clone/Name
Abbreviation V.sub.H Sequence (FIG. 2) V.sub.L Sequence (FIG. 2)
Mouse 12A11 Mu12A11 Mu12A11 V.sub.H SEQ ID NO: 31 Mu12A11 V.sub.L
SEQ ID NO: 41 Humanized 12A11.1b Hu12A11.1b Hu12A11.1b V.sub.H SEQ
ID NO: 32 Hu12A11.1a V.sub.L SEQ ID NO: 42 Humanized 12A11.2b
Hu12A11.2b1 Hu12A11.2b V.sub.H SEQ ID NO: 33 Hu12A11.1a V.sub.L SEQ
ID NO: 42 with mouse CDRs Humanized 12A11.2b Hu12A11.2b2 Hu12A11.2b
V.sub.H SEQ ID NO: 34 Hu12A11.1a V.sub.L SEQ ID NO: 42 with M99I in
CDR-H3 M99I variant Humanized 12A11.2b Hu12A11.2b3 Hu12A11.2b
V.sub.H SEQ ID NO: 35 Hu12A11.1a V.sub.L SEQ ID NO: 42 with M99V in
CDR-H3 M99V variant Humanized 12A11.2b Hu12A11.2b4 Hu12A11.2b
V.sub.H SEQ ID NO: 36 Hu12A11.1a V.sub.L SEQ ID NO: 42 with M99L in
CDR-H3 M99L variant
7.2. Definitions
[0026] Unless otherwise defined herein, scientific and technical
terms used in connection with the present disclosure shall have the
meanings that are commonly understood by those of ordinary skill in
the art.
7.3. Anti-PD-1 Antibodies and Binding Fragments
[0027] In one aspect, the disclosure concerns antibodies and/or
binding fragments thereof that specifically bind programmed cell
death protein 1 (PD-1) receptor (also known as PDCD1, CD279, PD1,
SLEB2, or SLE1).
[0028] As used herein, the term "antibody" (Ab) refers to an
immunoglobulin molecule that specifically binds to a particular
antigen--here, PD-1. In some embodiments, the anti-PD-1 antibodies
of the disclosure bind to human PD-1 and thereby modulate the
immune system. The resulting immune system response is cytotoxic to
tumor cells. Anti-PD-1 antibodies of the disclosure comprise
complementarity determining regions (CDRs), also known as
hypervariable regions, in both the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework (FR). As is known in the art, the
amino acid position/boundary delineating a hypervariable region of
an antibody can vary, depending on the context and the various
definitions known in the art. Some positions within a variable
domain may be viewed as hybrid hypervariable positions in that
these positions can be deemed to be within a hypervariable region
under one set of criteria while being deemed to be outside a
hypervariable region under a different set of criteria. One or more
of these positions can also be found in extended hypervariable
regions. The disclosure provides antibodies comprising
modifications in these hybrid hypervariable positions. The variable
domains of native heavy and light chains each comprise four FR
regions, largely by adopting a .beta.-sheet configuration,
connected by three CDRs, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The CDRs in each
chain are held together in close proximity by the FR regions and,
with the CDRs from the other chain, contribute to the formation of
the target binding site of antibodies. See Kabat et al., Sequences
of Proteins of Immunological Interest (National Institute of
Health, Bethesda, Md. 1987). As used herein, numbering of
immunoglobulin amino acid residues is done according to the
immunoglobulin amino acid residue numbering system of Kabat et al.
unless otherwise indicated.
[0029] The antibodies of the disclosure may be polyclonal,
monoclonal, genetically engineered, and/or otherwise modified in
nature, including but not limited to chimeric antibodies, humanized
antibodies, human antibodies, single chain antibodies, etc. In
various embodiments, the antibodies comprise all or a portion of a
constant region of an antibody. In some embodiments, the constant
region is an isotype selected from: IgA (e.g., IgA.sub.1 or
IgA.sub.2), IgD, IgE, IgG (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3 or
IgG.sub.4), and IgM. In specific embodiments, the antibodies
described herein comprise an IgG.sub.1. In other embodiments, the
anti-PD-1 antibodies comprise an IgG.sub.2. In yet other
embodiments, the anti-PD-1 antibodies comprise an IgG.sub.4. As
used herein, the "constant region" of an antibody includes the
natural constant region, allotypes or natural variants, such as
D356E and L358M, or A431G in human IgG.sub.1. See, e.g., Jefferis
and Lefranc, MAbs, 1(4): 332-338 (July-August 2009).
[0030] The light constant region of an anti-PD-1 antibody may be a
kappa (.kappa.) light region or a lambda (.lamda.) region. A light
region can be any one of the known subtypes, e.g., .lamda..sub.1,
.lamda..sub.2, .lamda..sub.3, or .lamda..sub.4. In some
embodiments, the anti-PD-1 antibody comprises a kappa (.kappa.)
light region.
[0031] The term "monoclonal antibody" as used herein is not limited
to antibodies produced through hybridoma technology. A monoclonal
antibody is derived from a single clone, including any eukaryotic,
prokaryotic, or phage clone, by any means available or known in the
art. Monoclonal antibodies useful with the present disclosure can
be prepared using a wide variety of techniques known in the art
including the use of hybridoma, recombinant, and phage display
technologies, or a combination thereof. In many uses of the present
disclosure, including in vivo use of the anti-PD-1 antibodies in
humans, chimeric, humanized, or human antibodies can suitably be
used.
[0032] The term "chimeric" antibody as used herein refers to an
antibody having variable sequences derived from a non-human
immunoglobulin, such as a rat or a mouse antibody, and human
immunoglobulin constant regions, typically chosen from a human
immunoglobulin template. Methods for producing chimeric antibodies
are known in the art. See, e.g., Morrison, 1985, Science
229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies
et al., 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816,397.
[0033] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins that contain minimal sequences derived
from non-human immunoglobulin. In general, a humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody can also comprise
at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin consensus sequence.
Methods of antibody humanization are known in the art. See, e.g.,
Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101;
5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No. 6,180,370 to
Queen et al.; EP239400; PCT publication WO 91/09967; U.S. Pat. No.
5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol.,
28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska
et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No.
5,565,332.
[0034] "Human antibodies" include antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from animals transgenic for
one or more human immunoglobulins and that do not express
endogenous immunoglobulins. 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 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. Human antibodies can also
be produced using transgenic mice which are incapable of expressing
functional endogenous immunoglobulins but which can express human
immunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO
92/01047; 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;
5,885,793; 5,916,771; and 5,939,598. In addition, companies such as
LakePharma, Inc. (Belmont, Calif.) or Creative BioLabs (Shirley,
N.Y.) can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described above.
Fully human antibodies that recognize a selected epitope can 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 (see, Jespers et al.,
1988, Biotechnology 12:899-903).
[0035] Anti-PD-1 antibodies of the disclosure include full-length
(intact) antibody molecules.
[0036] In some embodiments, the present disclosure also includes
anti-PD-1 binding fragments that are capable of specifically
binding PD-1. Examples of antibody binding fragments include by way
of example and not limitation, Fab, Fab', F(ab').sub.2, Fv
fragments, single chain Fv fragments and single domain
fragments.
[0037] An Fab fragment contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
F(ab') fragments are produced by cleavage of the disulfide bond at
the hinge cysteines of the F(ab').sub.2 pepsin digestion product.
Additional chemical couplings of antibody fragments are known to
those of ordinary skill in the art. Fab and F(ab').sub.2 fragments
lack the Fc fragment of an intact antibody, clear more rapidly from
the circulation of animals, and may have less non-specific tissue
binding than an intact antibody (see, e.g., Wahl et al., 1983, J.
Nucl. Med. 24:316).
[0038] An "Fv" fragment is the minimum fragment of an antibody that
contains a complete target recognition and binding site. This
region consists of a dimer of one heavy and one light chain
variable domain in a tight, non-covalent association
(V.sub.H-V.sub.L dimer). It is in this configuration that the three
CDRs of each variable domain interact to define a target binding
site on the surface of the V.sub.H-V.sub.L dimer. Often, the six
CDRs confer target binding specificity to the antibody. However, in
some instances even a single variable domain (or half of an Fv
comprising only three CDRs specific for a target) can have the
ability to recognize and bind target, although at a lower affinity
than the entire binding site.
[0039] "Single-chain Fv" or "scFv" antibody binding fragments
comprise the V.sub.H and V.sub.L domains of an antibody, where
these domains are present in a single polypeptide chain. Generally,
the Fv polypeptide further comprises a polypeptide linker between
the V.sub.H and V.sub.L domains which enables the scFv to form the
desired structure for target binding.
[0040] "Single domain antibodies" are composed of a single V.sub.H
or V.sub.L domains which exhibit sufficient affinity to PD-1. In a
specific embodiment, the single domain antibody is a camelized
antibody (See, e.g., Riechmann, 1999, Journal of Immunological
Methods 231:25-38).
[0041] The anti-PD-1 antibodies of the disclosure include
derivatized antibodies. For example, but not by way of limitation,
derivatized antibodies are typically modified by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein. Any of numerous chemical
modifications can be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally,
the derivative can contain one or more non-natural amino acids,
e.g., using ambrx technology (See, e.g., Wolfson, 2006, Chem. Biol.
13(10):1011-2).
[0042] The anti-PD-1 antibodies or binding fragments may be
antibodies or fragments whose sequences have been modified to alter
at least one constant region-mediated biological effector function.
For example, in some embodiments, an anti-PD-1 antibody may be
modified to reduce at least one constant region-mediated biological
effector function relative to the unmodified antibody, e.g.,
reduced binding to one or more of the Fc receptors (Fc.gamma.R)
such as Fc.gamma.RI, Fc.gamma.RIIA, Fc.gamma.RIIB, Fc.gamma.RIIIA
and/or Fc.gamma.RIIIB. Fc.gamma.R binding can be reduced by
mutating the immunoglobulin constant region segment of the antibody
at particular regions necessary for Fc.gamma.R interactions (See,
e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and
Lund et al., 1991, J. Immunol. 147:2657-2662). Reduction in
Fc.gamma.R binding ability of the antibody can also reduce other
effector functions which rely on Fc.gamma.R interactions, such as
opsonization, phagocytosis and antigen-dependent cellular
cytotoxicity ("ADCC").
[0043] The anti-PD-1 antibody or binding fragment described herein
include antibodies that have been modified to acquire or improve at
least one constant region-mediated biological effector function
relative to an unmodified antibody, e.g., to enhance Fc.gamma.R
interactions (See, e.g., US Patent Appl. No. 2006/0134709). For
example, an anti-PD-1 antibody of the disclosure can have a
constant region that binds Fc.gamma.RI, Fc.gamma.RIIA,
Fc.gamma.RIIB, Fc.gamma.RIIIA and/or Fc.gamma.RIIIB with greater
affinity than the corresponding wild type constant region.
[0044] Thus, antibodies of the disclosure may have alterations in
biological activity that result in increased or decreased
opsonization, phagocytosis, or ADCC. Such alterations are known in
the art. For example, modifications in antibodies that reduce ADCC
activity are described in U.S. Pat. No. 5,834,597. An exemplary
ADCC lowering variant corresponds to "mutant 3" (also known as
"M3," shown in FIG. 4 of U.S. Pat. No. 5,834,597) in which residues
234 and 237 (using EU numbering) are substituted with alanines. A
mutant 3 (also known as "M3") variation may be used in a number of
antibody isotypes, e.g., IgG.sub.2.
[0045] Additional substitutions that can modify Fc.gamma.R binding
and/or ADCC effector function of an anti-PD-1 antibody include the
K322A substitution or the L234A and L235A double substitution in
the Fc region. See, e.g., Hezareh, et al. J. Virol., 75 (24):
12161-12168 (2001).
[0046] In some embodiments, the anti-PD-1 antibodies of the
disclosure have low levels of, or lack, fucose. Antibodies lacking
fucose have been correlated with enhanced ADCC activity, especially
at low doses of antibody. See Shields et al., 2002, J. Biol. Chem.
277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73.
Methods of preparing fucose-less antibodies include growth in rat
myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells express low levels
of FUT8 mRNA, which encodes .alpha.-1,6-fucosyltransferase, an
enzyme necessary for fucosylation of polypeptides.
[0047] The anti-PD-1 antibodies of the disclosure can comprise
modified (or variant) CH2 domains or entire Fc domains that include
amino acid substitutions that increase binding to Fc.gamma.RIIB
and/or reduced binding to Fc.gamma.RIIIA as compared to the binding
of a corresponding wild-type CH2 or Fc region. Variant CH2 or
variant Fc domains have been described in U.S. Patent Appl. No.
2014/0377253. A variant CH2 or variant Fc domain typically includes
one or more substitutions at position 263, position 266, position
273, and position 305, wherein the numbering of the residues in the
Fc domain is that of the EU index as in Kabat. In some embodiments,
the anti-PD-1 antibodies comprise one or more substitutions
selected from V263L, V266L, V273C, V273E, V273F, V273L, V273M,
V273S, V273Y, V305K, and V305W, relative to the wild-type CH2
domain. In specific embodiments, the one or more substitutions of
the CH2 domain are selected from V263L, V273E, V273F, V273M, V273S,
and V273Y, relative to the CH2 domain of a human IgG.sub.1. For
example, the one or more substitutions of an IgG.sub.1 CH2 domain
can be V273E. In another specific embodiment, the anti-PD-1
antibody of the disclosure comprises a variant IgG.sub.1 hinge
region comprising the amino acid substitution V263L.
[0048] Other examples of variant CH2 or variant Fc domains that can
afford increased binding to Fc.gamma.RIIB and/or reduced binding to
Fc.gamma.RIIIA as compared to the binding of a corresponding
wild-type CH2 or Fc region include those found in Vonderheide, et
al. Clin. Cancer Res., 19(5), 1035-1043 (2013), such as S267E or
S267E/L328F in human IgG.sub.1.
[0049] Anti-PD-1 antibodies or binding fragments that comprise a
human IgG.sub.4 constant region can comprise the S228P mutation,
which has been reported to prevent Fab arm exchange. See, e.g.,
Silva, J P et al. Journal of Biological Chemistry, 290(9),
5462-5469 (2015).
[0050] In some embodiments, the anti-PD-1 antibodies or binding
fragments include modifications that increase or decrease their
binding affinities to the fetal Fc receptor, FcRn, for example, by
mutating the immunoglobulin constant region segment at particular
regions involved in FcRn interactions (see, e.g., WO 2005/123780).
In particular embodiments, an anti-PD-1 antibody of the IgG class
is mutated such that at least one of amino acid residues 250, 314,
and 428 of the heavy chain constant region is substituted alone, or
in any combinations thereof, such as at positions 250 and 428, or
at positions 250 and 314, or at positions 314 and 428, or at
positions 250, 314, and 428, with positions 250 and 428 a specific
combination. For position 250, the substituting amino acid residue
can be any amino acid residue other than threonine, including, but
not limited to, alanine, cysteine, aspartic acid, glutamic acid,
phenylalanine, glycine, histidine, isoleucine, lysine, leucine,
methionine, asparagine, proline, glutamine, arginine, serine,
valine, tryptophan, or tyrosine. For position 314, the substituting
amino acid residue can be any amino acid residue other than
leucine, including, but not limited to, alanine, cysteine, aspartic
acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine,
lysine, methionine, asparagine, proline, glutamine, arginine,
serine, threonine, valine, tryptophan, or tyrosine. For position
428, the substituting amino acid residues can be any amino acid
residue other than methionine, including, but not limited to,
alanine, cysteine, aspartic acid, glutamic acid, phenylalanine,
glycine, histidine, isoleucine, lysine, leucine, asparagine,
proline, glutamine, arginine, serine, threonine, valine,
tryptophan, or tyrosine. An exemplary substitution known to modify
Fc effector function is the Fc substitution M428L, which can occur
in combination with the Fc substitution T250Q. Additional specific
combinations of suitable amino acid substitutions are identified in
Table 1 of U.S. Pat. No. 7,217,797. Such mutations increase binding
to FcRn, which protects the antibody from degradation and increases
its half-life.
[0051] An anti-PD-1 antibody may have one or more amino acids
inserted into one or more of its CDRs, for example as described in
Jung and Pluckthun, 1997, Protein Engineering 10:9, 959-966; Yazaki
et al., 2004, Protein Eng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17;
and U.S. Pat. Appl. No. 2007/0280931.
[0052] Anti-PD-1 antibodies with high affinity for human PD-1 may
be desirable for therapeutic and diagnostic uses. Accordingly, the
present disclosure contemplates antibodies having a high binding
affinity to human PD-1. In specific embodiments, the anti-PD-1
antibodies that bind human PD-1 with an affinity of at least about
100 nM, but may exhibit higher affinity, for example, at least
about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20
nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM,
0.01 nM, or even higher. In some embodiments, the antibodies bind
human PD-1 with an affinity in the range of about 1 pM to about 100
nM, or an affinity ranging between any of the foregoing values.
[0053] Affinity of anti-PD-1 antibodies for human PD-1 can be
determined using techniques well known in the art or described
herein, such as for example, but not by way of limitation, ELISA,
isothermal titration calorimetry (ITC), surface plasmon resonance,
or fluorescent polarization assay.
[0054] Anti-PD-1 antibodies generally comprise a heavy chain
comprising a variable region (V.sub.H) having three complementarity
determining regions ("CDRs") referred to herein (in N.fwdarw.C
order) as V.sub.H CDR #1, V.sub.H CDR #2, and V.sub.H CDR #3, and a
light chain comprising a variable region (V.sub.L) having three
complementarity determining regions referred to herein (in
N.fwdarw.C order) as V.sub.L CDR #1, V.sub.L CDR #2, and V.sub.L
CDR #3. The amino acid sequences of exemplary CDRs, as well as the
amino acid sequence of the V.sub.H and V.sub.L regions of the heavy
and light chains of exemplary anti-PD-1 are provided herein.
Specific embodiments of anti-PD-1 antibodies include these
exemplary CDRs and/or V.sub.H and/or V.sub.L sequences, as well as
antibodies that compete for binding human PD-1 with such
antibodies.
[0055] In some embodiments, an anti-PD-1 antibody is suitable for
administration to humans. In a specific embodiment, the anti-PD-1
antibody is humanized. In some embodiments, the amino acid
sequences of the CDRs of an anti-PD-1 antibody are selected from
the following sequences:
TABLE-US-00004 CDR Sequence (N.fwdarw.C) Identifier V.sub.H CDR#1:
GYTFTHYGMN (SEQ ID NO: 11) V.sub.H CDR#2: WVNTYTGEPTYADDFKG (SEQ ID
NO: 12) V.sub.H CDR#3: EGEGLGFGD (SEQ ID NO: 13) EGEGIGFGD (SEQ ID
NO: 21) EGEGVGFGD (SEQ ID NO: 22) EGEGMGFGD (SEQ ID NO: 23) V.sub.L
CDR#1: RSSQSIVHSHGDTYLE (SEQ ID NO: 14) V.sub.L CDR#2: KVSNRFS (SEQ
ID NO: 15) V.sub.L CDR#3: FQGSHIPVT (SEQ ID NO: 16)
[0056] Specific exemplary embodiments of anti-PD-1 antibodies with
the above CDRs are described herein. In some embodiments, an
anti-PD-1 antibody has the CDRs of SEQ ID NOS: 11, 12, 13, 14, 15,
and 16. In some embodiments, an anti-PD-1 antibody has the CDRs of
SEQ ID NOS: 11, 12, 21, 14, 15, and 16. In some embodiments, an
anti-PD-1 antibody has the CDRs of SEQ ID NOS: 11, 12, 22, 14, 15,
and 16. In some embodiments, an anti-PD-1 antibody has the CDRs of
SEQ ID NOS: 11, 12, 23, 14, 15, and 16.
[0057] In some embodiments, each CDR of an anti-PD-1 antibody,
independently of the others, is selected to correspond in sequence
to the respective CDR of an antibody provided in TABLE 3. In some
embodiments, an anti-PD-1 antibody is an IgG, and has a V.sub.H and
V.sub.L corresponding in sequence to the V.sub.H and V.sub.L of an
antibody provided in TABLE 3.
[0058] In some embodiments, an anti-PD-1 antibody comprises a
V.sub.H chain corresponding in sequence to SEQ ID NO:31; and a
V.sub.L chain corresponding in sequence to SEQ ID NO:41. In some
embodiments, an anti-PD-1 antibody comprises a V.sub.H chain
corresponding in sequence to SEQ ID NO:32; and a V.sub.L chain
corresponding in sequence to SEQ ID NO:42. In some embodiments, an
anti-PD-1 antibody comprises a V.sub.H chain corresponding in
sequence to SEQ ID NO:33; and a V.sub.L chain corresponding in
sequence to SEQ ID NO:42. In some embodiments, an anti-PD-1
antibody comprises a V.sub.H chain corresponding in sequence to SEQ
ID NO:34; and a V.sub.L chain corresponding in sequence to SEQ ID
NO:42. In some embodiments, an anti-PD-1 antibody comprises a
V.sub.H chain corresponding in sequence to SEQ ID NO:35; and a
V.sub.L chain corresponding in sequence to SEQ ID NO:42. In some
embodiments, an anti-PD-1 antibody comprises a V.sub.H chain
corresponding in sequence to SEQ ID NO:36; and a V.sub.L chain
corresponding in sequence to SEQ ID NO:42.
[0059] Certain mutations of a V.sub.H or V.sub.L sequence in an
anti-PD-1 antibody described herein would be understood by a person
of skill to afford anti-PD-1 antibodies within the scope of the
disclosure. Mutations may include amino acid substitutions,
additions, or deletions from a V.sub.H or V.sub.L sequence as
disclosed herein while retaining significant anti-PD-1 activity.
Accordingly, in some embodiments, an anti-PD-1 antibody comprises a
V.sub.H sequence having at least 85%, at least 90%, at least 93%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identity to the V.sub.H sequence of any one of the
antibodies shown in TABLE 3. An anti-PD-1 antibody can comprise a
V.sub.H sequence having up to 8, up to 7, up to 6, up to 5, up to
4, up to 3, or up to 2 mutations compared with the V.sub.H sequence
of any one of the antibodies shown in TABLE 3. In some embodiments,
an anti-PD-1 antibody can comprise a V.sub.H sequence having 5 or
fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutations compared
with the V.sub.H sequence of any one of the antibodies shown in
TABLE 3. In some embodiments, an anti-PD-1 antibody comprises a
V.sub.L sequence having at least 85%, at least 90%, at least 93%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identity to the V.sub.L sequence of any one of the
antibodies shown in TABLE 3. An anti-PD-1 antibody can comprise a
V.sub.L sequence having up to 8, up to 7, up to 6, up to 5, up to
4, up to 3, or up to 2 mutations compared with the V.sub.L sequence
of any one of the antibodies shown in TABLE 3. In some embodiments,
an anti-PD-1 antibody can comprise a V.sub.L sequence having 5 or
fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutations compared
with the V.sub.L sequence of any one of the antibodies shown in
TABLE 3.
[0060] In some embodiments, an anti-PD-1 antibody comprises a heavy
chain amino acid sequence according to:
TABLE-US-00005 (SEQ ID NO: 51)
EIQLVQSGAEVKKPGSSVKVSCKASGYTFTHYGMNWVRQAPGQGLEWVGW
VNTYTGEPTYADDFKGRLTFTLDTSTSTAYMELSSLRSEDTAVYYCTREG
EGLGFGDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;
and a light chain amino acid sequence according to:
TABLE-US-00006 (SEQ ID NO: 61)
DVVMTQSPLSLPVTPGEPASISCRSSQSIVHSHGDTYLEWYLQKPGQSPQ
LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP
VTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC,
wherein the underlined amino acids represent the CDRs and the
italicized amino acids represent the constant regions.
[0061] Post-translational modifications to the sequences of an
anti-PD-1 antibody may occur, such as cleavage of one or more
(e.g., 1, 2, 3, or more) amino acid residues on the C-terminal end
of the antibody heavy chain.
[0062] In some embodiments, an anti-PD-1 antibody comprises a heavy
chain amino acid sequence according to:
TABLE-US-00007 (SEQ ID NO: 52)
EIQLVQSGAEVKKPGSSVKVSCKASGYTFTHYGMNWVRQAPGQGLEWVGW
VNTYTGEPTYADDFKGRLTFTLDTSTSTAYMELSSLRSEDTAVYYCTREG
EGLGFGDWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;
and a light chain amino acid sequence according to:
TABLE-US-00008 (SEQ ID NO: 61)
DVVMTQSPLSLPVTPGEPASISCRSSQSIVHSHGDTYLEWYLQKPGQSPQ
LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIP
VTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC,
wherein the underlined amino acids represent the CDRs and the
italicized amino acids represent the constant regions.
[0063] Additional post-translational modifications of an anti-PD-1
antibody may include glycosylation. Common biantennary complexes
can be composed of a core structure having two N-acetylglucosamine
(GlcNAc), three mannose, and two GlcNAc residues that are
.beta.-1,2 linked to .alpha.-6 mannose and .alpha.-3 mannose to
form two antennae. One or more fucose (Fuc), galactose (Gal), high
mannose glycans Man-5 or Man-9, bisecting GlcNAc, and sialic acid
including N-acetylneuraminic acid (NANA) or N-glycolylneuraminic
acid (NGNA) residues may be attached to the core. N-linked
glycoforms may include G0 (protein having a core biantennary
glycosylation structure), G0F (fucosylated G0), G0F GlcNAc, G1
(protein having a core glycosylation structure with one galactose
residue), G1F (fucosylated G1), G2 (protein having a core
glycosylation structure with two galactose residues), and/or G2F
(fucosylated G2). In some embodiments, an anti-PD-1 antibody has a
G0F glycan.
[0064] In some embodiments, the anti-PD-1 antibodies compete for
binding human PD-1 in in vitro assays with a reference antibody. In
some embodiments, the anti-PD-1 antibodies compete for binding
human PD-1 on cells expressing human PD-1. The reference antibody
may be any of the anti-PD-1 antibodies described herein. In some
embodiments, the reference antibody is an antibody provided in
TABLE 3. In specific embodiments, the reference antibody is
selected from mouse antibody 12A11 ("Mu12A11"). In some
embodiments, the reference antibody is a humanized version of
Mu12A11. In a specific embodiment, the reference antibody is
humanized antibody 12A11.1b ("Hu12A11.1b") or humanized antibody
12A11.2b M99L ("Hu12A11.2b4").
[0065] In some embodiments, the anti-PD-1 antibodies antagonize,
e.g., inhibit, human PD-1 (SEQ ID NO:1). PD-1 receptor antagonism
can occur by a number of mechanisms, for example, by inhibiting
binding of PD-1 by human PD-L1 (SEQ ID NO:3) or PD-L2 (SEQ ID
NO:4).
[0066] The anti-PD-1 antibodies described herein generally bind
specifically to human PD-1. Cross reactivity of the antibodies for
binding to PD-1 from other species, for example, from monkey, e.g.,
cynomolgus monkey, may offer advantages, such as the ability to
test in monkey animal models for biological activity. Such animal
model testing may be used to screen anti-PD-1 antibodies to select
properties related to efficacy, e.g., favorable pharmacokinetics,
or those related to safety, e.g., decreased hepatic toxicity. In
some embodiments, the anti-PD-1 antibodies bind to cynomolgus PD-1
as well as human PD-1.
[0067] Assays for competition include, but are not limited to, a
radioactive material labeled immunoassay (RIA), an enzyme-linked
immunosorbent assay (ELISA), a sandwich ELISA, fluorescence
activated cell sorting (FACS) assays, and surface plasmon resonance
assays.
[0068] In conducting an antibody competition assay between a
reference antibody and a test antibody (irrespective of species or
isotype), one may first label the reference with a detectable
label, such as a fluorophore, biotin or an enzymatic (or even
radioactive) label to enable subsequent identification. In this
case, cells expressing human PD-1 are incubated with unlabeled test
antibody, labeled reference antibody is added, and the intensity of
the bound label is measured. If the test antibody competes with the
labeled reference antibody by binding to an overlapping epitope,
the intensity will be decreased relative to a control reaction
carried out without test antibody.
[0069] In a specific embodiment of this assay, the concentration of
labeled reference antibody that yields 80% of maximal binding
("conc.sub.80%") under the assay conditions (e.g., a specified
density of cells) is first determined, and a competition assay
carried out with 10.times. conc.sub.80% of unlabeled test antibody
and conc.sub.80% of labeled reference antibody.
[0070] The inhibition can be expressed as an inhibition constant,
or K which is calculated according to the following formula:
K.sub.i=IC.sub.50/(1+[reference Ab concentration]/K.sub.d),
where IC.sub.50 is the concentration of test antibody that yields a
50% reduction in binding of the reference antibody and K.sub.d is
the dissociation constant of the reference antibody, a measure of
its affinity for human PD-1. Antibodies that compete with anti-PD-1
antibodies disclosed herein can have a K.sub.i from 10 pM to 10 nM
under assay conditions described herein.
[0071] In various embodiments, a test antibody is considered to
compete with a reference antibody if it decreases binding of the
reference antibody by at least about 20% or more, for example, by
at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even
more, or by a percentage ranging between any of the foregoing
values, at a reference antibody concentration that is 80% of
maximal binding under the specific assay conditions used, and a
test antibody concentration that is 10-fold higher than the
reference antibody concentration.
[0072] A specific assay and assay conditions useful for assessing
whether an antibody competes for binding human PD-1 with a
reference antibody as described herein is provided in Section
8.1.5.
7.4. Polynucleotides Encoding the Anti-PD-1 Antibodies, Expression
Systems and Methods of Making the Antibodies
[0073] The present disclosure encompasses nucleic acid molecules
encoding immunoglobulin light and heavy chain genes for anti-PD-1
antibodies, vectors comprising such nucleic acids, and host cells
capable of producing the anti-PD-1 antibodies of the
disclosure.
[0074] An anti-PD-1 antibody of the disclosure can be prepared by
recombinant expression of immunoglobulin light and heavy chain
genes in a host cell. To express an antibody recombinantly, a host
cell is transfected with one or more recombinant expression vectors
carrying DNA fragments encoding the immunoglobulin light and heavy
chains of the antibody such that the light and heavy chains are
expressed in the host cell and, optionally, secreted into the
medium in which the host cells are cultured, from which medium the
antibodies can be recovered. Standard recombinant DNA methodologies
are used to obtain antibody heavy and light chain genes,
incorporate these genes into recombinant expression vectors and
introduce the vectors into host cells, such as those described in
Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook,
Fritsch and Maniatis (eds), Cold Spring Harbor, N.Y., 1989),
Current Protocols in Molecular Biology (Ausubel, F. M. et al.,
eds., Greene Publishing Associates, 1989) and in U.S. Pat. No.
4,816,397.
[0075] To generate nucleic acids encoding such anti-PD-1
antibodies, DNA fragments encoding the light and heavy chain
variable regions are first obtained. These DNAs can be obtained by
amplification and modification of germline DNA or cDNA encoding
light and heavy chain variable sequences, for example using the
polymerase chain reaction (PCR). Germline DNA sequences for human
heavy and light chain variable region genes are known in the art
(See, e.g., the "VBASE" human germline sequence database; see also
Kabat, E. A. et al., 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J.
Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J. Immunol.
24:827-836; the contents of each of which are incorporated herein
by reference).
[0076] Once DNA fragments encoding anti-PD-1 antibody-related
V.sub.H and V.sub.L segments are obtained, these DNA fragments can
be further manipulated by standard recombinant DNA techniques, for
example to convert the variable region genes to full-length
antibody chain genes, to Fab fragment genes or to a scFv gene. In
these manipulations, a V.sub.L- or V.sub.H-encoding DNA fragment is
operatively linked to another DNA fragment encoding another
protein, such as an antibody constant region or a flexible linker.
The term "operatively linked," as used in this context, is intended
to mean that the two DNA fragments are joined such that the amino
acid sequences encoded by the two DNA fragments remain
in-frame.
[0077] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to another DNA molecule encoding heavy
chain constant regions (CH1, CH2, CH3 and, optionally, CH4). The
sequences of human heavy chain constant region genes are known in
the art (See, e.g., Kabat, E. A., et al., 1991, Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242) and DNA
fragments encompassing these regions can be obtained by standard
PCR amplification. The heavy chain constant region can be an
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA, IgE, IgM or IgD
constant region, but in certain embodiments is an IgG.sub.1 or
IgG.sub.4. For a Fab fragment heavy chain gene, the
V.sub.H-encoding DNA can be operatively linked to another DNA
molecule encoding only the heavy chain CH1 constant region.
[0078] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region, CL.
The sequences of human light chain constant region genes are known
in the art (See, e.g., Kabat, et al., 1991, Sequences of Proteins
of Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region, but in certain embodiments is a kappa
constant region. To create a scFv gene, the V.sub.H- and
V.sub.L-encoding DNA fragments are operatively linked to another
fragment encoding a flexible linker, e.g., encoding the amino acid
sequence (Gly.sub.4.about.Ser).sub.3 (SEQ ID NO:5), such that the
V.sub.H and V.sub.L sequences can be expressed as a contiguous
single-chain protein, with the V.sub.L and V.sub.H regions joined
by the flexible linker (See, e.g., Bird et al., 1988, Science
242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).
[0079] To express the anti-PD-1 antibodies of the disclosure, DNAs
encoding partial or full-length light and heavy chains, obtained as
described above, are inserted into expression vectors such that the
genes are operatively linked to transcriptional and translational
control sequences. In this context, the term "operatively linked"
is intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vectors or, more typically, both genes are inserted into the same
expression vector.
[0080] The antibody genes are inserted into the expression vector
by standard methods (e.g., ligation of complementary restriction
sites on the antibody gene fragment and vector, or blunt end
ligation if no restriction sites are present). Prior to insertion
of the anti-PD-1 antibody-related light or heavy chain sequences,
the expression vector can already carry antibody constant region
sequences. For example, one approach to converting the anti-PD-1
monoclonal antibody-related V.sub.H and V.sub.L sequences to
full-length antibody genes is to insert them into expression
vectors already encoding heavy chain constant and light chain
constant regions, respectively, such that the V.sub.H segment is
operatively linked to the CH segment(s) within the vector and the
V.sub.L segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the antibody chain gene. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0081] In addition to the antibody chain genes, the recombinant
expression vectors of the disclosure carry regulatory sequences
that control the expression of the antibody chain genes in a host
cell. The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990.
It will be appreciated by those skilled in the art that the design
of the expression vector, including the selection of regulatory
sequences may depend on such factors as the choice of the host cell
to be transformed, the level of expression of protein desired, etc.
Suitable regulatory sequences for mammalian host cell expression
include viral elements that direct high levels of protein
expression in mammalian cells, such as promoters and/or enhancers
derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see, e.g., U.S. Pat.
No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al.,
and U.S. Pat. No. 4,968,615 by Schaffner et al.
[0082] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the disclosure can
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (See, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Suitable selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in DHFR.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection). For expression of the light and heavy chains,
the expression vector(s) encoding the heavy and light chains is
transfected into a host cell by standard techniques. The various
forms of the term "transfection" are intended to encompass a wide
variety of techniques commonly used for the introduction of
exogenous DNA into a prokaryotic or eukaryotic host cell, e.g.,
electroporation, lipofection, calcium-phosphate precipitation,
DEAE-dextran transfection and the like.
[0083] It is possible to express the antibodies of the disclosure
in either prokaryotic or eukaryotic host cells. In certain
embodiments, expression of antibodies is performed in eukaryotic
cells, e.g., mammalian host cells, of optimal secretion of a
properly folded and immunologically active antibody. Exemplary
mammalian host cells for expressing the recombinant antibodies of
the disclosure include Chinese Hamster Ovary (CHO cells) (including
DHFR.sup.- CHO cells, described in Urlaub and Chasin, 1980, Proc.
Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable
marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol.
159:601-621), NSO myeloma cells, COS cells and SP2 cells. When
recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or secretion
of the antibody into the culture medium in which the host cells are
grown. Antibodies can be recovered from the culture medium using
standard protein purification methods. Host cells can also be used
to produce portions of intact antibodies, such as Fab fragments or
scFv molecules. It is understood that variations on the above
procedure are within the scope of the present disclosure. For
example, it can be desirable to transfect a host cell with DNA
encoding either the light chain or the heavy chain (but not both)
of an anti-PD-1 antibody of this disclosure.
[0084] Recombinant DNA technology can also be used to remove some
or all of the DNA encoding either or both of the light and heavy
chains that is not necessary for binding to human PD-1. The
molecules expressed from such truncated DNA molecules are also
encompassed by the antibodies of the disclosure.
[0085] For recombinant expression of an anti-PD-1 antibody of the
disclosure, the host cell can be co-transfected with two expression
vectors of the disclosure, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors can contain identical
selectable markers, or they can each contain a separate selectable
marker. Alternatively, a single vector can be used which encodes
both heavy and light chain polypeptides.
[0086] Once a nucleic acid encoding one or more portions of an
anti-PD-1 antibody has been obtained, further alterations or
mutations can be introduced into the coding sequence, for example
to generate nucleic acids encoding antibodies with different CDR
sequences, antibodies with reduced affinity to the Fc receptor, or
antibodies of different subclasses.
[0087] The anti-PD-1 antibodies of the disclosure can also be
produced by chemical synthesis (e.g., by the methods described in
Solid Phase Peptide Synthesis, 2.sup.nd ed., 1984 The Pierce
Chemical Co., Rockford, Ill.). Variant antibodies can also be
generated using a cell-free platform (See, e.g., Chu et al.,
Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et
al., 2013, Current Opinion in Chemical Biology, 17:420-426).
[0088] Once an anti-PD-1 antibody of the disclosure has been
produced by recombinant expression, it can be purified by any
method known in the art for purification of an immunoglobulin
molecule, for example, by chromatography (e.g., ion exchange,
affinity, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins. Further, the anti-PD-1 antibodies of the
present disclosure can be fused to heterologous polypeptide
sequences described herein or otherwise known in the art to
facilitate purification.
[0089] Once isolated, the anti-PD-1 antibody can, if desired, be
further purified, e.g., by high performance liquid chromatography
(see, e.g., Fisher, Laboratory Techniques In Biochemistry And
Molecular Biology, Work and Burdon, eds., Elsevier, 1980), or by
gel filtration chromatography on a Superdex.TM. 75 column
(Pharmacia Biotech AB, Uppsala, Sweden).
7.5. Pharmaceutical Compositions
[0090] The anti-PD-1 antibodies described herein may be in the form
of compositions comprising the antibody and one or more carriers,
excipients and/or diluents. The compositions may be formulated for
specific uses, such as for veterinary uses or pharmaceutical uses
in humans. The form of the composition (e.g., dry powder, liquid
formulation, etc.) and the excipients, diluents and/or carriers
used will depend upon the intended uses of the antibody and, for
therapeutic uses, the mode of administration.
[0091] For therapeutic uses, the compositions may be supplied as
part of a sterile, pharmaceutical composition that includes a
pharmaceutically acceptable carrier. This composition can be in any
suitable form (depending upon the desired method of administering
it to a patient). The pharmaceutical composition can be
administered to a patient by a variety of routes such as orally,
transdermally, subcutaneously, intranasally, intravenously,
intramuscularly, intratumorally, intrathecally, topically or
locally. The most suitable route for administration in any given
case will depend on the particular antibody, the subject, and the
nature and severity of the disease and the physical condition of
the subject. Typically, the pharmaceutical composition will be
administered intravenously or subcutaneously.
[0092] Pharmaceutical compositions can be conveniently presented in
unit dosage forms containing a predetermined amount of an anti-PD-1
antibody described herein per dose. The quantity of anti-PD-1
antibody included in a unit dose will depend on the disease being
treated, as well as other factors as are well known in the art.
Such unit dosages may be in the form of a lyophilized dry powder
containing an amount of antibody suitable for a single
administration, or in the form of a liquid. Dry powder unit dosage
forms may be packaged in a kit with a syringe, a suitable quantity
of diluent and/or other components useful for administration. Unit
dosages in liquid form may be conveniently supplied in the form of
a syringe pre-filled with a quantity of the anti-PD-1 antibody
suitable for a single administration.
[0093] The pharmaceutical compositions may also be supplied in bulk
form containing quantities of anti-PD-1 antibody suitable for
multiple administrations.
[0094] Pharmaceutical compositions may be prepared for storage as
lyophilized formulations or aqueous solutions by mixing an antibody
having the desired degree of purity with optional
pharmaceutically-acceptable carriers, excipients or stabilizers
typically employed in the art (all of which are referred to herein
as "carriers"), i.e., buffering agents, stabilizing agents,
preservatives, isotonifiers, non-ionic detergents, antioxidants,
and other miscellaneous additives. See, Remington's Pharmaceutical
Sciences, 16th edition (Osol, ed. 1980). Such additives should be
nontoxic to the recipients at the dosages and concentrations
employed.
[0095] Buffering agents help to maintain the pH in the range which
approximates physiological conditions. They may be present at a
wide variety of concentrations, but will typically be present in
concentrations ranging from about 2 mM to about 50 mM. Suitable
buffering agents for use with the present disclosure include both
organic and inorganic acids and salts thereof such as citrate
buffers (e.g., monosodium citrate-disodium citrate mixture, citric
acid-trisodium citrate mixture, citric acid-monosodium citrate
mixture, etc.), succinate buffers (e.g., succinic acid-monosodium
succinate mixture, succinic acid-sodium hydroxide mixture, succinic
acid-disodium succinate mixture, etc.), tartrate buffers (e.g.,
tartaric acid-sodium tartrate mixture, tartaric acid-potassium
tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.),
phosphate buffers (e.g., phosphoric acid-monosodium phosphate
mixture, phosphoric acid-disodium phosphate mixture, monosodium
phosphate-disodium phosphate mixture, etc.), gluconate buffers
(e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium
hydroxide mixture, gluconic acid-potassium gluconate mixture,
etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture,
oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate
mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate
mixture, lactic acid-sodium hydroxide mixture, lactic
acid-potassium lactate mixture, etc.) and acetate buffers (e.g.,
acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide
mixture, etc.). Additionally, fumarate buffers, histidine buffers
and trimethylamine salts such as
2-amino-2-hydroxymethyl-propane-1,3-diol (i.e., Tris, THAM, or
tris(hydroxymethyl)aminomethane) can be used.
[0096] Isotonicifiers sometimes known as "stabilizers" can be added
to ensure isotonicity of liquid compositions of the present
disclosure and include polyhydric sugar alcohols, for example
trihydric or higher sugar alcohols, such as glycerin, erythritol,
arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a
broad category of excipients which can range in function from a
bulking agent to an additive which solubilizes the therapeutic
agent or helps to prevent denaturation or adherence to the
container wall. Typical stabilizers can be polyhydric sugar
alcohols (enumerated above); amino acids such as arginine, lysine,
glycine, glutamine, asparagine, histidine, alanine, ornithine,
leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic
sugars or sugar alcohols, such as lactose, trehalose, stachyose,
mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol,
glycerol and the like, including cyclitols such as inositol;
polyethylene glycol; amino acid polymers; sulfur containing
reducing agents, such as urea, glutathione, thioctic acid, sodium
thioglycolate, thioglycerol, .alpha.-monothioglycerol and sodium
thiosulfate; low molecular weight polypeptides (e.g., peptides of
10 residues or fewer); hydrophilic polymers, such as
polyvinylpyrrolidone monosaccharides, such as xylose, mannose,
fructose, glucose; disaccharides such as lactose, maltose, sucrose
and trehalose; and trisaccacharides such as raffinose; and
polysaccharides such as dextran. Stabilizers may be present in
amounts ranging from 0.5 to 10 weight % per weight of anti-PD-1
antibody.
[0097] Non-ionic surfactants or detergents (also known as "wetting
agents") may be added to help solubilize the glycoprotein as well
as to protect the glycoprotein against agitation-induced
aggregation, which also permits the formulation to be exposed to
shear surface stressed without causing denaturation of the protein.
Suitable non-ionic surfactants include polysorbates (20, 80, etc.),
poloxamers (184, 188 etc.), and pluronic polyols. Non-ionic
surfactants may be present in a range of about 0.05 mg/mL to about
1.0 mg/mL.
[0098] A specific exemplary embodiment of an aqueous composition
suitable for administration via intravenous infusion comprises 20
mg/mL of an anti-PD-1 antibody comprising a heavy chain sequence of
SEQ ID NO:51 or SEQ ID NO:52, and a light chain sequence of SEQ ID
NO:61, 15 mM histidine pH 5.7, 8.0% (w/v) sucrose, and 0.05% (w/v)
polysorbate 80. The composition may be in the form of a lyophilized
powder that, upon reconstitution with sterile water or other
solution suitable for injection or infusion (for example, 0.9%
saline, Ringer's solution, lactated Ringer's solution, etc.)
provides the above aqueous composition. The composition, or other
embodiments of compositions, may also be in the form of a syringe
or other device suitable for injection and/or infusion pre-filled
with a quantity of composition suitable for a single administration
of the anti-PD-1 antibody.
7.6. Methods of Use
[0099] 7.6.1. Therapeutic Benefit
[0100] Data provided herein demonstrate that the disclosed
anti-PD-1 antibodies antagonize PD-1 in the presence of cancer
cells and exert potent anticancer activity against cancers such as
solid tumors and blood malignancies in vivo. Accordingly, the
anti-PD-1 antibodies, binding fragments, and/or pharmaceutical
compositions comprising the anti-PD-1 antibodies may be used
therapeutically to treat solid tumors or blood malignancies.
[0101] In some embodiments, the method involves administering to a
human patient having a solid tumor an amount of an anti-PD-1
antibody that antagonizes PD-1, and kills tumor cells at a rate
effective to provide therapeutic benefit. Solid tumors that may be
treated with the anti-PD-1 antibody include, but are not limited
to, adrenal cancers, bladder cancers, bone cancers, brain cancers,
breast cancers (e.g., triple negative breast cancer), cervical
cancers, colorectal cancers, endometrial cancers, esophageal
cancers, eye cancers, gastric cancers, head and neck cancers,
kidney cancers (e.g., advanced renal cell carcinoma), liver cancers
(e.g., hepatocellular carcinoma, cholangiocarcinoma), lung cancers
(e.g., non-small cell lung cancer, mesothelioma, small cell lung
cancer), head and neck cancers, melanomas (e.g., unresectable or
metastatic melanoma, advanced malignant melanoma), oral cancers,
ovarian cancers, penile cancers, prostate cancers, pancreatic
cancers, skin cancers (e.g., Merkel cell carcinoma), testicular
cancers, thyroid cancers, uterine cancers, vaginal cancers, and
tumors with evidence of DNA mismatch repair deficiency. The cancers
may be comprised of tumor cells that express PD-L1 or PD-L2,
cancers comprised of tumor cells that do not express PD-L1 or
PD-L2, or cancers comprised of tumor cells, some of which express
PD-L1 or PD-L2 and some of which do not. The cancer may be newly
diagnosed and naive to treatment, or may be relapsed, refractory,
or relapsed and refractory, or a metastatic form of a solid tumor.
In some embodiments, the solid tumor is selected from bladder
cancer, breast cancer, head and neck cancer, kidney cancer, lung
cancer, lymphoma, melanoma, and gastric cancer. In some
embodiments, the solid tumor is selected from: melanoma (e.g.,
unresectable or metastatic melanoma), lung cancer (e.g., non-small
cell lung cancer), and renal cell carcinoma (e.g., advanced renal
cell carcinoma). In some embodiments, the solid tumor is selected
from triple negative breast cancer, ovarian cancer, hepatocellular
carcinoma, gastric cancer, small cell lung cancer, mesothelioma,
cholangiocarcinoma, Merkel cell carcinoma and tumors with evidence
of DNA mismatch repair deficiency. In certain embodiments, the
melanoma is BRAF V600 wild-type unresectable or metastatic
melanoma. In other certain embodiments, the melanoma is BRAF V600
mutation-positive unresectable or metastatic melanoma. In certain
embodiments, the lung cancer is metastatic non-small cell lung
cancer with progression on or after platinum-based chemotherapy. In
certain embodiments, the lung cancer is locally advanced or
metastatic non-small cell lung cancer that has failed
platinum-based therapy and is naive to a PD-1 targeting agent. In
certain embodiments, the head and neck cancer is metastatic
(disseminated) head and neck squamous cell carcinoma of the oral
cavity, oropharynx, hypopharynx, and larynx that is considered
incurable by local therapies. In certain embodiments, the renal
cell carcinoma is advanced renal cell carcinoma that has received
prior anti-angiogenic therapy.
[0102] In some embodiments, the method of the disclosure involves
administering to a human patient having a blood malignancy an
amount of an anti-PD-1 antibody that antagonizes PD-1, and kills
malignant cells at a rate effective to provide therapeutic benefit.
The cancers may be comprised of malignant cells that express PD-L1
or PD-L2, cancers comprised of malignant cells that do not express
PD-L1 or PD-L2, or cancers comprised of malignant cells, some of
which express PD-L1 or PD-L2 and some of which do not. The cancer
may be newly diagnosed and naive to treatment, or may be relapsed,
refractory, or relapsed and refractory, or a metastatic form of a
blood malignancy. Blood-borne malignancies that may be treated with
the anti-PD-1 antibody include, but are not limited to, myelomas
(e.g., multiple myeloma), lymphomas (e.g., Hodgkin's lymphoma,
non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, mantle
cell lymphoma), leukemias (e.g., chronic lymphocytic leukemia,
acute myeloid leukemia, acute lymphocytic leukemia), and
myelodysplastic syndromes.
[0103] As discussed above, the presently disclosed anti-PD-1
antibodies modulate an immunological response. Accordingly,
patients having compromised immune systems may be excluded from
treatment. In some embodiments, a patient is excluded after meeting
one or more of the following criteria: (1) Active or prior
documented autoimmune disease (including, but not limited to,
inflammatory bowel disease, celiac disease, Wegener syndrome)
within the past 2 years. (Subjects with childhood atopy or asthma,
vitiligo, alopecia, Hashimoto syndrome, Grave's disease, or
psoriasis not requiring systemic treatment (within the past 2
years) are not excluded); (2) History of primary immunodeficiency,
bone marrow transplantation, chronic lymphocytic leukemia, solid
organ transplantation, or previous clinical diagnosis of
tuberculosis; (3) History of a coagulopathy or a platelet disorder;
(4) Confirmed positive test results for human immunodeficiency
virus (HIV), or subjects with chronic or active hepatitis B or C.
(Subjects who have a history of hepatitis B or C who have
documented cures after anti-viral therapy may be enrolled); (5)
Prior grade .gtoreq.3 immune-mediated neurotoxicity or pneumonitis
while receiving immunotherapy (including but not limited to agents
directed against CTLA-4, PD-L1, or PD-1). In addition, any other
prior grade .gtoreq.3 immune-mediated adverse event while receiving
immunotherapy that has not resolved or become asymptomatic within 3
months; (6) Receipt of live, attenuated vaccine within 28 days
prior to the first dose of the anti-PD-1 antibody.
[0104] An anti-PD-1 antibody of the disclosure may be administered
alone (monotherapy) or adjunctive to, or with, other anti-cancer
therapies and/or targeted or non-targeted anti-cancer agents. When
administered as an anti-PD-1 monotherapy, one or more antibodies
may be used. Whether administered as monotherapy or adjunctive to,
or with, other therapies or agents, an amount of anti-PD-1 antibody
is administered such that the overall treatment regimen provides
therapeutic benefit.
[0105] By therapeutic benefit is meant that the use of anti-PD-1
antibodies to treat cancer in a patient results in any demonstrated
clinical benefit compared with no therapy (when appropriate) or to
a known standard of care. Clinical benefit can be assessed by any
method known to one of ordinary skill in the art. In one
embodiment, clinical benefit is assessed based on objective
response rate (ORR) (determined using RECIST version 1.1), duration
of response (DOR), progression-free survival (PFS), and/or overall
survival (OS). In some embodiments, a complete response indicates
therapeutic benefit. In some embodiments, a partial response
indicates therapeutic benefit. In some embodiments, stable disease
indicates therapeutic benefit. In some embodiments, an increase in
overall survival indicates therapeutic benefit. In some
embodiments, therapeutic benefit may constitute an improvement in
time to disease progression and/or an improvement in symptoms or
quality of life. In other embodiments, therapeutic benefit may not
translate to an increased period of disease control, but rather a
markedly reduced symptom burden resulting in improved quality of
life. As will be apparent to those of skill in the art, a
therapeutic benefit may be observed using the anti-PD-1 antibodies
alone (monotherapy) or adjunctive to, or with, other anti-cancer
therapies and/or targeted or non-targeted anti-cancer agents.
[0106] Typically, therapeutic benefit is assessed using standard
clinical tests designed to measure the response to a new treatment
for cancer. To assess the therapeutic benefits of the anti-PD-1
antibodies described herein one or a combination of the following
tests can be used: (1) the Response Evaluation Criteria In Solid
Tumors (RECIST) version 1.1, (2) immune-related RECIST (irRECIST),
(3) the Eastern Cooperative Oncology Group (ECOG) Performance
Status, (4) immune-related response criteria (irRC), (5) disease
evaluable by assessment of tumor antigens, (6) validated patient
reported outcome scales, and/or (7) Kaplan-Meier estimates for
overall survival and progression free survival.
[0107] Assessment of the change in tumor burden is an important
feature of the clinical evaluation of cancer therapeutics. Both
tumor shrinkage (objective response) and time to the development of
disease progression are important endpoints in cancer clinical
trials. Standardized response criteria, known as RECIST (Response
Evaluation Criteria in Solid Tumors), were published in 2000. An
update (RECIST 1.1) was released in 2009. RECIST criteria are
typically used in clinical trials where objective response is the
primary study endpoint, as well as in trials where assessment of
stable disease, tumor progression or time to progression analyses
are undertaken because these outcome measures are based on an
assessment of anatomical tumor burden and its change over the
course of the trial. TABLE 4 provides the definitions of the
response criteria used to determine objective tumor response to a
study drug, such as the anti-PD-1 antibodies described herein.
TABLE-US-00009 TABLE 4 Response Criteria Complete Response
Disappearance of all target lesions. Any pathological lymph nodes
(CR) (whether target or non-target) must have reduction in short
axis to <10 mm. Partial Response At least a 30% decrease in the
sum of diameters of target lesions, taking as (PR) reference the
baseline sum diameters. Progressive Disease At least a 20% increase
in the sum of diameters of target lesions, taking as (PD) reference
the smallest sum on study (this includes the baseline sum if that
is the smallest on study). In addition to the relative increase of
20%, the sum must also demonstrate an absolute increase of at least
5 mm. (Note: the appearance of one or more new lesions is also
considered progression). Stable Disease Neither sufficient
shrinkage to qualify for PR nor sufficient increase to (SD) qualify
for PD, taking as reference the smallest sum diameters while on
study.
[0108] Secondary outcome measures that can be used to determine the
therapeutic benefit of the anti-PD-1 antibodies described herein
include, Objective Response Rate (ORR), Progression Free Survival
(PFS), Overall Survival (OS), Duration of Overall Response (DOR),
and Depth of Response (DpR). ORR is defined as the proportion of
the participants who achieve a complete response (CR) or partial
response (PR). PFS is defined as the time from the first dose date
of an anti-PD-1 antibody to either disease progression or death,
whichever occurs first. OS is defined as the length of time from
either the date of diagnosis or the start of treatment for a
disease, that patients diagnosed with the disease are still alive.
DOR is defined as the time from the participant's initial CR or PR
to the time of disease progression. DpR is defined as the
percentage of tumor shrinkage observed at the maximal response
point compared to baseline tumor load. Clinical endpoints for both
ORR and PFS can be determined based on RECIST 1.1 criteria
described above.
[0109] Additional criteria that may be used for clinical evaluation
specific to cancer patients undergoing immune therapy treatment
include the standardized immune-related RECIST (irRECIST) criteria.
See, e.g., Nishino, M. et al. Eur. J. Radiol., 84(7), pages
1259-1268 (2015 July). These guidelines modified the RECIST 1.1
criteria above with consideration of potential immunomodulatory
effects. TABLE 5 provides the definitions of the response criteria
used to determine objective tumor response to an immunomodulatory
drug, such as the anti-PD-1 antibodies described herein.
TABLE-US-00010 TABLE 5 Response Criteria Complete Response Complete
disappearance of all measurable and non-measurable lesions. (irCR)
Lymph nodes must decrease to <10 mm in short axis. Partial
Response Decrease of .gtoreq.30% in total measured tumor burden
relative to baseline, (irPR) non-target lesions are irNN, and no
unequivocal progression of new non- measurable lesions Progressive
Disease At least a 20% increase and at least 5 mm absolute increase
in TMTB (irPD) compared to nadir, or irPD for non-target or new
non-measurable lesions. Confirmation of progression is recommended
at least 4 weeks after the first irPD assessment. Non-irCR or non-
No target disease was identified at baseline and at follow-up the
patient irPD (irNN) fails to meet criteria for irCR or irPD Stable
Disease Neither sufficient shrinkage to qualify for irPR nor
sufficient increase to (irSD) qualify for irPD, taking as reference
the smallest sum diameters while on study. irNE Used in exceptional
cases where insufficient data exists.
[0110] The ECOG Scale of Performance Status shown in TABLE 6 is
used to describe a patient's level of functioning in terms of their
ability to care for themselves, daily activity, and physical
ability. The scale was developed by the Eastern Cooperative
Oncology Group (ECOG), now part of the ECOG-ACRIN Cancer Research
Group, and published in 1982.
TABLE-US-00011 TABLE 6 Grade ECOG Performance Status 0 Fully
active, able to carry on all pre-disease performance without
restriction 1 Restricted in physically strenuous activity but
ambulatory and able to carry out work of a light or sedentary
nature, e.g., light house work, office work 2 Ambulatory and
capable of all selfcare but unable to carry out any work
activities; up and about more than 50% of waking hours 3 Capable of
only limited selfcare; confined to bed or chair more than 50% of
waking hours 4 Completely disabled; cannot carry on any selfcare;
totally confined to bed or chair 5 Dead
[0111] Another set of criteria that can be used to characterize
fully and to determine response to immunotherapeutic agents, such
as antibody-based cancer therapies, is the immune-related response
criteria (irRC), which was developed for measurement of solid
tumors in 2009, and updated in 2013 (Wolchok, et al. Clin. Cancer
Res. 2009; 15(23): 7412-7420 and Nishino, et al. Clin. Cancer Res.
2013; 19(14): 3936-3943). The updated irRC criteria are typically
used to assess the effect of an immunotherapeutic agent, such as an
anti-PD-1 antibody described herein, on tumor burden, and defines
response according to TABLE 7.
TABLE-US-00012 TABLE 7 Response Criteria Complete Response
Disappearance of all target lesions in two consecutive observations
not less (CR) than 4 weeks apart Partial Response At least a 30%
decrease in the sum of the longest diameters of target (PR)
lesions, taking as reference the baseline sum diameters.
Progressive Disease At least a 20% increase in the sum of diameters
of target lesions, taking as (PD) reference the smallest sum on
study (this includes the baseline sum if that is the smallest on
study). (Note: the appearance of one or more new lesions is not
considered progression. The measurement of new lesions is included
in the sum of the measurements). Stable Disease Neither sufficient
shrinkage to qualify for PR nor sufficient increase to (SD) qualify
for PD, taking as reference the smallest sum diameters while on
study.
[0112] Tumor antigens that can be used to evaluate the therapeutic
benefit of the anti-PD-1 antibodies described herein include ApoE,
CD11c, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB,
Ly86, MS4A7, PIK3AP1, PIK3CD, CD74, CCL5, CCR5, CXCL10, IFNG,
IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG,
SERPINE1, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4,
EGLN, ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6,
PLOD2, KISS1R, STC2, DDIT4, OX40, OX40L, PFKFB3, PGK1, PDK1,
AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM,
WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2,
CD11b, ADGRE1 (EMR1, F4/80), CD86, CD68, MHC-Class II, CD3, HLA-DR,
CD4, CD3, CD5, CD19, CD7, CD8, CD16, TCR.alpha..beta.,
TCR.gamma..delta., PD-1, PD-L1, CTLA-4, acid phosphatase, ACTH,
alkaline phosphatase, alpha-fetoprotein CA-125, CA15-3, CA19-9,
CA-195, C-212, CA-549, calcitonin, catecholamines, cathepsin-D,
CEA, ERBB2 (HER2/neu), chromagranin-A, c-Myc, EGFR, ERA (estrogen
receptor assay), ferritin, gastrin, 5-HIAA, hCG, alpha-HCG,
beta-HCG, HVA, LDH1-5, NSE (neuron specific enolase), pancreatic
polypeptide, PLAP, PLP, PRA (progesterone receptor A), proinsulin
C-peptide, PSA, SMA, SCC, thyroglobulin, TDT, TPA, and alpha-TSH.
These tumor antigens can be assessed at the DNA, RNA or protein
level using DNA sequencing techniques, RNA sequencing techniques,
gene chip microarray, PCR based methods, flow cytometry or
immunohistochemistry methods as known to experts in the art.
[0113] One exemplary therapeutic benefit resulting from the use of
anti-PD-1 antibodies described herein to treat solid tumors,
whether administered as monotherapy or adjunctive to, or with,
other therapies or agents, is a complete response. Another
exemplary therapeutic benefit resulting from the use of anti-PD-1
antibodies described herein to treat solid tumors, whether
administered as monotherapy or adjunctive to, or with, other
therapies or agents, is a partial response.
[0114] Validated patient reported outcome scales can also be used
to denote response provided by each patient through a specific
reporting system. Rather than being disease focused, such outcome
scales are concerned with retained function while managing a
chronic condition. One non-limiting example of a validated patient
reported outcome scale is PROMIS.RTM. (Patient Reported Outcomes
Measurement Information System) from the United States National
Institutes of Health. For example, PROMIS.RTM. Physical Function
Instrument for adult cancer patients can evaluate self-reported
capabilities for the functioning of upper extremities (e.g.,
dexterity), lower extremities (e.g., walking or mobility), and
central regions (e.g., neck, back mobility), and includes routine
daily activities, such as running errands.
[0115] Kaplan-Meier curves (Kaplan and Meier, J. Am. Stat. Assoc.
1958; 53(282): 457-481) can also be used to estimate overall
survival and progression free survival for cancer patients
undergoing anti-PD-1 antibody therapy in comparison to standard of
care.
[0116] 7.6.2. Adjunctive Therapies
[0117] The anti-PD-1 antibodies may be used adjunctive to, or with,
other agents or treatments having anti-cancer properties. When used
adjunctively, the anti-PD-1 antibody and other agent(s) may be
formulated together in a single, combination pharmaceutical
formulation, or may be formulated and administered separately,
either on a single coordinated dosing regimen or on different
dosing regimens. Agents administered adjunctive to or with the
anti-PD-1 antibodies will typically have complementary activities
to the anti-PD-1 antibodies such that the antibodies and other
agents do not adversely affect each other.
[0118] Agents that may be used adjunctively with anti-PD-1
antibodies include, but are not limited to, alkylating agents,
angiogenesis inhibitors, antibodies, antimetabolites, antimitotics,
antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis
promoters (for example, Bcl-2 family inhibitors), activators of
death receptor pathway, Bcr-Abl kinase inhibitors, BiTE
(Bi-Specific T cell Engager) antibodies, antibody drug conjugates,
biologic response modifiers, Bruton's tyrosine kinase (BTK)
inhibitors, cyclin-dependent kinase inhibitors, cell cycle
inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral
oncogene homolog (ErbB2) receptor inhibitors, growth factor
inhibitors, heat shock protein (HSP)-90 inhibitors, histone
deacetylase (HDAC) inhibitors, hormonal therapies, immunologicals,
inhibitors of inhibitors of apoptosis proteins (IAPs),
intercalating antibiotics, kinase inhibitors, kinesin inhibitors,
Jak2 inhibitors, mammalian target of rapamycin inhibitors,
microRNAs, mitogen-activated extracellular signal-regulated kinase
inhibitors, multivalent binding proteins, non-steroidal
anti-inflammatory drugs (NSAIDs), poly ADP (adenosine
diphosphate)-ribose polymerase (PARP) inhibitors, platinum
chemotherapeutics, polo-like kinase (Plk) inhibitors,
phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors,
purine analogs, pyrimidine analogs, receptor tyrosine kinase
inhibitors, retinoids/deltoids plant alkaloids, small inhibitory
ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin
ligase inhibitors, and the like, as well as combinations of one or
more of these agents.
[0119] BiTE antibodies are bispecific antibodies that direct
T-cells to attack cancer cells by simultaneously binding the two
cells. The T-cell then attacks the target cancer cell. Examples of
BiTE antibodies include adecatumumab (Micromet MT201), blinatumomab
(Micromet MT103) and the like. Without being limited by theory, one
of the mechanisms by which T-cells elicit apoptosis of the target
cancer cell is by exocytosis of cytolytic granule components, which
include perforin and granzyme B.
[0120] SiRNAs are molecules having endogenous RNA bases or
chemically modified nucleotides. The modifications do not abolish
cellular activity, but rather impart increased stability and/or
increased cellular potency. Examples of chemical modifications
include phosphorothioate groups, 2'-deoxynucleotide,
2'-OCH.sub.3-containing ribonucleotides, 2'-F-ribonucleotides,
2'-methoxyethyl ribonucleotides, combinations thereof and the like.
The siRNA can have varying lengths (e.g., 10-200 bps) and
structures (e.g., hairpins, single/double strands, bulges,
nicks/gaps, mismatches) and are processed in cells to provide
active gene silencing. A double-stranded siRNA (dsRNA) can have the
same number of nucleotides on each strand (blunt ends) or
asymmetric ends (overhangs). The overhang of 1-2 nucleotides can be
present on the sense and/or the antisense strand, as well as
present on the 5'- and/or the 3'-ends of a given strand.
[0121] Multivalent binding proteins are binding proteins comprising
two or more antigen binding sites. Multivalent binding proteins are
engineered to have the three or more antigen binding sites and are
generally not naturally occurring antibodies. The term
"multispecific binding protein" means a binding protein capable of
binding two or more related or unrelated targets. Dual variable
domain (DVD) binding proteins are tetravalent or multivalent
binding proteins binding proteins comprising two or more antigen
binding sites. Such DVDs may be monospecific (i.e., capable of
binding one antigen) or multispecific (i.e., capable of binding two
or more antigens). DVD binding proteins comprising two heavy chain
DVD polypeptides and two light chain DVD polypeptides are referred
to as DVD Ig's. Each half of a DVD Ig comprises a heavy chain DVD
polypeptide, a light chain DVD polypeptide, and two antigen binding
sites. Each binding site comprises a heavy chain variable domain
and a light chain variable domain with a total of 6 CDRs involved
in antigen binding per antigen binding site.
[0122] Alkylating agents include, but are not limited to,
altretamine, AMD-473, AP-5280, apaziquone, bendamustine,
brostallicin, busulfan, carboquone, carmustine (BCNU),
chlorambucil, CLORETAZINE.RTM. (laromustine, VNP 40101M),
cyclophosphamide, dacarbazine, estramustine, fotemustine,
glufosfamide, ifosfamide, KW-2170, lomustine (CCNU), mafosfamide,
melphalan, mitobronitol, mitolactol, nimustine, nitrogen mustard
N-oxide, ranimustine, temozolomide, thiotepa, TREANDA.RTM.
(bendamustine), treosulfan, and trofosfamide.
[0123] Angiogenesis inhibitors include, but are not limited to,
endothelial-specific receptor tyrosine kinase (Tie-2) inhibitors,
epidermal growth factor receptor (EGFR) inhibitors, vascular
endothelial growth factor receptor (VEGF) inhibitors, delta-like
ligand 4 (DLL4) inhibitors, insulin growth factor-2 receptor
(IGFR-2) inhibitors, matrix metalloproteinase-2 (MMP-2) inhibitors,
matrix metalloproteinase-9 (MMP-9) inhibitors, platelet-derived
growth factor receptor (PDGFR) inhibitors, thrombospondin analogs,
and vascular endothelial growth factor receptor tyrosine kinase
(VEGFR) inhibitors.
[0124] Antibody drug conjugates include, but are not limited to,
those that target c-Met kinase (e.g., ADCs described in U.S. Pat.
No. 7,615,529), LRRC15, CD30 (e.g., ADCETRIS.RTM. (brentuximab
vedotin)), CS1 (e.g., ADCs described in US publication no.
20160122430), DLL3 (e.g., rovalpituzumab tesirine (ROVA-T)), HER2
(e.g., KADCYLA.RTM. (trastuzumab emtansine)), EGFR (e.g., ADCs
described in US publication no. 20150337042), and prolactin
receptor (e.g., ADCs described in US publication no.
20140227294).
[0125] Antimetabolites include, but are not limited to, ALIMTA.RTM.
(pemetrexed disodium, LY231514, MTA), 5-azacitidine, XELODA.RTM.
(capecitabine), carmofur, LEUSTAT.RTM. (cladribine), clofarabine,
cytarabine, cytarabine ocfosfate, cytosine arabinoside, decitabine,
deferoxamine, doxifluridine, eflornithine, EICAR
(5-ethynyl-1-.beta.-D-ribofuranosylimidazole-4-carboxamide),
enocitabine, ethnylcytidine, fludarabine, 5-fluorouracil alone or
in combination with leucovorin, GEMZAR.RTM. (gemcitabine),
hydroxyurea, ALKERAN.RTM. (melphalan), mercaptopurine,
6-mercaptopurine riboside, methotrexate, mycophenolic acid,
nelarabine, nolatrexed, ocfosfate, pelitrexol, pentostatin,
raltitrexed, ribavirin, triapine, trimetrexate, S-1, tiazofurin,
tegafur, TS-1, vidarabine, and UFT.
[0126] Antivirals include, but are not limited to, ritonavir,
acyclovir, cidofovir, ganciclovir, foscarnet, zidovudine,
ribavirin, and hydroxychloroquine.
[0127] Aurora kinase inhibitors include, but are not limited to,
ABT-348, AZD-1152, MLN-8054, VX-680, Aurora A-specific kinase
inhibitors, Aurora B-specific kinase inhibitors and pan-Aurora
kinase inhibitors.
[0128] Bcl-2 protein inhibitors include, but are not limited to,
ABT-263 (navitoclax), AT-101 ((-)gossypol), GENASENSE.RTM. (G3139
or oblimersen (Bcl-2-targeting antisense oligonucleotide)),
IPI-194, IPI-565,
N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazin-1-yl)benzoyl)-4--
(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobe-
nzene sulfonamide),
N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)pip-
erazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl-
)propyl)amino)-3-((trifluoromethyl)sulfonyl)benzene sulfonamide,
venetoclax and GX-070 (obatoclax).
[0129] Bcr-Abl kinase inhibitors include, but are not limited to,
DASATINIB.RTM. (BMS-354825) and GLEEVEC.RTM. (imatinib).
[0130] BTK inhibitors include, but are not limited to, ibrutinib
and acalabrutinib.
[0131] CDK inhibitors include, but are not limited to, AZD-5438,
BMI-1040, BMS-032, BMS-387, CVT-2584, flavopyridol, GPC-286199,
MCS-5A, PD0332991, PHA-690509, seliciclib (CYC-202, R-roscovitine),
and ZK-304709.
[0132] COX-2 inhibitors include, but are not limited to, ABT-963,
ARCOXIA.RTM. (etoricoxib), BEXTRA.RTM. (valdecoxib), BMS347070,
CELEBREX.RTM. (celecoxib), COX-189 (lumiracoxib), CT-3,
DERAMAXX.RTM. (deracoxib), JTE-522,
4-methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoylphenyl-1H-pyrrole),
MK-663 (etoricoxib), NS-398, parecoxib, RS-57067, SC-58125,
SD-8381, SVT-2016, S-2474, T-614, and VIOXX.RTM. (rofecoxib).
[0133] EGFR inhibitors include, but are not limited to, ABX-EGF,
anti-EGFR immunoliposomes, EGF-vaccine, EMD-7200, ERBITUX.RTM.
(cetuximab), HR3, IgA antibodies, IRESSA.RTM. (gefitinib),
TARCEVA.RTM. (erlotinib or OSI-774), TAGRISSO.RTM. (osimertinib),
TP-38, EGFR fusion protein, and TYKERB.RTM. (lapatinib).
[0134] ErbB2 receptor inhibitors include, but are not limited to,
CP-724-714, CI-1033 (canertinib), HERCEPTIN.RTM. (trastuzumab),
TYKERB.RTM. (lapatinib), OMNITARG.RTM. (2C4, pertuzumab), TAK-165,
GW-572016 (ionafarnib), GW-282974, EKB-569, PI-166, dHER2 (HER2
vaccine), APC-8024 (HER-2 vaccine), anti-HER/2neu bispecific
antibody, B7.her2IgG.sub.3, AS HER2 trifunctional bispecific
antibodies, mAB AR-209, and mAB 2B-1.
[0135] Histone deacetylase inhibitors include, but are not limited
to, depsipeptide, LAQ-824, MS-275, trapoxin, suberoylanilide
hydroxamic acid (SAHA), TSA, and valproic acid.
[0136] HSP-90 inhibitors include, but are not limited to,
17-AAG-nab, 17-AAG, CNF-101, CNF-1010, CNF-2024, 17-DMAG,
geldanamycin, IPI-504, KOS-953, MYCOGRAB.RTM. (human recombinant
antibody to HSP-90), NCS-683664, PU24FC1, PU-3, radicicol,
SNX-2112, STA-9090, and VER49009.
[0137] Inhibitors of apoptosis proteins include, but are not
limited to, HGS1029, GDC-0145, GDC-0152, LCL-161, and LBW-242.
[0138] Activators of death receptor pathway include, but are not
limited to, TRAIL, antibodies or other agents that target TRAIL or
death receptors (e.g., DR4 and DR5) such as Apomab, conatumumab,
ETR2-ST01, GDC0145 (lexatumumab), HGS-1029, LBY-135, PRO-1762 and
trastuzumab.
[0139] Kinesin inhibitors include, but are not limited to, Eg5
inhibitors such as AZD4877, ARRY-520; and CENPE inhibitors such as
GSK923295A.
[0140] JAK-2 inhibitors include, but are not limited to, CEP-701
(lesaurtinib), XL019 and INCB018424.
[0141] MEK inhibitors include, but are not limited to, ARRY-142886,
ARRY-438162, PD-325901, and PD-98059.
[0142] mTOR inhibitors include, but are not limited to, AP-23573,
CCI-779, everolimus, RAD-001, rapamycin, temsirolimus,
ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242,
PP30, and Torin 1.
[0143] Non-steroidal anti-inflammatory drugs include, but are not
limited to, AMIGESIC.RTM. (salsalate), DOLOBID.RTM. (diflunisal),
MOTRIN.RTM. (ibuprofen), ORUDIS.RTM. (ketoprofen), RELAFEN.RTM.
(nabumetone), FELDENE.RTM. (piroxicam), ibuprofen cream, ALEVE.RTM.
(naproxen) and NAPROSYN.RTM. (naproxen), VOLTAREN.RTM.
(diclofenac), INDOCIN.RTM. (indomethacin), CLINORIL.RTM.
(sulindac), TOLECTIN.RTM. (tolmetin), LODINE.RTM. (etodolac),
TORADOL.RTM. (ketorolac), and DAYPRO.RTM. (oxaprozin).
[0144] PDGFR inhibitors include, but are not limited to, C-451,
CP-673 and CP-868596.
[0145] Platinum chemotherapeutics include, but are not limited to,
cisplatin, ELOXATIN.RTM. (oxaliplatin) eptaplatin, lobaplatin,
nedaplatin, PARAPLATIN.RTM. (carboplatin), satraplatin, and
picoplatin.
[0146] Polo-like kinase inhibitors include, but are not limited to,
BI-2536.
[0147] Phosphoinositide-3 kinase (PI3K) inhibitors include, but are
not limited to, wortmannin, LY294002, XL-147, CAL-120, ONC-21,
AEZS-127, ETP-45658, PX-866, GDC-0941, BGT226, BEZ235, and
XL765.
[0148] Thrombospondin analogs include, but are not limited to,
ABT-510, ABT-567, ABT-898, and TSP-1.
[0149] VEGFR inhibitors include, but are not limited to, ABT-869,
AEE-788, ANGIOZYME.TM. (a ribozyme that inhibits angiogenesis
(Ribozyme Pharmaceuticals (Boulder, Colo.) and Chiron (Emeryville,
Calif.)), axitinib (AG-13736), AZD-2171, CP-547,632, CYRAMZA.RTM.
(ramucirumab), IM-862, MACUGEN.RTM. (pegaptamib), NEXAVAR.RTM.
(sorafenib, BAY43-9006), pazopanib (GW-786034), vatalanib (PTK-787,
ZK-222584), SUTENT.RTM. (sunitinib, SU-11248), STIVARGA.RTM.
(regorafenib), VEGF trap, and ZACTIMA.TM. (vandetanib,
ZD-6474).
[0150] Antibiotics include, but are not limited to, intercalating
antibiotics aclarubicin, actinomycin D, amrubicin, annamycin,
adriamycin, BLENOXANE.RTM. (bleomycin), daunorubicin, CAELYX.RTM.
or MYOCET.RTM. (liposomal doxorubicin), elsamitrucin, epirbucin,
glarbuicin, ZAVEDOS.RTM. (idarubicin), mitomycin C, nemorubicin,
neocarzinostatin, peplomycin, pirarubicin, rebeccamycin,
stimalamer, streptozocin, VALSTAR.RTM. (valrubicin), and
zinostatin.
[0151] Topoisomerase inhibitors include, but are not limited to,
aclarubicin, 9-aminocamptothecin, amonafide, amsacrine,
becatecarin, belotecan, BN-80915, CAMPTOSAR.RTM. (irinotecan
hydrochloride), camptothecin, CARDIOXANE.RTM. (dexrazoxine),
diflomotecan, edotecarin, ELLENCE.RTM. or PHARMORUBICIN.RTM.
(epirubicin), etoposide, exatecan, 10-hydroxycamptothecin,
gimatecan, lurtotecan, mitoxantrone, Onivyde.TM. (liposomal
irinotecan), orathecin, pirarbucin, pixantrone, rubitecan,
sobuzoxane, SN-38, tafluposide, and topotecan.
[0152] Antibodies include, but are not limited to, AVASTIN.RTM.
(bevacizumab), CD40-specific antibodies, chTNT-1/B, denosumab,
ERBITUX.RTM. (cetuximab), HUMAX-CD4.RTM. (zanolimumab),
IGF1R-specific antibodies, lintuzumab, OX-40 specific antibodies,
PANOREX.RTM. (edrecolomab), RENCAREX.RTM. (WX G250), RITUXAN.RTM.
(rituximab), ticilimumab, trastuzumab, pertuzumab, VECTIBIX.RTM.
(panitumumab) and CD20 antibodies types I and II.
[0153] Hormonal therapies include, but are not limited to,
ARIMIDEX.RTM. (anastrozole), AROMASIN.RTM. (exemestane),
arzoxifene, CASODEX.RTM. (bicalutamide), CETROTIDE.RTM.
(cetrorelix), degarelix, deslorelin, DESOPAN.RTM. (trilostane),
dexamethasone, DROGENIL.RTM. (flutamide), EVISTA.RTM. (raloxifene),
AFEMA.TM. (fadrozole), FARESTON.RTM. (toremifene), FASLODEX.RTM.
(fulvestrant), FEMARA.RTM. (letrozole), formestane,
glucocorticoids, HECTOROL.RTM. (doxercalciferol), RENAGEL.RTM.
(sevelamer carbonate), lasofoxifene, leuprolide acetate,
MEGACE.RTM. (megesterol), MIFEPREX.RTM. (mifepristone),
NILANDRON.TM. (nilutamide), NOLVADEX.RTM. (tamoxifen citrate),
PLENAXIS.TM. (abarelix), prednisone, PROPECIA.RTM. (finasteride),
rilostane, SUPREFACT.RTM. (buserelin), TRELSTAR.RTM. (luteinizing
hormone releasing hormone (LHRH)), VANTAS.RTM. (Histrelin implant),
VETORYL.RTM. (trilostane or modrastane), and ZOLADEX.RTM.
(fosrelin, goserelin).
[0154] Deltoids and retinoids include, but are not limited to,
seocalcitol (EB1089, CB1093), lexacalcitrol (KH1060), fenretinide,
PANRETIN.RTM. (aliretinoin), ATRAGEN.RTM. (liposomal tretinoin),
TARGRETIN.RTM. (bexarotene), and LGD-1550.
[0155] PARP inhibitors include, but are not limited to, ABT-888
(veliparib), KU-59436, AZD-2281 (olaparib), AG-014699 (rucaparib),
MK4827 (niraparib), BMN-673 (talazoparib), iniparib, BSI-201,
BGP-15, INO-1001, and ONO-2231.
[0156] Plant alkaloids include, but are not limited to,
vincristine, vinblastine, vindesine, and vinorelbine.
[0157] Proteasome inhibitors include, but are not limited to,
VELCADE.RTM. (bortezomib), KYPROLIS.RTM. (carfilzomib), MG132,
NPI-0052, and PR-171.
[0158] Examples of immunologicals include, but are not limited to,
interferons, immune checkpoint inhibitors, co-stimulatory agents,
and other immune-enhancing agents. Interferons include interferon
alpha, interferon alpha-2a, interferon alpha-2b, interferon beta,
interferon gamma-1a, ACTIMMUNE.RTM. (interferon gamma-1b) or
interferon gamma-n1, combinations thereof and the like. Immune
check point inhibitors include antibodies that target PD-L1 (e.g.,
durvalumab, atezolizumab, avelumab, MEDI4736, MSB0010718C and
MPDL3280A), and CTLA4 (cytotoxic lymphocyte antigen 4; e.g.,
ipilimumab, tremelimumab). Co-stimulatory agents include, but are
not limited to, antibodies against CD3, CD40, CD40L, CD27, CD28,
CSF1R, CD137 (e.g., urelumab), B7H1, GITR, ICOS, CD80, CD86, OX40,
OX40L, CD70, HLA-DR, LIGHT, LIGHT-R, TIM3, A2AR, NKG2A, KIR (e.g.,
lirilumab), TGF-.beta. (e.g., fresolimumab) and combinations
thereof.
[0159] Other agents include, but are not limited to,
ALFAFERONE.RTM. (IFN-.alpha.), BAM-002 (oxidized glutathione),
BEROMUN.RTM. (tasonermin), BEXXAR.RTM. (tositumomab), CAMPATH.RTM.
(alemtuzumab), dacarbazine, denileukin, epratuzumab, GRANOCYTE.RTM.
(lenograstim), lentinan, leukocyte alpha interferon, imiquimod,
melanoma vaccine, mitumomab, molgramostim, MYLOTARG.TM. (gemtuzumab
ozogamicin), NEUPOGEN.RTM. (filgrastim), OncoVAC-CL, OVAREX.RTM.
(oregovomab), pemtumomab (Y-muHMFG1), PROVENGE.RTM. (sipuleucel-T),
sargaramostim, sizofilan, teceleukin, THERACYS.RTM. (Bacillus
Calmette-Guerin), ubenimex, VIRULIZIN.RTM. (immunotherapeutic,
Lorus Pharmaceuticals), Z-100 (Specific Substance of Maruyama
(SSM)), WF-10 (Tetrachlorodecaoxide (TCDO)), PROLEUKIN.RTM.
(aldesleukin), ZADAXIN.RTM. (thymalfasin), ZINBRYTA.RTM.
(daclizumab high-yield process), and ZEVALIN.RTM.
(.sup.90Y-Ibritumomab tiuxetan).
[0160] Biological response modifiers are agents that modify defense
mechanisms of living organisms or biological responses, such as
survival, growth or differentiation of tissue cells to direct them
to have anti-tumor activity and include, but are not limited to,
krestin, lentinan, sizofiran, picibanil PF-3512676 (CpG-8954), and
ubenimex.
[0161] Pyrimidine analogs include, but are not limited to,
cytarabine (ara C or Arabinoside C), cytosine arabinoside,
doxifluridine, FLUDARA.RTM. (fludarabine), 5-FU (5-fluorouracil),
floxuridine, GEMZAR.RTM. (gemcitabine), TOMUDEX.RTM. (ratitrexed),
and TROXATYL.TM. (triacetyluridine troxacitabine).
[0162] Purine analogs include, but are not limited to, LANVIS.RTM.
(thioguanine) and PURINETHOL.RTM. (mercaptopurine).
[0163] Antimitotic agents include, but are not limited to,
batabulin, epothilone D (KOS-862),
N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide,
ixabepilone (BMS 247550), TAXOL.RTM. (paclitaxel), TAXOTERE.RTM.
(docetaxel), PNU100940 (109881), patupilone, XRP-9881 (larotaxel),
vinflunine, and ZK-EPO (synthetic epothilone).
[0164] Ubiquitin ligase inhibitors include, but are not limited to,
MDM2 inhibitors, such as nutlins, and NEDD8 inhibitors such as
MLN4924.
[0165] Anti-PD-1 antibodies may also be used to enhance the
efficacy of radiation therapy. Examples of radiation therapy
include external beam radiation therapy, internal radiation therapy
(i.e., brachytherapy) and systemic radiation therapy.
[0166] Anti-PD-1 antibodies may be administered adjunctive to or
with other chemotherapeutic agents such as ABRAXANE.TM. (ABI-007),
ABT-100 (farnesyl transferase inhibitor), ADVEXIN.RTM. (Ad5CMV-p53
vaccine), ALTOCOR.RTM. or MEVACOR.RTM. (lovastatin), AMPLIGEN.RTM.
(poly I:poly C12U, a synthetic RNA), APTOSYN.RTM. (exisulind),
AREDIA.RTM. (pamidronic acid), arglabin, L-asparaginase, atamestane
(1-methyl-3,17-dione-androsta-1,4-diene), AVAGE.RTM. (tazarotene),
AVE-8062 (combreastatin derivative) BEC2 (mitumomab), cachectin or
cachexin (tumor necrosis factor), canvaxin (vaccine), CEAVAC.RTM.
(cancer vaccine), CELEUK.RTM. (celmoleukin), CEPLENE.RTM.
(histamine dihydrochloride), CERVARIX.RTM. (human papillomavirus
vaccine), CHOP.RTM. (C: CYTOXAN.RTM. (cyclophosphamide); H:
ADRIAMYCIN.RTM. (hydroxydoxorubicin); O: Vincristine
(ONCOVIN.RTM.); P: prednisone), CYPAT.TM. (cyproterone acetate),
combrestatin A4P, DAB(389)EGF (catalytic and translocation domains
of diphtheria toxin fused via a His-Ala linker to human epidermal
growth factor) or TransMID-107R.TM. (diphtheria toxins),
dacarbazine, dactinomycin, 5,6-dimethylxanthenone-4-acetic acid
(DMXAA), eniluracil, EVIZON.TM. (squalamine lactate),
DIMERICINE.RTM. (T4N5 liposome lotion), discodermolide, DX-8951f
(exatecan mesylate), enzastaurin, EP0906 (epithilone B),
GARDASIL.RTM. (quadrivalent human papillomavirus (Types 6, 11, 16,
18) recombinant vaccine), GASTRIMMUNE.RTM., GENASENSE.RTM., GMK
(ganglioside conjugate vaccine), GVAX.RTM. (prostate cancer
vaccine), halofuginone, histrelin, hydroxycarbamide, ibandronic
acid, IGN-101, IL-13-PE38, IL-13-PE38QQR (cintredekin besudotox),
IL-13-pseudomonas exotoxin, interferon-.alpha., interferon-.gamma.,
JUNOVAN.TM. or MEPACT.TM. (mifamurtide), lonafarnib,
5,10-methylenetetrahydrofolate, miltefosine
(hexadecylphosphocholine), NEOVASTAT.RTM. (AE-941), NEUTREXIN.RTM.
(trimetrexate glucuronate), NIPENT.RTM. (pentostatin),
ONCONASE.RTM. (a ribonuclease enzyme), ONCOPHAGE.RTM. (melanoma
vaccine treatment), ONCOVAX.RTM. (IL-2 Vaccine), ORATHECIN.TM.
(rubitecan), OSIDEM.RTM. (antibody-based cell drug), OVAREX.RTM.
MAb (murine monoclonal antibody), paclitaxel, PANDIMEX.TM.
(aglycone saponins from ginseng comprising 20(S)protopanaxadiol
(aPPD) and 20(S)protopanaxatriol (aPPT)), panitumumab,
PANVAC.RTM.-VF (investigational cancer vaccine), pegaspargase, PEG
Interferon A, phenoxodiol, procarbazine, rebimastat, REMOVAB.RTM.
(catumaxomab), REVLIMID.RTM. (lenalidomide), RSR13 (efaproxiral),
SOMATULINE.RTM. LA (lanreotide), SORIATANE.RTM. (acitretin),
staurosporine (Streptomyces staurospores), talabostat (PT100),
TARGRETIN.RTM. (bexarotene), TAXOPREXIN.RTM. (DHA-paclitaxel),
TELCYTA.RTM. (canfosfamide, TLK286), temilifene, TEMODAR.RTM.
(temozolomide), tesmilifene, thalidomide, THERATOPE.RTM. (STn-KLH),
thymitaq
(2-amino-3,4-dihydro-6-methyl-4-oxo-5-(4-pyridylthio)quinazoline
dihydrochloride), TNFERADE.TM. (adenovector: DNA carrier containing
the gene for tumor necrosis factor-.alpha.), TRACLEER.RTM. or
ZAVESCA.RTM. (bosentan), tretinoin (Retin-A), tetrandrine,
TRISENOX.RTM. (arsenic trioxide), VIRULIZIN.RTM., ukrain
(derivative of alkaloids from the greater celandine plant), vitaxin
(anti-alphavbeta3 antibody), XCYTRIN.RTM. (motexafin gadolinium),
XINLAY.TM. (atrasentan), XYOTAX.TM. (paclitaxel poliglumex),
YONDELIS.RTM. (trabectedin), ZD-6126, ZINECARD.RTM. (dexrazoxane),
ZOMETA.RTM. (zolendronic acid), and zorubicin, as well as
combinations of any of these agents.
[0167] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with an antibody-drug conjugate targeting c-Met
kinase for treating non-small cell lung cancer, head and neck
cancer, pancreatic cancer, colorectal cancer, or gastric
cancer.
[0168] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with an antibody-drug conjugate targeting LRRC15
for treating non-small cell lung cancer, head and neck cancer,
pancreatic cancer, sarcoma, triple negative breast cancer, or
melanoma.
[0169] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with an antibody-drug conjugate targeting EGFR for
treating glioblastoma.
[0170] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with an antibody-drug conjugate targeting CS1 for
treating a blood malignancy such as multiple myeloma.
[0171] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with an antibody-drug conjugate targeting DLL3 for
treating small cell lung cancer or glioblastoma.
[0172] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with an anti-CD40 protein for treating head and
neck cancer, lung cancer (such as adenocarcinoma, non-small cell
lung cancer, mesothelioma, small cell lung cancer), melanoma,
ovarian cancer or pancreatic cancer.
[0173] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with venetoclax for treating a blood malignancy
such as chronic lymphocytic leukemia.
[0174] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with ibrutinib for treating a blood malignancy,
such as chronic lymphocytic leukemia, mantle cell lymphoma, or
Waldenstrom's macroglobulinemia, or a solid tumor.
[0175] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with ipilimumab and an antibody-drug conjugate
targeting c-Met kinase for treating non-small cell lung cancer.
[0176] In some embodiments, an anti-PD-1 antibody is administered
adjunctive to or with ipilimumab and an antibody-drug conjugate
targeting LRRC15 for treating non-small cell lung cancer.
7.7. Dosages and Administration Regimens
[0177] The amount of anti-PD-1 antibodies administered will depend
upon a variety of factors, including but not limited to, the
particular type of solid tumor treated, the stage of the solid
tumor being treated, the mode of administration, the frequency of
administration, the desired therapeutic benefit, and other
parameters such as the age, weight and other characteristics of the
patient, etc. Determination of dosages effective to provide
therapeutic benefit for specific modes and frequency of
administration is within the capabilities of those skilled in the
art.
[0178] Dosages effective to provide therapeutic benefit may be
estimated initially from in vivo animal models or clinical.
Suitable animal models for a wide variety of diseases are known in
the art.
[0179] The anti-PD-1 antibodies disclosed herein may be
administered by any route appropriate to the condition to be
treated. In some embodiments, the anti-PD-1 antibody is any one of
the humanized antibodies listed in TABLE 3. In a specific
embodiment, the anti-PD-1 antibody has a heavy chain according to
SEQ ID NO:51 or SEQ ID NO:52, and a light chain according to SEQ ID
NO:61. An anti-PD-1 antibody will typically be administered
parenterally, i.e., infusion, subcutaneous, intramuscular,
intravenous (IV), intradermal, intrathecal, bolus, intratumoral
injection or epidural ((Shire et al., 2004, J. Pharm. Sciences
93(6):1390-1402)). In one embodiment, an anti-PD-1 antibody is
provided as a lyophilized powder in a vial. The vials may contain
100 mg, 110 mg, 120 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 400 mg
of anti-PD-1 antibody. Prior to administration, the lyophilized
powder is reconstituted with sterile water for injection (SWFI) or
other suitable medium to provide a solution containing 20 mg/mL
anti-PD-1 antibody. In some embodiments, the resulting
reconstituted solution is further diluted with saline or other
suitable medium for infusion and administered via an IV infusion
twice every 7 days, once every 7 days, once every 14 days, once
every 21 days, once every 28 days, once every 35 days, once every
42 days, once every 49 days, or once every 56 days. In some
embodiments, for the first cycle, the infusion occurs over 90
minutes. In some embodiments, subsequent infusions are over 60
minutes.
[0180] In some embodiments, the anti-PD-1 antibody is administered
as an IV infusion once every 7 days at 0.1 mg/kg, 0.5 mg/kg, 1.0
mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 8.0
mg/kg, or 10.0 mg/kg.
[0181] In some embodiments, the anti-PD-1 antibody is administered
as an IV infusion once every 14 days at 0.1 mg/kg, 0.5 mg/kg, 1.0
mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 8.0
mg/kg, or 10.0 mg/kg.
[0182] In some embodiments, the anti-PD-1 antibody is administered
as an IV infusion once every 21 days at 0.1 mg/kg, 0.5 mg/kg, 1.0
mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 8.0
mg/kg, or 10.0 mg/kg.
[0183] In some embodiments, the anti-PD-1 antibody is administered
as an IV infusion once every 28 days at 0.1 mg/kg, 0.5 mg/kg, 1.0
mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 8.0
mg/kg, or 10.0 mg/kg.
[0184] In one exemplary embodiment, an anti-PD-1 antibody is used
adjunctive to ipilimumab (YERVOY.RTM.) to treat non-small cell lung
cancer. The anti-PD-1 antibody is administered via IV infusion once
every 21 days at 1.0 mg/kg or 3.0 mg/kg. Ipilimumab is administered
by intravenous infusion at a dose of 1 mg/kg once every three weeks
for four doses. Subsequent to the last ipilimumab dose, the
anti-PD-1 antibody is administered via IV infusion once every 14
days at 1.0 mg/kg or 3.0 mg/kg. The adjunctive anti-PD-1
antibody/ipilimumab therapy is continued until disease progression
or no longer tolerated by the patient.
[0185] In one exemplary embodiment, an anti-PD-1 antibody is used
adjunctive to ipilimumab (YERVOY.RTM.) to treat non-small cell lung
cancer. The anti-PD-1 antibody is administered via IV infusion once
every 14 days at 1.0 mg/kg or 3.0 mg/kg. Ipilimumab is administered
by intravenous infusion at a dose of 1 mg/kg once every six weeks
for four doses. The adjunctive anti-PD-1 antibody/ipilimumab
therapy is continued until disease progression or no longer
tolerated by the patient.
[0186] In one exemplary embodiment, an anti-PD-1 antibody is used
adjunctive to ipilimumab (YERVOY.RTM.) to treat non-small cell lung
cancer. The anti-PD-1 antibody is administered via IV infusion once
every 14 days at 1.0 mg/kg or 3.0 mg/kg. Ipilimumab is administered
by intravenous infusion at a dose of 1 mg/kg once every twelve
weeks for four doses. The adjunctive anti-PD-1 antibody/ipilimumab
therapy is continued until disease progression or no longer
tolerated by the patient.
[0187] When administered adjunctive to or with other agents, such
as other chemotherapeutic agents, the anti-PD-1 antibodies may be
administered on the same schedule as the other agent(s), or on a
different schedule. When administered on the same schedule, the
anti-PD-1 antibody may be administered before, after, or
concurrently with the other agent. In some embodiments where an
anti-PD-1 antibody is administered adjunctive to, or with,
standards of care, the anti-PD-1 antibody may be initiated prior to
commencement of the standard therapy, for example a day, several
days, a week, several weeks, a month, or even several months before
commencement of standard of care therapy. In some embodiments where
an anti-PD-1 antibody is administered adjunctive to, or with,
standards of care, the anti-PD-1 antibody may be initiated after
commencement of the standard therapy, for example a day, several
days, a week, several weeks, a month, or even several months after
commencement of standard of care therapy.
[0188] As will be appreciated by those of skill in the art, the
recommended dosages for the various agents described above may need
to be adjusted to optimize patient response and maximize
therapeutic benefit.
7.8. Exemplary Embodiments
[0189] The following are exemplary enumerated embodiments of the
present disclosure.
[0190] 1. An anti-PD-1 binding protein which comprises (i) a
V.sub.H chain comprising three CDRs; and (ii) a V.sub.L chain
comprising three CDRs, wherein: V.sub.H CDR #1 is GYTFTHYGMN (SEQ
ID NO:11); V.sub.H CDR #2 is WVNTYTGEPTYADDFKG (SEQ ID NO:12);
V.sub.H CDR #3 is EGEGLGFGD (SEQ ID NO:13); V.sub.L CDR #1 is
RSSQSIVHSHGDTYLE (SEQ ID NO:14); V.sub.L CDR #2 is KVSNRFS (SEQ ID
NO:15); and V.sub.L CDR #3 is FQGSHIPVT (SEQ ID NO:16).
[0191] 2. The anti-PD-1 binding protein of embodiment 1, which
comprises a V.sub.H chain corresponding in sequence to SEQ ID
NO:31; and a V.sub.L chain corresponding in sequence to SEQ ID
NO:41.
[0192] 3. The anti-PD-1 binding protein of embodiment 1, which is
humanized.
[0193] 4. The anti-PD-1 binding protein of embodiment 3, which
comprises a V.sub.H chain corresponding in sequence to SEQ ID
NO:36; and a V.sub.L chain corresponding in sequence to SEQ ID
NO:42.
[0194] 5. An anti-PD-1 binding protein which comprises (i) a
V.sub.H chain comprising three CDRs; and (ii) a V.sub.L chain
comprising three CDRs, wherein: V.sub.H CDR #1 is GYTFTHYGMN (SEQ
ID NO:11); V.sub.H CDR #2 is WVNTYTGEPTYADDFKG (SEQ ID NO:12);
V.sub.H CDR #3 is EGEGMGFGD (SEQ ID NO:23); V.sub.L CDR #1 is
RSSQSIVHSHGDTYLE (SEQ ID NO:14); V.sub.L CDR #2 is KVSNRFS (SEQ ID
NO:15); and V.sub.L CDR #3 is FQGSHIPVT (SEQ ID NO:16).
[0195] 6. The anti-PD-1 binding protein of embodiment 5, which
comprises a V.sub.H chain corresponding in sequence to SEQ ID
NO:33; and a V.sub.L chain corresponding in sequence to SEQ ID
NO:42.
[0196] 7. The anti-PD-1 binding protein of any one of embodiments 1
to 6, which is an IgG.
[0197] 8. The anti-PD-1 binding protein of embodiment 7, which is
an IgG.sub.1, optionally comprising a variant CH2 domain comprising
the amino acid substitutions L234A and L235A.
[0198] 9. The anti-PD-1 binding protein of embodiment 7, which is
an IgG.sub.4, optionally comprising a variant Fc region comprising
the amino acid substitution S228P.
[0199] 10. The anti-PD-1 binding protein of embodiment 8 which
comprises a heavy chain corresponding to the sequence of SEQ ID
NO:51 or SEQ ID NO:52, and a light chain corresponding to the
sequence of SEQ ID NO:61.
[0200] 11. The anti-PD-1 binding protein of any one of embodiments
1 to 10 which has a K.sub.D of less than about 100 nM.
[0201] 12. The anti-PD-1 binding protein of embodiment 11 which has
a K.sub.D of less than about 10 nM.
[0202] 13. A pharmaceutical composition comprising the anti-PD-1
binding protein of any one of embodiments 1 to 12, and a
pharmaceutically acceptable carrier.
[0203] 14. A nucleic acid comprising a nucleotide sequence encoding
the anti-PD-1 binding protein of any one of embodiments 1 to
12.
[0204] 15. A vector comprising the nucleic acid of embodiment
14.
[0205] 16. A prokaryotic host cell transformed with the vector of
embodiment 15.
[0206] 17. A eukaryotic host cell transformed with the vector of
embodiment 15.
[0207] 18. A eukaryotic host cell engineered to express the nucleic
acid of embodiment 14.
[0208] 19. The eukaryotic host cell of embodiment 18 which is a
mammalian host cell.
[0209] 20. A method of producing an anti-PD-1 binding protein
thereof, comprising: (a) culturing the host cell of embodiment 17
or embodiment 18 and (b) recovering the anti-PD-1 binding
protein.
[0210] 21. A method of activating the immune system, comprising
administering to a patient in need thereof the anti-PD-1 binding
protein of any one of embodiments 1 to 12, or the pharmaceutical
composition according to embodiment 13.
[0211] 22. A method of treating a cancer, comprising administering
to a patient in need thereof the anti-PD-1 binding protein of any
one of embodiments 1 to 12, or the pharmaceutical composition
according to embodiment 13.
[0212] 23. The method of embodiment 22, wherein the cancer is
selected from bladder cancer, breast cancer, head and neck cancer,
kidney cancer, lung cancer, lymphoma, melanoma, and stomach
cancer.
[0213] 24. The method of embodiment 23, wherein the lung cancer is
non-small cell lung cancer.
[0214] 25. The method of embodiment 22, in which the anti-PD-1
binding protein is administered as a monotherapy.
[0215] 26. The method of embodiment 22, in which the anti-PD-1
binding protein is administered adjunctive to or with another agent
commonly used to treat the cancer.
[0216] 27. The method of embodiment 26, in which the other agent is
selected from radiation, chemotherapy, an antibody drug conjugate,
an anti-CD40 antibody, an anti-CTLA-4 antibody and an anti-OX40
antibody.
[0217] 28. The method of embodiment 27, in which the chemotherapy
is cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine,
vinorelbine, vinblastine, irinotecan, etoposide, or pemetrexed, or
a pharmaceutically acceptable salt thereof.
[0218] 29. The method of embodiment 27, in which the antibody drug
conjugate targets c-Met kinase.
[0219] 30. The method of embodiment 27, in which the antibody drug
conjugate targets LRRC15.
[0220] 31. The method of embodiment 27, in which the antibody drug
conjugate targets EGFR.
[0221] 32. The method of embodiment 27, in which the antibody drug
conjugate targets CS1.
[0222] 33. The method of embodiment 27, in which the antibody drug
conjugate is rovalpituzumab tesirine.
[0223] 34. The method of embodiment 27, in which the anti-CTLA-4
antibody is ipilimumab.
8. Examples
[0224] The following Examples, which highlight certain features and
properties of embodiments of the anti-PD-1 antibodies described
herein are provided for purposes of illustration, and not
limitation.
Example 1: Materials and Methods
[0225] 8.1.1. Antibody Binding to Plate Bound Human PD-1 by
ELISA
[0226] Immunolon 4.times.HB 96 well plates were coated with 1 ug/ml
of human PD-1 Fc fusion in at 4.degree. C. overnight. Plates were
blocked with PBS containing 1% BSA for 30 minutes at room
temperature and then washed three times with PBS containing 0.1%
Tween 20 (PBST) using a plate washer. PD-1-coated plates were then
incubated with indicated concentrations of antibodies at room
temperature (RT) for one hour. Plates were washed four times with
PBST and then incubated for 1 hour at room temperature with 100
.mu.L of goat anti-human Fab fragment specific-biotin prepared to a
dilution of 1:5000 in PBS containing 1% BSA. Plates were washed
five times in PBST and 100 .mu.L of a 1:1000 dilution of
Streptavidin-HRP was added to each well and incubated for 30
minutes at RT. Plates were washed five times in PBST and 100 .mu.L
of TMB One Component were added to each well and incubated at RT
until color developed (approximately 5-10 minutes). Optical density
(OD) was read at 650 nm using Spectromax190 (Molecular
Devices).
[0227] 8.1.2. Antibody Binding to Cell Surface Expressed Human
PD-1
[0228] PD-1 expressing Jurkat cells were harvested from flasks and
resuspended to 2.times.10.sup.6 cells/mL in PBS containing 1% BSA.
100 .mu.L of cells were added to a round bottom 96 well plate
containing 100 .mu.L of titrated test antibody or isotype control.
Cells were incubated with antibodies at room temperature for 25
minutes and then washed twice with PBS containing 1% BSA. The cell
pellet was resuspended in 100 .mu.L of a 1:250 diluted secondary
antibody goat anti-human Fab PE. Following 25 minutes incubation at
RT, cells were washed twice with PBS containing 1% BSA and
resuspended in 200 .mu.L of 1% BSA. Cells were analyzed using
Becton Dickinson FACSCanto flow cytometer. Data was analyzed using
BD FACSDiva software (version 8.0.1).
[0229] 8.1.3. Antibody Binding to Plate Bound Cynomolgus Monkey
PD-1 by ELISA
[0230] Immunolon 4.times.HB 96-well plates were coated with 1
.mu.g/mL of cynomolgus PD-1 Fc fusion in DPBS at 4.degree. C.
overnight. Plates were blocked with PBS containing 1% BSA for 30
minutes at RT and then washed three times with PBST (PBS Tween 20
0.1%) using a plate washer. PD-1-coated plates were then incubated
with indicated concentrations of antibodies at room temperature for
one hour. Plates were washed four times with PBST and then
incubated for 1 hour at RT with 100 .mu.L of goat anti-human Fab
fragment specific biotin prepared to a dilution of 1:5000 in PBS
containing 1% BSA. Plates were washed five times in PBST and 100
.mu.L of a 1:1000 dilution of Streptavidin-HRP was added to each
well and incubated for 30 minutes at RT. Plates were subsequently
washed five times in PBST and 100 .mu.L of TMB. One component was
added to each well and incubated at RT until color developed
(approximately 5-10 minutes). Optical density (OD) was read at 650
nm using a Spectromax190 (Molecular Devices).
[0231] 8.1.4. Antibody Binding to Activated Human CD4+ T Cells
[0232] Human peripheral blood mononuclear cells (PBMCs) were
isolated from buffy coats purchased from Stanford Blood Center
(Palo Alto, Calif.). Briefly, buffy coats were diluted in a 1:1
ratio with PBS without magnesium and calcium. Diluted blood (30 mL)
was layered over 15 mL of 90% Ficoll-Paque Plus prepared in PBS
without magnesium and calcium contained in SepMate tubes. The tubes
were spun at 1200 g for 10 minutes. The interphase was collected
and washed twice in 1.times.PBS. PBMC were cultured at
2.times.10.sup.6 cells/mL for 48 hrs in RPMI media containing 10%
HI FCS with 1 .mu.g/mL PHA and 50 U/mL recombinant human IL-2. The
cells were collected, washed and incubated with antibodies at RT
for 25 minutes. Labeled cells were washed twice with PBS containing
1% BSA. Cells were resuspended in 100 .mu.L of PBS+1% BSA and a
1:250 dilution of PE conjugated goat anti-human Fab fragment and
anti-CD4-FITC were added. After 30 minutes, cells were washed twice
with PBS containing 1% BSA and resuspended in 200 .mu.L of 1% BSA.
Cells were analyzed using Becton Dickinson FACSCanto flow
cytometer. Data was analyzed using BD FACSDiva software (version
8.0.1).
[0233] 8.1.5. Binding Affinity for PD-1 by Surface Plasmon
Resonance
[0234] The binding kinetics of anti-PD-1 antibodies for recombinant
soluble PD-1 ECD (extracellular domain) were determined by surface
plasmon resonance-based measurements made on Biacore T200
instrument at 25.degree. C. using an anti-Fc capture assay
approach. Recombinant extracellular domains of human PD-1 (residues
1-167) and cynomolgus PD-1 (residues 21-167) were purchased from a
commercial source and further purified by gel filtration in 10 mM
HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA. Chip preparation and binding
kinetic measurements were made in the assay buffer HBS-EP+ (10 mM
HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20). For anti-Fc
capture chip preparation, approximately 2000 RU of goat anti-human
IgG Fc polyclonal antibody diluted to 25 .mu.g/mL in 10 mM sodium
acetate (pH 4.5), was directly immobilized across a CMS biosensor
chip using a standard amine coupling kit according to
manufacturer's instructions and procedures. Unreacted moieties on
the biosensor surface were blocked with ethanolamine. For binding
kinetics measurements, each assay cycle comprises the following
steps: (1) capture of test antibody on test surface only; (2)
analyte injection (PD-1 ECD or buffer only) over both reference and
test surface, 240 .mu.L at 80 .mu.L/min, after which the
dissociation was monitored for 900 seconds at 80 .mu.L/min; and (3)
regeneration of capture surface by 10 mM glycine hydrochloride, pH
1.5 injections over both reference and test surface. During the
assay, all measurements were referenced against the capture surface
alone (i.e., with no captured test antibody) and buffer-only
injections were used for double referencing. PD-1 injections ranged
in concentration from 900 nM to 11.1 nM in a randomized 3-fold
dilution series. Data were processed and fitted globally to a 1:1
binding model using Biacore T200 Evaluation software to determine
the binding kinetic rate constants, k.sub.a (M.sup.-1sec.sup.-1)
and k.sub.d (sec.sup.-1), and the equilibrium dissociation constant
K.sub.D (M).
[0235] 8.1.6. Blocking of PD-1 Interaction with PD-L1 and PD-L2
[0236] Human PD-1 expressing HEK 293G cells were harvested from
confluent flasks. Cells were resuspended in PBS at a concentration
of 2.times.10.sup.6 cells/mL. Cells (1.times.10.sup.5) were added
to each well of a 96-well V bottom plate and cells were blocked for
15 min at 4.degree. C. using human FcR block. In separate plates,
test antibodies and isotype control solutions were prepared using a
3-fold serial dilution of the 20 .mu.g/mL starting concentration.
Cells were washed in 1.times.PBS and 50 .mu.L of the prepared
antibody dilutions and 50 .mu.L of the PD-L1 His-tagged or PD-L2
His-tagged ligands (10 .mu.g/ml) were added to the plate containing
cells. Cells were incubated at 4.degree. C. for 30 minutes and
washed twice with 1.times.PBS. Anti-His APC antibody (50 .mu.L)
prepared at a dilution of 1:50 in PBS was added to each well and
incubated for 30 minutes at 4.degree. C. The cells were washed
twice, resuspended in PBS and acquired on LSR II Fortessa (BD
Biosciences, San Jose, Calif.).
[0237] 8.1.7. Allogeneic Human Mixed Lymphocyte Reaction (MLR)
Assay Using Purified CD4 T Cells and Dendritic Cells
[0238] Human PBMCs were isolated from buffy coats purchased from
Stanford Blood Center (Palo Alto, Calif.). Briefly, buffy coats
were diluted in a 1:1 ratio with PBS without magnesium and calcium.
Diluted blood (30 mL) was layered over 15 mL of 90% Ficoll-Paque
Plus prepared in PBS without magnesium and calcium contained in
SepMate tubes. The tubes were spun at 1200 g for 10 minutes. The
interphase was collected and washed twice in 1.times.PBS. Cells
were resuspended at 1.times.10.sup.8 per mL in AIM-V media
containing beta mercaptoethanol. Dendritic cells (DCs) were derived
by culturing plastic adherent PBMCs in T75 flasks in the presence
of 80 ng/mL GM-CSF and 50 ng/mL IL-4 for 7 days. On day 5, 50 pg/mL
IL-1.alpha. and 200 pg/mL TNF-.alpha. was added to the DC cultures.
On day 7, DCs were harvested from flasks, irradiated for 7.3
minutes at 414 R/min, and resuspended to a final concentration of
1.times.10.sup.5 cells/mL in complete media (RPMI with L Glutamine
containing 10% FBS, 1.times. non-essential vitamins, 1% Pen/Strep
solution, 1% sodium pyruvate, 1% HEPES). Allogeneic human CD4 T
cells were isolated using the CD4 T cell isolation kit. DCs
(1.times.10.sup.4/well) and purified CD4 T cells
(1.times.10.sup.5/well) were added to a U bottom plate. Test
antibody or isotype control (10 .mu.g/mL) was added to the plate
containing DCs and T cells. After five days of incubation,
supernatant was collected and analyzed for IL-2 and IFN-.gamma.
using Milliplex cytometric bead array kit. IFN-.gamma. was measured
using BioRad Bioplex System (Bioplex manager 6.0).
[0239] 8.1.8. Antigen Recall Response to Tetanus Toxoid
[0240] Human PBMCs were isolated from buffy coats purchased from
Stanford Blood Center as previously described. Cells were
resuspended to a final concentration of 2.times.10.sup.6 cells/mL
in AIM-V media containing beta mercaptoethanol. PBMCs
(2.times.10.sup.5) were used per well with 0.2 .mu.g/mL tetanus
toxoid. Antibody was titrated and the plates were incubated for
five days. Supernatants were collected on day 5 and assessed for
IFN-.gamma. using a Milliplex cytometric bead array kit.
IFN-.gamma. was measured using BioRad Bioplex System (Bioplex
manager 6.0).
Example 2: Generation and Humanization of Mouse Anti-PD-1
Antibodies
[0241] Mice were immunized according to the methods known in the
art (E. Harlow, D. Lane. Antibody: A Laboratory Manual, (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998)).
Isotype of each monoclonal antibody was determined using the Mouse
Isotyping kit (Roche). Hybridoma clones producing antibodies of
interest were purified and further characterized for affinity by
surface plasmon resonance and ligand competition (ELISA).
[0242] Cloning and construction of the expression vector were
accomplished by methods known in the art for expression of
recombinant monoclonal antibodies.
[0243] Humanization of the antibody V region was carried out as
outlined by Queen, C. et al. (Proc. Natl. Acad. Sci. USA, 1989;
86:10029-10033). The canonical structures of the CDRs were
determined according to Huang et al. (Methods, 2005; 36:35-42).
Human variable germline sequences with the same or most similar CDR
canonical structures were identified, and appropriate human
V.sub.H, V.sub.L, and J segment sequences were selected to provide
the frameworks for the anti-PD-1 variable region. At framework
positions in which the computer model suggested significant contact
with the CDRs, the amino acids from the murine anti-PD-1 V regions
were substituted for the original human framework amino acids
(back-mutations). Full amino acid sequences of the V.sub.H and
V.sub.L regions of exemplary mouse and humanized antibodies are
shown in FIG. 2.
[0244] Anti-PD-1 mouse antibody Mu12A11 was humanized according to
the method described above. The humanized versions of Mu12A11
V.sub.H were Hu12A11.1b V.sub.H and Hu12A11.2b V.sub.H. Hu12A11.1b
V.sub.H had V.sub.H 7-4-1 framework regions, with four back
mutations of V2I, M48V, F67L, and A93T. Hu12A11.2b V.sub.H had
V.sub.H 1-69 framework regions, with seven back mutations of V2I,
M48V, V67L, I69F, A71L, E73T, and A93T. Either of the two humanized
V.sub.H could be combined with humanized light chain Hu12A11.1a
V.sub.L which had V.sub.L 2-28 framework regions, with one back
mutation of I2V.
Example 3: Stability of the Anti-PD-1 Antibodies
[0245] Stability of exemplary antibodies were determined by
measuring PD-1 binding of the antibodies after treatment under
oxidative or variable temperature conditions.
[0246] An assessment of liability motifs identified a conserved
methionine residue at Kabat position 99 (M99) that was believed to
be both solvent exposed and prone to deamidation. Samples of the
M99 parent antibody Hu12A11.2b1 were exposed to accelerated
degradation conditions to enhance potential deamidation (FIG. 3A).
The samples treated with 1% hydrogen peroxide (1% HP) or 1%
tert-butyl hydroperoxide (1% TBHP) demonstrated loss of binding
affinity, suggesting the potential for oxidation of the
methionine-99 residue. A set of antibodies containing point
mutations at position M99 were constructed, including isoleucine
(M991, Hu12A11.2b2 having a V.sub.H according to SEQ ID NO:34),
valine (M99V, Hu12A11.2b3 having a V.sub.H according to SEQ ID
NO:35), and leucine (M99L, Hu12A11.2b4 having a V.sub.H according
to SEQ ID NO:36) mutations. The antibody variants were screened for
binding to human PD-1 transfected Jurkat cells and EC.sub.50 values
were determined (FIG. 3B). All three point mutation-containing
antibodies bound to cell surface PD-1 similarly, but the M99L
variant antibody showed higher binding activity as compared to M99V
or M991 variants.
[0247] The M99L variant Hu12A11.2b4 was found to retain fully
binding capacity after temperature stability testing (FIG. 3C).
Incubation of Hu12A11.2b4 at -80, 5, 25, or 40.degree. C. afforded
no significant loss of activity in terms of EC.sub.50 or maximal
fluorescence intensity (MFI).
Example 4: Binding Affinity of the Anti-PD-1 Antibodies
[0248] Table 4-1 below shows in vitro binding affinity data of
exemplary antibody Hu12A11.2b4 in comparison to literature
anti-PD-1 antibody nivolumab prepared according to the procedures
found in U.S. Pat. No. 9,073,994. Hu12A11.2b4 exhibited similar
binding properties to PD-1 as compared to nivolumab according to
surface plasmon resonance, ELISA assay with human (Hu) or
cynomolgus (Cyno) PD-1, or in human Jurkat cells as measured in the
assays of Example 1.
TABLE-US-00013 TABLE 4-1 Binding Properties of Select Antibodies
against PD-1 Hu ELISA Hu ELISA Cyno Hu Jurkat SPR EC.sub.50
OD.sub.650 ELISA EC.sub.50 Antibody K.sub.D (M)* (pM) max EC.sub.50
(pM) (pM) Nivolumab 2.9E-09 101 2.2 70 56 Hu12A11.2b4 2.1E-09 197
2.9 102 45 *SPR = surface plasmon resonance as determined according
to Example 1; exponential notation shown (e.g., 3.0E-09 = 3.0
.times. 10.sup.-9).
Example 5: Biological Activity of Anti-PD-1 Antibody
Hu12A11.2b4
[0249] Hu12A11.2b4 was evaluated for biological activity in a
number of in vitro human cell assays described in Example 1. As
shown in Table 5-1, Hu12A11.2b4 demonstrated 180 pM binding to PD-1
in CD4+ T cells. In addition, the anti-PD-1 activity of Hu12A11.2b4
was shown to be mediated at least in part by its ability to block
PD-L1 or PD-L2 interaction with PD-1 as assessed by flow cytometry.
This biological activity was consistent with the activity of
Hu12A11.2b4 in recombinant Jurkat T cells expressing firefly
luciferase gene under the control of NFAT response elements with
constitutive expression of human PD-1 (Jurkat NFAT assay).
TABLE-US-00014 TABLE 5-1 Biological Activity of Exemplary anti-PD-1
Antibodies CD4+ T-cell PD-L1 PD-L2 binding blocking blocking Jurkat
NFAT Antibody EC.sub.50 (pM) (.mu.g/mL) (.mu.g/mL) EC.sub.50
(.mu.g/mL) Nivolumab 180 0.106 0.233 15 .+-. 20 Hu12A11.2b4 180
0.045 0.132 4.5 .+-. 6.5
[0250] Anti-PD-1 antibody Hu12A11.2b4 further demonstrated an
enhancement in immunological response in in vitro assays. As shown
in FIG. 4A, treatment with 10 .mu.g/mL of Hu12A11.2b4 in mixed
leukocyte cultures effected a significant increase in IL-2 as well
as an IFN-.gamma. increase of about 7.4-fold. FIG. 4B shows that
Hu12A11.2b4 exhibited a tetanus toxoid recall response of about
6-fold over no antibody treatment at 10 .mu.g/mL, with an
EC.sub.50=161 ng/mL.
[0251] The in vitro biological activity observed for Hu12A11.2b4
was similar to that measured for the nivolumab used in Example 4.
With respect to immunological response, the comparison antibody
nivolumab exhibited an IFN-.gamma. increase of about 5.6-fold in
MLR, and a tetanus toxoid recall response of about 6-fold over no
antibody treatment at 10 .mu.g/mL, with an EC.sub.50=218 ng/mL.
[0252] All publications, patents, patent applications and other
documents cited in this application are hereby incorporated by
reference in their entireties for all purposes to the same extent
as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by
reference for all purposes.
[0253] While various specific embodiments have been illustrated and
described, it will be appreciated that various changes can be made
without departing from the spirit and scope of the invention(s).
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 61 <210> SEQ ID NO 1 <211> LENGTH: 288 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
1 Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1
5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro
Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr
Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr
Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser
Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser
Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro
Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg
Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala
Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135
140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val
Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val
Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly
Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala
Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln
Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val
Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255
Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260
265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro
Leu 275 280 285 <210> SEQ ID NO 2 <211> LENGTH: 288
<212> TYPE: PRT <213> ORGANISM: Mus sp. <400>
SEQUENCE: 2 Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val
Leu Gln 1 5 10 15 Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro
Asn Gly Pro Trp 20 25 30 Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu
Thr Val Ser Glu Gly Ala 35 40 45 Asn Ala Thr Phe Thr Cys Ser Leu
Ser Asn Trp Ser Glu Asp Leu Met 50 55 60 Leu Asn Trp Asn Arg Leu
Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala 65 70 75 80 Ala Phe Cys Asn
Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln 85 90 95 Ile Ile
Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp 100 105 110
Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115
120 125 His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val
Val 130 135 140 Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser
Pro Ser Pro 145 150 155 160 Lys Pro Glu Gly Arg Phe Gln Gly Met Val
Ile Gly Ile Met Ser Ala 165 170 175 Leu Val Gly Ile Pro Val Leu Leu
Leu Leu Ala Trp Ala Leu Ala Val 180 185 190 Phe Cys Ser Thr Ser Met
Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp 195 200 205 Asp Thr Leu Lys
Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala 210 215 220 Tyr Glu
Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235
240 Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly
245 250 255 Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly
Leu Gln 260 265 270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys
Ser Trp Pro Leu 275 280 285 <210> SEQ ID NO 3 <211>
LENGTH: 290 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 3 Met Arg Ile Phe Ala Val Phe Ile Phe
Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val
Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr
Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala
Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile
Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70
75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly
Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala
Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr
Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile
Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His
Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu
Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190
Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195
200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu
Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu
Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys
Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly
Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn
Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290
<210> SEQ ID NO 4 <211> LENGTH: 262 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Leu
Gln Leu His Gln Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys 1 5 10
15 Glu Leu Tyr Ile Ile Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn
20 25 30 Phe Asp Thr Gly Ser His Val Asn Leu Gly Ala Ile Thr Ala
Ser Leu 35 40 45 Gln Lys Val Glu Asn Asp Thr Ser Pro His Arg Glu
Arg Ala Thr Leu 50 55 60 Leu Glu Glu Gln Leu Pro Leu Gly Lys Ala
Ser Phe His Ile Pro Gln 65 70 75 80 Val Gln Val Arg Asp Glu Gly Gln
Tyr Gln Cys Ile Ile Ile Tyr Gly 85 90 95 Val Ala Trp Asp Tyr Lys
Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr 100 105 110 Arg Lys Ile Asn
Thr His Ile Leu Lys Val Pro Glu Thr Asp Glu Val 115 120 125 Glu Leu
Thr Cys Gln Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp 130 135 140
Pro Asn Val Ser Val Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu 145
150 155 160 Gly Leu Tyr Gln Val Thr Ser Val Leu Arg Leu Lys Pro Pro
Pro Gly 165 170 175 Arg Asn Phe Ser Cys Val Phe Trp Asn Thr His Val
Arg Glu Leu Thr 180 185 190 Leu Ala Ser Ile Asp Leu Gln Ser Gln Met
Glu Pro Arg Thr His Pro 195 200 205 Thr Trp Leu Leu His Ile Phe Ile
Pro Ser Cys Ile Ile Ala Phe Ile 210 215 220 Phe Ile Ala Thr Val Ile
Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu 225 230 235 240 Tyr Ser Ser
Lys Asp Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg 245 250 255 Glu
Val Asn Ser Ala Ile 260 <210> SEQ ID NO 5 <211> LENGTH:
15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 5 Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
<210> SEQ ID NO 6 <400> SEQUENCE: 6 000 <210> SEQ
ID NO 7 <400> SEQUENCE: 7 000 <210> SEQ ID NO 8
<400> SEQUENCE: 8 000 <210> SEQ ID NO 9 <400>
SEQUENCE: 9 000 <210> SEQ ID NO 10 <400> SEQUENCE: 10
000 <210> SEQ ID NO 11 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 11 Gly Tyr Thr
Phe Thr His Tyr Gly Met Asn 1 5 10 <210> SEQ ID NO 12
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 12 Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr
Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly <210> SEQ ID NO 13
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 13 Glu Gly Glu Gly Leu Gly Phe Gly Asp 1 5
<210> SEQ ID NO 14 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 14 Arg Ser Ser Gln Ser Ile
Val His Ser His Gly Asp Thr Tyr Leu Glu 1 5 10 15 <210> SEQ
ID NO 15 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 15 Lys Val Ser Asn Arg Phe Ser 1 5
<210> SEQ ID NO 16 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 16 Phe Gln Gly Ser His Ile
Pro Val Thr 1 5 <210> SEQ ID NO 17 <400> SEQUENCE: 17
000 <210> SEQ ID NO 18 <400> SEQUENCE: 18 000
<210> SEQ ID NO 19 <400> SEQUENCE: 19 000 <210>
SEQ ID NO 20 <400> SEQUENCE: 20 000 <210> SEQ ID NO 21
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 21 Glu Gly Glu Gly Ile Gly Phe Gly Asp 1 5
<210> SEQ ID NO 22 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 22 Glu Gly Glu Gly Val Gly
Phe Gly Asp 1 5 <210> SEQ ID NO 23 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 23 Glu
Gly Glu Gly Met Gly Phe Gly Asp 1 5 <210> SEQ ID NO 24
<400> SEQUENCE: 24 000 <210> SEQ ID NO 25 <400>
SEQUENCE: 25 000 <210> SEQ ID NO 26 <400> SEQUENCE: 26
000 <210> SEQ ID NO 27 <400> SEQUENCE: 27 000
<210> SEQ ID NO 28 <400> SEQUENCE: 28 000 <210>
SEQ ID NO 29 <400> SEQUENCE: 29 000 <210> SEQ ID NO 30
<400> SEQUENCE: 30 000 <210> SEQ ID NO 31 <211>
LENGTH: 118 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 31 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu
Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Met Ile Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Lys
Gln Ala Pro Gly Lys Gly Leu Lys Trp Val 35 40 45 Gly Trp Val Asn
Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly
Arg Leu Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Thr Arg Glu Gly Glu Gly Met Gly Phe Gly Asp Trp Gly Gln Gly
Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> SEQ ID NO
32 <211> LENGTH: 118 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 32 Glu Ile Gln Leu Val Gln Ser
Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly Met Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly
Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55
60 Lys Gly Arg Leu Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Met Gly Phe Gly Asp Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210>
SEQ ID NO 33 <211> LENGTH: 118 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 33 Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Met Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 34 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 34 Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Ile Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 35 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 35 Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Val Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 36 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 36 Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Leu Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 37 <400> SEQUENCE: 37 000 <210>
SEQ ID NO 38 <400> SEQUENCE: 38 000 <210> SEQ ID NO 39
<400> SEQUENCE: 39 000 <210> SEQ ID NO 40 <400>
SEQUENCE: 40 000 <210> SEQ ID NO 41 <211> LENGTH: 112
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 41
Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5
10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His
Ser 20 25 30 His Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Ile Pro Val
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210>
SEQ ID NO 42 <211> LENGTH: 112 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 42 Asp Val Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 His
Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 Ser His Ile Pro Val Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 <210> SEQ ID NO 43
<400> SEQUENCE: 43 000 <210> SEQ ID NO 44 <400>
SEQUENCE: 44 000 <210> SEQ ID NO 45 <400> SEQUENCE: 45
000 <210> SEQ ID NO 46 <400> SEQUENCE: 46 000
<210> SEQ ID NO 47 <400> SEQUENCE: 47 000 <210>
SEQ ID NO 48 <400> SEQUENCE: 48 000 <210> SEQ ID NO 49
<400> SEQUENCE: 49 000 <210> SEQ ID NO 50 <400>
SEQUENCE: 50 000 <210> SEQ ID NO 51 <211> LENGTH: 448
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 51
Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5
10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His
Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Val 35 40 45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr
Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp
Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu
Gly Leu Gly Phe Gly Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385
390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 445 <210> SEQ ID NO 52
<211> LENGTH: 447 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 52 Glu Ile Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly Met Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly
Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55
60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Leu Gly Phe Gly Asp Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310
315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 <210> SEQ ID NO 53 <400> SEQUENCE: 53 000
<210> SEQ ID NO 54 <400> SEQUENCE: 54 000 <210>
SEQ ID NO 55 <400> SEQUENCE: 55 000 <210> SEQ ID NO 56
<400> SEQUENCE: 56 000 <210> SEQ ID NO 57 <400>
SEQUENCE: 57 000 <210> SEQ ID NO 58 <400> SEQUENCE: 58
000 <210> SEQ ID NO 59 <400> SEQUENCE: 59 000
<210> SEQ ID NO 60 <400> SEQUENCE: 60 000 <210>
SEQ ID NO 61 <211> LENGTH: 219 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 61 Asp Val Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 His
Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 Ser His Ile Pro Val Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 61 <210>
SEQ ID NO 1 <211> LENGTH: 288 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Met Gln
Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20
25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly
Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu
Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln
Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro
Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly
Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp
Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys
Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr
Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150
155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly
Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala
Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg
Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro
Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg
Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu
Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly
Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270
Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275
280 285 <210> SEQ ID NO 2 <211> LENGTH: 288 <212>
TYPE: PRT <213> ORGANISM: Mus sp. <400> SEQUENCE: 2 Met
Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln 1 5 10
15 Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp
20 25 30 Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu
Gly Ala 35 40 45 Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser
Glu Asp Leu Met 50 55 60 Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn
Gln Thr Glu Lys Gln Ala 65 70 75 80 Ala Phe Cys Asn Gly Leu Ser Gln
Pro Val Gln Asp Ala Arg Phe Gln 85 90 95 Ile Ile Gln Leu Pro Asn
Arg His Asp Phe His Met Asn Ile Leu Asp 100 105 110 Thr Arg Arg Asn
Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 His Pro
Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val 130 135 140
Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro 145
150 155 160 Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met
Ser Ala 165 170 175 Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp
Ala Leu Ala Val 180 185 190 Phe Cys Ser Thr Ser Met Ser Glu Ala Arg
Gly Ala Gly Ser Lys Asp 195 200 205 Asp Thr Leu Lys Glu Glu Pro Ser
Ala Ala Pro Val Pro Ser Val Ala 210 215 220 Tyr Glu Glu Leu Asp Phe
Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235 240 Thr Ala Cys
Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly 245 250 255 Leu
Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln 260 265
270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285 <210> SEQ ID NO 3 <211> LENGTH: 290
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 3 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr
Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys
Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu
Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu
Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe
Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr
Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90
95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile
Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg
Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr
Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp
Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr
Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser
Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys
Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215
220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His
225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val
Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met
Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys
Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 <210> SEQ
ID NO 4 <211> LENGTH: 262 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 4 Leu Gln Leu His Gln
Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys 1 5 10 15 Glu Leu Tyr
Ile Ile Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn 20 25 30 Phe
Asp Thr Gly Ser His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu 35 40
45 Gln Lys Val Glu Asn Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu
50 55 60 Leu Glu Glu Gln Leu Pro Leu Gly Lys Ala Ser Phe His Ile
Pro Gln 65 70 75 80 Val Gln Val Arg Asp Glu Gly Gln Tyr Gln Cys Ile
Ile Ile Tyr Gly 85 90 95 Val Ala Trp Asp Tyr Lys Tyr Leu Thr Leu
Lys Val Lys Ala Ser Tyr 100 105 110 Arg Lys Ile Asn Thr His Ile Leu
Lys Val Pro Glu Thr Asp Glu Val 115 120 125 Glu Leu Thr Cys Gln Ala
Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp 130 135 140 Pro Asn Val Ser
Val Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu 145 150 155 160 Gly
Leu Tyr Gln Val Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly 165 170
175 Arg Asn Phe Ser Cys Val Phe Trp Asn Thr His Val Arg Glu Leu Thr
180 185 190 Leu Ala Ser Ile Asp Leu Gln Ser Gln Met Glu Pro Arg Thr
His Pro 195 200 205 Thr Trp Leu Leu His Ile Phe Ile Pro Ser Cys Ile
Ile Ala Phe Ile 210 215 220 Phe Ile Ala Thr Val Ile Ala Leu Arg Lys
Gln Leu Cys Gln Lys Leu 225 230 235 240 Tyr Ser Ser Lys Asp Thr Thr
Lys Arg Pro Val Thr Thr Thr Lys Arg 245 250 255
Glu Val Asn Ser Ala Ile 260 <210> SEQ ID NO 5 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 1 5 10 15 <210> SEQ ID NO 6 <400> SEQUENCE: 6 000
<210> SEQ ID NO 7 <400> SEQUENCE: 7 000 <210> SEQ
ID NO 8 <400> SEQUENCE: 8 000 <210> SEQ ID NO 9
<400> SEQUENCE: 9 000 <210> SEQ ID NO 10 <400>
SEQUENCE: 10 000 <210> SEQ ID NO 11 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 11 Gly
Tyr Thr Phe Thr His Tyr Gly Met Asn 1 5 10 <210> SEQ ID NO 12
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 12 Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr
Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly <210> SEQ ID NO 13
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 13 Glu Gly Glu Gly Leu Gly Phe Gly Asp 1 5
<210> SEQ ID NO 14 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 14 Arg Ser Ser Gln Ser Ile
Val His Ser His Gly Asp Thr Tyr Leu Glu 1 5 10 15 <210> SEQ
ID NO 15 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 15 Lys Val Ser Asn Arg Phe Ser 1 5
<210> SEQ ID NO 16 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 16 Phe Gln Gly Ser His Ile
Pro Val Thr 1 5 <210> SEQ ID NO 17 <400> SEQUENCE: 17
000 <210> SEQ ID NO 18 <400> SEQUENCE: 18 000
<210> SEQ ID NO 19 <400> SEQUENCE: 19 000 <210>
SEQ ID NO 20 <400> SEQUENCE: 20 000 <210> SEQ ID NO 21
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 21 Glu Gly Glu Gly Ile Gly Phe Gly Asp 1 5
<210> SEQ ID NO 22 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 22 Glu Gly Glu Gly Val Gly
Phe Gly Asp 1 5 <210> SEQ ID NO 23 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 23 Glu
Gly Glu Gly Met Gly Phe Gly Asp 1 5 <210> SEQ ID NO 24
<400> SEQUENCE: 24 000 <210> SEQ ID NO 25 <400>
SEQUENCE: 25 000 <210> SEQ ID NO 26 <400> SEQUENCE: 26
000 <210> SEQ ID NO 27 <400> SEQUENCE: 27 000
<210> SEQ ID NO 28 <400> SEQUENCE: 28
000 <210> SEQ ID NO 29 <400> SEQUENCE: 29 000
<210> SEQ ID NO 30 <400> SEQUENCE: 30 000 <210>
SEQ ID NO 31 <211> LENGTH: 118 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 31 Gln Ile Gln Leu Val
Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Met
Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr
Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Thr Arg Glu Gly Glu Gly Met Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115
<210> SEQ ID NO 32 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 32 Glu Ile Gln Leu Val
Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Val Phe Ser Leu Asp Thr Ser Val Ser Thr
Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Met Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 33 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 33 Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Met Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 34 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 34 Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Ile Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 35 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 35 Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Val Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 36 <211> LENGTH: 118 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 36 Glu Ile Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Leu Gly Phe Gly
Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
<210> SEQ ID NO 37 <400> SEQUENCE: 37 000 <210>
SEQ ID NO 38 <400> SEQUENCE: 38 000 <210> SEQ ID NO 39
<400> SEQUENCE: 39
000 <210> SEQ ID NO 40 <400> SEQUENCE: 40 000
<210> SEQ ID NO 41 <211> LENGTH: 112 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 41 Asp Val Leu Met Thr
Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 His
Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 Ser His Ile Pro Val Thr Phe Gly Ala Gly
Thr Lys Leu Glu Ile Lys 100 105 110 <210> SEQ ID NO 42
<211> LENGTH: 112 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 42 Asp Val Val Met Thr Gln Ser
Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 His Gly Asp
Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Ile Pro Val Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110 <210> SEQ ID NO 43 <400>
SEQUENCE: 43 000 <210> SEQ ID NO 44 <400> SEQUENCE: 44
000 <210> SEQ ID NO 45 <400> SEQUENCE: 45 000
<210> SEQ ID NO 46 <400> SEQUENCE: 46 000 <210>
SEQ ID NO 47 <400> SEQUENCE: 47 000 <210> SEQ ID NO 48
<400> SEQUENCE: 48 000 <210> SEQ ID NO 49 <400>
SEQUENCE: 49 000 <210> SEQ ID NO 50 <400> SEQUENCE: 50
000 <210> SEQ ID NO 51 <211> LENGTH: 448 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 51 Glu Ile
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20
25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Val 35 40 45 Gly Trp Val Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Asp Asp Phe 50 55 60 Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Leu
Gly Phe Gly Asp Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150
155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395
400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 445 <210> SEQ ID NO 52 <211>
LENGTH: 447 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 52 Glu Ile Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly Trp Val Asn
Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60
Lys Gly Arg Leu Thr Phe Thr Leu Asp Thr Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Arg Glu Gly Glu Gly Leu Gly Phe Gly Asp Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310
315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 <210> SEQ ID NO 53 <400> SEQUENCE: 53 000
<210> SEQ ID NO 54 <400> SEQUENCE: 54 000 <210>
SEQ ID NO 55 <400> SEQUENCE: 55 000 <210> SEQ ID NO 56
<400> SEQUENCE: 56 000 <210> SEQ ID NO 57 <400>
SEQUENCE: 57 000 <210> SEQ ID NO 58 <400> SEQUENCE: 58
000 <210> SEQ ID NO 59 <400> SEQUENCE: 59 000
<210> SEQ ID NO 60 <400> SEQUENCE: 60 000 <210>
SEQ ID NO 61 <211> LENGTH: 219 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 61 Asp Val Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 His
Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 Ser His Ile Pro Val Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215
* * * * *