HK40016537B - Anti-pd-1 antibodies and uses thereof - Google Patents

Description

Anti-PD-1 antibodies and their uses

Background Technology

The cDNA of programmed cell death 1 (PD-1) was isolated in 1992 from mouse T-cell hybridomas and apoptotic hematopoietic progenitor cell lines. Genetic ablation studies have shown that PD-1 deficiency leads to different autoimmune phenotypes in various mouse strains. PD-1-deficient allogeneic T cells with transgenic T-cell receptors (TCRs) exhibit enhanced responses to alloantigens, suggesting that PD-1 on T cells plays a negative regulatory role in antigen response.

Several studies have contributed to the discovery of molecules that interact with PD-1. In 1999, B7 homolog 1 (B7-H1, also known as programmed death ligand 1 [PD-L1]), based on its homology with B7 family molecules, was independently identified, using molecular cloning and human expression sequence tag databases, and showed that B7-H1 functions as an inhibitor of human T cell responses in vitro. A year later, the labs of Freeman, Wood, and Honjo showed that B7-H1 (hereinafter referred to as PD-L1) is a binding and functional chaperone of PD-1, converging these two independent lines of research. Next, it was determined that PD-L1-deficient mice (PD-L1 KO mice) are susceptible to induced autoimmune diseases, although this mouse strain does not spontaneously develop such diseases. Subsequently, it was elucidated that the PD-L1/PD-1 interaction plays a dominant role in suppressing T cell responses in vivo, particularly in the tumor microenvironment.

Preliminary studies have shown that tumor-associated PD-L1 promotes apoptosis of activated T cells (Dong H. et al., Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immunoevasion. Nature medicine. 2002; 8(8):793-800), and also stimulates the production of IL-10 in human peripheral blood T cells (Dong H. et al., B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nature medicine. 1999; 5(12):1365-9) to mediate immunosuppression. The role of PD-L1 in immunosuppression is now known to be much more complex. In addition to T-cell apoptosis and IL-10 induction, PD-L1 can also cause T-cell dysfunction through multiple mechanisms. In vitro and in vivo, the PD pathway has also been shown to promote T-cell anergy.

Recently, the FDA approved two PD-1 mAbs for the treatment of human cancers: one from Bristol-Myers Squibb (OpDivo, nivolumab, MDx1106, BMS-936558, ONO-4538) and the other from Merck (Keytruda, pembrolizumab, lambolizumab, MK-3475). Furthermore, multiple PD-1 or PD-L1 mAbs are actively being developed in hundreds of clinical trials involving thousands of patients. To date, anti-PD therapy has demonstrated significant clinical benefits by inducing regression in advanced and metastatic tumors and improving survival. More importantly, anti-PD therapy can have durable effects, tolerable toxicities, and shows applicability to a broad spectrum of cancer types, particularly solid tumors. These clinical findings further confirm the principles of PD pathway blockade and place anti-PD therapy in a unique category distinct from personalized or tumor type-specific therapies. Due to its unique and non-overlapping mechanisms compared to other cancer treatments, anti-PD therapy is being combined with almost all cancer treatments in an attempt to further expand therapeutic efficacy. In addition to combining it with various cancer immunotherapies (such as cancer vaccines, co-stimulatory and co-inhibitory antibodies, and adoptive cell therapy), various clinical trials have been initiated to combine anti-PD therapy with chemotherapy, radiotherapy, and targeted therapy.

Anti-PD therapy plays a central role in immunotherapy for human cancers, particularly solid tumors. This therapy differs from previous immunotherapies—whose primary aim was to enhance the systemic immune response or generate new immunity against cancer; instead, anti-PD therapy modulates the immune response at the tumor site, targets tumor-induced immune deficiencies, and repairs ongoing immune responses. While the clinical success of anti-PD therapy in treating various human cancers has validated this approach, we are still investigating this pathway and the associated immune responses, which will contribute to the discovery and design of new clinically applicable cancer immunotherapies.

Summary of the Invention

This disclosure provides an anti-PD-1 antibody that exhibits excellent binding and inhibitory activity against the PD-1 protein. One of the tested PD-1 antibodies even showed stronger binding activity than two regulatory-approved anti-PD-1 antibody products.

Therefore, according to one embodiment of the present disclosure, an isolated antibody or fragment thereof is provided, said isolated antibody or fragment thereof being specific for human programmed cell death protein 1 (PD-1), said antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, said light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3, wherein said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the group consisting of: (a) HCDR1: GFTFSSYT (SEQ ID NO:1), HCDR2: ISHGGGDT (SEQ ID NO:2), HCDR3: ARHSGYERGYYYVMDY (SEQ ID NO:3), LCDR1: ESVDYYGFSF (SEQ ID NO:4), LCDR2: AAS (SEQ ID NO:5), LCDR3: QQSK EVPW(SEQ ID NO:6); (b)HCDR1:GYTFTSYT(SEQ ID NO:7), HCDR2:INPTTGYT(SEQ ID NO:8), HCDR3:ARDDAYYS GY(SEQ ID NO:9), LCDR1:ENIYSNL(SEQ ID NO:10), LCDR2:AAK(SEQ ID NO:11), LCDR3:QHFWGTPWT(SEQ IDNO :12); and (c) HCDR1: GFAFSSYD (SEQ ID NO: 13), HCDR2: ITIGGGTT (SEQ ID NO: 14), HCDR3: AHRRYDYFAMDN (SEQ I D NO:15), LCDR1:ENVDNYGINF(SEQ ID NO:16), LCDR2:VSS(SEQ ID NO:17), LCDR3:QQSKDVPW(SEQ ID NO:18).

In some embodiments, the antibodies or fragments disclosed herein further include a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a κ chain or a λ chain constant region.

In some embodiments, the antibody or fragment thereof may be an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody.

In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39. In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, or an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45.

In another embodiment, this disclosure provides an isolated antibody or fragment thereof that is specific for human programmed cell death protein 1 (PD-1), wherein the antibody or fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the group consisting of... (a)HCDR1:GTFFSSYT(SEQ ID NO:1), HCDR2:ISHGGGDT(SEQ ID NO:2), HCDR3:ARHSGYERGYYYVMDY(SEQ ID NO:3), LCDR1: ESVDYYGFSF(SEQ ID NO:4), LCDR2:AAS(SEQ ID NO:5), LCDR3:QQSKEVPW(SEQ ID NO:6); (b)HCDR1:GYTFTSYT(SEQ ID NO :7), HCDR2:INPTTGYT(SEQ ID NO:8), HCDR3:ARDDAYYSGY(SEQ ID NO:9), LCDR1:ENIYSNL(SEQ ID NO:10), LCDR2:AAK(SE Q ID NO:11), LCDR3:QHFWGTPWT(SEQ ID NO:12); (c)HCDR1:GFAFSSYD(SEQ ID NO:13), HCDR2:ITIGGGTT(SEQ ID NO:14), HCDR3:ARHRYDYFAMDN (SEQ ID NO:15), LCDR1:ENVDNYGINF (SEQ ID NO:16), LCDR2:VSS (SEQ ID NO:17), LCDR3:QQSKDVPW (SEQ ID NO:18); and (d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in (a)-(c), but at least one of them contains one, two or three amino acid additions, deletions, conserved amino acid substitutions or combinations thereof.

In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in any one of (a)-(c), but one of them includes a conserved amino acid substitution. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in any one of (a)-(c), but two of them each contain a conserved amino acid substitution. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in any one of (a)-(c), but three of them each contain a conserved amino acid substitution.

One embodiment also provides a composition comprising an antibody or a fragment thereof of the present disclosure and a pharmaceutically acceptable carrier. In another embodiment, an isolated cell is also provided containing one or more polynucleotides encoding said antibody or a fragment thereof.

Uses and methods are also provided. In one embodiment, the use of the antibody or a fragment thereof of this disclosure in the preparation of a medicament for treating cancer is provided. The cancer may be selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. Methods of treating cancer in patients in need are also provided, comprising administering the antibody or a fragment thereof of this disclosure to the patient.

In another embodiment, this disclosure provides a method for treating cancer or infection in a patient in need, comprising (a) treating cells in vitro with an antibody or fragment thereof disclosed herein; and (b) administering the treated cells to the patient. In some embodiments, the cells are T cells.

In another embodiment, the use of any antibody or fragment thereof disclosed herein in the preparation of a medicament for treating an infection is provided. In some embodiments, the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

In another embodiment, the use of the antibody or fragment thereof disclosed herein in the preparation of a medicament for treating immune disorders is provided. In some embodiments, the immune disorder is selected from the group consisting of: infection, infection-related endotoxin shock, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peronis disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune-mediated central and extracellular disorders. Inflammatory diseases of the peripheral nervous system, multiple sclerosis, lupus and Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrotic alveolitis, Graf's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, pancreatitis, trauma, graft-versus-host disease, transplant rejection, ischemic diseases, myocardial infarction, atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypoacidity, and infertility associated with maternal-fetal intolerance.

Brief description of the attached diagram

Figure 1 shows that all five hPD-1 mAb isotypes can bind to hPD-1 with high specificity.

Figure 2 shows that anti-hPD-1 does not bind to hB7-1, hPD-L1, hB7-H3, hB7-H4, and hCD137.

Figure 3 shows that hPD-1 mAb can bind to both human and cynomolgus monkey PD-1 protein, but does not show cross-binding with mPD-1.

Figure 4 shows that hPD-1 mAb has a dose-dependent blocking effect on the binding of hPD-1 to hPD-L1.

Figure 5 shows the aborting and blocking effects of hPD-1 mAbs observed in a competitive binding environment.

Figure 6 shows the gel electrophoresis analysis results confirming the RACE products.

Figure 7 shows (A) the ability of recombinant DNA antibodies to bind to PD-1, and (B) their blocking effect on the binding ability of PD-1 and PD-L1.

Figure 8 shows that the nine humanized antibodies exhibit various binding affinities to PD-1, including those higher and lower than those of the parent antibodies.

Figure 9 shows that the humanized antibody can block the binding ability of PD-1 and PD-L1.

Figure 10 shows that the humanized antibody can block the binding ability of PD-1 and PD-L2.

Figure 11 shows that humanized mAb enhances the in vitro cytotoxicity of allogeneic CD8+ CTL cells against cancer cells.

Figure 12 shows the proliferation response of MLR against the antihPD-1 antibody.

Figure 13 shows the expression characteristics of IL-2 and IFNγ in the supernatant of MLR culture.

Figure 14 shows that PD-1 mAb inhibits the expression of PD-L1 on lymphocytes.

Figure 15 shows the in vivo antitumor activity of the humanized PD-1 antibody.

Figure 16 shows a comparison of antibody binding affinity and kinetics.

Figure 17 shows a comparison of PD-1 antibodies in terms of PD-1/PD-L1 blockade.

Figure 18 shows a comparison of mAbs in enhancing the in vitro cytotoxicity of allogeneic CD8+ CTL cells against cancer cells.

Figure 19 shows the binding of the test product to hPD-1 or mPD-1 (ELISA assay).

Figure 20 shows the binding of the test product to CHOK1 cells expressing hPD-1 or cPD-1 using flow cytometry.

Figure 21 shows the blocking activity of the test product against hPD-L1 (left) and hPD-L2 (right) binding to CHOK1 cells expressing hPD-1.

Figure 22 shows the levels of IL-2 (left) and IFN-γ (right) in human MLR assays.

Figure 23 shows the IFN-γ level in the co-culture assay of engineered tumors and T cells.

Figure 24 shows epitope binding based on ELISA results (top) and a schematic diagram of epitope overlap for different test articles (bottom).

Figure 25 shows the binding of nivolumab (top) or TY101-04-T3-05 antibody (bottom) to recombinant hPD-1 at low fixation levels (60 RU, left) and high fixation levels (960 RU, right).

Detailed Implementation

definition

It should be noted that the term “a” or “an” refers to one or more of that entity; for example, “an antibody” is understood to represent one or more antibodies. Therefore, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably in this document.

As used herein, the term "polypeptide" is intended to include both the singular and plural forms of "polypeptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, without specifying a particular length of the product. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" may be used in place of any of these terms or interchangeably with them. The term "polypeptide" also means a product of post-expression modifications of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or modification by amino acids not naturally occurring. Polypeptides may be derived from natural biological sources or produced by recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be produced in any manner, including by chemical synthesis.

As used herein, the term "isolated" refers to a molecule, for the purposes of cell and nucleic acid (such as DNA or RNA), that is separate from other DNA or RNA present in the natural source of the macromolecule. The term "isolated" also refers to nucleic acids or peptides that, when produced by recombinant DNA technology, are substantially free of cellular material, viral material, or culture medium, or, when chemically synthesized, are substantially free of chemical precursors or other chemical substances. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and are not found in their natural state. The term "isolated" is also used herein to refer to cells or peptides isolated from other cellular proteins or tissues. Isolated peptides are intended to encompass both purified and recombinant peptides.

As used herein, the term “recombinant” when referring to a polypeptide or polynucleotide means a form of polypeptide or polynucleotide that does not exist naturally, and non-limiting examples of which can be constructed by combining polynucleotides or polypeptides that do not normally coexist.

"Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When positions in the compared sequences are occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity with one of the sequences in this disclosure, although preferably less than 25%.

"Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence having a specific percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that, in a comparison of the two sequences, that percentage of bases (or amino groups) are identical when aligned. This alignment and percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al., (2007) Current Protocols in Molecular Biology. Preferably, alignment uses default parameters. One alignment program is BLAST, using default parameters. Specifically, the procedures are BLASTN and BLASTP, using the following default parameters: Genetic Code = Standard; Filter = None; Chain = Two strands; Truncation = 60; Expected = 10; Matrix = BLOSUM62; Description = 50 sequences; Selection Method = High Score; Database = Non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translated Sequences+SwissProtein+SPupdate+PIR. Bioequivalent polynucleotides are those polynucleotides that have the aforementioned specified percentage of homology and encode polypeptides with the same or similar biological activities.

The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that shares a degree of homology or sequence identity with the nucleotide sequence of the nucleic acid or its complement. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that shares a degree of homology with the nucleic acid or its complement. In one aspect, a homolog of a nucleic acid is capable of hybridizing with a nucleic acid or its complement. Similarly, "equivalent polypeptide" refers to a polypeptide that shares a degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, or combinations thereof compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

Hybridization reactions can be carried out under various “strict” conditions. Typically, low-strict hybridization reactions are performed at approximately 40°C in solutions with approximately 10x SSC, or equivalent ionic strength/temperature. Moderately strict hybridization reactions are typically performed at approximately 6x SSC at approximately 50°C, and high-strict hybridization reactions are typically performed at approximately 1x SSC at approximately 60°C. Hybridization reactions can also be carried out under “physiological conditions” well known to those skilled in the art. A non-limiting example of physiological conditions is temperature, ionic strength, pH, and ^Mg²⁺ concentration commonly found in cells.

Polynucleotides are composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), with uracil (U) replacing thymine in the case of RNA polynucleotides. Therefore, the term "polynucleotide sequence" is the letter representation of a polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one form of a gene or a portion thereof. A portion of a gene that has at least two different forms, i.e., two different nucleotide sequences, is called a "polymorphic region of the gene." A polymorphic region can be a single nucleotide whose identity differs across different alleles.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure can be conferred before or after the assembly of the polynucleotide, if present. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the polynucleotide disclosed herein includes both double-stranded forms and either of two complementary single-stranded forms known or intended to form double-stranded forms.

The term "encoding" applied to polynucleotides means that if a polynucleotide, in its native state or manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce mRNA of a polypeptide and/or fragments thereof, then the polynucleotide is said to "encode" a polypeptide. The antisense strand is the complement of such nucleic acid, from which the coding sequence can be inferred.

As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody or any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein or peptide comprising at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Examples of such molecules include, but are not limited to, the complementarity-determining region (CDR), variable region, constant region, frame (FR) region, or any portion thereof of the heavy or light chain or its ligand-binding moiety, or at least a portion of the binding protein.

As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spikelers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions by binding to a specific antigen to form a complex, just like an antibody.

"Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain ( _VH ) and light chain ( _VL ) of an immunoglobulin. In some aspects, these regions are linked by a short linker peptide of 10 to approximately 25 amino acids. This linker may be enriched with glycine to aid flexibility, and serine or threonine to aid solubility, and may link the N-terminus of _VH to the C-terminus of _VL , or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original immunoglobulin. scFv molecules are known in the art and described, for example, in U.S. Patent 5,892,019.

The term antibody encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will recognize that heavy chains are classified as γ, μ, α, δ, or ε, with several subclasses (such as γ1-γ4). It is the nature of this chain that determines the “class” of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgG5 , etc., are well characterized and are known to confer functional specificity. From the perspective of this disclosure, the modifications of each of these classes and isotypes are readily identifiable by those skilled in the art, and therefore, they are also covered within the scope of this disclosure. All immunoglobulin types are obviously within the scope of this disclosure, and the following discussion will generally pertain to the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000 to 70,000. These four chains are typically connected by disulfide bonds in a “Y” configuration, where the light chain starts at the “Y” opening and continues to enclose the heavy chain throughout the variable region.

The antibodies, antigen-binding peptides, variants, or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, humanized antibodies, humanized antibodies, primate-like antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab′, and F(ab′) ₂ , Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

Light chains are classified as κ or λ. Each heavy chain category can bind to either a κ or λ light chain. Generally, light and heavy chains are covalently bonded to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the “tail” portions of the two heavy chains are bonded by covalent disulfide bonds or non-covalent bonds. In heavy chains, the amino acid sequence extends from the N-terminus of the Y-configuration fork to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into multiple structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this respect, it will be recognized that the variable domains (VK) of the light chain moiety and the variable domains (VH) of the heavy chain moiety determine antigen recognition and specificity. Conversely, the constant domains (CK) of the light chain and the constant domains (CH1, CH2, or CH3) of the heavy chain confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of constant domains increases with their distance from the antigen-binding site or N-terminus of the antibody. The N-terminal portion is the variable region, while the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl termini of the heavy and light chains, respectively.

As described above, the variable region allows antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, a subset of the antibody's VK and VH domains, or complementarity-determining regions (CDRs), are combined to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites at the ends of each arm of the Y-chain. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VK chains. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered immunoglobulins based on camel immunoglobulins, the complete immunoglobulin molecule may consist only of the heavy chain, without the light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous amino acid sequences that specifically position to form the antigen-binding domain when the antibody forms its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain are called "framework" regions, exhibiting less intermolecular variability. Framework regions mostly adopt a β-sheet conformation, while CDRs form loops that connect to the β-sheet structure and, in some cases, form part of the β-sheet structure. Thus, the frame regions act as a scaffold that provides the correct orientation of the CDRs through interchain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its corresponding epitope. For any given heavy or light chain variable region, the amino acids constituting the CDR and framework regions, respectively, can be readily identified by a person skilled in the art because they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E. et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987).

Where two or more definitions of terms used and/or accepted in the art exist, the definitions of terms used herein are intended to include all such meanings unless expressly stated otherwise to the contrary. A concrete example is the use of “complementarity-determining region” (“CDR”) to describe discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described in Kabet et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J. MoI. Biol. 196:901-917 (1987) (both incorporated herein by reference in their entirety). The CDR definitions by Kabet and Chothia include overlaps or subsets of amino acid residues when compared with each other. However, any application of either definition to refer to the CDR of an antibody or its variants is within the scope of the terms defined and used herein. The appropriate amino acid residues for the CDRs covering each definition in the above-cited references are listed in the table below for comparison. The exact number of residues covering a particular CDR varies depending on the sequence and size of the CDR. Given the amino acid sequence of the antibody's variable region, those skilled in the art can routinely determine which residues constitute a particular CDR.

Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabet et al. also defined a numbering system applicable to the variable domain sequence of any antibody. Those skilled in the art can readily assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately the 31st amino acid (i.e., approximately 9 residues after the first cysteine residue), comprises approximately 5-7 amino acids, and terminates at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, comprises approximately 16-19 amino acids, and terminates at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2; comprises 3-25 amino acids; and terminates at the W-G-X-G sequence, where X is any amino acid. CDR-L1 begins at approximately the 24th residue (i.e., after the cysteine residue); comprises approximately 10-17 residues; and terminates at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1 and comprises approximately 7 residues. CDR-L3 begins at approximately the thirty-third residue following the end of CDR-L2 (i.e., after the cysteine residue); it consists of approximately 7-11 residues and terminates at sequence F or W-G-X-G, where X is any amino acid.

The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be derived from cartilaginous fish (e.g., from sharks).

As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide intended for use in this disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising both a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this disclosure comprises a polypeptide chain comprising a CH3 domain. Furthermore, antibodies intended for use in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant regions can be modified so that they differ from naturally occurring immunoglobulin molecules in terms of their amino acid sequences.

The heavy chain constant region of the antibodies disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an _IgG3 molecule. In another example, the heavy chain constant region can include a hinge region partially derived from an _IgG1 molecule and partially derived from an _IgG3 molecule. In yet another example, the heavy chain portion can include a chimeric hinge partially derived from an _IgG1 molecule and partially derived from an _IgG4 molecule.

As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region includes at least one of a constant κ domain or a constant λ domain.

A "light chain-heavy chain pair" refers to a combination of light and heavy chains that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

As previously mentioned, the subunit structures and three-dimensional conformations of the constant regions of various immunoglobulins are well known. As used herein, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" includes the first (closest to the N-terminus) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located at the N-terminus of the hinge region of the immunoglobulin heavy chain molecule.

As used herein, the term "CH2 domain" encompasses the portion of the heavy chain molecule extending from approximately residue 244 to residue 360 of the antibody using conventional numbering schemes (residues 244–360, Kabat numbering system, and residues 231–340, EU numbering system; see Kabet et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. The CH3 domain, reliably documented, extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term "hinge region" refers to the portion of the heavy chain molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be further subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4043 (1998)).

As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine includes a thiol group, which can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to positions 239 and 242 in the Kabat numbering system (positions 226 or 229, EU numbering system).

As used herein, the term "chimeric antibody" is to be understood to mean any antibody in which the immune-reacting region or site is obtained or derived from a first species, while the constant region (which may be complete, partial, or modified according to this disclosure) is obtained from a second species. In some embodiments, the target-binding region or site may be derived from a non-human source (e.g., mouse or primate), while the constant region is human.

As used in this paper, the "percentage of humanization" is calculated by subtracting the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain from the total number of amino acids, dividing that number by the total number of amino acids, and then multiplying by 100.

"Specific binding" or "specific to" generally means that an antibody binds to an epitope through its antigen-binding domain, and that this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope more readily through its antigen-binding domain than it would to a random, unrelated epitope. In this paper, the term "specificity" is used to qualitatively describe the relative affinity of an antibody for a given epitope. For example, antibody "A" might be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" could be described as binding to epitope "C" with higher specificity than the associated epitope "D."

As used herein, the term "treat" or "treatment" refers to both therapeutic actions and preventative or avoidant measures aimed at preventing or slowing (reducing) undesirable physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization of the condition (i.e., no worsening), delay or slowing of disease progression, improvement or reduction of the disease state, and remission (partial or complete), whether detectable or undetectable. "Treatment" also means extended survival compared to expected survival without treatment. People requiring treatment include those who already have a condition or condition, those who are susceptible to a condition or condition, or those who need to prevent a condition or condition.

"Subject," "individual," "animal," "patient," or "mammal" refers to any subject who requires diagnosis, prognosis, or treatment, especially mammalian subjects. Mammal subjects include humans, livestock, animals, zoo animals, sporting animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

As used herein, phrases such as “patient in need of treatment” or “subject in need of treatment” include subjects who would benefit from the administration of the antibodies or compositions disclosed herein, for example for detection, diagnosis and/or treatment, such as mammalian subjects.

Anti-PD-1 antibody

This disclosure provides anti-PD-1 antibodies with high affinity for the human PD-1 protein. The tested antibodies exhibit strong binding and inhibitory activity and can be used for therapeutic and diagnostic purposes. Furthermore, one of the humanized antibodies tested (TY101) exhibits significantly higher binding affinity than two FDA-approved anti-hPD-1 antibodies.

Therefore, one embodiment of this disclosure provides an anti-PD-1 antibody or a fragment thereof, said antibody or fragment thereof being capable of specifically binding to human programmed death 1 (PD-1) protein.

According to one embodiment of this disclosure, an antibody is provided comprising heavy chain and light chain variable regions having CDR regions of one of the CDR groups as shown in Table 1.

Table 1: Sequence of CDR region

For example, in one embodiment, an isolated antibody or fragment thereof specifically targeting human programmed cell death protein 1 (PD-1) is provided, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are HCDR1:GFTFSSYT (SEQ ID NO:1), HCDR2:ISHGGGDT (SEQ ID NO:2), HCDR3:ARHSGYERGYYYVMDY (SEQ ID NO:3), LCDR1:ESVDYYGFSF (SEQ ID NO:4), LCDR2:AAS (SEQ ID NO:5), and LCDR3:QQSKEVPW (SEQ ID NO:6).

For example, in one embodiment, an isolated antibody or fragment thereof specifically targeting human programmed cell death protein 1 (PD-1) is provided, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are HCDR1:GYTFTSYT (SEQ ID NO:7), HCDR2:INPTTGYT (SEQ ID NO:8), HCDR3:ARDDAYYSGY (SEQ ID NO:9), LCDR1:ENIYSNL (SEQ ID NO:10), LCDR2:AAK (SEQ ID NO:11), and LCDR3:QHFWGTPWT (SEQ ID NO:12).

For example, in one embodiment, an isolated antibody or fragment thereof specifically targeting human programmed cell death protein 1 (PD-1) is provided, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are HCDR1:GFAFSSYD (SEQ ID NO:13), HCDR2:ITIGGGTT (SEQ ID NO:14), HCDR3:ARHRYDYFAMDN (SEQ ID NO:15), LCDR1:ENVDNYGINF (SEQ ID NO:16), LCDR2:VSS (SEQ ID NO:17), and LCDR3:QQSKDVPW (SEQ ID NO:18).

As demonstrated in the experimental examples, antibodies containing these CDR regions, whether mouse, humanized, or chimeric, exhibit potent PD-1 binding and inhibitory activity. Further computer modeling shows that certain residues within the CDRs can be modified to preserve or improve the antibody's properties. In some embodiments, the anti-PD-1 antibodies of this disclosure include the VH and VL CDRs listed in Table 1 with one, two, or three further modifications. Such modifications may be the addition, deletion, or substitution of amino acids.

In some embodiments, the modification is a substitution at no more than one hotspot location per CDR. In some embodiments, the modification is a substitution at one, two, or three such hotspot locations. In one embodiment, the modification is a substitution at one of the hotspot locations. In some embodiments, such substitution is a conservative substitution.

"Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin peptides are preferably replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid string can be replaced with a structurally similar string with different sequences and/or compositions of members of the side chain family.

Non-limiting examples of conserved amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conserved substitution between two amino acids.

Amino acid similarity matrix

C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12  

Conservative amino acid substitution

Non-limiting examples of VH are provided in SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39. SEQ ID NO:27 is a mouse VH. SEQ ID NO:31 is a chimeric antibody VH, while SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39 are humanized.

Non-limiting examples of VLs are provided in SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45. SEQ ID NO:29 is a mouse VL. SEQ ID NO:33 is a chimeric antibody VL, while SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45 are humanized.

In some embodiments, the anti-PD-1 antibody of this disclosure comprises VH of SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39, VL of SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45, or their respective bioequivalents. A bioequivalent of VH or VL is a sequence containing the specified amino acids and having an overall sequence identity of 80%, 85%, 90%, 95%, 98%, or 99%. For example, a bioequivalent of SEQ ID NO: 27 may be a VH that has an overall sequence identity of 80%, 85%, 90%, 95%, 98%, or 99% with SEQ ID NO: 27 but retains the CDR.

Those skilled in the art will also understand that the antibodies disclosed herein can be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from the specified protein can be similar, for example, having a certain percentage of identity with the starting sequence, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

In some embodiments, the antibody includes an amino acid sequence or one or more portions that are not typically associated with the antibody. Exemplary modifications are described in more detail below. For example, the antibodies of this disclosure may contain a flexible linker sequence or may be modified to add a functional portion (e.g., a PEG, drug, toxin, or tag).

The antibodies, variants, or derivatives disclosed herein include derivatives that are modified, i.e., by covalently attaching any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, and not intended to be limiting, antibodies can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization of known protecting/blocking groups, proteolytic cleavage, or linkage to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, antibodies may contain one or more atypical amino acids.

In some implementations, antibodies may be conjugated with therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical formulations, or PEG.

Antibodies may be conjugated or fused with therapeutic agents, which may include detectable markers such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents that may be drugs or toxins, ultrasound enhancers, non-radiolabels, combinations thereof, and other such agents known in the art.

Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding peptide is then determined by detecting the presence of light emission during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, and oxalates.

Antibodies can also be detectably labeled using fluorescently emitting metals such as ^152Eu or other lanthanides. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various parts to antibodies are well known; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., eds., pp. 243-56 (Alan R. Liss, Inc. (1985)); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled Drug Delivery (2nd ed.), Robinson et al., Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., eds., pp. 475-506 (1985); “Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer”. "Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al., Academic Press, pp. 303-16 (1985), and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)).

Bifunctional molecules

PD-1 is an immune checkpoint molecule and also a tumor antigen. As a tumor antigen targeting molecule, antibodies or antigen-binding fragments specific to PD-1 can bind to second antigen-binding fragments specific to immune cells to produce bispecific antibodies.

In some embodiments, the immune cells are selected from the group consisting of: T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells. Molecules on targetable immune cells include, for example, CD3, CD16, CD19, CD28, and CD64. Other examples include PD-1, CTLA-4, LAG-3 (also known as CD233), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer cell immunoglobulin-like receptor (KIR), and CD47. Specific examples of bispecificity include, but are not limited to, PD-L1/PD-1, PD-1/LAG3, PD-1/TIGIT, and PD-1/CD47.

As immune checkpoint inhibitors, antibodies or antigen-binding fragments specific to PD-1 can bind to second antigen-binding fragments specific to tumor antigens to produce bispecific antibodies. A "tumor antigen" is an antigenic substance produced in tumor cells that triggers an immune response in the host. Tumor antigens are useful in identifying tumor cells and are potential candidates for cancer treatment. Normal proteins in the body are not antigenic. However, certain proteins are produced or overexpressed during tumorigenesis and therefore appear "exogenous" to the body. These may include normal proteins well isolated from the immune system, proteins that are usually produced in very small quantities, proteins that are typically produced only at certain developmental stages, or proteins whose structure has been modified due to mutations.

A wide range of tumor antigens are known in the art, and novel tumor antigens can be readily identified through screening. Non-limiting examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and Tenascin.

In some respects, the monovalent unit is specific for proteins overexpressed on tumor cells compared to their corresponding non-tumor cells. "Corresponding non-tumor cells" as used herein refers to non-tumor cells of the same cell type as tumor cells. It is noteworthy that this protein is not necessarily different from the tumor antigen. Non-restrictive examples include carcinoembryonic antigen (CEA), which is overexpressed in most colon, rectal, breast, lung, pancreatic, and gastrointestinal cancers; heregulin receptors (HER-2, neu, or c-erbB-2), which are commonly overexpressed in breast, ovarian, colon, lung, prostate, and cervical cancers; epidermal growth factor receptor (EGFR), which is highly expressed in a range of solid tumors, including breast, head and neck, non-small cell lung cancer, and prostate cancer; sialic acid glycoprotein receptor; transferrin receptor; and serine proteases. Serpin inhibitors of enzyme complex receptors, expressed on hepatocytes; fibroblast growth factor receptors (FGFR), overexpressed in pancreatic ductal adenocarcinoma cells; vascular endothelial growth factor receptors (VEGFR), used for anti-angiogenic gene therapy; folate receptors, selectively overexpressed in 90% of non-mucinous ovarian cancers; cell surface glycocalyx; carbohydrate receptors; and polymerase receptors, which can be used for gene delivery to respiratory epithelial cells and are an attractive means of treating lung diseases such as cystic fibrosis. Non-limiting examples of bispecificity in this regard include PD-1/EGFR, PD-1/Her2, PD-1/CD33, PD-1/CD133, PD-1/CEA, and PD-1/VEGF.

Different forms of bispecific antibodies are also provided. In some embodiments, each anti-PD-1 fragment and the second fragment are independently selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies. In some embodiments, the bispecific antibody also includes an Fc fragment.

Bifunctional molecules that include not only antibodies or antigen-binding fragments are also provided. As tumor antigen-targeting molecules, PD-1-specific antibodies or antigen-binding fragments (such as those described herein) can optionally be combined with immune cytokines or ligands via peptide linkers. The linked immune cytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. Such bifunctional molecules can combine immune checkpoint blockade effects with local immune modulation at the tumor site.

Polynucleotides encoding antibodies and methods for preparing antibodies

This disclosure also provides isolated polynucleotide or nucleic acid molecules (e.g., SEQ ID NO: 22, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46) encoding antibodies, variants, or derivatives of this disclosure. The polynucleotides of this disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, its variants, or derivatives, either on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of this disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, its variants, or derivatives, either on the same polynucleotide molecule or on separate polynucleotide molecules.

Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide of this disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and techniques described herein. For example, a fully human antibody against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such an antibody in response to antigen challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents 6,150,584; 6,458,592; and 6,420,140, which are incorporated herein by reference in their entirety.

In some embodiments, the prepared antibody does not elicit a harmful immune response in the animal to be treated, such as a human. In one embodiment, the antigen-binding peptide, its variants, or derivatives thereof are modified using techniques known in the art to reduce their immunogenicity. For example, the antibody may be humanized, primate-modified, deimmunized, or may be prepared as a chimeric antibody. These types of antibodies are derived from nonhuman antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but have lower immunogenicity in humans. This can be achieved by various methods, including (a) transplanting the entire nonhuman variable domain into a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more nonhuman complementarity-determining regions (CDRs) into a human framework and constant region, wherein key framework residues are retained or not; or (c) transplanting the entire nonhuman variable region domain but making it "invisible" by replacing surface residues with human-like segments. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762 and 6,190,370, all of which are incorporated herein by reference in their entirety.

Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term “deimmunization” includes altering antibodies to modify T-cell epitopes (see, for example, International Application Publication Nos. WO/9852976A1 and WO/0034317A2). For example, the variable heavy and light chain sequences from the starting antibody are analyzed, and a “map” of human T-cell epitopes from each V region is constructed, showing the epitopes associated with the complementarity-determining region (CDR) and the positions of other key residues within the sequence. Individual T-cell epitopes from the T-cell epitope map are analyzed to identify optional amino acid substitutions with a low risk of altering the final antibody activity. A series of optional variable heavy and light chain sequences, including combinations of amino acid substitutions, are designed, and these sequences are subsequently incorporated into a series of binding peptides. Typically, 12 to 24 variant antibodies are generated, and their binding and/or function is tested. The complete heavy and light chain genes containing the modified variable regions and human constant regions are then cloned into expression vectors, and the resulting plasmids are introduced into cell lines to produce complete antibodies. The antibodies were then compared using appropriate biochemical and bioassays, and the best variants were identified.

The binding specificity of the antigen-binding peptides disclosed herein can be determined by in vitro assays, such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

Alternatively, the single-chain units of this disclosure can be generated using the techniques described for producing single-chain units (US Patent No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989)). The single-chain units are formed by linking the heavy and light chain fragments of the Fv region via amino acid bridges, thereby producing single-chain fusion peptides. Techniques for assembling functional Fv fragments in *E. coli* can also be used (Skerra et al., Science 242:1038-1041 (1988)).

Examples of techniques that can be used to generate single-chain Fv (scFv) and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498, Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some applications, including in vivo application of antibodies in humans and in vitro assays, chimeric, humanized, or humanized antibodies are preferred. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as an antibody having a variable region derived from a mouse monoclonal antibody and a constant region of a human immunoglobulin. Methods for generating chimeric antibodies are known in the art. See, for example, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Patent Nos. 5,807,715, 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety.

Humanized antibodies are antibody molecules derived from antibodies of non-human species that bind to a desired antigen, having one or more complementarity-determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Typically, framework residues in the human framework region are replaced with corresponding residues from a CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are identified by methods known in the art, for example, by modeling the interaction between the CDR and framework residues to identify framework residues important for antigen binding, and by performing sequence comparisons to identify aberrant framework residues at specific positions. (See, for example, Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entirety.) Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR transplantation (EP 239400; PCT Publication No. WO 91/09967; U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814(1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973(1994)) and chain rearrangement (U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety).

Fully human antibodies are particularly desirable for the treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods that use antibody libraries derived from human immunoglobulin sequences. See also U.S. Patents 4,444,887 and 4,716,111; and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins but do express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be randomly or through homologous recombination introduced into mouse embryonic stem cells. Alternatively, in addition to human heavy and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be defunctionalized individually or simultaneously with the introduction of human immunoglobulin gene loci through homologous recombination. In particular, the loss of homozygosity in the JH region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then fed to produce homozygous offspring expressing human antibodies. The transgenic mice are immunized in a normal manner with a selected antigen (e.g., all or part of the desired target peptide). Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by transgenic mice rearrange during B cell differentiation, followed by class switching and somatic mutations. Therefore, this technology can be used to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies. For a review of this technology for generating human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of the techniques used to generate human antibodies and human monoclonal antibodies, and the procedures for generating such antibodies, see, for example, PCT Publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Patents 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, all of which are incorporated herein by reference in their entirety. Furthermore, companies such as Abgenix, Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) may be contacted to provide human antibodies against selected antigens using similar techniques described above.

A technique known as “guided selection” can also be used to generate fully human antibodies that recognize the selected epitope. In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope. (Jespers et al., Bio/Technology 72:899-903 (1988). See also U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety.)

In another embodiment, the DNA encoding the desired monoclonal antibody can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Isolated and subcloned hybridoma cells serve as a preferred source of this DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells, such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or otherwise non-immunoglobulin-producing myeloma cells. More specifically, the isolated DNA (which may be synthetic as described herein) can be used to clone constant and variable region sequences for antibody preparation, as described in U.S. Patent No. 5,658,570, filed January 25, 1995, by Newman et al., which is incorporated herein by reference. Essentially, this requires the extraction of RNA from the selected cells, conversion to cDNA, and PCR amplification using Ig-specific primers. Suitable primers for this purpose are also described in U.S. Patent No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibodies can be grown in relatively large numbers to provide clinical and commercial supply of immunoglobulins.

Furthermore, using conventional recombinant DNA techniques, one or more CDRs of the antigen-binding peptide of this disclosure can be inserted into a frame region, for example, into a human frame region to humanize a non-human antibody. The frame region can be a naturally occurring or common frame region, preferably a human frame region (for a list of human frame regions, see, for example, Chothia et al., J. Mol. Biol. 278:457-479 (1998)). Preferably, the polynucleotide resulting from the combination of the frame region and the CDR encodes an antibody that specifically binds to at least one epitope of the desired peptide (e.g., LIGHT). Preferably, one or more amino acid substitutions can be made within the frame region, and preferably, said amino acid substitutions enhance the binding of the antibody to its antigen. Additionally, these methods can be used to substitute or delete amino acids of one or more variable region cysteine residues involved in intrachain disulfide bonds to produce antibody molecules lacking one or more intrachain disulfide bonds. Other modifications of the polynucleotide are also covered in this disclosure and are within the scope of the art.

Furthermore, techniques for developing "chimeric antibodies" can be used (Morrison et al., Proc. Natl. Acad. Sci. USA: 851-855 (1984); Neuberger et al., Nature 372: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)), which involves splicing together a gene from a mouse antibody molecule with appropriate antigen specificity and a gene from a human antibody molecule with appropriate biological activity. As used herein, a chimeric antibody is a molecule in which different parts originate from different animal species, such as those molecules having a variable region derived from a mouse monoclonal antibody and a constant region from a human immunoglobulin.

To date, Newman, Biotechnology 10:1455-1460 (1992) discloses another efficient method for generating recombinant antibodies. Specifically, this technique results in the production of primate-like antibodies containing monkey variable domains and human constant sequences. This document is incorporated herein by reference in its entirety. Furthermore, this technique is also described in U.S. Patent Nos. 5,658,570, 5,693,780, and 5,756,096, which are commonly assigned to the same U.S. Patent, each of which is incorporated herein by reference.

Alternatively, techniques well known to those skilled in the art can be used to select and culture antibody-producing cell lines. Such techniques are described in various laboratory manuals and original publications. In this regard, the techniques applicable to this disclosure as described below are described in Current Protocols in Immunology, edited by Coligan et al., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which, including additions, is incorporated herein by reference in its entirety.

Furthermore, standard techniques known to those skilled in the art for introducing mutations into the nucleotide sequence encoding the antibodies disclosed herein include, but are not limited to, site-directed mutagenesis leading to amino acid substitutions and PCR-mediated mutagenesis. Preferably, the variants (including derivatives) encode fewer than 50, fewer than 40, fewer than 30, fewer than 25, fewer than 20, fewer than 15, fewer than 10, fewer than 5, fewer than 4, fewer than 3, or fewer than 2 amino acid substitutions relative to the reference heavy chain variable region, CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2, or CDR-L3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

Cancer treatment

As described herein, the antibodies, variants, or derivatives disclosed herein may be used in certain therapeutic and diagnostic approaches.

This disclosure further relates to antibody-based therapies, which involve administering the antibodies of this disclosure to patients, such as animals, mammals, and humans, for the treatment of one or more disorders or conditions described herein. The therapeutic compounds of this disclosure include, but are not limited to, the antibodies of this disclosure (including variants and derivatives thereof described herein) and nucleic acids or polynucleotides (including variants and derivatives thereof described herein) encoding the antibodies of this disclosure.

The antibodies disclosed herein can also be used to treat or suppress cancer. PD-1 can be overexpressed in tumor cells. Tumor-derived PD-1 can bind to PD-L1 on immune cells, thereby limiting anti-tumor T-cell immunity. Results in mouse tumor models using small molecule inhibitors or monoclonal antibodies targeting PD-1 indicate that PD-1-targeted therapy is an important and realistic option for effectively controlling tumor growth. As demonstrated in experimental examples, anti-PD-1 antibodies activate adaptive immune response mechanisms, which can lead to improved survival rates in cancer patients.

Accordingly, in some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering an effective amount of the antibody disclosed herein to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient expresses, overexpresses, or is induced to express PD-1. For example, PD-1 expression induction can be accomplished by administering a tumor vaccine or radiation therapy.

Tumors expressing PD-1 protein include those of bladder cancer, non-small cell lung cancer, kidney cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, and small cell lung cancer. Therefore, the antibody disclosed in this application can be used to treat any one or more of these cancers.

This disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy. Suitable cells can be used, contacted with the anti-PD-1 antibody of this disclosure (or engineered to express the anti-PD-1 antibody of this disclosure). Following such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer disclosed herein. For example, the cells (e.g., T cells) may be tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto.

In some implementations, the cells are isolated from the cancer patient themselves. In other implementations, the cells are provided by a donor or cell bank. When the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

The antibodies or variants or derivatives thereof disclosed herein may be used to treat, prevent, diagnose, and/or prognose other diseases or conditions associated with increased cell viability, including but not limited to the progression and/or metastasis of malignancies, and related conditions such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (e.g., medulloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia) and chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and bone marrow cancer). Primary sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Treatment of infection and immune disorders

As shown in the experimental examples, the antibodies disclosed herein can activate an immune response, thereby enabling their use in the treatment of infections.

Infection is the invasion of an organism's body tissues by pathogens that cause disease, the proliferation of these pathogens, and the host tissues' response to these organisms and the toxins they produce. Infections can be caused by infectious pathogens such as viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks, mites, fleas, and lice, fungi such as tinea, and other macroparasites such as tapeworms and other worms. In one aspect, infectious pathogens are bacteria, such as Gram-negative bacteria. In another aspect, infectious pathogens are viruses, such as DNA viruses, RNA viruses, and retroviruses. Non-limiting examples of viruses include adenovirus, Coxsackievirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, HIV, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, and varicella-zoster virus.

In some implementations, methods or uses of the antibody or fragment thereof for treating immune disorders are also provided. Non-limiting examples of immune disorders include infection, infection-associated endotoxin shock, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peronis disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory diseases of the central and peripheral nervous systems, multiple sclerosis, etc. Lupus and Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrotic alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, pancreatitis, trauma, graft-versus-host disease, transplant rejection, ischemic diseases, myocardial infarction, atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypoacidity, and infertility associated with maternal and fetal intolerance.

The antibodies disclosed herein can also be used to treat infectious diseases caused by microorganisms by targeting microorganisms and immune cells to eliminate microorganisms, or to kill microorganisms. In one aspect, microorganisms include viruses, including RNA and DNA viruses, Gram-positive bacteria, Gram-negative bacteria, protozoa, or fungi.

The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex, and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The judgment of these factors by healthcare professionals is a general technique in the art. The dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using pharmacological and pharmacokinetic principles known in the art.

Antibodies and variants can be administered via routes including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucosal skin linings (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides of this disclosure can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), sublingually, or as an oral or nasal spray.

As used in this article, the term "parenteral" refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

Administration can be systemic or local. Furthermore, it may be desirable to introduce the antibodies of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be assisted by, for example, a ventricular catheter attached to a reservoir (e.g., the Ommaya reservoir). Lung administration may also be used, for example, by using an inhaler or nebulizer and a formulation containing a nebulizer.

It may be necessary to locally apply the antibody peptides or compositions of this disclosure to the area requiring treatment; this can be achieved, for example, by local infusion during surgery, topical application (e.g., in conjunction with postoperative wound dressings), by injection, by catheter, by suppository, or by implantation (the implant being a porous, non-porous, or gel-like material, including membranes such as sialastic membranes or fibers), but is not limited thereto. Preferably, when administering the proteins (including antibodies) of this disclosure, care must be taken to use materials that do not absorb the protein.

In another embodiment, the antibody or composition may be delivered in vesicles, particularly liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, edited by Lopez-Berestein and Fidler, Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; general information also see above.)

In another embodiment, the antigen-binding peptide or composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise, eds., CRC, Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball, eds., Wiley, New York (1984); Langer and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In another embodiment, the controlled release system can be placed near the therapeutic target (i.e., the brain), thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115–138 (1984)). Other controlled release systems are discussed in Langer’s review (1990, Science 249:1527–1533).

In specific embodiments of the compositions disclosed herein comprising nucleic acids or polynucleotides encoding proteins, the nucleic acid may be administered in vivo to promote the expression of its encoded protein by: constructing it as part of a suitable nucleic acid expression vector and administering it, for example, by using a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., a gene gun; a biolistic, DuPont), or by coating it with lipids or cell surface receptors or transfection agents, or by attaching it to a homologous cassette peptide known to enter the cell nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, the nucleic acid may be introduced into the cell and incorporated into the host cell DNA for expression via homologous recombination.

The amount of the disclosed antibody that will be effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined using standard clinical techniques. Additionally, in vitro assays may be optionally used to help identify the optimal dose range. The precise dose to be used in the formulation will also depend on the route of administration, the severity of the disease, disorder, or condition, and should be determined based on the judgment of the physician and the individual patient's circumstances. The effective dose can be deduced from dose-response curves obtained from in vitro or animal model testing systems.

As a general recommendation, the dose of the antigen-binding peptide of this disclosure administered to patients is typically 0.1 mg/kg to 100 mg/kg of patient body weight, 0.1 mg/kg to 20 mg/kg of patient body weight, or 1 mg/kg to 10 mg/kg of patient body weight. Generally, due to the immune response to exogenous peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses of human antibodies and less frequent administration are often possible. Furthermore, the dosage and frequency of administration of the antibodies of this disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) (through modifications such as lipidation).

Methods for treating infections or malignancies, conditions, or disorders, including the administration of antibodies, variants, or derivatives of this disclosure, are typically tested in vitro before being used in humans, and then in vivo in acceptable animal models to test the desired therapeutic or preventative activity. Suitable animal models (including transgenic animals) are well known to those skilled in the art. For example, in vitro tests demonstrating the therapeutic efficacy of the antigen-binding peptides described herein include the effect of the antigen-binding peptide on cell lines or patient tissue samples. The effect of the antigen-binding peptide on cell lines and/or tissue samples can be determined using techniques known to those skilled in the art, such as the tests disclosed in other parts of this document. According to this disclosure, in vitro tests that can be used to determine whether a particular antigen-binding peptide has been administered are indicated, including in vitro cell culture tests in which patient tissue samples are grown in a culture and exposed to or otherwise administered a compound, and the effect of such a compound on the tissue sample is observed.

Various delivery systems are known for administering the antibodies of this disclosure or the polynucleotides encoding the antibodies of this disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing compounds, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), constructing nucleic acids as part of retroviruses or other vectors, etc.

Diagnostic methods

PD-1 overexpression has been observed in certain tumor samples, and patients with PD-1 overexpressing cells may respond to treatment with the anti-PD-1 antibody disclosed herein. Therefore, the antibody disclosed herein can also be used for diagnostic and prognostic purposes.

Samples, preferably comprising cells, can be obtained from a patient, who may be a cancer patient or a patient seeking a diagnosis. The cells are cells from tumor tissue or tumor masses, blood samples, urine samples, or any sample from the patient. After optional pretreatment of the sample, the sample can be incubated with the antibody of this disclosure under conditions that allow the antibody to interact with the PD-1 protein that may be present in the sample. The presence of the PD-1 protein in the sample can be detected using an anti-PD-1 antibody using methods such as ELISA.

The presence (optionally, amount or concentration) of PD-1 protein in a sample can be used for cancer diagnosis, as an indication that the patient is suitable for antibody therapy, or as an indication that the patient has responded to (or has not yet responded to) cancer treatment. For prognostic methods, one, two or more tests can be performed at certain stages after the initiation of cancer treatment to indicate the progress of treatment.

Composition

This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

In certain implementations, the term "pharmaceuticalally acceptable" refers to a drug approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and especially in humans. Furthermore, a "pharmaceuticalally acceptable carrier" is typically a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation adjuvant.

The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such a drug carrier can be a sterile liquid, such as water and oil, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous dextran and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and tension modifiers such as sodium chloride or dextran are also envisioned. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. E.W. Martin describes examples of suitable drug carriers in Remington's Pharmaceutical Sciences (incorporated hereby by reference). Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide (preferably in a purified form) along with an appropriate amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the route of administration. Parenteral formulations can be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

In one embodiment, the composition is formulated according to conventional procedures to a pharmaceutical composition suitable for intravenous administration to the human body. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to reduce pain at the injection site. Typically, the components are supplied separately or mixed together in unit doses, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be prepared using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules containing sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

The compounds disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions (e.g., anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, and tartaric acid), and those formed with cations (e.g., cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

Example

Example 1: Generation of human monoclonal antibodies against human PD-1

Cloning of full-length human PD-1 cDNA

Human T lymphocytes were isolated from human peripheral blood lymphocytes (PBMCs) using MACS beads (Miltenyi Biotec). Total RNA was extracted from human T cells using the RNeasy Mini Kit (QiAGEN), and cDNA was obtained by reverse transcription PCR (SuperScript First-Strand Synthesis System, Invitrogen). Full-length cDNA encoding hPD-1 was generated from human T cell mRNA via RT-PCR using sense primers (5’-CTGTCTAGAATGCAGATCCCACAGGCGCC, SEQ ID NO: 47) and antisense primers (5’GGATCCTCAGAGGGGCCAAGAGCAGT, SEQ ID NO: 48). The sequence was validated by DNA sequencing and comparison with the NCBI database (NM-005018.2).

Establishment of a stable hPD-1 expression cell line: After digestion with Xbal and BamHI, the hPD-1 PCR fragment was cloned into the PCDA3.1(-) vector (Invitrogen). Then, the full-length pcDNA-hPD-1 plasmid was transfected into Chinese hamster ovary (CHO) cells using lipofectamine 2000 (Invitrogen). The stable hPD-1 expression cell line (CHO/hPD-1) was selected using G418 and screened by flow cytometry.

Generation of human PD-1 Ig fusion protein: cDNA containing the hPD-1 extracellular domain and the hPD-1 hIg fusion protein was amplified from full-length pcDNA-hPD-1 using specific primers via PCR. The PCR fragment, digested with EcoRI and BglII, was fused into the mouse IgG2a heavy chain of the expression plasmid pmIgG or into the CH2-CH3 domain of the human IgG1 heavy chain of the expression plasmid phIgG (H Dong et al. Nat Med. 1999; 5: 1365-1369). The protein in the culture supernatant was purified by protein A agarose gel column (HiTrap Protein A HP, GE Healthcare). The purified protein was confirmed by SDA-PAGE electrophoresis.

Monoclonal antibody generation: 8–10-week-old female Balb/c mice were immunized subcutaneously (s.c.) at multiple sites with an emulsion containing 100 μg hPD-1 mIg fusion protein and complete Freund's adjuvant (CFA) (Sigma Aldrich). Three weeks later, the mice were immunized subcutaneously (s.c.) with 50–100 μg protein containing incomplete Freund's adjuvant (IFA) (Sigma Aldrich), for a total of three immunizations. Two weeks after each immunization, the mice were exsanguinated for serum titer determination. When the titer was sufficient, the mice were boosted by intraperitoneal (i.p.) injection of 60 μg protein in PBS. Hybridomas were obtained by fusing spleen cells from immunized mice with the SP2/0-Ag14 myeloma cell line (from ATCC). Immunized mice were euthanized with carbon dioxide, and the spleens were harvested aseptically. The entire spleen was dissociated into a single-cell suspension, and red blood cells were lysed with ACK buffer. SP2/0-Ag14 myeloma and spleen cells were mixed at a 1:1 ratio in 50 mL conical centrifuge tubes. After centrifugation, the supernatant was discarded, and cell fusion was performed with 50% polyethylene glycol (Roche). The fused cells were cultured in HAT selective medium for 8–10 days, and the supernatant of the hybridoma culture was screened for binding to hPD-1-expressing cells using a high-throughput transfection and screening system (S Yao et al. Immunity. 2011; 34(5):729-40), and positive clones were confirmed by flow cytometry. Subcloning of positive hybridomas was performed at least 5 times using limiting dilution techniques to achieve pure monoclonal cultures.

Example 2: Characterization of PD-1 monoclonal antibody

mAb isotypes: The isotypes of mAbs were identified using the Mouse Immunoglobutin Isotyping Kit (BD Biosciences). All five PD-1 mAbs were identified as IgG1 isotypes and κ chains.

Binding specificity of anti-hPD-1: The specificity of PD-1 mAbs was determined by flow cytometry using CHO cells expressing hPD-1 on their surface (CHO/hPD-1). CHO/hPD-1 cells were incubated with anti-PD-1 mAbs on ice. After incubation, the cells were washed and further incubated with anti-mIgG-APC (eBiosciences). Flow cytometry analysis was performed using FACSVerse (BD Biosciences). The data showed that all five hPD-1 mAbs bound to hPD-1 with high specificity (Figure 1). To rule out the possibility of hPD-1 mAbs binding to other proteins, CHO cells transfected with hB7-1, hPD-L1, hB7-H3, hB7-H4, hCD137, or other protein molecules were stained with anti-hPD-1 mAbs by flow cytometry analysis. These cells were also stained with their respective positive antibodies as positive controls. The data show that anti-PD-1 mAbs do not bind to the proteins tested in these tests (Figure 2).

Species Cross-Reactivity: To evaluate the species specificity of the anti-hPD-1 mAb, peripheral blood mononuclear cells (PBMCs) from cynomolgus monkeys (from Guangdong Landau Biotechnology Company) were isolated from peripheral blood using Ficoll (Sigma-Aldrich). PBMCs were suspended in RPMI 1640 medium containing 10% FCS and placed in 24-well plates pre-coated with 1 μg/ml anti-hCD3. Cells were cultured for 2 days. Cells were first stained with anti-hPD-1. After washing, cells were stained with anti-mIgG-APC and CD3-FITC;CD8-PerCP for flow cytometry analysis. Furthermore, the cross-reactivity of the mAb with mouse PD-1 transfected CHO cells (CHO/mPD-1) was determined by flow cytometry.

Data showed that the anti-hPD-1 mAb could bind to the PD-1 protein on both human and cynomolgus monkey T cells, but no cross-binding was found on mouse PD-1 (Figure 3).

Ligand blocking: To examine ligand binding blockade, 100 ng of hPD1hIg fusion protein was pre-incubated with specified doses of mAb (400 ng/10 μL, 300 ng/10 μL, 200 ng/10 μL, 100 ng/10 μL, 50 ng/10 μL) or control Ig at 4°C for 30 minutes, and then used to stain CHO/hB7-H1 cells. Cells were washed and further stained with goat anti-hIgG-APC. The blocking effect was detected by flow cytometry.

Data showed that anti-hPD-1 mAbs 1 and 2 (Ab1 and Ab2) had no effect on ligand blocking. Ab3, Ab4, and Ab5 could block the binding of the hPD-1 fusion protein to hPD-L1 in a dose-dependent manner (Figure 4).

Competitive binding assay: A competitive binding assay was performed to investigate whether these mAbs recognized the same or different binding sites of the hPD-1 protein. CHO/hPD-1 cells were pre-incubated for 30 minutes at 4°C with an excess (10 μg) of each of the five PD-1 mAbs. After washing, the cells were incubated for 20 minutes at 4°C with 50 ng of each of the different biotin-labeled mAbs. The binding activity of the mAbs was measured using flow cytometry.

Flow cytometry analysis showed that Ab4 and Ab5 completely eliminated each other's binding to hPD-1 protein, while a saturated dose of Ab3 partially blocked the binding of Ab4 and Ab5, and Ab1 and Ab2 did not block the binding of Ab4 and Ab5 to hPD-1 (Figure 5). Therefore, the binding sites of Ab4 and Ab5 on PD-1 may overlap. Ab1 or Ab2 binds to PD-1 through different interfaces with Ab4 or Ab5, which was also confirmed by ligand blocking assays.

Example 3: Sequencing and humanization of hybridomas producing anti-PD-1 antibodies

Sequencing of hybridomas producing anti-PD-1 antibodies: 1 × ^10⁷ hybridoma cells were harvested and washed with PBS. Messenger RNA was extracted from the hybridomas using the RAeasy Mini Kit (Qiagen). RACE-Ready first-strand cDNA was synthesized using the SMARTer RACEcDNA Amplification Kit (Clontech). After reverse transcription, 5' RACE PCR was performed using the prepared cDNA as a template with the 5' universal primers (UPM) provided in the kit and the 3' gene-specific primers (GSP1) designed based on the mouse IgG1 heavy chain variable region and κ light chain gene sequences. The RACE products were identified by gel electrophoresis (Figure 6). The PCR products were cloned into a T vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). After transformation, the plasmid was validated by sequencing analysis. The antibody gene fragment was analyzed using VBASE2 (http://www.vBas2.org). The sequence is disclosed in (Table 2).

Table 2: Mouse antibody sequences

Protein expression and function determination of recombinant antibodies: To ensure the accuracy of the recombinant antibody sequence, the full-length sequences of the heavy and light chains of the recombinant antibody were cloned into the pcDNA3.1 vector and transiently transfected into HEK 293T cells. Proteins from the cell culture supernatant were purified using a protein G agarose gel column (GE Healthcare) for functional evaluation.

Flow cytometry analysis showed that the recombinant antibody could bind to the hPD-1 protein and block the binding of the hPD-1 fusion protein to the PD-L1 protein (Figure 7, Figures A and B).

Humanization of anti-human PD-1 antibodies: Humanization was performed based on the variable heavy chain (VH) and variable light chain (VL) sequences of anti-hPD-1 hybridomas. Generally, a mouse-human chimeric mAb containing the parental mouse VH and VL sequences, the human IgG4-S228P constant region, and the human κ chain was constructed first. After characterizing the chimeric antibodies, three VL and three VL humanized sequences were designed and used to prepare nine humanized antibodies. The sequences are listed in Tables 3A and 3B.

Table 3A: Chimeric Antibody (Human IgG4-S228P Backbone)

Table 3B: Variable Regions of Humanized Heavy and Light Chains

Example 4: Characteristics and functions of humanized antibodies

Binding activity of humanized antibodies: CHO/hPD-1 cells were incubated with serially diluted mAbs. The binding of nine humanized antibodies to the PD-1 protein was evaluated by flow cytometry and compared with chimeric parental antibodies.

Flow cytometry analysis showed that some mutant combinations exhibited higher binding activity than the parental antibody, while others showed the same or slightly lower binding activity as the parental antibody (Figure 8). The mutant combinations are listed in Table 4 below.

Blocking ability of humanized antibodies: The ability of humanized antibodies to block the binding of hPD-1 to hPD-L1 was measured. 100 ng of hPD1 mIg was pre-incubated with different doses of humanized antibody in 10 μL PBS for 30 minutes at 4 °C, and then used to stain CHO/hB7-H1 cells. Cells were washed and further stained with goat anti-mIgG-APC. The blocking effect was evaluated by flow cytometry. The ability of humanized antibodies to block the binding of hPD-1 to hPD-L2 was measured using a similar method.

The results showed that all humanized antibodies inhibited the binding of hPD-1 mIgG to CHO/hPD-L1 cells in a dose-dependent manner. Some mutant combinations showed higher blocking ability than the chimeric parental antibody (Figure 9). The results also showed that the binding of hPD-1 mIgG to CHO/hPD-L2 cells was blocked (Figure 10).

Binding affinity and kinetics of humanized antibodies were determined: The binding affinity and kinetics of the interaction between humanized PD-1 mAb and hPD-1 protein were assessed using a Biacore T100 (GE Healthcare Life Sciences). hPD-1 mIg protein was immobilized on a sensor chip CM5 via amine coupling. Filtered humanized antibodies were diluted with HBS-EP buffer at pH 7.4 (GE Healthcare Life Sciences) and then injected onto the hPD-1 mIg-immobilized surface. Nine different concentrations were tested for each sample. Detailed binding kinetic parameters (association rate, Ka, dissociation rate, Kd, and affinity constant, KD) were determined by full kinetic analysis.

Analysis showed no significant difference in binding rate (Ka) between the mutant combinations and the chimeric parent antibody. In terms of dissociation rate (Kd), the three mutant combinations (3, 6, 9) were close to the chimeric parent antibody. All humanized antibodies exhibited strong affinity, with KD values in the low nanomolar range ( ^10⁻¹⁰ M). The KD values of the two mutant combinations (3, 6) were close to those of the chimeric parent antibody (9.89 × ^10⁻¹¹ M) (Table 4).

Table 4: Determination of binding affinity and kinetics of humanized antibodies

Enhancement of Anti-PD-1 on the In Vitro Killing of PD-L1-Positive Tumor Cells by Allogeneic CD8+ CTLs: Based on the anti-tumor mechanism of anti-PD-1 antibodies, this embodiment designed an in vitro model to determine the enhancing effect of anti-PD-1 antibodies on the killing of tumor cells by human allogeneic CD8 ⁺ cytotoxic lymphocytes (allogeneic CD8 ⁺ CTLs). First, CD8 ⁺ lymphocytes were isolated from human PMBCs and co-cultured with irradiated, hB7-1-transfected human melanoma cells (624Mel/B7-1) to generate allogeneic CD8 ⁺ cytotoxic CTLs. Then, in the presence of humanized antibody or control Ig, allogeneic CD8 ⁺ CTLs were co-cultured in 96-well plates with overnight-cultured 624Mel/hPD-L1 tumor cells for 5 days. Cells in the wells were stained with 0.5% crystal violet, and the plates were read at 540 nm using an ELISA reader. Killing activity was calculated based on tumor cell viability.

The results showed that certain mutant combinations could enhance the ability of allogeneic CTL cells to kill tumor cells in vitro (Figure 11).

The optimal mutant combination (variant 3) was selected, the protein-coding sequence was cloned into a suitable expression vector, and transferred to CHO cells to produce an anti-hPD-1 antibody, also known as TY101.

Example 5: Characteristics of TY101 in tumor immunotherapy

Cytokine-enhanced mixed lymphocyte response (MRL) in PBMCs. Human peripheral blood mononuclear cells (PBMCs) from healthy individuals were isolated using density gradient centrifugation with Ficoll-Hypaque. PBMCs from healthy donor 1 were irradiated with 40 Gy of X-rays as stimulators. T lymphocytes were isolated from healthy donor 2 as responders using the human Pan T cell Isolation Kit (MiltenylBiotec). Responders and stimulators were resuspended in complete RPMI medium containing 10% FCS, and seeded into 96-well plates with 2.5 × ^10⁵ responders and 1.25 × ^10⁵ stimulators per well (R/S = 2) in the presence of serially diluted TY101 or hIgG controls. Cells were cultured at 37°C in a humidified incubator containing 5% _CO₂ for 5 days. T cell proliferation activity was assessed on day 5 using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.). To detect cytokines, culture supernatants were collected on days 3 and 5. Cytokine analysis was performed using the HumanTh1/Th2/Th17 Cytometric Bead Array kit (CBA; BD Biosciences).

The results showed that the T cell proliferation response to TY101 was similar to that to hIgG (Figure 12). Interestingly, compared with hIgG, the production of cytokines IL-2 and IFNγ was significantly increased in the culture supernatant of MLRs treated with TY101 (Figure 13).

Blocking PD-1 expression on T lymphocytes. PD-L1 expression on tumor cells can induce PD-1 expression on tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment and trigger PD-1-dependent immunosuppression. This study designed an in vitro model to determine whether TY101 could inhibit hPD-1 expression on human lymphocytes when co-cultured with hPD-L1-transfected tumor cells. Human T lymphocytes isolated from human PBMCs and hPD-L1-transfected human melanoma (624/hPD-L1) cells were cultured for 4 days in the presence of 10 μg/ml TY101 or control IgG. hPD-1 expression on lymphocytes was detected by flow cytometry.

The results showed that, compared with culture medium alone and hIgG control, the addition of TY101 completely inhibited PD-1 expression on lymphocytes (Figure 14).

In vivo antitumor activity of humanized PD-1 antibody: The in vivo antitumor effect of TY101 was investigated. Eight-week-old female human PD-1 knock-in mice (purchased from Shanghai Model Organisms Center, Inc.) were subcutaneously transplanted with hPD-L1-transfected MC38 tumor cells (MC38/hPD-L1) in the right flank (sc) on day 0 (1× ^10⁶ /mouse). TY101 or control Ig (10 mg/kg) was administered intraperitoneally (ip) on days 6, 9, and 13. Tumor size and survival were monitored.

All animals initially had detectable tumors (4–5 mm on day 6). However, complete response occurred in 100% of mice after treatment with TY101 for MC38/hPD-L1 tumors. Tumors in all five mice treated with TY101 completely regressed by day 25. In contrast, two of the five mice treated with control IgG developed persistently growing tumors. In the other three mice treated with control IgG, although the tumors also regressed by day 32, the tumors recurred rapidly in two of the mice (Figure 15). The results indicate that TY101 enhances antitumor efficacy in vivo.

Example 6: Comparison of the functions of TY101 and commercially available PD-1 antibodies

This example selected two anti-hPD-1 antibodies that are currently approved for clinical treatment of cancer patients to compare with TY101: Merck's Keytruda (pembrolizumab) and Bristol-Myers Squibb's Opdivo (nivolumab).

Antibody binding affinity and kinetics: The affinity and kinetics of TY101 were analyzed using a Biacore T200 instrument (GE Healthcare LifeSciences) and compared with two commercially available antibodies. hPD-1 mIg protein was immobilized at a low concentration (33 RU) on a CM5 sensor chip, and the antibodies were used as analytes (mobile phase) to detect interactions. Data showed that the binding rates Ka of the three antibodies did not differ significantly. TY101 was slightly lower than the commercially available antibodies. The dissociation rate Kd of TY101 was up to one-tenth slower than the two commercially available antibodies, and the affinity KD of TY101 was 4–7 times stronger than the commercially available antibodies. These results indicate that TY101 exhibits stronger binding (Figure 16).

Comparison of PD-1 antibody performance in PD-1/PD-L1 blockade: PD-1/PD-L1 blockade bioassays were performed using the PD-1/PD-L1 Blockade Bioassays Kit (Promega). Jurkat-PD1 cells (1 × ^10⁵ cells/well) were cultured in opaque 96-well TC plates and stimulated overnight-cultured CHO-PD-L1 cells (culture started at 5 × ^10⁴ cells/well) for 5 h in the presence of serial dilutions (0–30 μg/ml) of TY101, pembrolizumab, nivolumab, or negative control hIgG4. After 5 h of incubation, Jurkat-PD-1 cell activation was detected by measuring the relative optical units (RLU) of luciferase activity using the ONE-Glo substrate (Promega) on a SpectraMAX L spectrophotometer.

Analysis showed that TY101 and two commercially available antibodies could block the PD-1/PD-L1 pathway. The blocking effect of TY101 was similar to that of pembrolizumab, but superior to that of nivolumab (Figure 17).

Comparison of in vitro inhibitory effects on tumor cell growth: As previously described, allogeneic CD8 ⁺ CTL cells were co-cultured with overnight-cultured 624Mel/PD-L1 tumor cells in 96-well plates for 5 days in the presence of different mAbs and control IgG. Cells were stained with 0.5% crystal violet, and the plates were read at 540 nm using an ELISA reader. Killing activity was calculated based on tumor cell viability.

The results showed that all three anti-PD-1MAb antibodies enhanced the tumor-killing activity of allogeneic CD8 ⁺ CTLs. The enhancing effect of TY101 was greater than that of the two commercially available antibodies (Figure 18).

Example 7: Development of TY101 clone and its activity

The TY101 sequence was cloned into a proprietary expression vector and transfected into CHO cells. Monoclonal cell lines were established using ClonePix and/or limiting dilution methods. Multiple clones were established, and antibodies produced by three such clones (TY101-01-09, TY101-04-T3-05, and TY101-4G1) were characterized.

ELISA (Electro-ELISA) tests for antibodies that bind to hPD-1 or mPD-1 proteins.

The binding of antibodies to hPD-1 and their cross-reactivity with mPD-1 protein were detected by ELISA. Serially diluted test antibodies were added to ELISA plates pre-coated with 1 μg/ml hPD-1 or mPD-1. HRP-conjugated goat anti-human IgG or goat anti-mouse IgG antibodies were then added, followed by the substrate tetramethylbenzidine (TMB), and quantification was performed at 450 nm using a SpectraMax Plus 384 Microplate Reader (Molecular Device, LLC., Sunnyvale, CA). The tested TY101 clones TY101-01-09, TY101-04-T3-05, and TY101-4G1 showed good binding to hPD-1 protein, with EC50 values ranging from 0.01 to 0.15 nM. The antibodies did not show binding to mPD-1 protein (Figure 19).

Testing the binding of antibodies to CHOK1 cells expressing hPD-1 and cPD-1 (flow cytometry)

The binding of antibodies to hPD-1 and cross-reactivity with cPD-1 were tested using CHOK1 cells expressing hPD-1 or cynomolgus monkey PD-1 (cPD-1) by flow cytometry. CHOK1-hPD-1, CHOK1-cPD-1, and CHOK1 blank cells were incubated with serially diluted test products, followed by incubation with Alexa 488-conjugated goat anti-human IgG (H+L) antibody, and analyzed using FACSCanto II (BD Biosciences, San Jose, CA). The tested TY101 clones TY101-01-09, TY101-04-T3-05, and TY101-4G1 showed good binding to CHOK1-hPD-1 at sub-nanomolar EC50 and to CHOK1-cPD-1 cells at several nanomolar EC50 (Figure 20).

Testing the antibody's blocking activity against hPD-1/hPD-L1 or hPD-1/hPD-L2 binding (flow cytometry)

The ability of these antibodies to block the binding of hPD-1/hPD-L1 and hPD-1/hPD-L2 was further tested, which could be key to the potential efficacy of treatment for cancer patients. CHOK1-hPD-1 cells were incubated with serially diluted assay products mixed with biotin-hPD-L1 or biotin-hPD-L2. The cells were then incubated with streptavidin labeled with Alexa 488 and analyzed using FACSCanto II. Anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1 blocked the binding of hPD-L1 to hPD-1-expressing CHOK1 cells, with IC50 values ranging from 1.15 to 1.47 nM. They also blocked the binding of hPD-L2 to hPD-1-expressing CHOK1 cells, with IC50 values ranging from 1.52 to 2.33 nM (Figure 21).

Testing the effect of antibodies on T cells using a human mixed leukocyte reaction (MLR) assay

The effects of these antibodies on T cell function were tested in a human MLR assay using T cells isolated from two donors. Adhering PBMCs (primarily monocytes, isolated from donor 1 and plated in cell culture dishes to allow adhesion) were cultured for 5 days in the presence of 100 ng/ml recombinant human (rh) GM-CSF and 50 ng/ml rhIL-4, with half the culture medium replaced after 3 days, and 1 μg/ml LPS added on day 6. On day 7, the resulting cells (primarily mature DCs) were harvested and treated with mitomycin C. CD3+ T cells were isolated from donors 2 and 3 using the EasySep ^™ Human T Cell Isolation Kit (negative selection, STEMCELL technology). DCs ^and T cells were co-cultured for 5 days in the presence of three concentrations (5 μg/ml, 0.5 μg/ml, and 0.05 μg/ml) of the test antibody. The supernatant was harvested after 3 days, and IL-2 levels were measured, and IFN-γ levels were measured after 5 days (100 μL). Anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1, compared with the isotype control hIgG4, promoted the secretion of IL-2 and IFN-γ from cells from the two donors in a dose-dependent manner (Figure 22).

Engineered tumor cell-human T cell co-culture assay to test the effect of antibodies on T cells

The effects of these antibodies on T cell function were tested in a human-T cell co-culture assay using engineered tumor cells isolated from four different donors. CD3 ⁺ T cells were isolated from PBMCs of the four donors using the EasySep ^™ Human T Cell Isolation Kit. Engineered tumor cells Hep3B-OSC-hPDL1 (Hep3B cells engineered to stably express OS8 (anti-CD3 single-chain variable fragment (scFv)) and hPD-L1 (KCLB, catalog number: 88064)) were treated with mitomycin C and co-cultured with CD3 ⁺ T cells for 3 days in the presence of three concentrations (5 μg/ml, 0.5 μg/ml, and 0.05 μg/ml) of the test antibody. The culture supernatant was harvested to determine IFN-γ levels. When compared with the isotype control hIgG4, anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1 promoted the secretion of IFN-γ from cells from all four donors in a dose-dependent manner (Figure 23).

Example 8: Compared with FDA-approved anti-hPD-1 antibodies, the TY101 clone showed better binding affinity.

Antibody epitope overlap was tested using a competitive ELISA.

In a competitive ELISA assay, these antibodies were tested to determine whether they bind to the same epitopes as the FDA-approved anti-hPD-1 antibodies nivolumab or pembrolizumab. Serial dilutions of the competitive antibodies and biotin-hPD-1 were added to ELISA plates pre-coated with 1 μg/ml of the test antibody. HRP-conjugated streptavidin was then added, followed by the substrate TMB, and quantification was performed at 450 nm using a SpectraMax Plus 384 Microplate Reader. The anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1 almost completely blocked each other's binding to hPD-1, indicating that they share similar epitopes. These three antibodies also almost completely (93% to 94%) blocked the binding of nivolumab and pembrolizumab to hPD-1, while nivolumab and pembrolizumab only partially blocked the binding of these antibodies to hPD-1 (77%-78% for nivolumab and 46%-49% for pembrolizumab). These data suggest that the TY101-01-09, TY101-04-T3-05, and TY101-4G1 antibodies bind to epitopes different from those of nivolumab and pembrolizumab, and their affinity for hPD-1 may be higher than that of nivolumab and pembrolizumab (Figure 24).

The binding affinity of the antibody to hPD-1 was tested using SPR assay.

To obtain accurate measurements of hPD-1 binding affinity, antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1, as well as nivolumab and pembrolizumab, were analyzed using the SPR method. Human PD-1 ECD protein was immobilized on a CM5 sensor chip for different durations to achieve low immobilization levels (60 RU) in flow cell 3 and high immobilization levels (960 RU) in flow cell 4. Serially diluted antibodies (0 nM, 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25, and 50 nM) were injected into the flow cells. The association time was 180 s, and the dissociation time was 600 s (for nivolumab and pembrolizumab) or 1500 s (for TY101-01-09, TY101-04-T3-05, and TY101-4G1). Binding kinetics were calculated using BiaCore T200 evaluation software version 1.0 and a 1:1 binding model for curve fitting after the reference (flow cell 1) and zero concentration signals were subtracted from the sample signals. Control human IgG4 did not bind to hPD-1. Based on data from low-immobilization levels of hPD-1 (~60 RU; Table 3; Figure 23), the association rates of anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1 with human PD-1 were slightly lower than those of nivolumab and pembrolizumab (half to a quarter). The dissociation rates of these three antibodies with human PD-1 were one-twelfth to one-thirtieth that of nivolumab and pembrolizumab, resulting in a 4-8 times higher affinity than nivolumab and pembrolizumab (lower _KD corresponds to better affinity, and vice versa; Table 3). The binding affinity of anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1 to hPD-1 was also tested at high levels of immobilized hPD-1. Antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1 showed very slow dissociation rates, with only minimal dissociation observed even after a dissociation time of 1500 seconds (Figure 23). Data show that TY101-01-09, TY101-04-T3-05 and TY101-4G1 have better binding affinity than nivolumab and pambumab, mainly due to their slower dissociation rates (Figure 25).

The scope of this disclosure is not limited to the specific embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specification are incorporated herein by reference, as if each individual publication or patent application were specifically and individually indicated to be incorporated herein by reference.

sequence list

<110> Dayou Huaxia Biomedical Group Co., Ltd.

<120> Anti-PD-1 antibodies and their uses

<130> DSP1V190787XN-JW

<140> PCT/CN2018/073383

<141> 2018-01-19

<150> 2017100461482

<151> 2017-01-20

<160> 48

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 1

Gly Phe Thr Phe Ser Ser Tyr Thr

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 2

Ile Ser His Gly Gly Gly Asp Thr

1 5

<210> 3

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 3

Ala Arg His Ser Gly Tyr Glu Arg Gly Tyr Tyr Tyr Val Met Asp Tyr

1 5 10 15

<210> 4

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 4

Glu Ser Val Asp Tyr Tyr Gly Phe Ser Phe

1 5 10

<210> 5

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<220>

<221> misc_feature

<222> (4)..(4)

<223> X is a space filler, and does not need to be an amino acid.

<400> 5

Ala Ala Ser Xaa

1

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 6

Gln Gln Ser Lys Glu Val Pro Trp

1 5

<210> 7

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 7

Gly Tyr Thr Phe Thr Ser Tyr Thr

1 5

<210> 8

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 8

Ile Asn Pro Thr Thr Gly Tyr Thr

1 5

<210> 9

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 9

Ala Arg Asp Asp Ala Tyr Tyr Ser Gly Tyr

1 5 10

<210> 10

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 10

Glu Asn Ile Tyr Ser Asn Leu

1 5

<210> 11

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<220>

<221> misc_feature

<222> (4)..(4)

<223> X is a space filler, and does not need to be an amino acid.

<400> 11

Ala Ala Lys Xaa

1

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 12

Gln His Phe Trp Gly Thr Pro Trp Thr

1 5

<210> 13

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 13

Gly Phe Ala Phe Ser Ser Tyr Asp

1 5

<210> 14

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 14

Ile Thr Ile Gly Gly Gly Thr Thr

1 5

<210> 15

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 15

Ala Arg His Arg Tyr Asp Tyr Phe Ala Met Asp Asn

1 5 10

<210> 16

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 16

Glu Asn Val Asp Asn Tyr Gly Ile Asn Phe

1 5 10

<210> 17

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<220>

<221> misc_feature

<222> (4)..(4)

<223> X is a space filler, and does not need to be an amino acid.

<400> 17

Val Ser Ser Xaa

1

<210> 18

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 18

Gln Gln Ser Lys Asp Val Pro Trp

1 5

<210> 19

<211> 135

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 19

Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly

1 5 10 15

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

20 25 30

Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Asn Pro Thr Thr Gly Tyr Thr Asn Tyr Asn Gln Lys

50 55 60

Phe Lys Asp Lys Ala Asn Pro Thr Thr Thr Gly Tyr Thr Asn Tyr Asn Gln

65 70 75 80

Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr

85 90 95

Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Asp Asp Ala Tyr Tyr Ser Gly Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Leu Thr Val Ser Ser

130 135

<210> 20

<211> 354

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 20

tcccaggtcc agctgcagca gtctggggct gaactggcaa gacctggggc ctcagtgaag 60

atgtcctgca aggcttctgg ctacaccttt actagttaca cgatgcactg ggtaaaacag 120

aggcctggac agggtctgga atggattgga tacattaatc ctactactgg ttatactaat 180

tacaatcaga agttcaagga caaggccaca ttgactgcag acaaatcctc cagcacagcc 240

tacatgcaat tgagcagcct gacatctgag gactctgcag tctattactg tgcaagagat 300

gatgcttact actcgggcta ctggggccaa ggcaccactc tcacagtctc ctca 354

<210> 21

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Ala Ala Lys Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 22

<211> 324

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 22

gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatcg gcagaaacag 120

ggaaaatctc ctcagctcct ggtctatgct gcaaaaaact tagcagatgg tgtgccatca 180

aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct 240

gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa acgg 324

<210> 23

<211> 140

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 23

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser

1 5 10 15

Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp

20 25 30

Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Val Trp Val Ala

35 40 45

Tyr Ile Thr Ile Gly Gly Gly Thr Thr Tyr Tyr Tyr Ser Asp Thr Val Lys

50 55 60

Arg Leu Val Trp Val Ala Tyr Ile Thr Ile Gly Gly Gly Thr Thr Thr Tyr

65 70 75 80

Tyr Ser Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg His Arg Tyr Asp Tyr Phe Ala Met Asp

115 120 125

Asn Trp Gly His Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 24

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 24

gaagtgcagc tggtggagtc ggggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cgctttcagt agctatgaca tgtcttgggt tcgccagact 120

ccggagaaga ggctggtgtg ggtcgcatac attactattg gtggtggcac cacctactat 180

tcagacactg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagacatagg 300

tacgattact tcgctatgga caactggggt catggaacct cagtcaccgt ctcctca 357

<210> 25

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 25

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Glu

1 5 10 15

His Arg Ala Thr Ile Ser Cys Gln Ala Ser Glu Asn Val Asp Asn Tyr

20 25 30

Gly Ile Asn Phe Met Asn Trp Phe Gln His Lys Pro Ala Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Val Ser Ser Asn Leu Gly Ser Gly Val Pro Ala

50 55 60

Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Asp Val Pro Trp Thr Phe Ser Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 26

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 26

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagagca cagggccacc 60

atctcctgcc aagccagcga aaatgttgat aattatggca ttaattttat gaactggttc 120

caacacaaac cagcacagcc acccaactc ctcatctatg tttcatccaa cctaggatcc 180

ggggtccctg ccaagtttag tggcagtggg tctggaacag acttcagcct caacatccat 240

cctatggaag aagatgatac tgcaatgtat ttctgtcagc aaagtaagga cgttccgtgg 300

acgttcagtg gaggcaccaa actggaaatc aaacgg 336

<210> 27

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 27

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Ile Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser His Gly Gly Gly Asp Thr Tyr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Ser Gly Tyr Glu Arg Gly Tyr Tyr Tyr Val Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 28

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 28

gaagtgaagt tggtggagtc tgggggaggt ttagtgcagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt agctatacca tgtcttggat tcgccagact 120

ccagagaaga ggctggagtg ggtcgcatac attagtcatg gtggtggtga cacctactat 180

ccagacactg taaagggccg attcaccatc tccagggaca atgccaagaa caccctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acggccatgt attactgtgc aagacatagt 300

ggttacgaga ggggatatta ctatgttatg gattactggg gtcaaggaac ctcagtcacc 360

gtctcctca 369

<210> 29

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 29

Asp Ile Val Leu Thr Gln Phe Pro Thr Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Phe Ser Phe Ile Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Gly Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 30

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 30

gacattgtgc tgacccaatt tccaacttct ttggctgtgt ctctagggca gaggccacc 60

atctcctgca gagccagcga aagtgttgat tactatggct ttagttttat aaactggttc 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccagggatcc 180

ggggtccctg ccaggtttgg tggcagtggg tctggggacag acttcagcct caacatccat 240

cctatggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg 300

acgttcggtg gaggcaccaa gctggaaatc aaacgg 336

<210> 31

<211> 450

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 31

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Ile Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser His Gly Gly Gly Asp Thr Tyr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Ser Gly Tyr Glu Arg Gly Tyr Tyr Tyr Val Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

210 215 220

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

435 440 445

Gly Lys

450

<210> 32

<211> 1357

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 32

cgaagtgaag ttggtggagt ctgggggagg tttagtgcag cctggagggt ccctgaaact 60

ctcctgtgca gcctctggat tcactttcag tagctatacc atgtcttgga ttcgccagac 120

tccagagaag aggctggagt gggtcgcata cattagtcat ggtggtggtg acacctacta 180

tccagacact gtaaagggcc gattcaccat ctccagggac aatgccaaga acaccctgta 240

cctgcaaatg agcagtctga agtctgagga cacggccatg tattactgtg caagacatag 300

tggttacgag aggggatatt actatgttat ggattactgg ggtcaaggaa cctcagtcac 360

cgtctcctca gctagcacca aggggccccag cgtgtttcct ctcgctccct gcagccggag 420

cacatccgag agcaccgctg ctctgggctg tctcgtgaag gactacttcc ctgaacccgt 480

caccgtcagc tggaatagcg gcgccctgac atccggcgtc cacacattcc ccgctgtcct 540

gcagagcagc ggcctgtaca gcctgagctc cgtggtcacc gtgcctagca gcagcctggg 600

aacaaagacc tacacctgca acgtggacca taagccctcc aacaccaagg tggacaagcg 660

ggtggaatcc aagtatggac cccctgtcc tccttgccct gctcctgaat ttctcggagg 720

cccctccgtc ttcctgtttc cccccaagcc caaggacacc ctgatgatct cccggacaccc 780

cgaagtcacc tgcgtcgtgg tggatgtcag ccaggaagat cccgaggtgc agttcaactg 840

gtacgtggac ggagtggagg tgcataacgc caaaaccaag cccagggaag agcagttcaa 900

cagcacctat cgggtcgtgt ccgtgctcac cgtcctgcat caggattggc tcaacggcaa 960

ggagtacaag tgcaaggtgt ccaacaaggg cctgccctcc tccatcgaga agaccatctc 1020

caaggctaag ggccaacctc gggagcccca agtgtatacc ctccctccca gccaggagga 1080

gatgaccaag aatcaagtga gcctgacctg cctcgtgaag ggattttacc cctccgacat 1140

cgctgtggaa tgggaaagca atggccaacc tgagaacaac tacaagacca caccccccgt 1200

gctggactcc gatggctcct tcttcctgta cagcaggctg accgtggaca aatcccggtg 1260

gcaagaggga aacgtgttca gctgctccgt gatgcacgag gctctccaca accactacac 1320

ccagaagagc ctctccctga gcctcggcaa gtagtaa 1357

<210> 33

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 33

Asp Ile Val Leu Thr Gln Phe Pro Thr Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Phe Ser Phe Ile Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Gly Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Thr 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

<210> 34

<211> 661

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 34

agacattgtg ctgacccaat ttccaacttc tttggctgtg tctctagggc agagggccac 60

catctcctgc agagccagcg aaagtgttga ttaactatggc tttagtttta taaactggtt 120

ccaacagaaa ccaggacagc cacccaaact cctcatctat gctgcatcca accagggatc 180

cggggtccct gccaggtttg gtggcagtgg gtctgggaca gacttcagcc tcaacatcca 240

tcctatggag gaggatgata ctgcaatgta tttctgtcag caaagtaagg aggttccgtg 300

gacgttcggt ggaggcacca agctggaaat caagcggacc gtggccgccc ccagcgtgtt 360

catcttccct cccagcgacg agcagctgaa gtctggcacc gccagcgtgg tgtgcctgct 420

gaacaacttc tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagag 480

cggcaacagc caggagagcg tgaccgagca acaggactcc aaggacagca cctacagcct 540

gaccagcacc ctgaccctga gcaaggccga ctacgagaag cacaaggtgt acgcctgcga 600

ggtgacccac cagggactgt ctagccccgt gaccaagagc ttcaaccggg gcgagtgcta 660

a 661

<210> 35

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser His Gly Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Ser Gly Tyr Glu Arg Gly Tyr Tyr Tyr Val Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 36

<211> 373

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 36

cgaagtgcag ctggtggaat ctggcggcgg actggtgcag cctggcggat ctctgagact 60

gtcttgtgcc gcctccggct tcaccttctc cagctacacc atgtcctggg tgcgacaggc 120

tcctggcaag ggcctggaat gggtgtccta catctctcac ggcggaggcg acacctacta 180

cgccgactct gtgaagggcc ggttcaccat ctcccgggac aactccaaga acaccctgta 240

cctgcagatg aactccctgc gggccgagga caccgccgtg tactactgtg ctcggcactc 300

tggctacgag cggggctact actacgtgat ggactactgg ggccagggca ccctcgtgac 360

cgtgtcatct gct 373

<210> 37

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser His Gly Gly Gly Asp Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Ser Gly Tyr Glu Arg Gly Tyr Tyr Tyr Val Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 38

<211> 373

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 38

cgaagtgcag ctggtggaat ctggcggcgg actggtgcag cctggcggat ctctgagact 60

gtcttgtgcc gcctccggct tcaccttctc cagctacacc atgtcctggg tgcgacaggc 120

tcctggcaag ggcctggaat gggtgtccta catctctcac ggcggaggcg acacctacta 180

ccccgactct gtgaagggcc ggttcaccat ctcccgggac aactccaaga acaccctgta 240

cctgcagatg aactccctgc gggccgagga caccgccgtg tactactgtg ctcggcactc 300

tggctacgag cggggctact actacgtgat ggactactgg ggccagggca ccctcgtgac 360

cgtgtcatct gct 373

<210> 39

<211> 146

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 39

Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser His Gly Gly Gly Asp Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Gly Gly Asp Thr

65 70 75 80

Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

85 90 95

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg His Ser Gly Tyr Glu Arg Gly Tyr

115 120 125

Tyr Tyr Val Met Asp Tyr Trp Gly Lys Gly Thr Thr Val Thr Val Ser

130 135 140

Ser Ala

145

<210> 40

<211> 372

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 40

gaagtgaagc tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60

tcttgtgccg cctccggctt caccttctcc agctacacca tgtcctgggt gcgacaggct 120

cctggcaagg gcctggaatg ggtgtcctac atctctcacg gcggaggcga cacctactac 180

cccgactctg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc tcggcactct 300

ggctacgagc ggggctacta ctacgtgatg gactactggg gcaagggcac caccgtgacc 360

gtgtcatctg ct 372

<210> 41

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 41

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Phe Ser Phe Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Arg Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 42

<211> 337

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 42

agacatcgtg atgacccagt cccccgactc cctggctgtg tctctgggcg agagagccac 60

catcaactgc aagtcctccg agtccgtgga ctactacggc ttctccttcc tgaactggtt 120

ccagcagaag cccggccagc cccctaagct gctgatctac gccgcctcca accgcgagtc 180

tggcgtgccc gatagattct ccggctctgg ctctggcacc gactttaccc tgaccatcag 240

ctccctgcag gccgaggatg tggccgtgta ctactgccag cagtccaaag aggtgccctg 300

gaccttcggc cagggcacaa agctggaaat caagcgg 337

<210> 43

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 43

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Phe Ser Phe Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Arg Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 44

<211> 337

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 44

agacatcgtg atgacccagt cccccgactc cctggctgtg tctctgggcg agagagccac 60

catcaactgc aaggcctccg agtccgtgga ctactacggc ttctccttcc tgaactggtt 120

ccagcagaag cccggccagc cccctaagct gctgatctac gccgcctcca accgcgagtc 180

tggcgtgccc gatagattct ccggctctgg ctctggcacc gactttaccc tgaccatcag 240

ctccctgcag gccgaggatg tggccgtgta ctactgccag cagtccaaag aggtgccctg 300

gaccttcggc cagggcacaa agctggaaat caagcgg 337

<210> 45

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 45

Asp Ile Gln Leu Thr Gln Ser Pro Asp Ser Leu Ser Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Phe Ser Phe Leu Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Arg Gln Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 46

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 46

gacatccagc tgacccagtc ccccgactcc ctgtctgtgt ctctgggcga gagagccacc 60

atcaactgca aggcctccga gtccgtggac tactacggct tctccttcct gaactggttc 120

cagcagaagc ccggccagcc ccctaagctg ctgatctacg ccgcctccaa ccgccagtct 180

ggcgtgcccg atagattctc cggctctggc tctggcaccg actttaccct gaccatcagc 240

tccctgcagg ccgaggatgt ggccgtgtac ttctgccagc agtccaaaga ggtgccctgg 300

accttcggcc agggcacaaa gctggaaatc aagcgg 336

<210> 47

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 47

ctgtctagaa tgcagatccc acaggcgcc 29

<210> 48

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 48

ggatcctcag aggggccaag agcagt 26

Claims (14)

1. A composition comprising an antibody or a fragment thereof and a pharmaceutically acceptable carrier; The antibody or fragment thereof is an isolated antibody or fragment thereof, the isolated antibody or fragment thereof being specific for human programmed cell death protein 1 (PD-1), wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as follows: HCDR1:GTFFSSYT(SEQ ID NO:1), HCDR2:ISHGGGDT(SEQ ID NO:2), HCDR3:ARHSGYERGYYYVMDY(SEQ ID NO:3), LCDR1:ESVDYYGFSF(SEQ ID NO:4), LCDR2:AAS(SEQ IDNO:5), LCDR3:QQSKEVPW(SEQ ID NO:6). 2. The composition according to claim 1, wherein the antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. 3. The composition according to claim 2, wherein the light chain constant region is a κ chain or λ chain constant region. 4. The composition according to claim 1, wherein the antibody or a fragment thereof is an isotype of IgG, IgM, IgA, IgE or IgD. 5. The composition according to claim 4, wherein the isotype is IgG1, IgG2, IgG3 or IgG4. 6. The composition according to any one of claims 1-5, wherein the antibody or fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody. 7. The composition according to claim 6, wherein the antibody or a fragment thereof is a humanized antibody. 8. The composition of claim 7, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39. 9. The composition of claim 8, wherein the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:41, SEQ ID NO:43 or SEQ ID NO:45. 10. An isolated cell comprising one or more polynucleotides encoding an antibody or a fragment thereof as defined in any one of claims 1-9. 11. Use of an antibody or fragment thereof as defined in any one of claims 1-9 in the preparation of a medicament for treating cancers expressing human programmed cell death protein 1 (PD-1), wherein the medicament comprises cells treated in vitro with the antibody or fragment thereof. 12. The use according to claim 11, wherein the cancer is selected from the group consisting of: bladder cancer, liver cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. 13. The use according to claim 12, wherein the gastrointestinal cancer is selected from the group consisting of: colon cancer, rectal cancer and gastric cancer. 14. The use according to any one of claims 11-13, wherein the cell is a T cell.