HK40016537A - Anti-pd-1 antibodies and uses thereof - Google Patents

Description

anti-PD-1 antibodies and uses thereof

Background

The cDNA for programmed cell death 1(PD-1) was isolated in 1992 from murine T cell hybridomas and hematopoietic progenitor cell lines that underwent apoptosis. Genetic ablation (genetic ablation) studies have shown that defects in PD-1 result in different autoimmune phenotypes in a variety of mouse strains. PD-1 deficient allogeneic T cells with transgenic T Cell Receptors (TCRs) showed an enhanced response to alloantigens (alloantegen), suggesting that PD-1 on T cells plays a negative regulatory role in responding to antigens.

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 to B7family molecules, was identified independently of PD-1 using molecular cloning and human expression sequence tag database searches, and showed that B7-H1 functions as an inhibitor of human T cell responses in vitro. One year later, the laboratories of Freeman, Wood and Honjo showed that B7-H1 (hereinafter PD-L1) is a binding and functional partner for PD-1, which were pooled from two separate lines of research. Next, it was determined that PD-L1 deficient mice (PD-L1KO mice) were susceptible to induction of autoimmune disease, although this mouse strain did not spontaneously develop such disease. Later, it was demonstrated that the PD-L1/PD-1 interaction plays a dominant role in inhibiting 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 proteins T-cell apoptosis: a potential mechanism of immunological evolution. Nature mechanism.2002; 8(8): 793) and also stimulates IL-10 production in human peripheral blood T cells (Dong H et al, B7-H1, a third mechanism of the B7family, co-stimulation and intercalary-10 secretion. Nature mechanism.1999; 5(12):1365-9) to mediate immunosuppression. The effect of PD-L1 on immunosuppression is now known to be much more complex. In addition to T cell apoptosis and IL-10 induction, PD-L1 may also cause T cell dysfunction through a variety of mechanisms. The PD pathway has also been shown to promote T cell anergy in vitro and in vivo.

Recently, the FDA has approved two PD-1 mAbs for the treatment of human cancer, one from Shibaobao (OpDivo, nivolumab, MDx1106, BMS-936558, ONO-4538) and the other from Merck (Keytruda, pembrolizumab, lambrolizumab, MK-3475). In addition, multiple mabs to PD-1 or PD-L1 are actively being developed in hundreds of clinical trials involving thousands of patients. To date, anti-PD therapy produces significant clinical benefit by inducing regression (regression) and increasing survival of advanced and metastatic tumors. More importantly, anti-PD therapy can have a long-lasting effect, tolerable toxicity, and appear to be applicable to a broad spectrum of cancer types, particularly solid tumors. These clinical findings further confirm the principle of PD pathway blockade and place anti-PD therapy in a distinct category distinct from personalized or tumor type-specific therapy. Because of their unique and non-overlapping mechanisms compared to other cancer treatments, anti-PD therapy is being combined with almost all cancer therapies in an attempt to further expand the efficacy of the treatment. In addition to combining with a variety of cancer immunotherapy approaches (such as cancer vaccines, co-stimulatory and co-inhibitory antibodies, and adoptive cell therapy), various clinical trials have begun to combine anti-PD therapy with chemotherapy, radiation therapy, and targeted therapies.

anti-PD therapy is central in the immunotherapy of human cancers, particularly solid tumors. This therapy differs from previous immunotherapeutics-its primary purpose is to enhance the systemic immune response or to generate new immunity to cancer; in contrast, anti-PD therapy modulates the immune response at the tumor site, targets the tumor-induced immunodeficiency, and restores the ongoing immune response. Although the clinical success of anti-PD therapy for the treatment of a variety of human cancers has demonstrated this approach, we are still studying this pathway and the associated immune responses, which will help in the discovery and design of new clinically applicable cancer immunotherapies.

Disclosure of Invention

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

Thus, according to one embodiment of the present disclosure, there is provided an isolated antibody or fragment thereof specific for human programmed cell death protein 1(PD-L1), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the 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: 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 (SEQ ID NO:11), LCDR3: QHFWGTPWT (SEQ ID NO: 12); and (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).

In some embodiments, the antibodies or fragments of the present disclosure further comprise 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 kappa chain or lambda chain constant region.

In some embodiments, the antibody or fragment thereof may be of an isotype IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG 4. In some embodiments, the antibody or fragment thereof is a chimeric, humanized, or fully human antibody.

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

In another embodiment, the present disclosure provides an isolated antibody or fragment thereof specific for human programmed cell death protein 1(PD-L1), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the 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: 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 (SEQ 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 which comprises one, two or three amino acid additions, deletions, conservative amino acid substitutions or combinations thereof.

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

Also provided in one embodiment is a composition comprising an antibody or fragment thereof of the present disclosure and a pharmaceutically acceptable carrier. In one embodiment, there is also provided an isolated cell comprising one or more polynucleotides encoding the antibody or fragment thereof.

Uses and methods are also provided. In one embodiment, there is provided a use of an antibody or fragment thereof of the present disclosure in the manufacture of a medicament for treating cancer. 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, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. Also provided are methods of treating cancer in a patient in need thereof, comprising administering to the patient an antibody or fragment thereof of the disclosure.

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

In another embodiment, there is provided a use of any one of the antibodies or fragments thereof of the present disclosure in the manufacture of a medicament for treating an infection. In some embodiments, the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

In yet another embodiment, there is provided a use of an antibody or fragment thereof of the present disclosure in the manufacture of a medicament for treating an immune disorder. In some embodiments, the immune disorder is selected from the group consisting of: infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, alzheimer's disease, inflammatory bowel disease, crohn's disease, ulcerative colitis, pelonetz's disease, celiac disease, gallbladder disease, pilocarcinosis (pilonidal disease), peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory diseases of the central and peripheral nervous system, multiple sclerosis, lupus and guillain-barre syndrome, atopic dermatitis, autoimmune hepatitis, fibroalveolar inflammation, glaff's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, wegener's granuloma, pancreatitis, trauma, wound, trauma, or cancer, Graft versus host disease, graft rejection, ischemic disease, myocardial infarction, atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, acid hypoxemia, and infertility associated with maternal-fetal intolerance.

Brief Description of Drawings

FIG. 1 shows that all five hPD-1mAb isoforms bind hPD-1 with high specificity.

FIG. 2 shows that anti-hPD-1 does not bind to hB7-1, hPD-L1, hB7-H3, hB7-H4, and hCD 137.

FIG. 3 shows that hPD-1mAb binds to both human and cynomolgus monkey cell PD-1 proteins, while cross-binding to mPD-1 is not shown.

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

FIG. 5 shows the abrogation (abrogating) and blocking effect of hPD-1mAb when observed in a competitive binding environment.

Figure 6 shows the results of gel electrophoresis analysis confirming the RACE product.

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

Figure 8 shows that the nine humanized antibodies exhibited various binding affinities for PD-1, including higher and lower affinities than the parent antibody.

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

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

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

FIG. 12 shows the proliferative response of MLR to anti-hPD-1 antibody.

FIG. 13 shows IL-2 and IFN γ expression profiles in MLR culture supernatants.

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

Figure 15 shows the in vivo anti-tumor activity of humanized PD-1 antibodies.

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

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

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

FIG. 19 shows binding of test articles to hPD-1 or mPD-1 (ELISA assay).

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

FIG. 21 shows the blocking activity of test preparations on hPD-L1 (left) and hPD-L2 (right) binding to hPD-1 expressing CHOK1 cells.

FIG. 22 shows the levels of IL-2 (left) and IFN- γ (right) in a human MLR assay.

Figure 23 shows IFN- γ levels in engineered tumor and T cell co-culture assays.

Figure 24 shows epitope binding according to ELISA results (upper panel) and a schematic representation of epitope overlap for different test preparations (lower panel).

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

Detailed Description

Definition of

It is noted that the terms "a" or "an" entity refer to one or more of the entity; for example, "an antibody(s)" is understood to represent one or more antibody(s). Thus, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

As used herein, the term "polypeptide" is intended to include both the singular "polypeptide" and the plural "polypeptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, without reference to a particular length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," 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 instead of, or interchangeably with, any of these terms. The term "polypeptide" also means the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization of known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis.

The term "isolated" as used herein with respect to a cell, nucleic acid (e.g., DNA or RNA), refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" as used herein also refers to the following nucleic acids or peptides: substantially free of cellular material, viral material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not naturally occur as fragments and that are not found in nature. The term "isolated" is also used herein to refer to cells or polypeptides that have been separated from other cellular proteins or tissues. Isolated polypeptides are intended to encompass both purified and recombinant polypeptides.

As used herein, the term "recombinant" when used in reference to a polypeptide or polynucleotide means a form of polypeptide or polynucleotide that does not occur naturally, non-limiting examples of which may be constructed by combining polynucleotides or polypeptides that do not normally occur in concert.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then 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 of the present disclosure, although less than 25% identity is preferred.

"sequence identity" of a polynucleotide or polynucleotide region (or polypeptide region) to another sequence in a specified percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that the percentage of bases (or amino groups) are identical when aligned in a comparison of the two sequences. Such alignments and percent 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, the alignment uses default parameters. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: the genetic code is standard; no filter; a strand is two strands; truncation is 60; intended as 10; BLOSUM 62; stated as 50 sequences; selecting a mode of high score; database-non redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation sequence + SwissProtein + SPupdate + PIR. Bioequivalent polynucleotides are those polynucleotides having the percent homology specified above and encoding polypeptides having the same or similar biological activity.

The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. Homologues of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence which has a degree of homology with either it or its complement. In one aspect, a homolog of the nucleic acid is capable of hybridizing to the nucleic acid or a complement thereof. Likewise, an "equivalent polypeptide" refers to a polypeptide that has a degree of homology or sequence identity to 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, an equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof, as compared to a reference polypeptide or polynucleotide. In certain 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 performed under different "stringent" conditions. Typically, low stringency hybridization reactions are performed in about 10x SSC, or equivalent ionic strength/temperature, solution at about 40 ℃. Medium stringency hybridization is typically performed at about 6 XSSC at about 50 ℃ and high stringency hybridization reactions are typically performed at about 1 XSSC at about 60 ℃. The hybridization reaction can also be carried out under "physiological conditions" well known to those skilled in the art. One non-limiting example of physiological conditions are temperature, ionic strength, pH and Mg, which are typically found in cells²⁺The concentration of (c).

The polynucleotide is composed of a specific sequence of four nucleotide bases as follows: adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) replacing thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is a letter representation of a polynucleotide molecule. The 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 co-existence of more than one form of a gene or a portion thereof. A portion of a gene having at least two different forms, i.e., two different nucleotide sequences, is referred to as "a polymorphic region of the gene". A polymorphic region may be a single nucleotide, the identity of which differs on different alleles.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, which may be deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polynucleotide. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling element. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, embodiments of any one polynucleotide of the present disclosure include both double stranded forms and any one of two complementary single stranded forms known or expected to form double stranded forms.

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

As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. The antibody may be a whole antibody and any antigen binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide comprising a molecule that comprises at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen. Examples include, but are not limited to, Complementarity Determining Regions (CDRs) of a heavy or light chain or ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a Framework (FR) region or any portion thereof, or at least a portion of a binding protein.

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

"Single chain variable fragment" or "scFv" refers to the heavy chain (V) of an immunoglobulin_H) And light chain (V)_L) Fusion proteins of variable regions. In some aspects, the regions are linked with a short linker peptide of 10 to about 25 amino acids. The linker may be glycine rich to aid flexibility, and serine or threonine to aid solubility, and V may be substituted_HIs connected to V_LOr vice versa. Despite removal of the constant region and introduction of the linker, the specificity of the original immunoglobulin is retained by the protein. scFv molecules are known in the art and described, for example, in U.S. patent 5,892,019.

The term antibody includes a wide variety of polypeptides that are biochemically distinct. Those skilled in the art will recognize that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, with some subclasses (e.g., gamma l-gamma 4). It is the nature of this chain that determines the "class" of antibodies as IgG, I, respectivelygM, IgA, IgG or IgE. Immunoglobulin subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgG₅Etc., and are known to confer functional specificity. From the perspective of the present disclosure, modified forms of each of these classes and isoforms are readily discernible by those of skill in the art and, therefore, are also encompassed within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion will be directed generally to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule includes two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of 53,000-70,000. These four chains are typically linked by disulfide bonds in a "Y" configuration, where the light chain begins at the "Y" opening and continues to sandwich the heavy chain throughout the variable region.

The antibodies, antigen binding polypeptides, variants or derivatives thereof of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab 'and F (ab')₂Fd, Fv, single chain Fv (scFv), single chain antibody, disulfide linked Fv (sdFv), fragment comprising VK or VH domain, fragment produced by Fab expression library, and anti-idiotypic (anti-Id) antibody (including anti-Id antibody to LIGHT antibody disclosed herein). The immunoglobulin or antibody molecules of the present disclosure may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

Light chains are classified as either kappa or lambda. Each heavy chain class may be associated with a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bonded by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence runs from the N-terminus of the fork of the Y configuration to the C-terminus of the bottom of each chain.

Both the light and heavy chains are divided into multiple regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domain of the light chain portion (VK) and the variable domain of the heavy chain portion (VH) determine antigen recognition and specificity. In contrast, the constant domain of the light Chain (CK) and the constant domain of the heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation, and the like. By convention, the numbering of the constant region domains increases as they move away from the antigen binding site or amino terminus of the antibody. The N-terminal part is a variable region and the C-terminal part is a constant region; the CH3 and CK domains actually comprise the carboxy-termini of the heavy and light chains, respectively.

As described above, the variable regions allow the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK domain and VH domain or a subset of Complementarity Determining Regions (CDRs) of an antibody are combined to form variable regions that define a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by 3 CDRs on each of the VH and VK chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some cases, such as certain immunoglobulin molecules derived from camelid species or engineered immunoglobulins based on camelid immunoglobulins, the entire immunoglobulin molecule may consist of only heavy chains, without light chains. See, for example, Hamers-Casterman et al, Nature 363: 446-.

In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen binding domain when the antibody forms its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain are called "framework" regions, showing less intermolecular variability. The framework regions largely adopt a β -sheet conformation, while the CDRs form loops that connect, and in some cases form part of, the β -sheet structure. Thus, the framework regions serve to form a scaffold that provides for the positioning of the CDRs in the correct orientation through inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on the immunoreactive antigen. The complementary surface facilitates non-covalent binding of the antibody to its corresponding epitope. The amino acids that make up the CDR and framework regions, respectively, for any given heavy or light chain variable region can be readily identified by those of ordinary skill in the art, as they have been precisely defined (see "Sequences of Proteins of immunological Interest," Kabat, E. et al, U.S. department of Health and HumanServices, (1983); and Chothia and Lesk, J.MoI. biol.,196: 901-.

Where there are two or more definitions of a term used and/or accepted in the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. One specific example is the use of "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. 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), which are all incorporated herein by reference in their entirety, describe this particular region. CDR definitions according to Kabat and Couthia include overlaps or subsets of amino acid residues when compared to each other. However, it is intended that any definition applied to refer to the CDRs of an antibody or variant thereof is within the scope of the terms defined and used herein. Suitable amino acid residues encompassing the CDRs defined in each of the above-cited references are listed in the following table for comparison. The exact number of residues covering a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues make up 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 define a numbering system for the variable domain sequences applicable to any antibody. One of ordinary skill in the art can unambiguously 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 set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of immunologicalcateInterest" (1983).

In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at about amino acid position 31 (i.e., about 9 residues after the first cysteine residue), includes about 5-7 amino acids, and terminates at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, comprising about 16-19 amino acids, and terminates at the next arginine or lysine residue. CDR-H3 begins at about the thirty-third 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 about residue 24 (i.e., after the cysteine residue); including about 10-17 residues; and terminates at the next tryptophan residue. CDR-L2 begins at about the sixteenth residue after the end of CDR-L1 and comprises about 7 residues. CDR-L3 begins at about the thirty-third residue (i.e., after the cysteine residue) after the end of CDR-L2; comprising about 7-11 residues and terminating in the sequence F or W-G-X-G, wherein X is any amino acid.

The antibodies disclosed herein can be from any animal source, including birds and mammals. Preferably, the antibody is a human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibody. 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 amino acid sequences derived from immunoglobulin heavy chains. The heavy chain constant region-containing polypeptide comprises at least one of a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use in the present disclosure can include a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptides of the present disclosure include polypeptide chains comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, it will be understood by those of ordinary skill in the art that the heavy chain constant regions may be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

The heavy chain constant regions of the antibodies disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a heavy chain constant region derived from an IgG₃The hinge region of the molecule. In another example, the heavy chain constant region may comprise a portion derived from an IgG₁The molecules and moieties are derived from IgG₃The hinge region of the molecule. In another example, the heavy chain portion may include a portion from IgG₁The molecules and moieties are derived from IgG₄Chimeric hinges of molecules.

As used herein, the term "light chain constant region" includes amino acid sequences derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

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

As previously mentioned, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH 1 domain" includes the first (amino-terminal-most) constant region domain of an immunoglobulin heavy chain. The CH1 domain is located adjacent to the VH domain, amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

The term "CH 2 domain" as used herein includes the portion of the heavy chain molecule that extends from about residue 244 to residue 360 of an antibody, for example, using conventional numbering schemes (residues 244 to 360, Kabat numbering system, and residues 231 and 340, EU numbering system; see Kabet et al, U.S. Dept. of Health and Humanservices, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique in that it does not pair closely with another domain. In contrast, two N-linked branched sugar chains were inserted between the two CH2 domains of the intact native IgG molecule. The CH3 domain is reliably recorded as extending from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

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

The term "disulfide bond" as used herein includes a covalent bond formed between two sulfur atoms. The amino acid cysteine includes a thiol group, which may 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 using Kabat numbering system (226 th or 229 th, EU numbering system).

As used herein, the term "chimeric antibody" will be taken to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species, while the constant region (which may be intact, partial or modified in accordance with the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate), while the constant region is human.

As used herein, "percent humanization" is calculated by determining the number of framework amino acid differences (i.e., non-CDR differences) between the humanized and germline domains, subtracting that number from the total number of amino acids, then dividing it by the total number of amino acids and multiplying by 100.

"specific binding" or "specific for … …" generally means that an antibody binds to an epitope through its antigen-binding domain, and that the binding requires some complementarity between the antigen-binding domain and the epitope. By this definition, an antibody, when referred to as "specifically binding" to an epitope, binds to the epitope more readily through its antigen binding domain than it binds to a random, unrelated epitope. The term "specificity" is used herein to characterize the relative affinity of an antibody for binding to an epitope. For example, antibody "a" may be considered to have a higher specificity for a given epitope than antibody "B", or antibody "a" may be said to bind epitope "C" with a higher specificity than the relevant epitope "D".

As used herein, the terms "treatment" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, with the purpose of preventing or slowing (reducing) the progression of an undesirable physiological change or disorder, such as cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) condition, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), whether detectable or undetectable. "treatment" also means an increase in survival compared to the expected survival when not receiving treatment. Persons in need of treatment include those already with the condition or disorder, as well as those susceptible to the condition or disorder, or those in need of prevention of the condition or disorder.

By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, domestic animals, livestock, zoo animals, sport animals or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, etc.

As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure, e.g., for detection, for diagnosis, and/or for treatment.

anti-PD-1 antibodies

The present disclosure provides anti-PD-1 antibodies with high affinity for human PD-1 protein. The antibodies tested exhibit potent binding and inhibitory activity and are useful for therapeutic and diagnostic purposes. In addition, one of the humanized antibodies tested (TY101) exhibited significantly higher binding affinity than the two FDA-approved anti-hPD-1 antibodies.

Accordingly, one embodiment of the present disclosure provides an anti-PD-1 antibody or fragment thereof that can specifically bind to human programmed death 1(PD-1) protein.

According to one embodiment of the present disclosure, there is provided an antibody comprising heavy and light chain variable regions having CDR regions of one of the CDR sets as in table 1.

Table 1: sequences of CDR regions

For example, in one embodiment, an isolated antibody or fragment thereof specific for human apoptosis protein 1(PD-1) is provided, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the 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), LCDR3: QQSVPW (SEQ ID NO: 6).

For example, in one embodiment, an isolated antibody or fragment thereof specific for human apoptosis protein 1(PD-1) is provided, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a 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: 3612).

For example, in one embodiment, an isolated antibody or fragment thereof specific for human apoptosis protein 1(PD-1) is provided, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a 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), LCDR3: QKDVPW (SEQ ID NO: 18).

As shown in the experimental examples, antibodies containing these CDR regions, whether murine, humanized or chimeric, have potent PD-1 binding and inhibitory activity. Further computer modeling suggests that certain residues within the CDRs can be modified to retain or improve the properties of the antibody. In some embodiments, an anti-PD-1 antibody of the present disclosure includes the VH and VL CDRs listed in table 1 with one, two, or three further modifications. Such modifications may be additions, deletions or substitutions of amino acids.

In some embodiments, the modification is a substitution at no more than one hotspot position 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 substitutions are conservative substitutions.

A "conservative amino acid substitution" is an amino acid substitution in which the amino acid residue is replaced 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). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, the amino acid string may be replaced with a structurally similar string that differs in the order and/or composition of the side chain family members.

Non-limiting examples of conservative amino acid substitutions are provided in the following table, wherein a similarity score of 0 or higher indicates a conservative 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 substitutions

Non-limiting examples of VH's are provided in SEQ ID NO 27, 31, 35, 37 and 39. SEQ ID NO:27 is the murine VH. 31 is the VH of the chimeric antibody, while SEQ ID NO: 35. SEQ ID NO: 37. 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 murine VL. 33 is the VL of the chimeric antibody, while SEQ ID NO: 41. SEQ ID NO: 43. SEQ ID NO:45 is humanized.

In some embodiments, an anti-PD-1 antibody of the present disclosure comprises SEQ ID NO: 27. SEQ ID NO: 31. SEQ ID NO: 35. SEQ ID NO:37 or SEQ ID NO:39, VH of SEQ ID NO: 29. VL of SEQ ID NO 33, 41, 43 or 45 or their respective bioequivalents. A VH or VL bioequivalence is a sequence comprising the specified amino acids, while overall having 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. For example, a bioequivalence of SEQ ID NO:27 can be a VH that has 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the entirety of SEQ ID NO:27, but retains the CDRs.

One of ordinary skill in the art will also appreciate that the antibodies disclosed herein can be modified such that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a given protein may be similar, e.g., have a certain percentage of identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

In certain embodiments, an antibody comprises an amino acid sequence or one or more portions that are not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, antibodies of the present disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, drug, toxin, or tag).

The antibodies, variants or derivatives thereof of the present disclosure 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 without intending to be limiting, antibodies may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization of known protecting/blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be made by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the antibody may contain one or more atypical amino acids.

In some embodiments, the antibody may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

The antibody may be conjugated or fused to a therapeutic agent, which may include a detectable label, such as a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, an optically active therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or toxin, an ultrasound enhancing agent, a nonradioactive label, combinations thereof and other such agents known in the art.

The antibody may be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts and oxalate esters.

Antibodies can also be made using fluorescence emitting metals such as¹⁵²Eu or other lanthanides are detectably labeled. These metals can be attached to the antibody with a metal chelating group such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA). Techniques For conjugating various moieties to Antibodies are well known, see, e.g., Arnon et al, "monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", monoclonal Antibodies And Cancer Therapy, edited by Reisfeld et al, pages 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al, "Antibodies For Drug Delivery", Controlled Drug Delivery (2 nd edition), edited by Robinson et al, Marcel Dekker, inc., pages 623-53 (1987); thorpe, "antibodies Of Cytotoxic Agents In Cancer Therapy: A Review", monoclonal antibodies'84: Biological And Clinical Applications, Pinchera et al eds., pp.475-; "Analysis, Results, And d Future productive Of The Therapeutic Use Of radioactive enhanced In Cancer Therapy", Monoclonal Antibodies For Cancer Therapy And Therapy, compiled by Baldwin et al, Academic Press, pp.303-16 (1985), And by Thorpe et al, "The Preparation Of Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.Rev. (52:119-58 (1982)).

Bifunctional molecules

PD-1 is an immune checkpoint molecule and is also a tumor antigen. As a tumor antigen targeting molecule, an antibody or antigen binding fragment specific for PD-1 can be conjugated to a second antigen binding fragment specific for immune cells to produce a bispecific antibody.

In certain embodiments, the immune cell is 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 immune cells that can be targeted include, for example, CD3, CD16, CD19, CD28, and CD 64. 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 immunoglobulin-like receptor (KIR), and CD 47. Specific examples of bispecific include, but are not limited to, PD-L1/PD-1, PD-1/LAG3, PD-1/TIGIT, and PD-1/CD 47.

As an immune checkpoint inhibitor, an antibody or antigen-binding fragment specific for PD-1 can be conjugated to a second antigen-binding fragment specific for a tumor antigen to produce a bispecific antibody. A "tumor antigen" is an antigenic substance produced in tumor cells, i.e., it triggers an immune response in the host. Tumor antigens are useful in identifying tumor cells and are potential candidates for cancer therapy. Normal proteins in vivo are not antigenic. However, certain proteins are produced or overexpressed during tumorigenesis and thus appear to be "foreign" to the body. This may include normal proteins that are well-isolated from the immune system, proteins that are usually produced in very small amounts, proteins that are usually produced only at certain developmental stages, or proteins that are structurally modified due to mutations.

Abundant tumor antigens are known in the art, and novel tumor antigens can be readily identified by screening. Non-limiting examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, α V β 3, α 5 β 1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and Tenascin (Tenascin).

In certain aspects, the monovalent units are specific for proteins that are overexpressed on tumor cells compared to corresponding non-tumor cells. As used herein, "corresponding non-tumor cell" refers to a non-tumor cell that is of the same cell type as the tumor cell origin. Notably, this protein is not necessarily distinct from the tumor antigen. Non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in most colon, rectal, breast, lung, pancreatic, and gastrointestinal cancers; heregulin receptor (HER-2, neu or c-erbB-2), which is frequently overexpressed in breast, ovarian, colon, lung, prostate and cervical cancers; epidermal Growth Factor Receptor (EGFR), which is highly expressed in a range of solid tumors, including breast, head and neck, non-small cell lung, and prostate cancers; a sialoglycoprotein receptor; a transferrin receptor; a serine protease inhibitor (serpin) enzyme complex receptor, which is expressed on hepatocytes; a Fibroblast Growth Factor Receptor (FGFR) that is overexpressed in pancreatic ductal adenocarcinoma cells; vascular Endothelial Growth Factor Receptor (VEGFR) for use in anti-angiogenic gene therapy; folate receptor, which is selectively overexpressed in 90% of non-mucinous ovarian cancers; cell surface glycocalyx (glycocalyx); a carbohydrate receptor; and polymeric immunoglobulin receptors, which are useful for gene delivery to respiratory epithelial cells, and are attractive means for treating pulmonary diseases, such as cystic fibrosis. Non-limiting examples of bispecific 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 of the anti-PD-1 fragment and the second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

Bifunctional molecules that include not only antibodies or antigen-binding fragments are also provided. As a tumor antigen targeting molecule, an antibody or antigen binding fragment specific for PD-1 (such as those described herein) can optionally be combined with an immunocytokine or ligand through a peptide linker. Linked immunocytokines 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 may combine immune checkpoint blockade effects with local immune modulation at the tumor site.

Polynucleotides encoding antibodies and methods of making antibodies

The present disclosure also provides isolated polynucleotides or nucleic acid molecules (e.g., SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46) encoding the antibodies, variants, or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, variants or derivatives thereof, on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, variants or derivatives thereof, on the same polynucleotide molecule or on separate polynucleotide molecules.

Methods of making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen binding polypeptides of the present disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and techniques described herein. For example, fully human antibodies against a particular antigen can be prepared by: antigens are administered to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Pat. nos. 6,150,584; 6,458,592, respectively; 6,420,140, which are herein incorporated by reference in their entirety.

In certain embodiments, the antibodies produced do not elicit a deleterious immune response in the animal to be treated, e.g., a human. In one embodiment, the antigen binding polypeptides of the present disclosure, variants or derivatives thereof are modified to reduce their immunogenicity using art-recognized techniques. For example, the antibody may be humanized, primatized, deimmunized or chimeric. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen binding properties of the parent antibody, but are less immunogenic in humans. This can be achieved by various methods, including (a) grafting the entire non-human variable domain to a human constant region to produce a chimeric antibody; (b) grafting at least a portion of one or more non-human Complementarity Determining Regions (CDRs) into a human framework and constant region, with or without retention of critical framework residues; or (c) transplanting the entire variable region domain of non-human origin, but "stealth" with a human-like segment by surface residue substitution. 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-; padlan, Molec.Immun.25:489-498 (1991); padlan, Molec. Immun.31:169-217(1994), and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are incorporated herein by reference in their entirety.

Deimmunization may also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes altering antibodies to modify T cell epitopes (see, e.g., International application publication Nos.: WO/9852976A 1 and WO/0034317A 2). For example, the variable heavy and variable light chain sequences from the starting antibody are analyzed, and a human T cell epitope "map" is constructed from each V region showing the epitopes associated with the Complementarity Determining Regions (CDRs) and the positions of other critical residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions with low risk of altering the final antibody activity. A series of alternative variable heavy and variable light chain sequences were designed that included combinations of amino acid substitutions and these sequences were subsequently incorporated into a series of binding polypeptides. Typically, 12 to 24 variant antibodies are generated and tested for binding and/or function. The complete heavy and light chain genes comprising 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 are then compared in appropriate biochemical and biological assays and the best variant identified.

The binding specificity of an antigen-binding polypeptide of the present disclosure can be determined by in vitro assays, such as immunoprecipitation, Radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

Alternatively, the single stranded units of the present disclosure can be produced using the techniques described for producing single stranded units (U.S. Pat. No. 4,694,778; Bird, Science 242: 423-. Single chain units are formed by connecting the heavy and light chain fragments of the Fv region via an amino acid bridge, thereby producing a single chain fusion peptide. The technique of assembling functional Fv fragments in E.coli (E.coli) can also be used (Skerra et al, Science 242:1038-1041 (1988)).

Examples of techniques that can be used to produce single chain fv (scFv) and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498, Huston et al, Methods in Enzymology 203:46-88 (1991); shu et al, Proc.Natl.Sci.USA 90:1995-1999 (1993); and those described in Skerra et al, Science 240: 1038-. For some applications, including in vivo applications of antibodies in humans and in vitro detection assays, it is preferred to use chimeric, humanized or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as an antibody having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods of producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); oi et al, BioTechniques 4:214 (1986); gillies et al, J.Immunol.methods 125:191-202 (1989); U.S. patent No. 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 the 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 regions will be substituted with corresponding residues from the CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction of the CDRs 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, e.g., Queen et al, U.S. Pat. No. 5,585,089; Riechmann et al, Nature 332:323(1988), which is incorporated herein by reference in its entirety). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), veneering (surfacing) or resurfacing (EP 592,106; EP 519, 596; Padlan, Molecular Immunology 28(4/5): 489-.

Fully human antibodies are particularly desirable for treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods, which use antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. nos. 4,444,887 and 4,716,111; and PCT publications nos. 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 express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells at random or by homologous recombination. 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 rendered non-functional either alone or simultaneously with introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice were then bred to produce homozygous progeny expressing the human antibody. The transgenic mice are immunized with a selected antigen (e.g., all or a portion of the desired polypeptide of interest) in a normal manner. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B-cell differentiation, followed by class switching and somatic mutation. Thus, using this technique, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced. For an overview of this technique for generating human antibodies, see Lonberg and Huszarrint. Rev. Immunol.73:65-93 (1995). For a detailed discussion of such techniques for producing human antibodies and human monoclonal antibodies and procedures for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. patent No. 5,413,923; 5,625,126, respectively; 5,633,425, respectively; 5,569,825; 5,661,016, respectively; 5,545,806; 5,814, 318; and 5,939,598, which is incorporated by reference herein in its entirety. In addition, companies such as Abgenix, inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) may be contacted to provide human antibodies to selected antigens using techniques similar to those described above.

Fully human antibodies recognizing selected epitopes can also be generated using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope. (Jespers et al, Bio/Technology 72:899-903(1988) see also U.S. Pat. No. 5,565,332, which is incorporated herein by reference in its entirety)

In another embodiment, DNA encoding the desired monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Isolated and subcloned hybridoma cells are used as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into prokaryotic or eukaryotic host cells, such as E.coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins. More specifically, isolated DNA (which may be synthetic as described herein) may be used to clone constant and variable region sequences for antibody production as described in Newman et al, U.S. Pat. No. 5,658,570, filed 1, 25, 1995, which is incorporated herein by reference. Basically, this requires extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig-specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody can be grown in relatively large numbers to provide a clinical and commercial supply of immunoglobulin.

Furthermore, using conventional recombinant DNA techniques, one or more CDRs of an antigen binding polypeptide of the present disclosure can be inserted into a framework region, e.g., into a human framework region to humanize a non-human antibody. The framework regions may be naturally occurring or shared framework regions, preferably human framework regions (see, e.g., Chothia et al, J.mol.biol.278: 457-. Preferably, the polynucleotides produced by the combination of framework regions and CDRs encode an antibody that specifically binds to at least one epitope of a desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions may be made within the framework regions, and preferably the amino acid substitutions enhance binding of the antibody to its antigen. In addition, these methods can be used to substitute or delete amino acids of one or more variable region cysteine residues involved in an intrachain disulfide bond to produce an antibody molecule lacking one or more intrachain disulfide bonds. Other variations of polynucleotides are encompassed by the present disclosure, which are also within the skill of the art.

Furthermore, techniques developed for the production of "chimeric antibodies" (Morrison et al, Proc. Natl. Acad. Sci. USA:851-855 (1984); Neuberger et al, Nature 372:604-608 (1984); Takeda et al, Nature 314:452-454(1985)) by splicing together genes from mouse antibody molecules with genes from human antibody molecules with appropriate biological activity can be used. As used herein, chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

Another efficient method for producing recombinant antibodies has been disclosed so far by Newman, Biotechnology 10:1455-1460 (1992). In particular, the technology results in the production of primatized antibodies comprising monkey variable domains and human constant sequences. This document is incorporated by reference herein in its entirety. In addition, this technique is also described in U.S. Pat. nos. 5,658,570, 5,693,780, and 5,756,096, each of which is incorporated herein by reference, having the same assignee (common assigned).

Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to those skilled in the art. This technique is described in various laboratory manuals and original publications. In this regard, techniques suitable for use in the present disclosure, as described below, are described in Current Protocols in Immunology, Coligan et al, Green publishing associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated by reference herein in its entirety, including supplements.

In addition, standard techniques known to those skilled in the art that can be used to introduce mutations in the nucleotide sequences encoding antibodies disclosed herein include, but are not limited to, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions. Preferably, the variant (including derivatives) encodes less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to a 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 of the disclosure are useful in certain therapeutic and diagnostic methods.

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

The antibodies of the present disclosure may also be used to treat or inhibit cancer. PD-1 may be overexpressed in tumor cells. Tumor-derived PD-1 can bind to PD-L1 on immune cells, thereby limiting anti-tumor T cell immunity. The results in murine tumor models with small molecule inhibitors, or monoclonal antibodies targeting PD-1, indicate that targeted PD-1 treatment is an important alternative and realistic approach to effectively control tumor growth. As shown in the experimental examples, anti-PD-1 antibodies activate adaptive immune response mechanisms, which may lead to improved survival of cancer patients.

Accordingly, in some embodiments, methods for treating cancer in a patient in need thereof are provided. In one embodiment, the method involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one cancer cell (e.g., stromal cell) in the patient expresses, overexpresses, or is induced to express PD-1. For example, the induction of PD-1 expression can be accomplished by administration of 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, urinary tract cancer, colorectal cancer, head and neck cancer, squamous cell cancer, Merck cell cancer, gastrointestinal tract cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, and small cell lung cancer. Accordingly, the antibodies disclosed herein may be used to treat any one or more of such cancers.

The disclosure also provides cell therapies, such as Chimeric Antigen Receptor (CAR) T cell therapies. Suitable cells can be used, contacted with (or engineered to express) an anti-PD-1 antibody of the disclosure. After such contacting or engineering, the cells can be introduced into a cancer patient in need of treatment. A cancer patient may have any type of cancer disclosed herein. For example, the cells (e.g., T cells) can be tumor infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof, but are not limited thereto.

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

Other diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and/or prognosed with an antibody or variant or derivative thereof of the present disclosure include, but are not limited to, progression and/or metastasis of malignancies, and related disorders such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelogenous leukemia (including medulloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia) and chronic leukemias (e.g., chronic myelogenous (myelogenous) leukemia) and chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., hodgkin's disease and non-hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, polycythemia, and leukemia, Myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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 cancer, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchial cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, angioblastoma, angioma, angiosarcoma, neuroblastoma, Acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

Treatment of infections and immune disorders

As shown in the experimental examples, the antibodies of the present disclosure can activate an immune response and thus can be used to treat an infection.

Infections are the invasion of the body tissues of organisms by pathogens (agents) causing disease, the proliferation of said pathogens, and the reaction of host tissues 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 large parasites (macroparasities) such as tapeworms and other worms. In one aspect, the infectious agent is a bacterium, such as a gram-negative bacterium. In one aspect, the infectious agent is a virus, such as a DNA virus, an RNA virus, and a retrovirus. Non-limiting examples of viruses include adenovirus, Coxsackievirus (Coxsackievirus), Epstein-Barr virus, hepatitis a virus, hepatitis b virus, hepatitis c virus, herpes simplex virus-1, herpes simplex virus-2, cytomegalovirus, human herpes virus-8, HIV, influenza virus, measles virus, mumps virus, human papilloma virus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, varicella zoster virus.

In certain embodiments, methods or uses of the antibodies or fragments thereof for treating immune disorders are also provided. Non-limiting examples of immune disorders include infection, infection-related endotoxic shock, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Pair's disease, celiac disease, gallbladder disease, Tibetan hair disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory diseases of the central and peripheral nervous system, multiple sclerosis, lupus and Guillain-Barre syndrome, atopic dermatitis, autoimmune hepatitis, fibroalveolar inflammation, Grave disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, inflammatory diseases of the prostate, inflammatory diseases of the bladder, inflammatory diseases of the, Pancreatitis, trauma, graft versus host disease, graft rejection, ischemic disease, myocardial infarction, atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, acid stomach deficiency, and infertility associated with fetal maternal tolerance deficiency.

The antibodies of the present disclosure may also be used to treat infectious diseases caused by microorganisms, or to kill microorganisms, by targeting microorganisms and immune cells to achieve elimination of microorganisms. In one aspect, the microorganism is a virus, gram positive bacterium, gram negative bacterium, protozoan, or fungus, including RNA and DNA viruses.

The specific dose and treatment regimen for any particular patient will depend upon a variety of factors including the particular antibody, variant or derivative thereof used, the age, body weight, general health, sex and diet of the patient, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of these factors by a medical caregiver is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease and the effect desired. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

Methods of administration of the antibodies, variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition may be administered by any convenient route, for example by infusion or bolus injection (bolus injection), absorbed through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other bioactive agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), buccally, or as an oral or nasal spray.

The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

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

It may be desirable to locally administer an antibody polypeptide or composition of the present disclosure to an area in need of treatment; this may be achieved, for example, by local infusion during surgery, local application (e.g. fitting in a wound dressing after surgery), by injection, by means of a catheter, by means of a suppository, or by means of an implant which is a porous, non-porous or gelatinous material including membranes such as silicone rubber (sialastic) membranes or fibres, but is not limited thereto. Preferably, when administering proteins of the present disclosure (including antibodies), 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-

In yet another embodiment, the antigen binding polypeptide 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, eds. Langer and Wise, CRC Press, Boca Raton, Fla. (1974); Controlled Drug bioavailability, Drug Product Design and Performance, Smolen and Ball eds., Wiley, NewYork (1984); Ranger and Peppas, J.,1983, Macromol. Sci. Rev. Macromol. Chem.23: 61; see also Levy et al, 1985, Science 228: 190; During et al, 1989, Ann. Neurol.25: 351; Howard et al, 1989, J. Neurosurg.71: 105). In yet another embodiment, the controlled Release system may be placed near the target of treatment (i.e., the brain), so that only a small fraction of the systemic dose is required (see, e.g., Goodson, in Medical Applications of controlled Release, supra, Vol.2, pp.115-138 (1984)). Other controlled release systems are discussed in the review by Langer (1990, Science 249: 1527) -1533).

In particular embodiments of the compositions of the present disclosure comprising a nucleic acid or polynucleotide encoding a protein, the nucleic acid can be administered in vivo to facilitate expression of the protein encoded thereby by: it is constructed as part of a suitable nucleic acid expression vector and administered, for example, by using a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by using microprojectile bombardment (e.g., gene gun; Biolistic, Dupont), or coated with lipid or cell surface receptors or transfection agents, or by ligating it to a cognate cassette-like peptide known to enter the nucleus of the cell (see, for example, Joliot et al, 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868) to allow it to enter the cell. Alternatively, the nucleic acid may be introduced into the cell and incorporated into the host cell DNA for expression by homologous recombination.

The amount of an antibody of the present disclosure that will be effective in treating, inhibiting, and preventing an inflammatory, immune, or malignant disease, disorder, or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the severity of the disease, disorder or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

As a general recommendation, the dose of an antigen-binding polypeptide of the present disclosure administered to a patient 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 10mg/kg of patient body weight. Generally, human antibodies have a longer half-life in humans than antibodies from other species due to the immune response to the foreign polypeptide. Thus, lower doses of human antibodies and less frequent administration are often possible. In addition, by enhancing uptake and tissue penetration (e.g., into the brain) of the antibody (modified by, e.g., lipidation), the dosage and frequency of administration of the antibodies of the disclosure can be reduced.

Methods of treating an infection or malignant disease, condition, or disorder comprising administering an antibody, variant, or derivative thereof of the present disclosure are typically tested in vitro and then in vivo for the desired therapeutic or prophylactic activity in an acceptable animal model, prior to use in humans. Suitable animal models, including transgenic animals, are well known to those of ordinary skill in the art. For example, in vitro assays to demonstrate the therapeutic utility of the antigen binding polypeptides described herein include the effect of the antigen binding polypeptides on cell lines or patient tissue samples. The effect of the antigen-binding polypeptide on a cell line and/or tissue sample can be determined using techniques known to those skilled in the art, such as the assays disclosed elsewhere herein. In accordance with the present disclosure, in vitro assays useful for determining whether to administer a particular antigen binding polypeptide are indicated, including in vitro cultured cell assays in which a patient tissue sample is grown in culture and exposed to or otherwise administered a compound, and the effect of such compound on the tissue sample is observed.

A variety of delivery systems are known and can be used to administer the antibodies of the present disclosure or polynucleotides encoding the antibodies of the present disclosure, e.g., embedded in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J.biol.chem.262: 4429. sup. 4432), nucleic acid construction as part of a retrovirus or other vector, and the like.

Diagnostic method

Over-expression of PD-1 is observed in certain tumor samples, and patients with PD-1 overexpressing cells may respond to treatment with the anti-PD-1 antibodies of the present disclosure. Thus, the antibodies of the present disclosure may also be used for diagnostic and prognostic purposes.

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

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

Composition comprising a metal oxide and a metal oxide

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

In certain embodiments, the term "pharmaceutically acceptable" refers to a drug that is approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In addition, a "pharmaceutically acceptable carrier" will typically be a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid of any type.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates or phosphates, if desired. Antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and tonicity adjusting agents such as sodium chloride or dextrose. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated into suppository with conventional binder and carrier such as triglyceride. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical carriers are described by martin Remington's Pharmaceutical Sciences (incorporated herein by reference). Such compositions will comprise a therapeutically effective amount of the antigen-binding polypeptide (preferably in purified form) together with an appropriate amount of carrier to provide a form suitable for administration to a patient. The formulation should be suitable for the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In one embodiment, the composition is formulated in accordance with conventional procedures as a pharmaceutical composition suitable for intravenous administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may further include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Typically, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container (e.g., ampoule or sachet) that indicates the quantity of active agent. Where the composition is administered by infusion, it may be administered using an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

The compounds of the present disclosure may be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, and tartaric acids, and the like, and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Examples

Example 1: generation of human monoclonal antibodies to human PD-1

Cloning of full-Length human PD-1cDNA

Human T lymphocytes were isolated from human peripheral blood lymphocytes (PBMC) using MACS beads (Miltenyi Biotec). Total RNA was extracted from human T cells using RNeasy Mini kit (Qiagen) and cDNA was obtained by reverse transcription PCR (SuperScriptfirst-Strand Synthesis System, Invitrogen). A full-length cDNA encoding hPD-1 was generated by RT-PCR from human T-cell mRNA using a sense primer (5 '-CTGTCTAGAATGCAGATCCCACAGGCGCC, SEQ ID NO: 47) and an antisense primer (5' GGATCCTCAGAGGGGCCAAGAGCAGT, SEQ ID NO: 48). The sequence was verified by DNA sequencing and comparison with the NCBI database (NM-005018.2).

Establishing hPD-1 stable expression cell line: after digestion with Xbal and BamHI, the hPD-1 PCR fragment was cloned into the PCDA3.1(-) vector (Invitrogen). The pcDNA-hPD-1 full-length plasmid was then transfected into Chinese Hamster Ovary (CHO) cells using lipofectamine 2000 (Invitrogen). Cell lines stably expressing hPD-1 (CHO/hPD-1) were selected by G418 and screened by flow cytometry.

Production of human PD-1Ig fusion protein: the cDNA of the hPD-1mIg and hPD-1 hIg fusion proteins comprising the hPD-1 ectodomain were amplified from full-length pcDNA-hPD-1 by PCR with specific primers. The PCR fragment digested with EcoRI and BglII was fused to the mouse IgG2a heavy chain in the expression plasmid pmIgG or to the CH2-CH3 domain in the human IgG1 heavy chain of the expression plasmid phIgG (H Dong et al Nat Med.1999; 5: 1365-. The proteins in the culture supernatant were purified by Protein a sepharose column (HiTrap Protein a HP, GE healthcare). The purified protein was confirmed by SDA-PAGE electrophoresis.

Production of monoclonal antibodies: female Balb/c mice, 8-10 weeks old, were immunized subcutaneously (s.c.) at various sites with 200 μ L of an emulsion containing 100 μ g of hPD-1mIg fusion protein and Complete Freund's Adjuvant (CFA) (Sigma Aldrich). After 3 weeks, mice were immunized three times with 50-100 μ g subcutaneous protein (s.c.) with Incomplete Freund's Adjuvant (IFA) (Sigma Aldrich). Mice were bled 2 weeks after each immunization for serum titer determination. When the titers were sufficient, mice were immunopotentiated (boost) by intraperitoneal (i.p.) injection of 60 μ g of protein in PBS. Hybridomas were obtained by fusing spleen cells of immunized mice with the SP2/0-Ag14 myeloma cell line (from ATCC). The immunocompetent mice were sacrificed with carbon dioxide and spleens were harvested aseptically. The whole spleen was dissociated into single cell suspensions and red blood cells were lysed with ACK buffer. SP2/0-Ag14 myeloma and splenocytes were mixed in a 1:1 ratio in a 50 ml conical centrifuge tube. After centrifugation, the supernatant was discarded and cell fusion was performed with 50% polyethylene glycol (Roche). The fused cells were cultured in HAT selection medium for 8-10 days, the supernatants of the hybridoma cultures were screened for binding to hPD-1 expressing cells in 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 analysis. Positive hybridomas were subcloned at least 5 times using limiting dilution techniques to achieve pure monoclonal cultures.

Example 2: characterization of the PD-1 monoclonal antibody

Isotype of mAb: the isotype of the mAb was identified using the Mouse Immunoglobutin Isotyping Kit (BD Biosciences). All five PD-1 mAbs were identified as IgG1 isotype and kappa chain.

Binding specificity against hPD-1: specificity of PD-1mAb was determined by flow cytometry using CHO cells expressing hPD-1 on the surface (CHO/hPD-1). CHO/hPD-1 cells were incubated with anti-PD-1 mAb on ice. After incubation, cells were washed and further incubated with anti-mIgG-APC (ebiosciences). Flow cytometry analysis was performed using facsverse (bd biosciences). The data show that all five hPD-1 mAbs bind hPD-1 with high specificity (FIG. 1). To exclude the possibility of binding of hPD-1mAb 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 mAb by flow cytometry analysis. These cells were also stained with their respective positive antibodies as positive controls. The data indicate that anti-PD-1 mAb did not bind to these tested proteins (FIG. 2).

Species cross-reactivity: to evaluate the species specificity of the anti-hPD-1 mAb, Peripheral Blood Mononuclear Cells (PBMC) from cynomolgus monkeys (from Guangdong landau Biotechnology Company) were isolated from peripheral blood using Ficoll (Sigma Aldrich). PBMC were suspended in RPMI 1640 medium containing 10% FCS and placed in 24-well plates precoated with 1. mu.g/ml of anti-hCD 3. The cells were cultured for 2 days. Cells were first stained with anti-hPD-1. After washing, cells were treated with anti-mIgG-APC and CD 3-FITC; CD8-PerCP staining for flow cytometry analysis. In addition, cross-reactivity of mAbs to mouse PD-1 was determined by flow cytometry using CHO cells transfected with mouse PD-1 (CHO/mPD-1).

The data indicate that the anti-hPD-1 mAb binds to PD-1 protein on both human and cynomolgus T cells, and no cross-binding was found to mouse PD-1 (fig. 3).

Ligand blocking: to examine the blocking of ligand binding, 100ng of hPD1hIg fusion protein was preincubated with the indicated doses of mAb (400ng/10ul, 300ng/10ul, 200ng/10ul, 100ng/10ul, 50ng/10ul) or control Ig at 4 ℃ for 30 min before being used to stain CHO/hB7-H1 cells. Cells were washed and further stained with goat anti-hIgG-APC. The blocking effect was examined by flow cytometry.

The data show that anti-hPD-1 mAbs 1 and 2(Ab1 and Ab2) had no effect on ligand blockade. Ab3, Ab4, and Ab5 blocked binding of hPD-1 fusion protein to hPD-L1 in a dose-dependent manner (fig. 4).

Competitive binding assay: competitive binding assays were 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 with an excess (10. mu.g) of 5 PD-1 mAbs, respectively, for 30 minutes at 4 ℃. After washing, cells were incubated with 50ng of different biotin-labeled mAbs for 20 minutes at 4 ℃. The binding of the mAb was measured using flow cytometry analysis.

Flow cytometry analysis showed that Ab4 and Ab5 completely abolished each other's binding to hPD-1 protein, that Ab3 at saturating doses had a partial blocking effect on Ab4 and Ab5 binding, and that Ab1 and Ab2 had no blocking effect on Ab4 and Ab5 binding to hPD-1 (fig. 5). Thus, the binding sites of Ab4 and Ab5 may overlap on PD-1. Ab1 or Ab2 and Ab4 or Ab5 bound to PD-1 through different interfaces, as also demonstrated by ligand blocking assays.

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

Sequencing of hybridomas producing anti-PD-1 antibodies: harvest 1X 10⁷Individual hybridoma cellAnd 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 RACECDNAamplification Kit (Clontech). After reverse transcription, 5 ' RACE PCR reactions were performed using the prepared cDNA as a template, 5 ' Universal Primers (UPM) provided with the kit, and 3' gene specific primers (GSP1) designed from the mouse IgG1 heavy chain variable region and kappa light chain gene sequences. RACE products were determined by gel electrophoresis analysis (FIG. 6). The PCR product was cloned into the T-vector using Zero Blunt TOPO PCR Cloning Kit (Invitrogen). After transformation, the plasmid was verified by sequencing analysis. Analysis of antibody gene fragments was performed using VBASE2(http:// www.vBas2.org). The sequences are disclosed in (table 2).

Table 2: murine antibody sequences

Protein expression and functional determination of recombinant antibodies: to ensure the correctness of the recombinant antibody sequences, the full-length sequences of the recombinant antibody heavy and light chains were cloned into pcdna3.1 vectors, respectively, and HEK 293T cells were transiently transfected. Proteins from cell culture supernatants were purified using protein G sepharose columns (GE healthcare) for functional evaluation.

Flow cytometry analysis data showed that recombinant antibody can bind hPD-1 protein and block binding of hPD-1 fusion protein to PD-L1 protein (FIG. 7, FIG. A, B)

Humanization of anti-human PD-1 antibodies: humanization was based on Variable Heavy (VH) and Variable Light (VL) sequences of anti-hPD-1 hybridoma. Generally, a mouse-human chimeric mAb comprising the mouse parental VH and VL sequences, as well as the human IgG4-S228P constant region and human kappa chain, is first constructed. After characterization of the chimeric antibodies, three VL and three VL humanized sequences were designed and used to make nine humanized antibodies. The sequences are listed in (tables 3A and 3B).

Table 3A: chimeric antibody (human IgG4-S228P frame)

Table 3B: humanized heavy and light chain variable regions

Example 4: characterization and function of humanized antibodies

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

Flow cytometry analysis showed that some mutant combinations had higher binding activity than the parent antibody, some were identical to or slightly lower than the parent antibody (figure 8). The mutant combinations are listed in table 4 below.

Blocking ability of humanized antibody: the ability of the humanized antibody to block hPD-1 binding to hPD-L1 was measured. 100ng of hPD1mIg were pre-incubated with different doses of humanized antibody in 10. mu.L of PBS for 30 minutes at 4 ℃ 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. Using a similar method, the ability of the humanized antibody to block binding of hPD-1 to hPD-L2 was measured.

The results show that all humanized antibodies inhibited hPD-1mIgG binding to CHO/hPD-L1 cells in a dose-dependent manner. Some mutant combinations had higher blocking capacity than the chimeric parent antibody (fig. 9). The results also show that hPD-1mIgG was also blocked from binding to CHO/hPD-L2 cells (FIG. 10).

Binding affinity and kinetic determination of humanized antibodies: biacore T100(GE Healthcare Life Sciences) was used to assess the binding affinity and kinetics of the interaction of humanized PD-1mAb with hPD-1 protein. hPD-1mIg protein was immobilized on sensor chip CM5 by amine coupling. The filtered humanized antibody was diluted with HBS-EP buffer pH7.4(GEHealthcare Life Sciences) and then injected onto hPD-1mIg immobilized surfaces. Nine different concentrations were tested for each sample. Detailed binding kinetic parameters (association rate, Ka, dissociation rate, Kd, and affinity constant, Kd) can be determined by full kinetic analysis.

The analytical data showed no significant difference in the binding rate (Ka) between the mutant combinations and the chimeric parent antibody. The three mutant combinations (3,6,9) approach the chimeric parent antibody in terms of off-rate (Kd). All humanized antibodies have strong affinity with a KD in the low nanomolar range (10)^-10M). The KD values of the two mutant combinations (3,6) are close to those of the chimeric parent antibody (9.89X 10)^-11M) (table 4).

Table 4: binding affinity and kinetic determination of humanized antibodies

The enhancement effect of the anti-PD-1 on killing PD-L1 positive tumor cells in vitro by allogeneic CD8+ CTL: based on the anti-tumor mechanism of the anti-PD-1 antibody, this example designed an in vitro model to determine that the anti-PD-1 antibody is a human allogeneic CD8⁺Cytotoxic stranguriaBarocyte (allogeneic CD 8)⁺CTL) killing of tumor cells. First, CD8 was isolated from human PMBC⁺Lymphocytes and cultured with irradiated hB7-1 transfected human melanoma cells (624Mel/B7-1) to produce allogeneic CD8⁺cytoCTL. The allogeneic CD8 is then combined in the presence of humanized antibodies or control Ig⁺CTL was co-cultured with overnight cultured 624Mel/hPD-L1 tumor cells in 96-well plates for 5 days. Cells in wells of the plate were stained with 0.5% crystal violet and the plate was read at 540nm with an ELISA reader. Killing activity was calculated based on the survival of tumor cells.

The results indicate that certain mutant combinations can enhance the ability of allogeneic CTL cells to kill tumor cells in vitro (fig. 11).

The best mutant combination set (variant 3) was selected, the protein coding sequence cloned into an appropriate expression vector and transferred into CHO cells to generate anti-hPD-1 antibody, also known as TY 101.

Example 5: characterization of TY101 in tumor immunotherapy

Cytokine-enhanced mixed lymphocyte reaction (MRL) in PBMC. Human Peripheral Blood Mononuclear Cells (PBMC) from healthy individuals were isolated by density gradient centrifugation using Ficoll-Hypaque. PBMCs from healthy donors 1 were irradiated with X-rays at a dose of 40Gy as stimulating cells. T lymphocytes were isolated as responder cells from healthy donor 2 using human Pan T cell Isolation Kit (Miltenyl Biotec). The responder and stimulator cells were resuspended in complete RPMI medium containing 10% FCS and 2.5X 10 cells per well in the presence of serially diluted TY101 or hIgG controls⁵Individual responding cells and 1.25X 10⁵One stimulated cell (R/S ═ 2) was seeded into 96-well plates. Cells were incubated at 37 ℃ in 5% CO₂The culture was carried out in a humidified incubator for 5 days. The proliferative activity of T cells was assessed on day 5 by Cell Counting Kit-8(Dojindo Molecular Technologies, Inc). For cytokine detection, on day 3And culture supernatants were collected on day 5. Cytokine analysis was performed using the HumanTh1/Th2/Th17 Cytometric Bead Array kit (CBA; BD Biosciences).

The results show that T cells respond similarly to TY101 proliferation as to hIgG (figure 12). Interestingly, the production of cytokines IL-2 and IFN γ was significantly increased in the culture supernatants of TY 101-administered MLRs compared to administration of hIgG (figure 13).

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

The results show that addition of TY101 completely inhibited PD-1 expression on lymphocytes compared to medium only and hIgG control (fig. 14).

In vivo anti-tumor activity of humanized PD-1 antibody: the in vivo anti-tumor effect of TY101 was investigated. 8-week-old female human PD-1 knock-in (knock-in) mice (purchased from Shanghai Model organics Center, Inc.), MC38 tumor cells (MC38/hPD-L1) transfected with hPD-L1 on day 0 were implanted (1X 10) subcutaneously (s.c) on the right side (right flash)⁶Mice). TY101 or control Ig (10mg/kg) was administered by intraperitoneal (i.p.) injection 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, after treatment of mice bearing MC38/hPD-L1 tumors with TY101, a complete response occurred in 100% of the mice. Tumors in all five mice treated with TY101 completely regressed on day 25. In contrast, two of the five control IgG-treated mice developed tumors that grew continuously. In three other mice treated with control IgG, tumors regressed rapidly in two mice, although they also regressed at day 32 (fig. 15). The results indicate that TY101 can enhance antitumor efficacy in vivo.

Example 6: comparison of TY101 function with commercially available PD-1 antibody

This example selects two anti-hPD-1 antibodies that have currently been approved for clinical treatment of cancer patients for comparison with TY 101: keytruda (pembrolizumab) by Merck and Opdivo (nivolumab) by Bristol-Myers Squibb.

Antibody binding affinity and kinetics: TY101 was analyzed for affinity and kinetics with a Biacore T200 instrument (GE Healthcare Life Sciences) and compared to two commercially available antibodies. hPD-1mIg protein was immobilized at low concentration (33RU) on sensor chip CM5, and the interaction was detected using antibodies as the analyte (mobile phase). The data show that the binding rates Ka of the three antibodies are not significantly different. TY101 was slightly lower than the commercial antibody. The dissociation rate Kd of TY101 is slower to one tenth than for the two commercial antibodies and the affinity Kd of TY101 is 4-7 times stronger than for the commercial antibodies. The results show that TY101 shows stronger binding (fig. 16).

Comparison of PD-1 antibodies in terms of PD-1/PD-L1 blockade: PD-1/PD-L1 blocking bioassay was performed with PD-1/PD-L1Block Bioassays Kit (Promega). Will be 1 × 10⁵Individual cell/well Jurkat-PD1 cells were plated in opaque 96-well TC plates with overnight cultured CHO-PD-L1 cells (culture started at 5X 10) in the presence of TY101, pembrolizumab, nivolumab, or serial dilutions of negative control hIgG4 (0-30. mu.g/ml)⁴Individual cells/well) for 5 h. After 5h incubation, Jurkat-PD-1 cell activation was detected by measuring Relative Light Units (RLU) of luciferase activity with ONE-Glo substrate (Promega) on a SpectramaX L luminometer.

Analytical data showed that TY101 and two commercially available antibodies blocked the PD-1/PD-L1 pathway. The blockade effect of TY101 was similar to that of pamitumumab but better than that of nivolumab (fig. 17).

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

The results show that all three anti-PD-1 MAbs enhance allogeneic CD8⁺Tumor killing activity of CTL. The enhancement of TY101 was higher than that of the two commercial antibodies (fig. 18).

Example 7: development of TY101 clones and their Activity

The TY101 sequence was cloned into a proprietary (prepracticary) expression vector and transfected into CHO cells. Monoclonal cell lines were established using ClonePix and/or limiting dilution. A number of clones were established and antibodies generated by 3 of these clones (TY101-01-09, TY101-04-T3-05 and TY101-4G1) were characterized.

Testing for antibodies binding to hPD-1 or mPD-1 proteins (ELISA)

The binding of the antibody to hPD-1 and cross-reactivity to mPD-1 protein were detected by ELISA. Serial dilutions of test antibody were added to ELISA plates pre-coated with 1. mu.g/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 quantitated at 450nm wavelength with a SpectraMax Plus 384Microplate Reader (Molecular Device, llc., Sunnyvale, CA). The tested TY101 clones TY101-01-09, TY101-04-T3-05 and TY101-4G1 showed good binding to the hPD-1 protein with EC50 in the range of 0.01-0.15 nM. The antibody did not exhibit binding to mPD-1 protein (FIG. 19).

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

Antibodies were tested for binding to hPD-1 and cross-reactivity to cPD-1 by flow cytometry using CHOK1 cells expressing hPD-1 or cynomolgus PD-1 (cPD-1). Blank cells of CHOK1-hPD-1, CHOK1-cPD-1 and CHOK1 were incubated with serially diluted test preparations, followed by Alexa488 conjugated goat anti-human IgG (H + L) antibodies were incubated together and FACSCANTO II (BD) was usedBiosciences, San Jose, CA). The TY101 clones tested TY101-01-09, TY101-04-T3-05 and TY101-4G1 showed good binding with sub-nanomolar EC50 to CHOK1-hPD-1 and with several nanomolar EC50 to CHOK1-cPD-1 cells (FIG. 20).

Test of blocking Activity of antibodies against hPD-1/hPD-L1 or hPD-1/hPD-L2 binding (flow cytometry)

The ability of these antibodies to block hPD-1/hPD-L1 and hPD-1/hPD-L2 binding was further tested, and would be critical to the potential effectiveness of cancer patient treatment. CHOK1-hPD-1 cells were incubated with serial dilutions of test article mixed with biotin-hPD-L1 or biotin-hPD-L2. Cells were then incubated with Alexa 488-labeled streptavidin and analyzed using facscan II. anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05 and TY101-4G1 blocked binding of hPD-L1 to CHOK1 cells expressing hPD-1 with an IC50 of 1.15-1.47 nM. They also blocked hPD-L2 binding to hPD-1 expressing CHOK1 cells with IC50 at 1.52-2.33nM (FIG. 21).

Testing of the Effect of antibodies on T cells Using a human Mixed Leukocyte Reaction (MLR) assay

The effect of these antibodies on T cell function was tested in a human MLR assay with T cells isolated from 2 donors. Adherent PBMCs (mainly monocytes, isolated from donor 1 and plated in cell culture dishes to allow adhesion) were cultured for 5 days in the presence of 100ng/ml recombinant human (rh) GM-CSF and 50ng/ml rhIL-4, half the volume of the medium was changed after 3 days and 1 μ g/ml LPS was added on day 6. On day 7, the resulting cells (mainly mature DCs) were harvested and treated with mitomycin C. From donors 2 and 3 by EasySep^TMSeparation of CD3 by Human T Cell Isolation Kit (negative selection, STEMCELL technique)⁺T cells. DCs and T cells were co-cultured for 5 days in the presence of 3 concentrations (5. mu.g/ml, 0.5. mu.g/ml, 0.05. mu.g/ml) of the test antibody. Supernatants were harvested after 3 days, IL-2 levels were determined, and IFN-. gamma.levels were determined after 5 days (100. mu.L). anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05 and TY101-4G1 promoted secretion of IL-2 and IFN- γ from cells from both donors in a dose-dependent manner compared to isotype control hIgG4 (FIG. 22).

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

The effect of these antibodies on T cell function was tested in an engineered tumor cell human-T cell co-culture assay using T cells isolated from 4 different donors. By EasySep^TMIsolation of CD3 from PBMC of 4 donors by Human T Cell Isolation Kit⁺T cells. The engineered tumor cells Hep3B-OSC-hPDL1 (which are Hep3B cells engineered to stably express OS8 (anti-CD 3 single-chain variable fragment (scFv)) and hPD-L1 (KCLB, Cat.: 88064)) were treated with mitomycin C and reacted with CD3 in the presence of 3 concentrations (5. mu.g/ml, 0.5. mu.g/ml, 0.05. mu.g/ml) of the test antibody⁺T cells were co-cultured for 3 days, and culture supernatants were harvested to determine IFN- γ levels. anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05 and TY101-4G1 promoted secretion of IFN-. gamma.by cells from all 4 donors in a dose-dependent manner when compared to isotype control hIgG4 (FIG. 23).

Example 8: the TY101 clone showed better binding affinity compared to the FDA-approved anti-hPD-1 antibody.

Testing of antibody epitope overlap by competitive ELISA

These antibodies were tested in a competitive ELISA assay for binding to the same epitope as the FDA-approved anti-hPD-1 antibody nivolumab or pembrolizumab. Serial dilutions of the competitive antibody and biotin-hPD-1 were added to ELISA plates pre-coated with 1. mu.g/ml of test antibody. HRP-conjugated streptavidin was then added, followed by substrate TMB, and quantified with a SpectraMax Plus 384Microplate Reader at 450nm wavelength. The anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05 and TY101-4G1 almost completely blocked each other from binding hPD-1, indicating that they share a similar epitope. These 3 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 indicate that the TY101-01-09, TY101-04-T3-05, and TY101-4G1 antibodies bind to epitopes other than those of nivolumab and pembrolizumab, and that their affinity for hPD-1 may be higher than for nivolumab and pembrolizumab (figure 24).

Testing the binding affinity of antibodies to hPD-1 by SPR assay

To obtain an accurate measurement of the binding affinity to hPD-1, antibodies TY101-01-09, TY101-04-T3-05 and TY101-4G1, as well as nivolumab and palimumab were analyzed by SPR. Human PD-1ECD protein was immobilized on a CM5 sensor chip for varying lengths of time to achieve a low level of immobilization in flow cell 3 (at 60RU) and a high level of immobilization in flow cell 4 (960 RU). Serially diluted (0nM, 1.5625nM, 3.125nM, 6.25nM, 12.5nM, 25 and 50nM) antibodies were injected into the flow cell. The association time was 180s and the dissociation time was 600s (for nivolumab and pembrolizumab) or 1500s (for TY101-01-09, TY101-04-T3-05 and TY101-4G 1). After subtraction of the reference (flow cell 1) and zero concentration signals from the sample signals, binding kinetics were calculated using the BiaCore T200 evaluation software version 1.0 and 1:1 binding model for curve fitting. Control human IgG4 did not bind to hPD-1. Based on data from low immobilization levels of hPD-1 (-60 RU; Table 3; FIG. 23), the association rates of anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05, and TY101-4G1 to human PD-1 were slightly lower than those of nivolumab and pembrolizumab (one-half to one-quarter). The 3 antibodies disassociated from human PD-1 at a rate of one twelfth to one thirty times that of nivolumab and pembrolizumab, resulting in their affinity up to 4-8 times higher (lower K) than nivolumab and pembrolizumab_DCorresponds to better affinity, and vice versa; table 3). The anti-hPD-1 antibodies TY101-01-09, TY101-04-T3-05 and TY101-4G1 were also tested for binding affinity to hPD-1 at high immobilization levels of hPD-1. The 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 (FIG. 23). The data indicate that the binding affinities of TY101-01-09, TY101-04-T3-05 and TY101-4G1 were superior to that of nivolumab and pembrolizumab, mainly due to the slow off-rate (FIG. 25).

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

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

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<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 (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 (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 (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 Ser Asp Thr Val Lys

50 55 60

Arg Leu Val Trp Val Ala Tyr Ile Thr Ile Gly Gly Gly 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 (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 (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 (Artificial Sequence)

<220>

<223> synthetic

<400> 26

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagagca cagggccacc 60

atctcctgcc aagccagcga aaatgttgat aattatggca ttaattttat gaactggttc 120

caacacaaac cagcacagcc accccaactc 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 (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 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 (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 (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 (Artificial Sequence)

<220>

<223> synthetic

<400> 30

gacattgtgc tgacccaatt tccaacttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca gagccagcga aagtgttgat tactatggct ttagttttat aaactggttc 120

caacagaaac caggacagcc acccaaactc ctcatctatg ctgcatccaa ccagggatcc 180

ggggtccctg ccaggtttgg tggcagtggg tctgggacag acttcagcct caacatccat 240

cctatggagg aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttccgtgg 300

acgttcggtg gaggcaccaa gctggaaatc aaacgg 336

<210> 31

<211> 450

<212> PRT

<213> Artificial Sequence (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 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 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 (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 agggccccag 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 ccccctgtcc tccttgccct gctcctgaat ttctcggagg 720

cccctccgtc ttcctgtttc cccccaagcc caaggacacc ctgatgatct cccggacacc 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 (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 (Artificial Sequence)

<220>

<223> synthetic

<400> 34

agacattgtg ctgacccaat ttccaacttc tttggctgtg tctctagggc agagggccac 60

catctcctgc agagccagcg aaagtgttga ttactatggc 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 (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 Ser Ala

115 120

<210> 36

<211> 373

<212> DNA

<213> Artificial Sequence (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> synthetic

<400> 47

ctgtctagaa tgcagatccc acaggcgcc 29

<210> 48

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic

<400> 48

ggatcctcag aggggccaag agcagt 26

Claims (24)

1. An isolated antibody or fragment thereof specific for human programmed cell death protein 1(PD-L1), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the 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 IDNO: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 (SEQ ID NO:11), LCDR3: QHFWGTPWT (SEQ ID NO: 12); and

(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 IDNO:17)，LCDR3:QQSKDVPW(SEQ ID NO:18)。

2. the antibody or fragment thereof of claim 1, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

3. The antibody or fragment thereof of claim 1, wherein the light chain constant region is a kappa chain or lambda chain constant region.

4. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof is of the isotype IgG, IgM, IgA, IgE, or IgD.

5. The antibody or fragment thereof of claim 4, wherein the isotype is IgG1, IgG2, IgG3, or IgG 4.

6. The antibody or fragment thereof of any one of claims 1-5, wherein the antibody or fragment thereof is a chimeric, humanized, or fully human antibody.

7. The antibody or fragment thereof of claim 6, wherein the antibody or fragment thereof is a humanized antibody.

8. The antibody or fragment thereof of claim 7, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO 35, SEQ ID NO 37, or SEQ ID NO 39.

9. The antibody or fragment thereof of claim 8, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO 41, SEQ ID NO 43, or SEQ ID NO 45.

10. An isolated antibody or fragment specific for human programmed cell death protein 1(PD-L1), wherein the antibody or fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the 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 IDNO: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(SEQ 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 which comprises one, two or three amino acid additions, deletions, conservative amino acid substitutions or combinations thereof.

11. The isolated antibody or fragment thereof of claim 10, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as set forth in any one of (a) - (c), but one of which comprises a conservative amino acid substitution.

12. The isolated antibody or fragment thereof of claim 10, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as set forth in any one of (a) - (c), but wherein each of two comprises a conservative amino acid substitution.

13. The isolated antibody or fragment thereof of claim 10, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as set forth in any one of (a) - (c), but wherein each of three comprises a conservative amino acid substitution.

14. A composition comprising the antibody or fragment thereof of any one of claims 1-13 and a pharmaceutically acceptable carrier.

15. An isolated cell comprising one or more polynucleotides encoding the antibody or fragment thereof of any one of claims 1-13.

16. Use of the antibody or fragment thereof of any one of claims 1-13 in the manufacture of a medicament for the treatment of cancer.

17. The use of claim 16, wherein the cancer is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer.

18. A method of treating cancer in a patient in need thereof, comprising administering to the patient the antibody or fragment thereof of any one of claims 1-13.

19. A method of treating cancer or infection in a patient in need thereof, comprising:

(a) treating a cell in vitro with the antibody or fragment thereof of any one of claims 1-13; and

(b) administering the treated cells to the patient.

20. The method of claim 19, wherein the cell is a T cell.

21. Use of the antibody or fragment thereof of any one of claims 1-13 in the manufacture of a medicament for treating an infection.

22. The use of claim 21, wherein the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

23. Use of an antibody or fragment thereof according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment of an immune disorder.

24. The use of claim 23, wherein the immune disorder is selected from the group consisting of: infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, alzheimer's disease, inflammatory bowel disease, crohn's disease, ulcerative colitis, peloth's disease, celiac disease, gallbladder disease, hirsutism, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory diseases of the central and peripheral nervous system, multiple sclerosis, lupus and guillain-barre syndrome, atopic dermatitis, autoimmune hepatitis, fibroalveolar disease, glaff's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, wegener's granuloma, pancreatitis, trauma, graft-versus-host disease, inflammatory bowel disease, stroke, inflammatory bowel disease, peyronie's disease, celiac disease, multiple sclerosis, graft rejection, ischemic disease, myocardial infarction, atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, acid hypo-gastric and infertility associated with maternal-fetal intolerance.