CN106243225B - Novel anti-PD-L1 antibodies - Google Patents

Abstract

The present application provides monoclonal antibodies directed against the protein programmed death ligand 1(PD-L1) which are capable of blocking the binding of PD-L1 to programmed death molecule 1(PD-1), thus blocking the inhibitory effect of PD-L1 on T cells expressing PD-1. The antibodies of the present invention provide very effective agents for the treatment of various cancers by modulating human immune function.

Description

Novel anti-PD-L1 antibodies

Technical Field

The present invention relates to novel anti-PD-L1 antibodies.

Background

Evidence of increasing preclinical and clinical outcomes suggests that targeting immune checkpoints is becoming the most promising approach to treating cancer patients. Programmed cell death molecule 1 is one of the immune checkpoint proteins that plays a major role in limiting T cell activity, which provides the major immune resistance mechanism by which tumor cells can evade immune surveillance. The interaction of PD-1 expressed on activated T cells with PD-L1 expressed on tumor cells down-regulates the immune response and attenuates anti-tumor immunity. The expression of PD-L1 on tumors is associated with a decreased survival rate in esophageal, pancreatic and other types of cancer, highlighting the potential of this pathway as a new promising target for tumor immunotherapy. Pharmaceutical companies have developed a variety of drugs for the PD-1 pathway, such as BMSs, merck, roche, and Glatiramer Smith (GSK). Data from clinical trials show early evidence of persistent clinical activity and good safety in patients with various tumor types. Nivolumab is a PD-1 drug developed by BMS that is being put into the central phase of the next generation field. Currently in 6 late stage studies, treatment promoted tumor shrinkage in 3 of the 5 cancer groups studied, including 18% in 72 patients with lung cancer, nearly one-third in 98 patients with melanoma, and 27% in 33 patients with renal cancer. Lambrolizumab, developed by merck corporation is a humanized monoclonal IgG4 antibody that acts on PD-1, seizing a new breakthrough indicator of FDA after impressive IB data obtained for skin cancer. Results from the phase IB study showed an objective anti-tumor response in 51% of 85 cancer patients and a complete response in 9% of patients. Experimental MPDL3280A from roche demonstrated shrinkage of tumors in 29 (21%) of 140 advanced cancer patients with tumors of various sizes.

However, existing therapeutic approaches are not entirely satisfactory, and there is still a need for new antibodies against PD-L1.

Brief description of the invention

The present application provides novel anti-PD-L1 monoclonal antibodies, polynucleotides encoding the same, and methods of using the same.

In one aspect, the present application provides an isolated monoclonal antibody or antigen binding fragment thereof, which can be present at no more than 10^-9M (e.g. ≦ 9x10^-10M、≤8x10^-10M、≤7x10^-10M、≤6x10^-10M、≤5x10^-10M、≤4x10^-10M、≤3x10^-10M、≤2x10^-10M、or≤10^-10M) specifically binds to human PD-L1, said Kd value being determined by plasmon resonance binding.

In some embodiments, the antibody or antigen-binding fragment thereof has an EC of no more than 10nM (e.g., no more than 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, or 0.01nM)₅₀Binds to monkey PD-L1. In certain embodiments, the antibody or antigen-binding fragment thereof does not bind to mouse PD-L1, but binds monkey PD-L1 with similar binding affinity as human PD-L1. In certain embodiments, the antibody or anti-antibody thereofThe antigen-binding fragment has an IC of no more than 100nM (e.g., no more than 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, or 0.01nM)₅₀Effectively inhibit the binding of human or monkey PD-L1 to its receptor (such as PD-1). In certain embodiments, the EC is₅₀Or IC₅₀The assay was performed by Fluorescence Activated Cell Sorting (FACS) analysis.

In certain embodiments, the antibody or antigen-binding fragment thereof has substantially reduced effector function. In certain embodiments, the antibody or antigen-binding fragment thereof does not mediate ADCC or CDC or both.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a heavy chain CDR sequence selected from the group consisting of: 1, 2 and 3 of SEQ ID NO.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a light chain CDR sequence selected from the group consisting of: 4,5 and 6 of SEQ ID NO.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises at least 1, 2, 3, 4,5, or 6 CDRs selected from the group consisting of SEQ ID NOs 1, 2, 3, 4,5, and 6.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise the heavy chain variable region as set forth in SEQ ID NO 1, SEQ ID NO 2, and/or SEQ ID NO 3.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise the light chain variable region as set forth in SEQ ID NO 4, SEQ ID NO 5, and/or SEQ ID NO 6.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise the heavy chain variable region as set forth in SEQ ID NO 1, SEQ ID NO 2, and/or SEQ ID NO 3; and a light chain variable region as set forth in SEQ ID NO 4, SEQ ID NO 5 and/or SEQ ID NO 6.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO 7, SEQ ID NO 11, and SEQ ID NO 18.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a light chain variable region, wherein the light chain variable region is selected from the group consisting of SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 16, and SEQ ID NO 22.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises:

a) a heavy chain variable region comprising SEQ ID NO 7; and a light chain variable region comprising SEQ ID NO 9;

b) a heavy chain variable region comprising SEQ ID NO 11; and a light chain variable region comprising SEQ ID NO 13;

c) a heavy chain variable region comprising SEQ ID NO 11; and a light chain variable region comprising SEQ ID NO 16;

d) a heavy chain variable region comprising SEQ ID NO 18; and a light chain variable region comprising SEQ ID NO 13;

and

e) a heavy chain variable region comprising SEQ ID NO 18; and a light chain variable region comprising SEQ ID NO 22.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein includes, for example, 2.74.15, 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb 8.

In certain embodiments, the antibody or antigen binding fragment thereof described herein competes for the same epitope as antibody 2.74.15, 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, or 2.74.15.hAb 8.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of blocking binding of human PD-L1 to its receptor and thus providing at least one of the following activities:

a) in CD4⁺Inducing IL-2 production in T cells;

b) in CD4⁺Inducing production of IFN γ in T cells;

c) induction of CD4⁺Proliferation of T cells; and

d) reversing the T reg inhibitory function.

In certain embodiments, the antibody described herein is a monoclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a diabody, or an anti-idiotypic antibody.

In certain embodiments, an antigen-binding fragment provided herein is a camelized single domain antibody (camelized single domain antibody), a diabody, a scFv dimer, a BsFv, a dsFv, (dsFv)2, dsFv-dsFv ', a Fv fragment, a Fab ', a F (ab ')2, a ds diabody, a nanobody, a domain antibody, or a diabody.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein further comprise an immunoglobulin constant region.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein further comprise a conjugate.

In certain embodiments, the conjugate can be a detectable label, a pharmacokinetic modifying moiety, or a purifying moiety.

In another aspect, the present application provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof as described herein. In certain embodiments, the polynucleotides provided herein encode the amino acid sequence of an antibody or antigen-binding fragment thereof as described herein. In certain embodiments, the present application provides vectors comprising these polynucleotides. In certain embodiments, the present application provides methods of expressing one or more antibodies or antigen-binding fragments described herein by culturing a host cell under conditions in which the antibody or antigen-binding fragment encoded by the polynucleotide is expressed in a vector. In certain embodiments, the polynucleotides provided herein are operably linked to a promoter, such as the SV40 promoter, in a vector. In certain embodiments, the host cell comprising the vector provided herein is a chinese hamster ovary cell, or 293 cell.

In another aspect, the present application provides a kit comprising an antibody or antigen-binding fragment thereof described herein.

In another aspect, the present application provides a method of detecting the presence or level of PD-L1 (e.g., human or monkey) in a biological sample, comprising contacting the biological sample with an antibody or antigen-binding fragment thereof described herein, and determining the presence or level of human or monkey PD-L1 in the sample.

In another aspect, the present application provides a method of identifying an individual having a disorder or condition that is likely to respond to a PD-L1 antagonist, comprising: determining the presence or level of PD-L1 (e.g., human or monkey) in a test biological sample from the individual using the antibodies or antigen binding fragments thereof described herein, wherein the presence or level of PD-L1 in the biological sample is indicative of the likelihood of a response. In certain embodiments, the method further comprises administering to the individual identified as having a disorder or condition that is likely to respond to a PD-L1 antagonist an effective amount of an antibody or antigen-binding fragment thereof described herein.

The present application further provides a method of monitoring a therapeutic response or disease progression in a subject treated with a PD-L1 antagonist, comprising determining the presence or level of PD-L1 (e.g., human or monkey) in a test biological sample from the individual using an antibody or antigen-binding fragment thereof described herein.

In another aspect, the present application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and one or more pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical carrier may be, for example, a diluent, an antioxidant, an adjuvant, an excipient, or a non-toxic auxiliary substance.

In another aspect, the present application provides a method of treating a condition in a subject that would benefit from an upregulated immune response, comprising administering to the subject an effective amount of an antibody, or antigen-binding fragment thereof, described herein. In certain embodiments, the subject has up-regulated PD-L1 expression.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof as described herein in the manufacture of a medicament for the treatment of a condition that would benefit from an upregulated immune response. In certain embodiments, the condition is cancer or a chronic viral infection.

Brief Description of Drawings

FIG. 1 shows binding of murine anti-human PD-L1 antibody to CHO cells expressing PD-L1 as determined by FACS analysis. EC (EC)₅₀Expressed in nM.

FIG. 2 shows FACS analysis determined binding of humanized PD-L1 antibody to CHO cells expressing PD-L1.

FIG. 3 shows FACS analysis determined binding of humanized PD-L1 antibody to activated Dendritic Cell (DC) cells expressing PD-L1. EC (EC)₅₀Expressed in nM.

FIG. 4 shows that the humanized PD-L1 antibody blocks the binding of PD-1 to CHO cells transfected with PD-L1 as determined by FACS analysis. EC (EC)₅₀Expressed in nM.

Figure 5 shows that humanized PD-L1 antibody specifically binds to PD-L1 but not to PD-L2 as determined by FACS analysis.

FIG. 6 shows that the PD-L1 antibody binds to cynomolgus monkey PD-L1. EC (EC)₅₀Expressed in nM.

FIG. 7 shows the complete kinetics of the binding affinity of the PD-L1 antibody to human PD-L1.

FIG. 8 shows the effect of humanized anti-PD-L1 antibody on IL-2 production in a Mixed Lymphocyte Reaction (MLR).

FIG. 9 shows the effect of humanized anti-PD-L1 antibody on IFN γ production in MLR.

FIG. 10 shows that humanized anti-PD-L1 antibody promotes T cell proliferation in MLR.

Figure 11 shows that humanized PD-L1 antibody increased IFN γ production in specific T cell responses. The specific T cell response was generated by the co-culture of Cytomegalovirus (CMV) specific T cells with CMV-pp65 peptide-loaded DCs for 5 days.

Figure 12 shows that humanized PD-L1 antibody promotes T cell proliferation in specific T cell responses.

Figure 13 shows that anti-PD-L1 antibody reversed Treg suppression function.

Figure 14 shows that anti-PD-L1 antibody lacks ADCC in mdcs.

Figure 15 shows that anti-PD-L1 antibody lacks CDC in activated T cells.

Detailed Description

The following description of the present application is intended to be illustrative of various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limitations on the scope of the application. Numerous equivalents, changes, and modifications will readily occur to those skilled in the art without departing from the scope of the present application, and it is intended that such equivalents be included within the scope of the present invention. All documents, including publications, patents, and patent applications, cited in this application are incorporated by reference in their entirety.

Definition of

The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds a particular antigen. A natural intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions; each light chain consists of a variable region and a constant region. Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains as λ or κ. The antibody is "Y" shaped, the neck of the "Y" structure consisting of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure includes the variable and first constant regions of one of the heavy chains, which are associated with the variable and constant regions of one of the light chains. The variable regions of the light and heavy chains determine the binding of the antigen. The variable region of each chain contains three hypervariable regions, called Complementarity Determining Regions (CDRs) (CDRs for the light chain (L) comprise LCDR1, LCDR2, LCDR3 and CDRs for the heavy chain (H) comprise HCDR1, HCDR2, HCDR 3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be named or identified by the Kabat, Chothia or Al-Lazikani nomenclature. (Al-Lazikani, B., Chothia, C., Lesk, A.M., J.mol.biol.,273(4),927 (1997)), Chothia, C., et Al, J Mol biol.Dec 5; 186(3):651-63 (1985); Chothia, C.and Lesk, A.M., J.mol.biol.,196,901 (1987); Chothia, C. et Al, Nature.Dec 21-28; 342(6252):877-83 (1989); Kabat E.A. et Al, National Institutes of Health, Bethesda, Md. (1991)). Where three CDRs are separated by flanking continuous portions called Framework Regions (FRs), which are more highly conserved than CDRs and form a scaffold-supported hypervariable loop. The constant regions of the heavy and light chains are not involved in antigen binding, but have multiple effector functions. Antibodies can be classified into several classes depending on the amino acid sequence of the heavy chain constant region. Depending on whether it contains alpha, delta, epsilon, gamma and mu heavy chains, antibodies can be classified into five major classes or isoforms, respectively: IgA, IgD, IgE, IgG and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1(γ 1 heavy chain), IgG2(γ 2 heavy chain), IgG3(γ 3 heavy chain), IgG4(γ 4 heavy chain), IgA1(α 1 heavy chain), or IgA2(α 2 heavy chain), among others.

The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds an antigen but does not have an intact antibody structure. Examples of antigen binding fragments include, but are not limited to, antibodies such as bifunctional antibodies (diabodies), Fab ', F (ab')₂Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)₂Bispecific dsFv (dsFv-dsFv'), disulfide stabilized bifunctional antibodies (ds diabody), single chain antibody molecules (scFv), scFv dimers (diabodies), bivalent single chain antibodies (BsFv), multispecific antibodies, camelized single domain antibodies (camelized single domain antibodies), nanobodies, domain antibodies, and bivalent domain antibodies. The antigen-binding fragment may bind to the same antigen as the maternal antibody. In certain embodiments, an antigen-binding fragment can comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

An "Fab" fragment of an antibody refers to the portion of the antibody molecule that is disulfide bonded to the variable and constant regions of a light chain (which includes both the variable and constant regions) and a heavy chain.

By "Fab'" fragment is meant a Fab fragment comprising part of the hinge region.

“F(ab')₂"refers to a dimer of Fab.

The "Fc" of an antibody refers to that portion of the antibody which consists of the second and third constant regions of the heavy chain bound via disulfide bonds. The Fc portion of antibodies is responsible for a variety of different effector functions such as ADCC and CDC, but is not involved in antigen binding.

The "Fv" segment of an antibody refers to the smallest antibody fragment that contains the entire antigen-binding site. The Fv fragment consists of the variable region of one light chain and the variable region of one heavy chain.

"Single chain Fv antibody" or "scFv" refers to an engineered antibody having a light chain variable region directly linked to a heavy chain variable region or linked via a peptide chain (Huston JS et al, Proc Natl Acad Sci USA,85:5879 (1988)).

"Single chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to an Fc portion of an antibody.

"Camelidized single domain antibodies", "Heavy chain antibodies" or "HCAb (Heavy-chain-only antibodies)" all refer to antibodies containing two V-chains_HAntibodies that do not contain a light chain in the domain (Riechmann L. and Muydermans S., J Immunol methods. Dec 10; 231(1-2):25-38 (1999); Muydermans S., J Biotechnol. Jun; 74(4):277-302 (2001); WO 94/04678; WO 94/25591; U.S. patent No.6,005,079). Heavy chain antibodies were originally derived from camelidae (camels, dromedary and llamas). Despite the absence of light chains, camelized antibodies (camelized antibodies) have all the functions that are confirmed for antigen binding (Hamers-Casterman C. et al, Nature. Jun 3; 363(6428):446-8 (1993); Nguyen VK. et al, "Heavy-chain antibodies in Camellia; a case of evolution innovation," immunogenetics. Apr; 54(1):39-47 (2002); Nguyen VK. et al, immunology. May; 109(1):93-101 (2003)). The variable region of the heavy chain antibody (VHH domain) is the smallest known antigen-binding unit produced by adaptive immunity (Koch-Nolte F. et al, FASEB J. Nov; 21(13):3490-8.Epub 2007Jun 15 (2007)).

By "nanobody" is meant an antibody fragment consisting of one VHH domain from a heavy chain antibody and two constant regions CH2 and CH 3.

"bifunctional antibodies" (diabodies) include antibodies with two antigen binding sitesSmall spotted antibody fragment wherein the fragment comprises V linked on the same polypeptide chain_HField and V_LDomain (V)_H-V_LOr V_H-V_L) (see, Holliger P. et al, Proc Natl Acad Sci U S.Jul 15; 90(14) 6444-8 (1993); EP 404097; WO 93/11161). The linker between the two domains is so short that the two domains on the same chain do not pair with each other, thereby forcing the two domains to pair with the complementary domains of the other chain, forming two antibody binding sites. The two antibody binding sites may be targeted to bind to the same or different antigens (or epitopes).

"Domain antibody" refers to an antibody fragment containing only one heavy chain variable region or one light chain variable region. In some cases, two or more V_HThe domains are covalently bound by a polypeptide linker and form bivalent domain antibodies. Two V of bivalent domain antibody_HThe domains may be targeted to the same or different antigens.

In certain embodiments, "(dsFv)₂"contains three peptide chains: two V_HGroups are linked by a polypeptide linker and linked to two V groups by disulfide bonds_LThe groups are combined.

In certain embodiments, a "bispecific ds bifunctional antibody" comprises V_L1-V_H2(connected by a polypeptide linker) and V_H1-V_L2(again, linked by a polypeptide linker) both at V_H1And V_L1The two are bonded through disulfide bonds.

A "bispecific dsFv" or "dsFv-dsFv" comprises three polypeptide chains: v_H1-V_H2Groups in which the heavy chains of both are linked by polypeptide linkers (e.g.long flexible linkers) and each linked to V by a disulfide bond_L1And V_L2Groups are combined, each pair of heavy and light chains paired by a disulfide bond having different antigen specificity.

In certain embodiments, an "scFv dimer" is a diabody or a diabody (BsFv), comprising two V s that dimerize_H-V_L(attached by a polypeptide linker) group, V of one of the groups_HWith V of another group_LThe two binding sites cooperate to form two binding sites that can be targeted to bind to the same antigen (or epitope) or to different antigens (or epitopes). In other embodiments, an "scFv dimer" is a bispecific diabody comprising interconnected V_L1-V_H2(ligated by polypeptide linker) and V_H1-V_L2(ligated by polypeptide linkers) wherein V_H1And V_L1Collaboration, V_H2And V_L2In cooperation, and each cooperative pair has a different antigen specificity.

The term "humanized" as used herein, when applied to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that includes CDRs derived from a non-human animal, FR regions derived from a human, and constant regions derived from a human, where applicable. Because the humanized antibody or antigen binding fragment has reduced immunogenicity, it may be used in certain embodiments as a therapeutic agent in humans. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment consists essentially entirely of human-derived sequences, except for CDR sequences that are non-human. In some embodiments, the human-derived FR region may include the same amino acid sequence as the human-derived antibody from which it is derived, or it may include some amino acid changes, e.g., no more than 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid change. In some embodiments, the amino acid change can be present in only the heavy chain FR region, only the light chain FR region, or both chains. In some preferred embodiments, the humanized antibody comprises human FR1-3 and human JH and jk.

The term "chimeric" as used herein refers to an antibody or antigen-binding fragment having a portion of a heavy and/or light chain derived from one species, and the remainder of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody can include constant regions derived from a human and variable regions derived from a non-human animal, such as a mouse.

"PD-L1" as used in this application refers to programmed cell death ligand 1(PD-L1, see, e.g., Freeman et al (2000) J.Exp.Med.192: 1027). The amino acid sequence of representative humanized PD-L1 is NCBI accession No. NP-054862.1, and the nucleic acid sequence of representative humanized PD-L1 is NCBI accession No. NM-014143.3. PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-L1 binds to the receptor PD-1 or B7-1 expressed on activated T cells, B cells and bone marrow cells. Binding of PD-L1 to its receptor triggers signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Thus, PD-L1 plays a major role in suppressing the immune system in specific events, such as pregnancy, autoimmune diseases, tissue transplantation, and it is thought to allow tumor or cancer cells to circumvent immune checkpoints and evade immune responses.

As used herein, an "anti-PD-L1 antibody" refers to an antibody that specifically binds to PD-L1 (e.g., human or monkey PD-L1) with sufficient affinity to provide diagnostic and/or therapeutic utility.

"specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application specifically bind to human PD-L1, and their binding affinity (K)_D)≤10^-6M (e.g.. ltoreq.5 x10^-7M，≤2x10^-7M，≤10^-7M，≤5x10^{- 8}M，≤2x10^-8M，≤10^-8M，≤5x10^-9M，≤2x10^-9M，≤10^-9M，≤10^-10M). KD in the present application refers to the ratio of dissociation rate to association rate (koff/kon), and can be determined by surface plasmon resonance methods, for example using instruments such as Biacore.

The ability to "block binding" or "compete for the same epitope" in this application refers to the ability of an antibody or antigen-binding fragment thereof to inhibit the interaction of two intermolecular bindings (e.g., human PD-L1 and anti-PD-L1 antibodies) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules can inhibit the interaction of binding between two molecules by at least 50%. In certain embodiments, such inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

As used herein, "epitope" refers to the portion of an amino acid or group of atoms of an antigenic molecule that binds to an antibody. If two antibodies exhibit competitive binding to the antigen, it is possible to bind to the same epitope on the antigen. For example, if an antibody or antigen-binding fragment thereof provided herein blocks the binding of an exemplified antibody, e.g., 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb8, to human PD-L1, then the antibody or antigen-binding fragment thereof can be considered to bind to the same epitope as those exemplified antibodies.

The present application describes "2.74.15" as a murine monoclonal antibody comprising the amino acid sequence represented by SEQ ID NO: 1.2 and 3, and 3 light chain CDRs consisting of 4,5 and 6, respectively.

As used herein, "2.74.15. hAb 4" refers to a humanized antibody having the heavy chain variable region shown in SEQ ID NO. 7, the light chain variable region shown in SEQ ID NO. 9, and a human IgG4 isotype constant region.

As used herein, "2.74.15. hAb 5" refers to a humanized antibody having the heavy chain variable region shown in SEQ ID NO. 11, the light chain variable region shown in SEQ ID NO. 13, and a human IgG4 isotype constant region.

As used herein, "2.74.15. hAb 6" refers to a humanized antibody having the heavy chain variable region shown in SEQ ID NO. 11, the light chain variable region shown in SEQ ID NO. 16, and a human IgG4 isotype constant region.

As used herein, "2.74.15. hAb 7" refers to a humanized antibody having the heavy chain variable region as set forth in SEQ ID NO. 18, the light chain variable region as set forth in SEQ ID NO. 13, and a human IgG4 isotype constant region.

As used herein, "2.74.15. hAb 8" refers to a humanized antibody having the heavy chain variable region shown in SEQ ID NO:18, the light chain variable region shown in SEQ ID NO:22, and a human IgG4 isotype constant region.

In the present application, "conservative substitution" when used in reference to an amino acid sequence means the substitution of one amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions may be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between directional side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions do not generally result in significant changes in the conformational structure of a protein, and therefore the biological activity of the protein can be retained.

"percent sequence identity," when used with respect to an amino acid sequence (or nucleic acid sequence), refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to a reference sequence to the amino acid (or nucleic acid) residues in the candidate sequence after alignment and, if necessary, introduction of a spacer to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of the amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine percent sequence identity of amino acid (or Nucleic acid) sequences by tools disclosed in the art, such as BLASTN, BLASTp (national center for Biotechnology information website (NCBI), see also, Altschul S.F. et al, J.mol.biol., 215: 403-. One skilled in the art can use default parameters for the tool or adjust the parameters appropriately as needed for the alignment, for example by choosing an appropriate algorithm.

"T cells" as used herein includes CD4⁺T cell, CD8⁺T cells, T helper type 1T cells, T helper type 2T cells, T helper type 17T cells, and suppressor T cells.

"effector function" as used herein refers to the biological activity of the Fc region of an antibody to bind its effectors such as the C1 complex and Fc receptor. Exemplary effector functions include Complement Dependent Cytotoxicity (CDC) induced by interaction of the antibody with C1q on the C1 complex, antibody dependent cell mediated cytotoxicity (ADCC) induced by binding of the Fc region of the antibody to Fc receptors on effector cells, and phagocytosis.

"cancer" or "cancerous state" in the present application refers to any medical condition which is mediated by the growth, proliferation or metastasis of neoplastic or malignant cells and which causes solid and non-solid tumors, such as leukemia. The term "tumor" as used herein refers to a solid substance of a tumor and/or malignant cells.

"treating" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof. For cancer, "treatment" or "therapy" may refer to inhibiting or slowing the growth, reproduction, or metastasis of a tumor or malignant cell, or some combination of the above. In the context of a tumor, "treating" or "therapy" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

The "isolated" material has been artificially altered from its natural state. If a "separated" substance or component occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not isolated, but is considered to be "isolated" if the polynucleotide or polypeptide is sufficiently isolated from the materials with which it naturally coexists in its natural state and is present in a sufficiently pure state. In certain embodiments, the antibodies and antigen-binding fragments are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure, as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (e.g., ion exchange chromatography or reverse phase HPLC).

A "vector" in the present invention refers to a vehicle into which a polynucleotide encoding a protein is operably inserted and the protein is expressed. The vector may be used to transform, transduce or transfect a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses, among others. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma vacuolium viruses (e.g., SV 40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into the cell, including, but not limited to, viral particles, liposomes, or protein coats.

The term "host cell" as used herein refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.

The term "PD-L1-related or associated diseases" as used herein refers to any disease caused, exacerbated or otherwise associated by increased or decreased expression or activity of PD-L1 (e.g., human PD-L1).

As used herein, a "therapeutically effective amount" or "effective dose" refers to a dose or concentration of a drug effective to treat a disease or condition associated with human PD-L1. For example, for use of the antibodies or antigen-binding fragments thereof disclosed in the present invention, a therapeutically effective amount is at that dose or concentration, and the antibody or antigen-conjugate can eliminate all or a portion of the tumor, inhibit or slow the growth of the tumor, inhibit the growth or proliferation of cells that mediate the cancerous state, inhibit the metastasis of tumor cells, reduce any symptoms or markers associated with the tumor or cancerous state, prevent or delay the progression of the tumor or cancerous state, or some combination thereof.

By "pharmaceutically acceptable" is meant a carrier, vehicle, diluent, adjuvant and/or salt that is, in general, chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

anti-PD-L1 antibodies

In one aspect, the invention provides anti-PD-L1 antibodies and antigen-binding fragments thereof. PD-1, also known as B7-1, is a known key immune checkpoint receptor expressed by activated T cells that modulates immunosuppression. PD-1 ligand 1(PD-L1) is a 40kDa transmembrane protein expressed on a variety of tumor cells, stromal cells, or both, that binds to PD-1. The interaction between PD-1 and PD-L1 is known to enhance T cell responses and thereby mediate anti-cancer activity.

In certain embodiments, the present application provides an exemplary murine monoclonal antibody 2.74.15, the CDR sequences of which are shown in table 1.

TABLE 1

In certain embodiments, the present application provides an exemplary humanized antibody 2.74.15 comprising: 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb8, the heavy or light chain variable region amino acid sequence and encoding nucleic acid sequence thereof are shown below.

2.74.15.hAb4-VH (SEQ ID NO:7 is amino acid SEQ ID NO:8 is nucleic acid):

section V: IGHV 4-59X 01

And (D) section: IGHD 3-16X 01

Segment J: IGHJ 1x 01

2.74.15.hAb4-VL (SEQ ID NO:9 is the amino acid SEQ ID NO:10 is the nucleic acid):

section V: IGKV 1-27X 01

Segment J: IGKJ2 x 01

2.74.15hAb5-VH (SEQ ID NO:11 is amino acid SEQ ID NO:12 is nucleic acid):

section V: IGHV 4-59X 06

And (D) section: IGHD 1-26X 01

Segment J: IGHJ 1x 01

2.74.15hAb5-VL (SEQ ID NO:13 is the amino acid SEQ ID NO:14 is the nucleic acid):

section V: IGKV1-9 x 01

Segment J: IGKJ2 x 01

2.74.15hAb6-VH (SEQ ID NO:11 is amino acid SEQ ID NO:15 is nucleic acid):

section V: IGHV 4-59X 06

And (D) section: IGHD 1-26X 01

Segment J: IGHJ 1x 01

2.74.15hAb6-VL (SEQ ID NO:16 is the amino acid SEQ ID NO:17 is a nucleic acid):

section V: IGKV 1-27X 01

Segment J: IGKJ2 x 01

2.74.15hAb7-VH (SEQ ID NO:18 is amino acid SEQ ID NO:19 is nucleic acid):

section V: IGHV 4-59X 01

And (D) section: IGHD 3-16X 01

Segment J: IGHJ 1x 01

2.74.15hAb7-VL (SEQ ID NO:13 is the amino acid SEQ ID NO:20 is a nucleic acid):

section V: IGKV1-9 x 01

Segment J: IGKJ2 x 01

2.74.15hAb8-VH (SEQ ID NO:18 is amino acid SEQ ID NO:21 is nucleic acid):

section V: IGHV 4-59X 01

And (D) section: IGHD 3-16X 01

Segment J: IGHJ 1x 01

2.74.15hAb8-VL (SEQ ID NO:22 is the amino acid SEQ ID NO:23 is the nucleic acid):

section V: IGKV 1-27X 01

Segment J: IGKJ2 x 01

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise heavy chain CDR sequences selected from the group consisting of: 1, 2 and 3 in SEQ ID NOs. In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise light chain CDR sequences selected from the group consisting of: SEQ ID NOs 4,5 and 6.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a heavy chain variable region, wherein the heavy chain variable region comprises SEQ ID NO:1, SEQ ID NO:2, and/or SEQ ID NO: 3.

In some embodiments, the anti-PD-L1 antibody and antigen-binding fragments thereof comprises a light chain variable region, wherein the light chain variable region comprises SEQ ID No. 4, SEQ ID No. 5, and/or SEQ ID No. 6.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof include a heavy chain variable region comprising SEQ ID NO:1, SEQ ID NO:2, and/or SEQ ID NO: 3; and a light chain variable region as set forth in SEQ ID NO 4, SEQ ID NO 5 and/or SEQ ID NO 6.

It will be appreciated by those skilled in the art that the CDR sequences provided in table 1 may be modified to include substitutions of one or more amino acids, thereby resulting in improved biological activity, such as improved binding affinity to human PD-L1. For example, libraries of antibody variants (e.g., Fab or FcFv variants) can be produced and expressed using phage display technology, and subsequently screened for antibodies having affinity for human PD-L1. In another example, computer software can be used to simulate the binding of the antibody to human PD-L1 and to identify the amino acid residues on the antibody that form the binding interface. Substitutions of these residues may be avoided to prevent a decrease in binding affinity, or may be targeted for substitution to form stronger binding. In certain embodiments, at least one (or all) substitution in a CDR sequence is a conservative substitution.

In certain embodiments, the antibodies and antigen-binding fragments comprise one or more CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences listed in table 1, while retaining similar or even higher binding affinity to human PD-L1 than its parent antibody, which has substantially the same sequences, but whose corresponding CDR sequences have 100% sequence identity to the sequences listed in table 1.

In certain embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof are humanized. The humanized antibodies have reduced immunogenicity in humans compared to their parent antibodies prior to humanization, but retain the binding affinity of the parent antibody. In certain embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof include CDR sequences of non-human origin, framework regions of human origin, and optionally constant regions of human origin. In certain embodiments, one or more amino acid residues of a non-human antibody (e.g., a mouse framework region) from which the CDR sequences are derived are substituted in the human framework region, e.g., to increase or retain binding affinity.

In some embodiments, the humanized anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a heavy chain variable region selected from the group consisting of SEQ ID No. 7, SEQ ID No. 11, SEQ ID No. 18, and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto; and/or a light chain variable region, wherein the light chain variable region is selected from the group consisting of SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 16, SEQ ID NO 22, and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto. These humanized antibodies retain binding affinity to human PD-L1, preferably with the following exemplary antibodies: levels of 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb8 were similar.

In some embodiments, the humanized anti-PD-L1 antibodies and antigen-binding fragments thereof comprise a) a heavy chain variable region comprising SEQ ID No. 7; and a light chain variable region comprising SEQ ID NO 9; b) a heavy chain variable region comprising SEQ ID NO 11; and a light chain variable region comprising SEQ ID NO 13; c) a heavy chain variable region comprising SEQ ID NO 11; and a light chain variable region comprising SEQ ID NO 16; d) a heavy chain variable region comprising SEQ ID NO 18; and a light chain variable region comprising SEQ ID NO 13; and e) a heavy chain variable region comprising SEQ ID NO 18; and a light chain variable region comprising SEQ ID NO 22.

Antibodies and antigen-binding fragments thereof that compete for the same epitope as the anti-PD-L1 antibodies and antigen-binding fragments thereof of the present application are also contemplated by the present application. In certain embodiments, the antibody is administered at less than 10^-6M, less than 10^-7M, less than 10^-7.5M, less than 10^-8M, less than 10^-8.5M or less than 10^-9M or less than 10^-10IC of M₅₀The value (i.e., half inhibitory concentration) blocks 2.74.15, 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7 or

2.74.15. binding of hAb8 to human or monkey PD-L1. IC (integrated circuit)₅₀Values are determined by competitive assays such as ELISA assays, radioligand competition binding assays, and FACS analysis.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein can have a binding affinity of ≦ 10^-6M(e.g.,≤5x10^-7M,≤2x10^-7M,≤10^-7M,≤5x10^-8M,≤2x10^-8M,≤10^-8M,≤5x10^-9M,≤2x10^-9M,≤10^-9M,10^-10M) binding affinity (Kd) specific for human PD-L1Binding, measured by plasmon resonance binding. Binding affinity can be defined by K_DValues are expressed as calculated by the ratio of off-rate to on-rate (koff/kon) when the binding of antigen and antigen binding molecule reaches equilibrium. The antigen binding affinity (e.g. K)_D) May suitably be determined by suitable methods known in the art, including the use of instruments such as the plasmon resonance binding method of Biacore (see, for example, Murphy, M.et al, Current protocols in protein science, Chapter 19, Unit 19.14,2006).

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein and human PD-L1 have an EC of 0.02nM to 100nM (e.g., 0.02nM to 50nM, 0.02nM to 30nM, 0.02nM to 20nM, or 0.02nM to 10nM, 0.02nM to 1nM, or 0.02nM to 0.1nM)₅₀(i.e., half the binding concentration) binding. Binding of the antibody to human PD-L1 can be determined by methods known in the art such as sandwich assays such as ELISA, Western blotting, FACS or other binding assays. In an illustrative example, a test antibody (i.e., a primary antibody) is bound to immobilized human PD-L1 or cells expressing human PD-L1, followed by washing away unbound antibody, and introducing a labeled secondary antibody that is capable of binding to the primary antibody and thus detecting the bound primary antibody. The detection can be performed on a microplate reader plate when immobilized PD-L1 is used, or can be performed using FACS analysis when cells expressing human PD-L1 are used.

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein have an IC of 0.2nM to 100nM (e.g., 0.2nM to 50nM, 0.2nM to 30nM, 0.2nM to 20nM, or 0.2nM to 10nM)₅₀Inhibits binding of human PD-L1 to its receptor, as measured by a competitive assay.

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein inhibit the binding of human PD-L1 to its receptor, and thereby provide methods that include, for example, inducing cytokine production by activated T cells (e.g., CD 4)⁺T cells and CD8⁺T cells), inducing proliferation of activated T cells (e.g., CD 4)⁺T cells and CD8⁺T cells) and biological activity to reverse the suppressive function of regulatory tregs. Exemplary cytokines include IL-2 and IFN gamma. The term "IL-2" refers to leukocytesInterleukin 2, which is a cytokine signaling molecule, modulates the activity of white blood cells (e.g., leukocytes) in the immune system. The term "interferon gamma (IFN γ)" is derived from Natural Killer (NK) cells, NK T cells, CD4⁺And CD8⁺T cells produce cytokines, which are important activators of macrophages and inducers of Major Histocompatibility Complex (MHC) molecule expression. Cytokine production can be determined by methods known in the art, such as ELISA. These methods can also be used to detect T cell proliferation, comprising³H]Thymidine incorporation assay.

The anti-PD-L1 antibodies and antigen-binding fragments thereof are specific for human PD-L1. In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to PD-L2 (e.g., human PD-L2). For example, the binding affinity to PD-L2 is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the binding affinity of human PD-L1.

In certain embodiments, the antibodies and antigen-binding fragments thereof have an EC of no greater than 10nM, e.g., no greater than 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, or 0.01nM₅₀Binding to monkey PD-L1 (determined by ELISA).

In certain embodiments, the antibodies and antigen-binding fragments thereof do not bind to murine PD-L1, but bind to monkey PD-L1 with similar binding affinity to human PD-L1. For example, binding of exemplary antibodies 2.74.15, 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, or 2.74.15.hAb8 to murine PD-L1 was not detectable using common binding assays such as ELISA or FACS analysis, which detected binding of these antibodies to monkey PD-L1 with similar affinity or EC to human PD-L1₅₀The values are combined.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof have reduced or depleted effector function. In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof have constant regions of the IgG4 isotype with reduced or depleted effector function. Equivalent functions such as ADCC and CDC can result in cytotoxicity to cells expressing PD-L1. Many cells, including healthy or normal cells, are capable of expressing PD-L1. To avoid possible unwanted toxicity to these health or normal, certain embodiments of the antibodies and antigen binding fragments thereof described herein have reduced or even depleted effector functions. Numerous assays are known for estimating ADCC or CDC activity, such as Fc receptor binding assays, complement C1q binding assays, and cell lysis methods, which can be readily selected by one skilled in the art. Without wishing to be bound by theory, it is believed that antibodies with reduced or depleted effector functions such as ADCC and CDC do not cause or minimize cytotoxicity of PD-L1 expressing cells (e.g., those healthy or normal cells), thus avoiding unwanted side effects. At the same time, tumor cells expressing PD-L1 bind to the anti-PD-L1 antibody and therefore fail to escape the immune checkpoint, and thus are recognized and eliminated by the immune system.

The anti-PD-L1 antibodies and antigen-binding fragments thereof described herein can be monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, bivalent antibodies, or anti-idiotypic antibodies. Recombinant antibodies are antibodies that are produced in vitro using recombinant methods rather than animals. Bispecific antibodies or diabodies are artificial antibodies having fragments of two different monoclonal antibodies, which are capable of binding two different antigens. A "bivalent" antibody and antigen-binding fragment thereof includes two antigen-binding sites. The two antigen binding sites may bind to the same antigen, or may each bind to a different antigen, in which case the antibody or antigen binding fragment is "bispecific".

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein are camelized single domain antibodies (camelized single chain domain antibodies), diabodies (diabodies), scfvs, scFv dimers, bsfvs, dsfvs, (dsFv)2, dsFv-dsFv ', Fv fragments, fabs, Fab ', F (ab ')2, ds diabodies (ds diabodies), nanobodies, domain antibodies, or diabodies.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and/or light chain constant region. The heavy chain constant region includes the CH1, CH1-CH2, or CH1-CH3 regions. In some embodiments, the immunoglobulin constant region may further comprise one or more modifications to achieve a desired property. For example, the constant region may be modified to reduce or deplete one or more effector functions to enhance FcRn receptor binding or to introduce one or more cysteines.

In certain embodiments, the anti-PD-L1 antibody and antigen-binding fragments thereof further comprise a conjugate. It is contemplated that the antibodies or antigen-binding fragments thereof of the present invention may be linked to a variety of conjugates (see, e.g., "Conjugate Vaccines", constraints to Microbiology and Immunology, j.m.cruse and r.e.lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be attached to the antibody or antigen conjugate by covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing or addition, among other means. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than the epitope-binding portion that can be used to bind to one or more conjugates. For example, such sites may comprise one or more reactive amino acid residues, such as cysteine and histidine residues, for facilitating covalent attachment to the conjugate. In certain embodiments, the antibody may be linked indirectly to the conjugate, or via another conjugate. For example, the antibody or antigen-binding fragment thereof can bind to biotin and then indirectly bind to a second conjugate, which is linked to avidin. The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purifying moiety, or a cytotoxic moiety. Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase, or β -D-hemi-componentLactase), radioactive isotopes (e.g., gamma-glucosidase,¹²³I、¹²⁴I、¹²⁵I、¹³¹I、³⁵S、³H、¹¹¹In、¹¹²In、¹⁴C、⁶⁴Cu、⁶⁷Cu、⁸⁶Y、⁸⁸Y、⁹⁰Y、¹⁷⁷Lu、²¹¹At、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、²¹²Bi、and ³²P, other lanthanides, luminescent labels), chromophore moieties, digoxin, biotin/avidin, DNA molecules, or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety such as PEG, which helps to extend the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A "cytotoxic moiety" may be any agent that is harmful to or may damage or kill a cell. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazide), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, doxycycline, and pharmaceutically acceptable salts thereof, Mitomycin C and cis-dichlorodiammineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and anthranilic Acid (AMC)), and antimitotics (e.g., vincristine and vinblastine).

Polynucleotides and recombinant methods

The present application provides isolated polynucleotides encoding anti-PD-L1 antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotide comprises one or more sequences encoding a CDR as in table 1, e.g., those provided in the polynucleotide sequences encoding the variable region.

In some embodiments, the isolated polynucleotide encodes a heavy chain variable region and comprises a sequence selected from the group consisting of seq id no: SEQ ID NO 8, SEQ ID NO 12, SEQ ID NO 15, SEQ ID NO 19, SEQ ID NO 21, and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto. In some embodiments, the isolated polynucleotide encodes a light chain variable region and comprises a sequence selected from the group consisting of seq id no: SEQ ID NO 10, SEQ ID NO 14, SEQ ID NO 17, SEQ ID NO 20, SEQ ID NO 23, and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto. In certain embodiments, the percentage of identity is derived from the degeneracy of the genetic code, while the encoded protein sequence remains unchanged.

Vectors comprising polynucleotides encoding the anti-PD-L1 antibodies and antigen-binding fragments thereof (e.g., comprising the sequences shown in table 1) can be introduced into host cells for cloning (amplification of DNA) or gene expression using recombinant techniques well known in the art. In another embodiment, the antibody can be made by methods of homologous recombination as are well known in the art. The DNA encoding the monoclonal antibody may be isolated and sequenced by conventional methods (e.g., oligonucleotide probes may be used which specifically bind to the genes encoding the heavy and light chains of the antibody). Various carriers can be selected. Carrier components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (e.g.SV 40, CMV, EF-1. alpha.) and a transcription termination sequence.

In some embodiments, the vector system comprises a mammalian, bacterial, yeast system, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2, etc., other vectors available from the laboratory or commercially available. Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

Vectors comprising polynucleotides encoding the antibodies and antigen-binding fragments thereof may be introduced into host cells for cloning or gene expression. The host cells suitable for cloning or expressing the DNA in the vector of the present invention are prokaryotic cells, yeast or the above-mentioned higher eukaryotic cells. Prokaryotic cells suitable for use in the present invention include eubacteria such as, for example, gram-negative or gram-positive bacteria, e.g., Enterobacteriaceae, e.g., E.coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and Shigella, and bacilli, e.g., Bacillus subtilis and Bacillus licheniformis, Pseudomonas, e.g., Pseudomonas aeruginosa, and Streptomyces.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast may also be used as host cells for cloning or expressing vectors encoding anti-PD-L1 antibodies. Saccharomyces cerevisiae, or Saccharomyces cerevisiae, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are more commonly used and are suitable for use in the present invention, such as Schizosaccharomyces pombe; kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces williamsii (ATCC 24,178), Kluyveromyces lactis (ATCC 56,500), Kluyveromyces drosophilus (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; yarrowia lipolytica (EP 402,226); pichia pastoris (EP 183,070); candida species; trichoderma reesei (EP 244,234); performing Neurospora; schwann yeast in western countries, such as: schwann yeast western; and filamentous fungi, such as: neurospora, Penicillium, Tolypocladium and Aspergillus, such as: aspergillus nidulans and Aspergillus niger.

The host cells provided herein that are suitable for expressing glycosylated antibodies or antigen binding fragments thereof are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Various baculovirus strains (bacterial strains) and variants thereof, as well as corresponding permissive insect host cells (permissive insect host cells), have been found to be derived from hosts such as: spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as Autographa californica nuclear polyhedrosis virus and Bm-5 variants of Bombyx mori nuclear polyhedrosis virus, all of which can be used in the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

However, the most interesting are the vertebral cells, and the culture of the vertebral cells (tissue culture) has become a routine procedure. As examples of mammalian host cells which may be used, there are SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cell subclones in suspension culture, Graham et al, j.gen virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse testicular support cells (TM4, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human liver cancer cell line (Hep G2). In certain preferred embodiments, the host cell is a 293F cell.

Host cells are transformed with the above-described expression or cloning vectors that produce anti-PD-L1 antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformed cells, or amplifying genes encoding the sequences of interest.

The host cells of the invention used to produce the antibodies or antigen-binding fragments thereof can be cultured in a variety of media. Commercially available culture media such as Ham's F10(Sigma), minimal essential Medium (MEM, (Sigma)), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma may be used to culture the host cells. Additionally, any of the methods described in Ham et al, meth.enz.58:44(1979), Barnes et al, anal. biochem.102:255(1980), U.S. patent No. 4,767,704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655, respectively; or 5,122,469; WO 90/03430; WO 87/00195; or the medium described in U.S. patent application Re.30,985, can be used as the medium for the host cells. These media may be supplemented with the necessary hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds, usually in the micromolar range, at final concentrations), and glucose or an equivalent energy source. The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the medium, such as temperature, pH, and the like, which have been previously used to select host cells for expression, are well known to those of ordinary skill.

When using recombinant techniques, the antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the particulate debris of the host cells or lysed fragments is first removed, for example, by centrifugation or sonication. Carter et al, Bio/Technology 10:163-167(1992) describes a method for isolating antibodies secreted into the membrane space of E.coli walls. Briefly, the cell paste (cell paste) was opened in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethanesulfonyl fluoride (PMSF) for about 30 minutes or more. Cell debris was removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant of the expression system is usually first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be added in any of the foregoing steps to inhibit proteolytic degradation, as well as antibiotics to prevent the growth of adventitious contaminants.

The antibody produced from the cells can be purified by purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique. The class of the antibody and the presence of the Fc domain of any immunoglobulin in the antibody determines whether protein a is suitable as an affinity ligand. Protein a can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, j. immunol. meth.62:1-13 (1983)). Protein G is applicable to all murine isoforms and human gamma 3(Guss et al, EMBO J.5:15671575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices may be used. Mechanically stable matrices such as controlled pore glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX. TM. resin (J.T.Baker, Phillipsburg, N.J.). Other techniques for protein purification may also be determined depending on the antibody to be obtained, such as fractionation in ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resins (e.g.polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification step, the mixture containing the antibody of interest and impurities can be treated by low pH hydrophobic interaction chromatography, using an elution buffer at a pH of about 2.5-4.5, preferably at low salt concentrations (e.g., from about 0 to 0.25M salt concentration).

Reagent kit

Kits comprising the anti-PD-L1 antibodies and antigen-binding fragments thereof are provided. In some embodiments, the kit is used to detect the presence or level of PD-L1 in a biological sample. The biological sample may comprise a cell or tissue.

In some embodiments, the kit comprises an anti-PD-L1 antibody and antigen-binding fragments thereof conjugated to a detectable label. In some embodiments, the kit comprises an unlabeled anti-PD-L1 antibody and antigen-binding fragments thereof, and further comprises a secondary antibody that is capable of binding to the unlabeled anti-PD-L1 antibody and antigen-binding fragments thereof to a label. The kit may further include instructions for use and packaging separating each component in the kit.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof are linked to a substrate or instrument for use in a sandwich assay such as an ELISA or immunochromatographic assay. Suitable substrates or instruments may be, for example, microplates and test strips.

Pharmaceutical compositions and methods of treatment

The present application further provides pharmaceutical compositions comprising the anti-PD-L1 antibodies and antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants or nontoxic auxiliary substances, other components well known in the art, or various combinations thereof.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in a composition comprising an antibody or antigen-binding fragment thereof disclosed herein will reduce oxidation of the antibody or antigen-binding fragment thereof. The reduction in oxidation prevents or reduces the reduction in binding affinity, thereby improving antibody stability and extending shelf life. Thus, in certain embodiments, the invention provides compositions comprising one or more of the antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. The present invention further provides methods of preventing oxidation, extending shelf life and/or increasing activity of an antibody or antigen-binding fragment thereof provided herein by admixing the antibody or antigen-binding fragment thereof with one or more antioxidants, such as methionine.

Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, ringer's solution injection, isotonic glucose injection, sterile water injection, or dextrose and lactate injection, non-aqueous media such as: fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as: sodium chloride or glucose, buffers such as: phosphate or citrate buffers, antioxidants such as: sodium bisulfate, local anesthetics such as: procaine hydrochloride, suspending and dispersing agents such as: sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifiers such as: polysorbate 80 (tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents as carriers may be added to the pharmaceutical composition in multi-dose containers, including phenolics or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzalkonium chloride. Suitable excipients may include, for example, water, salt, glucose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions may be prepared in any conventional form, for example, liquid solvents, suspending agents, emulsifying agents, or solid forms suitable for the production of liquid solvents, suspending agents or emulsifying agents. Injectable preparations may include ready-to-use sterile and/or pyrogen-free solutions, sterile dried solubles, e.g., lyophilized powders, combined with solvents prior to use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dried insoluble products, combined with vehicles prior to use, and sterile and/or pyrogen-free emulsions. The solvent may be aqueous or non-aqueous.

In certain embodiments, a unit dose of an injectable formulation is packaged in an ampoule, a manifold, or a syringe with a needle. It is well known in the art that all formulations for injection administration should be sterile pyrogen free.

In certain embodiments, sterile lyophilized powders can be prepared by dissolving an antibody or antigen-binding fragment thereof disclosed herein in an appropriate solvent. The solvent may contain a compound that enhances the stability of the powder or reconstituted solution prepared from the powder, or improves the pharmacological properties of the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable materials. The solvent may contain a buffer, such as citric acid buffer, sodium or potassium phosphate buffer or other buffers known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. The dissolution is followed by sterile filtration under standard conditions well known in the art and then lyophilized to produce the desired formulation. In one embodiment, the resulting solvent is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of the anti-PD-L1 antibody or antigen-binding fragment thereof or composition thereof. The loading per vial may be slightly higher than that required for each dose or for multiple doses (e.g., 10% excess), thereby ensuring accurate sampling and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as in the range of about 4 ℃ to room temperature.

And re-dissolving the freeze-dried powder with water for injection to obtain the preparation for injection administration. In one embodiment, the lyophilized powder can be reconstituted by addition to sterile pyrogen-free water or other suitable liquid carrier. The precise amount is determined by the selected therapy and can be determined empirically.

Also provided are methods of treatment comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein to a subject in need thereof, thereby treating or preventing a condition or disorder associated with PD-L1. In another aspect, there is also provided a method of treating a condition in a subject that would benefit from an upregulated immune response, comprising administering to the subject in need thereof a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, as described herein.

The therapeutically effective dose of the antibody or antigen-binding fragment thereof provided herein depends on a variety of factors well known in the art, such as body weight, age, past medical history, current therapy, the health status of the subject and the potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of tumor development. One skilled in the art (e.g., a physician or veterinarian) can proportionately lower or raise the dosage based on these or other conditions or requirements.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein can be administered at a therapeutically effective dose of between about 0.01mg/kg and about 100mg/kg (e.g., about 0.01mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg, about 95mg/kg, or about 100 mg/kg). In certain embodiments, the antibody or antigen-binding fragment thereof is administered at a dose of about 50mg/kg or less, and in certain embodiments, 10mg/kg or less, 5mg/kg or less, 1mg/kg or less, 0.5mg/kg or less, or 0.1mg/kg or less. A particular dose can be administered at multiple intervals, such as once daily, twice or more monthly, once weekly, once every two weeks, once every three weeks, once monthly, or once every two or more months. In certain embodiments, the dosage administered may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequent administered dose. In certain embodiments, the dosage administered is adjusted during the course of treatment according to the response of the subject to whom it is administered.

The dosage regimen may be adjusted to achieve an optimal response (e.g., therapeutic response). For example, administration can be carried out as a single dose or in multiple divided doses over a period of time.

The antibodies and antigen-binding fragments disclosed in the present invention can be administered by administration means well known in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection, or intradermal injection) or non-injection (e.g., oral, nasal, sublingual, rectal, or topical administration).

The conditions and disorders associated with PD-L1 may be immune-related diseases or disorders. Such conditions and disorders include tumors and cancers, e.g., non-small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides (mycoses fungoids), merkel cell carcinoma and other hematological malignancies, such as Classical Hodgkin Lymphoma (CHL), primary mediastinal large B cell lymphoma, B cell-rich lymphoma of T cells/histiocytes, EBV positive and negative PTLD and EBV-associated Diffuse Large B Cell Lymphoma (DLBCL), plasmablast lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal cancer and HHV 8-associated primary effusion lymphoma, hodgkin lymphoma; autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), autoimmune diabetes, chronic viral infections such as hepatitis B, hepatitis C, herpes virus, Epstein-Barr virus, AIDS virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus 2, human papilloma virus, viral infections of adenovirus, Kaposi's sarcoma-associated herpes virus epidemics, Torquenovirus, JC virus or BK virus, and the like.

Application method

The application further provides methods of using the anti-PD-L1 antibodies or antigen-binding fragments thereof.

In some embodiments, the present application provides methods of detecting the presence or level of PD-L1 in a biological sample comprising contacting the biological sample with an anti-PD-L1 antibody or antigen-binding fragment thereof described herein, and determining the presence or level of PD-L1 in the sample. The sample containing PD-L1 may be derived from a cell or tissue or body fluid of a subject. In certain embodiments, the sample comprising PD-L1 is derived from a tumor or cancer cell or tissue, such as a circulating tumor cell, a tumor mass, or a tissue suspected of containing a tumor cell. In certain embodiments, the sample containing PD-L1 is an immune cell that infiltrates a tumor, e.g., an immune cell within a tumor, an immune cell surrounding a tumor, or other tumor stromal cells such as fibroblasts. The tumor-infiltrating cells can be T cells (e.g., CD 4)⁺T cells or CD8⁺T cells), B cells, or other myeloid lineage cells, e.g., macrophages, natural killer cells, dendritic cells, monocytes, and the like. The presence or level of PD-L1 in a sample can be determined, for example, by detecting binding of an antibody to PD-L1 using methods such as Immunohistochemistry (IHC), ELISA, flow cytometry or any other suitable method known in the art.

The presence and level of PD-L1 in a target biological tissue may indicate whether the individual from which the biological sample was derived is likely to respond to a PD-L1 antagonist. Accordingly, the present invention further provides a method of identifying an individual having a disorder or condition that is likely to respond to a PD-L1 antagonist, comprising: determining the presence or level of PD-L1 in a test biological sample from the individual using the anti-PD-L1 antibody or antigen binding fragment thereof described herein, wherein the presence or level of PD-L1 in the test biological sample is upregulated as indicative of the likelihood of a response. In some embodiments, the test sample is derived from a cancer cell or tissue, or an immune cell that enters a tumor. The term "upregulation" as used herein means a total increase in the level of PD-L1 protein detected in a test sample of no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more, using an antibody or antigen binding fragment thereof as described herein, as compared to the level of PD-L1 protein in a reference sample detected using the same antibody. The reference sample may be a control sample obtained from a healthy or disease-free subject, or a healthy or disease-free sample obtained from an individual from which the test sample is derived. For example, the reference sample can be a disease-free sample that is near or adjacent to the test sample (e.g., tumor). In some embodiments, the method further comprises administering a therapeutically effective amount of an anti-PD-L1 antibody, or antigen-binding fragment thereof, to an individual identified as having a disorder or condition that is likely to respond to a PD-L1 antagonist.

The antibodies and antigen-binding fragments disclosed herein can be administered alone or in combination with one or more other therapeutic means or substances. For example, the antibodies and antigen-binding fragments disclosed herein may be used in combination with chemotherapy, radiation therapy, cancer treatment surgery (e.g., tumor resection), treatment of one or more anti-emetic drugs or other chemotherapy-induced complications, or any other therapeutic substance for cancer or any other condition mediated by PD-L1. In certain such embodiments, the antibodies and antigen-binding fragments disclosed herein, when used in combination with one or more therapeutic agents, may be administered concurrently with the one or more therapeutic agents, and in certain such embodiments, the antibodies and antigen-binding fragments may be administered concurrently as part of the same pharmaceutical composition. However, the antibody and antigen conjugate "in combination" with other therapeutic substances need not be administered simultaneously or in the same composition as the therapeutic substance. The meaning of "in combination" in the present invention also includes that an antibody and an antigen-binding agent administered before or after another therapeutic substance are also considered to be "in combination" with the therapeutic substance, even if the antibody or antigen-binding fragment thereof and the second substance are administered by different administration means. Where possible, other therapeutic agents to be used in combination with the antibodies or antigen-binding fragments thereof disclosed herein may be administered by Reference to the methods of the product specification for the other therapeutic agent, or by Reference to surgeon's docket No. 2003(Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (11 months 2002)), or by Reference to other methods known in the art.

In certain embodiments, the therapeutic substance is capable of inducing or enhancing an immune response against the cancer. For example, tumor vaccines can be used to induce an immune response to certain tumors or cancers. Cytokine therapy can be used to enhance the presentation of tumor antigens to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as interferon alpha, beta and gamma, colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte-CSF, interleukins such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor necrosis factors such as TNF-alpha and TNF-beta. Agents that inactivate immunosuppressive targets, such as TGF- β inhibitors, IL-10 inhibitors, and Fas ligand inhibitors, may also be used. Another group of agents include those that activate an immune response against a tumor or cancer cell, for example, those that increase T cell activation (e.g., T cell co-stimulatory molecule agonists such as CTLA-4, ICOS, and OX-40), and those that increase dendritic cell function and antigen presentation.

The present application further provides a method of monitoring a therapeutic response or disease progression in a subject treated with a PD-L1 antagonist, comprising determining the presence or level of PD-L1 in a test biological sample from the individual with an anti-PD-L1 antibody or antigen-binding fragment thereof described herein. In certain embodiments, the methods further comprise comparing the level of PD-L1 in the test biological sample to the level of PD-L1 in a comparable sample previously obtained from the same individual, wherein an increase in PD-L1 that is reduced or slowed or halted in the test biological sample indicates a positive therapeutic response or controlled disease progression. The comparable sample may be the same type of sample as the sample to be tested, but obtained from the same individual prior to or at the initial stage of treatment.

The following examples are intended to better illustrate the invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials and methods described below, in whole or in part, are within the scope of the invention. These specific compositions, materials and methods are not intended to limit the invention, but are merely illustrative of specific embodiments within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without adding inventive step and without departing from the scope of the invention. It will be appreciated that various modifications to the method of the invention may still be included within the scope of the invention. The inventors intend such variations to be included within the scope of the present invention.

Example 1: generation of antibody hybridomas

1.1 immunization: female Balb/c mice, 8 weeks old, were sensitized via foot pad injection of human PD-L1ECD protein in 10 μ g TiterMax, followed by foot pad challenge with PD-L1ECD protein in aluminum phosphate gel adjuvant every 3 days until fusion was appropriate. anti-PD-L1 antibody serum titers were detected by ELISA or FACS every two weeks.

1.2 cell fusion: animals received a final challenge of 10g of human PD-L1ECD protein in PBS intraperitoneally 3 days prior to fusion. On the day of fusion, lymph node cells were harvested and single cell suspensions were prepared. The obtained lymphocytes were mixed with myeloma cells (P3) at a ratio of 1: 1. The cell mixture was washed and washed in ECF solution at 2.0x 10⁶cell/mL density heavy suspension. Cell fusion was performed using a BTX 2000 electrofusion apparatus.

1.3 first and second hybridoma supernatant screens: after incubation at 37 ℃ for 7-14 days, a portion of the hybridoma supernatant was detected by Mirrorball assay. Briefly, hybridoma supernatantsThe solution was diluted 5-fold with 1 XPBS. CHO-K1 cells (1.25X 10) expressing PD-L1⁵cells/mL) was mixed with secondary fluorescently labeled antibody (400ng/mL) and DraQ5(1: 5000). Add 20. mu.L of the cell mixture and 20. mu.L of the diluted hybridoma supernatant sample to each well of the 384-well plate and incubate at room temperature in the dark for at least 2 hours until ready for use

And (4) carrying out analysis on a high-sensitivity microplate cytometer. Positive cells were verified by FACS using CHO-K1 cells expressing PD-L1. Cells were stained with a sample of hybridoma supernatant, followed by secondary antibody binding with goat anti-mouse IgG Fc conjugated with FITC. The corresponding maternal cell line was used as a negative control. Bound cells were analyzed using the FACSCANTO II and FlowJo version of software from BD Biosciences.

1.4 subcloning: subcloning was performed using hybridoma cell lines that were verified to be positive for PD-L1 binding. Briefly, for each hybridoma cell line, cells were counted and diluted to 5 or 1 cells per well in cloning media. Plating was performed in 96-well plates at 200. mu.L/well. The plates were placed at 37 ℃ in 5% CO₂Incubate until subsequent analysis.

1.5 isotype assay: the ELISA plates were coated with 50. mu.L/well of goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA and IgM antibodies at 1. mu.g/mL, respectively. After blocking, 50 μ L of hybridoma supernatant samples were added to each well and incubated for 2 hours at room temperature. Goat anti-mouse kappa light chain-HRP was used as the detection antibody. The color was developed using TMB substrate incubation for 10min and the reaction was stopped with 2M HCl. The plate was read at 450nm on an ELISA plate reader.

1.6 cell-based binding assays: to test the binding activity of the lead antibody to the target, CHO-K1 or mDC expressing human PD-L1 was conjugated with the lead antibody, followed by a secondary antibody conjugated with FITC-conjugated goat anti-human IgG Fc. The corresponding maternal cell line was used as a negative control. Bound cells were analyzed using the FACSCANTO II and FlowJo version of software from BD Biosciences.

Full-length human PD-L1 transfected with antibody binding to human PD-L1 from murine hybridoma cellsCHO cells, subsequently bound with a secondary antibody conjugated to FITC-conjugated goat anti-rat IgG Fc and analyzed with FACS. As shown in fig. 1, antibody 27.3; 74.15 and 17.42 with an EC of about 1nM₅₀Specifically binds to PD-L1 expressed on CHO cells.

Example 2: humanization and purification

Humanization: two mouse anti-human PD-L1 antibodies (designated 2.27.3 and 2.74.15, respectively) from hybridomas were selected for humanization based on their high affinity and specificity of binding to PD-L1 for increasing T cell proliferation and increasing secretion of the cytokines IFN γ and IL-2 in MLR. The humanization is performed using a technique known as CDR grafting. The FR and CDR regions are defined according to the Kabat system and the IMGT system. Combining the results of sequence homology with the structural similarity alignment, the mouse FR1-3 gene was replaced with the closest human FR1-3 gene, and the mouse FR4 gene was replaced with the closest human JH and J κ genes. After the template sequence and the optimized codons are verified, the heavy chain variable region and the light chain variable region are amplified and cloned into an expression vector, and then the humanized antibody is expressed. The obtained humanized antibodies of 2.74.15 were designated 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb 8. A humanized antibody of 2.27.3 was also prepared.

Antibody purification: DNA vectors containing humanized antibodies were used to transfect 293F cells for antibody expression and production. The antibody in the 293F cell culture supernatant was purified using a protein a affinity chromatography column.

Example 3: characterization of humanized antibodies

The humanized antibody of 2.74.15 was selected for further characterization. Humanized antibodies 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb8 were analyzed for binding to CHO cells transfected with full-length human PD-L1 using FACS according to the procedure of section 1.6 in example 1.

FIG. 2 shows staining of PD-L1 transfected CHO cells with humanized antibody against human PD-L1, and FACS analysis shows humanized PD-L1 antibody with an EC of about 1nM₅₀Specifically binds to PD-L1. FIG. 3 shows the generation of human dendritic cells from single cell cultures cultured for 7 days in GM-CSF and IL4,it was stained with humanized PD-L1 antibody (i.e., 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb 8). FACS analysis showed that the humanized antibody specifically binds to native PD-L1 expressed on DCs.

3.1 competitive assay by FACS assay: to examine whether the humanized antibody could block the binding of PD-L1 to PD-1, CHO-K1 cells expressing human PD-L1 were incubated with different concentrations of humanized antibody at 4 ℃ for 1 hour. Unbound antibody was eluted and mouse Fc-labeled human PD-1 was added to the cells. Binding of human PD-1 to cells expressing PD-L1 was detected using FITC-conjugated goat anti-mouse IgG and subsequently analyzed using the FACSCAnto II and FlowJo version of software from BD Biosciences.

CHO cells expressing human PD-L1 were incubated with different concentrations of humanized antibodies (2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb8) or control antibodies, and then mouse Fc-labeled human PD-1 was added to the cells. Binding of human PD-1 to cells expressing PD-L1 was detected using FITC-conjugated goat anti-mouse IgG, followed by FACS analysis. As shown in FIG. 4, all of the humanized PD-L1 antibodies tested blocked the binding of PD-1 to PD-L1 expressed on transformed CHO cells.

3.2 affinity testing for Surface Plasmon Resonance (SPR) assays: the affinity and binding kinetics of the antibodies (2.74, 2.74.15.cAb (chimeric antibody of 2.74.15), 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb8) to PD-L1 were characterized by the SPR method using ProteOnXPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized on a GLM sensor chip by amine coupling (Bio-Rad). The purified antibody was flowed over the sensor chip and captured by protein a. The chip was rotated 90 ℃ and washed with running buffer (1 XPBS/0.01% Tween20, Bio-Rad) until the baseline stabilized. 5 concentrations of human PD-L1 and running buffer were flowed through the antibody flow cell at a flow rate of 100 μ L/min, first for a bound phase flow of 240s, followed by a dissociation phase of 600 s. After each run with H pH 1.7₃PO₄Regenerating the chip. Binding and dissociation curves were fitted to a 1:1 Langmiur binding model using ProteOn software.

As shown in FIG. 7, the affinity of the humanized PD-L1 antibody for recombinant human PD-L1, detected by using surface plasmon resonance, was from 1.55E-10 to 1.18E-11 mol/L.

3.3 affinity test by FACS assay: antibody binding affinity assay to cell surface PD-L1 was performed by FACS analysis using CHO-K1 cells expressing human PD-L1. The test antibody was diluted 1:2 serially with elution buffer (1 XPBS/1% BSA) and incubated at 4 ℃ for 1 hour. Secondary antibody goat anti-human IgGFc FITC (3.0 moles FITC per mole IgG, (Jackson Immunoresearch Lab)) was added and incubated at 4 ℃ for 1 hour protected from light. The cells were then washed once and resuspended in 1 XPBS/1% BSA and analyzed using flow cytometry (BD). Fluorescence intensity will be converted to molecular/cellular correlation based on the qualitative beads quantumMESF Kit (Bangs Laboratories, Inc.). K was calculated using Graphpad Prism5_D。

3.4 in vitro functional assay: to estimate the ability of humanized antibodies to modulate T cell responses (including cytokine production and cell proliferation), the following three experiments were performed using antibodies 2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb8, as well as a control antibody.

3.4.1 allogeneic MLR: monocytes were isolated from healthy donors using a human monocyte enrichment kit according to instructions. The cells were cultured for 5-7 days to induce Dendritic Cells (DCs). 1 μ g/mL LPS was added to the cell culture to induce maturation of DCs 18 to 24 hours before use.

To examine the effect of the humanized anti-PD-L1 antibody on IL-2 production in MLR, human CD4 was used⁺T cell enrichment kit for isolating human CD4 according to instructions⁺T cells are then stimulated with mature or immature allogeneic DCs, with or without a humanized anti-PD-L1 antibody or control antibody. IL-2 levels in the culture supernatants were determined by ELISA on day 3. As shown in fig. 8, all the anti-PD-L1 antibodies tested (2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb8) increased IL-2 secretion in a dose-dependent manner.

To examine the production of IFN-. gamma.in MLR by the humanized anti-PD-L1 antibodyUsing human CD4⁺T cell enrichment kit according to the instruction for separating human CD4⁺T cells are then stimulated with mature or immature allogeneic DCs, with or without a humanized anti-PD-L1 antibody or control antibody. The level of IFN γ in the culture supernatants was determined by ELISA on day 5. As shown in fig. 9, all the anti-PD-L1 antibodies tested (2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb8) increased IFN γ secretion in a dose-dependent manner.

To examine the effect of the humanized anti-PD-L1 antibody on T cell proliferation in MLR, human CD4 was used⁺T cell enrichment kit according to the instruction for separating human CD4⁺T cells are then stimulated with mature or immature allogeneic DCs, with or without a humanized anti-PD-L1 antibody or control antibody. Pass [ 2 ]³H]Thymidine incorporation measures T cell proliferation. As shown in fig. 10, all the anti-PD-L1 antibodies tested (2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb8) increased concentration-dependent T cell proliferation.

3.4.2 autoantigen specific immune response: from the same CMV⁺Isolation of CD4 from donors⁺T cells and monocytes. Cultivation of CD4 in the Presence of CMV pp65 peptide and Low dose IL2(20U/ml)⁺T cells. While producing DCs by culturing monocytes according to the method described above. After 5 days, DCs were pulsed with pp65 peptide and then added to CD4 in the presence or absence of humanized or control antibodies⁺T cells. IL-2 and IFN γ levels in the culture supernatants were measured by ELISA on day 3. CMVpp 65-specific CD4⁺Proliferation of T cells³H]Thymidine incorporation assay.

As shown in fig. 11, IFN γ production in specific T cell responses was enhanced by humanized PD-L1 antibodies (2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb 8). FIG. 12 shows that humanized PD-L1 antibody increases autologous DC concentration dependent CMV loading with CMV pp65 peptide⁺-CD4⁺T cells proliferate.

3.4.3Treg suppression assay: regulatory T cells (Tregs) are a subset of T cells and are key immune regulators that play an important role in maintaining self-tolerance. CD4⁺CD25⁺Tregs are associated with tumors, as an increased number of tregs are found in patients with multiple cancers and are associated with a poorer prognosis. To directly evaluate the role of the humanized antibodies to human PD-L1 in inhibiting Treg suppressive function, the function of tregs was compared in the presence or absence of humanized or control antibodies. Briefly, CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cells were isolated by MACS. The function of tregs was compared in the presence or absence of different concentrations of humanized or control antibodies. Briefly, CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cells were isolated by MACS and CD4 co-cultured with allogeneic mature DCs⁺CD25⁺Tregs and CD4⁺CD25^-T cells (Treg: Teff ratio 1: 1). No antibody or isotype antibody was used as a negative control. Cytokine production and T cell proliferation were measured using the methods described previously.

FIG. 13 shows MACS-isolated CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cells. Co-culture of CD4 with allogeneic DCs in the presence or absence of varying concentrations of humanized or control antibodies⁺CD25⁺Tregs and CD4⁺CD25^-T cells (Treg: Teff ratio 1: 1). The results indicate that the antibody to PD-L1 abolished the suppressive function of tregs and restored the proliferation of reactive T cells and secretion of IFN γ.

3.5ADCC/CDC assay: since human PD-L1 is expressed in a variety of cell types, in healthy and tumor cells in order to be compared to healthy PD-L1⁺The unwanted toxicity of the cells was minimized, verifying that the selected anti-PD-L1 humanized antibody lacks ADCC and CDC functions.

3.5.1 ADCC: target cells (mdcs) and different concentrations of humanized antibody were preincubated in 96-well plates for 30min, followed by PBMC (effector) addition at an effector/target ratio of 50: 1. The plates were incubated at 37 ℃ with 5% CO₂Incubate in incubator for 6 hours. Lysis of the target cells was determined by a cytotoxicity assay kit (Roche). Use ofMolecular Devices spectra Max M5e microplate detector determined optical density. Herceptin (roche) and human breast cancer cell line BT474(HER2 positive) were used as positive controls.

Figure 14 shows that humanized PD-L1 antibodies (2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb8) do not mediate ADCC using IL-2-activated PBMC as a source of Natural Killer (NK) cells and mdcs expressing high levels of cell surface PD-L1 as target cells.

3.5.2 CDC: target cells (mdcs), diluted human serum complement (Quidel-a112), and different concentrations of humanized antibody were mixed in 96-well plates. The plates were incubated at 37 ℃ with 5% CO₂Incubate in incubator for 4 hours. Target cell lysis was determined using CellTiter glo (Promega-G7573). Rituximab (roche) and the human B-cell lymphoma cell line Raji (CD20 positive) were used as positive controls.

FIG. 15 shows mixed incubation of target cells (mDC), diluted human serum complement (Quidel-A112) and different concentrations of humanized PD-L1 antibody for 4 hours. Target cell lysis was determined using CellTiter glo (Promega-G7573). The data show that the humanized PD-L1 antibody does not mediate CDC.

3.6 sorting by FACS (Binning) test: this experiment was primarily to find out whether these antibodies bind to the same, overlapping, or completely different epitopes. To check whether the humanized antibody binds to the same epitope as the control antibody, CHO-K1 cells expressing human PD-L1 were incubated with different concentrations of humanized antibody at 4 ℃ for 1 hour. Unbound antibody was eluted and biotin-labeled control antibody was added to the cells. Binding of biotin-labeled control antibodies to PD-L1-expressing cells was detected using PE-linked streptomycin, followed by analysis using the facscan II and FlowJo version of software from BD Biosciences.

3.7 Cross-species binding assay: the cross-reactivity of the antibodies to cynomolgus monkey and murine PD-L1 was determined by ELISA. Human, cynomolgus monkey and mouse PD-L1 were coated on ELISA plates, respectively. After blocking, the humanized antibody was added to the plate and incubated at room temperature for at least 2 hours. Binding of the antibody to the coated antigen was detected with goat anti-human IgG Fc-HRP. The color was developed using TMB substrate incubation for 10min and the reaction was stopped with 2M HCl. The plate was read at 450nm on a Molecular Device M5e microplate reader.

As shown in FIG. 6, the results of ELISA experiments showed that the humanized PD-L1 to be tested bound to cynomolgus monkey PD-L1 in a dose-dependent manner. However, none of the antibodies tested (2.74.15.hAb4, 2.74.15.hAb5, 2.74.15.hAb6, 2.74.15.hAb7, and 2.74.15.hAb8) bound to murine PD-L1 (data not shown). In addition, 2.27.3 humanized antibodies were also tested, but the results indicated that these humanized antibodies lost binding affinity to monkey PD-L1 (data not shown).

3.8FACS Cross-family binding assay: to detect the cross-family binding activity of the humanized antibody, a cell line expressing PD-L2 was bound with the humanized antibody, followed by secondary antibody binding with a goat anti-human IgG Fc conjugated with FITC. PD-L1 expressing cells served as positive controls. The corresponding master cell line served as a negative control. Bound cells were analyzed using the FACSCANTO II and FlowJo version of software from BD Biosciences.

CHO cells transfected with PD-L1 or PD-L2 were stained with humanized PD-L1 antibody and analyzed by FACS. As shown in figure 5, the humanized PD-L1 antibody specifically bound PD-L1, but not PD-L2 of the PD-1 ligand family.

While the disclosure has been particularly shown and described with reference to specific embodiments, some of which are preferred, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising heavy chain CDR sequences as set forth in SEQ ID NOs:1, 2, and 3, respectively, and light chain CDR sequences as set forth in SEQ ID NOs:4, 5, and 6, respectively, capable of specifically binding to human PD-L1.

2. The antibody or antigen binding fragment thereof of claim 1, comprising a heavy chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO 7, SEQ ID NO 11, and SEQ ID NO 18;

it comprises a light chain variable region selected from the group consisting of SEQ ID NO 9, SEQ ID NO 13, SEQ ID NO 16 and SEQ ID NO 22.

3. The antibody or antigen binding fragment thereof of claim 1, which is one of:

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9;

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 11; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 13;

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 11; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 16;

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 13; and

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 22.

4. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-3.

5. A vector comprising the isolated polynucleotide of claim 4.

6. A host cell comprising the vector of claim 5.

7. A method of expressing the antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising culturing the host cell of claim 6 under conditions in which the isolated polynucleotide of claim 4 is expressed.

8. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-3.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-3 and one or more pharmaceutically acceptable carriers.

10. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-3 in the manufacture of a medicament for the treatment of nasopharyngeal carcinoma, non-small cell lung carcinoma, breast cancer, or head and neck cancer.

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