CN106432501B

CN106432501B - Novel anti-PD-L1 antibodies

Info

Publication number: CN106432501B
Application number: CN201610638134.5A
Authority: CN
Inventors: 郑勇; 李竞; 陈智胜
Original assignee: CStone Pharmaceuticals Shanghai Co Ltd; CStone Pharmaceuticals Suzhou Co Ltd; CStone Pharmaceuticals
Current assignee: CStone Pharmaceuticals Shanghai Co Ltd; CStone Pharmaceuticals Suzhou Co Ltd; CStone Pharmaceuticals
Priority date: 2015-08-06
Filing date: 2016-08-05
Publication date: 2021-07-30
Anticipated expiration: 2036-08-05
Also published as: CN106432501A

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 fully human monoclonal IgG4 antibody that acts on PD-1, seizing new breakthrough indicators of the 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 (particularly fully human 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 some embodiments, the antibody or antigen-binding fragment thereof 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, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39 and 41.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein comprises a light chain CDR sequence selected from the group consisting of: 7, 9, 11, 19, 21, 23, 31, 33 and 35.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a heavy chain variable region selected from the group consisting of

SEQ ID NOs

1, 3, 5, 7, 9, and 11; or selected from SEQ ID NO 13, 15, 17, 19, 21 and 23; or selected from SEQ ID NO 25, 27, 29, 31, 33 and 35; or at least 1, 2, 3, 4,5 or 6 CDRs selected from SEQ ID NOs 37, 39, 41, 19, 21 and 23.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region selected from the group consisting of:

a) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 3 and/or SEQ ID NO 5;

b) a heavy chain variable region comprising SEQ ID NO 13, SEQ ID NO 15 and/or SEQ ID NO 17;

c) a heavy chain variable region comprising SEQ ID NO 25, SEQ ID NO 27 and/or SEQ ID NO 29; and

d) a heavy chain variable region comprising SEQ ID NO 37, SEQ ID NO 39 and/or SEQ ID NO 41.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a light chain variable region selected from the group consisting of:

a) a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 9 and/or SEQ ID NO 11;

b) a light chain variable region comprising SEQ ID NO 19, SEQ ID NO 21 and/or SEQ ID NO 23; and

c) a light chain variable region comprising SEQ ID NO 31, SEQ ID NO 33 and/or SEQ ID NO 35.

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

a) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 3 and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 9 and/or SEQ ID NO 11;

b) a heavy chain variable region comprising SEQ ID NO 13, SEQ ID NO 15 and/or SEQ ID NO 17; and a light chain variable region comprising SEQ ID NO 19, SEQ ID NO 21 and/or SEQ ID NO 23;

c) a heavy chain variable region comprising SEQ ID NO 25, SEQ ID NO 27 and/or SEQ ID NO 29; and a light chain variable region comprising SEQ ID NO 31, SEQ ID NO 33, and/or SEQ ID NO 35; or

d) A heavy chain variable region comprising SEQ ID NO 37, SEQ ID NO 39 and/or SEQ ID NO 41; and a light chain variable region comprising SEQ ID NO 19, SEQ ID NO 21 and/or SEQ ID NO 23.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a heavy chain variable region selected from the group consisting of SEQ ID NO 43, SEQ ID NO 47, SEQ ID NO 51, and SEQ ID NO 55.

In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a light chain variable region selected from the group consisting of SEQ ID NO:45, SEQ ID NO:49, and SEQ ID NO: 53.

a) a heavy chain variable region comprising SEQ ID NO 43; and a light chain variable region comprising SEQ ID NO 45;

b) a heavy chain variable region comprising SEQ ID NO 47; and a light chain variable region comprising SEQ ID NO 49;

c) a heavy chain variable region comprising SEQ ID NO 51; and a light chain variable region comprising SEQ ID NO 53; or

d) A heavy chain variable region comprising SEQ ID NO: 55; and a light chain variable region comprising SEQ ID NO: 49.

In certain embodiments, an antibody or antigen-binding fragment thereof described herein includes, for example, 1.4.1, 1.14.4, 1.20.15, and 1.46.11.

In certain embodiments, the antibodies or antigen binding fragments thereof described herein compete for the same epitope as antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11. In certain embodiments, the epitope to which the antibody or antigen-binding fragment thereof described herein binds comprises at least one of the following PD-L1 amino acid residues: e58, E60, D61, K62, N63 and R113.

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 fully human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a diabody, 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 293F cell.

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

In another aspect, the PD-L1 antibodies of the present application, e.g., 1.4.1, 1.14.4, 1.20.15, and 1.46.11, have good tolerance and high in vivo anti-tumor activity in animals. In certain embodiments, the tumor volume of an animal having a tumor administered a PD-L1 antibody described herein is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a control animal having a similar baseline tumor volume administered solvent alone.

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 FACS analysis-determined binding of fully human PD-L1 antibody to PD-1 expressing CHO cells.

FIG. 2 shows that FACS analysis determined that fully human PD-L1 antibody blocked the binding of PD-1 to CHO cells transfected with PD-L1.

FIG. 3 shows that fully human PD-L1 antibody specifically binds to PD-L1 and not to PD-L2 as determined by FACS analysis.

FIG. 4 shows the binding of fully human PD-L1 antibody to human and cynomolgus monkey PD-L1.

FIG. 5 shows the complete kinetics of the binding affinity of the PD-L1 antibody to human PD-L1 as determined by plasmon resonance methods at from 2.26E-10 to 4.78E-10 mol/L.

Figure 6 shows that fully human PD-L1 antibody increased IFN γ production in a specific T cell response.

Figure 7 shows that fully human anti-PD-L1 antibody promotes specific T cell proliferation.

FIG. 8 shows that fully human PD-L1 antibody increased IFN γ production in a Mixed Lymphocyte Reaction (MLR).

FIG. 9 shows the effect of fully human anti-PD-L1 antibody on IL-2 production in MLR.

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

Figure 11 shows that anti-PD-L1 antibody reversed Treg suppression function.

Figure 12 shows that anti-PD-L1 antibody lacks ADCC in activated T cells.

Figure 13 shows that anti-PD-L1 antibody lacks CDC in activated T cells.

FIGS. 14A and 14B show cross-reactivity of anti-PD-L1 antibody with human/mouse PD-1. 2 μ g/ml of 1.14.4 antibody was coated on 96-well plates overnight and incubated with hPD-L1-His protein (FIG. 14A) and mPD-L1-His protein (FIG. 14B), followed by addition of HRP-anti-His antibody for detection.

Figure 15 shows the hot spot residues in the hPD-L1 structure, which are the binding sites for antibody 1.14.4. The data are from table 3. Colors in the figure are used to distinguish differences between epitopes.

Fig. 16 shows good tolerance of hPD-L1 antibody 1.14.4 in vivo. After multiple intraperitoneal injections of humanized B-hPD-1 mice inoculated with MC38-B7H1 colon cancer cells with three doses of 1.14.4 antibody (3 mg/kg, 10mg/kg, and 30mg/kg, respectively), the body weight of each group of mice did not change significantly during the experiment.

Figure 17 shows a significant inhibition of tumor cell growth in vivo by hPD-L1 antibody 1.14.4. Three doses of 1.14.4 antibody (3 mg/kg, 10mg/kg and 30mg/kg, respectively) all showed significant antitumor effects with Tumor Growth Inhibition (TGI) > 40% 19 days after antibody administration.

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 small antibody fragments with two antigen-binding sites, wherein the fragments comprise V's connected 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(by a polypeptideLinker attachment) 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 "fully human", when used in this application, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence corresponding to that of an antibody produced by a human or human immune cell or derived from a non-human source, e.g., a transgenic non-human animal utilizing a human antibody repertoire, or other sequence encoding a human antibody. In certain embodiments, a fully human antibody does not comprise amino acid residues (particularly antigen binding residues) derived from a non-human antibody.

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 and/or monkey PD-L1, and their binding affinity (K)_D)≤10^-6M (e.g.. ltoreq.5 x10^-7M，≤2x10^-7M，≤10^-7M，≤5x10^-8M，≤2x10^-8M，≤10^-8M，≤5x10^-9M，≤2x10^-9M，≤10^-9M，≤10^-10M, about 10^-10M、10^-10M to 10^-9M、10^-10M to 10^-8.5M or 10^-10M to 10^-8M). 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., 1.4.1, 1.14.4, 1.20.15, and 1.46.11, 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.

Mutations of specific amino acid residues in the epitope, e.g., by alanine scanning mutagenesis (alanine scanning mutagenesis), identify mutations that reduce or prevent protein binding. "alanine scanning mutation" is a method that can be used to identify certain residues or regions of a protein that affect the interaction of an epitope with other compounds or proteins to which it binds. A residue or group of target residues in a protein is substituted with a neutral or negatively charged amino acid (most preferably alanine or polyalanine, or a conservative amino acid substitution). Any mutation that reduces the binding of the protein or the codon encoding it to an extent that exceeds a threshold or that maximizes the reduction of binding of the protein compared to other mutations is likely to be in an epitope bound by the protein. In certain embodiments of the present application, epitopes important for PD-L1 antibodies include at least one of the following amino acid residues E58, E60, D61, K62, N63, and R113.

As used herein, "1.4.1" refers to a polypeptide having the sequence as set forth in SEQ ID NO:43, the heavy chain variable region as shown in SEQ ID NO:45 and a human IgG4 isotype constant region.

As used herein, "1.14.4" refers to a fully monoclonal human antibody having the heavy chain variable region shown in SEQ ID NO. 47, the light chain variable region shown in SEQ ID NO. 49, and a human IgG4 isotype constant region.

As used herein, "1.20.15" refers to a fully monoclonal human antibody having the heavy chain variable region shown in SEQ ID NO. 51, the light chain variable region shown in SEQ ID NO. 53, and a human IgG4 isotype constant region.

As used herein, "1.46.11" refers to a fully monoclonal human antibody having the heavy chain variable region shown in SEQ ID NO:55, the light chain variable region shown in SEQ ID NO:49, 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 condition" 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 "disease associated or associated with PD-L1" as used herein refers to any condition 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 condition, inhibit the metastasis of tumor cells, alleviate any symptoms or markers associated with the tumor or cancerous condition, prevent or delay the progression of the tumor or cancerous condition, 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 CD279, is a key immune checkpoint receptor known to be expressed by activated T cells, which 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. Inhibition of the interaction between PD-1 and PD-L1 can enhance T cell responses thereby mediating anticancer activity.

In certain embodiments, the present application provides exemplary fully human monoclonal antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11, whose CDR sequences are shown in table 1, and heavy or light chain variable region sequences are also listed below.

TABLE 1

1.4.1-VH (30511) (SEQ ID NO:43 is amino acid, SEQ ID NO:44 is nucleic acid) heavy chain CDR1-3 (SEQ ID NO:1, 3, 5 is amino acid sequence) and SEQ ID NO:2, 4,6 is nucleic acid sequence.

Section V: IGHV 4-39X 01

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

Segment J: IGHJ4 × 02

1.4.1-VL (30027) (SEQ ID NO:45 is an amino acid and SEQ ID NO:46 is a nucleic acid) light chain CDR1-3: 7, 9 and 11 are amino acid sequences and 8, 10 and 12 are nucleic acid sequences:

section V: IGLV3-1 x 01

Segment J: IGLJ2 x 01

1.14.4-VH (29812) (SEQ ID NO:47 is an amino acid, SEQ ID NO:48 is a nucleic acid) heavy chain CDR1-3: 13, 15 and 17 are amino acid sequences and 14, 16 and 18 are nucleic acid sequences:

section V: IGHV 3-23X 01

And (D) section: IGHD 5-5X 01

Segment J: IGHJ4 × 02

1.14.4-VL and 1.46.11-VL (29841) (SEQ ID NO:49 is an amino acid and SEQ ID NO:50 is a nucleic acid) light chain CDR1-3: 19, 21, 23 are amino acid sequences and 20, 22, 24 are nucleic acid sequences:

section V: IGLV3-21 x 02

Segment J: IGLJ2 x 01

1.20.15-VH (30712) (SEQ ID NO:51 is amino acid, SEQ ID NO:52 is nucleic acid) light chain CDR1-3: 25, 27, 29 are amino acid sequences and 26, 28, 30 are nucleic acid sequences:

section V: IGHV 4-39X 01

And (D) section: is not determined

Segment J: IGHJ4 × 02

1.20.15-VL (29907) (SEQ ID NO:53 is an amino acid and SEQ ID NO:54 is a nucleic acid) light chain CDR1-3: 31, 33, 35 are amino acid sequences and 32, 34, 36 are nucleic acid sequences:

section V: IGLV3-1 x 01

Segment J: IGLJ2 x 01

1.46.11-VH (30626) (SEQ ID NO:55 is amino acid, SEQ ID NO:56 is nucleic acid) light chain CDR1-3: 37, 39 and 41 are amino acid sequences and 38, 40 and 42 are nucleic acid sequences:

section V: IGHV 3-23X 01

And (D) section: IGHD 5-5X 01

Segment J: IGHJ4 × 02

1.46.11-VL (29841) (SEQ ID NO:49 is an amino acid and SEQ ID NO:50 is a nucleic acid) light chain CDR1-3: 19, 21, 23 are amino acid sequences and 20, 22, 24 are nucleic acid sequences:

section V: IGLV3-21 x 02

Segment J: IGLJ2 x 01

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof comprise heavy chain CDR sequences selected from the group consisting of seq id nos:1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39 and 41. In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof comprise light chain CDR sequences selected from the group consisting of: 7, 9, 11, 19, 21, 23, 31, 33 and 35.

In some embodiments, the anti-PD-L1 antibody and antigen-binding fragments thereof comprises a heavy chain variable region selected from the group consisting of: a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 3, and/or SEQ ID NO 5; a heavy chain variable region comprising SEQ ID NO 13, SEQ ID NO 15, and/or SEQ ID NO 17; a heavy chain variable region comprising SEQ ID NO 25, SEQ ID NO 27 and/or SEQ ID NO 29; and a heavy chain variable region comprising SEQ ID NO 37, SEQ ID NO 39 and/or SEQ ID NO 41.

In some embodiments, the anti-PD-L1 antibody and antigen-binding fragments thereof comprises a light chain variable region selected from the group consisting of: a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 9 and/or SEQ ID NO 11; a light chain variable region comprising SEQ ID NO 19, SEQ ID NO 21 and/or SEQ ID NO 23; and a light chain variable region comprising SEQ ID NO 31, SEQ ID NO 33 and/or SEQ ID NO 35.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof include: a) a heavy chain variable region comprising SEQ ID NO 1, SEQ ID NO 3, and/or SEQ ID NO 5; and a light chain variable region comprising SEQ ID NO 7, SEQ ID NO 9 and/or SEQ ID NO 11; b) a heavy chain variable region comprising SEQ ID NO 13, SEQ ID NO 15 and/or SEQ ID NO 17; and a light chain variable region comprising SEQ ID NO 19, SEQ ID NO 21 and/or SEQ ID NO 23; c) a heavy chain variable region comprising SEQ ID NO 25, SEQ ID NO 27 and/or SEQ ID NO 29; and a light chain variable region comprising SEQ ID NO 31, SEQ ID NO 33, and/or SEQ ID NO 35; and d) a heavy chain variable region comprising SEQ ID NO 37, SEQ ID NO 39 and/or SEQ ID NO 41; and a light chain variable region comprising SEQ ID NO 19, SEQ ID NO 21 and/or SEQ ID NO 23.

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 identical sequences, but whose corresponding CDR sequences have 100% sequence identity to the sequences listed in table 1.

In certain embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof are fully human. The fully human antibodies do not have problems in humans such as immunogenicity or reduced binding affinity as often observed in humanized antibodies.

In some embodiments, the fully human anti-PD-L1 antibody and antigen-binding fragments thereof comprises a heavy chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO 43, SEQ ID NO 47, SEQ ID NO 51, SEQ ID NO 55, 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:45, SEQ ID NO:49, SEQ ID NO:53, 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 fully human antibodies retain binding affinity to human PD-L1, preferably with the following exemplary antibodies: 1.4.1, 1.14.4, 1.20.15 and 1.46.11 were similar.

In some embodiments, the fully human anti-PD-L1 antibody and antigen-binding fragments thereof comprises a) a heavy chain variable region comprising SEQ ID NO 43; and a light chain variable region comprising SEQ ID NO 45; b) a heavy chain variable region comprising SEQ ID NO 47; and a light chain variable region comprising SEQ ID NO 49; c) a heavy chain variable region comprising SEQ ID NO 51; and a light chain variable region comprising SEQ ID NO 53; or d) a heavy chain variable region comprising SEQ ID NO 55; and a light chain variable region comprising SEQ ID NO: 49.

Also included are 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. 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 M₅₀Values (i.e., half inhibitory concentrations) blocked binding of 1.4.1, 1.14.4, 1.20.15, and 1.46.11 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, about 10^-10M、10^-10M to 10^-8.5M or 10^-10M to 10^-8M) binds specifically to human PD-L1, as 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) By any suitable method known in the artMethods include using instruments such as the plasmon resonance binding method of Biacore (see, e.g., Murphy, M.et al, Current protocols in protein science, Chapter 19, Unit19.14,2006).

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein and human PD-L1 have an EC of 0.1nM to 100nM (e.g., 0.1nM to 50nM, 0.1nM to 30nM, 0.1nM to 20nM, or 0.1nM to 10nM, or 0.1nM to 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 bind to human PD-L1 with an EC50 (i.e., an effective concentration of 50%) of 1nM to 10nM or 1nM to 5nM (as determined using FACS analysis).

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, 0.2nM to 10nM, or 1nM 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 interleukin 2, a cytokine signaling molecule that modulates the activity of white blood cells (e.g., leukocytes) in the immune system. The term "interferon gamma (IFN gamma)"Is prepared 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 are administered at an EC of no more than 100nM, e.g., no more than 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 0.01nM₅₀Binding to monkey PD-L1 (determined by ELISA). In certain embodiments, the antibodies and antigen-binding fragments thereof bind monkey PD-L1 with an EC50 of about 1nM-10 nM.

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 1.4.1, 1.14.4, 1.20.15, and 1.46.11 to murine PD-L1 was not detectable using common binding assays such as ELISA or FACS analysis, which detected 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 eliminated 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 eliminated effector function. Equivalent functions such as ADCC and CDC can result in cytotoxicity to cells expressing PD-L1. Many cells, including normal cells, are capable of expressing PD-L1. To avoid potential undesirable toxicity to these normal cells, certain embodiments of the antibodies and antigen-binding fragments thereof of the present invention have reduced or even eliminated effector function. 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 eliminated effector functions such as ADCC and CDC do not cause or minimize cytotoxicity to cells expressing PD-L1 (e.g., those normal cells), thus avoiding undesirable 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.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein have reduced side effects. For example, the anti-PD-L1 antibodies and antigen-binding fragments thereof can have fully human IgG sequences and are therefore less immunogenic than humanized antibodies. For another example, the anti-PD-L1 antibodies and antigen-binding fragments thereof can have an IgG4 format to abrogate ADCC and CDC.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein are advantageous in that they can be used in combination with immunogenic agents, such as tumor cells, purified tumor antigens, and cells transfected with a gene encoding an immunostimulatory factor, tumor vaccines. In addition, the anti-PD-L1 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemotherapy and radiation therapy, target-based small molecule therapy, other emerging immune checkpoint modulator therapies. In some embodiments, the antibodies and antigen-binding fragments thereof can be used as the base molecule for antibody-drug conjugates, bispecific or multivalent antibodies.

The anti-PD-L1 antibodies and antigen-binding fragments thereof described herein can be monoclonal antibodies, polyclonal antibodies, fully human 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-1 antibodies and antigen-binding fragments thereof described herein are fully human antibodies. In some embodiments, the fully human antibody is produced using recombinant methods. For example, a transgenic animal such as a mouse can be prepared to carry a transgene or transchromosome of a human immunoglobulin gene and thus be capable of producing fully human antibodies upon immunization with a suitable antigen such as human PD-1. Fully human antibodies can be isolated from such transgenic animals, or alternatively, can be prepared by hybridoma technology, the spleen cells of which are fused to an immortal cell line to produce hybridoma cells that secrete the fully human antibodies. Exemplary transgenic animals include, but are not limited to, Omni rats, in which the expression of endogenous rat immunoglobulin genes is inactivated and simultaneously genetically engineered to contain a functional recombinant human immunoglobulin locus; omni mice, whose endogenous mouse immunoglobulin gene expression is inactivated and at the same time genetically engineered to contain a recombinant human immunoglobulin locus with J-locus deletions and C-kappa mutations. OmniFilc, a transgenic rat, whose expression of endogenous rat immunoglobulin genes was inactivated and simultaneously genetically engineered to contain a recombinant human immunoglobulin locus with a single common, recombinant VkJk light chain and functional heavy chain. For specific information please see further: et al, Journal of Immunology,2013,190: 1481-90; et al, Journal of Immunological Methods 400-401 (2013) 78-86; geurts A.et al, Science,2009,325: 433; us patent 8,907,157; european patent 2152880B 1; european patent 2336329B1, each of which is incorporated by reference herein in its entirety. Other suitable transgenic animals may also be used, for example, HuMab mice (see in particular Lonberg, N.et al. Nature 368(6474): 856859 (1994)), Xeno-mice (Mendez et al. Nat Genet.,1997,15: 146-.

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 eliminate one or more effector functions to enhance FcRn receptor binding or to introduce one or more cysteine residues.

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 covalently bound, affinity bound, intercalated, coordinately bound, complexed, bound, mixed or added to the antibody or antigen in other waysThe conjugates are linked. 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-galactosidase), radioisotopes (e.g., gamma-glucosidase, gamma-glucosidase, etc.), and the like,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, cell relaxationB, 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, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), Anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthranilic Acid (AMC)), and antimitotic agents (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 nucleotide sequences as in table 1 that encode a CDR sequence as in table 1.

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 44, SEQ ID NO 48, SEQ ID NO 52, SEQ ID NO 56, 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:46, 50, 54, 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 (ATCC36,906), 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 (MMT 060562, 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. In certain embodiments, the conditions and disorders associated with PD-L1 include tumors and cancers, such as 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 fungoides), merkel cell carcinoma and other hematologic malignancies, such as Classical Hodgkin Lymphoma (CHL), primary mediastinal large B cell lymphoma, B-rich lymphoma of T cells/histiocytes, EBV positive and negative PTLD, and EBV-related Diffuse Large B Cell Lymphoma (DLBCL), plasmacytic lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal cancer, and HHV 8-related primary effusion lymphoma, Hodgkin's lymphoma, Central Nervous System (CNS) tumors, e.g. primary CNS lymphoma, spinal axis tumors, brain stem glioma. In certain embodiments, the tumors and cancers are metastatic, particularly metastatic tumors that express PD-L1. In certain embodiments, the conditions and disorders associated with PD-L1 include autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diabetes. In certain embodiments, the conditions and disorders associated with PD-L1 include 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 viral epidemics, Torquetenovirus, 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 a method of treating a condition or disorder associated with PD-L1 in an individual comprising administering a therapeutically effective amount of a PD-L1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the individual is identified as having a disorder or condition that is likely to respond to a PD-L1 antagonist.

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. Various methods may be used to determine the presence or level of PD-L1 in a test biological sample from the individual. For example, the biological sample to be tested may be exposed to an anti-PD-L1 antibody or antigen-binding fragment thereof, which binds to the expressed PD-L1 protein and detects the expressed PD-L1 protein. Alternatively, PD-L1 can be detected at the nucleic acid expression level using, e.g., qPCR, reverse transcription PCR, microarray, SAGE, FISH, and the like. In some embodiments, the test sample is derived from a cancer cell or tissue, or an immune cell that enters a tumor. In some embodiments, the presence or level of PD-L1 in the biological sample to be tested is upregulated indicating the likelihood of a response. 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 individual, 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).

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 a decrease or slowing or cessation of the increase in the level of PD-L1 in the test biological sample indicates an active 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 OMT rats (obtained from Open Monoclonal Technology, inc., parlo duo, usa) at 8 weeks of age were sensitized with human PD-L1ECD protein injected via footpad in 10 μ g TiterMax, followed by footpad 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 10 μ g of human PD-L1ECD protein in PBS by intraperitoneal injection 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) in an appropriate ratio. The cell mixture was washed and washed in ECF solution at 2.0x10⁶cell/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 supernatants were diluted 5-fold with 1 XPBS. CHO-K1 cells expressing PD-L1 were mixed with a secondary fluorescently labeled antibody and DraQ 5. 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 a hybridoma cell line that was verified to bind positively to PD-L1 expressing cells. Briefly, for each hybridoma cell line, cells were counted and diluted to 5 or 1 cells per 200 μ L in cloning medium. 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-rat 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-rat 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 examine the binding activity of the fully human antibody to the target, CHO-K1 expressing human PD-L1 or mature dendritic cells (mdcs) were conjugated with the fully human antibody, followed by binding with a secondary antibody conjugated with FITC-linked 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.

CHO cells transfected with full-length human PD-L1 were bound with antibody directed against human PD-L1 from rat hybridoma cells, and subsequently bound with a secondary antibody that binds to FITC-linked goat anti-rat IgG Fc and analyzed by FACS. As shown in FIG. 1, antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11 have an EC of about 1nM₅₀Specifically binds to PD-L1 expressed on CHO cells.

Example 2: modification of Fc portion and purification

The antibody in the 293F cell culture supernatant was purified using a protein a affinity chromatography column.

Example 3: characterization of fully human antibodies

3.1 competitive assay by FACS assay: to examine whether the fully human antibody was able to block the binding of PD-L1 to PD-1, CHO-K1 cells expressing human PD-L1 were incubated with different concentrations of fully human 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 fully human antibodies (1.4.1, 1.14.4, 1.20.15 and 1.46.11) 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. 2, all of the fully human PD-L1 antibodies tested blocked the binding of PD-1 to PD-L1 expressed on transformed CHO cells, and 1.4.1, 1.14.4, 1.20.15 and 1.46.11 showed IC50 values of about 10 nM.

3.2 affinity testing for Surface Plasmon Resonance (SPR) assays: tong (Chinese character of 'tong')The over-SPR method uses ProteOn XPR36(Bio-Rad) to characterize the affinity and binding kinetics of antibodies to PD-L1. 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. 5, the affinity of fully human PD-L1 antibody to recombinant human PD-L1 detected by using surface plasmon resonance was from 4.78E-10 to 2.26E-10 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 serially diluted 1 to 2 fold in elution buffer (1 XPBS/1% BSA) and incubated for 1 hour at 4 ℃. A secondary goat anti-human IgG Fc FITC (Jackson Immunoresearch Lab) was added and incubated at 4 ℃ for 1 hour with exclusion of 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 evaluate the ability of fully human antibodies to modulate T cell responses, including cytokine production and cell proliferation, the following three experiments were performed.

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 differentiate into 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.

Using human CD4⁺T cell enrichment kit for isolating CD4 according to instructions⁺T cells are then stimulated with mature or immature allogeneic DCs, with or without fully human anti-PD-L1 antibody or control antibody. IL-2 and IFN γ levels in the culture supernatants were measured by ELISA on day 3 and day 5, respectively. Pass [ 2 ]³H]Thymidine incorporation measures T cell proliferation.

As shown in fig. 9, all of the fully human PD-L1 antibodies tested (1.4.1, 1.14.4, 1.20.15, and 1.46.11) increased IL-2 secretion in a dose-dependent manner. As shown in fig. 8, all of the fully human PD-L1 antibodies tested (1.4.1, 1.14.4, 1.20.15, and 1.46.11) increased IFN γ secretion in a dose-dependent manner. As shown in fig. 10, all of the fully human PD-L1 antibodies tested (1.4.1, 1.14.4, 1.20.15, and 1.46.11) increased concentration-dependent T cell proliferation.

3.4.2 autoantigen specific immune response: PBMCs and monocytes were isolated from the same donor. PBMCs were cultured in the presence of CMV pp65 peptide and low dose IL2 (20U/ml). 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 fully human 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. 6, IFN γ production in specific T cell responses was enhanced by fully human PD-L1 antibodies (1.4.1, 1.14.4, 1.20.15 and 1.46.11). FIG. 7 shows that fully human PD-L1 antibody increases concentration-dependent CMV with autologous DC loaded with CMV pp65 peptide⁺-CD4⁺T cells proliferate.

3.4.3 Treg suppression assay: regulatory T cells (tregs) are key immune regulators and 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 effect of fully human anti-human PD-L1 antibodies on the inhibition of Treg function, the function of Tregs was compared in the presence or absence of fully human 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 varying concentrations of fully human or control antibodies. Briefly, CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cells were isolated by MACS and CD4 was co-cultured with allogeneic mDC⁺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.

As shown in fig. 11, PD-L1 antibody 1.20.15 abolished the suppressive function of tregs and restored the proliferation of reactive T cells and secretion of IFN γ.

3.5 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays: 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 undesired toxicity of the cells was minimized, verifying that the selected anti-PD-L1 fully human antibody lacks ADCC and CDC functions.

3.5.1 ADCC: target cells (mdcs) and different concentrations of fully human antibody were preincubated in 96-well plates for 30min, followed by IL-1 activated 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). Optical density was measured using a Molecular Devices spectra max M5e microplate detector. Control hAb (IgG1) and control hAb (IgG4) served as positive and negative controls, respectively.

Figure 12 shows that the fully human PD-L1 antibodies (1.4.1, 1.14.4, 1.20.15, and 1.46.11) 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 fully human antibody were mixed in 96-well plates. The plates were incubated at 37 ℃ with 5% CO₂Incubate in incubator for 4 hours. Use of CThe ellTiter glo (Promega-G7573) measures target cell lysis. Rituximab (roche) and the human B-cell lymphoma cell line Raji (CD20 positive) were used as positive controls. As shown in fig. 13, the fully human PD-L1 antibody did not mediate CDC.

3.6 sorting by FACS (Binning) test: to examine whether the fully human antibody has the same epitope classification as the reference antibody, CHO-K1 cells expressing human PD-L1 were incubated with different concentrations of fully human 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.

The results of the classification tests showed that the binding epitope of fully human PD-L1 antibodies (i.e., 1.4.1, 1.14.4, 1.20.15, and 1.46.11) on human PD-L1 was different from that of the known PD-L1 antibody (i.e., the reference antibody).

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, fully human antibodies were added to the plates 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. 4, the results of ELISA experiments showed that the tested fully human PD-L1 bound to cynomolgus monkey PD-L1 in a dose-dependent manner. However, none of the tested antibodies (1.4.1, 1.14.4, 1.20.15, and 1.46.11) bound to murine PD-L1 (data not shown).

3.8 FACS Cross-family binding assay: to detect cross-family binding activity of fully human antibodies, a cell line expressing PD-L2 was bound with fully human antibodies, 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 PD-L1 antibody of full human origin and analyzed by FACS. As shown in fig. 3, the fully human PD-L1 antibody specifically binds to PD-L1, but not PD-L2 of the PD-1 ligand family.

Example 4: epitope identification of fully human PD-L1 antibody

To determine the epitope of antibody 1.14.4 described herein, an alanine scanning mutagenesis experiment (alanine scanning experiment) and an assessment of the effect of antibody binding against hPD-L1 was performed using 1.14.4.

The alanine residue in hPD-L1 was mutated to a glycine codon and all other residues were mutated to alanine codons. Point substitutions of amino acids were made for each residue of the extracellular domain (ECD) of hPD-L1 using two-step sequential PCR. The plasmid pcDNA3.3-hPD-1_ ECD.His, encoding the ECD and C-terminal His-tag of human PD-L1, was used as a template, a set of mutagenic primers was used as a first step PCR, and a QuikChangeLighting multipoint site-directed mutagenesis kit (Agilent technologies, Palo Alto, Calif.) was used. The parent template was digested after the mutation combination reaction using the Dpn I endonuclease. In the second PCR, a linear DNA expression cassette containing the CMV promoter, PD-L1 extracellular domain (ECD), His-tag, and polyadenylation of herpes simplex virus Thymidine Kinase (TK) was amplified and transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD).

Monoclonal antibody 1.14.4 was coated on plates for ELISA binding assays. HRP-conjugated anti-His antibody (1: 5000; Rockland Immunochemicals, Pottstown, PA) was added as detection antibody after reaction with supernatant containing quantitative PD-L1 mutation or human/mouse PD-L1_ ecd. The absorbance was normalized according to the mean value of the control mutations. After additional thresholding of the binding fold changes (<0.55), the finally determined epitope residues are identified.

The binding activity of antibody 1.14.4 against human and mouse PD-L1 was performed (fig. 14). Antibody 1.14.4 was found to bind to human PD-L1 (fig. 14A), but not to mouse PD-L1 (fig. 14B).

The effect of the 131 point-substituted hPD-L1 point mutations on antibody binding is listed in Table 2. Checking the positions of all these residues on the hPD-L1 crystal structure (PDB codes 3RRQ and 4ZQK) shows that some amino acids (e.g. gly159, Tyr160, Pro161) are unlikely to be in direct contact with any antibody. The observed reduction in binding is most likely due to instability or even structural collapse of the hPD-L1 structure following alanine substitution. After additional thresholding of the fold change (<0.55) of binding, the finally determined epitope residues are listed in table 3. They are 6 positions for 1.14.4.

All data in table 3 were plotted on the crystal structure of hPD-L1 for better visualization and comparison (fig. 15).

TABLE 2 Effect of PD-L1 point mutations on antibody binding

1.14.4

^aFold changes in binding are relative to binding of several silent alanine substitutions.

TABLE 3 potential epitopes recognized

Critical value fold change <0.55

As shown in FIG. 15, the hotspot residues responsible for hPD-L1 binding were all concentrated in the C chain, CC' loop and F chain (FIG. 15). The position of the residues was checked on the crystal structure of the hPD-1/hPD-L1 complex (PDB code 4ZQK,

) These residues are shown to be located primarily in the A, C, F and G chains. The epitope of antibody 1.14.4 was mainly contributed by residues on the C chain that directly overlapped the hPD-1 and hPD-L1 interaction sites, suggesting a mechanism for binding hPD-L1 and blocking hPD-1.

Example 5: in vivo inhibition of tumor growth by fully human PD-L1 antibody

To evaluate the inhibition of tumor growth by fully human antibody hPD-L1, MC38-B7H1 tumor cells were treated at 5X10⁵Each 0.1mL of the cells was inoculated subcutaneously into the right anterior flank of a male B-hPD-1 humanized mouse, and 42 animals were transferred in total. When the tumor grows to about 100mm³The medicine is administered in groups, 7 of each group, 5 groups in total, respectively: solvent control, BMK6 antibody control (see description in WO2011066389A 1), 1.14.4 antibody 3mg/kg, 1.14.4 antibody 10mg/kg, and 1.14.4 antibody 30 mg/kg. All groups of administration routes are intraperitoneal injection, 1 time every two days, 6 times of continuous administration, and observation is continued for 2 weeks after the administration is finished. Tumor volume and body weight were measured 2 times per week and changes in mouse body weight and tumor volume were recorded as a function of time of administration. At the end of the experiment, the tumor volume ratio (T/C) and tumor growth inhibition ratio (TGI) of the treated versus solvent control groups were calculated and statistically analyzed. Statistical analysis of tumor volume was performed using the Graphpad Prism5 software for the T-test. P<0.05 was considered to have a significant difference.

Tumor volume was measured 2 times a week using a vernier caliper to measure the major and minor diameters of the tumor, and the volume calculation formula was: tumor volume is 0.5 × long diameter × short diameter². Calculating tumor increment rate T/C (%) (tumor volume of treatment group/tumor of negative control group) according to the measurement resultVolume x 100%. Tumor inhibition rate TGI (%) ([ 1- (Ti-T0)/(Vi-V0)]x100

(Ti: mean tumor volume of treatment group on day i of administration, T0: mean tumor volume of treatment group on day 0 of administration; Vi: mean tumor volume of control group on day i of administration, V0: mean tumor volume of control group on day 0 of administration).

TABLE 4 tumor suppression of fully human PD-L1 antibody 1.14.4 against MC38-B7H1 murine Colon cancer transplantation hPD-1 humanized mice

Note:^a.mean ± standard error;^b.comparing with control group

No significant reduction in weight average was seen for each group of experimental animals throughout the experiment (table 4 and figure 16), indicating good tolerance to the test compound. After 19 days of dosing (25 days after tumor cell inoculation), the tumor volume of the solvent control group grew to 2359mm³1.14.4 the low, medium and high dose groups all showed significant reduction in tumor volume (mean values of 949mm for tumor volume, respectively) compared to the control group³，1416mm³And 1115mm³) All three doses showed significant antitumor effects (tumor inhibition rates of 62.8%, 42.0% and 55.4%, respectively) (table 4 and fig. 17). The control antibody BMK6 also produced significant anti-tumor effects (mean tumor volume 1241 mm)³Tumor inhibition was 49.7%). The result shows that the 1.14.4 antibody has obvious anti-tumor effect, and the inhibition rate of low, medium and high dose groups to the tumor is above 40%.

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

1. An isolated antibody or antigen-binding fragment thereof, comprising:

a) heavy chain HCDR1(SEQ ID NO:1), HCDR2(SEQ ID NO:3) and HCDR3(SEQ ID NO:5) and light chain LCDR1(SEQ ID NO:7), LCDR2(SEQ ID NO:9) and LCDR3(SEQ ID NO: 11);

b) heavy chain HCDR1(SEQ ID NO:13), HCDR2(SEQ ID NO:15) and HCDR3(SEQ ID NO:17) and light chain LCDR1(SEQ ID NO:19), LCDR2(SEQ ID NO:21) and LCDR3(SEQ ID NO: 23);

c) heavy chain HCDR1(SEQ ID NO:25), HCDR2(SEQ ID NO:27) and HCDR3(SEQ ID NO:29) and light chain LCDR1(SEQ ID NO:31), LCDR2(SEQ ID NO:33) and LCDR3(SEQ ID NO: 35); or

d) Heavy chain HCDR1(SEQ ID NO:37), HCDR2(SEQ ID NO:39) and HCDR3(SEQ ID NO:41) and light chain LCDR1(SEQ ID NO:19), LCDR2(SEQ ID NO:21) and LCDR3(SEQ ID NO: 23);

wherein the antibody or antigen-binding fragment thereof specifically binds to PD-L1.

2. The antibody or antigen binding fragment thereof of claim 1, comprising a heavy chain variable region selected from the group consisting of: 43, 47, 51 and 55.

3. The antibody or antigen binding fragment thereof of claim 1, comprising a light chain variable region selected from the group consisting of: SEQ ID NO 45, SEQ ID NO 49 and SEQ ID NO 53.

4. The antibody or antigen-binding fragment thereof of claim 1, comprising:

a) the heavy chain variable region shown as SEQ ID NO. 43 and the light chain variable region shown as SEQ ID NO. 45;

b) the heavy chain variable region shown as SEQ ID NO. 47 and the light chain variable region shown as SEQ ID NO. 49;

c) the heavy chain variable region shown as SEQ ID NO. 51 and the light chain variable region shown as SEQ ID NO. 53; or

d) The heavy chain variable region shown as SEQ ID NO. 55 and the light chain variable region shown as SEQ ID NO. 49.

5. The isolated antibody or antigen binding fragment thereof of any one of claims 1-4, which can range from no more than 10^-8The Kd value of M, determined by plasmon resonance binding, specifically binds to human PD-L1.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which has an EC of no more than 10nM, or no more than 1nM₅₀Binding to monkey PD-L1, and/or no binding to mouse PD-L1.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which is capable of blocking binding of human or monkey PD-L1 to its receptor with an IC50 of no more than 100 nM.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which does not substantially bind to PD-L2.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which does not mediate ADCC or CDC or both.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which is a fully human monoclonal antibody.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein said fully human monoclonal antibody is produced by a transgenic rat.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which is capable of blocking binding of human PD-L1 to its receptor and thereby 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.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which is a camelized single domain antibody (camelized single chain domain antibody), a bifunctional antibody (diabody), a scFv dimer, a BsFv, a dsFv, (dsFv)2, a dsFv-dsFv ', a Fv fragment, a Fab ', a F (ab ')2, a ds bifunctional antibody (ds diabody), a nanobody, a domain antibody, or a bivalent domain antibody.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-4, further comprising an immunoglobulin constant region.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-4, further comprising a conjugate.

16. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-14.

17. A vector comprising the isolated polynucleotide of claim 16.

18. A host cell comprising the vector of claim 17.

19. A method of expressing the antibody or antigen-binding fragment thereof of any one of claims 1-14, comprising culturing the host cell of claim 18 under conditions in which the isolated polynucleotide of claim 16 is expressed.

20. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-15.

21. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-15 in the preparation of a kit for detecting the presence or level of human or monkey PD-L1 in a biological sample, said detecting comprising contacting the biological sample with the kit and determining the presence or level of human or monkey PD-L1 in the sample.

22. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-15 in the preparation of a kit for identifying an individual having a disorder or condition that is likely to respond to a PD-L1 antagonist, the identification comprising: determining the presence or level of PD-L1 in a test biological sample from the individual with the kit, wherein the presence or level of PD-L1 in the biological sample is upregulated as indicative of the likelihood of a response.

23. Use of the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-15 in the manufacture of a kit for monitoring treatment response or disease progression in a subject treated with a PD-L1 antagonist, the monitoring comprising determining with the kit the presence or level of PD-L1 in a test biological sample from the individual.

24. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-15 and one or more pharmaceutically acceptable carriers.

25. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-15 in the manufacture of a medicament for treating a condition in a subject who would benefit from an upregulated immune response, said treatment comprising administering to said subject a therapeutically effective amount of said medicament.

26. The use of claim 25, wherein the subject has up-regulated PD-L1 expression.

27. The use of claim 25, wherein the condition is cancer or a chronic viral infection.