HK1232555B - Novel anti-pd-l1 antibodies - Google Patents

Description

Novel anti-PD-L1 antibodies

Field of the Invention

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

Background Art

Accumulating preclinical and clinical evidence suggests that targeting immune checkpoints is emerging as a promising approach for treating cancer patients. Programmed cell death molecule 1 (PD-1) is one of the immune checkpoint proteins that plays a major role in limiting T cell activity, which provides a primary immune resistance mechanism by which tumor cells evade immune surveillance. The interaction between PD-1, expressed on activated T cells, and PD-L1, expressed on tumor cells, negatively regulates the immune response and attenuates anti-tumor immunity. PD-L1 expression on tumors is associated with decreased survival in esophageal cancer, pancreatic cancer, and other types of cancer, highlighting this pathway as a promising new target for tumor immunotherapy. Pharmaceutical companies such as Bristol-Myers Squibb (BMS), Merck, Roche, and GlaxoSmithKline (GSK) have developed a variety of drugs targeting the PD-1 pathway. Clinical trial data have shown early evidence of durable clinical activity and a favorable safety profile in patients with various tumor types. Nivolumab, a PD-1 drug developed by BMS, is being thrust into the forefront of the next generation of this field. Currently in six late-stage studies, the treatment induced tumor shrinkage in three of the five cancer groups studied, including 18% of 72 lung cancer patients, nearly a third of 98 melanoma patients, and 27% of 33 kidney cancer patients. Developed by Merck, lambrolizumab is a fully human monoclonal IgG4 antibody that targets PD-1, which grabbed the FDA's new breakthrough designation after impressive Phase IB data in skin cancer. Results from the Phase IB study showed an objective anti-tumor response in 51% of 85 cancer patients, with complete responses in 9%. Roche's experimental MPDL3280A demonstrated tumor shrinkage in 29 (21%) of 140 patients with advanced cancer who had tumors of various sizes.

However, existing treatments are not always satisfactory, and new anti-PD-L1 antibodies are still needed.

Summary 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 that can specifically bind to human PD-L1 with a Kd value of no more than ^10-9 M (e.g., ^≤9x10-10 M, ^≤8x10-10 M, ^≤7x10-10 M, ^≤6x10-10 M, ^≤5x10-10 M, ^≤4x10-10 M, ^≤3x10-10 M, ^≤2x10-10 M, or ^≤10-10 M), as determined by plasmon resonance binding.

In certain embodiments, the antibody or antigen-binding fragment thereof binds to monkey PD-L1 with an EC50 of no more than ₁₀ nM (e.g., no more than 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 nM). In certain embodiments, the antibody or antigen-binding fragment thereof does not bind to mouse PD-L1, but binds to monkey PD-L1 with a binding affinity similar to that of human PD-L1. In certain embodiments, the antibody or antigen-binding fragment thereof effectively inhibits the binding of human or monkey PD-L1 to its receptor (such as PD-1) with an IC50 of no more than 100 nM (such as no more than 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, ₅ nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 nM). In certain embodiments, the _EC50 or _IC50 is determined 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, the antibodies or antigen-binding fragments thereof described herein comprise a heavy chain CDR sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 13, 15, 17, 25, 27, 29, 37, 39, and 41.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 19, 21, 23, 31, 33, and 35.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise at least one, two, three, four, five or six CDRs selected from SEQ ID NOs: 1, 3, 5, 7, 9 and 11; or selected from SEQ ID NOs: 13, 15, 17, 19, 21 and 23; or selected from SEQ ID NOs: 25, 27, 29, 31, 33 and 35; or selected from SEQ ID NOs: 37, 39, 41, 19, 21 and 23.

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

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein include:

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, the antibodies or antigen-binding fragments thereof described herein include, 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 bound by the antibodies or antigen-binding fragments thereof described herein includes 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 the binding of human PD-L1 to its receptor and thereby providing at least one of the following activities:

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

b) induce IFNγ production in CD4 ⁺ T cells;

c) inducing proliferation of CD4 ⁺ T cells; and

d) Reversal of T reg suppressive function.

In certain embodiments, the antibodies described herein are monoclonal antibodies, fully human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, bivalent antibodies, or anti-idiotypic antibodies.

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

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 purification moiety.

On the other hand, the application provides isolated polynucleotides encoding antibodies or their antigen-binding fragments as described herein. In certain embodiments, the polynucleotides provided herein encode the amino acid sequences of antibodies or their antigen-binding fragments as described herein. In certain embodiments, the application provides vectors comprising these polynucleotides. In certain embodiments, the application provides methods for expressing one or more antibodies or antigen-binding fragments as described herein, which are achieved by culturing host cells under conditions where the antibodies or antigen-binding fragments encoded by the polynucleotides are expressed in a vector. In certain embodiments, the polynucleotides provided herein are operably linked to a promoter such as the SV40 promoter in the vector. In certain embodiments, the host cell comprising the vector provided herein is a Chinese hamster ovary cell, or a 293F cell.

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

On the other hand, the PD-L1 antibodies of the present application, such as 1.4.1, 1.14.4, 1.20.15 and 1.46.11, are well tolerated and have high in vivo anti-tumor activity in animals. In certain embodiments, the tumor volume of the tumor-bearing animal administered with the 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 group of animals with similar baseline tumor volume administered only with solvent.

In another aspect, the present application provides a method for detecting the presence or level of PD-L1 (e.g., human or monkey) in a biological sample, comprising contacting the biological sample with the 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 for identifying an individual suffering from a disorder or condition that may be responsive 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 an antibody or antigen-binding fragment thereof described herein, wherein an increased presence or level of PD-L1 in the biological sample indicates a likelihood of response. In certain embodiments, the method further comprises administering to the individual an effective amount of an antibody or antigen-binding fragment thereof described herein, wherein the individual is identified as suffering from a disorder or condition that may be responsive to a PD-L1 antagonist.

The present application further provides a method for monitoring treatment 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 the antibody or antigen-binding fragment thereof described herein.

In another aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein and one or more pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical carrier can 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 for 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 upregulated PD-L1 expression.

In another aspect, there is provided a use of the antibody or antigen-binding fragment thereof described herein in the preparation of a medicament for treating 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 THE DRAWINGS

Figure 1 shows the binding of fully human PD-L1 antibodies to PD-1 expressing CHO cells as determined by FACS analysis.

Figure 2 shows that a fully human PD-L1 antibody blocked the binding of PD-1 to PD-L1-transfected CHO cells as determined by FACS analysis.

FIG3 shows that the fully human PD-L1 antibody specifically binds to PD-L1 but not to PD-L2 as determined by FACS analysis.

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

Figure 5 shows the complete kinetics of the binding affinity of the PD-L1 antibody to human PD-L1 determined by plasmon resonance, ranging from 2.26E-10 to 4.78E-10 mol/L.

Figure 6 shows that fully human PD-L1 antibodies increase IFNγ production in specific T cell responses.

FIG7 shows that fully human anti-PD-L1 antibodies promote the proliferation of specific T cells.

Figure 8 shows that fully human PD-L1 antibodies increase IFNγ production in mixed lymphocyte reaction (MLR).

FIG9 shows the effect of fully human anti-PD-L1 antibodies on IL-2 production in the MLR.

FIG10 shows that anti-PD-L1 antibodies promote T cell proliferation in MLR.

Figure 11 shows that anti-PD-L1 antibodies reversed Treg suppressive function.

FIG12 shows the lack of ADCC of anti-PD-L1 antibodies in activated T cells.

FIG13 shows that anti-PD-L1 antibodies lack CDC in activated T cells.

Figures 14A and 14B show the cross-reactivity of anti-PD-L1 antibodies with human/mouse PD- 1. 2 μg/ml of 1.14.4 antibody was coated on a 96-well plate overnight and incubated with hPD-L1-His protein (Figure 14A) and mPD-L1-His protein (Figure 14B), followed by the addition of HRP-anti-His antibody for detection.

Figure 15 shows the hotspot residues in the hPD-L1 structure, which are the binding sites of antibody 1.14.4. Data are from Table 3. Colors are used to distinguish differences between epitopes.

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

Figure 17 shows the significant inhibitory effect of hPD-L1 antibody 1.14.4 on tumor cell growth in vivo. After 19 days of antibody administration, three doses of 1.14.4 antibody (3 mg/kg, 10 mg/kg and 30 mg/kg, respectively) all showed significant anti-tumor effects with tumor growth inhibition (TGI) > 40%.

Detailed Description of the Invention

The following description of the present application is intended only to illustrate various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A person skilled in the art can easily derive multiple equivalents, variations, and modifications without departing from the scope of the present application, and it should be understood that such equivalent embodiments are included within the scope of the present invention. All documents cited in this application, including publications, patents, and patent applications, are incorporated by reference in their entirety.

definition

The term "antibody" in the present invention includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that can bind to a specific antigen. A natural complete antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and the first, second and third constant regions; each light chain consists of a variable region and a constant region. The heavy chains of mammals can be divided into α, δ, ε, γ and μ, and the light chains of mammals can be divided into λ or κ. The antibody is "Y"-shaped, and the neck of the "Y" structure is composed 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 region and the first constant region of one of the heavy chains, which are combined with the variable region and constant region of one light chain. The variable regions of the light chain and the heavy chain determine the binding of the antigen. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs). The CDRs of the light chain (L) comprise LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, and HCDR3. The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be named or identified using 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)). The three CDRs are separated by flanking continuous portions called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but have various effector functions. Antibodies can be divided into several classes based on the amino acid sequence of the heavy chain constant region. Depending on whether they contain α, δ, ε, γ, and μ heavy chains, antibodies can be divided into five major classes or isoforms: IgA, IgD, IgE, IgG, and IgM. Several major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed by the portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but lacks the structure of a complete antibody. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab') ₂ , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bivalent single-chain antibodies (bsFv), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment can bind to the same antigen as the parent antibody. In certain embodiments, an antigen-binding fragment can contain one or more CDRs from a specific human antibody grafted to the framework regions of one or more different human antibodies.

The "Fab" fragment of an antibody refers to the portion of the antibody molecule consisting of a light chain (including the variable and constant regions) and the variable and constant regions of a heavy chain bound together by disulfide bonds.

The "Fab'" fragment refers to the Fab fragment including part of the hinge region.

"F(ab') ₂ " refers to a dimer of Fab.

The "Fc" portion of an antibody refers to the portion of the antibody consisting of the second and third constant regions of the heavy chain bound by disulfide bonds. The Fc portion of an antibody is responsible for various effector functions such as ADCC and CDC, but does not participate in antigen binding.

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

"Single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region linked directly to a heavy chain variable region or connected through 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 a scFv linked to the Fc region of an antibody.

"Camelized single domain antibody", "heavy chain antibody" or "HCAb (Heavy-chain-only antibodies, HCAb)" refers to an antibody containing two _VH domains but no light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4): 277-302 (2001); WO94/04678; WO94/25591; US Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries and llamas). Despite the absence of light chains, camelized antibodies have demonstrated full antigen-binding function (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable region (VHH domain) of a heavy-chain antibody is the smallest known antigen-binding unit produced by adaptive immunity (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

"Nanobody" refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant regions, CH2 and CH3.

"Diabodies" include small antibody fragments with two antigen-binding sites, wherein the fragment contains a _VH domain and a _VL domain linked on the same polypeptide chain ( _VH - _VL or _VH - _VL ) (see, Holliger P. et al., Proc Natl Acad Sci US A. Jul 15; 90(14): 6444-8 (1993); EP404097; WO93/11161). The linker between the two domains is very short, so that the two domains on the same chain cannot pair with each other, thereby forcing the two domains to pair with complementary domains of another chain to form two antibody binding sites. The two antibody binding sites can target the same or different antigens (or antigenic epitopes).

"Domain antibodies" are antibody fragments containing only one heavy chain variable region or one light chain variable region. In some cases, two or more _VH domains are covalently linked by a polypeptide linker to form a bivalent domain antibody. The two _VH domains of a bivalent domain antibody can target the same or different antigens.

In certain embodiments, "(dsFv) ₂ " contains three peptide chains: two V _H groups are connected by a polypeptide linker and are bound to two V _L groups by disulfide bonds.

In certain embodiments, a "bispecific ds diabody" comprises V _L1 -V _H2 (connected by a polypeptide linker) and V _H1 -V _L2 (also connected by a polypeptide linker), the two of which are linked by a disulfide bond between V _H1 and V _L1 .

"Bispecific dsFv" or "dsFv-dsFv" contains three polypeptide chains: a _VH1 - _VH2 group, wherein the two heavy chains are connected by a polypeptide linker (e.g., a long flexible linker) and are bound to the _VL1 and _VL2 groups, respectively, by disulfide bonds, and each pair of heavy and light chains paired by disulfide bonds has different antigenic specificity.

In certain embodiments, the "scFv dimer" is a bivalent diabody or a bivalent single-chain antibody (BsFv), comprising two dimerized _VH - _VL groups (connected by a polypeptide linker), wherein the _VH of one group cooperates with the _VL of the other group to form two binding sites, and these two binding sites can target and bind to the same antigen (or antigenic epitope) or different antigens (or antigenic epitopes). In other embodiments, the "scFv dimer" is a bispecific diabody, comprising _VL1 - _VH2 (connected by a polypeptide linker) and _VH1 - _VL2 (connected by a polypeptide linker), wherein _VH1 and _VL1 cooperate, and _VH2 and _VL2 cooperate, and each cooperating pair has a different antigenic specificity.

As used herein, the term "fully human" when applied to an antibody or antigen-binding fragment means that the antibody or antigen-binding fragment has or consists of an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human immune cell, or derived from a non-human source, such as a transgenic non-human animal utilizing a human antibody library, or other sequences encoding a human antibody. In certain embodiments, a fully human antibody does not contain amino acid residues (particularly antigen-binding residues) derived from a non-human antibody.

The term "humanization" as used in this application, when used for antibodies or antigen-binding fragments, refers to antibodies or antigen-binding fragments comprising CDRs derived from non-human animals, FR regions derived from humans, and constant regions (when applicable) derived from humans. Because humanized antibodies or antigen-binding fragments have reduced immunogenicity, they can be used as therapeutic agents for humans in certain embodiments. 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 is substantially entirely composed of human sequences except that the CDR sequence is non-human. In some embodiments, the FR region derived from humans may include the same amino acid sequence as the human antibody from which it is derived, or it may include some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid changes. In some embodiments, the amino acid changes may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains simultaneously. In some preferred embodiments, the humanized antibody includes human FR1-3 and human JH and Jκ.

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

As used herein, "PD-L1" 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 a representative human PD-L1 is NCBI Accession No.: NP_054862.1, and the nucleic acid sequence of a representative human PD-L1 is NCBI Accession No.: NM_014143.3. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumors or cancer cells. PD-L1 binds to receptors PD-1 or B7-1 expressed on activated T cells, B cells, and myeloid cells. Binding of PD-L1 to its receptor can trigger signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Therefore, PD-L1 plays a major role in suppressing the immune system in certain events, such as pregnancy, autoimmune diseases, and tissue transplantation, and is thought to allow tumor or cancer cells to circumvent immune checkpoints and escape the immune response.

As used herein, an "anti-PD-L1 antibody" refers to an antibody that can specifically bind to PD-L1 (eg, human or monkey PD-L1) with an affinity sufficient to provide diagnostic and/or therapeutic use.

As used herein, "specific binding" or "specific binding" 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 invention specifically bind to human and/or monkey PD-L1 with a binding affinity ( _KD ) of ^≤10-6 M (e.g., ^≤5x10-7 M, ^≤2x10-7 M, ^≤10-7 M, ^≤5x10-8 M, ^≤2x10-8 M, ^≤10-8 M, ^≤5x10-9 M, ^≤2x10-9 M, ^≤10-9 M, ^≤10-10 M, about ^10-10 M, ^10-10 M to ^10-9 M, ^10-10 M to ^10-8.5 M, or ^10-10 M to ^10-8 M). KD in this application refers to the ratio of the dissociation rate to the association rate (koff/kon), which can be determined by surface plasmon resonance, for example, using an instrument 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 binding interaction between two molecules (e.g., human PD-L1 and anti-PD-L1 antibody) to any detectable extent. In certain embodiments, the antibody or antigen-binding fragment that blocks binding between two molecules can inhibit the binding interaction between the two molecules by at least 50%. In certain embodiments, such inhibition can be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

As used herein, "epitope" refers to that portion of amino acids or atomic groups in an antigen molecule that binds to an antibody. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope on the antigen. For example, if the antibodies or antigen-binding fragments thereof provided herein block the binding of exemplary antibodies, such as 1.4.1, 1.14.4, 1.20.15, and 1.46.11, to human PD-L1, then the antibodies or antigen-binding fragments thereof can be considered to bind to the same epitope as those exemplary antibodies.

Specific amino acid residues in the epitope are mutated by, for example, alanine scanning mutagenesis, and mutations that reduce or prevent protein binding are identified. "Alanine scanning mutagenesis" 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 that bind thereto. A residue or a group of target residues in a protein is replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine, or a conservative amino acid substitution). Any amino acid residue mutation that reduces the binding of the protein or a codon mutation encoding it that exceeds a threshold or maximizes the reduction of protein binding compared to other mutations is likely to be in the epitope bound by the protein. In certain embodiments of the present application, an epitope important for PD-L1 antibodies includes 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 fully human monoclonal antibody having a heavy chain variable region as shown in SEQ ID NO: 43, a light chain variable region as shown in SEQ ID NO: 45, and a human IgG4 isotype constant region.

As used in this application, "1.14.4" refers to a fully monoclonal human antibody having a heavy chain variable region as shown in SEQ ID NO: 47, a light chain variable region as 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 a heavy chain variable region as shown in SEQ ID NO: 51, a light chain variable region as 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 a heavy chain variable region as shown in SEQ ID NO: 55, a light chain variable region as shown in SEQ ID NO: 49, and a human IgG4 isotype constant region.

In this application, when "conservative substitution" is used in relation to an amino acid sequence, it refers to the replacement of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can 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 generally do not cause significant changes in the conformational structure of the protein, and therefore can retain the biological activity of the protein.

When "percent sequence identity" is used in relation to amino acid sequences (or nucleic acid sequences), it refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after alignment and, if necessary, introduction of gaps to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine the percentage sequence identity of amino acid (or nucleic acid) sequences using tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see, Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tool or appropriately adjust the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

As used herein, “T cells” include CD4 ⁺ T cells, CD8 ⁺ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.

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

As used herein, "cancer" or "cancerous condition" refers to any medical condition mediated by the growth, proliferation, or metastasis of tumor or malignant cells, and which gives rise to solid tumors and non-solid tumors such as leukemia. As used herein, "tumor" refers to the physical mass of tumor and/or malignant cells.

"Treatment" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, causing complete or partial reversal of a condition, curing a condition, or some combination thereof. With respect to cancer, "treat" or "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. With respect to tumors, "treat" or "treatment" includes eliminating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor progression, or some combination thereof.

An "isolated" substance has been artificially altered from its natural state. If an "isolated" 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 can be considered "isolated" if the substance from which it coexists in nature is sufficiently separated and present in a sufficiently pure state. In certain embodiments, the purity of antibodies and antigen-binding fragments is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99%, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ion exchange chromatography or reversed-phase HPLC).

As used herein, a "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted, resulting in expression of the protein. Vectors can be used to transform, transduce, or transfect host cells, enabling expression of the genetic material elements they carry within the host cells. For example, vectors include 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. Examples of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). Vectors can contain a variety of expression-controlling elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate entry into cells, including, but not limited to, viral particles, liposomes, or protein coats.

In the present invention, "host cell" refers to a cell into which an exogenous polynucleotide and/or vector is introduced.

The "disease associated with or related to PD-L1" in the present invention refers to any condition caused, aggravated or otherwise related 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 that is effective in treating a disease or condition associated with human PD-L1. For example, with respect to the use of an antibody or antigen-binding fragment thereof disclosed herein, a therapeutically effective amount is a dose or concentration at which the antibody or antigen-binding compound can eliminate all or part of a tumor, inhibit or slow tumor growth, inhibit the growth or proliferation of cells that mediate a cancerous condition, inhibit tumor cell metastasis, alleviate any symptoms or markers associated with a tumor or cancerous condition, prevent or delay the progression of a tumor or cancerous condition, or some combination thereof.

"Pharmaceutically acceptable" means that the referenced carriers, vehicles, diluents, excipients and/or salts are generally 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 present 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, mediating immunosuppression. PD-1 ligand 1 (PD-L1) is a 40 kDa transmembrane protein expressed on a variety of tumor cells, stromal cells, or both, that binds to PD-1. Inhibiting the interaction between PD-1 and PD-L1 can enhance T cell responses, thereby mediating anti-cancer 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 whose 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 are amino acid sequences and SEQ ID NO:2, 4, 6 are nucleic acid sequences.

V segment: IGHV4-39*01

Section D: IGHD1-26*01

J segment: IGHJ4*02

1.4.1-VL(30027): (SEQ ID NO:45 for amino acid and SEQ ID NO:46 for nucleic acid) Light chain CDR1-3: SEQ ID NOs:7, 9, 11 for amino acid sequence and SEQ ID NOs:8, 10, 12 for nucleic acid sequence:

V segment: IGLV3-1*01

J segment: IGLJ2*01

1.14.4-VH(29812): (SEQ ID NO:47 for amino acids, SEQ ID NO:48 for nucleic acids) Heavy chain CDR1-3: SEQ ID NOs:13, 15, 17 for amino acid sequences and SEQ ID NOs:14, 16, 18 for nucleic acid sequences:

V segment: IGHV3-23*01

Section D: IGHD5-5*01

J segment: IGHJ4*02

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

V segment: IGLV3-21*02

J segment: IGLJ2*01

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

V segment: IGHV4-39*01

Section D: Undetermined

J segment: IGHJ4*02

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

V segment: IGLV3-1*01

J segment: IGLJ2*01

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

V segment: IGHV3-23*01

Section D: IGHD5-5*01

J segment: IGHJ4*02

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

V segment: IGLV3-21*02

J segment: IGLJ2*01

In some embodiments, the anti-PD-1 antibody and antigen-binding fragment thereof comprise a heavy chain CDR sequence 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 antibody and antigen-binding fragment thereof comprise a light chain CDR sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 19, 21, 23, 31, 33, and 35.

In some embodiments, the anti-PD-L1 antibody and antigen-binding fragment thereof include 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 fragment thereof include 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: ID NO: 19, SEQ ID NO: 21 and/or SEQ ID NO: 23.

Those skilled in the art will appreciate that the CDR sequences provided in Table 1 can be modified to include substitutions of one or more amino acids, thereby obtaining improved biological activity, such as improved binding affinity to human PD-L1. For example, phage display technology can be used to produce and express antibody variant libraries (e.g., Fab or FcFv variants), followed by screening for antibodies with affinity to human PD-L1. In another example, computer software can be used to simulate the binding of the antibody to human PD-L1 and identify the amino acid residues on the antibody that form the binding interface. Substitution of these residues can be avoided to prevent a decrease in binding affinity, or these residues can be targeted for substitution to form a stronger bond. In certain embodiments, at least one (or all) substitutions in the CDR sequence are conservative substitutions.

In certain embodiments, the antibodies and antigen-binding fragments comprise one or more CDR sequences that have 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 greater binding affinity to human PD-L1 than that of their parent antibodies, wherein the parent antibodies have substantially the same sequence, but their 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. Such fully human antibodies do not have the problems of immunogenicity or reduced binding affinity often observed in humanized antibodies.

In some embodiments, the fully human anti-PD-L1 antibodies and antigen-binding fragments thereof include a heavy chain variable region, wherein the heavy chain variable region is selected from 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 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 at levels similar to those of the exemplary antibodies: 1.4.1, 1.14.4, 1.20.15, and 1.46.11.

In some embodiments, the fully human anti-PD-L1 antibodies and antigen-binding fragments thereof include 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.

The present application also includes 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 blocks the binding of ^1.4.1 , 1.14.4, ^1.20.15 and 1.46.11 to human or monkey PD-L1 with an IC ₅₀ value (i.e., half-maximal inhibitory concentration) of less than 10 ^-6 M, less than 10 ^-7 M, less than 10 ^-7.5 M, less than ^{10 -8 M} , less than 10 -8.5 M or less than 10 -9 M or less than 10 -10 M. The IC ₅₀ value is 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 specifically bind to human PD-L1 with a binding affinity (Kd) of ^≤10-6 M (e.g., ^≤5x10-7 M, ^≤2x10-7 M, ^≤10-7 M, ^≤5x10-8 M, ^≤2x10-8 M, ^≤10-8 M, ^≤5x10-9 M, ^≤2x10-9 M, ^≤10-9 M, ^10-10 M, about ^10-10 M, ^10-10 M to ^10-8.5 M, or ^10-10 M to ^10-8 M) as measured by plasmon resonance binding. The binding affinity can be expressed as a _KD value, which is calculated as the ratio of the off-rate to the on-rate (koff/kon) when the binding of the antigen to the antigen-binding molecule reaches equilibrium. The antigen binding affinity (e.g. _KD ) can be suitably determined by any suitable method known in the art, including plasmon resonance binding using an instrument such as Biacore (see, e.g., Murphy, M. et al, Current protocols in protein science, Chapter 19, unit 19.14, 2006).

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein bind to human PD-L1 at an EC ₅₀ (i.e., half-binding concentration) of 0.1 nM-100 nM (e.g., 0.1 nM-50 nM, 0.1 nM-30 nM, 0.1 nM-20 nM, or 0.1 nM-10 nM, or 0.1 nM-1 nM). The binding of the antibody to human PD-L1 can be determined by methods known in the art, such as sandwich methods such as ELISA, Western blot, FACS, or other binding assays. In an exemplary example, the antibody to be tested (i.e., the primary antibody) is bound to immobilized human PD-L1 or cells expressing human PD-L1, followed by washing away unbound antibodies and introducing a labeled secondary antibody that is capable of binding to the primary antibody and thus detecting the bound primary antibody. When immobilized PD-L1 is used, the detection can be performed on an ELISA plate, or when cells expressing human PD-L1 are used, FACS analysis can be used for the detection. In certain embodiments, the antibodies and antigen-binding fragments thereof described herein bind to human PD-L1 with an EC50 (i.e., 50% effective concentration) of 1 nM to 10 nM or 1 nM to 5 nM (determined using FACS analysis).

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein inhibit the _{binding of} human PD-L1 to its receptor with an IC50 of 0.2nM-100nM (e.g., 0.2nM-50nM, 0.2nM-30nM, 0.2nM-20nM, 0.2nM-10nM, or 1nM-10nM), as measured by a competition 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 biological activities including, for example, inducing activated T cells to produce cytokines (such as CD4 ⁺ T cells and CD8 ⁺ T cells), inducing the proliferation of activated T cells (such as CD4 ⁺ T cells and CD8 ⁺ T cells), and reversing the inhibitory function of regulatory Tregs. Exemplary cytokines include IL-2 and IFNγ. The term "IL-2" refers to interleukin 2, which is a cytokine signaling molecule that regulates the activity of white blood cells (such as leukocytes) in the immune system. The term "interferon gamma (IFNγ)" is a cytokine produced by natural killer (NK) cells, NK T cells, CD4 ⁺ and CD8 ⁺ T cells, which is an important activator of macrophages and an inducer of major histocompatibility complex inducer (MHC) molecule expression. The production of cytokines can be determined by methods known in the art, such as ELISA. These methods can also be used to detect T cell proliferation, including [ ³ H] thymidine incorporation assays.

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 (such as 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 to human PD-L1.

In certain embodiments, the antibodies and antigen-binding fragments thereof bind to monkey PD-L1 with an EC50 (as determined by ELISA) of no greater than 100 nM, e.g., no greater than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, _0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM or 0.01 nM. In certain embodiments, the antibodies and antigen-binding fragments thereof bind to monkey PD-L1 with an EC50 of about 1 nM-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 a similar binding affinity as human PD-L1. For example, exemplary antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11 bind to murine PD-L1 undetectable by conventional binding assays such as ELISA or FACS analysis, whereas these antibodies bind to monkey PD-L1 with similar affinity or _EC50 values as human PD-L1 as detected by ELISA or FACS.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof have reduced or eliminated effector functions. In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof have a constant region of the IgG4 isotype, which has reduced or eliminated effector functions. Effector functions such as ADCC and CDC can lead to cytotoxicity against cells expressing PD-L1. Many cells, including normal cells, can express PD-L1. In order to avoid potential undesirable toxicity to these normal cells, certain embodiments of the antibodies and antigen-binding fragments thereof described herein have reduced or even eliminated effector functions. There are many known tests for assessing ADCC or CDC activity, such as Fc receptor binding assays, complement C1q binding assays, and cell lysis assays, which can be easily selected by those 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 against cells expressing PD-L1 (such as those normal cells), thereby avoiding undesirable side effects. At the same time, tumor cells expressing PD-L1 bind to anti-PD-L1 antibodies and are therefore unable to escape the immune checkpoints, allowing them to be 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 may have fully human IgG sequences, thereby being less immunogenic than humanized antibodies. As another example, the anti-PD-L1 antibodies and antigen-binding fragments thereof may have an IgG4 format to eliminate ADCC and CDC.

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein have the advantage that they can be used in combination with immunogenic substances, such as tumor cells, purified tumor antigens, cells transfected with encoded immunostimulatory factors, and tumor vaccines. In addition, the anti-PD-L1 antibodies and antigen-binding fragments thereof can be included in combination therapies, including standard chemotherapy and radiotherapy, target-based small molecule therapies, and other emerging immune checkpoint modulator therapies. In some embodiments, the antibodies and antigen-binding fragments thereof can be used as the base molecules of 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 produced in vitro using recombinant methods rather than animals. Bispecific antibodies or bivalent antibodies are artificial antibodies with fragments of two different monoclonal antibodies that can bind to two different antigens. "Bivalent" antibodies and antigen-binding fragments thereof include two antigen-binding sites. The two antigen-binding sites can bind to the same antigen, or they can 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 antibodies are prepared using recombinant methods. For example, transgenic animals, such as mice, can be prepared to carry a transgene or transchromosome containing human immunoglobulin genes and, thus, to produce fully human antibodies after immunization with an appropriate antigen, such as human PD-1. Fully human antibodies can be isolated from such transgenic animals, or alternatively, can be prepared using hybridoma technology, where spleen cells from the transgenic animals are fused with immortalized cell lines to generate 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 genetically engineered to contain functional recombinant human immunoglobulin loci; and Omni mice, in which the expression of endogenous mouse immunoglobulin genes is inactivated and genetically engineered to contain recombinant human immunoglobulin loci with J-locus deletions and C-kappa mutations. OmniFilc is a transgenic rat in which the expression of endogenous rat immunoglobulin genes is inactivated and simultaneously genetically engineered to contain a recombinant human immunoglobulin locus with a single, shared, recombinant VkJk light chain and a functional heavy chain. For further information, please refer to: Osborn M. et al, Journal of Immunology, 2013, 190: 1481-90; Ma B. et al, Journal of Immunological Methods 400–401 (2013) 78-86; Geurts A. et al, Science, 2009, 325: 433; U.S. Patent 8,907,157; European Patent 2152880B1; European Patent 2336329B1, all of which are incorporated herein by reference in their entirety. Other suitable transgenic animals can also be used, for example, HuMab mice (see Lonberg, N. et al. Nature 368(6474):856 859 (1994)), Xeno-mice (Mendez et al. Nat Genet., 1997, 15:146–156), TransChromo mice (Ishida et al. Cloning Stem Cells, 2002, 4:91–102) and VelocImmune mice (Murphy et al. Proc Natl Acad Sci USA, 2014, 111:5153–5158), Kymouse transgenic mice (Lee et al. Nat Biotechnol, 2014, 32:356–363), and transgenic rabbits (Flisikowska et al. PLoS One, 2011, 6:e21045).

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

In some embodiments, the anti-PD-L1 antibodies and antigen-binding fragments thereof described herein further include an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region includes a heavy chain and/or light chain constant region. The heavy chain constant region includes a CH1, CH1-CH2, or CH1-CH3 region. In some embodiments, the immunoglobulin constant region may further include one or more modifications to obtain desired properties. For example, the constant region may be modified to reduce or eliminate one or more effector functions to enhance FcRn receptor binding or introduce one or more cysteine residues.

In certain embodiments, the anti-PD-L1 antibody and antigen-binding fragment thereof further comprise a conjugate. It is conceivable that the antibodies or antigen-binding fragments thereof of the present invention can be linked to a variety of conjugates (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates can be linked to the antibody or antigen-binding compound by covalent binding, affinity binding, embedding, coordinate binding, complexation, binding, mixing or addition. In certain embodiments, the antibodies and antigen-binding fragments disclosed in the present invention can be engineered to contain specific sites other than epitope binding moieties, which can be used to bind one or more conjugates. For example, such sites can contain one or more reactive amino acid residues, such as cysteine residues and histidine residues, to facilitate covalent attachment to the conjugate. In certain embodiments, the antibody can be indirectly linked to the conjugate or linked through another conjugate. For example, the antibody or antigen-binding fragment thereof can be conjugated to biotin and then indirectly conjugated to a second conjugate, which is linked to avidin. The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purification 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, saccharide oxidase, or β-D-galactosidase), radioactive isotopes (e.g., ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, ¹¹¹ In, ¹¹² In, ¹⁴ C, 64 Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ²¹¹ At, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, and ^{32 P} , other lanthanides, luminescent labels), chromophore moieties, digoxigenin, biotin/avidin, DNA molecules, or gold for detection. In some 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol/propylene glycol copolymers, etc. In some embodiments, the conjugate can be a purification moiety such as magnetic beads. A "cytotoxic moiety" can be any agent that is harmful to cells or that may damage or kill cells. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, Examples include dacarbazine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatin (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine).

Polynucleotides and recombinant methods

The present application provides isolated polynucleotides encoding anti-PD-L1 antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotides comprise one or more nucleotide sequences as shown in Table 1, encoding the CDR sequences as shown 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: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, and homologous sequences thereof having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity. 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, SEQ ID NO: 50, SEQ ID NO: 54, and homologous sequences thereof having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity. In certain embodiments, the percentage of identity is due to the degeneracy of the genetic code, while the encoded protein sequence remains unchanged.

Using recombinant techniques known in the art, 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 (amplifying DNA) or gene expression. In another embodiment, the antibody can be made by homologous recombination methods known in the art. The DNA encoding the monoclonal antibody can be isolated and sequenced by conventional methods (e.g., oligonucleotide probes can be used that specifically bind to genes encoding the heavy and light chains of the antibody). A variety of vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancing sequence, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

In some embodiments, the vector system includes mammalian, bacterial, yeast systems, 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, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2 and other vectors that can be obtained from the laboratory or are commercially available. Suitable vectors can include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

The vector comprising the polynucleotide encoding the antibody and its antigen-binding fragment can be introduced into a host cell for cloning or gene expression. The host cell suitable for cloning or expressing the DNA in the vector in the present invention is a prokaryotic cell, yeast or the above-mentioned higher eukaryotic cell. Prokaryotic cells suitable for the purposes of the present invention include eubacteria such as, Gram-negative bacteria or Gram-positive bacteria, for example, Enterobacteriaceae, such as, Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as, Salmonella typhimurium, Serratia, such as, Serratia marcescens, and Shigella, and Bacillus such as, Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as, Pseudomonas aeruginosa and Streptomyces.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cells for cloning or expressing vectors encoding anti-PD-L1 antibodies. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species, and strains are commonly used and 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 weigensis (ATCC 24,178), Kluyveromyces krusonii (ATCC 56,500), Kluyveromyces drosophila (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora; Schwann yeast, such as Schwann yeast; and filamentous fungi, such as Neurospora, Penicillium, Tolylcoceras, and Aspergillus, such as Aspergillus nidulans and Aspergillus niger.

The host cells suitable for expressing glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculoviral strains and variants thereof, as well as corresponding permissive insect host cells, have been discovered 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 the Autographa californica nuclear polyhedrosis virus and the Bm-5 variant of the 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 can also be used as hosts.

However, the cells of greatest interest are spinal cord cells, and their cultivation (tissue culture) has become routine. Examples of mammalian host cells that can be used include SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or suspension cultured 293 cell subclones, 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 Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In certain preferred embodiments, the host cell is a 293F cell.

Host cells are transformed with the above-mentioned expression or cloning vectors capable of producing anti-PD-L1 antibodies and cultured in a conventional nutrient medium, which has been modified to be suitable for inducing promoters, selecting transformed cells, or amplifying genes encoding target sequences.

The host cells used to produce the antibodies or antigen-binding fragments thereof in the present invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM, (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) can be used to culture the host cells. In addition, any culture medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Application Re. 30,985 can be used as culture media for the host cells. These media may be supplemented with 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 adenylate and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds whose final concentrations are generally in the micromolar range), and glucose or an equivalent energy source. The media may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the media, such as temperature, pH, and the like, are those previously used for the host cells selected for expression and are well known to those of ordinary skill in the art.

When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, particulate debris from host cells or lysed fragments is first removed, for example, by centrifugation or ultrasound. Carter et al., Bio/Technology 10:163-167 (1992) describe a method for isolating antibodies secreted into the periplasmic space of Escherichia coli. Briefly, a cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes or more. Cell debris is removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant of the expression system is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be added during any of the aforementioned steps to inhibit protein degradation, as well as antibiotics to prevent the growth of incidental contaminants.

The antibodies produced from the cells can be purified using purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography columns, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique. The type of antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether protein A is suitable as an affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is suitable for all murine isoforms and human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). Agarose is the most commonly used matrix for attachment of affinity ligands, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrene)benzene can achieve faster flow rates and shorter processing times than using agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX.™ resin (J.T. Baker, Phillipsburg, N.J.) Other protein purification techniques can also be determined based on the desired antibody, such as fractionation on an ion exchange column, ethanol precipitation, reversed-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.

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

Reagent test kit

The present application provides a kit comprising the anti-PD-L1 antibody and its antigen-binding fragment. In some embodiments, the kit is used to detect the presence or level of PD-L1 in a biological sample. The biological sample may include cells or tissues.

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

In some embodiments, the anti-PD-L1 antibody and antigen-binding fragment thereof are linked to a substrate or instrument for sandwich assays such as ELISA or immunochromatographic assays. Suitable substrates or instruments can be, for example, microtiter plates and test strips.

Pharmaceutical compositions and methods of treatment

The present application further provides a pharmaceutical composition comprising the anti-PD-L1 antibody and the antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.

The pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic 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, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, including one or more antioxidants such as methionine in a composition containing an antibody or antigen-binding fragment thereof disclosed herein can reduce oxidation of the antibody or antigen-binding fragment thereof. Reducing oxidation can prevent or reduce a decrease in binding affinity, thereby improving antibody stability and extending shelf life. Therefore, in certain embodiments, the composition provided herein contains 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 for preventing oxidation of the antibodies or antigen-binding fragments thereof provided herein, extending their shelf life and/or enhancing their activity by mixing the antibodies or antigen-binding fragments thereof provided herein 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 dextrose injection, sterile water injection, or dextrose and lactated Ringer's 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 dextrose, 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, hydroxypropyl methylcellulose 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. Antimicrobial agents as carriers that can be added to pharmaceutical compositions in multiple-dose containers include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic 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 can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, saccharin sodium, cellulose, magnesium carbonate, etc.

In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, a liquid solvent, a suspending agent, an emulsifier, or a solid form suitable for producing a liquid solvent, a suspending agent, or an emulsifier. Injectable formulations can include ready-to-use sterile and/or pyrogen-free solutions, sterile dried solubles that are combined with a solvent before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dried insoluble products that are combined with a medium before use, and sterile and/or pyrogen-free emulsions. The solvent can be an aqueous phase or a non-aqueous phase.

In certain embodiments, the unit dose of the injection preparation is packaged in an ampoule, a tube or a syringe with a needle. As is known in the art, all preparations for injection should be sterile and pyrogen-free.

In certain embodiments, a sterile lyophilized powder can be prepared by dissolving the antibodies or antigen-binding fragments thereof disclosed herein in an appropriate solvent. The solvent may contain a component that improves the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components that improve 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 substances. The solvent may contain a buffer, such as a citrate buffer, sodium phosphate or potassium phosphate buffer, or other buffer known to those skilled in the art. In one embodiment, the buffer has a neutral pH. The dissolution is then sterilized by filtration under standard conditions known in the art, and then lyophilized to produce the desired formulation. In one embodiment, the resulting solution is dispensed into vials and lyophilized. Each vial can accommodate a single dose or multiple doses of the anti-PD-L1 antibody, or antigen-binding fragment thereof, or combination thereof. The amount filled into each vial can be slightly more than that required for each dose or multiple doses (eg, 10% excess) to ensure accurate sampling and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

The lyophilized powder is reconstituted with water for injection to obtain a formulation for injection. In one embodiment, the lyophilized powder can be reconstituted in sterile pyrogen-free water or other suitable liquid carrier. The exact amount is determined by the selected therapy and can be determined based on empirical values.

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

The therapeutically effective dose of the antibodies or antigen-binding fragments thereof provided herein depends on a variety of factors well known in the art, such as body weight, age, past medical history, current treatment, the subject's health status and potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of tumor development. A person skilled in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dose based on these or other conditions or requirements.

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

Dosage regimens can be adjusted to achieve the optimal response (e.g., therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over a period of time.

The antibodies and antigen-binding fragments disclosed in the present invention can be administered by administration methods 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 administration (e.g., oral administration, nasal administration, sublingual administration, rectal administration or topical administration).

Conditions and disorders associated with PD-L1 can 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, 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, thymic cancer, leukemia, lymphoma, myeloma, mycoses, mycoses, leukemia, lymphoma ... fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem glioma. In certain embodiments, the tumors and cancers are metastatic, particularly metastatic tumors expressing 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, and 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, HIV, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, human papilloma virus, adenovirus, Kaposi's sarcoma-associated herpes virus epidemic, Torquetenovirus, JC virus or BK virus, etc.

How to use

The present 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 for 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 as described herein. In some embodiments, the individual is identified as having a condition or disorder that may be responsive to a PD-L1 antagonist.

The presence and level of PD-L1 in the target biological tissue can indicate whether the individual from which the biological sample is derived is likely to respond to a PD-L1 antagonist. A variety of methods can be used to determine the presence or level of PD-L1 in the biological sample to be tested from the individual. For example, the biological sample to be tested can be exposed to an anti-PD-L1 antibody or an 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 methods such as qPCR, reverse transcription PCR, microarray, SAGE, FISH, etc. In some embodiments, the sample to be tested is derived from cancer cells or tissues, or immune cells that have entered a tumor. In some embodiments, the presence or level of PD-L1 in the biological sample to be tested indicates the possibility of a response. As used herein, the term "upregulation" refers to an increase in the level of PD-L1 protein detected in a test sample using an antibody or antigen-binding fragment thereof as described herein by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more compared to the level of PD-L1 protein in a reference sample detected using the same antibody. The reference sample can be a control sample obtained from a healthy or disease-free individual, or a healthy or disease-free sample obtained from an individual from whom 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., a tumor).

The antibodies and antigen-binding fragments disclosed in the present invention 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 in the present invention can be used in combination with chemotherapy, radiotherapy, cancer treatment surgery (such as tumor resection), one or more antiemetics or other chemotherapy-induced complications, or any other therapeutic substance for cancer or any PD-L1-mediated disease. In some such embodiments, when the antibodies and antigen-binding fragments disclosed in the present invention are used in combination with one or more therapeutic substances, they can be administered simultaneously with the one or more therapeutic substances. In some such embodiments, the antibodies and antigen-binding fragments can be administered simultaneously as part of the same pharmaceutical composition. However, antibodies and antigen-binding compounds that are "used in combination" with other therapeutic substances do not need to be administered at the same time or in the same composition as the therapeutic substance. The meaning of "used in combination" in the present invention also includes that antibodies and antigen-binding compounds that are administered before or after another therapeutic substance are also considered to be "used in combination" with the therapeutic substance, even if the antibody or antigen-binding fragment thereof and the second substance are administered by different administration methods. Where possible, other therapeutic substances used in combination with the antibodies or antigen-binding fragments thereof disclosed herein can be administered according to the methods in the product instructions of the other therapeutic substances, or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)), or according to other methods known in the art.

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

The present application further provides a method for monitoring treatment 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 using the anti-PD-L1 antibody or antigen-binding fragment thereof described herein. In certain embodiments, the method further comprises comparing the PD-L1 level in the test biological sample with the PD-L1 level in a comparable sample previously obtained from the same individual, wherein a decrease, slowing, or cessation of the increase in the PD-L1 level in the test biological sample indicates a positive treatment response or controlled disease progression. The comparable sample may be the same type of sample as the test sample, but is obtained from the same individual before treatment or in the early stages of treatment.

The following examples are intended to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All of the following specific compositions, materials, and methods, in whole or in part, are within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the present invention, but are merely intended to illustrate that specific embodiments are within the scope of the present invention. Those skilled in the art may develop equivalent compositions, materials, and methods without adding creativity and without departing from the scope of the present invention. It should be understood that various modifications made to the method of the present invention may still be within the scope of the present invention. The inventors intend that such variations 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., Palo Alto, USA) aged ∼8 weeks were sensitized with 10 μg of human PD-L1 ECD protein in TiterMax via footpad injection and subsequently challenged with PD-L1 ECD protein in aluminum phosphate gel adjuvant via footpad every 3 days until suitable for fusion. Anti-PD-L1 antibody serum titers were measured every 2 weeks by ELISA or FACS.

1.2 Cell Fusion: Three days before fusion, animals received a final challenge with 10 μg of human PD-L1 ECD protein in PBS via intraperitoneal injection. On the day of fusion, lymph node cells were harvested and a single-cell suspension was prepared. The obtained lymphocytes were mixed with myeloma cells (P3) at an appropriate ratio. The cell mixture was washed and resuspended in ECF solution at a density of 2.0 x 10 ⁶ cells/mL. Cell fusion was performed using a BTX 2000 electrofusion instrument.

1.3 Primary and Secondary Hybridoma Supernatant Screening: After 7–14 days of culture at 37°C, a portion of the hybridoma supernatant was assayed by Mirrorball analysis. Briefly, the hybridoma supernatant was diluted 5-fold with 1X PBS. PD-L1-expressing CHO-K1 cells were mixed with a secondary fluorescently labeled antibody and DraQ5. 20 μL of the cell mixture and 20 μL of the diluted hybridoma supernatant sample were added to each well of a 384-well plate and incubated in the dark at room temperature for at least 2 hours until ready for analysis on a high-sensitivity microplate cytometer. Positive cells were verified by FACS using PD-L1-expressing CHO-K1 cells. Cells were stained with the hybridoma supernatant sample and subsequently with FITC-conjugated goat anti-mouse IgG Fc for secondary antibody binding. The corresponding parental cell line was used as a negative control. Bound cells were analyzed using BD Biosciences FACSCanto II and FlowJo software.

1.4 Subcloning: Hybridoma cell lines that have been validated to positively bind to PD-L1-expressing cells were used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted to 5 or 1 cell per 200 μL of cloning medium. 200 μL/well of the medium was plated in a 96-well plate. The plates were incubated at 37°C with 5% _CO₂ until subsequent analysis.

1.5 Isotype Testing: Coat an ELISA plate with 50 μL/well of goat anti-rat IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM antibodies at 1 μg/mL. After blocking, add 50 μL of hybridoma supernatant sample to each well and incubate at room temperature for 2 hours. Use goat anti-rat kappa light chain-HRP as the detection antibody. Incubate with TMB substrate for 10 minutes to develop color, and stop the reaction with 2M HCl. Read the plate at 450 nm on an ELISA reader.

1.6 Cell-Based Binding Assay: To test the binding activity of the fully human antibody to the target, CHO-K1 cells or mature dendritic cells (mDCs) expressing human PD-L1 were bound to the fully human antibody, followed by binding to a secondary antibody conjugated to FITC-conjugated goat anti-human IgG Fc. The corresponding parental cell line was used as a negative control. Bound cells were analyzed using BD Biosciences FACSCanto II and FlowJo software.

Antibodies against human PD-L1 from rat hybridoma cells were used to bind to CHO cells transfected with full-length human PD-L1, followed by binding with a secondary antibody conjugated to FITC-conjugated goat anti-rat IgG Fc and analysis by FACS. As shown in Figure 1, antibodies 1.4.1, 1.14.4, 1.20.15, and 1.46.11 specifically bound to PD-L1 expressed on CHO cells with an _EC50 of approximately 1 nM.

Example 2: Modification of the Fc portion and purification

The antibodies in the 293F cell culture supernatant were purified using a protein A affinity chromatography column.

Example 3: Characterization of fully human antibodies

3.1 Competition test by FACS assay: To test whether fully human antibodies could block the binding of PD-L1 to PD-1, CHO-K1 cells expressing human PD-L1 were incubated with various concentrations of fully human antibodies for 1 hour at 4°C. Unbound antibody was eluted, and then mouse Fc-tagged human PD-1 was added to the cells. Binding of human PD-1 to PD-L1-expressing cells was detected using FITC-conjugated goat anti-mouse IgG and subsequently analyzed using BD Biosciences FACSCanto II and FlowJo software.

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-tagged human PD-1 was added to the cells. The binding of human PD-1 to cells expressing PD-L1 was detected using FITC-linked goat anti-mouse IgG, followed by FACS analysis. As shown in Figure 2, all 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 an IC50 value of approximately 10 nM.

3.2 Affinity testing by surface plasmon resonance (SPR) assay: The affinity and binding kinetics of the antibodies to PD-L1 were characterized by the SPR method using the ProteOn XPR36 (Bio-Rad). Protein A (Sigma) was immobilized on a GLM sensor chip (Bio-Rad) via amine coupling. The purified antibodies were flowed over the sensor chip and captured by Protein A. The chip was rotated 90° and washed with running buffer (1XPBS/0.01% Tween 20, Bio-Rad) until the baseline was stable. Five concentrations of human PD-L1 and running buffer were flowed through the antibody flow cell at a flow rate of 100 μL/min, initially for 240 s of the binding phase and then for 600 s of the dissociation phase. The chip was regenerated with H ₃ PO _4, pH 1.7, after each run. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using ProteOn software.

As shown in FIG5 , the affinity of the fully human PD-L1 antibodies to recombinant human PD-L1 detected by surface plasmon resonance ranged from 4.78E-10 to 2.26E-10 mol/L.

3.3 Affinity test by FACS assay: Antibody binding affinity to cell surface PD-L1 was determined by FACS analysis using CHO-K1 cells expressing human PD-L1. The antibody to be tested was serially diluted with elution buffer in a multiple of 1 to 2 (1XPBS/1% BSA) and incubated at 4°C for 1 hour. Secondary goat anti-human IgG Fc FITC (Jackson Immunoresearch Lab) was added and incubated at 4°C in the dark for 1 hour. The cells were then washed once and resuspended in 1XPBS/1% BSA and analyzed by flow cytometry (BD). Based on the quantitative beads QuantumTM MESF Kit (Bangs Laboratories, Inc.), the fluorescence intensity will be converted to molecules/cells. _KD was calculated using Graphpad Prism5.

3.4 In vitro functional assays: To assess 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: Isolate monocytes from healthy donors using a human monocyte enrichment kit according to the manufacturer's instructions. Culture the cells for 5-7 days to differentiate into dendritic cells (DCs). 18 to 24 hours before use, add 1 μg/mL LPS to the cell culture to induce DC maturation.

CD4 ⁺ T cells were isolated using a human CD4 ⁺ T cell enrichment kit according to the manufacturer's instructions and subsequently stimulated with mature or immature allogeneic DCs in the presence or absence of fully human anti-PD-L1 antibodies or control antibodies. IL-2 and IFNγ levels in culture supernatants were measured by ELISA on days 3 and 5, respectively. T cell proliferation was measured by [ ³ H]thymidine incorporation.

As shown in Figure 9, all 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 Figure 8, all fully human PD-L1 antibodies tested (1.4.1, 1.14.4, 1.20.15, and 1.46.11) increased IFNγ secretion in a dose-dependent manner. As shown in Figure 10, all fully human PD-L1 antibodies tested (1.4.1, 1.14.4, 1.20.15, and 1.46.11) increased T cell proliferation in a concentration-dependent manner.

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 IL-2 (20 U/ml). Simultaneously, DCs were generated by culturing monocytes as described above. Five days later, DCs were pulsed with pp65 peptide and subsequently added to CD4 ⁺ T cells in the presence or absence of fully human antibodies or control antibodies. IL-2 and IFNγ levels in culture supernatants were measured by ELISA on day 3. Proliferation of CMV pp65-specific CD4 ⁺ T cells was measured by [ ³ H]thymidine incorporation.

As shown in Figure 6, IFNγ 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). Figure 7 shows that fully human PD-L1 antibodies increased CMV ⁺ -CD4 ⁺ T cell proliferation in a concentration-dependent manner using autologous DC loaded with CMV pp65 peptide.

3.4.3 Treg Suppression Assay: Regulatory T cells (Tregs) are key immune regulators that play a crucial role in maintaining self-tolerance. CD4 ⁺ CD25 ⁺ Tregs are tumor-associated, as their numbers are found to be elevated in patients with multiple cancers and correlate with poor prognosis. To directly assess the role of fully human anti-human PD-L1 antibodies in inhibiting Treg suppressive function, Treg function was compared in the presence or absence of fully human antibodies or control antibodies. Briefly, CD4 ⁺ CD25 ⁺ Tregs and CD4 ^{+ CD25-} T cells were isolated by MACS. Treg function was compared in the presence or absence of varying ^{concentrations} of fully human antibodies or control antibodies. Briefly, CD4 ⁺ CD25 ⁺ Tregs and CD4 ^{+ CD25- T} cells were isolated by MACS ^and co ^- cultured with allogeneic mDCs (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 Figure 11, PD-L1 antibody 1.20.15 abolished the suppressive function of Tregs and restored the proliferation of reactive T cells and the secretion of IFNγ.

3.5 Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) testing: Since human PD-L1 is expressed on multiple cell types, in order to minimize the undesirable toxicity to healthy PD-L1 ⁺ cells in healthy and tumor cells, it was verified that the selected anti-PD-L1 fully human antibody has no ADCC and CDC functions.

3.5.1 ADCC: Target cells (mDCs) were preincubated with various concentrations of fully human antibodies in 96-well plates for 30 min, followed by the addition of IL-1-activated PBMCs (effectors) at an effector/target ratio of 50:1. The plates were incubated in a 37°C, 5% _CO2 incubator for 6 h. Target cell lysis was determined using a cytotoxicity assay kit (Roche). Optical density was determined using a Molecular Devices SpectraMax M5e microplate reader. Control hAb (IgG1) and control hAb (IgG4) served as positive and negative controls, respectively.

Figure 12 shows that 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 PBMCs 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 various concentrations of fully human antibodies were mixed in a 96-well plate. The plates were incubated in a 37°C, 5% _CO2 incubator for 4 hours. Target cell lysis was measured using CellTiter glo (Promega-G7573). Rituximab (Roche) and the human B-cell lymphoma cell line Raji (CD20 positive) served as positive controls. As shown in Figure 13, the fully human PD-L1 antibody did not mediate CDC.

3.6 Binning test determined by FACS: To examine whether the fully human antibody has the same epitope classification as the benchmark antibody, CHO-K1 cells expressing human PD-L1 were incubated with various concentrations of the fully human antibody at 4°C for 1 hour. Unbound antibody was eluted, and a biotinylated control antibody was added to the cells. Binding of the biotinylated control antibody to PD-L1-expressing cells was detected using PE-linked streptavidin and subsequently analyzed using BD Biosciences FACSCanto II and FlowJo software.

The results of the classification test showed that the binding epitopes of the fully human PD-L1 antibodies (i.e., 1.4.1, 1.14.4, 1.20.15 and 1.46.11) on human PD-L1 were different from those of known PD-L1 antibodies (i.e., benchmark antibodies).

3.7 Cross-species Binding Assay: Cross-reactivity of antibodies against cynomolgus macaque and murine PD-L1 was determined by ELISA. Human, cynomolgus macaque, and mouse PD-L1 were coated onto ELISA plates. After blocking, fully human antibodies were added to the plates and incubated at room temperature for at least 2 hours. Binding of the antibodies to the coated antigens was detected using goat anti-human IgG Fc-HRP. Color was developed using TMB substrate for 10 minutes and the reaction was terminated with 2M HCl. The plates were read at 450 nm on a Molecular Device M5e microplate reader.

As shown in Figure 4, the ELISA results showed that the fully human PD-L1 tested 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 test the cross-family binding activity of the fully human antibody, the fully human antibody was bound to a cell line expressing PD-L2, followed by secondary antibody binding using a FITC-conjugated goat anti-human IgG Fc. PD-L1-expressing cells served as a positive control. The corresponding parental cell line served as a negative control. Bound cells were analyzed using BD Biosciences FACSCanto II and FlowJo software.

CHO cells transfected with PD-L1 or PD-L2 were stained with a fully human PD-L1 antibody and analyzed by FACS. As shown in Figure 3, the fully human PD-L1 antibody specifically binds to PD-L1 but does not bind to PD-L2, a member of the PD-1 ligand family.

Example 4: Epitope Identification of Fully Human PD-L1 Antibodies

To determine the epitope of the antibody 1.14.4 described herein, an alanine scanning experiment against hPD-L1 and an evaluation of the antibody binding effect were performed using 1.14.4.

Alanine residues in hPD-L1 were mutated to glycine codons, and all other residues were mutated to alanine codons. Two-step sequential PCR was used to perform amino acid point substitutions for each residue in the hPD-L1 extracellular domain (ECD). The pcDNA3.3-hPD-1_ECD.His plasmid 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 the first PCR step, and the QuikChangeLighting multi-point site-directed mutagenesis kit (Agilent technologies, Palo Alto, CA) was used. The parent template was digested using Dpn I endonuclease after the mutation reaction. In the second PCR step, a linear DNA expression cassette containing a CMV promoter, the PD-L1 extracellular domain (ECD), a 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 a plate for ELISA binding assay. After reaction with supernatants containing quantified PD-L1 mutations or human/mouse PD-L1_ECD.His protein (Sino Biological, China), HRP-conjugated anti-His antibody (1:5000; Rockland Immunochemicals, Pottstown, PA) was added as a detection antibody. The absorbance was benchmarked according to the mean of the control mutations. After additional cutoff value setting for binding fold change (<0.55), the final determined epitope residues were identified.

The binding activity of antibody 1.14.4 against human and mouse PD-L1 was tested ( FIG14 ). Antibody 1.14.4 was found to bind to human PD-L1 ( FIG14A ), but not to mouse PD-L1 ( FIG14B ).

Table 2 lists the effects of 131 point substitutions on antibody binding in hPD-L1. Verification of the positions of all these residues on the hPD-L1 crystal structure (PDB codes 3RRQ and 4ZQK) revealed that some amino acids (e.g., Gly159, Tyr160, Pro161) are unlikely to directly contact any antibody. The observed reduction in binding is most likely due to destabilization or even structural collapse of the hPD-L1 structure after alanine substitution. After setting an additional threshold for binding fold change (<0.55), the final epitope residues are listed in Table 3. They target six positions at 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. Effects 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 identified

Critical value: fold change < 0.55

As shown in Figure 15, the hotspot residues responsible for hPD-L1 binding are all concentrated in the C chain, CC' loop, and F chain (Figure 15). Verification of the positions of the residues on the crystal structure of the hPD-1/hPD-L1 complex (PDB code 4ZQK) showed that these residues are mainly located in the A, C, F, and G chains. The epitope of antibody 1.14.4 is mainly contributed by residues on the C chain, which directly overlap with the hPD-1 and hPD-L1 interaction sites, suggesting a mechanism for binding to hPD-L1 and blocking hPD-1.

Example 5: In vivo inhibitory effect of fully human PD-L1 antibody on tumor growth

To evaluate the inhibitory effect of the fully human hPD-L1 antibody on tumor growth, MC38-B7H1 tumor cells (5× ^10⁵ cells/0.1mL) were inoculated subcutaneously in the right anterior flank of male B-hPD-1 humanized mice. A total of 42 animals were transferred. When tumors grew to approximately 100 ^mm³ , the mice were divided into five groups, each containing seven mice. These groups included: a vehicle control group, a BMK6 antibody control group (described in WO2011066389A1), a 1.14.4 antibody 3 mg/kg group, a 1.14.4 antibody 10 mg/kg group, and a 1.14.4 antibody 30 mg/kg group. All groups were administered via intraperitoneal injection every two days for six consecutive doses. Following the completion of dosing, the mice were observed for an additional two weeks. Tumor volume and body weight were measured twice weekly, and changes in body weight and tumor volume were recorded in relation to dosing time. At the end of the experiment, the tumor volume ratio (T/C) and tumor growth inhibition rate (TGI) of the treatment group compared to the solvent control group were calculated and statistically analyzed. Graphpad Prism 5 software was used to perform T-tests and statistical analysis of tumor volume. A P value < 0.05 was considered significant.

Tumor volume was measured twice weekly using a vernier caliper, measuring the long and short diameters. The volume was calculated using the formula: tumor volume = 0.5 × long diameter × short diameter ² . The tumor growth rate (T/C) (%) was calculated based on the measurement results: tumor volume in the treatment group / tumor volume in the negative control group x 100%. Tumor inhibition rate (TGI) (%) was calculated as [1-(Ti-T0)/(Vi-V0)] x 100.

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

Table 4. Antitumor effects of the fully human PD-L1 antibody 1.14.4 on MC38-B7H1 murine colon cancer transplanted hPD-1 humanized mice

Note: ^a. Mean ± standard error; ^b. Compared with the control group

Throughout the experiment, no significant weight loss was observed in any group of experimental animals (Table 4 and Figure 16), indicating good tolerance to the test compound. After 19 days of dosing (25 days after tumor cell inoculation), tumor volume in the solvent control group had grown to 2359 ^mm³ . Tumor volumes in the low, medium, and high dose groups of 1.14.4 were significantly reduced compared to the control group (mean tumor volumes were 949 ^mm³ , 1416 ^mm³ , and 1115 ^mm³ , respectively). All three doses demonstrated significant antitumor effects (tumor inhibition rates of 62.8%, 42.0%, and 55.4%, respectively) (Table 4 and Figure 17). The control antibody, BMK6, also produced significant antitumor effects (mean tumor volume of 1241 ^mm³ , tumor inhibition rate of 49.7%). The results demonstrate that the 1.14.4 antibody exhibits significant antitumor activity, with tumor inhibition rates exceeding 40% in the low, medium, and high dose groups.

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

Claims (27)

1. An isolated antibody or an antigen-binding fragment thereof, comprising: a) Heavy chains HCDR1 (SEQ ID NO:1), HCDR2 (SEQ ID NO:3) and HCDR3 (SEQ ID NO:5) and light chains LCDR1 (SEQ ID NO:7), LCDR2 (SEQ ID NO:9) and LCDR3 (SEQ ID NO:11); b) Heavy chains HCDR1 (SEQ ID NO:13), HCDR2 (SEQ ID NO:15) and HCDR3 (SEQ ID NO:17) and light chains LCDR1 (SEQ ID NO:19), LCDR2 (SEQ ID NO:21) and LCDR3 (SEQ ID NO:23); c) Heavy chains HCDR1 (SEQ ID NO:25), HCDR2 (SEQ ID NO:27), and HCDR3 (SEQ ID NO:29) and light chains LCDR1 (SEQ ID NO:31), LCDR2 (SEQ ID NO:33), and LCDR3 (SEQ ID NO:35); or d) Heavy chains HCDR1 (SEQ ID NO:37), HCDR2 (SEQ ID NO:39) and HCDR3 (SEQ ID NO:41) and light chains LCDR1 (SEQ ID NO:19), LCDR2 (SEQ ID NO:21) and LCDR3 (SEQ ID NO:23); The antibody or its antigen-binding fragment specifically binds to PD-L1. 2. The antibody or antigen-binding fragment thereof according to claim 1, comprising 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. 3. The antibody or antigen-binding fragment thereof according to 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 according to claim 1, comprising: a) The heavy chain variable region as shown in SEQ ID NO:43 and the light chain variable region as shown in SEQ ID NO:45; b) The heavy chain variable region as shown in SEQ ID NO:47 and the light chain variable region as shown in SEQ ID NO:49; c) The heavy chain variable region as shown in SEQ ID NO:51 and the light chain variable region as shown in SEQ ID NO:53; or d) The heavy chain variable region as shown in SEQ ID NO:55 and the light chain variable region as shown in SEQ ID NO:49. 5. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-4, which is capable of specifically binding to human PD-L1 with a Kd value not exceeding ^10⁻⁸ M, said Kd value being determined by plasmon resonance binding. 6. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which binds to monkey PD-L1 at an _EC50 of not more than 10 nM or not more than 1 nM, and/or does not bind to mouse PD-L1. 7. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which is capable of blocking the binding of human or monkey PD-L1 to its receptor with an IC50 of not more than 100 nM. 8. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which substantially does not bind to PD-L2. 9. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which does not mediate ADCC or CDC or neither. 10. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein it is a fully human monoclonal antibody. 11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the fully human monoclonal antibody is produced from transgenic rats. 12. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which is capable of blocking the binding of human PD-L1 to its receptor and thus provides at least one of the following activities: a) Induces IL-2 production in CD4 ⁺ T cells; b) Inducing the production of IFNγ in CD4 ⁺ T cells; c) Inducing CD4 ⁺ T cell proliferation; and d) Reverse the inhibitory function of T reg. 13. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein it is a camelized single-chain domain antibody, a diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody or bivalent domain antibody. 14. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, further comprising an immunoglobulin constant region. 15. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, further comprising a conjugate. 16. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-14. 17. A carrier comprising the isolated polynucleotide according to claim 16. 18. A host cell comprising the vector according to claim 17. 19. A method for expressing an antibody or an antigen-binding fragment thereof according to any one of claims 1-14, comprising culturing a host cell according to claim 18 under conditions of expressing the isolated polynucleotide according to claim 16. 20. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-15. 21. Use of the antibody or antigen-binding fragment thereof according to 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, the detection 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 the antibody or antigen-binding fragment thereof according to any one of claims 1-15 in the preparation of a kit for identifying an individual with a condition or disease that may 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 using the kit, wherein an upregulation of the presence or level of PD-L1 in the biological sample indicates 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 preparation of a kit for monitoring treatment response or disease progression in subjects treated with a PD-L1 antagonist, said monitoring comprising determining the presence or level of PD-L1 in a test biological sample from said individual using said kit. 24. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-15 and one or more pharmaceutically acceptable carriers. 25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-15 in the preparation of a medicament for treating a condition in a subject who would benefit from an upregulated immune response, said treatment comprising administering a therapeutically effective amount of said medicament to said subject. 26. The use according to claim 25, wherein the subject has upregulated PD-L1 expression. 27. The use according to claim 25, wherein the condition is cancer or a chronic viral infection.