WO2019129136A1 - Anti-pd-l1 antibody and uses thereof - Google Patents

Abstract

The invention provides an antibody and fragment thereof specifically binding to PD-L1, a composition containing the antibody or fragment thereof, a nucleic acid encoding the antibody or fragment thereof, a corresponding host cell and the uses thereof. Moreover, the invention also provides a type of therapy that uses a combination of the antibody and fragment thereof with other therapies.

Description

anti-PD-L1 antibody and use thereof

The present invention relates to novel antibodies and antibody fragments that specifically bind to PD-L1 and compositions containing the antibodies or antibody fragments. Furthermore, the invention relates to nucleic acids encoding the antibodies or antibody fragments thereof, and host cells comprising the same, and related uses. Furthermore, the invention relates to the therapeutic and diagnostic use of these antibodies and antibody fragments. In particular, the invention relates to the combination therapy of these antibodies and antibody fragments with other therapies, such as therapeutic modalities or therapeutic agents.

Background technique

Programmed Death Ligand 1 (PD-L1) is a protein involved in suppressing immune system responses during chronic infection, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 regulates the immune response by binding to an inhibitory receptor called programmed death 1 (PD-1) expressed on the surface of T cells, B cells, and monocytes. PD-L1 also negatively regulates T cell function through interaction with another receptor, B7.1 (also known as B7-1 or CD80). The formation of the PD-L1/PD-1 and PD-L1/B7.1 complex negatively regulates T cell receptor signaling, resulting in subsequent down-regulation of T cell activation and inhibition of anti-tumor immune activity. PD-L1 is overexpressed in many cancers, including a wide variety of solid tumors, such as bladder tumors, breast tumors, colon tumors, lung tumors, melanoma, ovarian tumors, salivary tumors, gastric tumors, and thyroid tumors. Overexpression of PD-L1 in tumor cells promotes tumor invasion and is often associated with poor prognosis.

Therefore, there is still a need in the art for new anti-PD-L1 antibodies that are better combined with PD-L1 and have good drug resistance.

Summary of invention

Disclosed herein are antibody molecules that bind to PD-L1. Also provided are nucleic acids encoding the antibodies or antibody fragments thereof, expression vectors, host cells and methods for producing antibody molecules. Immunoconjugates, multispecific or bispecific antibody molecules comprising an antibody molecule against PD-L1, and pharmaceutical compositions are also provided. The anti-PD-L1 antibody molecules disclosed herein can be used to treat, prevent, and/or diagnose neoplastic diseases as well as infectious diseases, either alone or in combination with other therapies, such as therapeutic agents or therapeutic modalities. Further, disclosed herein are compositions and methods for detecting PD-L1, and methods of preventing or treating a variety of diseases, including tumors and/or infectious diseases, using anti-PD-L1 antibody molecules.

Thus, in some embodiments, an antibody of the invention or a fragment thereof (specific) binds to PD-L1. In some embodiments, an antibody of the invention or a fragment thereof (specific) binds to human PD-L1.

In some embodiments, an anti-PD-L1 antibody or fragment thereof of the invention binds PD-L1 (eg, human PD-L1) with high affinity, eg, binds to PD-L1 with the following equilibrium dissociation constant (K _D ), The K _{D is} less than about 50 nM, preferably less than or equal to about 20 nM, more preferably less than or equal to about 15 nM, more preferably less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or 2 nM. most preferably, the K _D of less than or equal to about 1.5nM, 1.4nM, 1.3nM, 1.2nM, 1.1nM, 1nM, 0.9nM or 0.8nM. In some embodiments, an anti-PD-L1 antibody of the invention is 0.1-10 nM, preferably 0.5-10 nM, more preferably 0.6-10 nM, 0.7-8 nM, 0.7-5 nM, and most preferably 0.5-1.5 nM, 0.7- 1.5 dM, 0.7-1 nM K _D binds to PD-L1. In some embodiments, PD-L1 is human PD-L1. In some embodiments, antibody binding affinity is determined using a bio-optical interference assay (eg, Fortebio affinity measurement) assay.

In some embodiments, an antibody or fragment thereof of the invention binds to a cell expressing human PD-L1, eg, at less than or equal to about 4 nM, 3.5 nM, 3 nM, 2.9 nM, 2.8 nM, 2.7 nM, 2.6 nM, 2.5 nM EC50 of 2.4 nM, 2.3 nM, 2.2 nM, 2.1 nM, 2 nM, 1.9 nM, 1.8 nM, 1.7 nM or 1.6 nM. In some embodiments, the binding is determined by flow cytometry (eg, FACS). In some embodiments, the cell expressing human PD-L1 is a CHO cell expressing human PD-L1.

In some embodiments, an antibody or fragment thereof of the invention blocks the activity of PD-L1, for example, with an EC of less than or equal to about 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, or 0.7 nM. _50, preferably about 0.1-1nM, 0.5-1nM, 0.6-1nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM 1nM or the EC _50. In some embodiments, the related activity of PD-L1 is the binding of PD-L1 to PD-1. In some embodiments, the antibody or fragment of the invention in the MOA assay of less than or equal to about 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM or 0.7nM of EC _50, preferably from about 0.1 -1nM, 0.5-1nM, 0.6-1nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM EC ₅₀ of 1nM or inhibiting PD-L1 PD-1 binding with. In some embodiments, the cell is a CHO cell.

In some embodiments, an antibody or fragment thereof of the invention increases T cell function. In some embodiments, an antibody or fragment thereof of the invention increases T cell proliferation. In some embodiments, an antibody or fragment thereof of the invention increases IFN-[gamma] secretion. In some embodiments, an antibody or fragment thereof of the invention increases IL-2 secretion. In some embodiments, an antibody or fragment thereof of the invention increases IFN-[gamma] secretion and IL-2 secretion. In some embodiments, the increase is determined in a mixed lymphocyte reaction (MLR). In some embodiments, the ability of an antibody or fragment thereof of the invention to activate T cells is superior to known anti-PD-L1 antibodies, such as Tecentriq.

In some embodiments, an antibody of the invention or a fragment thereof has a lower viscosity than a known anti-PD-Ll antibody (eg, Tecentriq) and thus has better drug availability. In some embodiments, the antibody or fragment thereof of the invention, in a Zenix column assay, has a residence time (RT) of less than about 10 minutes, about 9 minutes, or about 8 minutes, preferably, a residence time of about 7 minutes. Between -9 minutes, preferably between about 7-8.5 minutes, about 7.5-8.5 minutes, about 7-8 minutes, or about 7.5-8 minutes, such as about 7.5 minutes, 7.6 minutes, 7.7 minutes, 7.8 minutes, 7.9 minutes, 8 minutes, 8.1 minutes, 8.2 minutes, 8.3 minutes, 8.4 minutes, 8.5 minutes.

In some embodiments, an antibody or fragment thereof of the invention inhibits one or more activities of PD-L1, for example, resulting in one or more of: increased tumor infiltrating lymphocytes, T cell receptor mediated proliferation Increase, or the immune evasion of cancer cells is reduced.

In some embodiments, an anti-PD-L1 antibody or fragment thereof of the invention is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC).

In some embodiments, an anti-PD-Ll antibody of the invention, alone or in combination with other therapies (eg, therapeutic modalities and/or therapeutic agents), is effective to treat a tumor (eg, a cancer) or an infection (eg, a chronic infection). In some embodiments, the tumor is a tumor immune escape. In some embodiments, the tumor is a cancer. In some embodiments, the tumor is a gastrointestinal tumor. In some embodiments, the cancer is colon cancer.

In some embodiments, the heavy and/or light chain of an anti-PD-L1 antibody or fragment thereof of the invention further comprises a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68).

In some embodiments, the antibodies of the invention also encompass variants of the amino acid sequence of an anti-PD-L1 antibody, as well as antibodies that bind to the same epitope as any of the anti-PD-L1 antibodies or fragments thereof described above.

In some embodiments, an anti-PD-L1 antibody of the invention further comprises a human or murine constant region. In some embodiments, an anti-PD-L1 antibody of the invention is an antibody in the form of IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE. In some embodiments, an anti-PD-L1 antibody of the invention comprises a heavy chain constant region selected from the group consisting of a heavy chain constant region such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; From the heavy chain constant region of the heavy chain constant region of, for example, IgG1, IgG2, IgG3, and IgG4, more specifically, the heavy chain constant region of IgG1 or IgG4, such as the heavy chain constant region of human IgG1 or IgG4. In one embodiment, the heavy chain constant region is a human IgGl or human IgG4 heavy chain constant region. In some embodiments, the anti-PD-L1 antibody of the invention comprises a murine constant region selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3.

In another embodiment, an anti-PD-L1 antibody molecule of the invention has, for example, a light chain constant region selected from the kappa or lambda light chain constant region, preferably a light chain constant region of kappa (eg, human kappa).

In yet another embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain constant region of IgG4 (eg, human IgG4). In one embodiment, human IgG4 comprises a substitution at position 228 according to the EU number (eg, Ser to Pro substitution). In yet another embodiment, human IgG4 is mutated to AA at positions 114-115 (EU numbering) (Armour KL1, Clark MR, Hadley AG, Williamson LM, Eur J Immunol. 1999 Aug; 29(8): 2613 24, Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities). In yet another embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain constant region of IgG1 (eg, human IgG1). In one embodiment, human IgGl comprises a substitution at position 297 according to the EU number (eg, Asn to Ala substitution). In one embodiment, human IgGl comprises a substitution at position 265 according to EU numbering, a substitution at position 329 according to EU numbering, or both substitutions (eg, Asp to Ala at position 265 according to EU numbering) Replacement and/or Pro to Ala replacement at position 329 according to EU numbering). In one embodiment, human IgGl comprises a substitution at position 234 according to EU numbering, a substitution at position 235 according to EU numbering, or both substitutions (eg, Leu to Ala at position 234 according to EU numbering) Replacement and/or replacement of Leu to Ala at position 235 according to EU numbering). In one embodiment, the heavy chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94 of the amino acid sequence set forth in SEQ ID NO: 64, 65 or 66 A sequence of %, 95%, 96%, 97%, 98%, 99% or more identity, or consists of the sequence.

In yet another embodiment, the anti-PD-L1 antibody molecule comprises a kappa light chain constant region, eg, a human kappa light chain constant region. In one embodiment, the light chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 of the amino acid sequence of SEQ ID NO:67 A sequence of %, 97%, 98%, 99% or more identity, or consists of the sequence.

In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain constant region of IgG1 (eg, a heavy chain constant region of human IgG1) and a kappa light chain constant region (eg, a human kappa light chain constant region). In one embodiment, human IgGl comprises a substitution at position 297 according to the EU number (eg, Asn to Ala substitution). In some embodiments, the human IgG1 heavy chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94 of the amino acid sequence set forth in SEQ ID NO: 64 or 65 A sequence of %, 95%, 96%, 97%, 98%, 99% or more identity, or consists of the sequence. In one embodiment, the human kappa light chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO:67 A sequence of 96%, 97%, 98%, 99% or more identity, or consists of the sequence.

In another embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain constant region of IgG4 (eg, a human IgG4 heavy chain constant region) and a kappa light chain constant region (eg, a human kappa light chain constant region). In one embodiment, the constant region is a mutated IgG4, eg, a mutated human IgG4 (eg, having a mutation at position 228 according to EU numbering (eg, S228P mutation) and/or having a position at positions 114-115 ( EU number) mutation to AA mutation). In some embodiments, the human IgG4 heavy chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence set forth in SEQ ID NO:66. A sequence of 95%, 96%, 97%, 98%, 99% or more identity, or consists of the sequence. In one embodiment, the human kappa light chain constant region comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO:67 A sequence of 96%, 97%, 98%, 99% or more identity, or consists of the sequence.

In one embodiment, the anti-PD-L1 antibody molecule is isolated or recombinant.

In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody or an antibody having monospecificity. The anti-PD-L1 antibody molecule can also be a humanized, chimeric, human antibody molecule. In some embodiments, the anti-PD-L1 antibody is a chimeric antibody. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody. In some embodiments, at least a portion of the framework sequence of the anti-PD-L1 antibody is a human consensus framework sequence. In one embodiment, an anti-PD-L1 antibody of the invention further encompasses an antibody fragment thereof, preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv) or ( Fab') ₂ , single domain antibody, double antibody (dAb) or linear antibody.

In certain embodiments, the anti-PD-L1 antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity for PD-L1 and a second binding specificity for LAG-3. In one embodiment, the bispecific antibody molecule binds to PD-L1 and LAG-3. A multispecific antibody molecule can have any combination of binding specificities for PD-L1 with other targets.

In one aspect, the invention provides a nucleic acid encoding any of the above anti-PD-L1 antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from the group consisting of a yeast cell, a mammalian cell (e.g., CHO cell or 293 cell) or other cell suitable for use in the preparation of an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic, such as an E. coli cell.

In one embodiment, the invention provides a method of making an anti-PD-L1 antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises, at an expression suitable for expression of the antibody or fragment thereof (preferably an antigen-binding fragment) The host cell is cultured under conditions of a nucleic acid, and the antibody or fragment thereof (preferably an antigen-binding fragment) is optionally isolated. In a certain embodiment, the method further comprises recovering an anti-PD-L1 antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

In some embodiments, the invention provides an immunoconjugate comprising any of the anti-PD-L1 antibodies and other materials provided herein, such as a cytotoxic agent or label. In some embodiments, the immunoconjugate is used to prevent or treat a tumor (eg, cancer) or an infectious disease. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer. Preferably, the infectious disease is a chronic infection.

In some embodiments, the invention provides compositions comprising any of the anti-PD-L1 antibodies or fragments thereof (preferably antigen-binding fragments thereof) described herein, or immunoconjugates thereof, preferably the compositions are pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, a composition, eg, a pharmaceutical composition, comprises an anti-PD-L1 antibody of the invention, or a fragment thereof, or an immunoconjugate thereof, and one or more additional therapeutic agents (eg, chemotherapeutic agents, other antibodies) A combination of a cytotoxic agent, a vaccine, an anti-infective active agent, or an immunomodulatory agent (eg, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule).

In some embodiments, the pharmaceutical composition is for use in preventing or treating a tumor (eg, cancer) or an infection. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer. Preferably, the infectious disease is a chronic infection. In another aspect, the invention relates to a method of preventing or treating a tumor (eg, cancer) or an infectious disease in a subject or individual, the method comprising administering to the subject an effective amount of any anti-PD described herein -L1 antibody or fragment thereof, pharmaceutical composition or immunoconjugate. In some embodiments, the tumor is a tumor immune escape. In one embodiment, the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer. In one embodiment, the infectious disease is a chronic infection. In another aspect, the invention relates to the use of any of the anti-PD-L1 antibodies or fragments thereof described herein for the manufacture of a medicament for treating a tumor (eg, cancer) or infection in a subject. In some embodiments, the tumor is a tumor immune escape. In one embodiment, the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer. In one embodiment, the infectious disease is a chronic infection.

In further some embodiments, in the prophylactic or therapeutic methods described herein, further comprising administering to the subject or individual one or more therapies (eg, a therapeutic modality and/or other therapeutic agent). In some embodiments, the treatment modality includes surgical treatment and/or radiation therapy. In some embodiments, the additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, a vaccine, an anti-infective active agent, other antibodies, or an immunomodulatory agent (eg, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule).

In some embodiments, the subject or individual is a non-human animal, such as a mammal, preferably a human.

In one aspect, the invention relates to a method of detecting PD-L1 in a sample, the method comprising (a) contacting a sample with any of the anti-PD-L1 antibodies or fragments thereof described herein; and (b) detecting anti-PD- Formation of a complex between the L1 antibody or a fragment thereof and PD-L1. In one embodiment, the anti-PD-L1 antibody is detectably labeled.

In some embodiments, the invention relates to a kit or article comprising any of the anti-PD-L1 antibodies or fragments thereof described herein. In some embodiments, the kit or article of manufacture comprises an anti-PD-L1 antibody or fragment thereof described herein, and an optional pharmaceutical excipient. In some embodiments, the kit or article further comprises instructions for administering a drug to treat a tumor or infection.

The invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein, or any combination thereof, are suitable for use in any and all of the anti-PD-L1 antibodies or fragments, methods and uses thereof of the invention described herein.

DRAWINGS

Figure 1 shows the binding of the anti-PD-L1 antibody of the present invention and CHO-PDL1 cells detected by FACS.

Figure 2 shows the binding of the anti-PD-L1 antibody of the present invention and CHO-PDL1 cells detected by FACS.

Figure 3 shows the blocking activity of the antibody of the present invention against PD-1/PD-L1 interaction detected by the MOA method.

Figures 4A and 4B show activation of T cells (relative expression of IL-2) by antibodies of the invention as detected by MLR assay.

Figures 5A and 5B show the activation of T cells (relative expression of IFN-γ) by the antibody of the present invention detected by the MLR assay.

Figure 6 shows the inhibitory effect of the antibody of the present invention on tumors.

Figure 7 shows the inhibition of tumors by the antibody of the present invention in combination with an anti-LAG-3 antibody.

Detailed description of the invention

abbreviation

Unless otherwise stated, the abbreviations in this specification have the following meanings:

Use the following abbreviation:

ADCC antibody-dependent cell-mediated toxicity

CDC complement dependent cytotoxicity

Complementarity determining region of CDRs in immunoglobulin variable regions

CHO Chinese Hamster Ovary

EC50 results in 50% potency or combined concentration

K _D equilibrium dissociation constant

ELISA enzyme-linked immunosorbent assay

FACS flow cytometry

MOA mechanism of action

MLR lymphocyte mixed reaction

FR antibody framework

IC50 produces a concentration of 50% inhibition

Ig immunoglobulin

Kabat's immunoglobulin alignment and numbering system established by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md.)

mAb or Mab or MAb monoclonal antibody

PCR polymerase chain reaction

IFN interferon

VL light chain variable region

VH heavy chain variable region

LC light chain

HC heavy chain

HCDR heavy chain complementarity determining region

LCDR light chain complementarity determining region

definition

Before the present invention is described in detail below, it is to be understood that the invention is not limited to the particular methodology, aspects, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiment Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

For the purpose of interpreting the specification, the following definitions will be used, and the terms used in the singular may also include the plural, and vice versa, as appropriate. It is understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

The term "about" when used in connection with a numerical value is meant to encompass a numerical value within the range of the lower limit of 5% less than the specified numerical value and the upper limit of 5% greater than the specified numerical value.

"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between a member of a binding pair (eg, an antibody and an antigen), unless otherwise indicated. The affinity of molecule X for its partner Y is generally expressed by the equilibrium dissociation constant (K _D ). Affinity can be measured by conventional methods known in the art, including those known in the art and described herein.

The term "programmed cell death 1 ligand 1", "PD-L1", "programmed death ligand 1", "differentiation cluster 274", "CD274" or "B7 homolog 1" as used herein refers to Any native PD-L1 of any vertebrate origin, including any mammalian species, such as primates (eg, humans) and rodents (eg, mice and rats). The terms encompass "full length", unprocessed PD-L1, and any form of PD-L1 produced by processing in a cell. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 includes four domains: an extracellular Ig-like V-domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. Additional information regarding the human PD-L1 gene (including genomic DNA sequences) can be found under NCBI Gene ID No. 29126. Additional information regarding the mouse PD-L1 gene (including genomic DNA sequences) can be found under NCBI Gene ID No. 60533. The amino acid sequence of an exemplary full length human PD-L1 protein can be found, for example, under NCBI Accession No. NP_001254653 or UniProt Accession No. Q9NZQ7, while an exemplary full length mouse PD can be found, for example, under NCBI Accession No. NP_068693 or Uniprot Accession No. Q9EP73. L1 protein sequence.

The term "anti-PD-L1 antibody", "anti-PD-L1", "PD-L1 antibody" or "antibody that binds PD-L1" as used herein refers to an antibody capable of binding PD with sufficient affinity. -L1 protein or a fragment thereof. In one embodiment, the anti-PD-L1 antibody binds to a non-PD-L1 protein to a lesser extent than about 10%, about 20%, about 30%, about 40%, about 50% of the binding of the antibody to PD-L1. About 60%, about 70%, about 80% or about 90% or more, as measured, for example, by radioimmunoassay (RIA) or biooptical interferometry or MSD assay.

As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to a single copy or cloned antibody derived from, for example, a eukaryotic, prokaryotic or phage clone, and does not refer to methods of its production. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant techniques, phage display technology, synthetic techniques such as CDR grafting, or a combination of such or other techniques known in the art.

"Native antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an isotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains bonded with a disulfide. From N to C, each heavy chain has a variable region (VH), also known as a variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C, each light chain has a variable region (VL), also known as a variable light or light chain variable domain, followed by a constant light (CL) domain. Depending on its constant domain amino acid sequence, the antibody light chain can be assigned to one of two types, called kappa (κ) and lambda (λ). A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. The native sequence human Fc region comprises the native sequence human IgGl Fc region (non-A and A allotype); the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region; and naturally occurring variants thereof.

"Antibody fragment" refers to a molecule that is distinct from an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single chain antibodies (eg, scFv); single domain antibodies; Specific antibodies or fragments thereof; camelid antibodies; and bispecific antibodies or multispecific antibodies formed from antibody fragments.

As used herein, the term "epitope" refers to a portion of an antigen (eg, human PD-L1) that specifically interacts with an antibody molecule. This portion (referred to herein as an epitope determinant) typically comprises an element such as an amino acid side chain or a sugar side chain or a component thereof. Epitope determinants can be defined by methods known in the art or disclosed herein (e.g., by crystallography or by hydrogen-oxime exchange). At least one or some portion of the antibody molecule that specifically interacts with an epitope determinant is generally located within the CDR. Typically, epitopes have specific three dimensional structural characteristics. Typically, epitopes have specific charge characteristics. Some epitopes are linear epitopes, while others are conformational epitopes.

An antibody that "binds to the same or overlapping epitope" as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% of the reference antibody in its competition assay Binding of the antigen, in other words, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the antibody to its antigen in a competition assay.

An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the reference antibody to its antigen in a competition assay. In other words, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether an antibody competes with another assay such as solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition. Assay (see for example Stahli et al, 1983, Methods in Enzymology 9: 242-253).

An antibody that inhibits (eg, competitively inhibits) binding of a reference antibody to its antigen refers to an antibody that inhibits binding of 50%, 60%, 70%, 80%, 90%, or 95% of the reference antibody to its antigen. . Conversely, the reference antibody inhibits binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90% or more. The binding of an antibody to its antigen can be measured by affinity (eg, equilibrium dissociation constant). Methods for determining affinity are known in the art.

An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of binding at least 50%, 60%, 70%, 80%, 90% or 95% of the reference antibody. Affinity and / or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.

A "complementarity determining region" or "CDR region" or "CDR" is a sequence that is hypervariable in an antibody variable domain and that forms a structurally defined loop ("hypervariable loop") and/or contains an antigen contact residue ( The area of the "antigen contact point"). The CDR is primarily responsible for binding to an epitope. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al, Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987) ), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (imgt.cines.fr/ on the World Wide Web), and affinity propagation clustering based on the use of a large number of crystal structures North CDR definition.

For example, depending on the CDR determination protocol, the residues of each CDR are as follows.

The CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence (e.g., any of the exemplary CDRs of the invention).

Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above manners.

Unless otherwise indicated, in the present invention, when referring to the position of a residue in the variable region of an antibody (including a heavy chain variable region residue and a light chain variable region residue), it is meant according to the Kabat numbering system ( The numbered positions of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

In one embodiment, the CDRs of the antibodies of the invention are bordered by Chothia rules or Kabat rules, for example, the sequences are shown in Table 1.

It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different. Thus, where an antibody is defined by a particular CDR sequence as defined by the present invention, the scope of the antibody also encompasses an antibody whose variable region sequence comprises the particular CDR sequence, but due to the application of a different protocol (eg Different assignment system rules or combinations result in different claimed CDR boundaries than the specific CDR boundaries defined by the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs (under the same assignment system). However, although the CDRs differ between antibodies and antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimal overlap region can be determined to provide a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of the CDR. As will be apparent to those skilled in the art, residues of the remainder of the CDR sequences can be determined by the structure of the antibody and protein folding. Accordingly, the invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues defined by Kabat or Chothia may be replaced by conservative amino acid residues.

There are five major classes of antibodies known in the art: IgA, IgD, IgE, IgG and IgM, and several of these antibodies can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

"Antibody in IgG form" refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions of all antibodies of the same type are identical, and the heavy chain constant regions differ between different types of antibodies. For example, an antibody in the IgGl form refers to an Ig domain whose heavy chain constant region Ig domain is IgG1.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to an Fc receptor (FcR) that is present on certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages). Secretory immunoglobulins enable these cytotoxic effector cells to specifically bind to target cells carrying the antigen, followed by cytotoxicity to kill the cytotoxic form of the target cells. The main cell that mediates ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991) summarizes FcR expression on hematopoietic cells. In order to assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as that described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Effector cells that can be used in such assays include PBMC and NK cells. Alternatively/in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal models such as those disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998).

The term "cytotoxic agent" or "cytotoxic factor" as used in the present invention refers to a substance which inhibits or prevents cellular function and/or causes cell death or destruction. Examples of cytotoxic agents are disclosed in WO2015/153513, WO2016/028672, WO2015/138920, WO2016/007235.

The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating a tumor (eg, cancer) and an infection (eg, a chronic infection), including a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, an anti-infective active agent, or An immunomodulator, such as any of the materials disclosed in WO2016/007235 or WO2010/077634 or US60/696426, which can be used in combination with an anti-PD-L1 antibody.

"Chemotherapeutic agents" include chemical compounds that are useful in the treatment of cancer. Examples of chemotherapeutic agents are disclosed in WO2016/007235, WO2010/077634, US60/696426 or WO2016/061142, US61/264061 or WO2016/007235.

The term "cytokine" is a generic term for a protein that is released by one cell population and acts as an intercellular medium on another cell. Examples of such cytokines are lymphokines, mononuclear factors; interleukins (IL) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL) and Γ-interferon. As used herein, the term cytokine includes biologically active equivalents of proteins and natural sequence cytokines from natural sources or from recombinant cell cultures, including small molecule entities produced by artificial synthesis, and their pharmaceutically acceptable Derivatives and salts.

The term "costimulatory molecule" refers to a related binding partner on a T cell that specifically binds to a costimulatory ligand, and thus is mediated by T cells, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for efficient immune responses. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activation molecules of signaling lymphocytes (SLAM proteins), NK cell activating receptors , BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS , ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2 CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55) , PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LA T, GADS, SLP-76, PAG/Cbp, CD19a and ligands that specifically bind to CD83.

The term "activator" or "agonist" includes a substance that increases certain parameters (eg, activity) of a given molecule (eg, a costimulatory molecule). For example, the term includes a substance that increases the activity of the given molecule by at least 5%, 10%, 25%, 50%, 75% or more (eg, costimulatory activity).

The term "immunoassay molecule" means a group of molecules on the cell surface of CD4 T cells and CD8 T cells. These molecules can effectively act as "brakes" that down-regulate or inhibit anti-tumor immune responses. Immunological checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40, and LAG-3, which directly inhibit Immune Cells.

The term "inhibitor" or "antagonist" includes a substance that reduces certain parameters (eg, activity) of a given molecule (eg, an immunological checkpoint inhibitor protein). For example, the term includes a substance that inhibits the given molecule by at least 5%, 10%, 20%, 30%, 40% or more of activity (eg, LAG-3 activity). Therefore, the inhibition does not have to be 100%.

The term "diabody" refers to an antibody fragment having two antigen binding sites comprising a heavy chain variable domain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). (VH). By using a linker that is too short to be able to pair between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain to create two antigen binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described, for example, in EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA ) 90:6444-6448 (1993). Tri- and tetra-antibodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).

A "functional Fc region" possesses an "effector function" of the native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (eg, B cell receptor; BCR), and the like. Such effector functions generally require that the Fc region be associated with a binding domain (eg, an antibody variable domain) and can be assessed using a variety of assays, such as those disclosed herein.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody that vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors) Down); and B cell activation.

"Human effector cell" refers to a leukocyte that expresses one or more FcRs and functions as an effector. In certain embodiments, the cell expresses at least FcyRIII and functions as an ADCC effector. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from their natural source, such as blood.

The term "effective amount" refers to an amount or dose of an antibody or fragment or conjugate or composition of the invention that, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. An effective amount can be readily determined by the attending physician as a person skilled in the art by considering various factors such as the species of the mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease; Response of an individual patient; specific antibody administered; mode of administration; bioavailability characteristics of the administered formulation; selected dosing regimen; and use of any concomitant therapy.

"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required. The therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof can vary depending on a variety of factors, such as the disease state, the age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition thereof are less than a therapeutically beneficial effect. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, a tumor growth rate) of at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70, relative to an untreated subject. % and still more preferably at least about 80% or 90%. The ability of a compound to inhibit measurable parameters (eg, cancer) can be evaluated in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit, in vitro by assays known to the skilled artisan.

By "prophylactically effective amount" is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, a prophylactically effective amount will be less than a therapeutically effective amount since the prophylactic dose is administered to a subject prior to the earlier stage of the disease or at an earlier stage of the disease.

"Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterozygous, chimeric Humanized (especially grafted with CDRs), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab') ₂ fragments, fragments produced by Fab expression libraries, Fd, Fv, II Sulfide-linked Fv (dsFv), single-chain antibody (eg, scFv), diabody or tetra-antibody (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (14), 6444-6448), Nanobody (nanobody) (also known as single domain antibody), anti-idiotypic (anti-Id) antibody (including, for example, an anti-Id antibody against an antibody of the invention), and an epitope-binding fragment of any of the above.

A "Fab" fragment includes a heavy chain variable domain and a light chain variable domain, and also includes a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the name for Fab' in which a cysteine residue of a constant domain carries a free thiol group. The F(ab') ₂ antibody fragment was originally produced as a pair of Fab' fragments with a hinge cysteine between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, which is also referred to as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National. Institutes of Health, Bethesda, MD, 1991.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of an antibody to an antigen. The variable domains of the heavy and light chains of a native antibody typically have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, eg, Kindt et ^{Kuby Immunology, 6 th ed.,} WHFreeman and page Co.91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind to the antigen can be isolated using a VH or VL domain from an antibody that binds to a particular antigen to separately screen a library of complementary VL or VH domains. See, for example, Portolano et al, J. Immunol. 150: 880-887 (1993); Clarkson et al, Nature 352: 624-628 (1991).

"Framework" or "F R" refers to a variable domain residue other than the CDR residues of the complementarity determining region. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, CDR and FR sequences typically appear in the following sequences of the heavy chain variable domain (VH) (or light chain variable domain (VL)):

FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

Unless otherwise indicated, the numbering of residues in each domain of an antibody is based on the EU numbering system, which is also referred to as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The terms "full length antibody", "intact antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

"Fv" is the smallest antibody fragment that contains the entire antigen binding site. In one embodiment, the double-stranded Fv species consists of one heavy chain variable domain and one light chain variable domain in a tight, non-covalently associated dimer. In a single-chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can be similar to a double-stranded Fv species. Dimer" structure association. In this configuration, it is the three CDRs of each variable domain that define the antigen binding site on the surface of the VH-VL dimer. In summary, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although the affinity is lower than the intact binding site. For a review of scFv see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York, 1994), pp. 269-315.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny of the same function or biological activity selected or selected in the originally transformed cells are included herein.

"Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source, which utilizes a human antibody library or other human Antibody coding sequence. This definition of a human antibody specifically excludes a humanized antibody comprising a non-human antigen-binding residue.

"Human consensus framework" refers to a framework that represents the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is selected from subtypes of variable domain sequences. In general, the subtype of this sequence is a subtype as disclosed in Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Vol. 1-3). In one embodiment, for VL, the subtype is subtype kappa I as in Kabat et al. (supra). In one embodiment, for VH, the subtype is subtype III as in Kabat et al. (supra).

A "humanized" antibody refers to a chimeric antibody comprising an amino acid residue from a non-human CDR and an amino acid residue from a human FR. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the CDRs (eg, CDRs) correspond to those of a non-human antibody, and all Or substantially all of the FRs correspond to those of human antibodies. The humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer. , hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, breast cancer, colon cancer, Rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, Malignant freckle-like melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as with phagomatoses, edema (such as those associated with brain tumors), and Meigs (Meigs) Significant vascular proliferation, brain tumors and brain Cancer, as well as head and neck cancer, and related metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include non-small cell lung cancer, squamous cell carcinoma, small cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glial Tumor, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid Cancer, liver cancer, leukemia and head and neck cancer, including those metastatic forms of cancer.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a certain degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder refers to cancer.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive when referred to herein.

The term "infectious disease" refers to a disease caused by a pathogen, including, for example, a viral infection, a bacterial infection, a fungal infection, or a protozoan such as a parasitic infection.

The term "tumor immune escape" refers to tumors escaping immune recognition and clearance. Thus, as a therapeutic concept, when the escape is weakened, tumor immunity is "treated" and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clarity.

The term "chronic infection" refers to an infection in which an infectious agent (eg, a pathogen such as a virus, a bacterium, a protozoan such as a parasite, a fungus, or the like) has induced an immune response in an infected host, but has not yet been as acutely infected. It is also removed or eliminated from the host as in the process. Chronic infections can be persistent, latent or slow. Although acute infections are usually resolved by the immune system for days or weeks (eg, flu), persistent infections can persist for months, years, decades, or lifetime (eg, hepatitis B) at relatively low levels. In contrast, latent infections are characterized by long-term asymptomatic activity, interrupted by high levels of rapid infection and elevated pathogen levels over time (eg, herpes simplex). Finally, slow infection is characterized by a gradual and continuous increase in disease symptoms, such as a long-term incubation period, followed by an onset of prolonged and progressive clinical processes following the onset of clinical symptoms. Unlike latent and persistent infections, chronic infections may not begin with the acute phase of viral proliferation (eg, picornaviruses infection, sheep visna virus, scrapie, Creutzfeldt-Jakobdisease). Exemplary infectious agents capable of inducing chronic infection include viruses (eg, cytomegalovirus, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type I and type II, human immunodeficiency virus types 1 and 2, Human papillomavirus, human T lymphocyte virus type 1 and 2, varicella-zoster virus, etc.), bacteria (eg, Mycobacterium tuberculosis, Listeria spp.) , Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Borrelia spp., Helicobacter pylori, etc., native Animals such as parasites (eg, Leishmania spp., Plasmodium falciparum, Schistosoma spp., Toxoplasma spp., Trypanosoma species) Trypanosoma spp.), Taenia carssiceps, etc., and fungi (eg, Aspergillus spp., Candida albicans, thick balls) Coccidioides immitis, Histoplasma capsulatum, Pneumocystis carinii, etc.). Additional infectious agents include prions or misfolded proteins that affect the brain or neuronal structure by further spreading protein misfolding in these tissues, resulting in the formation of amyloid plaques (which lead to cell death, tissue damage, and eventual death). Examples of diseases caused by prion infection include: Creutzfeldt-Jakob disease and its varieties, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (sFI) (fatal familial Insomnia (sFI)), kuru, scrapie, bovine spongiform encephalopathy (BSE) (also known as "mad cow" disease) (Bovine spongiformencephalopathy (BSE) in cattle (aka "mad cow") "Disease)), as well as various other animal forms of encephalopathy [eg, transmissible mink encephalopathy (TME), white-tailed deer, elk, and elk (" Mule deer) chronic wasting disease (CWD), feline spongiform encephalopathy, nyala, oryx (orx) and larger antelope (greater kudu) Hoofed encephalopathy (EUE) (exoungung encephalopathy (EUE), spongiform encephalopathy of theostrich].

An "immunoconjugate" is an antibody that is conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.

The term "label" as used herein, refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and that facilitates detection of the agent to which it is conjugated or fused. The label itself may be detectable (e.g., radioisotope label or fluorescent label) or, in the case of enzymatic labeling, may catalyze chemical alteration of a substrate compound or composition that is detectable. The term is intended to encompass the direct labeling of a probe or antibody by coupling (ie, physically linking) a detectable substance to a probe or antibody and indirectly labeling the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end labeling of a biotin-containing DNA probe such that it can be detected with a fluorescently labeled streptavidin protein.

An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, , mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one which has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) Determined by HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al, J. Chromatogr. B848: 79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that typically comprises the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-PD-L1 antibody or fragment thereof" refers to one or more nucleic acid molecules encoding an antibody heavy or light chain (or a fragment thereof), including such a single vector or a separate vector. Nucleic acid molecules, as well as such nucleic acid molecules that are present at one or more positions in a host cell.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. They refer to nucleotides of any length (deoxyribonucleotides or ribonucleotides) or analogs thereof in the form of a polymer. The polynucleotide may be single stranded or double stranded, and if single stranded, may be a coding strand or a non-coding (antisense) strand. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation to a labeling component. The nucleic acid can be a recombinant polynucleotide or a genomic, cDNA, semi-synthetic or synthetic source polynucleotide that is not found in nature or linked to another polynucleotide in an unnatural layout.

The terms "polypeptide", "peptide" and "protein" (if single stranded) are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be cleaved by non-amino acids. The term also includes amino acid polymers that have been modified (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component). Polypeptides can be isolated from natural sources, produced by eukaryotic or prokaryotic hosts by recombinant techniques, and can be the product of synthetic methods.

The calculation of sequence identity between sequences is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (eg, for optimal alignment, in the first and second amino acid sequences or nucleic acid sequences) Vacancies are introduced in one or both or non-homologous sequences can be discarded for comparison purposes. In a preferred embodiment, the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% for comparison purposes. , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at the corresponding amino acid position or nucleotide position are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecule is identical at this position.

Mathematical algorithms can be used to achieve sequence comparisons and percent identity calculations between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm in the GAP program that has been integrated into the GCG software package is used (at http://www.gcg.com) Obtained), using a Blossum 62 matrix or PAM250 matrix and vacancy weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6, to determine between the two amino acid sequences Percent identity. In yet another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and the vacancy weights of 40, 50, 60, 70 or 80 and The length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between the two nucleotide sequences. A particularly preferred set of parameters (and a set of parameters that should be used unless otherwise stated) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

You can also use the PAM120 weighted remainder table, the gap length penalty of 12, and the gap penalty 4), using the E. Meyers and W. Miller algorithms that have been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determine the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases to, for example, identify other family member sequences or related sequences. Such searches can be performed, for example, using the NBLAST and XBLAST programs of Altschul et al., (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain vacancy alignment results for comparison purposes, vacant BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

As used herein, the term "hybridizes under conditions of low stringency, medium stringency, high stringency or very high stringency" describes hybridization and washing conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6, incorporated by reference. Both aqueous and non-aqueous methods are described in the references and either method can be used. The specific hybridization conditions referred to herein are as follows: 1) Low stringency hybridization conditions are in about 45 C in 6X sodium chloride/sodium citrate (SSC) followed by at least 50 C (for low stringency conditions, washing can be increased) The temperature was up to 55 C) washed twice in 0.2X SSC, 0.1% SDS; 2) medium stringency hybridization conditions were washed once in 6X SSC at about 45 °C followed by 0.2X SSC, 0.1% SDS at 60 °C. Or multiple times; 3) High stringency hybridization conditions are one or more washes in 6X SSC at about 45 ° C followed by 0.2X SSC, 0.1% SDS at 65 ° C; and preferably 4) very high stringency hybridization The conditions were washed one or more times at 65 ° C in 0.5 M sodium phosphate, 7% SDS, followed by 65 ° C in 0.2X SSC, 0.1% SDS. Very high stringency conditions (4) are preferred conditions and one condition that should be used unless otherwise stated.

The term "pharmaceutical composition" refers to a composition that is present in a form that permits the biological activity of the active ingredient contained therein to be effective, and does not include additional toxicity to the subject to whom the composition is administered. Ingredients.

The term "pharmaceutically acceptable adjuvant" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, and the like, which are administered with the active substance.

As used herein, "treating" refers to slowing, interrupting, arresting, ameliorating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "prevention" includes inhibition of the occurrence or progression of a disease or condition or a symptom of a particular disease or condition. In some embodiments, a subject with a family history of cancer is a candidate for a prophylactic regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of a symptom or symptom of a cancer, particularly in a subject at risk for cancer.

The term "anti-infective active agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms at the concentration of administration and the interval of administration, but is not lethal to the host, such as viruses, bacteria, fungi or protozoa, such as parasites. As used herein, the term anti-infective active agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents. In a specific aspect, the anti-infective active agent is non-toxic to the host at the concentration of administration and the interval of administration.

Antibacterial anti-infective active agents or antibacterial agents can be broadly classified as either bactericidal (i.e., directly killed) or bacteriostatic (i.e., preventing division). Antibacterial anti-infective active agents can be further reclassified as narrow spectrum antibacterial agents (i.e., affecting only subtype bacterial subtypes, e.g., Gram negative, etc.) or broad spectrum antibacterial agents (i.e., affecting a wide variety). Examples include amikacin, gentamicin, geldanamycin, puromycin, mupirocin, nitrofurantoin, pyrazinamide, quinupristin/dalofopine, rifampicin/isofloxacin Amide or tinidazole.

The term "antiviral agent" includes any substance that inhibits or eliminates the growth, pathogenesis, and/or survival of a virus. This includes, for example, acyclovir, cidofovir, zidovudine, didanosine (ddI, VIDEX), zalcitabine (ddC, HIVID), stavudine (d4T, ZERIT), Lamy Fuding (3TC, EPIVIR)), azocavir (ZIAGEN), emtricitabine (EMTRIVA), and the like.

The term "antifungal agent" includes any substance that inhibits or eliminates the growth, pathogenesis and/or survival of fungi. This includes, for example, natamycin, bacteriocin, felofin, nystatin, amphotericin B, kandixin, patchouli, neem seed oil, coconut Oil (Coconut Oil) and the like.

The term "antigenic animal agent" includes any substance that inhibits or eliminates the growth, morbidity and/or survival of a protozoan organism (eg, a parasite). Examples of antiprotozoal agents include antimalarial agents such as quinine, quinidine, and the like.

Exemplary antibacterial, antiviral, antifungal, and antiprotozoal agents are described, for example, in WO 2010/077634 and the like.

Anti-infective active agents are also described, for example, in WO 2014/008218, WO 2016/028672, WO 2015/138920 or WO 2016/061142.

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes a vector that is a self-replicating nucleic acid structure and a vector that binds to the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

"Subject/patient sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be a solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample or a biopsy sample or a puncture sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from the subject's pregnancy or development at any time. Tissue samples may contain compounds that are naturally not intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples include, but are not limited to, tumor biopsy, fine needle aspirate, bronchial lavage fluid, pleural fluid (thoracic fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulation Plasma protein, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumors sample.

The term "package insert" is used to refer to instructions commonly included in commercial packages of therapeutic products containing information about indications, usage, dosage, administration, combination therapies, contraindications and/or warnings relating to the use of such therapeutic products. .

Antibody of the invention

Thus, in some embodiments, an antibody of the invention or a fragment thereof binds to PD-L1. In some embodiments, an antibody or fragment thereof of the invention binds to a mammalian PD-L1, such as human PD-L1. For example, an antibody molecule specifically binds to an epitope (eg, a linear or conformational epitope) on PD-L1. In some embodiments, the antibody molecule binds to one or more extracellular domains of PD-L1.

In some embodiments, an anti-PD-L1 antibody or fragment thereof of the invention has one or more of the following properties:

(1) The anti-PD-L1 antibody of the present invention or a fragment thereof binds PD-L1 (for example, human PD-L1) with high affinity, for example, binds to PD-L1 with the following equilibrium dissociation constant (K _D ), said K _{D is} less than about 50 nM, preferably less than or equal to about 20 M, more preferably less than or equal to about 15 nM, more preferably less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or 2 nM, most preferably , the K _D of less than or equal to about 1.5nM, 1.4nM, 1.3nM, 1.2nM, 1.1nM, 1nM, 0.9nM or 0.8nM. In some embodiments, an anti-PD-L1 antibody of the invention is 0.1-10 nM, preferably 0.5-10 nM, more preferably 0.6-10 nM, 0.7-8 nM, 0.7-5 nM, and most preferably 0.5-1.5 nM, 0.7- 1.5 dM, 0.7-1 nM K _D binds to PD-L1. In some embodiments, PD-L1 is human PD-L1. In some embodiments, antibody binding affinity is determined using a bio-optical interference assay (eg, Fortebio affinity measurement) assay.

(2) The antibody of the present invention or a fragment thereof binds to a cell expressing human PD-L1, for example, at less than or equal to about 4 nM, 3.5 nM, 3 nM, 2.9 nM, 2.8 nM, 2.7 nM, 2.6 nM, 2.5 nM, 2.4 nM. EC50 of 2.3 nM, 2.2 nM, 2.1 nM, 2 nM, 1.9 nM, 1.8 nM, 1.7 nM or 1.6 nM. In some embodiments, the binding is determined by flow cytometry (eg, FACS). In some embodiments, the cell expressing human PD-L1 is a CHO cell expressing human PD-L1.

(3) The antibody or fragment thereof of the present invention blocks the relative activity of PD-L1, for example, with an EC _{50 of} less than or equal to about 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM or 0.7 nM, preferably about 0.1-1nM, 0.5-1nM, 0.6-1nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM 1nM or the EC _50. In some embodiments, the related activity of PD-L1 is the binding of PD-L1 to PD-1. In some embodiments, the antibody or fragment of the invention in the MOA assay of less than or equal to about 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM or 0.7nM of EC _50, preferably from about 0.1 -1nM, 0.5-1nM, 0.6-1nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM EC ₅₀ of 1nM or inhibiting PD-L1 PD-1 binding with. In some embodiments, the cell is a CHO cell.

(4) The antibody or fragment thereof of the present invention enhances T cell function, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.

(5) The antibody or fragment thereof of the present invention enhances T cell proliferation, for example, in MLR, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.

(6) The antibody or fragment thereof of the present invention enhances IFN-γ secretion, for example, in MLR, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.

(7) The antibody or fragment thereof of the present invention enhances IL-2 secretion, for example, in MLR, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.

(8) The antibody of the present invention or a fragment thereof has a lower viscosity than a known anti-PD-L1 antibody (e.g., Tecentriq), and thus has better drug-forming properties. In some embodiments, the antibody or fragment thereof of the invention, in a Zenix column assay, has a residence time (RT) of less than about 10 minutes, about 9 minutes, or about 8 minutes, preferably, a residence time of about 7 minutes. Between -9 minutes, preferably between about 7-8.5 minutes, about 7.5-8.5 minutes, about 7-8 minutes, or about 7.5-8 minutes, such as about 7.5 minutes, 7.6 minutes, 7.7 minutes, 7.8 minutes, 7.9 minutes, 8 minutes, 8.1 minutes, 8.2 minutes, 8.3 minutes, 8.4 minutes, 8.5 minutes.

(9) The antibody or fragment thereof of the present invention inhibits one or more activities of PD-L1, for example, resulting in one or more of: an increase in tumor infiltrating lymphocytes, an increase in T cell receptor-mediated proliferation, or The immune escape of cancer cells is reduced.

(10) The anti-PD-L1 antibody of the present invention or a fragment thereof is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention has one or more of the following properties:

(i) showing the same or similar binding affinity and/or specificity to PD-L1 as an antibody of the invention (eg, any of the antibodies listed in Table 3);

(ii) inhibiting (eg, competitive inhibition) binding of an antibody of the invention (eg, any of the antibodies listed in Table 3) to PD-L1;

(iii) binding to the same or overlapping epitopes to an antibody of the invention (eg, any of the antibodies listed in Table 3);

(iv) competing for binding to PD-L1 with an antibody of the invention (such as any of the antibodies listed in Table 3);

(v) having one or more biological properties of an antibody of the invention, such as any of the antibodies listed in Table 3.

Exemplary antibody

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein said VH comprises

(i) three complementarity determining regions (CDRs) contained in the VH of any of the antibodies listed in Table B, or

(ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) on the three CDR regions relative to the sequence of (i).

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises:

(i) three complementarity determining regions (CDRs) contained in the VL of any of the antibodies listed in Table B;

(ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) on the three CDR regions relative to the sequence of (i).

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein

(a) The VH contains

(i) three complementarity determining regions (CDRs) contained in the VH of any of the antibodies listed in Table B, or

(ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) on said three CDR regions relative to the sequence of (i); and / or

(b) The VL contains:

(i) three complementarity determining regions (CDRs) contained in the VL of any of the antibodies listed in Table B;

(ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) on the three CDR regions relative to the sequence of (i).

In a preferred embodiment, the VH comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30 or 31.

In a preferred embodiment, VL comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 34, 35, 36 or 37.

In a preferred embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises

(i) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 26 or 30, and three complementarity determining regions of the light chain variable region set forth in SEQ ID NO: 32 or 36 LCDR, or

(ii) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 27, and three complementarity determining regions LCDR of the light chain variable region set forth in SEQ ID NO: 33, or

(iii) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 28, and three complementarity determining regions LCDR of the light chain variable region set forth in SEQ ID NO: 34, or

(iv) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 29 or 31, and three complementarity determining regions of the light chain variable region set forth in SEQ ID NO: 35 or 37 LCDR.

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein

(i) said VH comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence selected from SEQ ID NO: 1, 2, 3 or 4, or HCDR1 comprises An amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 or 4; HCDR2 comprising an SEQ ID NO: 5 Or the amino acid sequence of 6, 7, 8, or 9, or HCDR2 comprises one, two or more than the amino acid sequence selected from SEQ ID NO: 5, 6, 7, 8, or 9. An amino acid sequence of three alterations (preferably amino acid substitutions, preferably conservative substitutions); HCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 10, 11, 12 or 13 or HCDR3 comprises and is selected from the group consisting of SEQ ID NO: an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of 10, 11, 12 or 13;

and / or

(ii) wherein said VL comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence selected from SEQ ID NO: 14, 15 or 16, or LCDR1 comprises and An amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 14, 15 or 16; LCDR2 comprising a selected from the group consisting of SEQ ID NOs: 17, 18, 19 Or the amino acid sequence of 20 or consists of the amino acid sequence, or LCDR2 comprises one, two or three changes compared to the amino acid sequence selected from SEQ ID NO: 17, 18, 19 or 20 (preferably amino acid substitution, preferably Amino acid sequence of conservative substitution); LCDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 or 25, or LCDR3 comprises and is selected from the group consisting of SEQ ID NO: 21, 22, 23 The amino acid sequence of 24 or 25 has an amino acid sequence of one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions).

In a preferred embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein

(a) The VH contains

(i) a combination of HCDR1, HCDR2 and HCDR3 as shown in Table A; or

(ii) a variant of the HCDR combination of (i) comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably) on said three CDR regions Conservative substitution);

and / or

(ii) the VL contains

(i) a combination of LCDR1, LCDR2 and LCDR3 as shown in Table A; or

(ii) a variant of the LCDR combination of (i), said variant comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes on said three CDR regions (preferably amino acid substitution, preferably Conservative substitution).

In a preferred embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises a complementarity determining region ( CDR) HCDR1, HCDR2 and HCDR3 and the VL comprises (CDR) LCDR1, LCDR2 and LCDR3, wherein the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprised by the antibody or antigen-binding fragment thereof is as follows (Table A):

Table A: Exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in an antibody or antigen-binding fragment thereof of the present invention

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein

(a) Heavy chain variable region VH

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30 or 31 Or consisting of, or consisting of, an amino acid sequence of 98% or 99% identity; or

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30 or 31;

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30 or 31; 2, 1) amino acid sequence of amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, said amino acid change does not occur in the CDR region;

and / or

(b) Light chain variable region VL

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 34, 35, 36 or 37 Or consisting of, or consisting of, an amino acid sequence of 98% or 99% identity;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 34, 35, 36 or 37;

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 34, 35, 36 or 37; 2. The amino acid sequence of an amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR region.

In a preferred embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody or antigen-binding fragment thereof The combination of the heavy chain variable region VH and the light chain variable region VL included is shown in the following table (Table B):

Table B: Exemplary combinations of heavy chain variable region VH and light chain variable region VL in an antibody or antigen-binding fragment thereof of the invention

combination VH comprising the amino group represented by the following SEQ ID NO VL comprising the amino group represented by the following SEQ ID NO

Acid sequence or consists of Acid sequence or consists of (1) SEQ ID NO:26 SEQ ID NO:32 (2) SEQ ID NO:27 SEQ ID NO:33 (3) SEQ ID NO:28 SEQ ID NO: 34 (4) SEQ ID NO:29 SEQ ID NO: 35 (5) SEQ ID NO: 30 SEQ ID NO:36 (6) SEQ ID NO: 31 SEQ ID NO:37

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein

(a) heavy chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95 of the amino acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 42, 43, 44 or 45 Or consisting of amino acid sequences of %, 96%, 97%, 98% or 99% identity;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 42, 43, 44 or 45;

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no) than an amino acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 42, 43, 44 or 45 An amino acid sequence of more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR regions of the heavy chain, more preferably The amino acid change does not occur in the heavy chain variable region;

and / or

(b) Light chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence selected from the group consisting of SEQ ID NO: 46, 47, 48, 49, 50 or 51 Or consisting of 97%, 98% or 99% identical amino acid sequences;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 46, 47, 48, 49, 50 or 51;

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 46, 47, 48, 49, 50 or 51 Amino acid sequence of amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) of 3, 2, 1), preferably, said amino acid change does not occur in the CDR region of the light chain, more preferably, said amino acid The change does not occur in the light chain variable region.

In a preferred embodiment, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the combination of the heavy chain and the light chain comprised by the antibody or antigen-binding fragment thereof is as follows (Table C):

Table C: Exemplary combinations of heavy and light chains in an antibody or antigen-binding fragment thereof of the invention

In some embodiments, the heavy and/or light chain of an anti-PD-L1 antibody or fragment thereof of the invention further comprises a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68).

In one embodiment of the invention, the amino acid changes described herein include substitutions, insertions or deletions of amino acids. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in FR). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and/or outside the light chain variable region.

Optionally, an anti-PD-L1 antibody of the invention comprises a post-translational modification to a light chain variable region, a heavy chain variable region, a light chain or a heavy chain. Exemplary post-translational modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

In some embodiments, the substitution is a conservative substitution. A conservative substitution is one in which one amino acid is replaced by another amino acid in the same class, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is passed through another neutral amino acid. Replacement. An exemplary substitution is shown in Table D below:

Table D

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Addition or deletion of a glycosylation site to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

For example, one or more amino acid substitutions can be performed to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation increases the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861. Antibodies with altered types of glycosylation can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased aliquots of GlcNac structure. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell having an altered glycosylation system. Cells with altered glycosylation systems have been described in the art and can be used as host cells in which to express antibodies of the invention to thereby produce antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), thereby allowing expression of antibodies in the Ms704, Ms705, and Ms709 cell lines. It lacks fucose on its sugar. The Ms704, Ms705 and Ms709FUT8-/- cell lines were created by targeting the disruption of the FUT8 gene in CHO/DG44 cells using two alternative vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614 -twenty two). EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such cell lines exhibit low fucoal by reducing or eliminating alpha-1,6 bond-associated enzymes Saccharification. EP 1,176,195 also describes cell lines having low or no enzymatic activity of adding fucose to N-acetylglucosamine binding to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662) ). PCT publication WO 03/035835 describes a variant CHO cell line Lec13 cell in which the ability to attach fucose to Asn(297)-linked saccharide is reduced, thereby also resulting in expression in the host cell. Low fucosylation of antibodies (see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies with modified glycosylation profiles can also be produced in eggs as set forth in PCT Publication WO 06/089231. Alternatively, an antibody having a modified glycosylation profile can be produced in a plant cell (eg, Lemna). A method of producing antibodies in a plant system is disclosed in U.S. Patent Application Serial No. AU-A-A-A-A-A-A-A-A-A-A-A. PCT Publication WO 99/54342 describes cell lines engineered to express glycoprotein modified glycosyltransferases (eg, β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), thereby Antibodies expressed in this engineered cell line exhibit an increased aliquot of the GlcNac structure, which results in increased ADCC activity of the antibody (see also Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, the fucose residue of the antibody can be excised using a fucosidase; for example, the fucosidase alpha-L-fucosidase removes fucosyl residues from the antibody (Tarentino et al. 1975) Biochem. 14: 5516-23).

In one embodiment of the invention, the antibody or fragment of the invention is glycosylated with engineered yeast N-linked glycans or CHO N-linked glycans.

Another modification to the antibodies or fragments thereof described herein encompassed by the present invention is pegylation. The antibody can be PEGylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, typically the antibody or fragment thereof is attached to the antibody or one or more PEG groups thereof with polyethylene glycol (PEG) (eg, a reactive ester or aldehyde derivative of PEG) The reaction occurs under the conditions of the antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction using a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy polyethylene glycols or poly Ethylene glycol-maleimide. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention, see for example EP 0154316 and EP 0401384.

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to produce an Fc region variant to enhance the effectiveness of, for example, an antibody in treating cancer or a cell proliferative disorder. Anti-PD-L1 antibodies (eg, humanized antibodies or chimeric antibodies) and antigen-binding fragments thereof disclosed herein also include antibodies and fragments having modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Patent No. 5,624,821, WO2003/086310, WO2005/120571, WO2006/0057702. Such modifications can be used to enhance or inhibit various responses of the immune system, possibly with beneficial effects in diagnosis and treatment. Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and addition of multiple Fc. Modification of the Fc can also alter the half-life of the antibody in the therapeutic antibody, thereby enabling lower frequency administration and thus increased convenience and reduced material usage. See Presta (2005) J. Allergy Clin. Immunol. 116: 731, pp. 734-735.

In one embodiment, the number of cysteine residues of an antibody can be altered to modify antibody properties. For example, a modification is made to the hinge region of CH1 to alter (e.g., increase or decrease) the number of cysteine residues in the hinge region. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of CH1 can be altered to, for example, facilitate assembly of the light and heavy chains or increase or decrease the stability of the antibody.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein portions known in the art and readily available. Portions suitable for antibody derivatization include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene) Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on factors such as, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions. .

In some embodiments, the invention encompasses fragments of an anti-PD-L1 antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabody, linear antibody, single chain antibody (eg, scFv), single domain antibody; Multispecific antibodies formed.

For example, antibody molecules can include heavy chain (HC) variable domain sequences and light chain (LC) variable domain sequences. In one embodiment, the antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody molecule comprises two heavy chain variable domain sequences and two light chain variable domain sequences, thereby forming two antigen binding sites. Such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibody (eg scFv), single domain antibody, diabody (Dab) (bivalent and bispecific) and inlay Antibodies (eg, humanized), which can be produced by modifying intact antibodies, or those that are synthesized de novo using recombinant DNA techniques. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be from any antibody class including, but not limited to, IgG, IgA, IgM, IgD, and IgE and from any antibody subclass (eg, IgGl, IgG2, IgG3, and IgG4). Preparation of antibody molecules can be monoclonal or polyclonal. The antibody may also be a human antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, or an antibody produced in vitro. The antibody may have, for example, a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3 or IgG4. The antibody may also have, for example, a light chain selected from kappa or lambda.

The antibodies of the invention may also be single domain antibodies. Single domain antibodies can include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack a light chain, single domain antibodies derived from conventional 4-chain antibodies, or engineered antibodies. A single domain antibody can be any antibody of the prior art, or any single domain antibody of the future. Single domain antibodies can be derived from any species including, but not limited to, mice, humans, camels, alpaca, fish, sharks, goats, rabbits, and cattle. According to another aspect of the invention, a single domain antibody is a naturally occurring single domain antibody, referred to as a heavy chain antibody lacking a light chain. Such single domain antibodies are disclosed, for example, in WO 94/04678. Single domain antibodies or Nanobodies can be antibodies produced from Camelidae species such as camels, alpacas, dromedaries, llamas and guanaco. Other species than camels can produce heavy chain antibodies that naturally lack light chains; such single domain antibodies are within the scope of the invention.

In some embodiments, an anti-PD-L1 antibody of the invention is a humanized antibody. Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in its entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633). Almagro & Fransson distinguish between rational approaches and empirical approaches. A rational approach is characterized by the generation of a few engineered antibody variants and assessing their binding or any other property of interest. If the design variant does not produce the expected results, then a new round of design and combined assessment is initiated. Rational approaches include CDR grafting, Resurfacing, Superhumanization, and Human String Content Optimization. In contrast, empirical approaches are based on generating large populations of human variants and using enrichment techniques or high-throughput screening to select the best clones. Thus, empirical approaches rely on reliable selection and/or screening systems that are capable of searching for a large number of antibody variants. In vitro display techniques, such as phage display and ribosome display, meet these requirements and are well known to the skilled artisan. Empirical approaches include FR libraries, Guided selection, Framework-shuffling, and Humaneering.

In some embodiments, an anti-PD-L1 antibody of the invention is a human antibody. Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol 20: 450-459 (2008). For example, a human monoclonal antibody can be produced using a transgenic mouse carrying a human immunoglobulin gene rather than a mouse system (see, for example, Wood et al., International Application WO 91/00906; Kucherlapati et al., PCT Publication WO 91/ 10741; Lonberg et al., International Application WO 92/03918; Kay et al., International Application 92/03917; Lonberg, N. et al., 1994 Nature 368: 856-859; Green, LL et al., 1994 Nature Genet. 7: 13. -21; Morrison, SL et al, 1994 Proc. Natl. Acad. Sci. USA 81: 6851-6855; Bruggeman et al, 1993 Year Immunol 7: 33-40; Tuaillon et al, 1993 PNAS 90: 3720-3724; Bruggeman Et al., 1991 Eur J Immunol 21: 1323-1326).

In some embodiments, an anti-PD-L1 antibody of the invention is a non-human antibody, such as a rodent (mouse or rat) antibody, a goat antibody, a primate (eg, a monkey) antibody, a camelid antibody. Preferably, the non-human antibody is a rodent (mouse or rat) antibody. Methods of producing rodent antibodies are known in the art.

In certain embodiments, an antibody of the invention is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA. 81:6851-6855, 1984. In one embodiment, the chimeric antibody comprises a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In yet another embodiment, the chimeric antibody is a "class switching" antibody, wherein the class or subclass has been altered compared to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains, wherein, for example, a CDR (or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of the human constant region. In some embodiments, some of the FR residues in the humanized antibody are replaced by corresponding residues from a non-human antibody (eg, an antibody from which the CDR residues are derived), eg, to restore or improve antibody specificity or affinity.

An antibody of the invention can be isolated by screening a combinatorial library for antibodies having the desired activity. For example, a variety of methods are known in the art for generating phage display libraries and screening for antibodies having the desired binding characteristics in these libraries. These methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further, for example, in McCafferty et al, Nature 348:552-554. Clackso et al, Nature 352: 624-628 (1991); Marks et al, J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (edited by Lo, Human Press, Totowa, NJ, 2003); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et al, J. Mol. Biol 340(5): 1073-1093 (2004) Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284 (1-2): 119-132 (2004).

In one embodiment, the antibody molecule is a monospecific antibody molecule and binds to a single epitope. For example, a monospecific antibody molecule has multiple immunoglobulin variable domain sequences that each bind to the same epitope.

In one embodiment, the antibody molecule is a multispecific antibody molecule, eg, comprising a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable structure of the plurality of immunoglobulin variable domain sequences The domain sequence has binding specificity for the first epitope and the second immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for the second epitope. In one embodiment, the first and second epitopes are on the same antigen (eg, the same protein (or subunit of a multimeric protein)). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens (eg, different proteins (or different subunits of a multimeric protein)). In one embodiment, the multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule or a tetraspecific antibody molecule.

In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for the first epitope and a second immunoglobulin variable domain sequence having binding specificity for the second epitope. In one embodiment, the first and second epitopes are on the same antigen (eg, the same protein (or subunit of a multimeric protein)). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens (eg, different proteins (or different subunits of a multimeric protein)). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain having binding specificity for a second epitope Variable domain sequences and light chain variable domain sequences. In one embodiment, the bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a scFv having binding specificity for a first epitope or a fragment thereof and a scFv having binding specificity for a second epitope or a fragment thereof. In one embodiment, the first epitope is on PD-L1 and the second epitope is on LAG-3, OX40, TIM-3, CEACAM (eg, CEACAM-1 and/or CEACAM-5) or PD-L2 .

In some embodiments, the invention also encompasses anti-PD-L1 monoclonal antibodies ("immunoconjugates") conjugated to other substances, such as therapeutic modules or labels, such as cytotoxic agents or immunomodulators. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for the formation of immunoconjugates are known in the art, see for example WO2015/153513 or WO2015/138920. For example, cytotoxic agents include, but are not limited to, radioisotopes (eg, iodine (131I or 125I), hydrazine (90Y), hydrazine (177Lu), hydrazine (225Ac), hydrazine, hydrazine (211At), hydrazine (186Re), hydrazine (212Bi or 213Bi), indium (111In), antimony (99mTc), phosphorus (32P), antimony (188Rh), sulfur (35S), carbon (14C), antimony (3H), chromium (51Cr), chlorine (36Cl) , cobalt (57Co or 58Co), iron (59Fe), selenium (75Se) or gallium (67Ga). Examples of cytotoxic agents also include chemotherapeutic agents or other therapeutic drugs, such as paclitaxel, cytochalasin B, gramicidin D, Ethidium bromide, ipecaine, mitomycin, epipodophyllotoxin, epipodophyllotoxin, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, Dihydroxy anthraquinone dione, mitoxantrone, glomerin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol And puromycin and their analogs or homologs. Cytotoxic agents also include, for example, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, Aminomethamine (decarbazine)), an alkylating agent (eg, nitrogen mustard, thioepachlorambucil, phenylalanine mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, Busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthranil (eg, daunorubicin) (formerly known as daunorubicin) and doxorubicin), antibiotics (eg, actinomycin D (formerly known as actinomycin), bleomycin, phosfomycin, and amphotericin (AMC) ), and anti-mitotic agents (eg, vincristine and vinblastine).

Substances which can be conjugated/conjugated with an anti-PD-L1 antibody are also described, for example, in WO2010/077634, US60/696426, WO2016/007235 or WO2016/061142.

Nucleic acid of the present invention and host cell containing the same

In one aspect, the invention provides a nucleic acid encoding any of the above anti-PD-L1 antibodies or fragments thereof. The nucleic acid may encode an amino acid sequence comprising a light chain variable region and/or a heavy chain variable region of an antibody, or an amino acid sequence comprising a light chain and/or a heavy chain of an antibody.

For example, an exemplary nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 26 to 51, or encoding a nucleic acid selected from any one selected from the group consisting of SEQ ID NOs: 26 to 51. A nucleic acid having an amino acid sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the amino acid sequence.

The invention also encompasses a nucleic acid that hybridizes under stringent conditions with a nucleic acid as described below or a nucleic acid having one or more substitutions (eg, conservative substitutions), deletions, or insertions with a nucleic acid comprising: encoding from SEQ ID NO: 26 to a nucleic acid of a nucleic acid sequence of the amino acid sequence of any one of 51; or comprising at least 85%, 90%, 91%, 92% of the amino acid sequence selected from any one of SEQ ID NOS: 26 to 51; A nucleic acid of a nucleic acid sequence of an amino acid sequence of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC). A variety of carrier systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV) or SV40 virus. . Another type of vector utilizes RNA elements derived from RNA viruses such as Semliki forest virus, oriental equine encephalitis virus, and flavivirus. In a preferred embodiment, the expression vector of the invention is a pTT5 expression vector.

In addition, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of the host cells that have been transfected. The marker can, for example, provide prototrophic, biocidal (eg, antibiotic) or heavy metal (eg, copper) resistance, etc., to the auxotrophic host. The selectable marker gene can be ligated directly to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional components may also be required in order to optimally synthesize mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

Once an expression vector or DNA sequence has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to accomplish this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be based on the methods described in the present specification and the prior art, depending on the particular expression vector used and Mammalian host cell changes or optimization.

In one embodiment, a host cell comprising a nucleic acid encoding an antibody molecule described herein or a vector described herein is provided. Suitable host cells for cloning or expressing a nucleic acid or vector encoding the antibody include prokaryotic or eukaryotic cells as described herein. Antibodies can be produced, for example, in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523, also to Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003). , pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified. In one embodiment, the host cell is an E. coli cell.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from the group consisting of a yeast cell, a mammalian cell (eg, a human cell), an insect cell, a plant cell, or other cell suitable for use in the preparation of an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including glycosylation pathways that have been "humanized" resulting in the production of antibodies having partial or complete human glycosylation patterns. Fungal and yeast strains. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al, Nat. Biotech. 24: 210-215 (2006). Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth can be used. Further examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 HEK or 293 cells, eg eg Graham et al, J. Gen Virol. 36: 59 (1977) (described in) and so on. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 216 (1980)); and myeloma cell lines such as Y0. , NS0 and Sp2/0.

For a review of certain mammalian host cell lines suitable for producing antibodies, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). . Other useful host cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, MDCKII cells, PerC6 cell lines (eg, PERC6 cells from Crucell) oocytes, and from transgenic animals. Cells, such as mammary epithelial cells. Suitable insect cells include, but are not limited to, Sf9 cells.

Method for preparing antibody of the present invention and antigen-binding fragment thereof

The anti-PD-L1 antibodies disclosed herein can be recombinantly produced. There are several methods known in the art for producing recombinant antibodies. One example of a method for recombinant production of antibodies is disclosed in U.S. Patent No. 4,816,567.

In one embodiment, a method of making an anti-PD-L1 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, such as provided above, under conditions suitable for expression of the antibody, and The antibody is optionally recovered from the host cell (or host cell culture medium). For recombinant production of an anti-PD-L1 antibody, a nucleic acid encoding an antibody (such as an antibody described above) is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

In one embodiment, the host cell comprises a vector comprising a nucleic acid encoding the amino acid sequence of the VL of the antibody and a nucleic acid encoding the amino acid sequence of the VH of the antibody. In one embodiment, the host cell comprises a first vector comprising a nucleic acid encoding an amino acid sequence of the VL of the antibody and a second vector comprising a nucleic acid encoding the amino acid sequence of the VH of the antibody.

Assay

The anti-PD-L1 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, the antibody of the present invention is tested for its antigen binding activity, for example, by known methods such as ELISA, Western blot, flow cytometry, magnetic beads of antibody molecules, and the like. PD-L1 binding can be assayed using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, biophotonic interferometry (eg, Fortebio affinity measurement) or MSD assay or flow cytometry is used.

In another aspect, a competition assay can be used to identify antibodies that compete with any of the anti-PD-L1 antibodies disclosed herein for binding to PD-L1. In certain embodiments, such a competitive antibody binds to the same epitope (eg, a linear or conformational epitope) as the epitope to which any of the anti-PD-L1 antibodies disclosed herein bind. A detailed exemplary method for locating epitopes bound by antibodies is found in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

The invention also provides assays for identifying anti-PD-L1 antibodies having one or more of the properties described above. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for one or more of the properties described above.

Cells for use in any of the above in vitro assays include cells or cell lines that naturally express PD-L1 or that are engineered to express PD-L1.

It will be appreciated that any of the above assays can be performed by replacing or supplementing the anti-PD-Ll antibody with an immunoconjugate of the invention.

It will be appreciated that any of the above assays can be performed using an anti-PD-Ll antibody and other therapeutic agents.

Pharmaceutical composition and pharmaceutical preparation

The present invention also encompasses a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising an anti-PD-L1 antibody or a fragment thereof or an immunoconjugate thereof, and a composition comprising a nucleic acid encoding an anti-PD-L1 antibody or a fragment thereof. In certain embodiments, the composition comprises one or more antibodies that bind to PD-L1, or a fragment thereof, or an immunoconjugate thereof, or one or more antibodies that encode one or more antibodies that bind to PD-L1 or Fragment of nucleic acid. These compositions may also contain suitable pharmaceutical excipients such as pharmaceutically acceptable carriers, excipients and the like known in the art, including buffers.

Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions.

Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin , propylene, glycol, water, ethanol, etc. For the use of excipients and their use, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R. C. Rowe, P. J. Seskey and S. C. Owen, Pharmaceutical Press, London, Chicago.

If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

The anti-PD-L1 antibody of the present invention having the desired purity can be mixed with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980)). Pharmaceutical formulations comprising the anti-PD-L1 antibodies described herein are prepared, preferably in the form of a lyophilized formulation or an aqueous solution.

Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latter including a histidine-acetate buffer.

The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient which is required for the particular indication being treated, preferably those having complementary activities which do not adversely affect each other. For example, it is desirable to provide other anti-cancer active ingredients such as chemotherapeutic agents and/or cytotoxic agents. The active ingredient is suitably present in combination in an amount effective for the intended use. The active ingredient can be any substance known in the art to be capable of combination with an anti-PD-L1 antibody, including chemotherapeutic agents, other antibodies, and other therapeutic agents. Examples of such active ingredients are described, for example, in WO2010/077634, WO2016/061142, US61/264061, US60/696426, WO2016/007235 and the like.

In some embodiments, the active ingredient is an anti-LAG-3 antibody, eg, a human anti-LAG-3 antibody, preferably, the anti-LAG-3 antibody is humanized

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.

Use of antibodies

In one aspect, the invention relates to a method of modulating an immune response in a subject. The method comprises administering to the subject an effective amount of an antibody molecule (eg, an anti-PD-L1 antibody) or a pharmaceutical composition or immunoconjugate disclosed herein to modulate an immune response in the subject. In one embodiment, an antibody molecule (eg, a therapeutically effective amount of an anti-PD-Ll antibody molecule) or a pharmaceutical composition or immunoconjugate disclosed herein restores, enhances, stimulates, or increases an immune response in a subject.

In another aspect, the invention relates to a method of preventing or treating a tumor (eg, cancer) in a subject, the method comprising administering to the subject an effective amount of an antibody molecule disclosed herein (eg, anti-PD-L1 Antibody) or a pharmaceutical composition or immunoconjugate. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is a gastrointestinal tumor (eg, a cancer), such as colon cancer.

In another aspect, the invention relates to a method of preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an antibody molecule disclosed herein (eg, an anti-PD-L1 antibody) Or a pharmaceutical composition or immunoconjugate. In one embodiment, the infectious disease is a chronic infection.

In another aspect, the invention relates to a method of eliciting antibody-dependent cell-mediated cytotoxicity in a subject, the method comprising administering to the subject an effective amount of an antibody molecule disclosed herein (eg, anti-PD -L1 antibody) or a pharmaceutical composition or immunoconjugate.

The subject can be a mammal, for example, a primate, preferably a higher primate, for example, a human (eg, a patient having the disease described herein or at risk of having the disease described herein). In one embodiment, the subject is in need of an enhanced immune response. In some embodiments, an anti-PD-L1 antibody molecule described herein increases T cell proliferation. In some embodiments, an anti-PD-L1 antibody molecule described herein restores, enhances or stimulates an antigen-specific T cell response in a subject, eg, an interleukin-2 (IL-2) in an antigen-specific T cell response Or interferon-gamma (IFN-γ) production. In some embodiments, the immune response is an anti-tumor response. In one embodiment, the subject has or is at risk of having a disease described herein (eg, a tumor or infectious disease as described herein). In certain embodiments, the subject is immunocompromised or at risk of immunocompromised. For example, the subject has received or has received chemotherapy treatment and/or radiation therapy. Alternatively or in combination, the subject is immunocompromised due to infection or has a risk of being immunocompromised by the infection.

In some embodiments, the tumors described herein, eg, cancer, include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.

Examples of solid tumors include malignant tumors, for example, sarcomas and carcinomas of multiple organ systems (including adenocarcinoma and squamous cell carcinoma), such as invasive liver, lung, breast, lymph, gastrointestinal (eg, colon), reproduction Those of the urinary tract (eg, kidney, bladder epithelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma in lung cancer, small bowel cancer, and esophageal cancer. Squamous cell carcinomas include malignant tumors such as those in the lungs, esophagus, skin, head and neck area, mouth, anus, and cervix. In one embodiment, the cancer is melanoma, eg, advanced melanoma. In one embodiment, the cancer is a gastrointestinal cancer, such as colon cancer. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention.

Non-limiting examples of preferred cancers for treatment include lymphomas (eg, diffuse large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma), breast cancer (eg, metastatic breast cancer), Lung cancer (eg, non-small cell lung cancer (NSCLC), eg, stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma), myeloma (eg, multiple myeloma), leukemia (eg, , chronic myeloid leukemia), skin cancer (eg, melanoma (eg, stage III or IV melanoma) or Merkel cell carcinoma), head and neck cancer (eg, head and neck squamous cell carcinoma (HNSCC)), spinal cord development Adverse syndrome, bladder cancer (eg, transitional cell carcinoma), renal cancer (eg, renal cell carcinoma, eg, clear cell renal cell carcinoma, eg, advanced or metastatic clear cell renal cell carcinoma) and colon cancer. In addition, refractory or recurrent malignancies can be treated using the antibody molecules described herein.

Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach-esophage cancer, stomach cancer, Testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer , thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, children Solid tumor, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodysplastic syndrome, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, ridge Axon tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, including A combination of those cancers (eg, mesothelioma) induced by asbestos and the cancer.

Treatment of metastatic cancer (e.g., metastatic carcinoma expressing PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144) can be achieved using the antibody molecules described herein.

Tumor immune escape can also be treated using the antibody molecules described herein.

In one embodiment, the tumor is a cancer that expresses elevated levels of PD-L1.

In some embodiments, the cancer described herein is colon cancer and metastatic cancer thereof.

In some embodiments, the infection is acute or chronic. In some embodiments, the chronic infection is a persistent infection, a latent infection, or a slow infection. In some embodiments, the chronic infection is caused by a pathogen selected from the group consisting of bacteria, viruses, fungi, and protozoa.

In some embodiments, the pathogen is a bacterium. In one embodiment, the bacterium is selected from the group consisting of Mycobacterium spp., Salmonella spp., Listeria spp, and Streptococcus spp. ), Haemophilus (spp.), Neisseria spp., Klebsiella spp., Borrelia spp., fragile Bacterioides fragillis, Treponema spp., and Helicobacter pylori.

In some embodiments, the pathogen is a virus. In one embodiment, the virus is selected from the group consisting of an infectious virus, such as hepatitis B virus or hepatitis C virus (hepatitis-B, -C), Herpes simplex virus-I (herpes simplex virus-I, -II), human immunodeficiency virus-I, -II, cytomegalovirus, Eppstein Barr virus, human papillomavirus, human T Human T lymphotrophic viruses (I, -II), varicallazoster.

In some embodiments, the pathogen is a fungus. In one embodiment, the condition caused by the fungus is selected from the group consisting of: aspergilosis, blastomycosis, candidiasis albicans, coccidioiodmycosis immitis, histoplasmosis ( Histoplasmosis), paracoccidioiomycosis, microsporidiosis.

In some embodiments, the pathogen is a protozoan, such as a parasite. In one embodiment, the condition caused by the protozoan is selected from the group consisting of: leishmaniasis, plasmodiosis (ie, malaria), cryptosporidiosis, toxoplasma Toxoplasmosis, trypanosomiasis, and helminth infection, including those caused by trematodes (such as schistosomiasis), cestodes (such as echinococcosis), and nematodes (nemotodes) (eg, diseases caused by trchinosis, ascariasis, filariosis, and strongylodiosis).

In another embodiment, the infection is a hepatitis infection, such as a hepatitis B or hepatitis C infection. Anti-PD-L1 antibody molecules can be combined with conventional treatments for hepatitis B infection or hepatitis C infection for therapeutic advantages. In certain embodiments, the anti-PD-L1 antibody molecule is administered in combination with a hepatitis B antigen (eg, Engerix B) or a vaccine, and optionally in combination with an aluminum-containing adjuvant.

In another embodiment, the infectious disease is influenza. In certain embodiments, the anti-PD-L1 antibody molecule is administered in combination with an influenza antigen or vaccine.

A disease suitable for the prevention or treatment of an antibody against PD-L1 of the present invention or a fragment thereof can be further described in WO2010/077634, WO2016/061142, US60/696426, WO2016/007235 or US61/264061.

In other aspects, the invention provides the use of an anti-PD-Ll antibody or fragment thereof or immunoconjugate thereof for the manufacture or preparation of a medicament for the prevention or treatment of a related disease or condition as mentioned above.

In some embodiments, an antibody or antibody fragment or immunoconjugate of the invention delays the onset of a condition and/or a condition associated with the condition.

Combination therapy

In some embodiments, the prophylactic or therapeutic methods described herein further comprise administering to the subject or individual a combination of an antibody molecule (eg, an anti-PD-L1 antibody or fragment thereof) or a pharmaceutical composition or immunoconjugate disclosed herein. And one or more other therapies, such as therapeutic modalities and/or other therapeutic agents.

In some embodiments, the treatment modality includes surgery (eg, tumor resection); radiation therapy (eg, external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the illumination region is designed), local illumination (eg, pointing to a pre-selected target) Or irradiation of organs) or focused illumination). The focused illumination can be selected from stereotactic radiosurgery, segmented stereotactic radiosurgery, and intensity modulated radiation therapy. The focused illumination may have a radiation source selected from the group consisting of a particle beam (proton), a cobalt-60 (photon), and a linear accelerator (X-ray), for example as described in WO 2012/177624.

Radiation therapy can be administered by one or a combination of several methods including, but not limited to, external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiosurgery, whole body radiation therapy, radiotherapy, and permanent or transient Interstitial brachytherapy. The term "brachytherapy" refers to radiation therapy delivered by spatially constrained radioactive material that is inserted into the body at or near the site of a tumor or other proliferative tissue disease. The term is intended to be, without limitation, including exposure to radioisotopes (eg, At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and Lu's radioisotope). Suitable sources of radiation include solids and liquids. By way of non-limiting example, the source of radiation may be a radionuclide such as I-125, I-131, Yb-169, Ir-192 as a solid state source, I-125 as a solid source or emitting photons, beta particles, gamma radiation Or other radionuclides of other therapeutic rays. The radioactive material may also be a fluid made from any radionuclide solution, for example, an I-125 or I-131 solution, or a suitable fluid containing small particles of a solid radionuclide (such as Au-198, Y-90) may be used. The slurry produces a radioactive fluid. Additionally, the radionuclide can be contained in a gel or radioactive microspheres.

In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, an anti-infective active agent, or an immunomodulatory agent (eg, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule).

Exemplary cytotoxic agents include anti-microtubule drugs, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinblastine alkaloids, intercalating agents, capable of interfering with signal transduction Routes of active agents, pro-apoptotic active agents, proteasome inhibitors, and irradiation (eg, local or systemic exposure (eg, gamma radiation).

或

)、曲妥珠单抗

托西莫单抗

替伊莫单抗

阿来组单抗

依帕珠单抗

贝伐珠单抗

厄洛替尼

西妥昔单抗

等等。 Exemplary other antibodies include, but are not limited to, immunological checkpoint inhibitors (eg, anti-CTLA-4, anti-TIM-3, anti-CEACAM, or anti-LAG-3); antibodies that stimulate immune cells (eg, agonistic GITR antibodies or CD137) Antibody); an anti-cancer antibody (eg, rituximab (

or

),Trastuzumab

Tosimo monoclonal antibody

Imomozumab

Alemizumab

Epalizumab

Bevacizumab

Erlotinib

Cetuximab

and many more.

比卡鲁胺

硫酸博来霉素

白消安

白消安注射剂

卡培他滨

N4-戊氧羰基-5-脱氧-5-氟胞苷、卡铂

卡莫司汀

苯丁酸氮芥

顺铂

克拉立滨

环磷酰胺(

或

)、阿糖胞苷、胞嘧啶阿拉伯糖苷

阿糖胞苷脂质体注射剂

达卡巴嗪

更生霉素(dactinomycin)(放线菌素D、Cosmegan)、盐酸道诺霉素

柠檬酸道诺霉素脂质体注射剂

地塞米松、多西紫杉醇

盐酸多柔比星

依托泊苷

磷酸氟达拉滨

5-氟尿嘧啶

氟他胺

tezacitibine、吉西他滨(双氟脱氧胞苷)、羟基脲

伊达比星

异环磷酰胺

伊立替康

L-天冬酰胺酶

亚叶酸钙、美法仑

6-巯基嘌呤

甲氨蝶呤

米托蒽醌(米托蒽醌)、米罗他(mylotarg)、紫杉醇

phoenix(钇90/MX-DTPA)、喷司他丁、聚苯丙生20联用卡莫司汀植入物

柠檬酸他莫昔芬

替尼泊苷

6-硫鸟嘌呤、塞替派、替拉扎明

注射用盐酸拓扑替康

长春碱

长春新碱

长春瑞滨(长春瑞滨)、依鲁替尼、吉利德(idelalisib)和贝伦妥单抗-维多汀(brentuximab vedotin)。 Exemplary chemotherapeutic agents include, but are not limited to, anastrozole

Bicalutamide

Bleomycin sulfate

Bai Xiaoan

Baixiaoan injection

Capecitabine

N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin

Carmustine

Chlorambucil

Cisplatin

Clarity

Cyclophosphamide

or

), cytarabine, cytosine arabinoside

Cytarabine liposome injection

Dacarbazine

Dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride

Citric acid citrinomycin liposome injection

Dexamethasone, docetaxel

Doxorubicin hydrochloride

Etoposide

Fludarabine phosphate

5-fluorouracil

Flutamide

Tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea

Idabis

Ifosfamide

Irinotecan

L-asparaginase

Calcium leucovorin, melphalan

6-巯基嘌呤

Methotrexate

Mitoxantrone (mitoxantrone), mylotarg, paclitaxel

Phoenix (钇90/MX-DTPA), pentastatin, polyphenylphenanthrene 20 combined with carmustine implant

Tamoxifen citrate

Teniposide

6-thioguanine, thiotepa, tirapazamine

Topotecan hydrochloride for injection

Vinblastine

Vincristine

Vinorelbine (vinorelbine), ibrutinib, idelalisib, and brentuximab vedotin.

Exemplary vaccines include, but are not limited to, cancer vaccines. The vaccine can be a DNA based vaccine, an RNA based vaccine or a virus transduced vaccine. Cancer vaccines can be prophylactic or therapeutic. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, a multiple peptide, a peptide mixture, a hybrid peptide, or a peptide pulsed dendritic cell vaccine (see, eg, Yamada et al, Cancer Sci, 104: 14-21) , 2013).

Exemplary anti-infective active agents include, but are not limited to, antiviral, antifungal, antiprotozoal, antibacterial agents such as the nucleoside analog zidovudine (AST), ganciclovir, foscarnet or cidovir As described above.

Immunomodulators include immunological checkpoint molecular inhibitors and costimulatory molecule activators.

In some embodiments, the inhibitor of the immunological checkpoint molecule is PD-1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM (eg, CEACAM-1, -3, and/or -5) Inhibitors of VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR β. Inhibition of the molecule can be carried out at the DNA, RNA or protein level. In some embodiments, an inhibitory nucleic acid (eg, dsRNA, siRNA, or shRNA) can be used to inhibit expression of an immunological checkpoint molecule. In other embodiments, the inhibitor of the immunological checkpoint molecule is a polypeptide that binds to an immunological checkpoint molecule, eg, a soluble ligand (eg, PD-1-Ig or CTLA-4Ig), or an antibody or antigen-binding fragment thereof; , with PD-1, PD-L2, CEACAM (eg, CEACAM-1, -3, and/or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 An antibody or fragment thereof that binds to and/or TGFR β or a combination thereof. In other embodiments, the immunomodulatory agent is an inhibitor of CEACAM (eg, CEACAM-1, -3, and/or -5) (eg, human CEACAM (eg, CEACAM-1, -3, and/or -5)) . In other embodiments, the immunomodulatory agent is an inhibitor of LAG-3 (eg, human LAG-3). In one embodiment, the inhibitor of LAG-3 is an antibody molecule directed against LAG-3, for example, a human anti-LAG-3 antibody, preferably, the anti-LAG-3 antibody is humanized. In other embodiments, the immunomodulatory agent is an inhibitor of TIM-3 (eg, human TIM-3). In one embodiment, the inhibitor of TIM-3 is an antibody molecule directed against TIM-3.

In some embodiments, the immunomodulatory agent is an activator or agonist of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from an agonist of the following molecule (eg, an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion): OX40, CD2, CD27, CD28, CDS, ICAM-1 , LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 body. In other embodiments, the immunomodulatory agent is a GITR agonist. In one embodiment, the GITR agonist is an antibody molecule directed against GITR. In other embodiments, the immunomodulatory agent is an OX40 agonist. In one embodiment, the OX40 agonist is an antibody molecule directed against OX40.

In further some embodiments, an anti-PD-L1 antibody or fragment thereof of the invention can also be used in combination with a tyrosine kinase inhibitor (eg, a receptor tyrosine kinase (RTK) inhibitor). Exemplary tyrosine kinase inhibitors include, but are not limited to, epidermal growth factor (EGF) pathway inhibitors (eg, epidermal growth factor receptor (EGFR) inhibitors), vascular endothelial growth factor (VEGF) pathway inhibitors (eg, blood vessels) Endothelial growth factor receptor (VEGFR) inhibitors (eg, VEGFR-1 inhibitors, VEGFR-2 inhibitors, VEGFR-3 inhibitors), platelet-derived growth factor (PDGF) pathway inhibitors (eg, platelet-derived growth factor receptors) A body (PDGFR) inhibitor (eg, a PDGFR-β inhibitor), a RAF-1 inhibitor, a KIT inhibitor, and a RET inhibitor.

In some embodiments, an anti-PD-L1 antibody or fragment thereof of the invention can also be used in combination with a PI3K inhibitor, an mTOR inhibitor, a BRAF inhibitor, a MEK inhibitor, and/or a JAK2 inhibitor, and the like.

In some embodiments of any of the methods of the invention, administration of an anti-PD-L1 antibody or fragment thereof of the invention is combined with administration of an antigen. The antigen may for example be a tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen. In some embodiments, the tumor antigen comprises a protein. In some embodiments, the tumor antigen comprises a nucleic acid. In some embodiments, the tumor antigen is a tumor cell.

In some embodiments, an anti-PD-L1 antibody or fragment thereof of the invention can be administered in combination with treatment comprising a T cell (eg, a cytotoxic T cell or CTL) that adopts a adoptive transfer of a chimeric antigen receptor (CAR).

In some embodiments, an anti-PD-L1 antibody of the invention, or a fragment thereof, can be administered in combination with an anti-neoplastic agent.

In some embodiments, an anti-PD-L1 antibody of the invention, or a fragment thereof, can be administered in combination with an oncolytic virus. In some embodiments, the oncolytic virus is capable of selectively replicating in cancer cells and triggers cancer cell death or delays its growth. In some cases, oncolytic viruses have no or minimal effect on non-cancerous cells. Oncolytic viruses include, but are not limited to, oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sindbis virus, oncolytic influenza virus, or oncolytic RNA Virus (eg, oncolytic reovirus, oncolytic Newcastle disease virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)). In some embodiments, the oncolytic virus is a virus described in US 2010/0178684 A1, for example, a recombinant oncolytic virus.

In some embodiments, an anti-PD-L1 antibody of the invention, or a fragment thereof, can be administered in combination with a cytokine.

In some embodiments, an antibody of the invention or a fragment thereof can be combined with cancer therapy conventional in the art, including but not limited to: (i) radiation therapy (eg, radiation therapy, X-ray therapy, irradiation) or Kill cancer cells with ionizing radiation and shrink the tumor. Radiation therapy can be administered by external beam radiation therapy (EBRT) or by internal brachytherapy; (ii) chemotherapy, or the application of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or specific effects An agent that deregulates cancer cell proteins (eg, tyrosine kinase inhibitor imatinib, gefitinib; monoclonal antibody, photodynamic therapy); (iv) immunotherapy, or enhancement Host immune response (eg, vaccine); (v) hormonal therapy, or blocking hormones (eg, when the tumor is hormone sensitive), (vi) angiogenesis inhibitors, or blocking angiogenesis and growth, and (vii Palliative care, or treatments that involve improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding, including pain medications such as morphine and oxycodone, anti-emetics such as Ondans Ondansetron and aprepitant, allowing for more aggressive treatment options.

In some embodiments, an antibody of the invention or a fragment thereof can be combined with conventional methods of enhancing host immune function, including but not limited to: (i) APC enhancement, such as (a) injection of a heterologous MHC into a tumor. DNA of an allogeneic antigen, or (b) a tumor cell transfected with a gene that increases the likelihood of recognition of an immune antigen (eg, immunostimulatory cytokine, GM-CSF, costimulatory molecule B7.1, B7.2), Iii) Adoptive cellular immunotherapy or treatment with activated tumor-specific T cells. Adoptive cellular immunotherapy includes isolating tumor-infiltrating host T lymphocytes, such as by in vitro expansion of the population by IL-2 or tumor or both; in addition, dysfunctional isolated T cells can also be used in vitro by applying the antibodies of the invention To activate, the thus activated T cells can then be re-administered to the host.

The various combination therapies described above can be further combined for treatment.

Further examples of combinations of anti-PD-L1 antibodies with other therapeutic modalities or therapeutic agents can be found in WO2016/061142, WO2010/077634, US60/696426, US61/264061 or WO2016/007235.

Such combination therapies encompasses combined administration (wherein two or more therapeutic agents are included in the same formulation or separate formulations), and administered separately, in which case other therapies, such as treatments and Administration of the antibodies of the invention occurs before, simultaneously with, and/or after the therapeutic agent. Antibody molecules and/or other therapies, such as therapeutic agents or treatment modalities, can be administered during an active disease or during a period of lesser or less active disease. The antibody molecule can be administered prior to other treatments, simultaneously with other treatments, after treatment, or during disease remission.

In one embodiment, administration of the anti-PD-L1 antibody and administration of another therapy (eg, a therapeutic or therapeutic agent) within about one month, or within about one, two or three weeks, or about 1, 2, Occurs within 3, 4, 5, or 6 days.

In some embodiments, the antibody combinations described herein can be administered separately, eg, as separate antibodies, or when ligated (eg, as a bispecific or trispecific antibody molecule).

It will be appreciated that any treatment can be performed by replacing or supplementing the anti-PD-Ll antibody with the immunoconjugate of the invention.

Combination therapy with anti-LAG-3 antibody

In some embodiments, an anti-PD-L1 antibody of the invention can be used in combination with an anti-LAG-3 antibody.

In some embodiments, an anti-LAG-3 antibody of the invention is an anti-human LAG-3 antibody. In some embodiments, an anti-LAG3 antibody of the invention is an antibody in IgGl form or an antibody in IgG2 form or an antibody in IgG4 form. In some embodiments, the anti-LAG-3 antibody is a monoclonal antibody. In some embodiments, the anti-LAG-3 antibody is humanized. In some embodiments, the anti-LAG-3 antibody is a chimeric antibody. In some embodiments, at least a portion of the framework sequence of the anti-LAG-3 antibody is a human consensus framework sequence. In one embodiment, an anti-LAG-3 antibody of the invention further encompasses an antibody fragment thereof, preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv) or ( Fab') ₂ , single domain antibody, double antibody (dAb) or linear antibody.

In some specific embodiments, an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises

(i) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 75, and/or

(ii) three complementarity determining regions LCDR of the light chain variable region set forth in SEQ ID NO:76.

In some embodiments, an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein

(i) said VH comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 69; HCDR2 comprises an amino acid selected from the group consisting of SEQ ID NO: a sequence consisting of or consisting of the amino acid sequence; HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71;

and / or

(ii) wherein said VL comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 72; LCDR2 comprises the amino acid sequence of SEQ ID NO: 73 or Composition of the amino acid sequence; LCDR3 comprises or consists of the amino acid sequence selected from SEQ ID NO:74.

In some embodiments, an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein

(a) Heavy chain variable region VH

(i) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: a sequence consisting of or consisting of; or

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 75;

(iii) comprising an amino acid change having one or more (preferably no more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid sequences selected from SEQ ID NO: 75 (preferably) An amino acid sequence of an amino acid substitution, more preferably a conservative substitution of an amino acid, preferably, the amino acid change does not occur in the CDR region;

and / or

(b) Light chain variable region VL

(i) comprising an amino acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:76 a sequence or consisting of;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 76;

(iii) comprising an amino acid change having one or more (preferably no more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid sequences selected from SEQ ID NO: 76 (preferably) The amino acid sequence of an amino acid substitution, more preferably a conservative substitution of an amino acid, preferably does not occur in the CDR regions.

In some embodiments, an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein

(a) heavy chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from SEQ ID NO:77 Or consisting of a sequence of amino acids;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 77;

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 77 Amino acid sequence which changes (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, said amino acid change does not occur in the CDR region of the heavy chain, more preferably, said amino acid change does not occur in the heavy chain variable region ;

and / or

(b) Light chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence selected from SEQ ID NO:78 Or consisting of a sequence of amino acids;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 78;

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared to an amino acid sequence selected from the group consisting of SEQ ID NO:78 Amino acid sequence which changes (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, said amino acid change does not occur in the CDR region of the light chain, more preferably, said amino acid change does not occur in the light chain variable region .

In some embodiments, the modifications of the invention against an anti-PD-L1 antibody are equally applicable to an anti-LAG-3 antibody.

Route of administration and dosage

The antibodies of the invention (and pharmaceutical compositions or immunoconjugates comprising the same, as well as any additional therapeutic agents) can be administered by any suitable method, including parenteral, intrapulmonary and intranasal, Also, if local treatment is required, it is administered intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on the short-term or long-term nature of the administration, it can be administered by any suitable route, for example by injection, for example intravenous or subcutaneous injection. Various medication schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of a disease, a suitable dose of an antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity of the disease, and the Whether the antibody is administered for prophylactic purposes or for therapeutic purposes, prior treatment, clinical history of the patient and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient in one treatment or through a series of treatments.

In some embodiments, the dosage regimen is adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time or may be proportionally reduced or increased as indicated by the acute condition of the treatment situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity. Dosage unit form as used herein refers to physically discrete units suitable as a single dose for the subject to be treated; each unit contains a predetermined amount of active compound, the predetermined amount being calculated in association with the required pharmaceutical carrier. Produce the desired therapeutic effect. The specifications for the dosage unit form of the present invention are directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the incorporation of such active compounds for use in the field of sensitive treatment in individuals. limit.

The dosage and treatment regimen of the anti-PD-L1 antibody molecule can be determined by the skilled artisan. In certain embodiments, the anti-PD-L1 antibody molecule is injected (eg, subcutaneously or intravenously) at about 1 to 30 mg/kg, for example, about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to A dose of 5 mg/kg, or about 3 mg/kg is administered. The dosage regimen can vary, for example, from once a week to once every 2, 3 or 4 weeks. In one embodiment, the anti-PD-L1 antibody molecule is administered every other week at a dose of about 10 to 20 mg/kg. In one embodiment, the anti-PD-L1 antibody molecule is less than or equal to about 5 mg/kg; less than or equal to about 4 mg/kg; less than or equal to about 3 mg/kg; less than or equal to about 2 mg/kg; less than or equal to about A dose of 1 mg/kg is administered alone or in combination (e.g., in combination with an anti-LAG-3 antibody molecule) every other week. In one embodiment, the anti-PD-L1 antibody molecule is administered at a dose of about 1 to 5 mg/kg every other week, at a dose of about 1 to 4 mg/kg every other week, at a dose of about 1 to 3 mg/kg every other week or A dose of about 1 to 2 mg/kg is administered every other week. In one embodiment, the anti-LAG-3 antibody molecule is administered at a dose of about 1 to 5 mg/kg every other week, at a dose of about 1 to 4 mg/kg every other week, at a dose of about 1 to 3 mg/kg every other week or A dose of about 1 to 2 mg/kg is administered alone or in combination (e.g., in combination with an anti-LAG-3 antibody molecule) every other week.

Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of PD-L1 in a biological sample. The term "detection" as used herein, includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic binding of antibody molecules, ELISA assays. Method, PCR-technology (for example, RT-PCR). In certain embodiments, the biological sample is a blood, serum or other liquid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

In one embodiment, an anti-PD-L1 antibody for use in a diagnostic or detection method is provided. In another aspect, a method of detecting the presence of PD-L1 in a biological sample is provided. In certain embodiments, the methods comprise detecting the presence of a PD-L1 protein in a biological sample. In certain embodiments, PD-L1 is human PD-L1. In certain embodiments, the method comprises contacting a biological sample with an anti-PD-L1 antibody as described herein under conditions that permit binding of the anti-PD-L1 antibody to PD-L1, and detecting the anti-PD-L1 antibody Whether a complex is formed between PD and L1. Formation of the complex indicates the presence of PD-L1. The method can be an in vitro or in vivo method. In one embodiment, an anti-PD-L1 antibody is used to select a subject suitable for treatment with an anti-PD-L1 antibody, for example wherein PD-L1 is a biomarker for selection of the subject.

In one embodiment, a cancer or tumor can be diagnosed using an antibody of the invention, eg, to assess (eg, monitor) the treatment or progression of a disease (eg, a hyperproliferative or cancerous disease) described herein in a subject, its diagnosis, and/or Staging. In certain embodiments, a labeled anti-PD-L1 antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected portions, such as enzymes or ligands, for example, By enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbrella Umbelliferone, Luciferiferase, for example, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydropyridazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lytic enzyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin/avidin , spin labeling, phage labeling, stable free radicals, and more.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with an anti-PD-L1 antibody. In some embodiments, the sample is obtained prior to treatment with a cancer drug. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (eg, a core biopsy), a surgical specimen (eg, from a surgically resected specimen), or a fine needle aspirate.

In some embodiments, PD-L1 is detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after a treatment interval.

In some embodiments, a method of treating a tumor or infection is provided, the method comprising: examining a subject (eg, a sample) (eg, a sample of a subject comprising cancer cells) for detecting the presence of PD-L1, The PD-L1 value is thus determined, the PD-L1 value is compared to a control value, and if the PD-L1 value is greater than the control value, the subject is administered a therapeutically effective amount of an anti-antibody, optionally in combination with one or more other therapies. A PD-L1 antibody (eg, an anti-PD-L1 antibody described herein), thus treating a tumor or infection.

Sequence of an exemplary anti-PD-L1 antibody of the invention

These and other aspects and embodiments of the present invention are described in the drawings (hereinafter briefly described below) and the following detailed description of the invention and are illustrated in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the invention. The invention is further illustrated by the following examples, which are to be understood by way of illustration and not limitation

Example

Example 1. Preparation of hybridoma cells

Hybridoma technology is achieved by fusing two cells while maintaining the main features of both. These two cells are antigen-immunized mouse spleen cells and mouse myeloma cells, respectively. The main feature of mouse spleen cells (B lymphocytes) immunized by specific antigens is its antibody secretion function, but it cannot be continuously cultured in vitro, and mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, The so-called immortality. Under the action of the selective medium, only the hybrid cells in which the B cells are fused with the myeloma cells can have the ability to continue to culture, and form a cell clone having both the secretory function of the antibody and the immortality of the cells. In this experiment, mice were immunized with hPD-L1 protein, and then spleen cells and myeloma cells of mice were fused to obtain hybridoma cells capable of expressing positive antibodies.

Hybridoma fusion

Laboratory animals and immune information

Prepare the electrofusion dish: Thoroughly soak the electrofusion dish with 70% ethanol and blow dry in a clean bench.

Isolation of spleen cells: The mice were sacrificed by cervical dislocation, and the body surface was sterilized with 75% alcohol for 5 min, and then placed on the anatomical plate of the mouse in the ultra-clean table, and the left side was placed, and the limbs were fixed with a 7-gauge needle. The spleen was removed by aseptic opening of the abdominal cavity, washed with a basal medium (configuration method as follows), and the connective tissue attached thereto was carefully removed. The spleen was then transferred to another dish containing the basal medium. Press the spleen with a elbow needle, insert a small needle into the spleen, and squeeze with the scorpion to fully release the spleen cells to make a spleen cell suspension. The cell suspension was filtered through a 100 μM cell sieve, washed once with 30 ml of basal medium, and centrifuged at 1200 rpm for 6 min.

Lysis of red blood cells: The supernatant was removed and the cells were resuspended in 10 ml RBC lysis buffer (GIBCO). Then add 20 ml of RBC lysis buffer. The suspension was allowed to stand for 5 min and centrifuged at 1100 rpm for 6 min. After removing the supernatant, the cells were resuspended in 10 ml of basal medium, and then 30 ml of basal medium was added, and centrifuged at 1100 rpm for 6 min. After removing the supernatant, the cells were resuspended in 20 ml of basal medium and counted.

Electrofusion: Mouse myeloma cells SP2/0 cells (ATCC) were resuspended in 20 ml of basal medium and counted. SP2/0 and spleen cells were mixed at a ratio of 1:2 to 1:1 and centrifuged at 100 rpm for 6 min. After removing the supernatant, the mixed cells were resuspended in 10 ml of fusion buffer (BTXpress). Further, 15 ml of the fusion buffer was added, and the mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. After repeating the above steps, the cells were re-selected with an appropriate amount of fusion buffer, and the mixed cell density was adjusted to 1 × 10 ⁷ cells/ml. The parameters of the electrofusion amplifier are set as follows. 2 ml of the cell suspension was added to each electrofusion dish for electrofusion.

Condition Mouse(SP2/0-ECF-F) Alignment: 60v, 30sec Membrane breaking: 1500V, 30μs, 3X Post-fusion pulse: 60V, 3sec

Plate after electrofusion: The cells were allowed to stand in an electric fusion dish for 5 min at room temperature. The cells were transferred to a centrifuge tube, and the cells were diluted to 1 to 2 x 104 cells/ml using a screening medium (configuration method as follows). 100 μl of the cell suspension was added to each well of a 96-well plate. The screening medium was changed on the 7th day after the fusion. Screening was performed 10 days after culture (or longer, depending on the state of cell growth). Hybridoma cells expressing a specific anti-PD-L1 antibody were screened by FACS (FACS ARIA (BD Biosciences)).

Positive hybridoma cell subcloning

Subcloning step: A 96-well plate was prepared, and 200 μl of the basal medium as described above was added to each of the 2nd to 8th columns. The cells of the positive wells selected by the above fusion were made into cell suspensions and added to the first column. 100 μl of the cell suspension of column 1 was added to the second column, and after thorough mixing, 100 μl was added to the next column. The above procedure was repeated until the last column volume became 300 μl; the 96-well plate was allowed to stand for 15 min, and the count was observed under a microscope. The corresponding volume of 100 cells was added to 20 ml of the basal medium as described above, and the plates were mixed and mixed at 200 μl per well. After one week, the microscope was observed under the microscope, and the monoclonal wells were judged and labeled, and the positive wells were picked out.

Cryopreservation of cells: Observe the state of the cells, and wait for the cells to grow well. When the viability is >90%, centrifuge at 1000 rpm for 5 min to remove the supernatant. Resuspend the cells to 1×107 cells/ml with cryopreservation solution (45.5% FBS, 44.5% RPMI-1640, 10% DMSO), dispense into a cryotube, place in a programmed cooling box, and freeze at -80 °C. .

Example 2. Production and purification of chimeric antibodies

The present invention utilizes molecular biology techniques to obtain antibody sequences in anti-PD-L1 positive hybridoma cells, and uses the same to construct human and mouse chimeric antibodies.

Hybridoma sequencing

RNA extraction: fresh cells, centrifuged at 300 g for 5 min, the supernatant was removed, 500 μl of LY buffer (Biomiga) was added to the pellet (20 μl of β-mercaptoethanol was added per 1 ml before use), and mixed until clear. It was added to a DNA removal tube, centrifuged at 13,000 rpm for 2 min, and the flow-through was collected. 100% ethanol was added to the flow through solution at a ratio of 1/2, and mixed 5 times to clarify. Add the clarified solution to the RNA collection tube, centrifuge at 13000 rpm for 1 min to remove the liquid, add 500 μl RB (Recovery Buffer, Recovery Buffer) (Takara), centrifuge at 13000 rpm for 30 s, and add 500 μl RNA Wash Buffer (Biomiga). Ethanol), centrifugation for 30 s, repeat the above process, centrifuge to completely evaporate the ethanol, then add 30 μl of DEPC water to the collection column, centrifuge at 12000 g for 2 min, and collect the eluate. The RNA concentration was determined.

Reverse transcription to obtain cDNA:

The reaction system I is configured as follows:

*From PrimeScript II 1 ^st Strand cDNA Synthesis Kit, purchased from Takara.

After incubation at 65 ° C for 5 min, it was quickly cooled on ice. The following reverse transcription system was added to Reaction System I in a total amount of 20 μl:

*From PrimeScript II 1 ^st Strand cDNA Synthesis Kit, purchased from Takara.

After slowly mixing, reverse transcription translation was carried out under the following conditions: 42 ° C for 60 min → 95 ° C for 5 min, and then cooled on ice to obtain cDNA.

Link the cDNA to the T vector:

The heavy and light chain variable regions were amplified by PCR, and the PCR reaction system was as follows:

4.5 μl of the PCR product obtained by the above PCR reaction was added, 0.5 μl of pMD20-T vector (Clontech), 5 μl of Ligation Mighty Mix (Takara), gently mixed, and reacted at 37 ° C for 2 hours to obtain a ligation product.

Transformed cells:

The TOP10 competent state (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was taken out at -80 °C, and thawed on ice. 5 μl of the ligation product obtained above was added to the melted TOP10 competent state, and the mixture was incubated on ice for 30 min. After heat shock at 42 °C for 90 s, it was rapidly cooled on ice for 2 min, and 900 μl of LB medium (Biotech (Shanghai) Co., Ltd.) was added to the EP tube, and cultured at 37 ° C for 1 h at 220 rpm shaker. After centrifugation at 3000 g for 2 min, 800 μl of the supernatant was aspirated, and the bacteria were suspended and coated on the ampicillin-resistant plate with the remaining medium. Incubate overnight at 37 ° C and pick and clone.

Construction of chimeric antibodies

The murine anti-PD-L1 antibody VH and VL regions produced by the hybridoma of Example 1 which had been sequenced were PCR-amplified: the upstream and downstream primer sequences are shown in Table 5 and Table 6.

Table 5. Heavy chain variable region (VH) primers for mouse anti-PD-L1 antibody (Primer Mix 1)

After mixing in the above ratio, Primer Mix 1 was obtained for subsequent PCR amplification of VH.

Table 6. Light chain variable region (VL) primers (Primer Mix 2) of mouse anti-PD-L1 antibody:

After mixing in the above ratio, Primer Mix 2 was obtained for subsequent PCR amplification of VL.

The PCR system is as follows:

* Primer Mix 1 was applied for VH chain amplification; Primer Mix 2 was applied for VL chain amplification.

The PCR amplification product was recovered by gel cutting.

Homologous recombination reaction:

The homology recombination system is as follows:

The reaction was carried out at 37 ° C for 30 min to obtain a recombinant product. The recombinant product was transformed into TOP10 competent state, and monoclonal sequencing was picked. The clone containing the plasmid with the correct insertion direction was selected as a positive clone, and the positive clone was preserved.

Expression and purification of chimeric antibodies

A plasmid containing an anti-PD-L1 antibody was extracted from the positive clone obtained above.

293F cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1.5 × 10 ⁶ cells/ml one day before transfection. The cell density on the day of transfection was approximately 3 x 10 ⁶ cells/ml. A final volume of 1/10 of F17 medium (Gibco, A13835-01) was used as a transfection buffer, and an appropriate plasmid was added and mixed. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI is 1:3 in 293F cells), mix and incubate for 10 min at room temperature to obtain a DNA/PEI mixture. After resuspending the cells with the DNA/PEI mixture, 36.5 ° C, 8% CO ₂ . After 24 h, a transfection volume of 2% FEED (Sigma) was added and cultured at 36.5 ° C, 120 rpm, 8% CO ₂ . When the culture was continued until day 6 or when the cell viability was ≤ 60%, the cell supernatant was collected for purification.

The gravity column used for purification was treated with 0.5 M NaOH overnight, and the glass bottle and the like were washed with distilled water and then dry-baked at 180 ° C for 4 hours to obtain a purification column. The collected medium was centrifuged at 4500 rpm for 30 min before purification, and the cells were discarded. The supernatant was then filtered using a 0.22 μl filter. Each tube was filled with 1 ml of Protein A and equilibrated with 10 ml of binding buffer (sodium phosphate 20 mM. NaCl 150 mM, pH 7.0). The filtered supernatant was added to the purification column and re-equilibrated with 15 ml of binding buffer. 5 ml of elution buffer (citric acid + sodium citrate 0.1 M, pH 3.5) was added, and the eluate was collected, and 80 μl of Tris-HCl was added per 1 ml of the eluate. The collected antibodies were concentrated by ultrafiltration into PBS (Gibco, 70011-044), and the concentration was measured.

The CDRs, the light chain variable region and the heavy chain variable region, the amino acid sequences of the light chain and the heavy chain of the four chimeric antibodies obtained by the present invention, and the sequence numbers are shown in Tables 1-3 above.

The control antibody used in the present invention is Roche's PD-L1 antibody Atezolizumab (hereinafter referred to as ATE or Ate, trade name Tecentriq), and its CDR, light chain variable region and heavy chain variable region, and the amino acid sequences of the light chain and the heavy chain are also See Table 1-3 above.

Example 2 Bioluminescence Interferometry for the Binding Kinetics of the Chimeric Antibody of the Invention to Antigen

The equilibrium dissociation constant (KD) of the antibody of the present invention in combination with human PD-L1 was determined by bioluminescence interferometry (ForteBio). The ForteBio affinity assay was performed according to the existing method (Estep, P et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8).

Half an hour before the start of the experiment, according to the number of samples, take the appropriate amount of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensor soaked in SD buffer (PBS 1 ×, BSA 0.1%, Tween-20 0.05%).

100 μl of SD buffer, antibody, antigen (including human PD-L1, mouse PD-L1, and cynomolgus PD-L1, purchased from Acrobiosystems) were added to 96-well black polystyrene half-well microplate (Greiner). , 675076). Select the sensor location based on the sample position layout. The instrument setting parameters are as follows: running steps: Baseline, Loading ~ 1 nm, Baseline, Association and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the rotation speed is 400 rpm, and the temperature is 30 °C. KD values were analyzed using ForteBio analysis software.

In the experiments described in the above assays, the affinities of antibodies 3-266.1, 4-79.2, 4-26.6, 4-48.5 are shown in Table 7:

Table 7. Affinity constants (equilibrium dissociation constants) for ForteBio detection of antigen-antibody binding

In the above experiments, the KD values of the chimeric antibodies 3-266.1, 4-79.2, 4-26.6, and 4-48.5 were 9.80E-10M, 7.56E-09M, 3.85E-09M, and 1.23E-09M, respectively. compared group, in this study the antibody has a K _D value of a similar or better.

Example 3 Binding antibody and binding assay of cells overexpressing PD-L1

In this study, the binding of a gradient-diluted chimeric antibody of the present invention to a CHO-stabilized cell strain overexpressing human PD-L1 was detected by flow cytometry.

The CHO-S cells were transfected into the CHO-S cells by the pCHO1.0 vector (Invitrogen) carrying the human PD-L1 cDNA (Sino Biological) cloned into the multiple cloning site (MCS) (Invitrogen, ExpiCHO ^TM Expression System Kit, Cat. No. A29133 ), CHO cells (CHO-PDL1) overexpressing human PD-L1 were produced.

CHO-PDL1 cells were counted and diluted to 1 × 10 ⁶ cells/ml, and 100 μl/well was added to a U-bottom 96-well plate. 400 g for 5 min, centrifuge, and remove the cell culture medium. Samples (chimeric antibodies 3-266.1, 4-79.2, 4-26.6, 4-48.5, and positive control antibody Ate, respectively) (antibody dilution method: maximum antibody concentration 500 nM, triple dilution in 0.1% cattle) Serum albumin (BSA) in PBS, a total of 8 concentrations were tested.) U-plates were added and the cells were resuspended, 100 μl/well, and allowed to stand on ice for 30 min. The supernatant was removed by 400 g for 5 min, and the cells were washed 1 time with PBS. 400 g of PBS was removed for 5 min, 100 μl of anti-mouse Fab FITC-labeled secondary antibody (Jackson Immuno Research) (1:500 dilution in PBS) was added to each well, and 100 μl of anti-human Fab FITC-labeled was added to the cells to which the positive control antibody was added. Secondary antibody (Jackson Immuno Research). Incubate on ice for 30 min in the dark. The supernatant was removed by 400 g for 5 min, and the cells were washed 1 time with PBS. The cells were resuspended in 100 μl of 1×PBS and detected by FACS.

In the experiments described in the above assay, the binding of antibodies 3-266.1, 4-79.2, 4-26.6, 4-48.5 and CHO-PDL1 cells is shown in Figure 1.

In the above experiments, antibodies 3-266.1, 4-79.2, 4-26.6, and 4-48.5 all bind to human PD-L1 overexpressed on CHO cells with EC50 of 2.139 nM, 2.598 nM, 1.985 nM, and 1.995 nM, respectively. Compared with the antibody ATE, the binding ability is better, and the binding ability of some antibodies is more than twice that of the control antibody.

Example 4 Humanization of chimeric antibodies

The chimeric antibody obtained in Example 1 was humanized. The antibody humanization process was performed using the Macromtek SmrtMol Humanize proprietary software program. First enter the sequence into the software, the system will generate a three-dimensional model of the sequence, and then humanized through the following steps:

1 determining the structure of the CDR ring;

2 Find the closest homologous sequence in each V/J region of the heavy and light chains in the human germline sequence database;

3 screening the human germline that best matches the heavy chain light chain and the lowest amount of back mutation;

4 constructing the CDR regions of the chimeric antibody onto the human framework region;

5 using sequence and structural features to determine the position of the amino acid in the framework region that serves to maintain CDR function;

6 performing a back mutation at the sequence position determined to be important (returning to the input amino acid type);

7 generating a three-dimensional model of the humanized sequence;

8 Manually check the sequence and structure to identify risk sites that may cause misfolding or reduce stability;

9 Optimize the amino acids at the risk site.

The CDRs, light chain variable region and heavy chain variable region of the four humanized antibodies (HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-IgG4PAK and HZ4485-IgG1N297A) obtained by the present invention, the amino acid sequences of the light and heavy chains, please See Tables 1-3 as described above.

Example 5 Determination of Binding Kinetics of Humanized Antibody to Antigen by ForteBio

The equilibrium dissociation constant (KD) of humanized antibodies of different Fc isoforms of the invention in combination with human PD-L1 was determined using the ForteBio assay. The ForteBio affinity determination method was the same as in Example 2. In the experiments described in the above assays, the affinities of the antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-IgG4PAAK, HZ4485-IgG1N297A are shown in Table 8:

Table 8. Affinity constants for antigen-antibody binding by ForteBio

In the above test, described herein, humanized antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-G4PAAK, K D values were HZ4485-IgG1N297A 7.24E-10M, 9.35E-10M, 1.32E-09M, 3.17 E-09M, compared with the control group, the present study of humanized antibodies having a K _D value of a similar or better.

Example 6 Binding experiment of humanized antibody and cells overexpressing PD-L1

In this study, the binding of a gradient-diluted humanized antibody of the present invention to a CHO-stabilized cell line overexpressing human PD-L1 (CHO-PDL1) was detected by flow cytometry. The test method was the same as in Example 3 except that the antibodies used were humanized antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-G4PAAK, HZ4485-IgG1N297A, and the antibody dilution method was as follows: the highest antibody concentration was 500 nM, and the three-fold dilution was 0.1. A total of 8 concentrations of humanized antibody HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-G4PAAK, HZ4485-IgG1N297A and CHO-PDL1 cells were tested in PBS of % bovine serum albumin (BSA) as shown in Figure 2. Show.

In the above experiments, humanized antibodies HZ3266-IgG1, HZ3266-IgG1N297A, HZ3266-G4PAAK, HZ4485-IgG1N297A bind to human PD-L1 overexpressing CHO cells with EC50 of 1.813 nM, 1.784 nM, 1.862 nM, 1.561 nM, respectively. Compared with the control antibody ATE, it has stronger binding ability.

Example 7 Detection of Biological Activity of Antibodies by MOA Method

Anti-PD-1/PD-L1 antibodies can block the inhibition of downstream NFAT signaling pathway by blocking the binding of PD-1 and PD-L1. In this study, the MOA detection system (PD-1/PD-L1 Blockade Bioassay, Cell Propagation Model, Catalog J1252) provided by Promega was used to detect the activation of NFAT signal by detecting the expression of the fluorescent reporter gene according to the method provided in the specification. Thus, the inhibitory effect of the antibody on PD-1/PD-L1 binding was examined.

CHOK1-PDL1 cells (from the above MOA detection system) were placed one day before the activity test: the cells were passaged 1-2 days before the plating of CHOK1-PDL1. The culture supernatant was discarded, and the cells were washed once with PBS (Gibco). Add appropriate amount of Trypsin (Gibco) and digest at 37 ° C, 5% CO _{2 for} 3-5 min. Add 4 times Trypsin volume of medium, transfer the cells to a 50 ml centrifuge tube and count. Take the required volume of cells, 230g, and centrifuge for 10min. 1640 medium (Gibco) was added and the cells were resuspended to 4 x 105 cells/mL. The cells were added to a 96-well white cell culture plate (Nunclon) at 100 μl/well. The side holes were added with PBS, 200 μl/well. The cells were cultured overnight in a 37 ° C / 5% CO 2 incubator.

Jurkat-PD1 cells (from the MOA detection system described above) were treated: cell passage was performed two days prior to activity assay. After counting, take the required volume of cells, 170 g, and centrifuge for 5 min. The cells were resuspended to 1.25 x 10 ⁶ cells/ml with assay buffer (1640 medium (Gibco) + 1% FBS).

Samples and Jurkat-PD1 cells were added to the assay plate (from the MOA detection system described above): 95 μl/well of CHOK1-PDL1 cell supernatant was discarded. Add 40 μl of sample (humanized antibody HZ3266-IgG1, HZ3266-IgG1N297A, HZ4485-IgG1N297A prepared according to the invention) and positive control (Ate), negative control (IgG1) (antibody dilution method: maximum antibody concentration 100 nM, triple Dilute in assay buffer for a total of 8 concentrations). 40 μl of Jurkat-PD1 cells were added. Incubate for 6 hours in a 37 ° C / 5% CO ₂ incubator.

Detection: Bio-GloTM buffer (from the MOA detection system described above) was thawed in advance, Bio-GloTM substrate (from the above MOA detection system) was added and mixed. After 6 hours, Bio-GloTM reagent (from the MOA detection system described above) was added at 80 μl/well. Leave at room temperature for 5 to 10 minutes. reading.

In the above experiments, the experimental results are shown in Figure 3. The antibodies HZ3266-IgG1, HZ3266-IgG1N297A, and HZ4485-IgG1N297A all effectively blocked the interaction of PD1/PD-L1.

Example 8 Mixed lymphocyte experiment

In this study, the antibody was incubated with mature DC cells derived from different donors and CD4+ T cells in vitro, and the relative expression of IL2 and IFN-γ in the system was detected to reflect the activation of T cells by different antibodies. .

PBMC separation: Take 50 ml of fresh blood from donors, add 2.5 times PBS, gently add to FiColl (Thermo), divide into 4 tubes, 12.5 ml per tube, 400 g, centrifuge for 30 min, and stop at 0 deceleration. Pipette the middle white strip into PBS and wash twice with PBS.

DC cell isolation: PBMC cells isolated as described above were added with 5 ml T cell culture medium (the preparation method is as follows), cultured at 37 ° C, 6% CO ₂ , adherent culture for 2 h, and the suspension cell suspension was aspirated for CD4+ cell separation, leaving the cells Add 3 ml of DC medium (preparation method is shown in the following table), add 3 ml of DC medium after 2 days of culture, and then culture for 5 days, then add rTNFa (R&D Systems) (1000 U/ml), IL-1b (R&D Systems) (5 ng/ Ml), IL-6 (R&D Systems) (10 ng/ml) and 1 μM PGE2 (Tocris) were cultured for 2 days as DC cells for lymphocyte mixed reaction (MLR).

CD4+ T cell isolation: The kit was operated according to the 'Untouched CD4+ T cell isolation' kit instructions (11346D, Invitrogen) instructions. PBMC was cultured for 2 h, and the suspended cell liquid was aspirated into a 15 ml centrifuge tube, centrifuged at 200 g for 10 min, and 500 μl of the separation solution, 100 μl of AB-type serum, 100 μl of purified antibody were resuspended in the pellet, incubated at 4 ° C for 20 min, and washed once with the separation solution. After incubating for 15 min by adding 500 μl Bead Buffer (Invitrogen), Bead was removed in a magnetic field, and the T cell medium was washed once, resuspended in 8 ml of medium, and cultured at 37 ° C, 6% CO ₂ .

MLR experiment: The mature DC cells obtained above were mixed with CD4+ cells at a volume of 200 μl per well, 10000 DC cells, and 100000 CD4+ cells, and the antibody of the present invention was added (concentration: 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16). nM, 0.032 nM), DC cells prepared above (represented as DC in the table below), CD4+ T cells (represented as CD4 in the table below), DC cells mixed with CD4+ cells (represented as Cell in the table below) and IgG1 disclosed in Table 3 was used as a negative control, DC+CD4+ T cells + anti-CD3/CD28 magnetic beads (QIAGEN) (shown as Beads in the table below) as a positive control, mixed culture for 5 days, using cisbio kit (Human) IL2 Kit 1000 Test, Human IFN gamma 1000test) The IL2 and IFN-γ concentrations were measured (relative expression levels in DeltaF%).

The experimental results are shown in Tables 9, 10, 11 and 12 and Figures 4 and 5. The data unit in the table is DeltaF%.

Table 9. Relative expression levels of IL2 in donor 1

* Negative control without any antibodies or beads added.

Table 10. Relative expression levels of IL2 in donor 2

* Negative control without any antibodies or beads added.

Table 11. Relative expression levels of IFN-γ in donor 1

* Negative control without any antibodies or beads added.

Table 12. Relative expression levels of IFN-γ in donor 2

* Negative control without any antibodies or beads added.

Therefore, the antibodies of the present invention can effectively activate T cells in vitro, and the partial activation effect is superior to the positive control antibody.

Example 9 Zenix column detection antibody drug-forming

This experiment examined the drug-forming properties of antibodies by recording the residence time of an exemplary humanized antibody of the invention in a Zenix column (Sepax Technologies, Inc.). The shorter the residence time, the lower the viscosity of the reacted antibody and the better the drug-forming property.

Chromatographic conditions: detection wavelength: 214 nm, column temperature: 25 ° C, flow rate: 0.35 ml / min, injection amount: 10 μl.

Sample preparation: Take 100 μl of sample (antibody to be detected HZ3266-IgG1, HZ3266-IgG4PAAK, HZ3266-IgG1N297A, HZ4485-IgG1N297A; ATE as positive control, antibody concentration: 1 mg/ml), centrifuge at 13000 rpm for 5 min, and take 80 μl of supernatant in liquid phase. In the inner cannula, it is placed in the liquid sample tray for injection detection.

The experimental results are shown in Table 13. The different subtypes of HZ3266 and the retention time of HZ4485 on the column were shorter than the control antibody, indicating that the HZ3266 and HZ4485 were better in drug formation.

Table 13. Zenix column detection antibody drug-forming properties

Example 10. Antitumor efficacy test

In this experiment, the anti-tumor effect of the PD-L1 antibody of the present invention was measured in hPD-L1 transgenic mice using MC38 cells (MC38-hPDL1) expressing human PD-L1 (Nanjing Yinhe Co., Ltd.).

Human PD-L1 transgenic mice:

Female C57B1/6 background human PDL-1 transgenic mice (about 8 weeks old) were purchased from Shanghai Southern Experimental Animal Technology Co., Ltd. The mice were domesticated for 7 days after arrival and the study was started.

cell:

MC38 cells (MC38-hPDL1) expressing human PD-L1 were purchased from Nanjing Yinhe Biomedical Co., Ltd. and routinely subcultured for subsequent in vivo experiments in strict accordance with the instructions. The cells were collected by centrifugation, resuspended in sterile PBS and adjusted to a cell density of 5 x 106 cells/ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdomen region of human PD-L1 transgenic mice to establish a MC38-hPDL1 tumor-bearing mouse model.

Dosing:

Six days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volume ranging from 87.4 mm ³ to 228.4 mm ³ were selected and grouped according to tumor volume (8 mice per group, one group administered with IgG1, one) The group administered the antibody HZ3266-IgG1N297A). The administration was carried out on days 6, 10, 14, 17, 21, 24, 28, 31 and 35, respectively, in which the frequency of administration was 2 times/week, and the dosage and manner of administration were as shown in Table 12. Tumor volume and body weight changes were monitored during the administration period, and the monitoring frequency was 2 times/week for 5 weeks. Body weight and tumor volume were determined before each dose. Tumor volume determination: The maximum long axis (L) and the largest broad axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated as follows: V = L x W ² /2. Body weight was measured using an electronic balance twice a week.

Table 14. Experimental Design Table

The antibody of the present invention showed a remarkable antitumor effect after one week of administration (Fig. 6), and by the 35th day, one mouse of the antibody group of the present invention completely ablatedly disappeared. Body weight results showed that different antibody dose groups had no effect on the weight of tumor-bearing mice.

Therefore, the antibody of the present invention has a significant inhibitory effect on tumors.

Example 11. Combination of an anti-PD-L1 antibody of the present invention and an anti-human LAG-3 antibody

This study investigated the antitumor activity of the anti-PD-L1 antibody (HZ3266-IgG1N297A) of the present invention in combination with an anti-human LAG-3 antibody (ADI-31853) using a humanized mouse model.

In this study, A375 (ATCC) human skin cancer cells were used to measure the anti-tumor effect of anti-PD-L1 antibodies on NCG mice. Human PBMC (AllCells) (2×10 ⁶ cells/mouse) was pre-injected intravenously, and then A375 tumor-bearing mouse model was established by subcutaneous vaccination, grouped into tumors, treated with different antibodies, and monitored during drug administration. The tumor volume and body weight of each group were changed, the frequency of administration was 2 times/week, and the administration was 2 weeks, and a total of 5 doses were administered. The monitoring frequency was 2 times/week, and the monitoring was continued for 4 weeks. The dosage and mode of administration were as follows. The relative tumor inhibition rate (TGI%) was calculated after the end of administration.

Anti-human LAG-3 antibody ADI-31853

The cDNA encoding the light chain amino acid sequence and the heavy chain amino acid sequence (Table 3) of the anti-LAG-3 antibody ADI-31853 was separately cloned into the expression vector pTT5 according to a conventional method in the art.

The above expression vector containing the antibody gene of interest and the transfection reagent PEI (Polysciences) were transiently transfected into cultured human kidney blast cell 293 cells (Invitrogen) according to the protocol provided by the manufacturer, after transfection, the medium was discarded and fresh The cells were diluted to 4 × 10 ⁶ /ml in EXPI293 medium (Gibco). The cells were cultured for 7 days at 37 ° C, 5% CO ₂ , and fresh medium was added every 48 hours. After 7 days, centrifuge at 1300 rpm for 20 min. The supernatant was taken and the supernatant was purified with Protein A to give the antibody a purity of >95%.

The equilibrium dissociation constant (KD) of ADI31853 bound to human LAG-3 (hLAG-3) was determined using a Biote Interferometry (ForteBio) assay. The ForteBio affinity assay was performed according to the existing method (Estep, P et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8). Briefly, the sensor was equilibrated in assay buffer for 30 minutes, then on-line for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto an AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was then exposed to 100 nM human LAG-3 antigen (Arco Biosystems) for 5 minutes, after which the sensor was transferred to assay buffer for 5 minutes for dissociation rate measurement. Analysis of the kinetics was performed using a 1:1 binding model.

In the experiments performed as described in the above assay, the affinity of ADI-31853 is as follows:

Mouse:

NOG mice, female, 7-8 weeks (the age of mice at the time of tumor cell inoculation), weighing 17.6-24.2 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The mice were domesticated for 7 days after arrival and the study was started.

cell:

Human skin cancer cells A375 (ATCC #CRL-1619) were purchased from ATCC and routinely subcultured for subsequent in vivo experiments in strict accordance with ATCC requirements. The cells were collected by centrifugation, resuspended in sterile PBS, and adjusted to a cell density of 30 × 10 ⁶ /ml. After NOG mice have been intravenously injected with human PBMC (AllCells), the right back is shaved and subcutaneously injected with A375 cells 0.2 ml/mouse. Tumor cells were examined for tumor volume 7 days after inoculation, and mice with an average tumor volume in the range of 70-71 mm ³ were randomly grouped according to tumor volume.

Dosing:

Each group was injected subcutaneously with the following doses of antibody:

(1) human IgG (equitech-Bio), 20 mg/kg;

(2) LAG-3 (ADI-31853), 10 mg/kg;

(3) PD-L1 (HZ3266-IgG1N297A), 10 mg/kg;

(4) LAG-3 (ADI-31853), 10 mg/kg + PD-L1 (HZ3266-IgG1 N297A), 10 mg/kg.

On the 7th day after inoculation, mice with an average tumor volume meeting the above requirements were randomly divided into groups of 8 animals each. Each group of mice was dosed as above on days 7, 10, 14 and 17 with the above four groups of reagents, respectively.

Analysis: Tumors and body weight were measured twice weekly throughout the study, and mice were euthanized when the tumor reached the endpoint or when the mice had >20% weight loss. The maximum long axis (L) and the largest broad axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated as follows: V = L × W ² /2. Tumor size from each group of mice was plotted against time. Analysis of variance (ANOVA) was used to determine statistical significance. A P value of <0.05 was considered to be statistically significant in all analyses.

The experimental results are shown in Figure 7. It can be seen that the anti-LAG-3 monoclonal antibody ADI-31853 (31853) and the anti-PD-L1 monoclonal antibody (HZ3266-IgG1N297A) (HZ3266) are used in combination with human IgG (equitech-Bio) (hIgG). ) and the two antibodies can significantly inhibit tumor growth compared to the use of these two antibodies.

Claims (28)

An antibody that binds to PD-L1 or an antigen-binding fragment thereof, the antibody comprising (i) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 26 or 30, and three complementarity determining regions of the light chain variable region set forth in SEQ ID NO: 32 or 36 LCDR, or (ii) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 27, and three complementarity determining regions LCDR of the light chain variable region set forth in SEQ ID NO: 33, or (iii) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 28, and three complementarity determining regions LCDR of the light chain variable region set forth in SEQ ID NO: 34, or (iv) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 29 or 31, and three complementarity determining regions of the light chain variable region set forth in SEQ ID NO: 35 or 37 LCDR. An antibody or antigen-binding fragment thereof that binds to PD-L1, the antibody comprising three complementarity determining region HCDRs of the heavy chain variable region, and three complementarity determining regions LCDR of the light chain variable region, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, 2, 3 or 4, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, 6, 7, 8 or 9, and HCDR3 comprises SEQ ID NO: 10, 11, The amino acid sequence shown in 12 or 13, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, 15 or 16, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, 18, 19 or 20, and LCDR3 comprises SEQ ID NO: amino acid sequence shown in 21, 22, 23, 24 or 25. An antibody or antigen-binding fragment thereof that binds to PD-L1, the antibody comprising a heavy chain variable region and/or a light chain variable region, wherein The heavy chain variable region comprises: (i) three complementarity determining regions (HCDRs) contained in the VH of any of the antibodies listed in Table B; (ii) a combination of HCDR1, HCDR2 and HCDR3 shown in Table A; and / or The light chain variable region comprises: (i) three complementarity determining regions (LCDR) contained in the VL of any of the antibodies listed in Table B; (ii) A combination of LCDR1, LCDR2 and LCDR3 shown in Table A. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody comprises a light chain variable region and/or a heavy chain variable region, wherein (i) the heavy chain variable region comprises or consists of an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 26 or 30; and/or The light chain variable region comprises or consists of an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NO: 32 or 36; (ii) the heavy chain variable region comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:27; and/or The light chain variable region comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:33; (iii) the heavy chain variable region comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 28; and/or The light chain variable region comprises or consists of an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:34; (iv) the heavy chain variable region comprises or consists of an amino acid sequence at least 90% identical to the amino acid sequence selected from SEQ ID NO: 29 or 31; and/or The light chain variable region comprises or consists of an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NO: 35 or 37. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody comprises a light chain variable region and/or a heavy chain variable region, wherein (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26 or 30; and/or The light chain variable region comprises the amino acid sequence of SEQ ID NO: 32 or 36; (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 27; and/or The light chain variable region comprises the amino acid sequence of SEQ ID NO:33; (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28; and/or The light chain variable region comprises the amino acid sequence of SEQ ID NO:34; (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29 or 31; and/or The light chain variable region comprises the amino acid sequence of SEQ ID NO: 35 or 37. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which antibody comprises (a) heavy chain (i) comprising or consisting of an amino acid sequence having at least 85% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 38, 42, 43 or 44; (ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 38, 42, 43 or 44; and / or (b) Light chain (i) comprising or consisting of an amino acid sequence having at least 85% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 46 or 50; (ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 46 or 50; (a) heavy chain (i) comprising or consisting of an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 39; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:39; and / or (b) Light chain (i) comprising or consisting of an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 47; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 47; or (a) heavy chain (i) comprising or consisting of an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 40; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:40; and / or (b) Light chain (i) comprising or consisting of an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 48; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 48; or (a) heavy chain (i) comprising or consisting of an amino acid sequence having at least 85% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 41 or 45; (ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 41 or 45; and / or (b) Light chain (i) comprising or consisting of an amino acid sequence having at least 85% identity to an amino acid sequence selected from SEQ ID NO: 49 or 51; (ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 49 or 51. The PD-L1 binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which has one or more of the following characteristics: (1) high affinity binding to PD-L1 (e.g. human PD-L1), for example, in the following equilibrium dissociation constant (K _D) binding to PD-L1, the K _D of less than about 2nM; (2) an antibody or fragment thereof of the invention binds to a cell expressing human PD-L1, for example, with an EC50 of less than or equal to about 2 nM; (3) antibody or fragment thereof of the present invention block the PD-L1-related activity, for example, less than or equal to about 0.7nM EC _50; (4) an antibody or fragment thereof of the invention enhances T cell function, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq; (5) an antibody or fragment thereof of the invention increases T cell proliferation, for example in MLR, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq; (6) an antibody or fragment thereof of the present invention increases IFN-γ secretion, for example, in MLR, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq; (7) an antibody or fragment thereof of the invention increases IL-2 secretion, for example in an MLR, for example, over a known anti-PD-L1 antibody, such as Tecentriq; (8) In the Zenix column assay, the residence time (RT) is less than about 10 minutes; (9) inhibiting one or more activities of PD-L1, for example, resulting in one or more of: an increase in tumor infiltrating lymphocytes, an increase in T cell receptor-mediated proliferation, or a decrease in immune evasion of cancer cells; (10) being capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC); (11) showing the same or similar binding affinity and/or specificity to PD-L1 as any of the antibodies listed in Table 3; (12) inhibiting (eg, competitive inhibition) the binding of any of the antibodies listed in Table 3 to PD-L1; (13) an epitope that binds to the same or overlaps with any of the antibodies shown in Table 3; (14) competing with any of the antibodies shown in Table 3 to bind PD-L1; (15) having one or more of the biological properties of any of the antibody molecules listed in Table 3. The anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1 or IgG4, optionally the anti-PD-L1 antibody or Its antigen-binding fragment comprises a kappa light chain constant region, such as a human kappa light chain constant region. The anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody. The anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody such as scFv, ( Fab') 2 fragment, single domain antibody, diabody (dAb) or linear antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody is a bispecific or multispecific antibody, preferably the bispecific antibody molecule binds to PD-L1 and LAG-3 . An isolated nucleic acid encoding the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12. A vector comprising the nucleic acid of claim 13, preferably the vector is an expression vector, such as a pTT5 vector. A host cell comprising the nucleic acid of claim 13 or the vector of claim 14, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from E. coli cells, yeast cells, mammalian cells or suitable for preparation Other cells of the antibody or antigen-binding fragment thereof, most preferably, the host cell is a 293 cell or a CHO cell. A method of producing an anti-PD-L1 antibody or antigen-binding fragment thereof, the method comprising cultivating a right under conditions suitable for expressing a nucleic acid encoding the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 The host cell of claim 15, optionally isolating the antibody or antigen-binding fragment thereof, optionally the method further comprising recovering the anti-PD-L1 antibody or antigen-binding fragment thereof from the host cell. An immunoconjugate comprising the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and other substances, such as a cytotoxic agent. A pharmaceutical composition comprising the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or the immunoconjugate of claim 17, and optionally a pharmaceutically acceptable excipient. A pharmaceutical composition comprising the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or the immunoconjugate of claim 17, and other therapeutic agents, and optionally a pharmaceutically acceptable adjuvant; The other therapeutic agent is selected from the group consisting of a chemotherapeutic agent, other antibodies (eg, an anti-LAG-3 antibody, eg, a human anti-LAG-3 antibody, preferably, the anti-LAG-3 antibody is humanized), a cytotoxic agent , vaccines, anti-infective agents or immunomodulators (eg activators of costimulatory molecules or inhibitors of immunological checkpoint molecules). The pharmaceutical composition according to claim 19, wherein said anti-LAG-3 antibody comprises (i) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 75, and/or (ii) three complementarity determining regions LCDR of the light chain variable region set forth in SEQ ID NO:76. The pharmaceutical composition according to claim 19, wherein the anti-LAG-3 antibody comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein (i) said VH comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 69; HCDR2 comprises an amino acid selected from the group consisting of SEQ ID NO: a sequence consisting of or consisting of the amino acid sequence; HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71; and / or (ii) wherein said VL comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 72; LCDR2 comprises the amino acid sequence of SEQ ID NO: 73 or Composition of the amino acid sequence; LCDR3 comprises or consists of the amino acid sequence selected from SEQ ID NO:74. An effective amount of the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the immunoconjugate of claim 17, in the preparation for preventing or treating a subject or an individual in a subject The use of drugs for tumors or infectious diseases. An effective amount of the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the immunoconjugate of claim 17, or the pharmaceutical composition of claim 18 or 19, and an anti-LAG-3 antibody ( For example, in combination with a human anti-LAG-3 antibody, preferably an anti-LAG-3 antibody is humanized, for use in the preparation of a medicament for preventing or treating a subject or individual in a subject A tumor or infectious disease. The use according to claim 22 or 23, wherein the tumor is a cancer, such as a gastrointestinal tumor, such as a gastrointestinal cancer, such as colon cancer; or the infectious disease is a chronic infection. The use according to any one of claims 22 to 24, wherein the medicament is further capable of administering one or more other therapies in combination, the therapy comprising, for example, a treatment mode and/or other therapeutic agent, preferably the treatment Means include surgical treatment and/or radiation therapy, or the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, a vaccine, an anti-infective active agent, other antibodies (eg, an anti-LAG-3 antibody, eg, a human anti-LAG-3 antibody) Preferably, the anti-LAG-3 antibody is a humanized or immunomodulatory agent (eg, an activator of a costimulatory molecule or an inhibitor of an immunological checkpoint molecule). The use according to any one of claims 23 to 25, wherein the anti-LAG-3 antibody comprises (i) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 75, and/or (ii) three complementarity determining regions LCDR of the light chain variable region set forth in SEQ ID NO:76. The use according to any one of claims 23 to 25, wherein the anti-LAG-3 antibody comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein (i) said VH comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 69; HCDR2 comprises an amino acid selected from the group consisting of SEQ ID NO: a sequence consisting of or consisting of the amino acid sequence; HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 71; and / or (ii) wherein said VL comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 72; LCDR2 comprises the amino acid sequence of SEQ ID NO: 73 or Composition of the amino acid sequence; LCDR3 comprises or consists of the amino acid sequence selected from SEQ ID NO:74. A method of detecting PD-L1 in a sample, the method comprising (a) contacting the sample with any of the anti-PD-L1 antibodies or antigen-binding fragments thereof of any one of claims 1 to 12; (b) detecting the formation of a complex between the anti-PD-L1 antibody or antigen-binding fragment thereof and PD-L1; optionally, the anti-PD-L1 antibody is detectably labeled.

Images