Anti-pd-l1 antibodies and uses thereof

Abstract

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 present invention relates to nucleic acids encoding the antibodies or antibody fragments thereof and host cells comprising the same, and related uses. Furthermore, the present invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments. In particular, the present invention relates to the combination therapy of these antibodies and antibody fragments with other therapies.

Description

[0001] This application is a divisional application of Chinese patent application CN201811567281.3, filed on December 19, 2018, entitled "Anti-PD-L1 Antibody and Its Use Thereof".

[0002] This invention relates to novel antibodies and antibody fragments that specifically bind to PD-L1, as well as compositions containing said antibodies or antibody fragments. Furthermore, this invention relates to nucleic acids encoding said antibodies or antibody fragments thereof, host cells containing them, and related uses. Additionally, this invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments. In particular, this invention relates to the combination therapy of these antibodies and antibody fragments with other therapies, such as modes of treatment or therapeutic agents. Background Technology

[0003] Programmed death-ligand 1 (PD-L1) is a protein involved in suppressing the immune system response during chronic infection, pregnancy, tissue allogeneic transplantation, 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 its interaction with another receptor, B7.1 (also known as B7-1 or CD80). The formation of PD-L1 / PD-1 and PD-L1 / B7.1 complexes negatively regulates T cell receptor signaling, leading to subsequent downregulation of T cell activation and suppression of antitumor 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. PD-L1 overexpression in tumor cells can promote tumor invasion and is often associated with poor prognosis.

[0004] Therefore, there is still a need in the field for new anti-PD-L1 antibodies that bind better to PD-L1 and have better druggability. Invention Overview

[0005] This document discloses antibody molecules that bind to PD-L1. It also provides nucleic acids encoding said antibodies or antibody fragments thereof, expression vectors for generating antibody molecules, host cells, and methods. Immunoconjugates comprising anti-PD-L1 antibody molecules, multispecific or bispecific antibody molecules, and pharmaceutical compositions are also provided. The anti-PD-L1 antibody molecules disclosed herein can be used alone or in combination with other therapies, such as therapeutic agents or modalities, for the treatment, prevention, and / or diagnosis of oncological and infectious diseases. Furthermore, this document discloses compositions and methods for detecting PD-L1, and methods for using anti-PD-L1 antibody molecules to prevent or treat various diseases, including oncological and / or infectious diseases.

[0006] Therefore, in some embodiments, the antibody or fragment thereof of the present invention (specifically) binds to PD-L1. In some embodiments, the antibody or fragment thereof of the present invention (specifically) binds to human PD-L1.

[0007] In some embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention binds to PD-L1 (e.g., human PD-L1) with high affinity, for example, with the following equilibrium dissociation constant (K). D Combined with PD-L1, the K D Less than about 50 nM, preferably less than or equal to about 20 nM, more preferably less than or equal to about 15 nM, even 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 Less than or equal to approximately 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, or 0.8 nM. In some embodiments, the anti-PD-L1 antibody of the present invention is in the form of 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 nM, or 0.7-1 nM K. D Binding to PD-L1. In some embodiments, PD-L1 is human PD-L1. In some embodiments, antibody binding affinity is determined using a bio-optical interferometry assay (e.g., Fortebio affinity measurement).

[0008] In some embodiments, the antibody or fragment thereof of the present invention binds to cells expressing human PD-L1, for example, at an EC50 of 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, 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 (e.g., FACS). In some embodiments, the cells expressing human PD-L1 are CHO cells expressing human PD-L1.

[0009] In some embodiments, the antibody or fragment thereof of the present invention blocks PD-L1-related activity, for example, at EC50 concentrations 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, EC is present at concentrations of approximately 0.1-1 nM, 0.5-1 nM, 0.6-1 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, or 1 nM. 50In some embodiments, the PD-L1-related activity is the binding of PD-L1 to PD-1. In some embodiments, the antibody or fragment thereof of the present invention is expressed in an MOA assay at EC50 values ​​less than or equal to approximately 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, or 0.7 nM. 50 Preferably, EC is present at concentrations of approximately 0.1-1 nM, 0.5-1 nM, 0.6-1 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, or 1 nM. 50 Inhibits the binding of PD-L1 to PD-1. In some implementations, the cells are CHO cells.

[0010] In some embodiments, the antibodies or fragments thereof of the present invention enhance T cell function. In some embodiments, the antibodies or fragments thereof of the present invention enhance T cell proliferation. In some embodiments, the antibodies or fragments thereof of the present invention enhance IFN-γ secretion. In some embodiments, the antibodies or fragments thereof of the present invention enhance IL-2 secretion. In some embodiments, the antibodies or fragments thereof of the present invention enhance both IFN-γ and IL-2 secretion. In some embodiments, the enhancement is measured in a mixed lymphocyte reaction (MLR). In some embodiments, the antibodies or fragments thereof of the present invention have a superior ability to activate T cells compared to known anti-PD-L1 antibodies, such as Tecentriq.

[0011] In some embodiments, the antibodies or fragments of the present invention have lower viscosity than known anti-PD-L1 antibodies (e.g., Tecentriq), and therefore better druggability. In some embodiments, the antibodies or fragments of the present invention have a residence time (RT) of less than about 10 minutes, about 9 minutes, or about 8 minutes in Zenix column assays, preferably between about 7 minutes and 9 minutes, preferably between about 7-8.5 minutes, about 7.5-8.5 minutes, about 7-8 minutes, or between about 7.5-8 minutes, for example, 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, or 8.5 minutes.

[0012] In some embodiments, 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 the following: an increase in tumor-infiltrating lymphocytes, an increase in T-cell receptor-mediated proliferation, or a reduction in immune evasion of cancer cells.

[0013] In some embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention can induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0014] In some embodiments, the anti-PD-L1 antibody of the present invention, alone or in combination with other therapies (e.g., treatment modalities and / or therapeutic agents), is effective in treating tumors (e.g., cancer) or infections (e.g., chronic infections). In some embodiments, the tumor is a tumor immune evader. In some embodiments, the tumor is cancer. In some embodiments, the tumor is a gastrointestinal tumor. In some embodiments, the cancer is colon cancer.

[0015] In some embodiments, the heavy and / or light chains of the anti-PD-L1 antibody or fragment thereof of the present invention further comprise a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68).

[0016] In some embodiments, the antibodies of the present invention also cover variants of the amino acid sequence of anti-PD-L1 antibodies, as well as antibodies that bind to the same epitopes as any of the anti-PD-L1 antibodies or fragments thereof described above.

[0017] In some embodiments, the anti-PD-L1 antibody of the present invention further comprises a human or mouse constant region. In some embodiments, the anti-PD-L1 antibody of the present invention is an antibody in the form of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, or IgE. In some embodiments, the anti-PD-L1 antibody of the present invention comprises a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, and IgG4, more specifically, a 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 the heavy chain constant region of human IgG1 or human IgG4. In some embodiments, the mouse constant region comprised by the anti-PD-L1 antibody of the present invention is selected from IgG1, IgG2A, IgG2B, and IgG3.

[0018] In another embodiment, the anti-PD-L1 antibody molecule of the present invention has, for example, a light chain constant region selected from the κ or λ light chain constant region, preferably a κ (e.g., human κ) light chain constant region.

[0019] In yet another embodiment, the anti-PD-L1 antibody molecule comprises the heavy chain constant region of IgG4 (e.g., human IgG4). In one embodiment, human IgG4 contains a substitution at position 228 according to EU number (e.g., Ser to Pro substitution). In yet another embodiment, human IgG4 is mutated to AA at positions 114-115 (EU number) (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 the heavy chain constant region of IgG1 (e.g., human IgG1). In one embodiment, human IgG1 contains a substitution at position 297 according to EU number (e.g., Asn to Ala substitution). In one embodiment, human IgG1 includes a substitution at position 265 according to EU number, a substitution at position 329 according to EU number, or both (e.g., an Asp to Ala substitution at position 265 according to EU number and / or a Pro to A1a substitution at position 329 according to EU number). In one embodiment, human IgG1 includes a substitution at position 234 according to EU number, a substitution at position 235 according to EU number, or both (e.g., a Leu to A1a substitution at position 234 according to EU number and / or a Leu to Ala substitution at position 235 according to EU number). In one embodiment, the heavy chain constant region comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 64, 65, or 66, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity with, said sequence.

[0020] In yet another embodiment, the anti-PD-L1 antibody molecule comprises a κ light chain constant region, for example, a human κ light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with it, or is composed of said sequence.

[0021] In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain constant region of IgG1 (e.g., the heavy chain constant region of human IgG1) and a κ light chain constant region (e.g., the human κ light chain constant region). In one embodiment, human IgG1 includes a substitution at position 297 according to EU number (e.g., an Asn to Ala substitution). In some embodiments, the human IgG1 heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 64 or 65, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with it, or is composed of said sequence. In one embodiment, the human κ light chain constant region comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with it, or is composed of said sequence.

[0022] In another embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain constant region of IgG4 (e.g., the human IgG4 heavy chain constant region) and a κ light chain constant region (e.g., the human κ light chain constant region). In one embodiment, the constant region is mutated IgG4, for example, mutated human IgG4 (e.g., having a mutation at position 228 according to EU number (e.g., the S228P mutation) and / or having a mutation to AA at positions 114-115 (EU number)). In some embodiments, the human IgG4 heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 66, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with, or is composed of said sequence. In one embodiment, the human κ light chain constant region comprises the amino acid sequence of SEQ ID NO: 67, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with it, or is composed of said sequence.

[0023] In one implementation, the anti-PD-L1 antibody molecule is isolated or recombinant.

[0024] In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody or an antibody with monospecificity. The anti-PD-L1 antibody molecule may 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 anti-PD-L1 antibody's framework sequence is a human common framework sequence. In one embodiment, the anti-PD-L1 antibody of the present invention also encompasses its antibody fragment, preferably selected from antibody fragments such as Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv) or (Fab')2, single-domain antibodies, bispecific antibodies (dAbs), or linear antibodies.

[0025] In some 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 against PD-L1 and a second binding specificity against LAG-3. In one embodiment, the bispecific antibody molecule binds to both PD-L1 and LAG-3. The multispecific antibody molecule can have any combination of binding specificities against PD-L1 and other targets.

[0026] In one aspect, the present invention provides a nucleic acid encoding any of the above-described anti-PD-L1 antibodies or fragments thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells), or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic, such as *E. coli* cells.

[0027] In one embodiment, the present invention provides a method for preparing an anti-PD-L1 antibody or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing host cells under conditions suitable for expressing a nucleic acid encoding the antibody or a fragment thereof (preferably an antigen-binding fragment), and optionally isolating the antibody or a fragment thereof (preferably an antigen-binding fragment). In one embodiment, the method further comprises recovering the anti-PD-L1 antibody or a fragment thereof (preferably an antigen-binding fragment) from the host cells.

[0028] In some embodiments, the present invention provides immune conjugates comprising any anti-PD-L1 antibody provided herein and other substances, such as cytotoxic agents or markers. In some embodiments, the immune conjugates are used for the prevention or treatment of tumors (e.g., cancer) or infectious diseases. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is a gastrointestinal tumor (e.g., cancer), such as colon cancer. Preferably, the infectious disease is a chronic infection.

[0029] In some embodiments, the present invention provides compositions comprising any anti-PD-L1 antibody described herein or a fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof, preferably pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example, a pharmaceutical composition, comprises an anti-PD-L1 antibody of the present invention or a fragment thereof or an immunoconjugate thereof, and a combination of one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules)).

[0030] In some embodiments, the pharmaceutical composition is used to prevent or treat tumors (e.g., cancer) or infections. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is a gastrointestinal tumor (e.g., cancer), such as colon cancer. Preferably, the infectious disease is a chronic infection. In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer) or infectious disease in a subject or individual, the method comprising administering to the subject an effective amount of any anti-PD-L1 antibody or fragment thereof described herein, a pharmaceutical composition, or an immunoconjugate. In some embodiments, the tumor is a tumor immune escape. In one embodiment, the tumor is a gastrointestinal tumor (e.g., cancer), such as colon cancer. In one embodiment, the infectious disease is a chronic infection. In another aspect, the invention also relates to the use of any anti-PD-L1 antibody or fragment thereof described herein in the preparation of a medicament for treating tumors (e.g., cancer) or infections in a subject. In some embodiments, the tumor is a tumor immune escape. In one embodiment, the tumor is a gastrointestinal tumor (e.g., cancer), such as colon cancer. In one embodiment, the infectious disease is a chronic infection.

[0031] In further embodiments, the preventive or therapeutic methods described herein also include administration of one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to the subject or individual. In some embodiments, treatment modalities include surgical treatment and / or radiation therapy. In some embodiments, other therapeutic agents are selected from chemotherapeutic agents, cytotoxic agents, vaccines, anti-infective agents, other antibodies, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules).

[0032] In some implementations, the subject or individual is a non-human animal, such as a mammal, preferably a human.

[0033] In one aspect, the present invention relates to a method for detecting PD-L1 in a sample, the method comprising (a) contacting the sample with any anti-PD-L1 antibody or fragment thereof described herein; and (b) detecting the formation of a complex between the anti-PD-L1 antibody or fragment thereof and PD-L1. In one embodiment, the anti-PD-L1 antibody is detectably labeled.

[0034] In some embodiments, the present invention relates to a kit or product comprising any anti-PD-L1 antibody or fragment thereof described herein. In some embodiments, the kit or product comprises an anti-PD-L1 antibody or fragment thereof described herein with optional pharmaceutical excipients. In some embodiments, the kit or product further comprises instructions for administration of the drug to treat a tumor or infection.

[0035] This invention also covers any combination of any embodiments described herein. Any embodiment described herein, or any combination thereof, is applicable to any and all anti-PD-L1 antibodies, fragments thereof, methods, and uses of the invention described herein.

[0036] In one aspect, the present invention relates to the following specific embodiments:

[0037] 1. An antibody or antigen-binding fragment thereof that binds to PD-L1, said antibody comprising

[0038] (i) the three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 26 or 30, and the three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 32 or 36, or

[0039] (ii) the three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 27, and the three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 33, or

[0040] (iii) The three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 28, and the three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 34, or

[0041] (iv) The three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 29 or 31, and the three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 35 or 37.

[0042] 2. An antibody or antigen-binding fragment of PD-L1, wherein the antibody comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein...

[0043] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 5, 6, 7, 8 or 9; HCDR3 contains the amino acid sequence shown in SEQ ID NO: 10, 11, 12 or 13; LCDR1 contains the amino acid sequence shown in SEQ ID NO: 14, 15 or 16; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 17, 18, 19 or 20; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25.

[0044] 3. An antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody comprises a heavy chain variable region and / or a light chain variable region, wherein

[0045] The heavy chain variable region includes:

[0046] (i) The three complementarity-determining regions (HCDRs) contained in the VH of any antibody listed in Table B; or

[0047] (ii) The combinations of HCDR1, HCDR2 and HCDR3 shown in Table A;

[0048] and / or

[0049] The light chain variable region includes:

[0050] (i) The three complementarity-determining regions (LCDRs) contained in the VL of any antibody listed in Table B; or

[0051] (ii) The combination of LCDR1, LCDR2 and LCDR3 shown in Table A.

[0052] 4. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, wherein the antibody comprises a light chain variable region and / or a heavy chain variable region, wherein,

[0053] (i) The heavy chain variable region contains or is composed of an amino acid sequence having at least 90% identity with an amino acid sequence selected from SEQ ID NO: 26 or 30; and / or

[0054] The light chain variable region contains or is composed of an amino acid sequence that has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 32 or 36;

[0055] (ii) The heavy chain variable region contains or is composed of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 27; and / or

[0056] The light chain variable region contains or is composed of an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 33;

[0057] (iii) The heavy chain variable region contains or is composed of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 28; and / or

[0058] The light chain variable region contains or is composed of an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 34;

[0059] (iv) The heavy chain variable region contains or is composed of an amino acid sequence having at least 90% identity with an amino acid sequence selected from SEQ ID NO: 29 or 31; and / or

[0060] The light chain variable region contains or is composed of an amino acid sequence that has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 35 or 37.

[0061] 5. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, wherein the antibody comprises a light chain variable region and / or a heavy chain variable region, wherein,

[0062] (i) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26 or 30; and / or

[0063] The light chain variable region contains the amino acid sequence of SEQ ID NO: 32 or 36;

[0064] (ii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27; and / or

[0065] The light chain variable region contains the amino acid sequence of SEQ ID NO: 33;

[0066] (iii) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 28; and / or

[0067] The light chain variable region contains the amino acid sequence of SEQ ID NO: 34;

[0068] (iv) The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 29 or 31; and / or

[0069] The light chain variable region contains the amino acid sequence of SEQ ID NO: 35 or 37.

[0070] 6. An antibody or antigen-binding fragment thereof from any one of embodiments 1 to 5, wherein the antibody comprises

[0071] (a) Heavy chain

[0072] (i) Contains or consists of an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 38, 42, 43 or 44; or

[0073] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 38, 42, 43 or 44;

[0074] and / or

[0075] (b) Light chain

[0076] (i) Contains or consists of an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 46 or 50; or

[0077] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 46 or 50; or

[0078] (a) Heavy chain

[0079] (i) Contains or is composed of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 39; or

[0080] (ii) Contains or consists of the amino acid sequence of SEQ ID NO: 39;

[0081] and / or

[0082] (b) Light chain

[0083] (i) Contains or consists of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 47; or

[0084] (ii) Contains or consists of the amino acid sequence of SEQ ID NO: 47; or

[0085] (a) Heavy chain

[0086] (i) Contains or is composed of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 40; or

[0087] (ii) Contains or consists of the amino acid sequence of SEQ ID NO: 40;

[0088] and / or

[0089] (b) Light chain

[0090] (i) Contains or is composed of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 48; or

[0091] (ii) Contains or consists of the amino acid sequence of SEQ ID NO: 48; or

[0092] (a) Heavy chain

[0093] (i) Contains or is composed of an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 41 or 45; or

[0094] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 41 or 45;

[0095] and / or

[0096] (b) Light chain

[0097] (i) Contains or is composed of an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 49 or 51; or

[0098] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 49 or 51.

[0099] 7. The antibody or antigen-binding fragment thereof that binds to PD-L1 according to any one of embodiments 1 to 6 has one or more of the following characteristics:

[0100] (1) Binding PD-L1 with high affinity (e.g., human PD-L1), for example, with the following equilibrium dissociation constant (K D Combined with PD-L1, the K D Less than approximately 2 nM;

[0101] (2) The antibody or fragment thereof of the present invention binds to cells expressing human PD-L1, for example, at an EC50 of less than or equal to about 2 nM;

[0102] (3) The antibody or fragment thereof of the present invention blocks PD-L1-related activity, for example, at an EC50 concentration of less than or equal to about 0.7 nM. 50 ;

[0103] (4) The antibodies or fragments thereof of the present invention enhance T cell function, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq;

[0104] (5) The antibodies or fragments thereof of the present invention enhance T cell proliferation, for example in MLR, and are superior to known anti-PD-L1 antibodies, such as Tecentriq;

[0105] (6) The antibodies or fragments thereof of the present invention enhance IFN-γ secretion, for example in MLR, and are superior to known anti-PD-L1 antibodies, such as Tecentriq;

[0106] (7) The antibodies or fragments thereof of the present invention enhance IL-2 secretion, for example in MLR, and are superior to known anti-PD-L1 antibodies, such as Tecentriq;

[0107] (8) In the Zenix column assay, the residence time (RT) is less than approximately 10 minutes;

[0108] (9) Inhibit one or more activities of PD-L1, for example, resulting in one or more of the following: an increase in tumor-infiltrating lymphocytes, an increase in T-cell receptor-mediated proliferation, or a decrease in immune evasion of cancer cells;

[0109] (10) It can induce antibody-dependent cell-mediated cytotoxicity (ADCC);

[0110] (11) Shows the same or similar binding affinity and / or specificity to PD-L1 as any of the antibodies listed in Table 3;

[0111] (12) Inhibit (e.g., competitively inhibit) the binding of any of the antibodies listed in Table 3 to PD-L1;

[0112] (13) Epitopes that bind to the same or overlapping antibodies as any of the antibodies shown in Table 3;

[0113] (14) Compete with any of the antibodies shown in Table 3 to bind to PD-L1;

[0114] (15) Possesses one or more biological properties of any of the antibody molecules listed in Table 3.

[0115] 8. An anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1 or IgG4, and optionally the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a κ light chain constant region, such as a human κ light chain constant region.

[0116] 9. An anti-PD-L1 antibody or its antigen-binding fragment thereof from any one of embodiments 1 to 8, wherein the antibody is a monoclonal antibody.

[0117] 10. An anti-PD-L1 antibody or its antigen-binding fragment thereof from any one of embodiments 1 to 9, wherein the antibody is a humanized antibody, a human antibody, or a chimeric antibody.

[0118] 11. An antibody or an antigen-binding fragment thereof from any one of embodiments 1 to 10, wherein the antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody such as scFv, (Fab')2 fragment, single-domain antibody, bispecific antibody (dAb), or linear antibody.

[0119] 12. An antibody or antigen-binding fragment thereof of any one of embodiments 1 to 11, wherein the antibody is a bispecific or multispecific antibody, preferably, the bispecific antibody molecule binds to PD-L1 and LAG-3.

[0120] 13. The isolated nucleic acid, which encodes an anti-PD-L1 antibody or its antigen-binding fragment in any one of embodiments 1 to 12.

[0121] 14. A vector containing the nucleic acid of embodiment 13, preferably an expression vector, such as the pTT5 vector.

[0122] 15. A host cell comprising the nucleic acid of embodiment 13 or the vector of embodiment 14, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or their antigen-binding fragments, and most preferably, the host cell is a 293 cell or a CHO cell.

[0123] 16. A method for preparing an anti-PD-L1 antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell of embodiment 15 under conditions suitable for expressing a nucleic acid encoding an anti-PD-L1 antibody or an antigen-binding fragment thereof of any one of embodiments 1 to 12, optionally isolating the antibody or the antigen-binding fragment thereof, and optionally further comprising recovering the anti-PD-L1 antibody or the antigen-binding fragment thereof from the host cell.

[0124] 17. An immunoconjugate comprising an anti-PD-L1 antibody or its antigen-binding fragment thereof from any one of embodiments 1 to 12 and other substances, such as a cytotoxic agent.

[0125] 18. A pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof from any one of embodiments 1 to 12 or an immunoconjugate of embodiment 17, and optionally a pharmaceutical excipient.

[0126] 19. A pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof from any one of embodiments 1 to 12 or an immunoconjugate of embodiment 17, and other therapeutic agents, and optionally pharmaceutical excipients; preferably, the other therapeutic agents are selected from chemotherapeutic agents, other antibodies (e.g., anti-LAG-3 antibodies, such as human anti-LAG-3 antibodies, preferably, the anti-LAG-3 antibody is humanized), cytotoxic agents, vaccines, anti-infective agents, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules).

[0127] 20. The pharmaceutical composition according to embodiment 19, wherein the anti-LAG-3 antibody comprises

[0128] (i) the three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 75, and / or

[0129] (ii) The three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 76.

[0130] 21. The pharmaceutical composition of embodiment 19, wherein the anti-LAG-3 antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein

[0131] (i) The VH includes complementarity-determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 69; HCDR2 contains or is composed of the amino acid sequence selected from SEQ ID NO: 70; and HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 71.

[0132] and / or

[0133] (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 72; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 73; and LCDR3 comprises or is composed of the amino acid sequence selected from SEQ ID NO: 74.

[0134] 22. Use of an effective amount of the anti-PD-L1 antibody or its antigen-binding fragment from any one of embodiments 1 to 12, or the immunoconjugate of embodiment 17, in the preparation of a medicament for the prevention or treatment of a subject or individual with a tumor or infectious disease.

[0135] 23. Use in the preparation of a medicament in an effective amount of any one of embodiments 1 to 12 of the anti-PD-L1 antibody or its antigen-binding fragment, or the immunoconjugate of embodiment 17, or the pharmaceutical composition of embodiment 18 or 19, and an anti-LAG-3 antibody (e.g., a human anti-LAG-3 antibody, preferably, the anti-LAG-3 antibody is humanized), wherein the medicament is intended for the prevention or treatment of a tumor or infectious disease in a subject or individual.

[0136] 24. The use of embodiments 22 or 23, wherein the tumor is cancer, such as a gastrointestinal tumor, such as gastrointestinal cancer, such as colon cancer; or the infectious disease is a chronic infection.

[0137] 25. Use according to any one of embodiments 22 to 24, wherein the drug can also be administered in combination with one or more other therapies, said therapies including, for example, treatment modalities and / or other therapeutic agents, preferably, said treatment modalities including surgical treatment and / or radiotherapy, or said therapeutic agents selected from chemotherapeutic agents, cytotoxic agents, vaccines, anti-infective agents, other antibodies (e.g., anti-LAG-3 antibodies, such as human anti-LAG-3 antibodies, preferably, anti-LAG-3 antibodies are humanized) or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules).

[0138] 26. Use according to any one of embodiments 23 to 25, wherein the anti-LAG-3 antibody comprises

[0139] (i) the three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 75, and / or

[0140] (ii) The three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 76.

[0141] 27. Use according to any one of embodiments 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

[0142] (i) The VH includes complementarity-determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 69; HCDR2 contains or is composed of the amino acid sequence selected from SEQ ID NO: 70; and HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 71.

[0143] and / or

[0144] (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 72; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 73; and LCDR3 comprises or is composed of the amino acid sequence selected from SEQ ID NO: 74.

[0145] 28. A method for detecting PD-L1 in a sample, the method comprising:

[0146] (a) Contacting the sample with any anti-PD-L1 antibody or its antigen-binding fragment as described in any of embodiments 1 to 12; and

[0147] (b) Detection of the formation of a complex between an anti-PD-L1 antibody or its antigen-binding fragment and PD-L1; optionally, the anti-PD-L1 antibody is detectably labeled.

[0148] More specifically, the present invention relates to the following embodiments:

[0149] 1. An antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein

[0150] HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 3, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 8, HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 13, LCDR1 consists of the amino acid sequence shown in SEQ ID NO: 16, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 24.

[0151] 2. The antibody or antigen-binding fragment of PD-L1 as described in Embodiment 1, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises or is composed of an amino acid sequence having at least 90% identity with an amino acid sequence selected from or represented by SEQ ID NO: 29 or 31.

[0152] 3. The antibody or antigen-binding fragment of PD-L1 as described in Embodiment 1, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from or composed of the sequence shown in SEQ ID NO: 29 or 31.

[0153] 4. An antibody or antigen-binding fragment thereof that binds to PD-L1 as described in any one of embodiments 1-3, wherein the antibody comprises a light chain variable region, wherein the light chain variable region comprises or is composed of an amino acid sequence having at least 90% identity with an amino acid sequence selected from or represented by SEQ ID NO: 35 or 37.

[0154] 5. An antibody or antigen-binding fragment thereof that binds to PD-L1 as described in any one of embodiments 1-3, wherein the antibody comprises a light chain variable region, wherein the light chain variable region comprises or consists of an amino acid sequence selected from or composed of the sequence shown in SEQ ID NO: 35 or 37.

[0155] 6. The antibody or its antigen-binding fragment as described in Implementation Scheme 1, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein

[0156] (i) the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 29, and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 35; or

[0157] (ii) The heavy chain variable region contains or is composed of the amino acid sequence of SEQ ID NO: 31, and the light chain variable region contains or is composed of the amino acid sequence of SEQ ID NO: 37.

[0158] 7. The antibody or antigen-binding fragment of embodiment 1, wherein the antibody comprises a heavy chain containing or consisting of an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 41 or 45.

[0159] 8. The antibody or antigen-binding fragment of embodiment 1, wherein the antibody comprises a heavy chain comprising or consisting of an amino acid sequence selected from or composed of SEQ ID NO: 41 or 45.

[0160] 9. An antibody or antigen-binding fragment thereof according to embodiment 1, 7 or 8, said antibody comprising a light chain comprising or consisting of an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 49 or 51.

[0161] 10. An antibody or antigen-binding fragment thereof according to embodiment 1, 7 or 8, wherein the antibody comprises a light chain comprising or consisting of an amino acid sequence selected from or composed of SEQ ID NO: 49 or 51.

[0162] 11. The antibody or its antigen-binding fragment as described in Embodiment 1, wherein the antibody comprises a heavy chain and a light chain, wherein

[0163] (i) The heavy chain contains or is composed of the amino acid sequence of SEQ ID NO: 41; and the light chain contains or is composed of the amino acid sequence of SEQ ID NO: 49; or

[0164] (ii) The heavy chain contains or is composed of the amino acid sequence of SEQ ID NO: 45; and the light chain contains or is composed of the amino acid sequence of SEQ ID NO: 51.

[0165] 12. The antibody or antigen-binding fragment thereof that binds to PD-L1 as described in Embodiment 1 or 6 has one or more of the following characteristics:

[0166] (1) Binding to human PD-L1;

[0167] (2) Binding to cells expressing human PD-L1;

[0168] (3) Enhance T cell proliferation;

[0169] (4) Increase IFN-γ secretion;

[0170] (5) Increase IL-2 secretion;

[0171] (6) In the Zenix column detection method, the residence time is less than 10 minutes;

[0172] (7) Inhibition of one or more activities of PD-L1 may result in one or more of the following: an increase in tumor-infiltrating lymphocytes, an increase in T-cell receptor-mediated proliferation, or a decrease in immune evasion of cancer cells.

[0173] (8) It can induce antibody-dependent cell-mediated cytotoxicity;

[0174] (9) Demonstrates the same binding affinity and / or specificity to PD-L1 as any antibody described in Embodiment 11;

[0175] (10) Inhibit the binding of any antibody described in embodiment 11 to PD-L1;

[0176] (11) Binds to the same or overlapping epitopes as any antibody described in Implementation Scheme 11; or

[0177] (12) Compete with any of the antibodies described in Implementation Scheme 11 for binding to PD-L1.

[0178] 13. The antibody or antigen-binding fragment thereof that binds to PD-L1 as described in Implementation Scheme 12, wherein:

[0179] (1) The following equilibrium dissociation constant (K) D Combined with PD-L1, the K D Less than 2nM;

[0180] (2) Using EC50 of less than or equal to 2 nM to bind cells expressing human PD-L1;

[0181] (3) It enhances T cell proliferation more effectively than the known anti-PD-L1 antibody Tecentriq;

[0182] (4) It enhances IFN-γ secretion more effectively than the known anti-PD-L1 antibody Tecentriq;

[0183] (5) It enhances IL-2 secretion more effectively than the known anti-PD-L1 antibody Tecentriq; or

[0184] (6) Competitively inhibit the binding of any antibody described in implementation scheme 11 to PD-L1.

[0185] 14. The antibody or its antigen-binding fragment described in Implementation Scheme 13, wherein T cell proliferation, IFN-γ secretion or IL-2 secretion are detected in MLR.

[0186] 15. The antibody or antigen-binding fragment thereof described in embodiments 1, 6 or 11, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment in the form of IgG1 or IgG4.

[0187] 16. The antibody or antigen-binding fragment thereof as described in embodiments 1, 6 or 11, wherein the antibody or antigen-binding fragment thereof comprises a constant region of a κ light chain.

[0188] 17. The antibody or antigen-binding fragment thereof as described in Embodiment 15, wherein the antibody or antigen-binding fragment thereof comprises a constant region of the human κ light chain.

[0189] 18. The antibody or antigen-binding fragment thereof as described in embodiments 1, 6 or 11, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof in the form of IgG1 or IgG4, and contains a human κ light chain constant region.

[0190] 19. The antibody or antigen-binding fragment thereof described in embodiments 1, 6 or 11, wherein the antibody is a monoclonal antibody.

[0191] 20. The antibody or antigen-binding fragment thereof described in embodiments 1, 6 or 11, wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody.

[0192] 21. The antibody or antigen-binding fragment thereof described in embodiments 1, 6 or 11, wherein the antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody, (Fab')2 fragment, or biantibody (dAb).

[0193] 22. The antibody or antigen-binding fragment thereof described in Implementation Scheme 21, wherein the single-chain antibody is scFv.

[0194] 23. The antibody or antigen-binding fragment thereof described in embodiments 1, 6 or 11, wherein the antibody is a bispecific or multispecific antibody.

[0195] 24. The antibody or antigen-binding fragment thereof described in Implementation Scheme 23, wherein the bispecific antibody binds to PD-L1 and LAG-3.

[0196] 25. The antibody or antigen-binding fragment thereof described in Implementation Scheme 23, wherein the bispecific antibody binds to human PD-L1 and human LAG-3.

[0197] 26. An isolated nucleic acid that encodes an anti-PD-L1 antibody or its antigen-binding fragment from any one of embodiments 1 to 25.

[0198] 27. An expression vector containing the nucleic acid of implementation scheme 26.

[0199] 28. The expression vector of implementation scheme 27, wherein the expression vector is a pTT5 vector.

[0200] 29. A host cell containing the nucleic acid of implementation scheme 26 or the expression vector of implementation scheme 27 or 28.

[0201] 30. The host cell described in Implementation Scheme 29, wherein the host cell is prokaryotic or eukaryotic.

[0202] 31. The host cell described in Implementation Scheme 29, wherein the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0203] 32. The host cell described in Implementation Scheme 29, wherein the host cell is a 293 cell or a CHO cell.

[0204] 33. A method for preparing an anti-PD-L1 antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell of any one of embodiments 29-32 under conditions suitable for expressing a nucleic acid encoding an anti-PD-L1 antibody or an antigen-binding fragment thereof of any one of embodiments 1 to 25, isolating the antibody or the antigen-binding fragment thereof, and recovering the anti-PD-L1 antibody or the antigen-binding fragment thereof from the host cell.

[0205] 34. An immunoconjugate comprising an anti-PD-L1 antibody or its antigen-binding fragment thereof from any one of embodiments 1 to 25 and other substances.

[0206] 35. The immunoconjugate of embodiment 34, wherein the other substance is a cytotoxic agent.

[0207] 36. A pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof from any one of embodiments 1 to 25 or an immunoconjugate of embodiment 34 or 35, and optionally a pharmaceutical excipient.

[0208] 37. A pharmaceutical composition comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof from any one of embodiments 1 to 25 or an immunoconjugate of embodiment 34 or 35, and other therapeutic agents, and optionally pharmaceutical excipients.

[0209] 38. The pharmaceutical composition of embodiment 37, wherein the other therapeutic agent is selected from chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, or immunomodulators.

[0210] 39. The pharmaceutical composition of embodiment 38, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

[0211] 40. The pharmaceutical composition of embodiment 37, wherein the other therapeutic agent is a LAG-3 antibody or an antigen-binding fragment thereof.

[0212] 41. The pharmaceutical composition of embodiment 40, wherein the LAG-3 antibody or its antigen-binding fragment is an anti-LAG-3 antibody or its antigen-binding fragment that binds to human LAG-3.

[0213] 42. The pharmaceutical composition of embodiment 41, wherein the other therapeutic agent is a humanized anti-LAG-3 antibody or an antigen-binding fragment thereof.

[0214] 43. The pharmaceutical composition according to any one of embodiments 40-42, wherein the anti-LAG-3 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0215] (i) The VH includes complementarity-determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 is composed of the amino acid sequence of SEQ ID NO: 69; HCDR2 is composed of the amino acid sequence of SEQ ID NO: 70; and HCDR3 is composed of the amino acid sequence of SEQ ID NO: 71.

[0216] and

[0217] (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 is composed of the amino acid sequence of SEQ ID NO: 72; LCDR2 is composed of the amino acid sequence of SEQ ID NO: 73; and LCDR3 is composed of the amino acid sequence of SEQ ID NO: 74.

[0218] 44. The pharmaceutical composition of embodiment 43, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO: 75, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO: 76.

[0219] 45. Use of an effective amount of an anti-PD-L1 antibody or antigen-binding fragment thereof of any one of embodiments 1 to 25, or an immunoconjugate of embodiment 34 or 35, or a pharmaceutical composition of any one of embodiments 36 to 44 in the preparation of a medicament for the prevention or treatment of a tumor in a subject or individual.

[0220] 46. ​​Use of an effective amount of an anti-PD-L1 antibody or antigen-binding fragment thereof of any one of embodiments 1 to 25, or an immunoconjugate of embodiment 34 or 35, or a pharmaceutical composition of any one of embodiments 36 to 39, and an anti-LAG-3 antibody or antigen-binding fragment thereof in the preparation of a medicament, wherein the medicament is intended for the prevention or treatment of a tumor in a subject or individual.

[0221] 47. The use described in embodiment 46, wherein the anti-LAG-3 antibody is an anti-LAG-3 antibody that binds to human LAG-3.

[0222] 48. The use described in Implementation Scheme 47, wherein the anti-LAG-3 antibody is humanized.

[0223] 49. The use of any one of embodiments 45-48, wherein the tumor is cancer.

[0224] 50. The use of any one of embodiments 45-48, wherein the tumor is a gastrointestinal tumor.

[0225] 51. The use described in Implementation Scheme 49, wherein the cancer is a gastrointestinal cancer.

[0226] 52. The use described in Implementation Scheme 49, wherein the cancer is colon cancer.

[0227] 53. The use described in Implementation Scheme 45, wherein the drug can also be used in combination with one or more other therapies.

[0228] 54. The use described in embodiment 53, wherein the therapy includes treatment methods and / or other therapeutic agents.

[0229] 55. The use described in embodiment 54, wherein the treatment method includes surgical treatment and / or radiotherapy, or the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, vaccines, anti-infective agents, other antibodies or immunomodulators.

[0230] 56. The use described in embodiment 55, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

[0231] 57. The use described in embodiment 54, wherein the other therapeutic agent is a LAG-3 antibody or an antigen-binding fragment thereof.

[0232] 58. The use described in embodiment 57, wherein the LAG-3 antibody or its antigen-binding fragment is an anti-LAG-3 antibody or its antigen-binding fragment that binds to human LAG-3.

[0233] 59. The use described in embodiment 58, wherein the anti-LAG-3 antibody or its antigen-binding fragment is a humanized anti-LAG-3 antibody or its antigen-binding fragment.

[0234] 60. Use according to any one of embodiments 46 to 48, 57 to 59, wherein the anti-LAG-3 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0235] (i) The VH includes complementarity-determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 is composed of the amino acid sequence of SEQ ID NO: 69; HCDR2 is composed of the amino acid sequence of SEQ ID NO: 70; and HCDR3 is composed of the amino acid sequence of SEQ ID NO: 71.

[0236] and

[0237] (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 is composed of the amino acid sequence of SEQ ID NO: 72; LCDR2 is composed of the amino acid sequence of SEQ ID NO: 73; and LCDR3 is composed of the amino acid sequence of SEQ ID NO: 74.

[0238] 61. Use of embodiment 60, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO: 75, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO: 76.

[0239] 62. The use of the drug as described in any one of embodiments 46 to 48, wherein the drug can also be administered in combination with one or more other therapies.

[0240] 63. The use described in embodiment 62, wherein the therapy includes treatment methods and / or other therapeutic agents.

[0241] 64. The use described in Implementation Scheme 63, wherein the treatment method includes surgical treatment and / or radiotherapy, or the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, vaccines, anti-infective agents, other antibodies or immunomodulators.

[0242] 65. The use described in embodiment 64, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

[0243] 66. Use of any one of embodiments 1 to 25 of the anti-PD-L1 antibody or its antigen-binding fragment in the preparation of a detection reagent for detecting PD-L1 in a sample.

[0244] 67. The use described in embodiment 66, wherein the anti-PD-L1 antibody or its antigen-binding fragment is detectably labeled. Attached Figure Description

[0245] Figure 1 The binding of the anti-PD-L1 antibody of the present invention to CHO-PDL1 cells as detected by FACS is shown.

[0246] Figure 2 The binding of the present invention's anti-PD-L1 antibody to CHO-PDL1 cells was shown using FACS.

[0247] Figure 3 The blocking activity of the antibody of the present invention against PD-1 / PD-L1 interaction was demonstrated by MOA assay.

[0248] Figure 4A and 4B The activation effect of the antibody of the present invention on T cells (relative IL-2 expression level) is shown by MLR assay.

[0249] Figure 5A and 5B The activation effect of the antibody of the present invention on T cells (relative expression level of IFN-γ) is shown by MLR assay.

[0250] Figure 6 The antibody of the present invention was shown to have an inhibitory effect on tumors.

[0251] Figure 7This demonstrates the inhibitory effect of the antibody of the present invention combined with the anti-LAG-3 antibody on tumors. Invention Details

[0252] abbreviation

[0253] Unless otherwise stated, the abbreviations in this specification have the following meanings:

[0254] Use the following abbreviations:

[0255] ADCC antibody-dependent cell-mediated toxicity

[0256] CDC complement-dependent cytotoxicity

[0257] CDR in the immunoglobulin variable region complementarity-determining region

[0258] CHO Chinese hamster ovary

[0259] EC50 results in a concentration that yields 50% potency or binding.

[0260] K D Equilibrium dissociation constant

[0261] ELISA (Enzyme-Linked Immunosorbent Assay)

[0262] FACS Flow Cytometry

[0263] MOA Mechanism of Action

[0264] MLR mixed lymphocyte reaction

[0265] FR antibody framework region

[0266] IC50 produces a concentration that inhibits 50% of the concentration.

[0267] Ig Immunoglobulin

[0268] Kabat established an immunoglobulin matching and numbering system based on Elvin A. Kabat's work (1991, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md.).

[0269] mAb or Mab or MAb monoclonal antibody

[0270] PCR Polymerase Chain Reaction

[0271] IFN interferon

[0272] VL light chain variable region

[0273] VH Heavy Chain Variable Region

[0274] LC light chain

[0275] HC heavy chain

[0276] HCDR heavy chain complementarity determinant region

[0277] LCDR Light Chain Complementary Determinant Region

[0278] definition

[0279] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0280] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0281] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0282] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, when used herein, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed using the equilibrium dissociation constant (K0). D Affinity can be expressed as a definite affinity. It can be measured using methods commonly known in the art, including those known in the prior art and those described herein.

[0283] As used herein, the terms “programmed cell death 1-ligand 1,” “PD-L1,” “programmed death ligand 1,” “differentiation cluster 274,” “CD274,” or “B7 homologue 1” refer to any naturally occurring PD-L1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The terms encompass “full-length,” unprocessed PD-L1, and any form of PD-L1 produced by cellular processing. PD-L1 can exist as a transmembrane protein or as a soluble protein. The terms also encompass variants of naturally occurring PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 comprises four domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. Further information about the human PD-L1 gene (including the genomic DNA sequence) can be found under NCBI Gene ID No. 29126. Additional information about the mouse PD-L1 gene (including the genomic DNA sequence) can be found under NCBI Gene ID No. 60533. An exemplary full-length human PD-L1 protein amino acid sequence can be found, for example, under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7, while an exemplary full-length mouse PD-L1 protein sequence can be found, for example, under NCBI accession number NP_068693 or UniProt accession number Q9EP73.

[0284] As used herein, the terms "anti-PD-L1 antibody," "anti-PD-L1," "PD-L1 antibody," or "PD-L1-binding antibody" refer to antibodies that are capable of binding to the PD-L1 protein or fragments thereof with sufficient affinity. In one embodiment, the anti-PD-L1 antibody binds to non-PD-L1 proteins to a degree less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the antibody's binding to PD-L1, as measured, for example, by radioimmunoassay (RIA), bioluminescent interferometry, or MSD assay.

[0285] As used herein, “monoclonal antibody” or “mAb” or “Mab” refers to an antibody derived from a single copy or clone of, for example, a eukaryotic, prokaryotic, or phage clone, and not to the method of its production. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or a combination of such or other techniques known in the art.

[0286] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfides. From the N to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of their constant domains, antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ). The "natural sequence Fc region" contains the same amino acid sequence as the Fc region found in nature. The natural sequence human Fc region includes the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region; and their naturally occurring variants.

[0287] An "antibody fragment" refers to a molecule distinct from the intact antibody that contains a portion of the intact antibody and binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies; and bispecific or multispecific antibodies formed from antibody fragments.

[0288] As used herein, the term "epitaxy" refers to a portion of an antigen (e.g., human PD-L1) that specifically interacts with an antibody molecule. This portion (referred to herein as an epitope determinant) generally contains elements such as amino acid or sugar side chains or components thereof. Epitope determinants can be defined by methods known in the art or disclosed herein (e.g., by crystallography or by hydrogen-deuterium exchange). At least one or more portions of an antibody molecule that specifically interact with an epitope determinant are generally located within a CDR. Typically, epitopes have specific three-dimensional structural features. Typically, epitopes have specific charge features. Some epitopes are linear epitopes, while others are conformational epitopes.

[0289] "Antibody that binds to the same or overlapping epitopes as the reference antibody" means an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay; conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competitive assay.

[0290] An antibody that competes with a reference antibody for binding to its antigen is one that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competitive assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another; these assays include, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), and sandwich competitive assays (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253).

[0291] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0292] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90%, or 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for measuring binding affinity and / or specificity.

[0293] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those 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 precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (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)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web at imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0294] For example, depending on the different CDR determination schemes, the residues of each CDR are as follows.

[0295]

[0296] CDRs can also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0297] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0298] Unless otherwise stated, in this invention, when referring to the position of a residue in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0299] In one embodiment, the CDR of the antibody of the present invention is defined by the Chothia rule or the Kabat rule, for example, its sequence is shown in Table 1.

[0300] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined by the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0301] Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs (within the same assignment system). However, although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining portion of the CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0302] Five main classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, and several of these antibodies can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

[0303] "IgG form antibody" refers to an antibody whose heavy chain constant region belongs to the IgG form. All antibodies of the same type have the same heavy chain constant region, while antibodies of different types have different heavy chain constant regions. For example, an IgG1 form antibody means that its heavy chain constant region Ig domain is the Ig domain of IgG1.

[0304] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted immunoglobulins binding to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxicity. The main cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991), page 464, Table 3 summarizes FcR expression on hematopoietic cells. To assess the ADCC activity of a target molecule, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 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 PBMCs and NK cells. Optionally / additionally, ADCC activity of the target molecule can be assessed in vivo, for example in animal models, such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0305] The term "cytotoxic agent" or "cytotoxic factor" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or destruction. Examples of cytotoxic agents can be found in those disclosed in WO2015 / 153513, WO2016 / 028672, WO2015 / 138920, and WO2016 / 007235.

[0306] The term "therapeutic agent" as used herein encompasses any substance effective in the prevention or treatment of tumors (e.g., cancer) and infections (e.g., chronic infections), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, or immunomodulatory agents, such as any substance disclosed in WO2016 / 007235 or WO2010 / 077634 or US60 / 696426 that can be used in combination with anti-PD-L1 antibodies.

[0307] "Chemotherapy agents" include chemical compounds that are useful in the treatment of cancer. Examples of chemotherapeutic agents can be found in those disclosed in WO2016 / 007235, WO2010 / 077634, US60 / 696426, WO2016 / 061142, US61 / 264061, or WO2016 / 007235.

[0308] The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators to another cell. Examples of such cytokines include lymphokines, monokines; interleukins (ILs), 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, and IL-15; tumor necrosis factors, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL) and gamma interferon. As used herein, the term cytokine includes proteins of natural origin or derived from recombinant cell cultures and biologically active equivalents of naturally occurring sequence cytokines, including small molecule entities produced through artificial synthesis and their pharmaceutically acceptable derivatives and salts.

[0309] The term "co-stimulatory molecule" refers to a binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby inducing a T cell-mediated co-stimulatory response (such as, but not limited to, proliferation). Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Co-stimulatory molecules include, but are not limited to: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), NK cell activation receptors, BTLA, Toll ligand receptors, 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, and IL2R. β, 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, ​​LAT, GADS, SLP_76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0310] The term "activator" or "agonist" includes substances that increase certain parameters (e.g., activity) of a given molecule (e.g., a co-stimulatory molecule). For example, this term includes substances that increase the activity (e.g., co-stimulatory activity) of a given molecule by at least 5%, 10%, 25%, 50%, 75%, or more.

[0311] The term "immune checkpoint molecules" refers to a group of molecules on the cell surface of CD4 T cells and CD8 T cells. These molecules can effectively act as "brakes" to downregulate or inhibit anti-tumor immune responses. Immune 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.

[0312] The term "inhibitor" or "antagonist" includes substances that reduce certain parameters (e.g., activity) of a given molecule (e.g., an immune checkpoint inhibitory protein). For example, this term includes substances that inhibit the given molecule by at least 5%, 10%, 20%, 30%, 40%, or more of its activity (e.g., LAG-3 activity). Therefore, the inhibitory effect does not have to be 100%.

[0313] The term "biantibody" refers to an antibody fragment having two antigen-binding sites, wherein the fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL). Two antigen-binding sites are created by using a linker that is too short to pair between the two domains on the same chain, forcing the domain to pair with a complementary domain of the other chain. Biantibodies can be bivalent or bispecific. Biantibodies 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., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 90:6444-6448 (1993). Triantibodies and tetraantibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0314] The “functional Fc region” possesses the “effector function” of the native Fc region. Exemplary “effector functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). These effector functions generally require the Fc region to be coupled with a binding domain (e.g., antibody variable domain) and can be assessed using various assays, such as those disclosed herein.

[0315] "Effective functions" refer to biological activities that can be attributed to the Fc region of an antibody and vary with antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0316] "Human effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from their natural source, such as blood.

[0317] The term "effective amount" refers to such an amount or dose of the antibody, fragment, conjugate, or composition of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration method; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0318] "Therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. Therapeutic effective amount of an antibody or antibody fragment, or its conjugate or composition, can vary depending on various factors such as disease state, individual age, sex, weight, and the ability of the antibody or antibody fraction to elicit the desired response in the individual. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or antibody fragment, or its conjugate or composition, are less than the beneficial therapeutic effect. Relative to an untreated subject, "therapeutic effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be evaluated by testing the inhibitory ability of the compound, said inhibition being measured in vitro using methods known to a skilled craftsman.

[0319] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0320] The “antibodies and antigen-binding fragments thereof” applicable to this invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric, humanized (especially grafted with CDR), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, fragments generated from Fab expression libraries, Fd, Fv, disulfide-linked Fv (dsFv), single-chain antibodies (e.g., scFv), biantibodies or tetraantibodies (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (14), 6444-6448), nanobodies (also known as single-domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of this invention), and epitope-binding fragments of any of the above.

[0321] The “Fab” fragment comprises a variable domain in the heavy chain and a variable domain in the light chain, and also includes a constant domain in the light chain and a first constant domain (CH1) in the heavy chain. The Fab’ fragment differs from the Fab fragment by the addition of several residues (including one or more cysteine ​​residues from the antibody hinge region) at the carboxyl terminus of the CH1 domain in the heavy chain. Fab’-SH is the designation for Fab’ fragments in which the cysteine ​​residues in the constant domain carry a free thiol group. The F(ab’)2 antibody fragment was originally generated as paired Fab’ fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0322] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some 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 residue (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0323] The term "variable region" or "variable domain" refers to a domain of the heavy or light chain of an antibody that participates in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6) th See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991). A single VH or VL domain may be sufficient to provide antigen binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using VH or VL domains derived from antibodies that bind to that antigen, to screen libraries for complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0324] "Framework" or "FR" refers to the variable domain residues other than the CDR residues in the complementarity-determining region. A variable domain's FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0325] Unless otherwise stated, the residues in the various domains of the antibody are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0326] The terms “full-length antibody,” “intact antibody,” and “complete antibody” are used interchangeably in this document to refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing an Fc region as defined herein.

[0327] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. In one embodiment, the double-chain Fv species consists of a heavy chain variable domain and a light chain variable domain in a tightly bound, non-covalently associated dimer. In the single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that of the double-chain Fv species. 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 an Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind antigens, although with a lower affinity than the complete binding site. For a review of scFv, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York, 1994), pp. 269–315.

[0328] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in their nucleic acid content and may contain mutations. This document includes mutant progeny with the same function or biological activity screened or selected from the initially transformed cells.

[0329] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source, utilizing a human antibody library or other human antibody encoding sequences. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0330] "Human common framework" refers to a framework that represents the most frequently occurring amino acid residues in the selected human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is based on a subtype of the variable domain sequence. This subtype is generally the one disclosed in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Vols. 1-3). In one embodiment, for VL, this subtype is subtype κI as described in Kabat et al. (see above). In one embodiment, for VH, this subtype is subtype III as described in Kabat et al. (see above).

[0331] "Humanized" antibodies are chimeric antibodies comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. 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 (e.g., CDRs) correspond to those of nonhuman antibodies, and all or substantially all of the FRs correspond to those of human antibodies. Humanized antibodies may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized.

[0332] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals 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, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, 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 fibroids, and other cancers. Disseminated melanoma, malignant lentigines melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with phakomatoses, edema (such as that associated with brain tumors) and Meigs syndrome, brain tumors and brain cancers, and head and neck cancers, and related metastases. In some embodiments, cancers suitable for treatment with the antibodies of the present invention include non-small cell lung cancer, squamous cell carcinoma, small cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, 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 metastatic forms of those cancers.

[0333] The terms "cellular proliferative disorder" and "proliferative disorder" refer to disorders associated with a certain degree of abnormal cell proliferation. In one implementation, cellular proliferative disorder refers to cancer.

[0334] The term "tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "proliferative disorder," "proliferative lesion," and "tumor" are not mutually exclusive when used in this document.

[0335] The term "infectious disease" refers to a disease caused by a pathogen, including, for example, viral infections, bacterial infections, fungal infections, or protozoan infections such as parasitic infections.

[0336] The term "tumor immune escape" refers to a tumor evading immune recognition and clearance. Therefore, as a therapeutic concept, when this evasion is reduced, 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 clearance.

[0337] The term "chronic infection" refers to an infection in which the infectious agent (e.g., pathogens such as viruses, bacteria, protozoa such as parasites, fungi, or the like) has induced an immune response in the infected host but has not yet been cleared or eliminated from the host as in the course of an acute infection. Chronic infections can be persistent, latent, or slow-growing. While acute infections are typically resolved by the immune system within days or weeks (e.g., influenza), persistent infections can persist at relatively low levels for months, years, decades, or even a lifetime (e.g., hepatitis B). In contrast, latent infections are characterized by prolonged asymptomatic activity, interrupted from time to time by periods of rapidly increasing high levels of infection and elevated pathogen levels (e.g., herpes simplex). Finally, slow-growing infections are characterized by a gradual and progressive increase in disease symptoms, such as a long incubation period, followed by an extended and progressive clinical course following the onset of clinical symptoms. Unlike latent and persistent infections, chronic infections may not begin with an acute phase of viral replication (e.g., picovirus infection, visna virus, scrapie, Creutzfeldt-Jakobdisease).Exemplary infectious agents capable of inducing chronic infection include viruses (e.g., cytomegalovirus, Epstein-Barr virus, hepatitis B virus, hepatitis C virus, herpes simplex virus types I and II, human immunodeficiency virus types I and II, human papillomavirus, human T-lymphoblastic virus types I and II, varicella-zoster virus, etc.), bacteria (e.g., Mycobacterium tuberculosis, Listeria species, Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Borrelia species, Helicobacter pylori, etc.), and protozoa such as parasites (e.g., Leishmania species, Plasmodium). *Schistosoma* species, *Toxoplasma* species, *Trypanosoma* species, *Taenia carssiceps*, etc., and fungi (e.g., *Aspergillus* species, *Candida albicans*, *Coccidioides immitis*, *Histoplasma capsulatum*, *Pneumocystis carinii*, etc.). Other infectious agents include prions or misfolded proteins that spread further in these tissues. Protein misfolding affects brain or neuronal structures, leading to the formation of amyloid plaques (which result in cell death, tissue damage, and eventual death).Examples of diseases caused by prions include: Creutzfeldt-Jakob disease and its varieties, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (sFI), kuru, scrapie, bovine spongiform encephalopathy (BSE) in cattle (aka "mad cow" disease), and various other animal forms of encephalopathy [e.g., transmissible mink encephalopathy (TME), chronic wasting disease (CWD) in white-tailed deer, elk, and mule deer, feline spongiform encephalopathy]. encephalopathy, an invasive ungulate encephalopathy (EUE) in nyala, oryx and greater kudu (spongiform encephalopathy of theostrich).

[0338] "Immune conjugates" are antibodies conjugated to one or more other substances (including but not limited to cytotoxic agents or markers).

[0339] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugating (i.e., physically linking) a detectable substance to the probe or antibody and indirect labeling of probes or antibodies by reacting with another directly labeled reagent. Examples of indirect labeling include the detection of primary antibodies using fluorescently labeled secondary antibodies and the end labeling of biotinylated DNA probes so that they can be detected using fluorescently labeled streptomycin proteins.

[0340] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0341] "Isolated" antibodies are antibodies that have been separated from components of their native environment. In some embodiments, the antibody is purified to a purity exceeding 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0342] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain them, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0343] "Isolated nucleic acid encoding anti-PD-L1 antibody or a fragment thereof" refers to one or more nucleic acid molecules that encode the antibody heavy or light chain (or a fragment thereof), including such nucleic acid molecules in a single or separate vector, and such nucleic acid molecules present at one or more locations in a host cell.

[0344] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably with “polynucleotide.” They refer to nucleotides (deoxyribonucleotides or ribonucleotides) of any length in polymer form or similar. Polynucleotides can be single-stranded or double-stranded, and if single-stranded, can be coding or non-coding (antisense) strands. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of a nucleotide can be broken down by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components. Nucleic acids can be recombinant polynucleotides or polynucleotides of genomic, cDNA, semi-synthetic, or synthetic origin that do not exist in nature or are linked to another polynucleotide in a non-natural layout.

[0345] The terms “polypeptide,” “peptide,” and “protein” (if single-chain) are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components). Polypeptides can be isolated from natural sources, produced from eukaryotic or prokaryotic hosts via recombinant technologies, and may be products of synthetic methods.

[0346] The following is a calculation of sequence identity between sequences.

[0347] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0348] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. The particularly preferred set of parameters (and a set of parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0349] Alternatively, the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4) can be used to determine the percentage of identity between two amino acid sequences or nucleotide sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0350] Additionally or alternatively, the nucleic acid and protein sequences described herein can be further used as “query sequences” to perform searches against public databases to, for example, identify sequences of other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST procedures described in Altschul et al., (1990) J.Mol.Biol.215:403-10. BLAST nucleotide searches can be performed using the NBLAST procedure with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST procedure with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. For comparison purposes, vacancy-based alignment results can be obtained using vacancy BLAST as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When using BLAST and vacancy BLAST procedures, the default parameters of the respective procedures (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0351] As used herein, the term "hybridization under low, medium, high, or very high strictness conditions" describes the hybridization and washing conditions. Instructions for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which are incorporated herein by reference. Aqueous and non-aqueous methods are described in the references, and either method may be used. The specific hybridization conditions mentioned herein are as follows: 1) Low-toughness hybridization conditions are washing twice at about 45°C in 6X sodium chloride / sodium citrate (SSC), followed by washing twice at at least 50°C (for low-toughness conditions, the washing temperature can be increased to 55°C) in 0.2X SSC, 0.1% SDS; 2) Medium-toughness hybridization conditions are washing once or more at about 45°C in 6X SSC, followed by washing once or more at 60°C in 0.2X SSC, 0.1% SDS; 3) High-toughness hybridization conditions are washing once or more at about 45°C in 6X SSC, followed by washing once or more at 65°C in 0.2X SSC, 0.1% SDS; and preferably 4) Very high-toughness hybridization conditions are washing once or more at 65°C in 0.5M sodium phosphate, 7% SDS, followed by washing once or more at 65°C in 0.2X SSC, 0.1% SDS. Very high-toughness condition (4) is the preferred condition and, unless otherwise stated, should be used.

[0352] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0353] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0354] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0355] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0356] The term "anti-infective active agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms, such as viruses, bacteria, fungi, or protozoa, such as parasites, at the applied concentration and dosing interval. When used herein, the term anti-infective active agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoan agents. In one specific aspect, the anti-infective active agent is non-toxic to the host at the applied concentration and dosing interval.

[0357] Antibacterial agents, or antimicrobial agents, can be broadly classified as bactericidal (i.e., directly killing bacteria) or bacteriostatic (i.e., inhibiting cell division). Antimicrobial agents can be further subdivided into narrow-spectrum antimicrobial agents (i.e., affecting only a small number of bacterial subtypes, such as Gram-negative bacteria) or broad-spectrum antimicrobial agents (i.e., affecting a wide range of species). Examples include amikacin, gentamicin, geldmycin, atrazine, mupirocin, nitrofurantoin, pyrazinamide, quinupristin / dalfopristin, rifampin / isofopristin, or tinidazole.

[0358] The term "antiviral agent" includes any substance that inhibits or eliminates viral growth, pathogenicity, and / or survival. This includes, for example, acyclovir, cidofovir, zidovudine, doxorinosine (ddI, VIDEX), zalcitabine (ddC, HIVID), stavudine (d4T, ZERIT), lamivudine (3TC, EPIVIR), abacavir (ZIAGEN), emtricitabine (EMTRIVA), etc.

[0359] The term "antifungal agent" includes any substance that inhibits or eliminates the growth, pathogenicity, and / or survival of fungi. This includes, for example, natamycin, sclerotinib, ferulic acid, nystatin, amphotericin B, candisin, patchouli, neem seed oil, coconut oil, etc.

[0360] The term "antiprotozoan agent" includes any substance that inhibits or eliminates the growth, disease, and / or survival of protozoan organisms (such as parasites). Examples of antiprotozoan agents include antimalarial agents such as quinine and quinidine.

[0361] Examples of antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents can be found in, for example, WO2010 / 077634.

[0362] See also, for example, WO2014 / 008218, WO2016 / 028672, WO2015 / 138920 or WO2016 / 061142 for anti-infective agents.

[0363] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0364] "Subject / Patient Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of tissue or cell samples can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds naturally occurring and not contaminated with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of tumor samples in this document include, but are not limited to, tumor biopsies, fine-needle aspirations, bronchoalveolar lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like characteristics, and preserved tumor samples, such as formalin-fixed, paraffin-embedded, or frozen tumor samples.

[0365] The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of such a therapeutic product.

[0366] The antibody of the present invention

[0367] Therefore, in some embodiments, the antibody or fragment thereof of the present invention binds to PD-L1. In some embodiments, the antibody or fragment thereof of the present invention binds to mammalian PD-L1, such as human PD-L1. For example, the antibody molecule specifically binds to an epitope (e.g., a linear or conformational epitope) on PD-L1. In some embodiments, the antibody molecule binds to one or more extracellular domains of PD-L1.

[0368] In some embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention has one or more of the following properties:

[0369] (1) The anti-PD-L1 antibody or fragment thereof of the present invention binds to PD-L1 (e.g., human PD-L1) with high affinity, for example, with the following equilibrium dissociation constant (K). D Combined with PD-L1, the K DLess than about 50 nM, preferably less than or equal to about 20 M, more preferably less than or equal to about 15 nM, even 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 Less than or equal to approximately 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1.1 nM, 1 nM, 0.9 nM, or 0.8 nM. In some embodiments, the anti-PD-L1 antibody of the present invention is in the form of 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 nM, or 0.7-1 nM K. D Binding to PD-L1. In some embodiments, PD-L1 is human PD-L1. In some embodiments, antibody binding affinity is determined using a bio-optical interferometry assay (e.g., Fortebio affinity measurement).

[0370] (2) The antibody or fragment thereof of the present invention binds to cells expressing human PD-L1, for example, at an EC50 of 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, 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 (e.g., FACS). In some embodiments, the cells expressing human PD-L1 are CHO cells expressing human PD-L1.

[0371] (3) The antibody or fragment thereof of the present invention blocks PD-L1-related activity, for example, at EC50 concentrations less than or equal to approximately 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, or 0.7 nM. 50 Preferably, EC is present at concentrations of approximately 0.1-1 nM, 0.5-1 nM, 0.6-1 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, or 1 nM. 50 In some embodiments, the PD-L1-related activity is the binding of PD-L1 to PD-1. In some embodiments, the antibody or fragment thereof of the present invention is expressed in an MOA assay at EC50 values ​​less than or equal to approximately 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, or 0.7 nM. 50 Preferably, EC is present at concentrations of approximately 0.1-1 nM, 0.5-1 nM, 0.6-1 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, or 1 nM. 50Inhibits the binding of PD-L1 to PD-1. In some implementations, the cells are CHO cells.

[0372] (4) The antibodies or fragments thereof of the present invention enhance T cell function, for example, superior to known anti-PD-L1 antibodies, such as Tecentriq.

[0373] (5) The antibodies or fragments thereof of the present invention enhance T cell proliferation, for example in MLR, and are superior to known anti-PD-L1 antibodies, such as Tecentriq.

[0374] (6) The antibodies or fragments thereof of the present invention enhance IFN-γ secretion, for example in MLR, and are superior to known anti-PD-L1 antibodies, such as Tecentriq.

[0375] (7) The antibodies or fragments thereof of the present invention enhance IL-2 secretion, for example in MLR, and are superior to known anti-PD-L1 antibodies, such as Tecentriq.

[0376] (8) The antibodies or fragments thereof of the present invention have lower viscosity than known anti-PD-L1 antibodies (e.g., Tecentriq), and therefore have better druggability. In some embodiments, the antibodies or fragments thereof of the present invention have a residence time (RT) of less than about 10 minutes, about 9 minutes, or about 8 minutes in the Zenix column assay, preferably between about 7 minutes and 9 minutes, preferably between about 7-8.5 minutes, about 7.5-8.5 minutes, about 7-8 minutes, or between about 7.5-8 minutes, for example, 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, or 8.5 minutes.

[0377] (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 the following: an increase in tumor-infiltrating lymphocytes, an increase in T-cell receptor-mediated proliferation, or a decrease in immune evasion of cancer cells.

[0378] (10) The anti-PD-L1 antibody or fragment thereof of the present invention can induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0379] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention has one or more of the following characteristics:

[0380] (i) Showing the same or similar binding affinity and / or specificity to PD-L1 as the antibodies of the present invention (e.g., any of the antibodies listed in Table 3);

[0381] (ii) Inhibit (e.g., competitively inhibit) the binding of the antibodies of the present invention (e.g., any of the antibodies listed in Table 3) to PD-L1;

[0382] (iii) Epitopes that bind to the same or overlapping epitopes as the antibodies of the present invention (e.g., any of the antibodies listed in Table 3);

[0383] (iv) Competing with the antibodies of the present invention (e.g., any of the antibodies listed in Table 3) to bind to PD-L1;

[0384] (v) Having one or more biological characteristics of the antibodies of the present invention (e.g., any of the antibodies listed in Table 3).

[0385] Exemplary antibodies

[0386] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH), wherein the VH comprises

[0387] (i) The three complementarity-determining regions (CDRs) contained in the VH of any antibody listed in Table B, or

[0388] (ii) A sequence that, relative to the sequence in (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions.

[0389] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention comprises a light chain variable region (VL), wherein the VL comprises:

[0390] (i) The three complementarity-determining regions (CDRs) contained in the VL of any antibody listed in Table B; or

[0391] (ii) A sequence that, relative to the sequence in (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions.

[0392] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein

[0393] (a) The VH includes

[0394] (i) The three complementarity-determining regions (CDRs) contained in the VH of any antibody listed in Table B, or

[0395] (ii) a sequence that, relative to the sequence in (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three CDR regions; and / or

[0396] (b) The VL includes:

[0397] (i) The three complementarity-determining regions (CDRs) contained in the VL of any antibody listed in Table B; or

[0398] (ii) A sequence that, relative to the sequence in (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions.

[0399] In a preferred embodiment, VH comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 26, 27, 28, 29, 30 or 31.

[0400] In a preferred embodiment, VL comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 32, 33, 34, 35, 36 or 37.

[0401] In a preferred embodiment, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention comprises...

[0402] (i) the three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 26 or 30, and the three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 32 or 36, or

[0403] (ii) the three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 27, and the three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 33, or

[0404] (iii) The three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 28, and the three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 34, or

[0405] (iv) The three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 29 or 31, and the three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 35 or 37.

[0406] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein

[0407] (i) The VH comprises complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, or 4, or is composed of said amino acid sequence, or HCDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitution, preferably conservative substitution) compared to an amino acid sequence selected from SEQ ID NO: 1, 2, 3, or 4; HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 5, 6, 7, 8, or 9, or is composed of said amino acid sequence, or HCDR2 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitution, preferably conservative substitution) compared to an amino acid sequence selected from SEQ ID NO: 5, 6, 7, 8, or 9; HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 10, 11, 12, or 13, or is composed of said amino acid sequence, or HCDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitution, preferably conservative substitution) compared to an amino acid sequence selected from SEQ ID NO: 10, 11, 12, or 13;

[0408] and / or

[0409] (ii) wherein the VL comprises complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 14, 15, or 16, or LCDR1 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 SEQ ID NO: 14, 15, or 16; LCDR2 comprises or consists of an amino acid sequence selected from SEQ ID NO: 17, 18, 19, or 20, or LCDR2 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 SEQ ID NO: 17, 18, 19, or 20; LCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24, or 25, or LCDR3 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 SEQ ID NO: 14, 15, or 16; LCDR2 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 SEQ ID NO: 17, 18, 19, or 20; LCDR3 ... NO: 21, 22, 23, 24 or 25 have one, two or three altered (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequences compared to the amino acid sequences of NO: 21, 22, 23, 24 or 25.

[0410] In a preferred embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein

[0411] (a) The VH includes

[0412] (i) The combination of HCDR1, HCDR2 and HCDR3 shown in Table A; or

[0413] (ii)(i) variants of the HCDR combination, wherein the variants contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions;

[0414] and / or

[0415] (ii) The VL includes

[0416] (i) The combination of LCDR1, LCDR2 and LCDR3 shown in Table A; or

[0417] (ii)(i) variants of the LCDR combination, wherein the variants contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions.

[0418] In a preferred embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises (CDRs) LCDR1, LCDR2, and LCDR3. The combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprised in the antibody or antigen-binding fragment are shown in the following table (Table A):

[0419] Table A: Exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the antibodies or antigen-binding fragments of the present invention.

[0420]

[0421] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region VH and / or a light chain variable region VL, wherein,

[0422] (a) Heavy chain variable region VH

[0423] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 29, 30, or 31; or

[0424] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 26, 27, 28, 29, 30 or 31; or

[0425] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 26, 27, 28, 29, 30, or 31, preferably, the amino acid changes do not occur in the CDR region;

[0426] and / or

[0427] (b) Light chain variable region VL

[0428] (i) Containing or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 32, 33, 34, 35, 36, or 37;

[0429] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 32, 33, 34, 35, 36 or 37; or

[0430] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 32, 33, 34, 35, 36, or 37, preferably, the amino acid changes do not occur in the CDR region.

[0431] In a preferred embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the combination of the heavy chain variable region VH and the light chain variable region VL contained in the antibody or the antigen-binding fragment thereof is shown in the following table (Table B):

[0432] Table B: Exemplary combinations of heavy chain variable region VH and light chain variable region VL in the antibody or antigen-binding fragment of the present invention

[0433]

[0434] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment of the present invention comprises a heavy chain and / or a light chain, wherein

[0435] (a) Heavy chain

[0436] (i) Containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, or 45;

[0437] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 38, 39, 40, 41, 42, 43, 44 or 45; or

[0438] (iii) An amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, or 45, preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, the amino acid changes do not occur in the variable region of the heavy chain;

[0439] and / or

[0440] (b) Light chain

[0441] (i) Containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 46, 47, 48, 49, 50, or 51;

[0442] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 46, 47, 48, 49, 50 or 51; or

[0443] (iii) An amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 46, 47, 48, 49, 50, or 51, preferably, no more than 5, 4, 3, 2, or 1 amino acid changes, preferably, the amino acid changes do not occur in the CDR region of the light chain, more preferably, the amino acid changes do not occur in the variable region of the light chain.

[0444] In a preferred embodiment, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the combination of heavy and light chains comprising the antibody or the antigen-binding fragment thereof is shown in the following table (Table C):

[0445] Table C: Exemplary combinations of heavy and light chains in the antibodies or antigen-binding fragments of the present invention

[0446]

[0447] In some embodiments, the heavy and / or light chains of the anti-PD-L1 antibody or fragment thereof of the present invention further comprise a signal peptide sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68).

[0448] In one embodiment of the invention, the amino acid alteration described herein includes amino acid substitution, insertion, or deletion. Preferably, the amino acid alteration described herein is an amino acid substitution, and more preferably a conservative substitution.

[0449] In a preferred embodiment, the amino acid alteration described in this invention occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid alteration described in this invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region.

[0450] Optionally, the anti-PD-L1 antibody of the present invention comprises post-translational modifications to the light chain variable region, the heavy chain variable region, or the light or heavy chain. Exemplary post-translational modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, such as conjugation with a labeled component.

[0451] In some implementations, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in Table D below:

[0452] Table D

[0453]

[0454] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0455] For example, one or more amino acid substitutions can be performed to eliminate one or more variable region glycosylation sites, thereby eliminating glycosylation at those sites. Such glycosylation-free processes can increase the antibody's affinity for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861. Antibodies with altered types of glycosylation can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosylated residues or antibodies with increased isomeric GlcNac structures. These altered glycosylation patterns have been shown to increase the antibody's ADCC ability. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation system. Cells with altered glycosylation systems have been described in the art and can be used as host cells in which the antibodies of the present invention are expressed to thereby produce antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), resulting in antibodies expressed in these cell lines lacking fucosylation in their carbohydrates. Ms704, Ms705, and Ms709 FUT8- / - cell lines were created by targeting and disrupting the FUT8 gene in CHO / DG44 cells using two alternative vectors (see US Patent Publication 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene encoding fucosyltransferase, resulting in antibodies expressed in these cell lines exhibiting hypofucosylation by reducing or eliminating α-1,6 bond-associated enzymes. EP1,176,195 also describe cell lines with low or no enzymatic activity for adding fucose to the N-acetylglucosamine binding region of antibody Fc, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT publication WO 03 / 035835 describes a variant CHO cell line Lec13 in which the ability to attach fucose to Asn(297)-linked sugars is reduced, resulting in low fucosylation of antibodies expressed in the host cells (see also Shields et al. (2002) J. Biol. Chem. 277: 26733-26740). Antibodies with modified glycosylation profiles can also be produced in eggs, as described in PCT publication WO 06 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells (e.g., Lemna). Methods for generating antibodies in plant systems are disclosed in U.S. Patent Application filed August 11, 2006, corresponding to Alston and Bird LLP Attorney Number: 040989 / 314911.PCT publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)), in which antibodies expressed exhibit increased isomeric GlcNac structures, leading to increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, fucosidases can be used to cleave the fucose residues of the antibody; for example, fucosidase α-L-fucosidase removes fucose residues from the antibody (Tarentino et al. (1975) Biochem. 14: 5516-23).

[0456] 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.

[0457] Another modification of the antibody or its fragment described herein, as covered by this invention, is pegylation. Antibodies can be pegylated to, for example, increase their biological (e.g., serum) half-life. To pegylate an antibody, the antibody or its fragment is typically reacted with polyethylene glycol (PEG) (e.g., a reactive ester or aldehyde derivative of PEG) under conditions where one or more PEG groups become attached to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation or alkylation reaction using reactive PEG molecules (or similar reactive water-soluble polymers). As used herein, the term "polyethylene glycol" is intended to cover any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is a glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention, see, for example, EP 0154316 and EP 0401384.

[0458] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to create Fc region variants, thereby enhancing the efficacy of the antibody for treating, for example, cancer or proliferative diseases. The anti-PD-L1 antibodies (e.g., humanized or chimeric antibodies) and their antigen-binding fragments disclosed herein also include antibodies and fragments having modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Patent Nos. 5,624,821, WO2003 / 086310, WO2005 / 120571, and WO2006 / 0057702. Such modifications can be used to enhance or suppress various responses of the immune system, potentially with beneficial effects in diagnosis and treatment. Modifications to the Fc region include amino acid changes (substitution, deletion, and insertion), glycosylation or deglycosylation, and the addition of multiple Fc regions. Modifications to the Fc region can also alter the half-life of the antibody in a therapeutic antibody, thereby enabling less frequent dosing and thus increased convenience and reduced material usage. See Presta (2005) J. Allergy Clin. Immunol. 116: 731, 734-735.

[0459] In one embodiment, the number of cysteine ​​residues in an antibody can be altered to modify antibody properties. For example, modification of the hinge region of CH1 can alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region. This method 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, promote the assembly of light and heavy chains or increase or decrease antibody stability.

[0460] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known and readily available in the art. Suitable moieties 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, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polymers may have any molecular weight and may be branched or unbranched. The number of polymers linked to the antibody may vary, and if more than one polymer is linked, they may be the same or different molecules. Generally speaking, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used in a therapy under certain conditions.

[0461] In some embodiments, the present invention covers fragments of anti-PD-L1 antibodies. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, biantibodies, linear antibodies, single-chain antibodies (e.g., scFv), single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0462] For example, antibody molecules may 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 hapten). In another example, the antibody molecule comprises two heavy chain variable domain sequences and two light chain variable domain sequences, thus forming two antigen-binding sites. Antibodies such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single-domain antibodies, bivalent antibodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies can be generated by modifying intact antibodies or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their corresponding antigens or receptors. Antibodies and antibody fragments can originate from any antibody class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and from any antibody subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Antibody molecules can be prepared monoclonal or polyclonal. Antibodies can be human antibodies, humanized antibodies, chimeric antibodies, CDR-transplanted antibodies, or in vitro generated antibodies. Antibodies may have a heavy chain constant region, for example, selected from IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain, for example, selected from κ or λ.

[0463] The antibodies of this invention can also be single-domain antibodies. Single-domain antibodies can include antibodies whose complementarity-determining regions are components of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, naturally occurring antibodies lacking a light chain, single-domain antibodies derived from conventional 4-chain antibodies, or engineered antibodies. Single-domain antibodies can be any prior art antibody or any future single-domain antibody. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, alpacas, fish, sharks, goats, rabbits, and cattle. According to another aspect of the invention, single-domain antibodies are naturally occurring single-domain antibodies, referred to as heavy-chain antibodies 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 (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camels can produce naturally occurring heavy-chain antibodies lacking a light chain; such single-domain antibodies are within the scope of this invention.

[0464] In some embodiments, the anti-PD-L1 antibody of the present invention is a humanized antibody. Different methods for humanizing antibodies are known to those skilled in the art, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633). Almagro & Fransson distinguish between rational and empirical methods. Rational methods are characterized by generating a small number of engineered antibody variants and evaluating their binding or any other properties of interest. If the designed variant does not produce the expected results, a new round of design and binding evaluation is initiated. Rational methods include CDR grafting, resurfacing, superhumanization, and human string content optimization. In contrast, empirical methods are based on generating a large library of humanized variants and using enrichment techniques or high-throughput screening to select the best clones. Thus, empirical methods rely on reliable selection and / or screening systems capable of searching 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 those skilled in the art. Empirical methods include FR libraries, guided selection, framework-shuffling, and humaneering.

[0465] In some embodiments, the anti-PD-L1 antibody of the present invention is a human antibody. Human antibodies can be prepared using various 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, human monoclonal antibodies can be produced using transgenic mice carrying human immunoglobulin genes instead of mouse systems (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).

[0466] In some embodiments, the anti-PD-L1 antibody of the present invention is a non-human antibody, such as a rodent (mouse or rat) antibody, a goat antibody, a primate (e.g., monkey) antibody, or a camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat) antibody. Methods for generating rodent antibodies are known in the art.

[0467] In some embodiments, the antibody of the present 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 (e.g., 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 changed compared to the class or subclass of the parent antibody. The chimeric antibody includes its antigen-binding fragment.

[0468] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains, wherein, for example, the CDR (or a portion thereof) is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may optionally also contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from the non-human antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0469] The antibodies of the present invention can be isolated by screening for antibodies with the desired activity in a combinatorial library. For example, various methods for generating phage display libraries and screening for antibodies with the desired binding characteristics in these libraries are known in the art. These methods are described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, 2001), and further described, 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, 2001). 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).

[0470] 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, each binding to the same epitope.

[0471] In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first and second epitopes are located on the same antigen (e.g., the same protein (or a 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 located on different antigens (e.g., 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.

[0472] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody is specific to no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity against a first epitope and a second immunoglobulin variable domain sequence having binding specificity against a second epitope. In one embodiment, the first and second epitopes are on the same antigen (e.g., the same protein (or a 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 (e.g., 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 against a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity against a second epitope. In one embodiment, the bispecific antibody molecule comprises a hapten having binding specificity against a first epitope and a hapten having binding specificity against a second epitope. In one embodiment, the bispecific antibody molecule comprises a hapten or a fragment thereof having binding specificity against a first epitope and a hapten or a fragment thereof having binding specificity against a second epitope. In one embodiment, the bispecific antibody molecule comprises an scFv or a fragment thereof having binding specificity against a first epitope and an scFv or a fragment thereof having binding specificity against a second epitope. In one embodiment, the first epitope is located on PD-L1 and the second epitope is located on LAG-3, OX40, TIM-3, CEACAM (e.g., CEACAM-1 and / or CEACAM-5), or PD-L2.

[0473] In some embodiments, the invention also covers anti-PD-L1 monoclonal antibodies (“immunoconjugates”) conjugated with other substances, such as therapeutic modules or markers, like cytotoxic agents or immunomodulators. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art, see, for example, WO2015 / 153513 or WO2015 / 138920, etc. For example, cytotoxic agents include, but are not limited to: radioactive isotopes (e.g., iodine (131I or 125I), yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (211At), rhenium (186Re), bismuth (212Bi or 213Bi), indium (111In), technetium (99mTc), phosphorus (32P), rhodium (188Rh), sulfur (35S), carbon (14C), tritium (3H), chromium (51Cr), chlorine (3... 6Cl), cobalt (57Co or 58Co), iron (59Fe), selenium (75Se), or gallium (67Ga). Examples of cytotoxic agents also include chemotherapeutic agents or other therapeutic agents, such as paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucoside, epipodophyllotoxin thiophene glycoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, scintillan, actinomycin D. 1-Dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. Cytotoxic agents also include, for example: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., nitrogen mustard, thioepachlorambucil, phenylalanine mustard, etc.). Carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptomycin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP), cisplatin, ampicillin derivatives (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., actinomycin D (formerly known as actinomycin), bleomycin, scintillans and atrazomycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0474] Other substances that can be conjugated / coupled with anti-PD-L1 antibodies include, for example, WO2010 / 077634, US60 / 696426, WO2016 / 007235, or WO2016 / 061142.

[0475] The nucleic acid of the present invention and the host cell containing it.

[0476] In one aspect, the present invention provides a nucleic acid encoding any of the above-mentioned anti-PD-L1 antibodies or fragments thereof. The nucleic acid may encode an amino acid sequence comprising the light chain variable region and / or the heavy chain variable region of the antibody, or an amino acid sequence comprising the light chain and / or the heavy chain of the antibody.

[0477] 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 NO: 26 to 51, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 26 to 51.

[0478] The present invention also covers nucleic acids that hybridize under stringent conditions or that have one or more substitutions (e.g., conservative substitutions), deletions, or insertions with the following nucleic acids: nucleic acids comprising a nucleic acid sequence encoding an amino acid sequence selected from any one of SEQ ID NO: 26 to 51; or nucleic acids comprising a nucleic acid sequence encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 26 to 51.

[0479] In one embodiment, one or more vectors containing 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, kinases, λ phages, or yeast artificial chromosomes (YAC). Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rouse sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki forest virus, Eastern equine encephalitis virus, and flaviviruses. In a preferred embodiment, the expression vector of the present invention is a pTT5 expression vector.

[0480] Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0481] Once the expression vector or DNA sequence for expression has been prepared, it can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector used and the mammalian host cells.

[0482] In one embodiment, a host cell is provided comprising a nucleic acid encoding an antibody molecule described herein or a vector described herein. Suitable host cells for cloning or expressing the nucleic acid or vector encoding the antibody include prokaryotic or eukaryotic cells as described herein. Antibodies may be produced, for example, in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523, and also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, editor, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*. After expression, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified. In one embodiment, the host cell is an *E. coli* cell.

[0483] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., human cells), insect cells, plant cells, or other cells suitable for preparing antibodies or their antigen-binding fragments. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathways have been “humanized” to result in the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates).

[0484] Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to suit suspension growth can be used. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney lines (293HEK or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36: 59 (1977)); and others. 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, NSO, and Sp2 / 0.

[0485] A review of suitable mammalian host cell lines for antibody production can be found, 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 (e.g., PERC6 cells from Crucell), oocytes, and cells from transgenic animals, such as mammary epithelial cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0486] The method for preparing the antibody and its antigen-binding fragment of the present invention

[0487] The anti-PD-L1 antibody disclosed herein can be produced by recombinant synthesis. Several methods for producing recombinant antibodies are known in the art. One example of a method for producing antibodies by recombinant synthesis is disclosed in U.S. Patent No. 4,816,567.

[0488] In one embodiment, a method for preparing an anti-PD-L1 antibody is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium). To recombinantly generate an anti-PD-L1 antibody, the nucleic acid encoding the antibody (e.g., the antibody described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0489] In one embodiment, the host cell comprises a vector containing nucleic acid encoding the amino acid sequence of VL (antibody) and the amino acid sequence of VH (antibody). In another embodiment, the host cell comprises a first vector containing nucleic acid encoding the amino acid sequence of VL (antibody) and a second vector containing nucleic acid encoding the amino acid sequence of VH (antibody).

[0490] Determination method

[0491] The anti-PD-L1 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. On one hand, the antigen-binding activity of the antibodies of the present invention can be tested, for example by known methods such as ELISA, Western blotting, flow cytometry, and magnetic beads containing antibody molecules. PD-L1 binding can be measured using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, bio-optical interferometry (e.g., Fortebio affinity measurement), MSD assay, or flow cytometry is used.

[0492] On the other hand, competitive assays can be used to identify antibodies that compete with any of the anti-PD-L1 antibodies disclosed herein for binding to PD-L1. In some embodiments, such competitive antibodies bind to the same epitope (e.g., linear or conformational epitopes) as any of the anti-PD-L1 antibodies disclosed herein. Detailed illustrative methods for locating the epitopes bound by antibodies are given in Morris (1996), “Epitope Mapping Protocols,” Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0493] The present invention also provides an assay 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.

[0494] In some embodiments, the antibodies of the present invention are tested for one or more of the properties described above.

[0495] Cells used for any of the above in vitro assays include cells or cell lines that naturally express PD-L1 or are engineered to express PD-L1.

[0496] It is understood that the immunoconjugates of the present invention can be used to replace or supplement the anti-PD-L1 antibody for any of the above-described assays.

[0497] Understandably, anti-PD-L1 antibodies and other therapeutic agents can be used to perform any of the above assays.

[0498] Pharmaceutical compositions and pharmaceutical preparations

[0499] The present invention also includes compositions comprising anti-PD-L1 antibodies or fragments thereof or their immunoconjugates (including pharmaceutical compositions or pharmaceutical formulations) and compositions comprising nucleic acids encoding anti-PD-L1 antibodies or fragments thereof. In some embodiments, the compositions comprise one or more antibodies or fragments thereof that bind to PD-L1 or their immunoconjugates thereof, or one or more nucleic acids encoding one or more antibodies or fragments thereof that bind to PD-L1. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, excipients, etc., known in the art, including buffers.

[0500] The pharmaceutical carriers suitable for use in this invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0501] Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. For further information on the use and applications of excipients, see "Handbook of Pharmaceutical Excipients," 5th Edition, R.C. Rowe, P.J. Seskey, and S.C. Wen, Pharmaceutical Press, London, Chicago.

[0502] If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers.

[0503] These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard carriers and / or excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, and saccharin.

[0504] Pharmaceutical formulations containing the anti-PD-L1 antibody described herein can be prepared by mixing the anti-PD-L1 antibody of the present invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

[0505] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, the latter comprising a histidine-acetate buffer.

[0506] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anticancer active ingredients, such as chemotherapeutic agents and / or cytotoxic agents. The active ingredients are suitably combined in amounts effective for the intended use. The active ingredient can be any substance known in the art capable of combining with antiPD-L1 antibodies, including chemotherapeutic agents, other antibodies, and other therapeutic agents. Examples of such active ingredients can be found, for example, WO2010 / 077634, WO2016 / 061142, US61 / 264061, US60 / 696426, WO2016 / 007235, etc.

[0507] In some embodiments, the active ingredient is an anti-LAG-3 antibody, such as a human anti-LAG-3 antibody; preferably, the anti-LAG-3 antibody is humanized.

[0508] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0509] Uses of antibodies

[0510] In one aspect, the present invention relates to a method for modulating an immune response in a subject. The method comprises administering an effective amount of an antibody molecule (e.g., an anti-PD-L1 antibody) or pharmaceutical composition or immunoconjugate disclosed herein to the subject, thereby modulating an immune response in the subject. In one embodiment, the antibody molecule (e.g., a therapeutically effective amount of an anti-PD-L1 antibody molecule) or pharmaceutical composition or immunoconjugate disclosed herein restores, enhances, stimulates, or increases an immune response in the subject.

[0511] In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of an antibody molecule (e.g., an anti-PD-L1 antibody) or a pharmaceutical composition or immunoconjugate disclosed herein. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is a gastrointestinal tumor (e.g., cancer), such as colon cancer.

[0512] In another aspect, the present invention relates to a method for preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an antibody molecule (e.g., an anti-PD-L1 antibody) or a pharmaceutical composition or immunoconjugate disclosed herein. In one embodiment, the infectious disease is a chronic infection.

[0513] In another aspect, the present invention relates to a method for inducing antibody-dependent cell-mediated cytotoxicity in a subject, the method comprising administering to the subject an effective amount of an antibody molecule (e.g., an anti-PD-L1 antibody) or a pharmaceutical composition or immunoconjugate disclosed herein.

[0514] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient with or at risk of having the disease described herein). In one embodiment, the subject is required to enhance an immune response. In some embodiments, the anti-PD-L1 antibody molecule described herein enhances T cell proliferation. In some embodiments, the anti-PD-L1 antibody molecule described herein restores, enhances, or stimulates an antigen-specific T cell response in the subject, such as the production of interleukin-2 (IL-2) or interferon-γ (IFN-γ) in an antigen-specific T cell response. In some embodiments, the immune response is an anti-tumor response. In one embodiment, the subject has or is at risk of having the disease described herein (e.g., a tumor or infectious disease as described herein). In some embodiments, the subject is immunocompromised or at risk of being immunocompromised. For example, the subject has received or has received chemotherapy and / or radiation therapy. Alternatively or in combination, the subject is immunocompromised or at risk of being immunocompromised due to an infection.

[0515] In some implementations, the tumors described herein, such as cancers, include, but are not limited to, solid tumors, hematologic malignancies, soft tissue tumors, and metastatic lesions.

[0516] Examples of solid tumors include malignant tumors, such as sarcomas and carcinomas (including adenocarcinoma and squamous cell carcinoma) affecting multiple organ systems, such as those invading the liver, lungs, breast, lymph nodes, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., kidney, bladder epithelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell 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 region, oral cavity, anus, and cervix. In one embodiment, the cancer is melanoma, for example, 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 present invention.

[0517] Non-limiting examples of preferred cancers for treatment include lymphomas (e.g., diffuse large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma), breast cancers (e.g., metastatic breast cancer), lung cancers (e.g., non-small cell lung cancer (NSCLC), such as stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma), myelomas (e.g., multiple myeloma), leukemias (e.g., chronic myeloid leukemia), skin cancers (e.g., melanomas (e.g., stage III or IV melanoma) or Merkel cell carcinoma), head and neck cancers (e.g., head and neck squamous cell carcinoma (HNSCC)), spinal dysplasia syndromes, bladder cancers (e.g., transitional cell carcinoma), kidney cancers (e.g., renal cell carcinoma, such as clear cell renal cell carcinoma, such as advanced or metastatic clear cell renal cell carcinoma), and colon cancer. Additionally, refractory or recurrent malignancies may be treated with the antibody molecules described herein.

[0518] Examples of other treatable cancers include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastroesophageal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, and chronic or acute leukemia, including acute myeloid leukemia. Chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodysplastic syndrome, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos (e.g., mesothelioma), and combinations of the aforementioned cancers.

[0519] The antibody molecules described herein can be used to treat metastatic cancers (e.g., metastatic cancers expressing PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144)).

[0520] The antibody molecules described in this article can also be used to treat tumor immune escape.

[0521] In one implementation, the tumor is a cancer that expresses elevated levels of PD-L1.

[0522] In some implementations, the cancer described herein is colon cancer and its metastatic cancers.

[0523] In some embodiments, the infection is acute or chronic. In some embodiments, the chronic infection is persistent, latent, or slow-growing. In some embodiments, the chronic infection is caused by a pathogen selected from bacteria, viruses, fungi, and protozoa.

[0524] In some embodiments, the pathogen is a bacterium. In one embodiment, the bacteria are selected from: species of the genera *Mycobacterium*, *Salmonella*, *Listeria*, *Streptococcus*, *Haemophilus*, *Neisseria*, *Klebsiella*, *Borrelia*, *Bacterioides fragillis*, *Treponema*, and *Helicobacter pylori*.

[0525] In some embodiments, the pathogen is a virus. In one embodiment, the virus is selected from: infectious viruses, such as hepatitis B virus or hepatitis C virus, herpes simplex virus-I,-II, human immunodeficiency virus-I,-II, cytomegalovirus, Epstein-Barr virus, human papillomavirus, human T-lymphoblastic viruses-I or-II, and varicella-zoster virus.

[0526] In some embodiments, the pathogen is a fungus. In one embodiment, the fungal disease is selected from: aspergilosis, blastomycosis, candidiasis albicans, coccidioidomycosis immitis, histoplasmosis, paracoccidioiomycosis, and microsporidiosis.

[0527] In some embodiments, the pathogen is a protozoan, such as a parasite. In one embodiment, the disease caused by the protozoan is selected from leishmaniasis, plasmodiosis (i.e., malaria), cryptosporidiosis, toxoplasmosis, trypanosomiasis, and helminth infection, including those caused by trematodes (e.g., schistosomiasis), cestodes (e.g., echinococcosis), and nemotodes (e.g., trichinosis, ascariasis, filariosis, and strongylodiosis).

[0528] In another embodiment, the infection is a hepatitis infection, such as hepatitis B or hepatitis C. The anti-PD-L1 antibody molecule can be combined with conventional treatment for hepatitis B or hepatitis C infection for therapeutic advantage. In some embodiments, the anti-PD-L1 antibody molecule is administered in combination with hepatitis B antigen (e.g., Engerix B) or a vaccine, and optionally in combination with an aluminum-containing adjuvant.

[0529] In another implementation, the infectious disease is influenza. In some implementations, anti-PD-L1 antibody molecules are administered in combination with influenza antigens or vaccines.

[0530] For diseases suitable for prevention or treatment with the anti-PD-L1 antibody or fragment thereof of the present invention, see further references to WO2010 / 077634, WO2016 / 061142, US60 / 696426, WO2016 / 007235 or US61 / 264061.

[0531] In other respects, the present invention provides the use of anti-PD-L1 antibodies or fragments thereof or their immunoconjugates in the production or preparation of medicaments for the prevention or treatment of the aforementioned related diseases or conditions.

[0532] In some embodiments, the antibodies or antibody fragments or immune conjugates of the present invention may delay the onset of symptoms and / or symptoms associated with the disease.

[0533] combination therapy

[0534] In some embodiments, the preventive or treatment methods described herein also include administration to the subject or individual in combination of an antibody molecule (e.g., an anti-PD-L1 antibody or a fragment thereof) or a pharmaceutical composition or immunoconjugate disclosed herein, and one or more other therapies, such as modes of treatment and / or other therapeutic agents.

[0535] In some implementations, the treatment modality includes surgery (e.g., tumor resection); radiotherapy (e.g., external beam therapy, which involves three-dimensional conformal radiotherapy in which the irradiation area is designed), local irradiation (e.g., irradiation directed at a pre-selected target or organ), or focused irradiation, etc. Focused irradiation can be selected from stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiotherapy. Focused irradiation can have a radiation source selected from particle beams (protons), cobalt-60 (photons), and linear accelerators (X-rays), for example, as described in WO 2012 / 177624.

[0536] Radiation therapy can be administered through one or a combination of several methods, including but not limited to external 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 confined radioactive material inserted into or near a tumor or other site of proliferative tissue disease. This term is intended, but is not limited to, exposure to radioactive isotopes (e.g., At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and Lu). Suitable radiation sources include solids and liquids. By way of non-limiting example, the radiation source can be a radionuclide, such as I-125, I-131, Yb-169, Ir-192 as a solid source, I-125 as a solid source, or other radionuclides emitting photons, beta particles, gamma radiation, or other therapeutic rays. Radioactive materials can also be fluids made from any radionuclide solution, such as I-125 or I-131 solutions, or radioactive fluids can be generated from slurries of suitable fluids containing small particles of solid radionuclides (such as Au-198, Y-90). Additionally, radionuclides can be contained in gels or radioactive microspheres.

[0537] In some implementations, the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules).

[0538] Exemplary cytotoxic agents include antimicrotubule drugs, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vincristine alkaloids, intercalating agents, active agents that can interfere with signal transduction pathways, pro-apoptotic active agents, proteasome inhibitors, and irradiation (e.g., local or systemic irradiation, such as gamma radiation).

[0539] Other exemplary antibodies include, but are not limited to, immune checkpoint inhibitors (e.g., anti-CTLA-4, anti-TIM-3, anti-CEACAM, or anti-LAG-3); antibodies that stimulate immune cells (e.g., agonistic GITR antibodies or CD137 antibodies); and anticancer antibodies (e.g., rituximab). or trastuzumab Tosimomumab Temozolomide Alpha-1 monoclonal antibody Iprazizumab bevacizumab Erlotinib cetuximab etc.

[0540] Exemplary chemotherapy agents include, but are not limited to, anastrozole. Bicalutamide Bleomycin sulfate Bai Xiaoan Busulfan Injection Capecitabine N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin Camustin Chlorinated nitrogen mustard Cisplatin Klaribine Cyclophosphamide ( or ), cytarabine, cytosine arabinoside Cytarabine liposome injection Dacarbazine Dactinomycin (actinomycin D, Cosmegan), Daunomycin hydrochloride Donomycin Citrate Liposome Injection Dexamethasone, Docetaxel Doxorubicin Hydrochloride Etoposide Fludarabine phosphate 5-Fluorouracil Flutamide tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea Idabi Star Ifosfamide Irinotecan L-asparaginase Calcium leucovorin, melphalan 6-Mercaptopurine Methotrexate Mitoxantrone, Mylotarg, Paclitaxel Phoenix (Yttrium 90 / MX-DTPA), Pentostatin, and Carmustine 20 combined implants Tamoxifen Citrate teniposide 6-Thioguanine, thiotepa, telazamine Topotecan Hydrochloride for Injection Vincristine Changchun New Alkali Vinorelbine, ibrutinib, Gilead (idelalisib), and belentuximab vedotin.

[0541] Exemplary vaccines include, but are not limited to, cancer vaccines. Vaccines can be DNA-based, RNA-based, or virus-transduced. Cancer vaccines can be preventative 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, multiple peptides, peptide mixtures, hybrid peptides, or a dendritic cell vaccine via peptide pulses (see, for example, Yamada et al., Cancer Sci, 104:14-21, 2013).

[0542] Exemplary anti-infective agents include, but are not limited to, antiviral agents, antifungal agents, antiprotozoal agents, and antimicrobial agents, such as nucleoside analogs zidovudine (AST), ganciclovir, phosphonoformic acid, or cidovir, as described above.

[0543] Immunomodulators include immune checkpoint molecule inhibitors and co-stimulatory molecule activators.

[0544] In some embodiments, inhibitors of immune checkpoint molecules are inhibitors of PD-1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM (e.g., CEACAM-1, -3, and / or -5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and / or TGFR β. Inhibition of the molecules can be performed at the DNA, RNA, or protein level. In some embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA) can be used to inhibit the expression of immune checkpoint molecules. In other embodiments, the inhibitor of the immune checkpoint molecule is a polypeptide that binds to the immune checkpoint molecule, such as a soluble ligand (e.g., PD-1-Ig or CTLA-4Ig), or an antibody or its antigen-binding fragment; for example, an antibody or fragment thereof bound to PD-1, PD-L2, CEACAM (e.g., CEACAM-1, -3 and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and / or TGFRβ or combinations thereof. In other embodiments, the immunomodulator is an inhibitor of CEACAM (e.g., CEACAM-1, -3 and / or -5) (e.g., human CEACAM (e.g., CEACAM-1, -3 and / or -5)). In other embodiments, the immunomodulator is an inhibitor of LAG-3 (e.g., human LAG-3). In one embodiment, the LAG-3 inhibitor is an antibody molecule targeting LAG-3, such as a human anti-LAG-3 antibody, preferably humanized. In other embodiments, the immunomodulator is an inhibitor of TIM-3 (e.g., human TIM-3). In one embodiment, the TIM-3 inhibitor is an antibody molecule targeting TIM-3.

[0545] In some embodiments, the immunomodulator is an activator or agonist of a co-stimulatory molecule. In one embodiment, the agonist of the co-stimulatory molecule is selected from agonists of the following molecules (e.g., agonistic antibodies or their antigen-binding fragments, or soluble fusions): 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 ligands. In other embodiments, the immunomodulator is a GITR agonist. In one embodiment, the GITR agonist is an antibody molecule targeting GITR. In other embodiments, the immunomodulator is an OX40 agonist. In one embodiment, the OX40 agonist is an antibody molecule targeting OX40.

[0546] In further embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention can also be used in combination with tyrosine kinase inhibitors (e.g., receptor tyrosine kinase (RTK) inhibitors). Exemplary tyrosine kinase inhibitors include, but are not limited to, epidermal growth factor (EGF) pathway inhibitors (e.g., epidermal growth factor receptor (EGFR) inhibitors), vascular endothelial growth factor (VEGF) pathway inhibitors (e.g., vascular endothelial growth factor receptor (VEGFR) inhibitors (e.g., VEGFR-1 inhibitors, VEGFR-2 inhibitors, VEGFR-3 inhibitors), platelet-derived growth factor (PDGF) pathway inhibitors (e.g., platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., PDGFR-β inhibitors)), RAF-1 inhibitors, KIT inhibitors, and RET inhibitors.

[0547] In some embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention can also be used in combination with PI3K inhibitors, mTOR inhibitors, BRAF inhibitors, MEK inhibitors and / or JAK2 inhibitors.

[0548] In some embodiments of any method of the present invention, the administration of the anti-PD-L1 antibody or a fragment thereof is combined with the administration of an antigen. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived 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.

[0549] In some embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention may be administered in combination with a treatment comprising adoptive transfer of T cells expressing chimeric antigen receptors (CARs), such as cytotoxic T cells or CTLs.

[0550] In some embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention may be administered in combination with an antitumor agent.

[0551] In some embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention may be administered in combination with an oncolytic virus. In some embodiments, the oncolytic virus is capable of selectively replicating in cancer cells and triggering cancer cell death or delaying their growth. In some cases, the oncolytic virus has no effect or minimal effect on non-cancer cells. Oncolytic viruses include, but are not limited to, oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic synderby virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle disease virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)). In some embodiments, the oncolytic virus is the virus described in US 2010 / 0178684A1, for example, a recombinant oncolytic virus.

[0552] In some embodiments, the anti-PD-L1 antibody or fragment thereof of the present invention may be administered in combination with cytokines.

[0553] In some embodiments, the antibodies or fragments thereof of the present invention may be combined with conventional cancer therapies in the art, including but not limited to: (i) radiotherapy (e.g., radiotherapy, X-ray therapy, irradiation) or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy may be administered via external beam radiation therapy (EBRT) or internal brachytherapy; (ii) chemotherapy, or the application of cytotoxic drugs that generally affect rapidly dividing cells; (iii) targeted therapy, or agents that specifically affect the deregulation of cancer cell proteins (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, or agents that enhance the host's immune response (e.g., vaccines); (v) hormone therapy, or blocking hormones (e.g., when the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, or agents that block angiogenesis and growth; and (vii) palliative care, or treatments that involve improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding, where pain medications such as morphine and oxycodone, and antiemetics such as ondansetron and aprepitant, allow for more aggressive treatment regimens.

[0554] In some embodiments, the antibodies or fragments thereof of the present invention can be combined with conventional methods for enhancing host immune function, including but not limited to: (i) APC enhancement, such as (a) injecting DNA encoding heterologous MHC alloantigens into the tumor, or (b) transfecting biopsied tumor cells with genes that increase the likelihood of immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, co-stimulatory molecules 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 IL-2 or tumor stimulation or both, to expand this population in vitro; furthermore, dysfunctional isolated T cells can also be activated in vitro by applying the antibodies of the present invention, and the activated T cells can then be re-administered to the host.

[0555] The various combination therapies described above can be further combined for treatment.

[0556] More examples of combinations of anti-PD-L1 antibodies with other treatment modalities or agents can be found in WO2016 / 061142, WO2010 / 077634, US60 / 696426, US61 / 264061, or WO2016 / 007235, etc.

[0557] Such combination therapies encompass combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration, in which case the antibody of the present invention may be administered before, simultaneously with, and / or after the administration of other therapies, such as modalities and / or therapeutic agents. The antibody molecule and / or other therapies, such as therapeutic agents or modalities, may be administered during active disease or during remission or less active disease. The antibody molecule may be administered before, simultaneously with, after, or during disease remission of other treatments.

[0558] In one implementation, the administration of the anti-PD-L1 antibody and the administration of other therapies (e.g., treatment modalities or therapeutic agents) occur within each other within about one month, or about one, two, or three weeks, or about one, two, three, four, five, or six days.

[0559] In some implementations, the antibody combinations described herein can be administered separately, for example, as individual antibodies, or conjugated (e.g., as bispecific or trispecific antibody molecules).

[0560] It is understood that the immune conjugates of the present invention can be used to replace or supplement anti-PD-L1 antibodies for any treatment.

[0561] Combination therapy with anti-LAG-3 antibodies

[0562] In some embodiments, the anti-PD-L1 antibody of the present invention can be used in combination with the anti-LAG-3 antibody for treatment.

[0563] In some embodiments, the anti-LAG-3 antibody of the present invention is an anti-human LAG-3 antibody. In some embodiments, the anti-LAG-3 antibody of the present invention is an antibody in the form of IgG1, IgG2, or IgG4. 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 common framework sequence. In one embodiment, the anti-LAG-3 antibody of the present invention also encompasses its antibody fragments, preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv) or (Fab')2, single-domain antibody, bispecific antibody (dAb), or linear antibody.

[0564] In some specific embodiments, the anti-LAG-3 antibody or its antigen-binding fragment of the present invention comprises...

[0565] (i) the three complementary determinant regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 75, and / or

[0566] (ii) The three complementary determinant regions LCDR of the light chain variable region as shown in SEQ ID NO: 76.

[0567] In some embodiments, the anti-LAG-3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein

[0568] (i) The VH includes complementarity-determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 69; HCDR2 contains or is composed of the amino acid sequence selected from SEQ ID NO: 70; and HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 71.

[0569] and / or

[0570] (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 72; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 73; and LCDR3 comprises or is composed of the amino acid sequence selected from SEQ ID NO: 74.

[0571] In some embodiments, the anti-LAG-3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region VH and / or a light chain variable region VL, wherein,

[0572] (a) Heavy chain variable region VH

[0573] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 75; or

[0574] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 75; or

[0575] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 75, preferably, the amino acid changes do not occur in the CDR region;

[0576] and / or

[0577] (b) Light chain variable region VL

[0578] (i) Containing or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from SEQ ID NO: 76;

[0579] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 76; or

[0580] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 76, preferably, the amino acid changes do not occur in the CDR region.

[0581] In some embodiments, the anti-LAG-3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain and / or a light chain, wherein

[0582] (a) Heavy chain

[0583] (i) Containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 77;

[0584] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 77; or

[0585] (iii) An amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 77, preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, the amino acid changes do not occur in the variable region of the heavy chain.

[0586] and / or

[0587] (b) Light chain

[0588] (i) Containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 78;

[0589] (ii) Contains or consists of an amino acid sequence selected from or composed of SEQ ID NO: 78; or

[0590] (iii) An amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 78, preferably, the amino acid changes do not occur in the CDR region of the light chain, more preferably, the amino acid changes do not occur in the variable region of the light chain.

[0591] In some embodiments, the modifications of the present invention for anti-PD-L1 antibodies are also applicable to anti-LAG-3 antibodies.

[0592] Route of administration and dosage

[0593] The antibodies of the present invention (and pharmaceutical compositions or immunoconjugates comprising them, and any other therapeutic agents) may be administered by any suitable method, including parenteral administration, intrapulmonary administration, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Depending to some extent on whether the administration is short-term or long-term, it may be administered via any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various administration schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0594] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient as a single treatment or after a series of treatments.

[0595] In some embodiments, the dosing regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of dosing and uniformity. As used herein, unit dosage form refers to physically discrete units suitable as a single dose for the subject to be treated; each unit contains a predetermined amount of the active compound, which, when calculated, produces the desired therapeutic effect when combined with the required pharmaceutical carrier. The specifications of the unit dosage form used in this invention depend directly on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations specific to the field of mixing such active compounds for sensitive treatment in individuals.

[0596] The dosage and treatment regimen of the anti-PD-L1 antibody molecule can be determined by a technician. In some embodiments, the anti-PD-L1 antibody molecule is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 30 mg / kg, for example, about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The dosing regimen can be varied from, for example, 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 administered alone or in combination (e.g., in combination with an anti-LAG-3 antibody molecule) at doses 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; or less than or equal to about 1 mg / kg every other week. In one embodiment, the anti-PD-L1 antibody molecule is administered every other week 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, or every other week at a dose of about 1 to 2 mg / kg. In one embodiment, the anti-LAG-3 antibody molecule is administered alone or in combination (e.g., in combination with the anti-LAG-3 antibody molecule) at a dose of about 1 to 5 mg / kg every other week, 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, or every other week at a dose of about 1 to 2 mg / kg.

[0597] Methods and compositions for diagnosis and detection

[0598] In some embodiments, any anti-PD-L1 antibody or its antigen-binding fragment 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 (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is derived from hyperplastic or cancerous lesions.

[0599] In one embodiment, an anti-PD-L1 antibody is provided for use in a diagnostic or detection method. In another aspect, a method for detecting the presence of PD-L1 in a biological sample is provided. In some embodiments, the method includes detecting the presence of the PD-L1 protein in the biological sample. In some embodiments, PD-L1 is human PD-L1. In some embodiments, the method includes contacting the biological sample with an anti-PD-L1 antibody as described herein under conditions that allow the anti-PD-L1 antibody to bind to PD-L1, and detecting whether a complex is formed between the anti-PD-L1 antibody and PD-L1. The formation of the complex indicates the presence of PD-L1. This method can be in vitro or in vivo. In one embodiment, the anti-PD-L1 antibody is used to select subjects suitable for treatment utilizing the anti-PD-L1 antibody, for example, where PD-L1 is a biomarker used for selecting said subjects.

[0600] In one embodiment, the antibodies of the present invention can be used to diagnose cancer or tumors, such as to evaluate (e.g., monitor) the treatment or progression, diagnosis, and / or staging of the disease described herein (e.g., hyperplastic or cancerous disease) in a subject. In some embodiments, labeled anti-PD-L1 antibodies are provided. Labeling includes, but is not limited to, labels or portions that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioactive isotopes 32P, 14C, 125I, 3H, and 131I; fluorophores such as rare earth chelates or luciferin and its derivatives; rhodamine and its derivatives; dansyl; umbelliferone; luciferase, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456); luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HR); alkaline phosphatase; β-galactosidase; glucosylamylase; lysozyme; carbohydrate oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as uricase and xanthine oxidase; and enzymes utilizing hydrogen peroxide dye precursors such as HR, lactoperoxidase, or microperoxidase; biotin / avidin; spin labeling; phage labeling; stable free radicals; and so on.

[0601] 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 and paraffin-coated (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine-needle aspirate.

[0602] In some implementations, PD-L1 is detected before treatment, for example, before initiating treatment or before a treatment after a treatment interval.

[0603] In some embodiments, a method for treating a tumor or infection is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample containing cancer cells) for the presence of PD-L1, thereby determining a PD-L1 value; comparing the PD-L1 value with a control value; and if the PD-L1 value is greater than the control value, administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody (e.g., the anti-PD-L1 antibody described herein) optionally in combination with one or more other therapies, thereby treating the tumor or infection.

[0604] The sequence of an exemplary anti-PD-L1 antibody of the present invention

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612] These and other aspects and embodiments of the invention are described in the accompanying drawings (briefly described below) and the following detailed description of the invention, and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art. Example

[0613] Example 1. Preparation of hybridoma cells

[0614] Hybridoma technology involves fusing two cell types while simultaneously preserving the key characteristics of both: mouse spleen cells immunized with a specific antigen and mouse myeloma cells. Mouse spleen cells (B lymphocytes) immunized with a specific antigen are characterized by their antibody-secreting function but cannot be continuously cultured in vitro. Mouse myeloma cells, on the other hand, can proliferate indefinitely under culture conditions, exhibiting so-called immortality. Under selective culture conditions, only hybrid cells resulting from the fusion of B cells and myeloma cells can be continuously cultured, forming cell clones that simultaneously possess both antibody-secreting function and immortality. In this experiment, mice were immunized with hPD-L1 protein, and then mouse spleen cells and myeloma cells were fused to obtain hybridoma cells capable of expressing positive antibodies.

[0615] Hybridoma fusion

[0616] Laboratory animals and immune information

[0617]

[0618] Preparation of electrofusion dish: Thoroughly soak the electrofusion dish in 70% ethanol and dry it in a laminar flow hood for later use.

[0619] Spleen cell isolation: Mice were euthanized by cervical dislocation, and their bodies were disinfected with 75% alcohol for 5 minutes. They were then placed on a dissecting board in a laminar flow hood in a left lateral decubitus position, with their limbs secured using a No. 7 needle. The spleen was aseptically removed from the abdominal cavity and washed with basal culture medium (preparation method shown in the table below), carefully removing any surrounding connective tissue. The spleen was then transferred to another petri dish containing basal culture medium. A curved needle was used to hold the spleen in place, and a small needle was used to make a puncture in the spleen. The spleen cells were then squeezed with forceps to fully release them, creating a spleen cell suspension. The cell suspension was filtered through a 100 μM cell strainer, washed once with 30 ml of basal culture medium, and centrifuged at 1200 rpm for 6 minutes.

[0620]

[0621] Lysing red blood cells: Remove the supernatant and resuspend the cells in 10 ml of RBC lysis buffer (GIBCO). Then add 20 ml of RBC lysis buffer. After incubating the suspension for 5 min, centrifuge at 1100 rpm for 6 min. Remove the supernatant and resuspend the cells in 10 ml of basal medium, then add 30 ml of basal medium and centrifuge at 1100 rpm for 6 min. Remove the supernatant, resuspend the cells in 20 ml of basal medium, and count them.

[0622] Electrofusion: Mouse myeloma SP2 / 0 cells (ATCC) were resuspended in 20 ml of basal culture medium and counted. SP2 / 0 cells 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). Then, 15 ml of fusion buffer was added, and the mixture was centrifuged at 1000 rpm for 5 min, and the supernatant was removed. After repeating the above steps once, the cells were reselected with an appropriate amount of fusion buffer to adjust the mixed cell density to 1×10⁶ cells / mL. 7 Cells / ml. The parameters for the electrofusion apparatus are set as follows. 2 ml of cell suspension is added to each electrofusion dish for electrofusion.

[0623] Alignment: 60V, 30sec Membrane breaking: 1500V, 30μs, 3X Post-fusion pulse: 60V, 3sec

[0624] Electrofusion followed by plating: Cells were incubated in an electrofusion dish at room temperature for 5 min. Cells were then transferred to centrifuge tubes and diluted to 1–2 × 10⁴ cells / ml with selection medium (preparation method shown in the table below). 100 μl of cell suspension was added to each well of a 96-well plate. The selection medium was replaced on day 7 post-fusion. Selection was performed on day 10 (or longer, depending on cell growth status). Hybridoma cells expressing specific anti-PD-L1 antibodies were screened using FACS (FACS ARIA (BD Biosciences)).

[0625]

[0626] Positive hybridoma cell subclones

[0627] Subcloning procedure: Prepare a 96-well plate. Add 200 μl of the basal culture medium as described above to each well in columns 2 to 8. Prepare a cell suspension from the positive wells selected by fusion screening and add it to column 1. Take 100 μl of the cell suspension from column 1 and add it to column 2. Mix thoroughly, then take another 100 μl and add it to the next column. Repeat the above steps until the volume of the last column is 300 μl. Let the 96-well plate stand for 15 min and observe and count under a microscope. Take the volume corresponding to 100 cells and add it to 20 ml of the basal culture medium as described above, mix well, and plate the plate, 200 μl per well. Observe under a microscope after one week, identify and mark the single-clone wells, and pick out the positive wells to be tested.

[0628] Cell cryopreservation: Observe cell status. When cells grow well and viability is >90%, centrifuge at 1000 rpm for 5 min and remove the supernatant. Resuspend cells in cryopreservation buffer (45.5% FBS, 44.5% RPMI-1640, 10% DMSO) to 1×10⁷ cells / ml, aliquot into cryovials, and place in a programmed cooling box for cryopreservation at -80℃.

[0629] Example 2. Production and purification of chimeric antibodies

[0630] This invention utilizes molecular biology techniques to obtain antibody sequences in anti-PD-L1 positive hybridoma cells and uses them to construct human-mouse chimeric antibodies.

[0631] Hybridoma sequencing

[0632] RNA extraction: Fresh cells were centrifuged at 300g for 5 min, the supernatant was removed, and 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. The mixture was transferred to a DNA removal tube, centrifuged at 13000 rpm for 2 min, and the flow-through was collected. 100% ethanol was added to the flow-through at a 1 / 2 ratio, and the mixture was mixed 5 times until clear. The clear solution was transferred to an RNA collection tube, centrifuged at 13000 rpm for 1 min to remove the liquid, 500 μl of RB (Recovery Buffer) (Takara) was added, centrifuged at 13000 rpm for 30 s, and then 500 μl of RNA washing buffer (Biomiga) (ethanol was added before use) was added, centrifuged for 30 s, and the above process was repeated once. After centrifugation to completely evaporate the ethanol, 30 μl of DEPC water was added to the collection column, centrifuged at 12000g for 2 min, and the eluent was collected. The RNA concentration was determined.

[0633] cDNA obtained by reverse transcription:

[0634] The reaction system I is configured as follows:

[0635]

[0636] *From PrimeScript II 1 st Strand cDNA Synthesis Kit, purchased from Takara.

[0637] After incubating at 65°C for 5 minutes, immediately cool on ice. Add the following reverse transcription mixture to reaction system I, in a total volume of 20 μl:

[0638]

[0639] *From PrimeScript II 1 st Strand cDNA Synthesis Kit, purchased from Takara.

[0640] After slow mixing, reverse transcription translation was performed under the following conditions: 42℃ for 60 min → 95℃ for 5 min, then cooled on ice to obtain cDNA.

[0641] Ligate cDNA to a T vector:

[0642] PCR was used to amplify the variable regions of the heavy and light chains separately. The PCR reaction system is as follows:

[0643]

[0644]

[0645] Take 4.5 μl of the PCR product obtained from the above PCR reaction, add 0.5 μl of pMD20-T vector (Clontech) and 5 μl of Ligation Mighty Mix (Takara), mix gently, and react at 37℃ for 2 h to obtain the ligation product.

[0646] Transformed cells:

[0647] Remove TOP10 competent cells (Tiangen Biotech (Beijing) Co., Ltd.) at -80℃, thaw on ice, and add 5 μl of the ligation product obtained above to the thawed TOP10 competent cells. Mix well and incubate on ice for 30 min. Heat shock at 42℃ for 90 s, then rapidly cool on ice for 2 min. Add 900 μl of LB medium (Sangon Biotech (Shanghai) Co., Ltd.) to the EP tube and incubate at 37℃, 220 rpm for 1 h on a shaker. Centrifuge at 3000g for 2 min, remove 800 μl of supernatant, resuspend the bacterial body in the remaining medium, and plate on an ampicillin-resistant plate. Incubate overnight at 37℃, and select clones for sequencing.

[0648] Constructing chimeric antibodies

[0649] PCR amplification of the VH and VL regions of the mouse anti-PD-L1 antibody generated from the hybridoma in Example 1, which has been sequenced: upstream and downstream primer sequences are shown in Tables 5 and 6.

[0650] Table 5. Heavy chain variable region (VH) primers for mouse anti-PD-L1 antibody (Primer Mix 1)

[0651]

[0652]

[0653] After mixing in the above proportions, Primer Mix 1 is obtained for subsequent VH PCR amplification.

[0654] Table 6. Light chain variable region (VL) primers for mouse anti-PD-L1 antibody (Primer Mix 2):

[0655]

[0656]

[0657] After mixing in the above proportions, Primer Mix 2 is obtained for subsequent VL PCR amplification.

[0658] The PCR system is as follows:

[0659]

[0660] *For VH chain amplification, use Primer Mix 1; for VL chain amplification, use Primer Mix 2.

[0661] The PCR amplification products were recovered by gel cutting.

[0662] Homologous recombination reaction:

[0663] The homologous recombination system is as follows:

[0664]

[0665] The reaction was carried out at 37℃ for 30 min to obtain the recombinant product. The recombinant product was transformed into TOP10 competent cells, and single clones were selected for sequencing. Clones containing plasmids with the correct insertion direction were selected as positive clones and preserved.

[0666] Expression and purification of chimeric antibodies

[0667] Plasmids containing anti-PD-L1 antibodies were extracted from the positive clones obtained above.

[0668] Passage 293F cells (Invitrogen) to the required transfection volume, and adjust the cell density to 1.5 × 10⁻⁶ cells the day before transfection. 6 Cells / ml. The cell density on the day of transfection was approximately 3 × 10⁻⁶ cells / ml. 6 Cells / ml. Take 1 / 10 of the final volume of F17 medium (Gibco, A13835-01) as transfection buffer, add an appropriate amount of plasmid, and mix well. Add a suitable amount of polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the plasmid to PEI ratio is 1:3 in 293F cells), mix well, and incubate at room temperature for 10 min to obtain a DNA / PEI mixture. Resuspend the cells in the DNA / PEI mixture and incubate at 36.5℃ with 8% CO2. After 24 h, add 2% of the transfection volume of FEED (Sigma) and culture at 36.5℃, 120 rpm, and 8% CO2. Continue culturing until day 6 or when cell viability is ≤60%, collect the cell supernatant for purification.

[0669] The gravity column used for purification was treated overnight with 0.5M NaOH. Glass bottles and other equipment were washed with distilled water and then dried at 180℃ for 4 hours to obtain the purification column. Before purification, the collected culture medium was centrifuged at 4500 rpm for 30 min, and the cells were discarded. The supernatant was then filtered through a 0.22 μl filter. Each tube was loaded with 1 ml of Protein A and equilibrated with 10 ml of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). The filtered supernatant was added to the purification column and reequilibrated with 15 ml of binding buffer. 5 ml of elution buffer (0.1 M citric acid + sodium citrate, pH 3.5) was added, and the eluent was collected. 80 μl of Tris-HCl was added to every 1 ml of eluent. The collected antibody was ultrafiltered and concentrated, then transferred to PBS (Gibco, 70011-044), and the concentration was determined.

[0670] Please refer to Tables 1-3 above for the CDR, light chain variable region, and heavy chain variable region of the four chimeric antibodies obtained in this invention, as well as the amino acid sequences and sequence numbers of the light and heavy chains.

[0671] The control antibody used in this invention is Roche's PD-L1 antibody Atezolizumab (hereinafter referred to as ATE or Ate, trade name Tecentriq), whose CDR, light chain variable region and heavy chain variable region, and the amino acid sequences of the light chain and heavy chain are also shown in Tables 1-3 above.

[0672] Example 2: Determination of the binding kinetics between the chimeric antibody and antigen of the present invention using bio-optical interferometry.

[0673] The equilibrium dissociation constant (KD) of the antibody of this invention binding to human PD-L1 was determined using the ForteBio method. The ForteBio affinity assay was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).

[0674] Half an hour before the start of the experiment, depending on the number of samples, take an appropriate number of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors and immerse them in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

[0675] 100 μl of SD buffer, antibody, and antigen (including human PD-L1, mouse PD-L1, and cynomolgus monkey PD-L1, all purchased from Acrobiosystems) were added to 96-well black polystyrene semi-mass microplates (Greiner, 675076). The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: Run steps: Baseline, Loading ~1 nm, Baseline, Association, and Dissociation; the run time for each step depended on the sample binding and dissociation rates, the rotation speed was 400 rpm, and the temperature was 30℃. KD values ​​were analyzed using ForteBio analysis software.

[0676] In the experiments described above, the affinities of antibodies 3-266.1, 4-79.2, 4-26.6, and 4-48.5 are shown in Table 7.

[0677] Table 7. Affinity constants (equilibrium dissociation constants) for ForteBio detection of antigen-antibody binding

[0678]

[0679] In the above experiments, the KD values ​​of 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 with the control group, the antibodies in this study had similar or better KD values. D value.

[0680] Example 3: Binding experiment of chimeric antibody and cells overexpressing PD-L1

[0681] This study used flow cytometry to detect the binding of serially diluted chimeric antibodies of the present invention to CHO stable cell lines overexpressing human PD-L1.

[0682] CHO cells overexpressing human PD-L1 (CHO-PDL1) were generated by transfecting CHO-S cells (Invitrogen, ExpiCHO™ Expression System Kit, catalog number: A29133) with pCHO1.0 vector (Invitrogen) containing human PD-L1 cDNA cloned to the multiple cloning site (MCS) (Sino Biological) into CHO-S cells.

[0683] CHO-PDL1 cells were counted and diluted to 1×10⁻⁶. 6Cells / ml, add 100 μl / well to a U-bottom 96-well plate. Centrifuge at 400g for 5 min to remove cell culture medium. Add samples (chimeric antibodies 3-266.1, 4-79.2, 4-26.6, 4-48.5, and positive control antibody Ate) (antibody dilution method: the highest antibody concentration was 500 nM, three-fold diluted in PBS containing 0.1% bovine serum albumin (BSA), a total of 8 concentrations were tested) to the U-shaped plate and resuspend the cells, 100 μl / well, incubate on ice for 30 min. Remove supernatant at 400g for 5 min, wash cells once with PBS. Remove PBS after incubating at 400g for 5 min. Add 100 μl of FITC-labeled anti-mouse Fab secondary antibody (Jackson Immuno Research) (1:500 diluted in PBS) to each well. Add 100 μl of FITC-labeled anti-human Fab secondary antibody (Jackson Immuno Research) to cells containing the positive control antibody. Incubate on ice in the dark for 30 min. Remove the supernatant after incubating at 400g for 5 min. Wash cells once with PBS. Resuspend cells in 100 μl of 1×PBS and analyze using FACS.

[0684] In the experiments described above, the binding of antibodies 3-266.1, 4-79.2, 4-26.6, 4-48.5 to CHO-PDL1 cells was as follows: Figure 1 As shown.

[0685] In the above experiments, antibodies 3-266.1, 4-79.2, 4-26.6, and 4-48.5 all bound to human PD-L1 overexpressed on CHO cells, with EC50 values ​​of 2.139 nM, 2.598 nM, 1.985 nM, and 1.995 nM, respectively. Compared with the control antibody ATE, they showed superior binding ability, with some antibodies exhibiting more than twice the binding capacity of the control antibody.

[0686] Example 4: Humanization of chimeric antibodies

[0687] The chimeric antibody obtained in Example 1 was humanized. The antibody humanization process was performed using Macromoltek's proprietary software program, SmrtMolHumanize. First, the sequence was input into the software, which generated a three-dimensional model of the sequence, and then humanized it through the following steps:

[0688] ① Determine the CDR ring structure;

[0689] ② Find the closest homologous sequence for each V / J region of the heavy and light chains in the phylogenetic sequence database;

[0690] ③ Screen for the most closely matched human lineages and the lowest possible amount of reversion mutations to the heavy and light chains;

[0691] ④ Construct the CDR region of the chimeric antibody onto the human backbone region;

[0692] ⑤ Use sequence and structural features to determine the amino acid positions in the backbone region that maintain CDR function;

[0693] ⑥ Perform a reverse mutation (reverting to the input amino acid type) at the identified important sequence positions;

[0694] ⑦ Generate a three-dimensional model of the humanized sequence;

[0695] ⑧ Manually examine sequences and structures to identify risk sites that may cause misfolding or reduced stability;

[0696] ⑨ Optimize amino acids at risk sites.

[0697] The CDR, light chain variable region, and heavy chain variable region of the four humanized antibodies (HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-IgG4PAK, and HZ4485-IgG1N297A) obtained in this invention are shown in Tables 1-3 above. The amino acid sequences of the light chain and heavy chain are shown in Tables 1-3 above.

[0698] Example 5: ForteBio determines the binding kinetics of humanized antibodies and antigens.

[0699] The equilibrium dissociation constant (KD) of the humanized antibodies of different Fc isotypes of this invention binding to human PD-L1 was determined using the ForteBio assay. The ForteBio affinity assay method was the same as in Example 2. In the experiments described above, the affinities of antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-IgG4PAAK, and HZ4485-IgG1N297A are shown in Table 8.

[0700] Table 8. Affinity constants for antigen-antibody binding detected by ForteBio

[0701]

[0702] In the above experiments, the K of the humanized antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-G4PAAK, and HZ4485-IgG1N297A described in this article was... D The values ​​were 7.24E-10M, 9.35E-10M, 1.32E-09M, and 3.17E-09M, respectively. Compared with the control group, the humanized antibody in this study had similar or better K values. D value.

[0703] Example 6: Binding experiment of humanized antibody and cells overexpressing PD-L1

[0704] This study used flow cytometry to detect the binding of serially diluted humanized antibodies of the present invention to CHO stable cell line (CHO-PDL1) overexpressing human PD-L1. The experimental method was the same as in Example 3, except that the antibodies used were humanized antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-G4PAAK, and HZ4485-IgG1N297A. The antibody dilution method was as follows: the highest antibody concentration was 500 nM, and it was three-fold diluted in PBS containing 0.1% bovine serum albumin (BSA). A total of eight concentrations of humanized antibodies HZ3266-IgG1N297A, HZ3266-IgG1, HZ3266-G4PAAK, HZ4485-IgG1N297A were tested for binding to CHO-PDL1 cells. Figure 2 As shown.

[0705] In the above experiments, the humanized antibodies HZ3266-IgG1, HZ3266-IgG1N297A, HZ3266-G4PAAK, and HZ4485-IgG1N297A bound to human PD-L1 overexpressed on CHO cells, with EC50 values ​​of 1.813 nM, 1.784 nM, 1.862 nM, and 1.561 nM, respectively, demonstrating stronger binding ability compared to the control antibody ATE.

[0706] Example 7: Detection of antibody biological activity using the MOA method

[0707] Anti-PD-1 / PD-L1 antibodies can relieve the inhibition of downstream NFAT signaling pathways by blocking the binding of PD-1 and PD-L1. This study used the MOA detection system (PD-1 / PD-L1 BlockadeBioassay, Cell Propagation Model, Catalog J1252) provided by Promega, following the method described in the manufacturer's instructions. The expression of fluorescent reporter genes was used to detect the activation of NFAT signaling, thereby assessing the inhibitory effect of the antibody on PD-1 / PD-L1 binding.

[0708] CHOK1-PDL1 cells (from the MOA assay system described above) were seeded one day before activity assay: Cells were passaged 1-2 days prior to seeding. The culture supernatant was discarded, and the cells were washed once with PBS (Gibco). An appropriate amount of Trypsin (Gibco) was added, and the cells were digested at 37°C and 5% CO2 for 3-5 minutes. Four times the volume of Trypsin-containing culture medium was added, and the cells were transferred to a 50ml centrifuge tube and counted. The desired volume of cells was taken, 230g, and centrifuged for 10 minutes. 1640 medium (Gibco) was added, and the cells were resuspended to 4 × 10⁵ cells / mL. Cells were added to 96-well Nunclon white cell culture plates, 100 μl / well. PBS was added to the side wells, 200 μl / well. The cells were cultured overnight at 37°C / 5% CO2.

[0709] Jurkat-PD1 cells (from the MOA assay system described above): Cell passage was performed two days prior to the viability assay. After counting, the required cell volume was taken, 170g, and centrifuged for 5 min. Cells were resuspended in assay buffer (1640 medium (Gibco) + 1% FBS) to a final volume of 1.25 × 10⁻⁶. 6 Cells / ml

[0710] Add samples and Jurkat-PD1 cells to the assay plate (from the above MOA assay system): Discard 95 μl / well CHOK1-PDL1 cell supernatant. Add 40 μl of sample (humanized antibodies HZ3266-IgG1, HZ3266-IgG1N297A, HZ4485-IgG1N297A prepared in this invention) and positive control (Ate), negative control (IgG1) (antibody dilution method: the highest antibody concentration is 100 nM, three-fold dilution in assay buffer, a total of 8 concentrations were tested). Add 40 μl of Jurkat-PD1 cells. Incubate at 37℃ / 5% CO2 for 6 hours.

[0711] Assay: Melt the Bio-Glo™ buffer (from the MOA detection system described above) beforehand, add the Bio-Glo™ substrate (from the MOA detection system described above), and mix well. After 6 hours, add the Bio-Glo™ reagent (from the MOA detection system described above), 80 μl / well. Incubate at room temperature for 5–10 min. Read the values.

[0712] In the above experiments, the experimental results are as follows: Figure 3 As shown, antibodies HZ3266-IgG1, HZ3266-IgG1N297A, and HZ4485_IgG1N297A can all effectively block the interaction between PD1 and PD-L1.

[0713] Example 8 Mixed Lymphocyte Experiment

[0714] This study co-incubated antibodies with mature dendritic cells (DCs) and CD4+ T cells cultured in vitro from different donors. By detecting the relative expression levels of IL2 and IFN-γ in the system, the activation effect of different antibodies on T cells was observed.

[0715] PBMC separation: Take 50ml of fresh blood from the donor, add 2.5 times the amount of PBS, and gently add to FiColl (Thermo). Divide into 4 tubes, 12.5ml each, centrifuge at 400g for 30min, and stop at zero deceleration. Aspirate the middle white band into PBS and wash twice with PBS.

[0716] DC cell isolation: Take the PBMC cells isolated as described above and add 5 ml of T cell culture medium (preparation method as shown in the table below). Incubate at 37℃, 6% CO2, and adhere for 2 hours. Take the suspended cell fluid for CD4+ cell isolation. Add 3 ml of DC culture medium (preparation method as shown in the table below) to the remaining cells. After culturing for 2 days, add 3 ml of DC culture medium and culture for another 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 GE2 (Tocris) and culture for 2 days to prepare DC cells for lymphocyte mixed reaction (MLR).

[0717]

[0718] CD4+ T cell isolation: The procedure was performed using the 'Untouched CD4+ T cell isolation' kit instructions (11346D, Invitrogen). PBMCs were incubated statically for 2 hours. The suspended cell solution was transferred to a 15 ml centrifuge tube and centrifuged at 200 g for 10 min. The pellet was resuspended in 500 μl of separation buffer, 100 μl of AB serum, and 100 μl of purified antibody. The pellet was incubated at 4 °C for 20 min, washed once with separation buffer, and then incubated for 15 min with 500 μl of Bead Buffer (Invitrogen). The bead was removed using a magnetic field, and the pellet was washed once with T cell culture medium. The pellet was resuspended in 8 ml of culture medium and incubated at 37 °C with 6% CO2.

[0719] MLR experiment: The mature DC cells obtained above were mixed with CD4+ cells, with a volume of 200 μl per well, containing 10,000 DC cells and 100,000 CD4+ cells. The antibodies of this invention (concentrations: 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM) were added. The DC cells prepared above (referred to as DC in the table below), CD4+ T cells (referred to as CD4 in the table below), the mixture of DC cells and CD4+ cells (referred to as Cell in the table below), and IgG1 disclosed in Table 3 of this invention were used as negative controls. DC+CD4+ T cells + anti-CD3 / CD28 magnetic beads (QIAGEN) (referred to as Beads in the table below) were used as positive controls. The mixture was cultured for 5 days, and the concentrations of IL2 and IFN-γ (relative expression levels expressed as DeltaF%) were detected using cisbio kits (Human IL2 Kit 1000 Test, Human IFN gamma 1000 test).

[0720] The experimental results are shown in Tables 9, 10, 11, and 12, and Figures 4 and 5. The data in the tables are in Delta (F%).

[0721] Table 9. Relative IL2 expression levels of donor 1

[0722]

[0723] *Negative control, no antibodies or magnetic beads were added.

[0724] Table 10. Relative IL2 expression levels of donor 2

[0725]

[0726] *Negative control, no antibodies or magnetic beads were added.

[0727] Table 11. Relative expression levels of IFN-γ in donor 1

[0728]

[0729] *Negative control, no antibodies or magnetic beads were added.

[0730] Table 12. Relative expression levels of IFN-γ in donor 2

[0731]

[0732] *Negative control, no antibodies or magnetic beads were added.

[0733] Therefore, the antibodies of the present invention can effectively activate T cells in vitro, and some of them have a better activation effect than the positive control antibody.

[0734] Example 9: Zenix column detection of antibody druggability

[0735] This experiment assessed the druggability of the exemplary humanized antibody of this invention by recording its residence time in a Zenix column (Sepax Technologies, Inc.). A shorter residence time indicates lower antibody viscosity and better druggability.

[0736] Chromatographic conditions: Detection wavelength: 214 nm, column temperature: 25 ℃, flow rate: 0.35 ml / min, injection volume: 10 μl.

[0737] Sample preparation: Take 100 μl of sample (antibodies to be tested: HZ3266-IgG1, HZ3266-IgG4PAAK, HZ3266-IgG1N297A, HZ4485-IgG1N297A; ATE as a positive control, antibody concentration: 1 mg / mL), centrifuge at 13000 rpm for 5 min, take 80 μl of supernatant into the liquid chromatography tube, and place it in the liquid chromatography sample tray for injection and detection.

[0738] The experimental results are shown in Table 13. The residence time of different subtypes of HZ3266 and HZ4485 on the column was shorter than that of the control antibody, indicating that HZ3266 and HZ4485 have good drug-like properties.

[0739] Table 13. Zenix column assay for antibody drugability

[0740]

[0741] Example 10. Antitumor efficacy test

[0742] In this experiment, the antitumor effect of the PD-L1 antibody of the present invention was determined in hPD-L1 transgenic mice using MC38 cells expressing human PD-L1 (MC38-hPDL1) (Nanjing Yinhe Company).

[0743] Human PD-L1 transgenic mice:

[0744] Female human PDL-1 transgenic mice with a C57B1 / 6 background (approximately 8 weeks old) were purchased from Shanghai Southern Laboratory Animal Technology Co., Ltd. The mice were acclimatized for 7 days after arrival before the study began.

[0745] cell:

[0746] MC38 cells expressing human PD-L1 (MC38-hPDL1) were purchased from Nanjing Yinhe Biopharmaceutical Co., Ltd., and were passaged and cultured strictly according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 5 × 10⁶ cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of human PD-L1 transgenic mice to establish an MC38-hPDL1 tumor-bearing mouse model.

[0747] Administration:

[0748] Six days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of 87.4 mm were selected. 3 ~228.4mm 3 Mice within the range were divided into groups of 8 mice each, based on average tumor volume; one group received IgG1, and the other received the antibody HZ3266-IgG1N297A of this invention. The mice were administered the antibody twice weekly on days 6, 10, 14, 17, 21, 24, 28, 31, and 35 post-inoculation. The dosage and administration method are shown in Table 12. Tumor volume and body weight changes were monitored twice weekly for 5 consecutive weeks during the administration period. Body weight and tumor volume were measured before each administration. Tumor volume was determined using calipers to measure the maximum long axis (L) and maximum wide axis (W) of the tumor. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance twice a week.

[0749] Table 14. Experimental Design Table

[0750]

[0751] The antibody of this invention showed significant antitumor activity one week after administration. Figure 6 By day 35, one mouse in the antibody group of this invention had completely regressed tumor. Body weight results showed that different antibody dosages had no effect on the body weight of tumor-bearing mice.

[0752] Therefore, the antibody of this invention has a significant inhibitory effect on tumors.

[0753] Example 11. Combination of the anti-PD-L1 antibody and anti-human LAG-3 antibody of the present invention

[0754] This study investigated the antitumor activity of the combined use of the anti-PD-L1 antibody (HZ3266-IgG1N297A) and the anti-human LAG-3 antibody (ADI-31853) of this invention using a humanized mouse model.

[0755] This study used A375 (ATCC) human skin cancer cells in NCG mice to determine the antitumor effect of anti-PD-L1 antibodies. Human PBMCs (AllCells) (2 × 10⁻⁶ cells) were pre-injected intravenously. 6 A375 tumor-bearing mouse models were established by subcutaneous inoculation (using cells / mouse). After tumor formation, mice were divided into groups and treated with different antibodies. Tumor volume and body weight changes were monitored during treatment. Dosing was administered twice weekly for two weeks, for a total of five administrations. Monitoring was conducted twice weekly for four consecutive weeks, using the following dosage and administration method. The relative tumor inhibition rate (TGI%) was calculated after treatment.

[0756] Anti-human LAG-3 antibody ADI-31853

[0757] According to conventional methods in the art, the light chain amino acid sequence and heavy chain amino acid sequence (Table 3) encoding the anti-LAG-3 antibody ADI-31853 were cloned into the expression vector pTT5, respectively.

[0758] The expression vector containing the target antibody gene was transiently transfected into cultured human kidney embryonic cell 293 cells (Invitrogen) using PEI (Polysciences) transfection reagent according to the manufacturer's instructions. After transfection, the culture medium was discarded, and the cells were diluted to 4 × 10⁻⁶ cells in fresh EXPI 293 medium (Gibco). 6 / ml. Culture cells at 37℃ and 5% CO2 for 7 days, adding fresh culture medium every 48 hours. After 7 days, centrifuge at 1300 rpm for 20 min. Collect the supernatant and purify it with Protein A to achieve an antibody purity >95%.

[0759] The equilibrium dissociation constant (KD) of ADI31853 binding to human LAG-3 (hLAG-3) was determined using ForteBio biointerferometry. ForteBio affinity assays were performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitopebinning. MAbs, 2013.5(2): p.270-8). In short, the sensor was equilibrated offline in the analysis buffer for 30 minutes, then detected online for 60 seconds to establish a baseline. The purified antibody obtained as described above was then loaded online onto the AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was then exposed to 100 nM human LAG-3 antigen (ArcoBiosystems) for 5 minutes, followed by dissociation in the analysis buffer for 5 minutes for dissociation rate measurement. Kinetic analysis was performed using a 1:1 binding model.

[0760] In the experiments conducted as described above, the affinity of ADI-31853 was as follows:

[0761]

[0762] Mice:

[0763] NOG mice, female, 7-8 weeks old (at the time of tumor cell inoculation), weighing 17.6-24.2 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were acclimatized for 7 days after arrival before the study began.

[0764] cell:

[0765] Human skin cancer cell line A375 (ATCC#CRL-1619) was purchased from ATCC and passaged strictly according to ATCC requirements for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 30 × 10⁶ cells / mL. 6 NOG mice were intravenously injected with human PBMCs (AllCells), then shaved on the right back and subcutaneously injected with 0.2 ml of A375 cells per mouse. Tumor volume was measured 7 days after inoculation, and mice with an average tumor volume of 70-71 mm² were selected. 3 Mice within the range were randomly grouped according to tumor volume.

[0766] Administration:

[0767] Each group received a subcutaneous injection of the following dose of antibody:

[0768] (1) Human IgG (equitech-Bio), 20 mg / kg;

[0769] (2) LAG-3 (ADI-31853), 10 mg / kg;

[0770] (3) PD-L1 (HZ3266-IgG1N297A), 10mg / kg;

[0771] (4) LAG-3 (ADI-31853), 10 mg / kg + PD-L1 (HZ3266-IgG1N297A), 10 mg / kg.

[0772] On day 7 post-inoculation, mice with tumors meeting the above-mentioned average volume were randomly divided into groups of 8. Each group of mice was then administered the above four reagents at the above-mentioned dosages on days 7, 10, 14, and 17.

[0773] Analysis: Tumor size and body weight were measured twice weekly throughout the study. Mice were euthanized when the tumor reached its endpoint or when they experienced a >20% weight loss. The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using calipers. Tumor volume was calculated using the following formula: V = L × W 2 / 2. Plot tumor size versus time for mice from each group. Use analysis of variance (ANOVA) to determine statistical significance. A p-value < 0.05 was considered statistically significant in all analyses.

[0774] The experimental results are shown in Figure 7 It can be seen that when the anti-LAG-3 monoclonal antibody ADI-31853 (31853) and the anti-PD-L1 monoclonal antibody (HZ3266-IgG1N297A) (HZ3266) are used in combination, compared with the human IgG control (equitech-Bio) (hIgG) and the use of these two antibodies separately, the growth of tumors can be significantly inhibited.

Claims

1. An antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment comprises three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein HCDR1 consists of the amino acid sequence shown in SEQ ID NO:3, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:8, HCDR3 consists of the amino acid sequence shown in SEQ ID NO:13, LCDR1 consists of the amino acid sequence shown in SEQ ID NO:16, LCDR2 consists of the amino acid sequence shown in SEQ ID NO:19, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

24.

2. The antibody binding to PD-L1 or its antigen-binding fragment as claimed in claim 1, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises or is composed of an amino acid sequence having at least 90% identity with an amino acid sequence selected from or represented by SEQ ID NO: 29 or 31.

3. The antibody binding to PD-L1 or its antigen-binding fragment as described in claim 1, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from or composed of the sequence shown in SEQ ID NO: 29 or 31.

4. The antibody or antigen-binding fragment thereof that binds to PD-L1 as described in any one of claims 1-3, wherein the antibody comprises a light chain variable region, wherein the light chain variable region comprises or is composed of an amino acid sequence having at least 90% identity with an amino acid sequence selected from or represented by SEQ ID NO: 35 or 37.

5. The antibody or antigen-binding fragment of PD-L1 as described in any one of claims 1-3, wherein the antibody comprises a light chain variable region, wherein the light chain variable region comprises or is composed of an amino acid sequence selected from or consisting of the sequence shown in SEQ ID NO: 35 or 37.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein (i) The heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:29, and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:35; or (ii) The heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:31, and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:

37.

7. The antibody of claim 1 or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain comprising or consisting of an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 41 or 45.

8. The antibody of claim 1 or an antigen-binding fragment thereof, said antibody comprising a heavy chain comprising or consisting of an amino acid sequence selected from or composed of SEQ ID NO:41 or 45.

9. The antibody of claim 1, 7 or 8 or an antigen-binding fragment thereof, said antibody comprising a light chain comprising or consisting of an amino acid sequence having at least 85% identity with an amino acid sequence selected from SEQ ID NO: 49 or 51.

10. The antibody of claim 1, 7 or 8 or an antigen-binding fragment thereof, said antibody comprising a light chain comprising or consisting of an amino acid sequence selected from or composed of SEQ ID NO: 49 or 51.

11. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody comprises a heavy chain and a light chain, wherein (i) The heavy chain contains or is composed of the amino acid sequence of SEQ ID NO: 41; and the light chain contains or is composed of the amino acid sequence of SEQ ID NO: 49; or (ii) The heavy chain contains or is composed of the amino acid sequence of SEQ ID NO:45; and the light chain contains or is composed of the amino acid sequence of SEQ ID NO:

51.

12. The antibody or antigen-binding fragment thereof that binds to PD-L1 as described in claim 1 or 6, having one or more of the following characteristics: (1) Binding to human PD-L1; (2) Binding to cells expressing human PD-L1; (3) Enhance T cell proliferation; (4) Increase IFN-γ secretion; (5) Increase IL-2 secretion; (6) In the Zenix column detection method, the residence time is less than 10 minutes; (7) Inhibition of one or more activities of PD-L1 may result in one or more of the following: an increase in tumor-infiltrating lymphocytes, an increase in T-cell receptor-mediated proliferation, or a decrease in immune evasion of cancer cells; (8) It can induce antibody-dependent cell-mediated cytotoxicity; (9) Exhibiting the same binding affinity and / or specificity for PD-L1 as any antibody of claim 11; (10) Inhibits the binding of any antibody of claim 11 to PD-L1; (11) An epitope that binds to the same or overlapping epitopes as any antibody of claim 11; or (12) Competes with any of the antibodies of claim 11 for binding to PD-L1.

13. The antibody or antigen-binding fragment thereof that binds to PD-L1 according to claim 12, wherein: (1) The following equilibrium dissociation constant (K) D Combined with PD-L1, the K D Less than 2nM; (2) Binding of cells expressing human PD-L1 with EC50 less than or equal to 2 nM; (3) It enhances T cell proliferation more effectively than the known anti-PD-L1 antibody Tecentriq; (4) It enhances IFN-γ secretion more effectively than the known anti-PD-L1 antibody Tecentriq; (5) It enhances IL-2 secretion more effectively than the known anti-PD-L1 antibody Tecentriq; or (6) Competitively inhibit the binding of any antibody of claim 11 to PD-L1.

14. The antibody of claim 13 or its antigen-binding fragment thereof, wherein T cell proliferation, IFN-γ secretion or IL-2 secretion is detected in MLR.

15. The antibody or antigen-binding fragment thereof of claim 1, 6 or 11, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment in the form of IgG1 or IgG4.

16. The antibody or antigen-binding fragment thereof according to claim 1, 6 or 11, wherein the antibody or antigen-binding fragment thereof comprises a κ light chain constant region.

17. The antibody or antigen-binding fragment thereof of claim 15, wherein the antibody or antigen-binding fragment thereof comprises a constant region of a human κ light chain.

18. The antibody or antigen-binding fragment thereof according to claim 1, 6 or 11, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof in the form of IgG1 or IgG4, and comprises a human κ light chain constant region.

19. The antibody or antigen-binding fragment thereof according to claim 1, 6 or 11, wherein the antibody is a monoclonal antibody.

20. The antibody or antigen-binding fragment thereof according to claim 1, 6 or 11, wherein the antibody is a humanized antibody or a chimeric antibody.

21. The antibody or antigen-binding fragment thereof according to claim 1, 6 or 11, wherein the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody, (Fab')2 fragment, or biantibody (dAb).

22. The antibody of claim 21 or an antigen-binding fragment thereof, wherein the single-chain antibody is scFv.

23. The antibody or antigen-binding fragment thereof according to claim 1, 6 or 11, wherein the antibody is a bispecific antibody.

24. The antibody of claim 23 or an antigen-binding fragment thereof, wherein the bispecific antibody binds to PD-L1 and LAG-3.

25. The antibody of claim 23 or an antigen-binding fragment thereof, wherein the bispecific antibody binds to human PD-L1 and human LAG-3.

26. The antibody or antigen-binding fragment thereof according to claim 1, 6 or 11, wherein the antibody is a multispecific antibody.

27. An isolated nucleic acid encoding an anti-PD-L1 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 26.

28. An expression vector comprising the nucleic acid of claim 27.

29. The expression vector of claim 28, wherein the expression vector is a pTT5 vector.

30. A host cell comprising the nucleic acid of claim 27 or the expression vector of claim 28 or 29.

31. The host cell of claim 30, wherein the host cell is prokaryotic or eukaryotic.

32. The host cell of claim 30, wherein the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

33. The host cell of claim 30, wherein the host cell is a 293 cell or a CHO cell.

34. A method for preparing an anti-PD-L1 antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell of any one of claims 30-33 under conditions suitable for expressing a nucleic acid encoding an anti-PD-L1 antibody or an antigen-binding fragment thereof of any one of claims 1 to 26, isolating the antibody or the antigen-binding fragment thereof, and recovering the anti-PD-L1 antibody or the antigen-binding fragment thereof from the host cell.

35. An immunoconjugate comprising an anti-PD-L1 antibody or an antigen-binding fragment thereof of any one of claims 1 to 26 and other substances, wherein said other substances are markers.

36. A pharmaceutical composition comprising an anti-PD-L1 antibody of any one of claims 1 to 26 or an antigen-binding fragment thereof, or an immunoconjugate of claim 35.

37. The pharmaceutical composition of claim 36, further comprising pharmaceutical excipients.

38. A pharmaceutical composition comprising an anti-PD-L1 antibody of any one of claims 1 to 26 or an antigen-binding fragment thereof or an immunoconjugate of claim 35, and other therapeutic agents.

39. The pharmaceutical composition of claim 38, further comprising pharmaceutical excipients.

40. The pharmaceutical composition of claim 38, wherein the other therapeutic agent is selected from chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, or immunomodulators.

41. The pharmaceutical composition of claim 40, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

42. The pharmaceutical composition of claim 38, wherein the other therapeutic agent is a LAG-3 antibody or an antigen-binding fragment thereof.

43. The pharmaceutical composition of claim 42, wherein the LAG-3 antibody or its antigen-binding fragment is an anti-LAG-3 antibody or its antigen-binding fragment that binds to human LAG-3.

44. The pharmaceutical composition of claim 38, wherein the other therapeutic agent is a humanized anti-LAG-3 antibody or an antigen-binding fragment thereof.

45. The pharmaceutical composition of any one of claims 42-44, wherein the anti-LAG-3 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) The VH includes complementarity-determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 is composed of the amino acid sequence of SEQ ID NO:69; HCDR2 is composed of the amino acid sequence of SEQ ID NO:70; and HCDR3 is composed of the amino acid sequence of SEQ ID NO:

71. and (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 is composed of the amino acid sequence of SEQ ID NO:72; LCDR2 is composed of the amino acid sequence of SEQ ID NO:73; and LCDR3 is composed of the amino acid sequence of SEQ ID NO:

74.

46. ​​The pharmaceutical composition of claim 45, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:75, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:

76.

47. Use of an effective amount of the anti-PD-L1 antibody of any one of claims 1 to 26 or its antigen-binding fragment, or the immunoconjugate of claim 35, or the pharmaceutical composition of any one of claims 36 to 46 in the preparation of a medicament for the prevention or treatment of a tumor in a subject or individual, wherein the tumor is a solid tumor.

48. Use of an effective amount of the anti-PD-L1 antibody or its antigen-binding fragment of any one of claims 1 to 26, or the immunoconjugate of claim 35, or the pharmaceutical composition of any one of claims 36 to 41, and the anti-LAG-3 antibody or its antigen-binding fragment in the preparation of a medicament, wherein the medicament is intended to prevent or treat a tumor in a subject or individual, wherein the tumor is a solid tumor.

49. The use according to claim 48, wherein the anti-LAG-3 antibody is an anti-LAG-3 antibody that binds to human LAG-3.

50. The use according to claim 49, wherein the anti-LAG-3 antibody is humanized.

51. The use according to any one of claims 47-50, wherein the tumor is cancer.

52. The use according to any one of claims 47-50, wherein the tumor is a gastrointestinal tumor.

53. The use according to claim 51, wherein the cancer is a gastrointestinal cancer.

54. The use according to claim 51, wherein the cancer is colon cancer.

55. The use as described in claim 47, wherein the drug can also be administered in combination with one or more other therapies.

56. The use of claim 55, wherein the therapy comprises a treatment method and / or other therapeutic agents.

57. The use of claim 56, wherein the treatment method includes surgical treatment and / or radiotherapy, or the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, vaccines, anti-infective agents, other antibodies or immunomodulators.

58. The use of claim 57, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

59. The use according to claim 56, wherein the other therapeutic agent is a LAG-3 antibody or an antigen-binding fragment thereof.

60. The use according to claim 59, wherein the LAG-3 antibody or its antigen-binding fragment is an anti-LAG-3 antibody or its antigen-binding fragment that binds to human LAG-3.

61. The use according to claim 60, wherein the anti-LAG-3 antibody or its antigen-binding fragment is a humanized anti-LAG-3 antibody or its antigen-binding fragment.

62. The use according to any one of claims 48 to 50, 59 to 61, wherein the anti-LAG-3 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (i) The VH includes complementarity-determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 is composed of the amino acid sequence of SEQ ID NO:69; HCDR2 is composed of the amino acid sequence of SEQ ID NO:70; and HCDR3 is composed of the amino acid sequence of SEQ ID NO:

71. and (ii) wherein the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 is composed of the amino acid sequence of SEQ ID NO:72; LCDR2 is composed of the amino acid sequence of SEQ ID NO:73; and LCDR3 is composed of the amino acid sequence of SEQ ID NO:

74.

63. The use of claim 62, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:75, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:

76.

64. The use according to any one of claims 48 to 50, wherein the drug can also be administered in combination with one or more other therapies.

65. The use of claim 64, wherein the therapy comprises a treatment method and / or other therapeutic agents.

66. The use of claim 65, wherein the treatment method includes surgical treatment and / or radiotherapy, or the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, vaccines, anti-infective agents, other antibodies or immunomodulators.

67. The use of claim 66, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

68. Use of the anti-PD-L1 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 26 in the preparation of a detection reagent for detecting PD-L1 in a sample.

69. The use of claim 68, wherein the anti-PD-L1 antibody or its antigen-binding fragment is detectably labeled.

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