Anti-PD-L1 / anti-4-1BB natural antibody structure-mimicking heterodimer bispecific antibody and method for producing the same

Abstract

The present invention provides an anti-PD-L1 / anti-4-1BB natural antibody structure-like heterodimer bispecific antibody and a preparation method thereof. Specifically, the present invention provides a highly stable anti-PD-L1 / anti-4-1BB heterodimer bispecific antibody that has the structural characteristics of natural IgG and has no heavy chain-light chain mismatch, and a preparation method thereof, which can simultaneously bind to two target molecules and has better efficacy and fewer side effects in treating complex diseases.

Description

[Technical Field]

[0001] The present invention relates to an anti-PD-L1 / anti-4-1BB native antibody structure-like heterodimeric bispecific antibody and a method for producing the same. Specifically, the present invention provides a highly stable heterodimeric anti-PD-L1 / anti-4-1BB bispecific antibody that has the characteristics of native IgG and has no heavy chain-light chain mismatch, and a method for producing the same. [Background technology]

[0002] Programmed death ligand 1 (PD-L1) is a ligand for the immune checkpoint, programmed death-1 (PD-1). It belongs to the B7 family and is inducibly expressed on the surface of various immune cells, including T cells, B cells, monocytes, macrophages, DCs, endothelial cells, and epidermal cells. When PD-L1 binds to PD-1, it is primarily involved in negative regulation of T cell activation, thereby regulating the strength and duration of immune responses. PD-L1, as a ligand for PD-1 and CD80, transmits negative regulatory signals to T cells and induces T cell immune tolerance (Autoimmun Rev, 2013, 12(11):1091-1100; Front Immunol, 2013, 4:481; Nat Rev Cancer, 2012, 12(4):252-264; Trends Mol Med. 2015 Jan;21(1):24-33; Clin Cancer Res. 2012 Dec 15;18(24):6580-7). Under normal circumstances, PD-L1 and PD-1 can induce and maintain immune tolerance in living tissues, prevent the immune system from becoming overly activated and damaging self-tissues during inflammatory responses, and play a positive role in preventing the development of autoimmune diseases. In pathological situations, they are involved in tumor immunity and the onset and development of various autoimmune diseases. Numerous studies have shown that PD-L1 is highly expressed in various tumor tissues, while PD-1 is highly expressed in tumor-infiltrating lymphocytes. Overexpression of PD-L1 and PD-1 is closely associated with poor clinical tumor prognosis (Anticancer Agents Med Chem. 2015;15(3):307-13. Hematol Oncol Stem Cell Ther. 2014 Mar;7(1):1-17. Trends Mol Med. 2015 Jan;21(1):24-33. Immunity. 2013 Jul 25;39(1):61-73. J Clin Oncol. 2015 Jun 10;33(17):1974-82.).Blocking the PD-L1 / PD-1 and CD80 / PD-L1 interactions with PD-L1 monoclonal antibodies has shown favorable antitumor effects in preclinical experimental studies and clinical trials. PD-L1 monoclonal antibodies have now been approved for the treatment of many tumors, including non-small cell lung cancer and urothelial carcinoma.

[0003] 4-1BB (also called CD137, TNFRSF9, etc.) is a member of the tumor necrosis factor receptor superfamily (TNFRS F 4-1BB is a transmembrane protein of the 4-1BB family. It is expressed on DCs, activated monocytes, NK cells, neutrophils, eosinophils, and mast cells. 4-1BBL (CD137L) is a glycoprotein member of the TNF superfamily and is primarily expressed on activated B cells, macrophages, dendritic cells, and myeloid cells. 4-1BB is a costimulatory molecule on CD8+ and CD4+ T cells, regulatory T cells (Tregs), natural killer T cells (NK(T) cells), B cells, and neutrophils. When 4-1BB binds to 4-1BBL, intracellular signaling pathways are activated. Studies have shown that several 4-1BB agonist mAbs increase the expression of costimulatory molecules in multiple models, significantly enhance cytolytic T lymphocyte responses, and induce antitumor effects.

[0004] Therefore, in this field, PD-L1 and 4-1BB and simultaneously combine New therapeutic agents need to be researched. Summary of the Invention

[0005] The present invention provides PD-L1 and 4-1BB antibodies that have the structural characteristics of natural IgG and are free of heavy-light chain mismatches. and simultaneously combined The present invention provides a novel, highly stable heterodimeric bifunctional antibody capable of inhibiting PD-L1 of Highly expressing tumor cells and 4-1BB-expressing T cells simultaneously They tend to bind to the cytotoxic T cells, thereby exerting an efficient and specific killing effect with low toxic side effects.

[0006] A first aspect of the present invention relates to a bispecific antibody comprising a first antigen-binding functional region that specifically binds to PD-L1 and a second antigen-binding functional region that specifically binds to 4-1BB, wherein the first antigen-binding functional region that specifically binds to PD-L1 is: (A) a heavy chain variable region; (B) a light chain variable region, The heavy chain variable region (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15; (b) an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16; (c) an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; The light chain variable region (a) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18; (b) an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19; (c) LCDR3 comprising the amino acid sequence shown in SEQ ID NO:20.

[0007] In some embodiments, the second antigen-binding functional region of the bispecific antibody that specifically binds to 4-1BB is (A) a heavy chain variable region; (B) a light chain variable region, The heavy chain variable region (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:21; (b) an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:22; (c) an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:23; The light chain variable region (a) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:24; (b) an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:25; (c) LCDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0008] In some embodiments, the first antigen-binding functional region that specifically binds to PD-L1 of the bispecific antibody comprises: (A) a heavy chain variable region; (B) a light chain variable region; The heavy chain variable region (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15; (b) an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16; (c) an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; The light chain variable region (a) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18; (b) an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19; (c) an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:20; The second antigen-binding functional region that specifically binds to 4-1BB is (A) a heavy chain variable region; (B) a light chain variable region, The heavy chain variable region (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:21; (b) an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:22; (c) an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:23; The light chain variable region (a) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:24; (b) an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:25; (c) LCDR3 comprising the amino acid sequence shown in SEQ ID NO:26.

[0009] The six CDR sequences of the first antigen-binding functional domain that specifically binds to PD-L1 shown in SEQ ID NOs. 15-20 and the six CDR sequences of the second antigen-binding functional domain that specifically binds to 4-1BB shown in SEQ ID NOs. 21-26 are the CDRs of the heavy and light chain variable domains of monoclonal antibodies obtained by the present inventors using hybridoma technology with human PD-L1 and 4-1BB as antigens. These monoclonal antibodies differ from the PD-L1 antibodies and 4-1BB antibodies known in the prior art and possess higher biological activities, such as higher binding specificity and anti-tumor activity.

[0010] The six CDR sequences of the first antigen-binding domain that specifically binds to PD-L1 each have at least 70% identity, for example at least 75%, 80%, 85%, 90%, 95% or more identity, to the sequences set forth in SEQ ID NOs. 15-20, and retain the biological activity of the corresponding parent sequence, or are obtained by deleting, substituting, and / or adding one or more amino acid residues, for example one, two, three or more, to the sequences set forth in SEQ ID NOs. 15-20, and retaining the biological activity of the corresponding parent sequence.

[0011] The six CDR sequences of the second antigen-binding functional region that specifically binds to 4-1BB each have at least 70% identity, for example, at least 75%, 80%, 85%, 90%, 95% or more identity, to the sequences shown in SEQ ID NOs. 21-26, and retain the biological activity of the corresponding parent sequences, or are obtained by deleting, substituting, and / or adding one or more amino acid residues, for example, one, two, three or more amino acid residues, from the sequences shown in SEQ ID NOs. 21-26, and retaining the biological activity of the corresponding parent sequences.

[0012] In some embodiments, the first antigen-binding functional domain of the bispecific antibody that specifically binds to PD-L1 comprises a heavy chain variable domain and a light chain variable domain, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:6, or has at least 70% identity, for example at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, to the sequence set forth in SEQ ID NO:6, and retains the biological activity of the corresponding parent sequence, or is a variant sequence obtained by deleting, substituting, and / or adding one or more amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, to the sequence set forth in SEQ ID NO:6, and retains the biological activity of the corresponding parent sequence. The light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO:2, or may have at least 70% identity, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, to the sequence set forth in SEQ ID NO:2, and retain the biological activity of the corresponding parent sequence; or may be a variant sequence obtained by deleting, substituting, and / or adding one or more amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, from the sequence set forth in SEQ ID NO:2, and retaining the biological activity of the corresponding parent sequence.

[0013] In some embodiments, the second antigen-binding functional region of the bispecific antibody that specifically binds to 4-1BB comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, or has at least 70% identity, for example at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, to the sequence set forth in SEQ ID NO: 12 and retains the biological activity of the corresponding parent sequence, or is a variant sequence obtained by deleting, substituting, and / or adding one or more amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, to the sequence set forth in SEQ ID NO: 12, and retains the biological activity of the corresponding parent sequence. The light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO:10, or may have at least 70% identity, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, to the sequence set forth in SEQ ID NO:10, and retain the biological activity of the corresponding parent sequence; or may be a variant sequence obtained by deleting, substituting, and / or adding one or more amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, from the sequence set forth in SEQ ID NO:10, and retaining the biological activity of the corresponding parent sequence.

[0014] In some embodiments, the first antigen-binding functional region of the bispecific antibody that specifically binds to PD-L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:6 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2. The second antigen-binding functional region that specifically binds to 4-1BB comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:12 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10.

[0015] It is clear to those skilled in the art that the amino acid sequence combination should comply with biological rules, i.e., the light chain and heavy chain or light chain and heavy chain variable regions and antigen-binding fragments should match each other. For example, SEQ ID NO:6 and SEQ ID NO:2 match each other, and SEQ ID NO:12 and SEQ ID NO:10 match each other.

[0016] In some embodiments, the first antigen-binding functional region and the second antigen-binding functional region of the bispecific antibody are selected from a Fab fragment, an scFv fragment, and a variable domain fragment Fv.

[0017] In some embodiments, the first antigen-binding functional region and the second antigen-binding functional region of the bispecific antibody are both Fab fragments.

[0018] In some embodiments, the Fab fragment of the bispecific antibody comprises a first heavy chain variable region and a second heavy chain variable region that are different, and a first light chain variable region and a second light chain variable region that are different.

[0019] In some embodiments, one of the first antigen-binding functional region and the second antigen-binding functional region of the bispecific antibody is a Fab fragment, and the other is an scFv.

[0020] In some embodiments, the bispecific antibody comprises a first Fc chain and a second Fc chain, and a first antigen-binding functional region capable of specifically binding to PD-L1 and a second antigen-binding functional region capable of specifically binding to 4-1BB.

[0021] Here, the first Fc chain and the second Fc chain are both Fc fragments of immunoglobulin G containing amino acid substitutions, and the first Fc chain and the second Fc chain together form a heterodimer that can bind to an Fc receptor.

[0022] Here, the first Fc chain and the second Fc chain are linked to the first antigen-binding functional region and the second antigen-binding functional region, respectively, via a covalent bond or a linker. and one of the first Fc chain and the second Fc chain comprises amino acid substitutions at positions 366 and 399, and the other comprises amino acid substitutions at positions 351, 407, and 409, wherein the amino acid positions are numbered according to the Kabat EU index numbering system.

[0023] In some embodiments, the amino acid substitutions in the first and second Fc chains of the bispecific antibody are as follows: a) L351G, L351Y, L351V, L351P, L351D, L351E, L351K or L351W; b) T366L, T366P, T366W or T366V; c) D399C, D399N, D399I, D399G, D399R, D399T or D399A; d) Y407L, Y407A, Y407P, Y407F, Y407T or Y407H; and e) K409C, K409P, K409S, K409F, K409V, K409Q or K409R.

[0024] In some embodiments, the amino acid substitutions of the bispecific antibody include the following: a) one of the first Fc chain and the second Fc chain has T366L and D399R substitutions, and the other has L351E, Y407L, and K409V substitutions; b) one of the first Fc chain and the second Fc chain has T366L and D399C substitutions, and the other has L351G, Y407L and K409C substitutions; c) one of the first Fc chain and the second Fc chain has T366L and D399C substitutions, and the other has L351Y, Y407A and K409P substitutions; d) one of the first Fc chain and the second Fc chain has T366P and D399N substitutions, and the other has L351V, Y407P and K409S substitutions; e) one of the first Fc chain and the second Fc chain has T366W and D399G substitutions, and the other has L351D, Y407P, and K409S substitutions; f) one of the first Fc chain and the second Fc chain has T366P and D399I substitutions, and the other has L351P, Y407F and K409F substitutions; g) one of the first Fc chain and the second Fc chain has T366V and D399T substitutions, and the other has L351K, Y407T and K409Q substitutions; h) Either the first Fc chain or the second Fc chain has substitutions of T366L and D399A, and the other has substitutions of L351W, Y407H and K409R.

[0025] In some embodiments, the amino acid substitutions of the bispecific antibody include the following: a) one of the first Fc chain and the second Fc chain has substitutions of T366L and K409V, and the other has substitutions of L351E, Y407L, and D399R; b) one of the first Fc chain and the second Fc chain has T366L and K409C substitutions, and the other has L351G, Y407L and D399C substitutions; c) one of the first Fc chain and the second Fc chain has T366L and K409P substitutions, and the other has L351Y, Y407A and D399C substitutions; d) one of the first Fc chain and the second Fc chain has T366P and K409S substitutions, and the other has L351V, Y407P and D399N substitutions; e) one of the first Fc chain and the second Fc chain has T366W and K409S substitutions, and the other has L351D, Y407P and D399G substitutions; f) one of the first Fc chain and the second Fc chain has T366P and K409F substitutions, and the other has L351P, Y407F and D399I substitutions; g) one of the first Fc chain and the second Fc chain has T366V and K409Q substitutions, and the other has L351K, Y407T, and D399T substitutions; h) Either the first Fc chain or the second Fc chain has substitutions of T366L and K409R, and the other has substitutions of L351W, Y407H and D399A.

[0026] In some embodiments, the amino acid substitutions in one of the first Fc chain and the second Fc chain of the bispecific antibody are T366L and D399R, and the amino acid substitutions in the other are L351E, Y407L, and K409V.

[0027] In some embodiments, the weight percentage of the first Fc chain and the first antigen-binding functional region linked thereto via a covalent bond, and the second Fc chain and the second antigen-binding functional region linked thereto via a covalent bond of the bispecific antibody, based on all polypeptide chains, of the formed homodimer is less than 50%, for example, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, or less, when the solution contains no polypeptides other than the first Fc chain and the first antigen-binding functional region linked thereto via a covalent bond and the second Fc chain and the second antigen-binding functional region linked thereto via a covalent bond in the presence of a reducing agent.

[0028] In some embodiments, the bispecific antibody comprises a first heavy chain / first light chain pair that specifically binds to PD-L1, wherein the first heavy chain has a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:8, and the first light chain has a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:4.

[0029] In some embodiments, the bispecific antibody comprises a second heavy chain / second light chain pair that specifically binds to 4-1BB, wherein the second heavy chain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:12 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1. 4 The first light chain has a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4 and a light chain constant region comprising the amino acid sequence set forth in

[0030] A second aspect of the present invention relates to an isolated polynucleotide encoding a heterodimeric bispecific antibody as described above.

[0031] In some embodiments, the nucleotide sequence encoding the amino acids of the first antigen-binding functional region comprises the nucleotide sequence of SEQ ID NOs: 1 and 5.

[0032] In some embodiments, the nucleotide sequence encoding the amino acids of the second antigen-binding functional region comprises the nucleotide sequence of SEQ ID NOs: 11 and 9.

[0033] In some embodiments, the nucleotide sequences encoding the amino acids of the first light chain and the second light chain both comprise the nucleotide sequence of SEQ ID NO:3.

[0034] In some embodiments, the nucleotide sequence encoding the amino acids of one of the first heavy chain and the second heavy chain comprises the nucleotide sequence of SEQ ID NO:7, and the nucleotide sequence encoding the other comprises the nucleotide sequence of SEQ ID NO:13.

[0035] A third aspect of the present invention relates to a recombinant expression vector comprising an isolated polynucleotide as described above.

[0036] In some embodiments, the expression vector is the plasmid vector X0GC obtained by modification based on pCDNA.

[0037] A fourth aspect of the invention relates to a host cell comprising an isolated polynucleotide as described above or a recombinant expression vector as described above.

[0038] In some embodiments, the host cell is selected from human embryonic kidney cells HEK293 or HEK293T, HEK293E, and HEK293F obtained by modification based on HEK293 cells; hamster ovary cells CHO or CHO-S, CHO-dhfr-, CHO / DG44, and ExpiCHO obtained by modification based on CHO cells; Escherichia coli or Escherichia coli BL21, BL21(DE3), Rosetta, and Origami obtained by modification based on E. coli; yeast or Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, and Hansenula polymorpha obtained by modification based on yeast; insect cells or cells High5 and SF9 obtained by modification based on insect cells; plant cells; mammalian mammary gland cells; and somatic cells.

[0039] A fifth aspect of the present invention relates to a composition comprising a bispecific antibody as described above, or an isolated polynucleotide as described above, or a recombinant expression vector as described above, or a host cell as described above, and a pharmaceutically acceptable carrier. In a further embodiment, the composition further comprises at least one second therapeutic agent, preferably wherein the second therapeutic agent and the bispecific antibody, isolated polynucleotide, recombinant expression vector, or host cell are in different parts of the composition, and preferably wherein the second therapeutic agent is an anti-PD-1 antibody and / or a STING agonist.

[0040] A sixth aspect of the invention relates to a method for producing a bispecific antibody as described above, comprising the steps of: 1) expressing in a host cell the isolated polynucleotide as described above or the recombinant expression vector as described above, respectively; 2) reducing each expressed protein in the host cell; 3) mixing the reduced protein and then oxidizing the mixture.

[0041] In some embodiments, the host cell is selected from human embryonic kidney cells HEK293 or HEK293T, HEK293F, or HEK293F obtained by modification based on HEK293 cells; hamster ovary cells CHO or CHO-S, CHO-dhfr, CHO / DG44, or ExpiCHO obtained by modification based on CHO cells; Escherichia coli or Escherichia coli BL21, BL21(DE3), Rosetta, or Origami obtained by modification based on E. coli; yeast or Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, or Hansenula polymorpha obtained by modification based on yeast; insect cells or High5 cells or SF9 cells obtained by modification based on insect cells; plant cells; mammalian mammary gland cells; and somatic cells.

[0042] In some embodiments, the reduction step comprises: 1) carrying out the reduction reaction in the presence of a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine, or other chemical derivatives; and 2) removing the reducing agent. In some embodiments, the reducing agent is dithiothreitol at a concentration of 0.1 mM or greater, e.g., 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, or greater, and the reaction conditions are at 4°C for at least 3 hours, e.g., 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, or more.

[0043] In some embodiments, the oxidation step is performed in air, but further includes performing the oxidation reaction in the presence of an oxidizing agent selected from L-dehydroascorbic acid or a chemical derivative thereof. In some embodiments, the oxidizing agent is L-dehydroascorbic acid at a concentration of 0.5 mM or greater, e.g., 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.2 mM, 1.5 mM, or greater, and the reaction conditions are at 4°C for at least 5 hours, e.g., 5, 6, 7, 8, 9 hours, or more.

[0044] In some embodiments, the method further comprises the step of isolation and purification, in some embodiments, by cation exchange resin, anion exchange resin, reverse phase chromatography, affinity chromatography, size exclusion chromatography, and combinations thereof.

[0045] A seventh aspect of the present invention relates to the use of a bispecific antibody as described above, and / or an isolated polynucleotide as described above, and / or a recombinant expression vector as described above, and / or a host cell as described above, and / or a composition as described above in the manufacture of a medicament for preventing and / or treating a disease in a subject.

[0046] An eighth aspect of the present invention relates to a bispecific antibody as described above, and / or an isolated polynucleotide as described above, and / or a recombinant expression vector as described above, and / or a host cell as described above, and / or a composition as described above, for use as a medicament for preventing and / or treating a disease in a subject.

[0047] A ninth aspect of the present invention relates to a method for preventing and / or treating a disease comprising the step of administering to a subject in need thereof a bispecific antibody as described above, and / or an isolated polynucleotide as described above, and / or a recombinant expression vector as described above, and / or a host cell as described above, and / or a composition as described above.

[0048] In some embodiments, the subject is a mammal, preferably a human.

[0049] In some embodiments, the disease is selected from leukemia, lymphoma, myeloma, brain cancer, squamous cell carcinoma of the head and neck, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, and bone cancer.

[0050] In other words, the present invention has designed a novel, highly stable, heterodimeric anti-PD-L1 / anti-4-1BB bispecific antibody that mimics the natural antibody structure, possessing the characteristics of natural IgG but lacking heavy-light chain mismatches. The bispecific antibody produced in this invention can simultaneously bind to two target molecules, PD-L1 and 4-1BB, and when applied to the treatment of complex diseases, it can exert superior efficacy and have fewer side effects than single therapeutic agents. Furthermore, compared to combination therapy using multiple drugs, this bispecific antibody acts as a single therapeutic molecule, making it easier for patients and medical professionals to use and simplifying the complex new drug development process. [Brief explanation of the drawings]

[0051] [Figure 1] Chromatograms of the elution peaks of anti-PD-L1 expression products are shown. [Figure 2] Chromatogram of the elution peak of the anti-4-1BB expression product is shown. [Figure 3] Figure 1 shows the structure of the anti-PD-L1 / anti-4-1BB bispecific antibody molecule. [Figure 4] 1 shows the structure of a half antibody molecule containing one heavy chain and one light chain. [Figure 5] Figure 5 shows the results of SEC-HPLC analysis of half antibody molecules containing one heavy chain and one light chain, where Figure 5A and Figure 5B show the results for an anti-PD-L1 half antibody molecule and an anti-4-1BB half antibody molecule, respectively. [Figure 6]Figure 1 shows the results of SEC-HPLC analysis of anti-PD-L1 / anti-4-1BB bispecific antibody molecules. [Figure 7] Figure 1 shows the results of CE analysis of anti-PD-L1 / anti-4-1BB bispecific antibody molecules. [Figure 8] Figure 8A shows the affinity of the anti-PD-L1 / anti-4-1BB bispecific antibody for PD-L1, Figure 8B shows the affinity of the anti-PD-L1 / anti-4-1BB bispecific antibody for 4-1BB, and Figure 8C shows the affinity of the anti-PD-L1 / anti-4-1BB bispecific antibody for 4-1BB. [Figure 9] Figure 9A shows the blocking activity of anti-PD-L1 / anti-4-1BB bispecific antibodies on PD-L1 / PD-1 binding, and Figure 9B shows the blocking activity of anti-PD-L1 / anti-4-1BB bispecific antibodies on PD-L1 / CD80 binding. [Figure 10] Figure 1 shows the regulatory activity of anti-PD-L1 / anti-4-1BB bispecific antibodies on T cells. [Figure 11] Figure 1 shows 4-1BB agonist activity mediated by anti-PD-L1 / anti-4-1BB bispecific antibody. [Figure 12] Figure 1 shows the anti-tumor effect of anti-PD-L1 / anti-4-1BB bispecific antibodies in a gene knock-in mouse syngeneic tumor model. [Figure 13] This shows the changes in tumor volume in BALB / c-hPD1 / hPDL1 / hCD137 mice. [Figure 14] Tumor volume data from h4-1BB / hPD-1 mice are shown. DETAILED DESCRIPTION OF THE INVENTION

[0052] Definition: As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen-binding site and includes natural and artificial antibodies of various structures, including, but not limited to, complete antibodies and antigen-binding fragments of antibodies.

[0053] As used herein, the term "bispecific antibody" refers to an antibody that contains antigen-binding domains that specifically bind to epitopes on two different biological molecules. Unless otherwise specified, the order of the antigens to which the bispecific antibody binds in the name of the described bispecific antibody is arbitrary. That is, in some embodiments, the terms "anti-PD-L1 / 4-1BB bispecific antibody" and "anti-4-1BB / PD-L1 bispecific antibody" may be used interchangeably. In some embodiments, a bispecific antibody comprises two half antibodies, each comprising a single heavy chain variable region and at least a portion of any heavy chain constant region, and a single light chain variable region and at least a portion of any light chain constant region. In some embodiments, a bispecific antibody comprises two half antibodies, each comprising a single heavy chain variable region and a single light chain variable region, and does not comprise more than one single heavy chain variable region or more than one single light chain variable region. In some embodiments, a bispecific antibody comprises two half antibodies, where each half antibody comprises a single heavy chain variable region and a single light chain variable region, and where the first half antibody binds to a first antigen but not to a second antigen, and the second half antibody binds to the second antigen but not to the first antigen.

[0054] The terms "complementarity determining region" or "CDR region" or "CDR" (which may be used interchangeably herein with hypervariable region "HVR") refer to the regions of an antibody variable domain that form highly variable sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding to an antigen epitope. Herein, the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0055] It should be noted that the CDR boundaries of the variable regions of the same antibody obtained based on different numbering schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined by different numbering schemes will be different. Therefore, when an antibody is defined using a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences contain the specific CDR sequences, but whose so-called CDR boundaries differ from the specific CDR boundaries defined in the present invention by applying a different scheme (e.g., a different numbering scheme rule or combination).

[0056] "Covalent bond" refers to the formation of a single molecule by covalently linking two Fc chains or one of the Fc chains and its associated antigen-binding domain in a heterodimeric bispecific antibody. Here, the Fc chain comprises a first antigen-binding domain and a second antigen-binding domain linked by one or more covalent bonds (e.g., disulfide bonds), and the first Fc chain and the second Fc chain are each linked to their antigen-binding domains via covalent bonds (e.g., imine or amide bonds).

[0057] An antigen-binding functional region refers to a region that can specifically interact with a target molecule such as an antigen. This action is highly selective. Typically, a sequence that recognizes one target molecule cannot recognize sequences of other molecules. Representative antigen-binding functional regions include antibody variable regions, structural variants of antibody variable regions, receptor-binding domains, ligand-binding domains, and enzyme-binding domains.

[0058] "Interchain bonded by one or more disulfide bonds" means that the first Fc chain and the second Fc chain are linked by one or more disulfide bonds to form a heterodimer fragment. In the present invention, the one or more disulfide bonds can be formed when the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto, are synthesized in the same cell, or can be formed by an in vitro reduction-oxidation process after the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto, are separately synthesized in different cells.

[0059] The first Fc chain and the second Fc chain refer to linked fragments formed by a covalent bond involving a disulfide bond, and each chain comprises at least a portion of an immunoglobulin heavy chain constant region. The first Fc chain and the second Fc chain differ in amino acid sequence, differing by at least one amino acid. In the first Fc chain and the second Fc chain of the present invention, strong repulsive forces exist between same chains, while attractive forces exist between different chains. Therefore, the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional regions linked thereto, tend to form heterodimers when expressed in cells. When a first Fc chain and a second Fc chain, or a first Fc chain and a second Fc chain and an antigen-binding functional region linked thereto, are separately expressed in two host cells, the first Fc chain or the first Fc chain and an antigen-binding functional region linked thereto do not tend to form homodimers, and the second Fc chain or the second Fc chain and an antigen-binding functional region linked thereto do not tend to form homodimers. In the present invention, when a first Fc chain and a second Fc chain, or a first Fc chain and a second Fc chain and an antigen-binding functional region linked thereto, are separately expressed in two host cells in the presence of a reducing agent, the proportion of homodimers is less than 50%, i.e., the proportion of monomers (one Fc chain or one Fc chain and an antigen-binding functional region linked thereto) is greater than 50%.

[0060] Immunoglobulins have a symmetrical structure with four polypeptide chains: two identical heavy chains, which are relatively long and have a relatively large relative molecular weight, containing 450 to 550 amino acid residues and a relative molecular weight between 55,000 and 70,000 Da; and two identical light chains (L chains), which are relatively short and have a relatively small relative molecular weight, containing approximately 210 amino acid residues and a relative molecular weight of approximately 24,000 Da. The sequence of approximately 110 amino acids near the N-terminus varies significantly between the heavy and light chains of different immunoglobulins and is called the variable region (V region). In contrast, the remaining amino acid sequence near the C-terminus is relatively stable and is called the constant region (C region). Each heavy chain of an antibody is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH), while each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). In the heavy chain, the variable region occupies approximately one-quarter of the length of the heavy chain, while the constant region occupies approximately three-quarters of the length. Regarding the five known classes of Ig, namely, IgG (γ), IgA (α), IgD (δ), IgM (μ), and IgE (ε), the H chains of the first three Ig types have three constant regions, namely, CH1, CH2, and CH3. The H chains of the last two types (IgM and IgE) have one VH region and four constant regions, namely, CH1 to CH4. The constant region is both the framework of the immunoglobulin molecule and one of the regions that activates the immune response. Although the examples of the present invention relate to IgG, it will be clear to those skilled in the art that the class of the antibody of the present invention can be converted by known methods. For example, the initial IgM antibody of the present invention can be class-converted to the IgG antibody of the present invention. Furthermore, class conversion techniques can be used to convert IgG subclasses to other subclasses, for example, IgG1 to IgG2. Thus, the effector functions of the antibodies of the invention can be converted by isotype switching to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies for various therapeutic uses.In one example, an antibody of the invention is an IgG1 antibody, eg, IgG1,κ.

[0061] In the present invention, the portion of the constant region includes at least the region where the first Fc chain and the second Fc chain interact. For IgG, this region is a portion of amino acids located in the CH3 region, including at least GLN347, TYR349, THR350, LEU351, SER354, ARG355, ASP356, GLU357, LYS360, SER364, THR366, LEU368, LYS370, ASN390, LYS392, THR394, PR0395, VAL397, ASP399, SER400, PHE405, TYR407, LYS409, and LYS439.

[0062] "The first Fc chain and the second Fc chain are each linked to a single antigen-binding functional region via a covalent bond or a linker" means that the first Fc chain and the second Fc chain are each linked via a covalent bond or a linker to an antigen-binding fragment of an antibody, a single-chain antibody capable of recognizing an antigen, a structural variant of another antibody fragment capable of recognizing an antigen, a receptor capable of recognizing a ligand, or a ligand capable of recognizing a receptor. Here, the covalent bond is a type of chemical bond in which two or more atoms share their outer electrons, ideally reaching a state of electronic saturation, thereby forming a relatively stable chemical structure called a covalent bond. In other words, a covalent bond is an interaction formed between atoms by sharing electron pairs. Atoms of the same element or different elements may be linked via a covalent bond. Examples of covalent bonds between the first and second Fc chains of the present invention include, but are not limited to, amide bonds formed by dehydration between the amino group of one amino acid molecule and the carboxyl group of another amino acid molecule, and amide or imine bonds formed between the amino group of one amino acid molecule and an aldehyde group of ethylene glycol, polyethylene glycol, or other compounds or polymers thereof. Here, the linker is an amino acid sequence, compound, or compound polymer capable of covalently linking two polypeptide chains, and the amino acid sequence includes, but is not limited to, a small peptide segment such as GGGGSGGGSGGGGGS. The first or second Fc chain can be linked to a single-chain antibody capable of recognizing an antigen or a structural variant of another antibody fragment capable of recognizing an antigen via an amide bond.

[0063] The tendency of the first Fc chain and the second Fc chain to form heterodimers rather than homodimers refers to the tendency of the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto, to form heterodimers when coexpressed in a cell, because a repulsive force exists between the same polypeptide chains in the first Fc chain and the second Fc chain, while an attractive force exists between different polypeptide chains. When the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and the antigen-binding functional region linked thereto, are separately expressed in two host cells, the first Fc chain or the first Fc chain and the antigen-binding functional region linked thereto do not tend to form homodimers, and the second Fc chain or the second Fc chain and the antigen-binding functional region linked thereto do not tend to form homodimers.

[0064] The Kabat EU index numbering system refers to assigning a number to each amino acid in an antibody sequence based on Kabat's method, and this method of assigning numbers to each residue has become standard in the art. Kabat's scheme can be extended to other antibodies beyond his work. Based on conserved amino acids, an antibody of interest is matched to one of the consensus sequences identified by Kabat. Unless otherwise specified, all amino acid positions in an antibody are numbered based on the Kabat EU index numbering system.

[0065] Fc domain refers to the fragment crystallizable region (Fc) corresponding to the CH2 and CH3 domains of Ig, which is the portion of Ig that interacts with effector molecules or cells.

[0066] IgG, short for immunoglobulin G (IgG), is the major antibody component in serum. Human IgG is classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on antigenic differences in the r chain of the IgG molecule.

[0067] A half antibody molecule is a structure formed by one heavy chain and one light chain of an antibody, which may or may not be linked via a covalent bond, and is a monovalent antibody structure that recognizes an antigen.

[0068] A Fab fragment is an antigen-binding fragment (Fab) and has molecular recognition sequences corresponding to the two arms of an antibody molecule, consisting of an intact light chain and the VH and CHI domains of the heavy chain. An scFv is a structural variant of an antibody fragment obtained by genetically engineering the light and heavy chain variable regions of an antibody. The extracellular region of a membrane receptor is a molecular recognition sequence. A membrane receptor typically comprises an extracellular region located on the outside of a cell that can recognize and bind to the corresponding antigen or ligand, a transmembrane region that anchors the receptor to the cell surface, and an intracellular region located inside the cell that has kinase activity or can transmit signals. A ligand for a cell membrane receptor refers to a protein, small peptide, or compound that is recognized and bound by the extracellular region of the membrane receptor. Cytokines are low-molecular-weight soluble proteins produced by various cells in response to immunogens, mitogens, or other stimuli and have various functions, such as innate immunity, adaptive immunity, hematopoiesis, cell growth and regulation of adult pluripotent stem cells (APSCs), and repair of damaged tissue. Cytokines are classified into interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc. Protein expression tags refer to amino acid sequences, either small peptides or long amino acids, that are added to the N- or C-terminus of a protein of interest. Addition of tags can be advantageous for correct protein folding, protein isolation and purification, and reduced intracellular degradation of proteins. Commonly used tags include, but are not limited to, HA, SUMO, His, GST, GFP, and Flag.

[0069] There are no limitations on the antibodies that can be used for the heterodimeric bispecific antibodies of the present invention. Preferably, any antibody known in the prior art that can be used for the treatment and / or prevention of a disease may be used in the present invention.

[0070] The heterodimeric bispecific antibodies of the present invention may have one or more substitutions, deletions, additions, and / or insertions. For example, some amino acids can be substituted for other amino acids in the protein structure without significantly losing the ability to bind to other polypeptides (e.g., antigens) or cells. Because the biological functional activity of a protein is determined by its binding ability and protein characteristics, some amino acid sequence substitutions can be made in the protein sequence without significantly losing their biological effect or activity.

[0071] In many cases, a polypeptide variant will contain one or more conservative substitutions, which refers to the substitution of an amino acid with another amino acid having similar characteristics, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophilicity of the polypeptide to remain substantially unchanged.

[0072] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account various such characteristics are known to those of skill in the art and include arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, and valine, leucine, and isoleucine.

[0073] The term "identity," as used herein, has the meaning commonly known in the art, and the rules and criteria for measuring identity between different sequences are also known to those skilled in the art. It refers to the percentage of identical residues between a polynucleotide or polypeptide sequence variant and a non-variant sequence after aligning the sequences and introducing gaps (if necessary to achieve the maximum % homology). In the present invention, if the identity constraint is met, the resulting variant sequence is also required to have the biological activity possessed by the parent sequence. Methods and means for screening variant sequences for the above-mentioned activities are known to those skilled in the art. Those skilled in the art can easily obtain such variant sequences from the teachings disclosed herein. In specific embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% polynucleotide or polypeptide identity to the polynucleotides or polypeptides described herein. Due to redundancy in the genetic code, variants exist that encode the same amino acid sequences as these sequences.

[0074] In another embodiment of the present invention, polynucleotide compositions are provided that are capable of hybridizing to the polynucleotide sequences provided by the present invention, or fragments thereof, or complementary sequences thereof, under moderately to highly stringent conditions. Hybridization techniques are well known in the field of molecular biology. For illustrative purposes, moderately stringent conditions suitable for testing hybridization of the polynucleotides of the present invention with other polynucleotides include prewashing with a solution of 5x SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0), hybridizing overnight at 5x SSC at 50-60°C, and then washing twice for 20 minutes at 65°C with 2x, 0.5x, and 0.2x SSC containing 0.1% SDS. Those skilled in the art will appreciate that the stringency of hybridization can be easily manipulated, for example, by varying the salt content of the hybridization solution and / or the hybridization temperature. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, except that the hybridization temperature is increased, for example, to 60-65°C or 65-70°C.

[0075] The host cells of the present invention may be any cells for expressing foreign genes, including, but not limited to, E. coli, yeast, insect cells, plant cells, and mammalian cells.

[0076] Vectors of the present invention include vectors that can replicate in any type of cell or organism, including, for example, plasmids, phages, cosmids, and minichromosomes. In some embodiments, vectors containing polynucleotides of the present invention are vectors suitable for propagation or replication of the polynucleotide or for expression of a polypeptide of the present invention. Such vectors are known in the art and commercially available.

[0077] "Vector" includes shuttle vectors and expression vectors. Typically, a plasmid construct further comprises an origin of replication (e.g., the CoE1 origin of replication) for plasmid replication and selection in bacteria, as well as a selectable marker (e.g., ampicillin or tetracycline resistance). "Expression vector" refers to a vector containing control sequences or regulatory elements necessary for expressing an antibody, including an antibody fragment, of the invention in bacteria or eukaryotic cells.

[0078] The vector of the present invention may be any vector for expressing a foreign gene, including, but not limited to, a plasmid vector, which contains at least a replication origin, a promoter, a gene of interest, a multicloning site, and a selectable marker gene. Preferably, the vector of the present invention includes, but is not limited to, a plasmid vector obtained by modification based on pCDNA, such as the X0GC vector.

[0079] The compositions of the invention further comprise at least one second therapeutic agent, preferably wherein said second therapeutic agent and said bispecific antibody, isolated polynucleotide, recombinant expression vector, or host cell are in different parts of the composition, and preferably wherein said second therapeutic agent is an anti-PD-1 antibody and / or a STING agonist.

[0080] The second therapeutic agent contained in the composition of the present invention is not particularly limited, but it need only be physiologically compatible with the bispecific antibody, isolated polynucleotide, recombinant expression vector, or host cell of the present invention. Physiological compatibility means that when administered to a subject, adverse reactions such as excessive irritation or toxicity do not occur. In some embodiments, the second therapeutic agent is present in the composition as a mixture with the bispecific antibody, isolated polynucleotide, recombinant expression vector, or host cell. In some embodiments, the second therapeutic agent and the bispecific antibody, isolated polynucleotide, recombinant expression vector, or host cell are present in different portions of the composition. Being present in different portions of the composition means that the composition is composed of mutually isolated portions. The mutually isolated portions may be present in different containers, such as different vials, syringes, or small tubes, or may be present in different compartments separated from each other within the same container. The isolated portions may be mixed together prior to administration, or may be administered sequentially at intervals of 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 5 hours, 12 hours, 24 hours, 48 ​​hours, 1 week, 1 month, or more. The second therapeutic agent may be a drug for treating the same or a different disease as the bispecific antibody, isolated polynucleotide, recombinant expression vector, or host cell, for example, both drugs for treating leukemia, or one drug for treating leukemia and the other for treating colon tumors. In some embodiments, the second therapeutic agent comprises an anti-inflammatory agent, such as an IL-6 inhibitor, tumor necrosis factor alpha (TNF-α) inhibitor, interferon gamma (IFN-γ) inhibitor, corticosteroid, antihistamine, antipyretic, and / or antibiotic. In some embodiments, the second therapeutic agent is selected from the group consisting of a second antibody, an immunotherapeutic agent, a targeted therapeutic agent, or a chemotherapeutic agent.

[0081] In some embodiments, the second therapeutic agent is an antibody, and the target of the antibody is CD47, CD70, CD200, CD154, CD223 (LAG-3), KIR (killer cell immunoglobulin-like receptor), GITR, CD20, CD28, CD40, CD86, CD160, CD258, CD270, CD275, CD276, OX40L, B7-H4, GITRL, 4-1BBL, CD3 , CD25, CD48, CD66a, CD80, CD94, CD96, CD112, CD115, CD205, CD226, CD244, CD262, CD284, CD288, leukemia inhibitory factor (LIF), TNFSF15, TDO2, IGF-1R, GD2, TMIGD2, RGMB, VISTA, BTNL2, Btn, TIGIT, Siglecs (sialic acid-binding Ig-like lectins, i.e., SIGLEC 15), VEGFR, ILT family, MICA, TGFβ, STING pathway (stimulator of interferon gene pathway), arginase, EGFRvIII, HHLA2, PD-1, PD-L1, PD-L2, CTLA-4, BTLA, indoleamine 2,3-dioxygenase (IDO, IDO1), TIM3, A2A adenosine receptor (ADO receptor), CD39, CD73, CD27, ICOS (CD278), CD137 (4-1BB), OX40, TNFSF25, IL-10, galectin, NKp3 0, NKp40, NKp44, NKp46, NKG2A, NKG2D, DNAM1, DAP10, CD16 (CD16a, CD16b or both), CRTAM, CD27, PSGL1, CD96, CD100 (also known as SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160.

[0082] In some embodiments, the antibody is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, camrelizumab, toripalimab, sintilimab, tislelizumab, penpulimab, or zimberelimab.

[0083] In some embodiments, the second therapeutic agent is an immunotherapeutic agent. Non-limiting examples of immunotherapeutic agents include antibodies, small molecule immunomodulators, chimeric antigen receptor (CAR) T cells, NK cells, dendritic cells (DCs), adoptive cell transfer (ACT), immune checkpoint modulators, cytokines, cancer vaccines, adjuvants, oncolytic viruses, or combinations thereof. In some embodiments, the immunotherapeutic agent is a STING agonist.

[0084] In some embodiments, the second therapeutic agent is a targeted therapeutic agent. A "targeted therapeutic agent" refers to a molecule that specifically targets and inhibits one or more oncogenic signaling proteins (e.g., those involved in cell growth and survival). Non-limiting examples of such targeted therapeutic agents include tyrosine kinase inhibitors (e.g., imatinib (GLEEVEC®), gefitinib (IRESSA®), erlotinib (TARCEVA®), sorafenib (NEXAVAR®), sunitinib (SUTENT®), dasatinib (SPRYCL®), lapatinib (TYKERB®), nilotinib (TASIGNA®), bortezomib (VELJANZ®), tamoxifen (NOLVADEX®), tofacitinib (XELJANZ®), and the like. target), ALK inhibitors (e.g., crizotinib), Bcl-2 inhibitors (e.g., obatoclax, navitoclax, gossypol), PARP inhibitors (e.g., iniparib, olaparib), PI3K inhibitors (e.g., perifosine), apatinib, AN-152, Braf inhibitors (e.g., trametinib, MEK162), CDK inhibitors (e.g., PD-0332991, LEE011) , Hsp90 inhibitors (salinomycin, VAL-083); small molecule drug conjugates (e.g., vintafolide); serine-threonine kinase inhibitors (e.g., temsirolimus (TORISEL®), everolimus (AFINITOR®), vemurafenib (ZELBORAF®), trametinib (MEKINIST®), dabrafenib (TAFINLAR®);Antibodies include antibodies (e.g., anti-CD20 antibodies (e.g., rituximab (RITUXAN®)), anti-HER2 / neu antibodies (e.g., trastuzumab (HERCEPTIN®)), alemtuzumab (CAMPATH®), anti-EGFR antibodies (e.g., cetuximab, panitumumab), anti-VEGF antibodies (e.g., bevacizumab (AVASTIN®))); antibody conjugates, such as antibody drug conjugates (ADCs), immune-stimulating antibody conjugates (ISACs), and antibody-oligonucleotide conjugates (AOCs). In some embodiments, the antibody conjugate is an ADC-type drug. Non-limiting examples of ADC-type drugs include Her2-ADC, Trop2-ADC, CD22-ADC, BCMA-ADC, CD19-ADC, and Nectin-4-ADC.

[0085] In some embodiments, the second therapeutic agent is a chemotherapeutic agent, non-limiting examples of which include alkylating agents such as Tepadina, CYTOXAN®, and cyclophosphamide; temozolomide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedepa, and uredepa; ethylenimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (synthetic including the synthetic analog topotecan; bryostatin; callystatin; CC-1065 (including the synthetic analogs adozelesin, carzelesin, and biscelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); aplysiatoxin; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatins; such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, isocyclophosphamide, dichloroethylmethylamine, mechlorethamine oxide hydrochloride oxide hydrochloride), mechlorethamine such as melphalan, novembichin, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., antibiotics such as calicheamicin, particularly calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; diphosphonates such as chlorophosphonates; esperamicin, neocarzinostatin chromophore, and related chromoprotein-based enediyne antibiotic chromophores);Aclacinomycins, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinyl-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin , marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rhodrubicin, rufochromomycin, streptozocin, tubercidin, ubenimex, zinostatin, zolirebicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, deoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergics, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; acetonitrile Graton; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestravcil; Bisantrene; Edatrexate; Defofamine; Demecolcine; Diaziquone; Eflornithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxycarbamide; Lentinan; Lonidamine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone;Podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizomycin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipoproman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; e.g., TAXOL® (paclitaxel; Bristol Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, NY) and TAXOTERE® docetaxel (Rhone-Poulenc Paclitaxel analogues such as cyclosporine (Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); isocyclophosphamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE); novantrone; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine; ibandronate; irinotecan (Camptosar, CPT-11) (a regimen containing irinotecan with 5-FU and formyltetrahydrofolate); the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; formyltetrahydrofolate (LV); oxaliplatin, including oxaliplatin therapy (FOLFOX); lapatinib (TYKERB);PKC-α, Raf, H-Ras, EGFR inhibitors (e.g., Tarceva®) and VEGF-A that reduce cell proliferation, including pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0086] Subjects of the present invention include birds, reptiles, mammals, etc. Preferably, mammals include rodents and primates, and more preferably, primates include humans.

[0087] The range of diseases according to the present invention includes, but is not limited to, tumors, preferably leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, and bone cancer.

[0088] Pharmaceutically acceptable carriers refer to common pharmaceutical carriers in the pharmaceutical field, such as diluents, excipients, water, etc., fillers such as starch, sucrose, lactose, microcrystalline cellulose, etc., binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone, wetting agents such as glycerin, disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, and sodium bicarbonate, absorption promoters such as quaternary ammonium compounds, surfactants such as hexadecanol and sodium dodecyl sulfate, adsorption carriers such as kaolinite and bentonite, lubricants such as talc powder, calcium stearate, magnesium stearate, micronized silica gel, and polyethylene glycol. In addition, other adjuvants such as flavorings and sweeteners may be added to the composition.

[0089] The present invention will now be further described by the following non-limiting examples. Those skilled in the art will recognize that various modifications can be made to the present invention without departing from the gist of the invention. Such modifications are also within the scope of the present invention.

[0090] Unless otherwise specified, the following experimental methods are all conventional methods. The experimental materials used are readily available from commercial companies unless otherwise specified. The antibodies used in the following examples of the present invention are all commercially available standard antibodies. [Example]

[0091] Example 1 Construction of vector for anti-PD-L1 / anti-4-1BB heterodimer antibody molecule X0GC expression vectors containing the heavy and light chains of anti-human PD-L1 antibodies were constructed. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2. The nucleotide sequence of the light chain constant region is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4. The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6. The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8. The light chain variable region and light chain constant region, and the heavy chain variable region and heavy chain constant region were amplified by PCR. NEB Phusion High-Fidelity DNA Polymerase (F-530L) was used in all PCR reactions in this application. PCR primers were conventionally designed according to the principles of base pair complementarity and enzyme cleavage site requirements. The reaction system consisted of 8.9 μL of HO, 4 μL of 5x Phusion High-Fidelity DNA Polymerase Buffer, 4 μL of 1 mM dNTPs, 1 μL of upstream primer, 1 μL of downstream primer, 0.1 μL of Phusion High-Fidelity DNA Polymerase, and 1 μL of template. The PCR products for the variable and constant regions were subjected to 1.5% agarose gel electrophoresis, and the corresponding fragments were recovered using a DNA recovery kit (Promega, A9282). The recovered variable and constant region fragments were used as templates for further PCR reactions using the upstream primer for the variable region and the downstream primer for the constant region. The corresponding fragments were then recovered to obtain the full-length light or heavy chain fragments. The X0GC vector and full-length fragments were enzymatically digested with EcoRI (Thermo, Cat. No. FD0275) and HindIII (Thermo, Cat. No. FD0505). The enzyme cleavage reaction system consisted of 6 μL of 10× buffer, 2 μL each of EcoRI and HindIII, 40 μL of the full-length fragment recovered from the gel, and 10 μL of H2O, and was reacted at 37°C for 1 hour.The enzyme cleavage products were ligated using T4 DNA ligase (Thermo, catalog no. EL0011) (hereinafter the same). The reaction system consisted of 1μL of 10x ligase buffer, 0.5μL of ligase, 7.5μL of gel-recovered full-length fragment, and 1μL of gel-recovered X0GC vector. Ligation was carried out at 22°C for 30 minutes. E. coli DH5α competent cells (Tiangen, CB104) were transformed with the ligation products. X0GC expression vectors for the heavy and light chains of anti-human PD-L1 antibodies were obtained for expression of the heavy and light chains of anti-human PD-L1 antibodies, respectively, in eukaryotic cells.

[0092] In the present invention, X0GC expression vectors containing the heavy and light chain variable region sequences of anti-human 4-1BB antibody were simultaneously constructed. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO: 10. The nucleotide sequence of the light chain constant region is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 11, and the amino acid sequence is shown in SEQ ID NO: 12. The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO. 13, and the amino acid sequence is shown in SEQ ID NO: 14. X0GC expression vectors for the heavy and light chains of anti-human 4-1BB antibody were obtained to express the heavy and light chains of anti-human 4-1BB antibody, respectively, in eukaryotic cells.

[0093] Example 2 Expression of anti-PD-L1 / anti-4-1BB heterodimeric antibody molecules An expression vector containing the heavy and light chains of the anti-human PD-L1 antibody was co-transfected into ExpiCHO cells (ExpiCHOTM cells, catalog no. A29127, Invitrogen), and an expression vector containing the heavy and light chains of the anti-human 4-1BB antibody was also co-transfected into ExpiCHO cells.

[0094] The day before transfection, cells were diluted to 3.5 x 10 6On the day of transfection, cells were inoculated at an inoculation density of 1000 cells / mL. TM Expression Medium (Cat. No. A29100-01, Invitrogen) with 6 × 10 cells 6 The plasmid was diluted to a density of 100 cells / mL according to the transfection volume, and the final concentration of the plasmid was adjusted to 0.5 μg / mL. TM SFM medium (OptiPRO TM The plasmid was diluted with SFM (Invitrogen, Cat. No. 12309-019) to 4% of the transfection volume and mixed by inversion. TM Transfection reagent (ExpiFectamine TM CHO Transfection Kit (Cat. No. A29129, Invitrogen) and OptiPRO TM The transfection reagent was diluted with SFM medium to 4% of the transfection volume and mixed by inversion. The diluted transfection reagent was added to the diluted plasmid, mixed gently, and left at room temperature for 1-5 minutes before being added dropwise to the cells. The cells were then placed in a cell incubator (CO2 concentration 8%) and incubated at 125 rpm on a shaker at 37°C for 20 hours. ExpiCHO was added to 0.006x the transfection volume. TM Enhancer (ExpiFectamine TM CHO Transfection Kit, Cat. No. A29129, Invitrogen) and 0.24x the transfection volume of ExpiCHO TM Feed(ExpiCHO TM Feed (Cat. No. A29101-02, Invitrogen) was slowly added dropwise to the cells. The cells were incubated at 32°C in a shaker at 125 rpm. The cell culture supernatant after 10 days of transfection was collected by centrifugation.

[0095] The expression level was measured by the Protein A method. Before purification by chromatography column, precipitates were removed by filtration through a 0.22 μm membrane filter. This step was carried out at 4°C.

[0096] Example 3 Purification of anti-PD-L1 / anti-4-1BB heterodimer antibody molecule expression product Purification was performed at room temperature using an AKTA explorer 100 protein purification system (GE Healthcare) and a MabSelect SuRe affinity chromatography column (GE Healthcare). The chromatography column was first equilibrated with mobile phase A (20 mM sodium phosphate buffer, pH 7.4). After stabilizing the baseline, the supernatant of the cells treated as described above was loaded. After loading, mobile phase A was used for equilibration. The samples were anti-PD-L1 expression product and anti-4-1BB expression product, respectively. The column was then washed with 2-3 column volumes of mobile phase B (mobile phase A containing 1 M sodium chloride), followed by 2-3 column volumes of mobile phase A. The elution peaks, i.e., the peaks of the target proteins, were collected. The chromatograms of the elution peaks of the anti-PD-L1 expression product are shown in Figure 1, and the elution peaks of the anti-4-1BB expression product are shown in Figure 2. The indicated elution peak (gray area in the figure) was collected, and the pH was adjusted to 7.2 by dropwise addition of 1 M Tris alkaline solution, NaCl was added to a final concentration of 0.15 M, and the solution was sterile filtered and stored at 2-8°C.

[0097] Example 4: Preparation and purification of anti-PD-L1 / anti-4-1BB heterodimeric antibody molecules The structure of the anti-PD-L1 / anti-4-1BB heterodimer antibody molecule is shown in Figure 3.

[0098] Heterodimers were obtained by in vitro recombination of the products purified using the MabSelect SuRe (GE Healthcare) method described above. First, the protein solution purified and collected as described above was reduced with cysteine, resulting in cleavage of disulfide bonds, including cleavage of disulfide bonds in the hinge regions of the homodimeric antibody molecules contained in the anti-PD-L1 and anti-4-1BB products, resulting in the formation of half antibody molecules containing one heavy chain and one light chain, the structures of which are shown in Figure 4. The reduced samples were analyzed by SEC-HPLC (Shodex, Protein KW-803) using 1 mM DTT reducing agent in the mobile phase buffer. The results are shown in Figure 5A and B, respectively. The proportions of anti-PD-L1 and anti-4-1BB homodimeric molecules were both less than 10%, and the proportions of half antibody molecules were both greater than 90%.

[0099] The reduced anti-PD-L1 and anti-4-1BB half antibody molecules were then mixed in an equal molar ratio and the recombination reaction was carried out at room temperature for 0.5 hours. During the recombination process, a heterodimeric bispecific antibody containing both anti-PD-L1 and anti-4-1BB half antibody molecules was formed from the anti-PD-L1 and anti-4-1BB half antibody molecules via non-covalent interactions between their CH2-CH3 domains. The protein solution was then concentrated by ultrafiltration (10 kDa nominal molecular weight cutoff), and the solution was substituted with 20 mM phosphate buffer, 0.15 M NaCl, and 0.1 mM cystine. The oxidation reaction was carried out at room temperature overnight to reform the disulfide bonds of the heterodimeric bispecific antibody.

[0100] The anti-PD-L1 / anti-4-1BB heterodimer antibody molecules obtained by reduction and oxidation of the anti-PD-L1 and anti-4-1BB expression products were concentrated by ultrafiltration (10 kDa nominal molecular weight cutoff), and the solution was eluted in 20 mM citrate buffer (pH 6.0). Purification was performed at room temperature using an AKTA explorer 100 protein purification system (GE Healthcare) and a Poros XS ion chromatography column (ThermoFisher). The chromatography column was first equilibrated with mobile phase A (20 mM citric acid, pH 6.0) and allowed to stabilize. After the baseline was stabilized, the protein solution treated as described above was loaded. After loading, mobile phase A was used for equilibration. The column was then washed with a 15-column-volume gradient from A (20 mM citric acid, pH 6.0) to B (20 mM citric acid, 200 mM arginine, pH 6.0) (0% B to 100% B, 80 min). The main elution peak was collected, and the collected protein solution was concentrated by ultrafiltration (10 kDa nominal molecular weight cutoff). The solution was then substituted with 20 mM citric acid, 140 mM arginine (pH 6.0), sterilized by filtration, and stored at 4°C with 0.02% Tween 80. The purified anti-PD-L1 / anti-4-1BB heterodimer antibody molecule BH3120h was analyzed for purity by SEC-HPLC; the results are shown in Figure 6; the purity was 98.54%. CE analysis was also performed; the results are shown in Figure 7; the purity was 96.44%.

[0101] Example 5 Target binding activity of anti-PD-L1 / anti-4-1BB heterodimer antibodies The binding ability of anti-PD-L1 / anti-4-1BB heterodimer antibodies to single antigens was measured by enzyme-linked immunosorbent assay (ELISA). The specific procedure was as follows: Recombinant human PD-L1 (Beijing Sino Biological Co., Ltd., Catalog No. 10084-H08H) or human 4-1BB (Beijing Sino Biological Co., Ltd., Catalog No. 10041-H08H) was coated onto a 96-well high-binding ELISA plate (Costar, Catalog No. 42592) using a carbonate buffer solution at pH 9.6 at a coating concentration of 1 μg / mL in 100 μL / well. The coating was performed overnight at 4°C. The plate was washed five times with PBST. The plate was blocked with 300 μL / well of PBST containing 1% BSA and incubated at 25°C for 1 hour. The plate was washed five times with PBST. Serially diluted heterodimer antibody samples and controls in PBST containing 1% BSA were added at 100 μL / well and incubated at 25°C for 1 hour. After washing five times with PBST, horseradish peroxidase-conjugated anti-human IgG antibody (Chemicon, catalog no. AP309P) diluted 1:10,000 in PBST containing 1% BSA was added at 100 μL / well and incubated at 25°C for 1 hour. After washing five times with PBST, 100 μL / well of the chromogenic substrate TMB was added and allowed to develop at room temperature for 10 minutes. Color development was stopped by adding 100 μL / well of 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader.

[0102] Simultaneously, the binding ability of the anti-PD-L1 / anti-4-1BB heterodimer antibody to the 4-1BB antigen was measured using GS-H2 / 4-1BB cells. The specific procedure was as follows: GS-H2 / 4-1BB cells (purchased from GenScript) were harvested and washed once with cold DPBS (GIBCO, catalog number 14190-136) containing 2% FBS (Hyclone, catalog number SH30084.03). GS-H2 / 4-1BB cells were then cultured in cold DPBS containing 2% FBS at a concentration of 5 x 10 6The cells were resuspended at a density of 100 cells / mL. 100 μL of the cell suspension was added to each flow cytometry tube, along with 100 μL of serially diluted heterodimer antibody samples and controls. The flow cytometry tubes were incubated on ice for 30 minutes. The cells were washed twice with DPBS containing 2% FBS. The cells were resuspended in 200 μL of cold DPBS containing 2% FBS and 488A-Fab-anti-human IgG (final concentration: 5 μg / mL). The cells were incubated on ice for 30 minutes, protected from light. The cells were washed twice with DPBS containing 2% FBS. The cells were resuspended in 500 μL of cold DPBS. The cell suspension was detected and analyzed by flow cytometry (BD, FACS Calibur) to read the intracellular fluorescence intensity.

[0103] The results are shown in Figure 8A. Anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120h has high affinity for PD-L1, with slightly weaker antigen-binding activity than PD-L1 bivalent monoclonal antibodies. As shown in Figure 8B, anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120h has low affinity for 4-1BB, with weaker antigen-binding activity than 4-1BB bivalent monoclonal antibodies. As shown in Figure 8C, anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120h has high affinity for 4-1BB, with stronger antigen-binding activity than anti-4-1BB bivalent monoclonal antibodies.

[0104] Example 6 Blocking activity of anti-PD-L1 / anti-4-1BB heterodimer antibodies against ligand-receptor binding The detection process for blocking the binding of PD-L1 to PD-1 and CD80 was as follows: Recombinant human PD-L1-Fc (Acro Biosystems Co., Ltd., Beijing, catalog no. PD1-H5258) was coated onto a 96-well high-binding ELISA plate using a carbonate buffer solution (pH 9.6) at a coating concentration of 1 μg / mL and a coating volume of 100 μL / well. The plate was then coated overnight at 4°C and washed five times with PBST. Blocking was performed with 300 μL / well of PBST containing 1% BSA, followed by incubation at 25°C for 1 hour. The plate was then washed five times with PBST. Serial dilutions of heterodimer antibody samples and controls in PBST containing 1% BSA were added at 50 μL per well. 50 μL of biotin-labeled PD-1-Fc (Beijing Hanmi Pharm) was added at a concentration of 40 nM (final concentration of 20 nM) or 50 μL of biotin-labeled CD80-Fc (Acro Biosystems Co., Ltd., Beijing, catalog number B71-H82F2) was added at a concentration of 100 nM (final concentration of 50 nM) and incubated at 25°C for 90 minutes. The plate was washed five times with PBST. Next, 100 μL of Streptavidin-HRP (BD Pharmingen, catalog number 554066) diluted 1:1000 in PBST containing 1% BSA was added at 1:1000 and incubated at 25°C for 1 hour. The plate was then washed five times with PBST. The colorimetric substrate TMB was added at 100 μL / well and allowed to develop for 10 minutes at room temperature. Color development was stopped by adding 100 μL / well of 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader.

[0105] The results are shown in Figure 9A. The anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120h can block the binding of PD-L1 to PD-1, but slightly less than the anti-PD-L1 bivalent monoclonal antibody. As shown in Figure 9B, the anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120h can block the binding of PD-L1 to CD80.

[0106] Example 7 Regulatory activity of anti-PD-L1 / anti-4-1BB heterodimer antibodies on T cells The modulating activity of anti-PD-L1 / anti-4-1BB heterodimer antibodies on T cell immune responses was measured using mixed lymphocyte reaction (MLR).

[0107] Obtaining human dendritic cells (DCs): Human monocytes were isolated by following the instructions of the Monocyte Cell Isolation Kit (Miltenyi Biotech, Catalog No. 130091153). Briefly, PBMCs (Allcells, Catalog No. PB0005-C) were first washed once with DPBS (GIBCO, Catalog No. 14190-136) and then diluted with 10% PBS. 7 Resuspend the cells in 40 μL of isolation buffer (PBS containing 2 mM EDTA and 0.5% BSA, pH 7.2) per cell (the amounts used below are all 10 7 10 μL of FcR blocking reagent and 10 μL of Biotin Antibody Cocktail (a mixture of biotin-labeled antibodies) were added and incubated at 4°C for 5 minutes. 30 μL of isolation buffer and 20 μL of Anti-Biotin MicroBeads were added and incubated at 4°C for 10 minutes. Human mononuclear cells were obtained by passing through a MACS isolation column. 5 × 10 human mononuclear cells were collected. 6The cells were resuspended in serum-free RPMI 1640 medium (GIBCO, catalog no. 22400-089) at a cell density of 1 / mL, seeded into cell culture flasks, and incubated in complete medium (RPMI 1640 containing 10% FBS) supplemented with 200 ng / mL GM-CSF (Beijing Sino Biological Co., Ltd., catalog no. 10015-HNAH) and 100 ng / mL IL-4 (Beijing Sino Biological Co., Ltd., catalog no. 11846-HNAE). After incubation for 3 days, the medium was replaced and incubated for another 3 days. The medium was then replaced with complete medium (RPMI 1640 containing 10% FBS) containing 200 ng / mL GM-CSF, 100 ng / mL IL-4, and 20 ng / mL TNF-α (Beijing Sino Biological Co., Ltd., catalog no. 10602-HNAE) and incubated for 1 day to obtain DC cells.

[0108] Obtaining human T cells: Human PBMCs were resuscitated and collected to ensure that these PBMCs and the PBMCs used to derive DC cells were derived from different individuals. Human T cells were isolated according to the instructions of the Pan T Cell Isolation Kit (Miltenyi Biotech, Cat. No. 130096535). Briefly, PBMCs were first washed once with DPBS and then collected at 10 7 Resuspend the cells in 40 μL of isolation buffer (PBS containing 2 mM EDTA and 0.5% BSA, pH 7.2) per cell (the amounts used below are all 10 7 10 μL of Pan T cell Biotin Antibody Cocktail (calculated in cells) and 10 μL of Pan T cell Biotin-labeled antibody cocktail were added and incubated at 4°C for 5 minutes. 30 μL of isolation buffer and 20 μL of Pan T cell MicroBead Cocktail (Pan T cell microbead cocktail) were then added and incubated at 4°C for 10 minutes. T cells were obtained by passing through a MACS isolation column.

[0109] The collected human DC cells and human T cells were resuspended in complete medium (RPMI 1640 containing 10% FBS) and seeded into a 96-well plate. The seeded DC cells and T cells were each 1 × 10 4 cells / well, 1 x 10 5 The cells were mixed and incubated. Serially diluted heterodimer antibody samples and controls were then added in complete medium. The culture plate was placed in a carbon dioxide incubator at 37°C and incubated for 5 days. After incubation, the supernatants in the wells were removed and the cytokine content was detected using an IL-2 detection kit (RayBiotech, catalog number ELH-IL2) according to the manufacturer's instructions. Briefly, 100 μL of the standard and diluted samples were added to the sample detection plate and incubated at 25°C for 2.5 hours. The plate was washed five times with PBST, and 100 μL of biotin-conjugated detection antibody was added and incubated at 25°C for 1 hour. The plate was washed five times with PBST, followed by the addition of 100 μL of horseradish peroxidase-conjugated streptavidin per well and incubation at 25°C for 45 minutes. The plate was then washed five times with PBST. 100 μL of the chromogenic substrate TMB was added per well and allowed to develop for 10 minutes at room temperature. Color development was stopped by adding 100 μL / well of 1 M H2SO4, and the absorbance at 450 nm was read using a microplate reader.

[0110] The results are shown in Figure 10. Human T cells were activated by stimulation with allogeneic DC cells and secreted IL-2. The addition of anti-PD-L1 and anti-4-1BB bivalent antibodies enhanced T cell activation and promoted cytokine secretion. The anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120h also exhibited strong T cell regulatory activity and significantly promoted the secretion of the cytokine IL-2.

[0111] Example 8 4-1BB agonist activity mediated by anti-PD-L1 / anti-4-1BB heterodimer antibody GS-H2 / 4-1BB cells (purchased from GenScript) were harvested and washed once with MEM+Glu medium (GIBCO, catalog number 41090101) containing 2% FBS (Hyclone, catalog number SH30084.03). GS-H2 / 4-1BB cells were cultured at 1 × 10 in MEM+Glu medium containing 2% FBS. 5 The cells were resuspended at a density of 1 x 10 cells / mL. A 96-well flat-bottom cell culture plate (Costar, catalog no. 3599) was taken, and the cell suspension was added at 100 μL per well. The culture plate was placed in a carbon dioxide incubator at 37°C and incubated for 2 hours to allow the cells to adhere to the culture plate. Heterodimer antibody samples and controls were diluted in MEM + Glu medium containing 2% FBS, and 50 μL was added to the cell culture plate containing GS-H2 / 4-1BB cells. At the same time, DLD-1 / PD-L1 cells (DLD-1 purchased from the Chinese Academy of Sciences) were harvested and diluted to 1 x 10 cells / mL in MEM + Glu medium containing 2% FBS. 5 A cell suspension was prepared at 100 cells / mL, and 50 μL was added to cell culture plates containing GS-H2 / 4-1BB cells and containing the heterodimer antibody samples and controls. The cell culture plates were incubated at 37°C in a carbon dioxide incubator for 24 hours. The cell culture supernatants were collected and the cytokine content was detected using an IL-8 detection kit (Dakewe Biotech Co., Ltd., catalog number 1110802) according to the manufacturer's instructions. Briefly, 100 μL of the standard and diluted samples were added to the sample detection plate, and simultaneously, 50 μL of biotin-labeled detection antibody was added and incubated at 25°C for 1 hour. The plate was washed five times with PBST, followed by the addition of 100 μL / well of horseradish peroxidase-labeled streptavidin and incubation at 25°C for 1 hour. The plate was then washed five times with PBST. The colorimetric substrate TMB was added at 100 μL / well and allowed to develop for 10 minutes at room temperature. Color development was stopped by adding 100 μL / well of 1 M H2SO4. The absorbance at 450 nm was read using a microplate reader.

[0112] The results are shown in Figure 11. Addition of anti-4-1BB bivalent monoclonal antibody induced some activation of GS-H2 / 4-1BB cells, resulting in the secretion of small amounts of IL-8, whereas addition of anti-PD-L1 bivalent antibody failed to activate GS-H2 / 4-1BB cells. In contrast, the anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120h was able to crosslink PD-L1 on DLD-1 / PD-L1 cells with 4-1BB on GS-H2 / 4-1BB cells, thereby causing 4-1BB clustering and strong activation of GS-H2 / 4-1BB cells, resulting in the production of large amounts of IL-8.

[0113] Example 9 Pharmacodynamic study of anti-MC38 tumors in gene knock-in mice with anti-PD-L1 / anti-4-1BB heterodimer antibodies Six- to eight-week-old female hPD-L1 / h4-1BB dual-targeted humanized mice (Jiangsu Biocytogen Co., Ltd.) were used as experimental subjects. After 1 week of adaptation, 5 × 10 IgG antibodies were administered to each mouse. 5 MC38 / hPD-L1 mouse colon tumor cells (MC38 were purchased from Shanghai Shunran Biotech Co., Ltd.) were inoculated subcutaneously into the right dorsal region. Tumor volumes were approximately 100 mm. 3 When tumor volume reached 100%, tumor-bearing mice were divided into groups of six mice each based on tumor volume. Vehicle (DPBS, GIBCO, catalog number 14190-136), anti-PD-L1 monoclonal antibody 35 nmol / kg, anti-4-1BB monoclonal antibody 35 nmol / kg, and anti-PD-L1 / anti-4-1BB heterodimer antibody 70 nmol / kg (considering that the monoclonal antibody is a bivalent antibody, both the anti-PD-L1 and anti-4-1BB bispecific antibodies are monovalent) were administered three times a week for two consecutive weeks via intraperitoneal injection. Tumor volume was measured twice a week from the day of administration, and its major axis (a) and minor axis (b) were measured. Tumor volume was calculated using the formula: tumor volume (mm 3 )=(a×b 2) / 2. The duration of tumor volume measurement was 4 weeks, i.e., after administration was stopped, observation was continued for another 2 weeks.

[0114] The results are shown in Figure 12. In the allogeneic tumor model, the anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120h had a stronger anti-tumor effect than the anti-PD-L1 bivalent antibody and the anti-4-1BB bivalent antibody.

[0115] Example 10 Pharmacodynamic study of anti-CT-26 tumors in gene knock-in mice using a combination of anti-PD-L1 / anti-4-1BB heterodimer antibody and anti-PD-1 antibody Six- to eight-week-old female hPD1 / hPDL1 / hCD137 triple-targeted humanized mice (Jiangsu Jiangsu Pharmaceutical Co., Ltd.) were selected. After allowing the mice to adapt to the environment for 1 week, 5 × 10 IgG per mouse were administered. 5 CT-26 / hPD-L1 mouse colon tumor cells (Jiangsu Jiangsu Pharmaceutical Co., Ltd.) were subcutaneously inoculated into the right dorsal region of the neck. Tumor volumes were approximately 100 mm. 3 Once tumor volume reached this level, the mice were randomly assigned to groups based on tumor volume. Vehicle (DPBS), anti-PD-L1 / 4-1BB dual antibody 10 mg / kg, anti-PD-1 monoclonal antibody (BH2917b) 5 mg / kg, or the combination group (10 mg / kg + 5 mg / kg) were administered every two days for four consecutive doses. Administration was intraperitoneal. Tumor volume was measured three times a week from the day of administration, and the long and short diameters were recorded. Tumor volume was calculated using the following formula: Tumor volume = [(short diameter^2 × long diameter) / 2]. Tumor growth inhibition rate was calculated using the following formula: Tumor growth inhibition rate = [1 - RTV (experimental group) / RTV (control group)] × 100%. RTV: relative tumor volume, RTV = V t / V0, V t: tumor volume at time t, V0: initial tumor volume. The results are shown in Figure 13. In an allogeneic tumor model, the combination of the anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120 and an anti-PD-1 antibody demonstrated synergistic anti-tumor activity. Twenty days after inoculation of CT-26-hPD-L1 cells, tumor volumes were reduced in both the BH2917b (5 mg / kg) and BH3120 (10 mg / kg) groups compared to the control group, with tumor growth inhibition rates of 44.1% and 37.3%, respectively. BH3120 + BH2917b (10 mg / kg + 5 mg / kg) significantly inhibited tumor growth, with tumor growth inhibition rate of 79.6%. Analysis by t-test showed that tumor volume was significantly reduced in the combination therapy group compared to BH3120 (10 mg / kg), with a statistically significant difference (P<0.05). Compared with the BH2917b (5 mg / kg) group, the tumor volume in the combination therapy group was reduced, but the difference was not statistically significant (P>0.05).

[0116] Example 11 Pharmacodynamic study of anti-MC38 tumors in gene knock-in mice using a combination of anti-PD-L1 / anti-4-1BB heterodimer antibody and anti-PD-1 antibody Six- to eight-week-old female hPD1 / hCD137 double-targeted humanized mice (Jiangsu Jiangsu Pharmaceutical Co., Ltd. and Biocytogen Co., Ltd.) were selected. After allowing the mice to adapt to the environment for 1 week, 1 × 10 IgG was administered per mouse. 6 MC38 / hPD-L1 mouse colon tumor cells (Jiangsu Jiangsu Pharmaceutical Co., Ltd.) were subcutaneously inoculated into the right dorsal cervical region. Tumor volumes were approximately 100 mm. 3Once tumor volume reached this level, the mice were randomly assigned to groups based on tumor volume. Vehicle (DPBS), anti-PD-L1 / 4-1BB dual antibody 1 and 3 mg / kg, anti-PD-1 monoclonal antibody (BH2917b) 5 mg / kg, and the combination group (1 + 5 mg / kg) were administered twice weekly for six consecutive days. Administration was intraperitoneal. Tumor volume was measured three times weekly from the day of administration, and the long and short diameters were recorded. Tumor volume was calculated using the following formula: tumor volume = [(short diameter^2 × long diameter) / 2]. Tumor growth inhibition rate was calculated using the following formula: tumor growth inhibition rate = [1 - RTV (experimental group) / RTV (control group)] × 100%. RTV: relative tumor volume; RTV = V t / V0, V t : tumor volume at time t, V0: initial tumor volume. The results are shown in Figure 14. In an allogeneic tumor model, the combination of anti-PD-L1 / anti-4-1BB heterodimer antibody BH3120 and anti-PD-1 antibody demonstrated better synergistic anti-tumor activity. 33 days after inoculation of hPD-L1 / MC38 cells, the antibody monotherapy group exhibited a consistent tumor-inhibitory effect that was dose-dependent, with tumor growth inhibition rates of 76.58% for BH2917b (5 mg / kg), 47.07% for BH3120 (3 mg / kg), and 30.95% for BH3120 (1 mg / kg), respectively. The combination therapy of BH3120 and BH2917b (1 + 5 mg / kg) significantly inhibited tumor growth and reduced TGI. TV The % was 122.40%. Analysis by t-test showed that there was a statistically significant difference compared to the BH3120 (1 mg / kg) group (P<0.05) and the BH2917b (5 mg / kg) group (P<0.01).

Claims

1. A bispecific antibody comprising a first antigen-binding functional region that specifically binds to PD-L1 and a second antigen-binding functional region that specifically binds to 4-1BB, The first antigen-binding functional region that specifically binds to PD-L1 is (A) a heavy chain variable region; (B) a light chain variable region, The heavy chain variable region (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15; (b) an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (c) an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; The light chain variable region (a) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18; (b) an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19; (c) an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; The second antigen-binding functional region that specifically binds to 4-1BB is (A) a heavy chain variable region; (B) a light chain variable region, The heavy chain variable region (a) an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; (b) an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22; and (c) an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; The light chain variable region (a) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24; (b) an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; (c) an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26; Bispecific antibodies.

2. The bispecific antibody of claim 1, wherein the first antigen-binding functional region that specifically binds to PD-L1 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

2.

3. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding functional region that specifically binds to 4-1BB comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

10.

4. The first antigen-binding functional region that specifically binds to PD-L1 is a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2; The second antigen-binding functional region that specifically binds to 4-1BB is The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

10. A bispecific antibody according to any one of claims 1 to 3.

5. The bispecific antibody according to any one of claims 1 to 4, wherein the first antigen-binding functional region and the second antigen-binding functional region are selected from a Fab fragment, an scFv fragment, and a variable domain fragment Fv.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the first antigen-binding functional region and the second antigen-binding functional region are both Fab fragments.

7. 7. The bispecific antibody of claim 1 , wherein the Fab fragment comprises a first heavy chain variable region and a second heavy chain variable region that are different, and a first light chain variable region and a second light chain variable region that are different.

8. The bispecific antibody according to any one of claims 1 to 5, wherein one of the first antigen-binding functional region and the second antigen-binding functional region is a Fab fragment, and the other is an scFv.

9. 1. A bispecific antibody comprising a first Fc chain and a second Fc chain, the first Fc chain and the second Fc chain are both Fc fragments of immunoglobulin G containing amino acid substitutions, and the first Fc chain and the second Fc chain together form a heterodimer capable of binding to an Fc receptor; the first Fc chain and the second Fc chain are linked to the first antigen-binding functional region and the second antigen-binding functional region, respectively, via a covalent bond or a linker; and wherein one of the first Fc chain and the second Fc chain comprises amino acid substitutions of T366L and D399R at positions 366 and 399, and the other comprises amino acid substitutions of L351E, Y407L, and K409V at positions 351, 407, and 409, wherein the amino acid positions are numbered according to the Kabat EU index numbering system. A bispecific antibody according to any one of claims 1 to 8.

10. 10. The bispecific antibody according to any one of claims 1 to 9, wherein the weight percentage of the formed homodimer based on all polypeptide chains of the first Fc chain and the first antigen-binding functional region linked thereto via a covalent bond, and the second Fc chain and the second antigen-binding functional region linked thereto via a covalent bond is less than 50% in a solution in the presence of a reducing agent, when the solution does not contain any polypeptides other than the first Fc chain and the first antigen-binding functional region linked thereto via a covalent bond, and the second Fc chain and the second antigen-binding functional region linked thereto via a covalent bond.

11. A bispecific antibody comprising a first heavy chain / first light chain pair that specifically binds to PD-L1, the first heavy chain has a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6 and a heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 8; The first light chain has a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2 and a light chain constant region comprising the amino acid sequence shown in SEQ ID NO:

4. A bispecific antibody according to any one of claims 1 to 10.

12. A bispecific antibody comprising a second heavy chain / second light chain pair that specifically binds to 4-1BB, the second heavy chain has a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 14; the second light chain has a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10 and a light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 4; A bispecific antibody according to any one of claims 1 to 11.

13. An isolated polynucleotide encoding the bispecific antibody of any one of claims 1 to 12.

14. A recombinant expression vector comprising the isolated polynucleotide of claim 13.

15. 15. A host cell comprising the isolated polynucleotide of claim 13 or the recombinant expression vector of claim 14.

16. HEK293T, HEK293E, HEK293F obtained by modification based on human embryonic kidney cells HEK293 or HEK293 cells, CHO-S obtained by modification based on hamster ovary cells CHO or CHO cells, CHO-dhfr-, CHO / DG44, ExpiCHO, E. coli or E. coli obtained by modification based on E. coli BL21, BL21 (DE3), Rosetta, Origami, Pichia pastoris obtained by modification based on yeast or yeast, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, insect cells or insect cell-based modifications obtained by modification based on cells High5, SF9, plant cells, mammalian mammary gland cells, somatic cells, the host cell according to claim 15 selected from the group consisting of.

17. 17. A composition comprising a bispecific antibody according to any one of claims 1 to 12, or an isolated polynucleotide according to claim 13, or a recombinant expression vector according to claim 14, or a host cell according to claim 15 or 16, and a pharmaceutically acceptable carrier.

18. further comprising at least one second therapeutic agent; the second therapeutic agent and the bispecific antibody, isolated polynucleotide, recombinant expression vector, or host cell are in different parts of the composition.

18. The composition of claim 17.

19. 20. The composition of claim 18, wherein the second therapeutic agent is selected from the group consisting of a second antibody, an immunotherapeutic agent, a targeted therapeutic agent, or a chemotherapeutic agent.

20. The composition of claim 19, wherein the second therapeutic agent is an anti-PD-1 antibody and / or a STING agonist.

21. 1) expressing the isolated polynucleotide of claim 13 or the recombinant expression vector of claim 14 in a host cell, respectively; 2) reducing each expressed protein in the host cell; 3) mixing the reduced protein and then oxidizing the mixture; A method for producing a bispecific antibody according to any one of claims 1 to 12.

22. The method according to claim 21, wherein the reduction step comprises: 1) performing a reduction reaction in the presence of a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine, and cysteine; and 2) removing the reducing agent.

23. 23. The method according to claim 21 or 22, wherein the oxidation step is oxidation in air, and further comprises a step of carrying out the oxidation reaction in the presence of an oxidizing agent selected from L-dehydroascorbic acid or a chemical derivative thereof.

24. 24. The method of any one of claims 21 to 23, further comprising an isolation and purification step.

25. 21. Use of a bispecific antibody according to any one of claims 1 to 12, and / or an isolated polynucleotide according to claim 13, and / or a recombinant expression vector according to claim 14, and / or a host cell according to claim 15 or 16, and / or a composition according to any one of claims 17 to 20 in the manufacture of a medicament for the prevention and / or treatment of a disease in a subject.

26. 21. A bispecific antibody according to any one of claims 1 to 12, and / or an isolated polynucleotide according to claim 13, and / or a recombinant expression vector according to claim 14, and / or a host cell according to claim 15 or 16, and / or a composition according to any one of claims 17 to 20, for use as a medicament for the prevention and / or treatment of a disease in a subject.

27. the subject is a mammal; 26. The use according to claim 25.

28. 28. The use of claim 27, wherein the subject is a human.

29. 26. The use of claim 25, wherein the disease is selected from leukemia, lymphoma, myeloma, brain tumor, squamous cell carcinoma of the head and neck, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer, and bone cancer.

30. The subject is a mammal.

27. The bispecific antibody, isolated polynucleotide, recombinant expression vector, host cell or composition of claim 26.

31. The bispecific antibody, isolated polynucleotide, recombinant expression vector, host cell or composition of claim 30, wherein the subject is a human.

32. The bispecific antibody, isolated polynucleotide, recombinant expression vector, host cell or composition of claim 26, wherein the disease is selected from leukemia, lymphoma, myeloma, brain tumor, squamous cell carcinoma of the head and neck, non-small cell lung cancer, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, pancreatic adenocarcinoma, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma, small cell lung cancer and bone cancer.

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