Combination therapy of claudin 18.2 antagonist and PD-1 / PD-L1 inhibitor

Abstract

Combination therapy of a CLDN18.2 antagonist and a PD-1 / PD-L1 system inhibitor is provided for a subject having expression of CLDN18.2 and low or no expression of PD-L1 in diseased tissue (e.g., tumor tissue) obtained from the subject.

Description

[Technical field]

[0001] The present disclosure relates generally to combination therapies comprising claudin 18.2 (CLDN18.2) antagonists and PD-1 / PD-L1 axis inhibitors for CLDN18.2-associated diseases. [Background technology]

[0002] Claudin-18 (CLDN18) molecular splice variant 2 (CLDN18A2 or CLDN18.2): NM_001002026, NP_001002026) is an integral transmembrane protein with a molecular weight of approximately 27.72 kD. In healthy cells, CLDN18.2 is expressed only in gastric cells. Most importantly, CLDN18.2 expression is restricted to differentiated short-lived cells of the gastric epithelium, but is spared from the stem cell region of the stomach. CLDN18.2 is undetectable in any other normal human organs, even when using highly sensitive RT-PCR. CLDN18.2 is highly expressed in several cancer types, including gastric, esophageal, pancreatic, and lung tumors, as well as in human cancer cell lines (see Matsuda Y, Semba S, Ueda J, et al. Gastric and intestinal claudin expression at the invasive front of gastric carcinoma[J]. Cancer science, 2007, 98(7):1014-1019).

[0003] CLDN18.2-expressing cancers can be treated with combination therapy of antibodies targeting CLDN18.2 and immune checkpoint inhibitors, such as PD-1 / PD-L1 inhibitors, as disclosed in WO2021 / 025177, the disclosure of which is incorporated by reference in its entirety. However, such combination therapy still faces challenges in achieving satisfactory therapeutic efficacy in clinical trials. Summary of the Invention

[0004] Therefore, there is a great need for improved combination therapies for CLDN18.2-expressing cancers to meet clinical needs.

[0005] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or more than one antibody.

[0006] The present disclosure relates, inter alia, to a method of treating a CLDN18.2-associated disease or condition in a subject in need thereof, comprising: administering to a subject a therapeutically effective amount of a CLDN18.2 antagonist in combination with a therapeutically effective amount of a PD-1 / PD-L1 inhibitor; A method is provided in which a subject is determined to have CLDN18.2 expression in diseased tissue.

[0007] In certain embodiments, the method further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent.

[0008] In certain embodiments, CLDN18.2 expression in diseased tissue is higher than or equivalent to expression in healthy or non-cancerous gastric cells or tissue.

[0009] In certain embodiments, CLDN18.2 expression in diseased tissue is lower than expression in healthy or non-cancerous gastric cells or tissue, but higher than expression in healthy or non-cancerous tissues or organs other than the stomach.

[0010] In certain embodiments, CLDN18.2 expression in diseased tissue is comparable to expression in healthy or non-cancerous tissues or organs other than the stomach and is detectable by an anti-CLDN18.2 diagnostic antibody.

[0011] In certain embodiments, expression of CLDN18.2 is on the cell surface or membrane-associated.

[0012] In certain embodiments, the subject is determined to have PD-L1 expression in the diseased tissue.

[0013] In certain embodiments, the subject is determined to have low or no PD-L1 expression in the affected tissue.

[0014] In certain embodiments, PD-L1 expression in the diseased tissue is lower than the reference level.

[0015] In certain embodiments, no more than 20% of the cells in the affected tissue are positive for PD-L1 expression.

[0016] In certain embodiments, cells of the diseased tissue include disease cells and immune cells in the diseased tissue.

[0017] In certain embodiments, PD-L1 expression in diseased tissue is similar to that in healthy or non-cancerous tissue.

[0018] In certain embodiments, PD-L1 expression in diseased tissues is low or not detectable by anti-PD-L1 diagnostic antibodies.

[0019] In another aspect, the disclosure also provides a method of sensitizing a disease or condition to treatment with a PD-1 / PD-L1 axis inhibitor in a subject in need thereof, the subject being determined to have low or absent expression of PD-L1 in an affected tissue; a) determining the presence or expression level of CLDN18.2 in diseased tissue obtained from the subject; and b) administering to the subject a therapeutically effective amount of a CLDN18.2 antagonist, optionally in combination with a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor, if the presence of CLDN18.2 is determined in step a) or if the expression level of CLDN18.2 reaches a threshold level in step a); thereby sensitizing the disease or condition to treatment with a PD-1 / PD-L1 axis inhibitor. Also provided is a method comprising:

[0020] In certain embodiments, step b) further comprises administering to the subject a chemotherapeutic agent.

[0021] In certain embodiments, expression of CLDN18.2 is on the cell surface or membrane-associated.

[0022] In certain embodiments, PD-L1 expression in the diseased tissue is lower than the reference level.

[0023] In certain embodiments, no more than 20% of the cells in the affected tissue are positive for PD-L1 expression.

[0024] In certain embodiments, cells of the diseased tissue include disease cells and immune cells in the diseased tissue.

[0025] In certain embodiments, PD-L1 expression in diseased tissue is similar to that in healthy or non-cancerous tissue.

[0026] In certain embodiments, PD-L1 expression in diseased tissues is low or not detectable by anti-PD-L1 diagnostic antibodies.

[0027] In another aspect, the disclosure also provides a method of increasing the responsiveness of a tumor to treatment with a PD-1 / PD-L1 inhibitor in a subject, the subject being determined to have a tumor that is resistant or refractory to treatment with a PD-1 / PD-L1 inhibitor; a) determining the presence or expression level of CLDN18.2 in a tumor sample obtained from a subject; and b) administering to the subject a therapeutically effective amount of a CLDN18.2 antagonist, optionally in combination with a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor, if the presence of CLDN18.2 is determined in step a) or if the expression level of CLDN18.2 reaches a threshold level in step a); Thereby, increasing the responsiveness of a tumor to treatment with a PD-1 / PD-L1 inhibitor in a subject. The present invention provides a method comprising:

[0028] In certain embodiments, step b) further comprises administering to the subject a chemotherapeutic agent.

[0029] In certain embodiments, expression of CLDN18.2 is on the cell surface or membrane-associated.

[0030] In certain embodiments, PD-L1 expression in the tumor tissue is lower than the reference level.

[0031] In certain embodiments, no more than 20% of the cells in the tumor tissue are positive for PD-L1 expression.

[0032] In certain embodiments, the cells of the tumor tissue comprise cancer cells and immune cells in the tumor tissue.

[0033] In certain embodiments, PD-L1 expression in tumor tissue is similar to that in healthy or non-cancerous tissue.

[0034] In certain embodiments, PD-L1 expression in tumor tissue is low or not detectable by an anti-PD-L1 diagnostic antibody.

[0035] In another aspect, the disclosure also provides a method for determining the likelihood of a subject having low or no expression of PD-L1 to be eligible for or responsive to treatment with a CLDN18.2 antagonist, optionally in combination with a PD-1 / PD-L1 axis inhibitor, comprising: a) contacting a sample obtained from a subject with a CLDN18.2 diagnostic agent under conditions that allow for detection of expression of CLDN18.2; b) determining the presence or expression level of CLDN18.2 in the sample; If the sample has positive expression of CLDN18.2 in the sample, the subject is determined to be eligible for or likely to respond to treatment with a CLDN18.2 antagonist, optionally in combination with a PD-1 / PD-L1 axis inhibitor; or If no expression of CLDN18.2 is detected in the sample, the subject is determined to be ineligible for or not likely to respond to treatment with a CLDN18.2 antagonist, optionally in combination with a PD-1 / PD-L1 inhibitor. The present invention provides a method comprising:

[0036] In certain embodiments, the CLDN18.2 diagnostic agent is an anti-CLDN18.2 diagnostic antibody.

[0037] In certain embodiments, the CLDN18.2 antagonist comprises an anti-CLDN18.2 antibody, e.g., a monoclonal anti-CLDN18.2 antibody, a bispecific antibody targeting CLDN18.2 and a second antigen (e.g., CD3, 4-1BB, TGFβ, SIRPα and IL15), or an immune cell expressing a chimeric antigen receptor (CAR) or genetically modified TCR comprising an anti-CLDN18.2 antigen-binding domain.

[0038] In certain embodiments, the anti-CLDN18.2 antibody comprises heavy chain HCDR1, HCDR2, and HCDR3 and / or light chain LCDR1, LCDR2, and LCDR3 sequences, The HCDR1 sequence comprises GYNMN (SEQ ID NO:1) or a homologous sequence thereof with at least 80% sequence identity; The HCDR2 sequence comprises NIDPYYGGTSYNQKFKG (SEQ ID NO: 2) or a homologous sequence thereof of at least 80% sequence identity; the HCDR3 sequence comprises MYHGNAFDY (SEQ ID NO:3) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence comprises KSSQSLLNSGNLKNYLT (SEQ ID NO: 4) or a homologous sequence thereof with at least 80% sequence identity; the LCDR2 sequence comprises WASTRKS (SEQ ID NO:5) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence includes QNDYSYPLT (SEQ ID NO:6) or a homologous sequence thereof with at least 80% sequence identity.

[0039] In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:8.

[0040] In certain embodiments, the anti-CLDN18.2 antibody further comprises an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region.

[0041] In certain embodiments, the anti-CLDN18.2 antibody further comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0042] In certain embodiments, the constant region of human IgG1 comprises SEQ ID NO: 9 or a homologous sequence thereof having at least 80% sequence identity.

[0043] In certain embodiments, the constant region contains a substitution or modification of one or more amino acid residues that confers increased CDC or ADCC relative to the wild-type constant region.

[0044] In certain embodiments, the constant region comprises one or more amino acid residue substitutions relative to SEQ ID NO:9 selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof.

[0045] In certain embodiments, the constant region comprises the sequence of SEQ ID NO:11.

[0046] In certain embodiments, the constant region further comprises the sequence of SEQ ID NO:10.

[0047] In certain embodiments, the anti-CLDN18.2 antibody is humanized.

[0048] In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14; and The light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:16.

[0049] In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO:39; The light chain comprises the amino acid sequence of SEQ ID NO:40.

[0050] In certain embodiments, the anti-CLDN18.2 antibody is capable of inducing the expression of PD-L1 in affected tissues of a subject.

[0051] In certain embodiments, the anti-CLDN18.2 antibody is linked to one or more conjugate moieties.

[0052] In certain embodiments, the conjugate moiety comprises a clearance modifier, a chemotherapeutic drug, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a cytokine (e.g., IL-15, IL-2, IL-7) or other anti-cancer drug.

[0053] In certain embodiments, the PD-1 / PD-L1 axis inhibitor comprises a PD-1 inhibitor selected from the group consisting of an antibody, a small molecule, and a combination thereof.

[0054] In certain embodiments, the PD-1 inhibitor is selected from the group consisting of nivolumab (OPDIVO; BMS-936558), dostallimab (TSR-042), pembrolizumab (KEYTRUDA; MK-3475), MEDI0680 (AMP-514), MEDI4736, BI 754091, pidilizumab (CT-011), cemiplimab (LIBTAYO, REGN2810), spartalizumab (PDR001), cetrelimab (JNJ 63723283), Toripalimab (JS001), PF-06801591, Tislelizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, Lambrolizumab, Camrelizumab (SHR-1210), Sintilimab (Tyvyt, IBI308), Penpulimab (AK105), Zimberelimab, Retifanlimab, Serplulimab mab, Balstilimab, Geptanolimab, Prolgolimab, Ezabenlimab, Sasanlimab, Pimivalimab, Budigalimab, Nofazinlimab, Sindelizumab, MGA404, Sym021, BAT1306, and HX008.

[0055] In certain embodiments, the PD-1 / PD-L1 axis inhibitor comprises a PD-L1 inhibitor selected from the group consisting of an antibody, a small molecule, and a combination thereof.

[0056] In certain embodiments, the PD-L1 inhibitor is atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446), BMS-936559, avelumab (bavencio), lodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, envafolimab (KN035), MDX-1105, STI-1040, CS1001, avelum ... The anti-PD-L1 antibody is selected from the group consisting of Adebrelimab (SHR-1316), SHR-1701, TOB2450, Bintrafusp, LP002, STI-3031, Cosibelimab, Pacmilimab, NM01, LDP, AMP-224, Garibulimab (BGB-A333), A167, SCD-135, Opucolimab, and GR1405.

[0057] In certain embodiments, the PD-L1 inhibitor comprises a bispecific antibody that targets both PD-L1 and another checkpoint molecule selected from the group consisting of PD-1, PD-L1, PD-L2, CLTA-4, SIRP, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, or CD83.

[0058] In certain embodiments, the checkpoint molecule is TGFβ, 4-1BB, CTLA4, LAG3, or TIGIT.

[0059] In certain embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody that comprises a heavy chain HCDR1, HCDR2, and HCDR3, and / or a light chain LCDR1, LCDR2, and LCDR3 sequence; The HCDR1 sequence comprises DYYMN (SEQ ID NO: 22) or a homologous sequence thereof of at least 80% sequence identity; The HCDR2 sequence comprises DINPNNAETLYNHKFKG (SEQ ID NO: 23) or a homologous sequence thereof of at least 80% sequence identity; The HCDR3 sequence comprises WGDGPFAY (SEQ ID NO: 24) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence comprises KASQNVGAAVA (SEQ ID NO:25) or a homologous sequence thereof of at least 80% sequence identity; The LCDR2 sequence comprises SVSDRYT (SEQ ID NO:26) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence includes QQYSNYPT (SEQ ID NO:27) or a homologous sequence thereof with at least 80% sequence identity.

[0060] In certain embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17; The light chain variable region comprises the amino acid sequence of SEQ ID NO:18.

[0061] In certain embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20; The light chain comprises the amino acid sequence of SEQ ID NO:21.

[0062] In certain embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody that comprises a heavy chain HCDR1, HCDR2, and HCDR3, and / or a light chain LCDR1, LCDR2, and LCDR3 sequence; The HCDR1 sequence comprises TYWMH (SEQ ID NO: 32) or a homologous sequence thereof of at least 80% sequence identity; The HCDR2 sequence comprises MIQPNSGGTKYNEKFKK (SEQ ID NO: 33) or a homologous sequence thereof of at least 80% sequence identity; The HCDR3 sequence comprises GAGTVDYFDY (SEQ ID NO: 34) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence comprises RASESVDIYGNSFMH (SEQ ID NO: 35) or a homologous sequence thereof of at least 80% sequence identity; The LCDR2 sequence comprises RASNLES (SEQ ID NO:36) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence includes QQSTEDPYT (SEQ ID NO: 37) or a homologous sequence thereof with at least 80% sequence identity.

[0063] In certain embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:28; The light chain variable region comprises the amino acid sequence of SEQ ID NO:29.

[0064] In certain embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 30; The light chain comprises the amino acid sequence of SEQ ID NO:31.

[0065] In certain embodiments, the subject is a human.

[0066] In certain embodiments, administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0067] In certain embodiments, administration of a composition comprising an anti-CLDN18.2 antibody precedes, is concomitant with, or follows administration of a composition comprising a PD-1 / PD-L1 axis inhibitor.

[0068] In certain embodiments, the disease or condition is cancer.

[0069] In certain embodiments, the diseased tissue comprises cancer cells.

[0070] In certain embodiments, the cancer is selected from the group consisting of gastric cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain tumors, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, and adenocarcinoma.

[0071] In certain embodiments, the cancer is gastric cancer, lung cancer, colon cancer, cholangiocarcinoma, or a combination thereof.

[0072] In certain embodiments, the disease or condition is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor.

[0073] In certain embodiments, the resistance is de novo resistance or acquired resistance.

[0074] In certain embodiments, the disease or condition is further resistant or refractory to a second therapy selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, and combinations thereof.

[0075] In certain embodiments, the disease or condition is resistant or refractory to combination therapy with a PD-1 / PD-L1 axis inhibitor and a chemotherapeutic agent.

[0076] In certain embodiments, the chemotherapeutic agent is an antimetabolite, e.g., methotrexate and 5-fluorouracil (5-FU), oxaliplatin, alkylating agents (e.g., thiotepa and cyclophosphamide (Cytoxan™)), alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan), aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa), emylerumine, and memylamelamine (e.g., altretamine, triemylenmelamine, and cyclophosphamide), cyclosulfonates (e.g., cyclosulfonate ... lenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine), acetogenins, camptothecins (e.g., the synthetic analog topotecan), bryostatin, callystatin, CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin), cryptophycins (especially cryptophycin and cryptophycin), dolastatins, duocarmycins (including the synthetic analog KW-2189 and CBI-TMI), erytherobin, pancratistatin, sarcodictyin, spongistatin, cisplatin, nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard), nitrosoureas (e.g., carmustine, chlorozotocin, foremustine, lomustine, nimustine, ranimustine, folic acid analogues (e.g., demopterin, methotrexate, pteropterin, trimetrexate), purine analogues (e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine), pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine), androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone), anti-adrenal (e.g., aminoglutethimide, mitotane, trilostane), folic acid supplements (e.g., frolinic acid), aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, hestrabucil, bi santrene, edatrexate, defofamine, demecolcine, diaziquone, elformthine, elliptinium acetateacetate), epothilones, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids (e.g., maytansine and ansamitocin), mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSKTM, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triazicon, 2,2,2″-trichlorotriemylamine, trichothecenes, urethanes, vindesine, dacarbazine, mannomustine, mitobronitol, and mitolactol.

[0077] In another aspect, the disclosure also provides a kit useful in treating a disease or condition in a subject in need thereof, comprising a first container comprising a CLDN18.2 antagonist, a second container comprising a PD-1 / PD-L1 axis inhibitor, and optionally instructions for use of the kit, Kits are provided in which the disease or condition is characterized by a) having CLDN18.2 expression in the diseased tissue, and / or b) having low or absent expression of PD-L1 in the diseased tissue.

[0078] In another aspect, the present disclosure also provides a kit comprising a CLDN18.2 antagonist and a package insert containing instructions for using the CLDN18.2 antagonist in combination with a PD-1 / PD-L1 axis inhibitor to treat a disease or condition in a subject in need thereof, comprising: Kits are provided in which the disease or condition is characterized by a) having CLDN18.2 expression in the diseased tissue, and / or b) having low or absent expression of PD-L1 in the diseased tissue.

[0079] In another aspect, the disclosure also provides a kit comprising a PD-1 / PD-L1 axis inhibitor and a package insert containing instructions for using the PD-1 / PD-L1 axis inhibitor in combination with a CLDN18.2 antagonist to treat a disease or condition in a subject in need thereof, comprising: Kits are provided in which the disease or condition is characterized by a) CLDN18.2 expression in the diseased tissue, and / or b) low or absent expression of PD-L1 in the diseased tissue.

[0080] In another aspect, the present disclosure also provides a kit for predicting a subject's responsiveness to treatment with a CLDN18.2 antagonist in combination with a PD-1 / PD-L1 system inhibitor, the kit comprising one or more reagents for detecting the presence of CLDN18.2 and / or PD-L1 in a biological sample obtained from a subject, or one or more reagents for measuring the expression level of CLDN18.2 and / or PD-L1 in a biological sample obtained from a subject, wherein optionally the biological sample is tumor tissue.

[0081] In another aspect, the disclosure also provides for the use of a pharmaceutical composition comprising a therapeutically effective amount of a) a CLDN18.2 antagonist, b) a PD-1 / PD-L1 axis inhibitor, or c) both, and one or more pharma- ceutically acceptable carriers, in the manufacture of a medicament for treating a disease or condition in a subject in need thereof, wherein the disease or condition is characterized by a) CLDN18.2 expression in the affected tissue, and / or b) low or absent expression of PD-L1 in the affected tissue. [Brief description of the drawings]

[0082] [Figure 1A] FIG. 1A is a bar graph showing PD-L1 levels of NUGC-4 when co-cultured with different supernatants. [Figure 1B] FIG. 1B is a bar graph showing the PD-L1 levels of KATOIII when co-cultured with different supernatants. [Figure 1C] Figure 1C is a bar graph showing PD-L1 levels of SNU620 when co-cultured with different supernatants. [Diagram 2] FIG. 2 shows tumor volume in NCI-H460-CLDN18.2 xenograft model with different treatments. [Diagram 3] FIG. 3 shows tumor volume in CT26-CLDN18.2 syngeneic tumor model with different treatments. [Figure 4] FIG. 4 shows tumor volumes in the MC38-CLDN18.2 syngeneic tumor model with different treatments. [Figure 5A-5B] 5A and 5B show tumor volumes in the CT26-CLDN18.2 syngeneic tumor model with different treatments. [Figure 6A] Figure 6A shows representative IHC images of CLDN18.2 and corresponding PD-L1 staining in various cancer tissues in clinical samples with different staining intensities. [Figure 6B] Figure 6B shows representative IHC images of CLDN18.2 and corresponding PD-L1 staining in various cancer tissues in patient-derived xenograft (PDX) samples with different staining intensities. [Figure 7] FIG. 7 shows tumor volume in the NUGC4-hCLDN18.2 and human PBMC co-inoculated tumor model with different treatments. [Figure 8] FIG. 8 shows tumor volume in MFC-hCLDN18.2 syngeneic tumor model with different treatments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] The following description of the present disclosure is intended to merely illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limitations on the scope of the present disclosure. It is believed that various equivalents, modifications, and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included in this specification. All references cited in this specification, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0084] definition As used herein, the terms "a," "an," "the," and similar terms as used in the context of the present invention (especially in the context of the claims) are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0085] The term "CLDN18.2" refers to Claudin-18 splice variant 2 from mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice). In certain embodiments, CLDN18.2 is human CLDN18.2. Exemplary sequences of human CLDN18.2 include human CLDN18.2 protein (NCBI Reference SEQ ID NO: NP_001002026.1, or SEQ ID NO: 38). Exemplary sequences of CLDN18.2 include Mus musculus (mouse) CLDN18.2 protein (NCBI Reference SEQ ID NO: NP_001181852.1), Macaca fascicularis (cynomolgus monkey) CLDN18.2 protein (NCBI Reference SEQ ID NO: XP_015300615.1). CLDN18.2 is expressed in cancer cells. In one embodiment, CLDN18.2 is expressed on the surface of cancer cells.

[0086] As used herein, the term "antagonist" in relation to CLDN18.2 refers to any molecule that partially or completely inhibits, blocks or neutralizes the biological activity of CLDN18.2.Suitable CLDN18.2 antagonists can include, but are not limited to, antibodies, antisense oligonucleotides, peptides and small organic molecules.In certain embodiments, CLDN18.2 antagonists are anti-CLDN18.2 antibodies.

[0087] An "anti-CLDN18.2 antibody," as used herein, refers to an antibody that can specifically bind to CLDN18.2 (e.g., human or non-human CLDN18.2) with sufficient affinity to provide, for example, diagnostic and / or therapeutic uses.

[0088] The term "antibody", as used herein, includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, bispecific antibody or antibody variant (e.g., affinity variants, glycosylation variants, cysteine ​​engineered variants, Fc variants, antigen-binding fragments, antibody drug conjugates) that binds to a specific antigen.

[0089] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and can bind to two different epitopes, which may be on the same antigen or on two different antigens.

[0090] The term "antibody drug conjugate" as used herein refers to the linkage of an antibody or antigen-binding fragment thereof to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, and the like. The linkage may be a covalent bond or a non-covalent interaction, such as by electrostatic forces. A variety of linkers known in the art can be used to form antibody drug conjugates. In addition, the antibody drug conjugates can be provided in the form of a fusion protein that can be expressed from a polynucleotide encoding the conjugate.

[0091] As used herein, "fusion protein" refers to a protein created by the joining of two or more genes or gene fragments that originally encoded separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein having functional properties derived from each of the original proteins.

[0092] As used herein, the term "antigen-binding fragment" refers to a fragment of an antibody that contains one or more CDRs, or an antibody fragment formed from any other antibody portion that binds to an antigen but does not contain an intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen that the parent antibody binds. In certain embodiments, antigen-binding fragments can contain one or more CDRs from a particular human antibody.

[0093] "Fab" with respect to an antibody refers to a monovalent antigen-binding fragment of an antibody consisting of a single light chain (both variable and constant regions) linked by disulfide bonds to a single heavy chain variable region and a first constant region. Fab can be obtained by papain digestion of an antibody at residues proximal to the N-terminus of the inter-heavy chain disulfide bond in the hinge region.

[0094] "Fab'" refers to a Fab fragment containing a portion of the hinge region, which can be obtained by pepsin digestion of an antibody at residues proximal to the C-terminus of the inter-heavy chain disulfide bond in the hinge region, and thus differs from Fab by a small number of residues in the hinge region, including one or more cysteines.

[0095] "F(ab')2" refers to a dimer of Fab' containing two light chains and a portion of two heavy chains.

[0096] "Fc" with respect to an antibody refers to the portion of an antibody that consists of the second and third constant regions of a first heavy chain bound via disulfide bonds to the second and third constant regions of a second heavy chain. IgG and IgM Fc regions contain three heavy chain constant regions (the second, third, and fourth heavy chain constant regions in each chain). The Fc region can be obtained by papain digestion of an antibody. The Fc portion of an antibody is responsible for various effector functions such as ADCC, ADCP, and CDC, but does not function in antigen binding.

[0097] "Fv" with respect to an antibody refers to the smallest fragment of an antibody that retains a complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0098] "Single chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)). "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions together with a linker. In certain embodiments, an "scFv dimer" is a dimer of the V of one of the moieties. H The part is the V of the other part L to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L V dimerized with the moiety H -V L In another embodiment, the "scFv dimer" is a bivalent diabody or a bivalent ScFv (BsFv) comprising V H1 and V L1 In cooperation with V H2 and V L2 and each cooperating pair has a different antigen specificity. L1 -V H2 V bound to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0099] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0100] A "camelized single domain antibody", "heavy chain antibody", "nanobody" or "HCAb" is a camelized single domain antibody that is composed of two V HIt refers to antibodies that contain heavy chain domains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4): 277-302 (2001); WO 94 / 04678; WO 94 / 25591; U.S. Pat. No. 6,005,079). Heavy chain antibodies were originally obtained from camelids (camels, dromedaries, and llamas). Camelized antibodies lack light chains but have a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1): 93-101 (2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)). "Diabodies" include small antibody fragments with two antigen-binding sites, which are separated into V and V subunits in a single polypeptide chain. L Domain and connected V H Domain (V H -V L or V L -V H) (see, for example, Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90(14): 6444-8 (1993), EP 404097, WO 93 / 11161). The two domains on the same chain cannot pair because the linker is too short, and are therefore forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites can target the same or different antigens (or epitopes).

[0101] A "domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In certain embodiments, two or more V H The domains are covalently linked with peptide linkers to form bivalent or multivalent domain antibodies. The two V H The domains can target the same or different antigens.

[0102] In certain embodiments, a "(dsFv)2" is a Fv comprising three peptide chains, linked by a peptide linker, and two V L Two V's attached to the part H Includes parts.

[0103] In certain embodiments, a "bispecific ds diabody" is H1 and V L1 V via a disulfide bridge between L1 -V H2 V bound to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0104] In certain embodiments, a "bispecific dsFv" or "dsFv-dsFv'" is a compound that comprises three peptide chains, the heavy chains of which are linked by a peptide linker (e.g., a long flexible linker) and each V L1 and V L2Partially paired V H1 -V H2 Each disulfide-paired heavy and light chain has a different antigen specificity.

[0105] The term "humanized" as used herein means that the antibody or antigen-binding fragment comprises CDRs derived from a non-human animal, FR regions derived from a human, and, if applicable, a constant region derived from a human. In certain embodiments, amino acid residues in the variable region framework of a humanized CLDN18.2 antibody are replaced for sequence optimization. In certain embodiments, the variable region framework sequences of the humanized CLDN18.2 antibody chains are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the corresponding human variable region framework sequences.

[0106] The term "chimeric" as used herein refers to an antibody or antigen-binding fragment having a portion of the heavy and / or light chain derived from one species and the remaining portion of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0107] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum correspondence. Alignment for the purposes of determining percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available at the National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website, see also Higgins D Get al, Methods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool or customize them as appropriate for alignment, for example by selecting a suitable algorithm. In certain embodiments, non-identical residue positions may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution does not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference.

[0108] As used herein, "homologous sequence" and "homologous sequence" are used interchangeably and refer to a polynucleotide sequence (or its complementary strand) or amino acid sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence, when aligned as necessary.

[0109] An "isolated" material is altered from its natural state by the hand of man. When an "isolated" composition or material occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated", but the same polynucleotide or polypeptide is "isolated" if it is sufficiently separated from the coexisting materials of its natural state so that it exists in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (e.g., ion exchange chromatography or reverse phase HPLC).

[0110] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where otherwise noted, the terms "patient" and "subject" are used interchangeably herein.

[0111] The term "diseased tissue", as used herein, broadly encompasses diseased cells (eg, cancer cells) and tissues (eg, tissue sections).

[0112] The term "anti-tumor activity" refers to the reduction of tumor cell proliferation, survival rate, or metastatic activity. For example, anti-tumor activity can be shown by a reduction in the growth rate of abnormal cells or a stable or reduced tumor size during therapy, or a longer survival time due to therapy compared to a control not receiving therapy. Such activity can be evaluated using any acceptable in vitro or in vivo tumor model, including but not limited to xenograft models, allograft models, mouse mammary tumor virus (MMTV) models, and other known models known in the art for studying anti-tumor activity.

[0113] "Effector function" or "antibody effector function", as used herein, refers to a biological activity that is believed to result from the binding of the Fc region of an antibody to its effector, such as the C1 complex and Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is induced by the interaction of an antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC), which is induced by the binding of the Fc region of an antibody to an Fc receptor on an effector cell; and antibody-dependent cellular phagocytosis (ADCP), in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells. Effector functions include both those that act after antigen binding and those that act independently of antigen binding.

[0114] "Treating" a condition or "treatment" of a condition, as used herein, includes preventing or alleviating the condition, delaying the onset or slowing the rate of occurrence of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or eliminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0115] A "CLDN18.2-associated" disease or condition, as used herein, refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of CLDN18.2. In some embodiments, the CLDN18.2-associated disease or condition is, for example, cancer.

[0116] "Cancer," as used herein, refers to any medical condition characterized by malignant cell growth or neoplasia, abnormal proliferation, invasion, or metastasis, and includes both solid tumors and non-solid cancers (e.g., hematological malignancies), such as leukemia. As used herein, a "solid tumor" refers to a solid mass of neoplastic and / or malignant cells.

[0117] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation, and physiologically compatible with the recipient thereof.

[0118] "Targeted therapy" is a type of therapy that acts on specific molecules associated with cancer that are present in cancer cells but not in normal cells or that are more abundant in cancer cells, such as specific proteins, or target molecules in the cancer microenvironment that contribute to the growth and survival of the cancer. Targeted therapy directs therapeutic agents to the tumor, thereby avoiding normal tissues from the effects of the therapeutic agent.

[0119] Reference herein to a value or parameter of "about" includes (and describes) an embodiment that is directed to that value or parameter itself. For example, a reference to "about X" includes the reference to "X". A numerical range includes the numbers that define the range. Generally speaking, the term "about" refers to a stated value of a variable and all values ​​of the variable that are within experimental error of the stated value (e.g., within a 95% confidence interval of the mean) or within 10 percent of the stated value, whichever is greater. When the term "about" is used in the context of a time period (years, months, weeks, days, etc.), the term "about" refers to the period plus or minus one subquantity of time (e.g., about 1 year means 11-13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6-8 days, etc.) or within 10 percent of the stated value, whichever is greater.

[0120] I. How to use In one aspect, the present disclosure provides a method of treating a CLDN18.2-related disease or condition in a subject in need thereof. The method may comprise administering to the subject a therapeutically effective amount of a CLDN18.2 antagonist in combination with a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor.

[0121] In certain embodiments, the subject is determined to have CLDN18.2 expression in diseased tissue.In certain embodiments, the subject is further determined to have PD-L1 expression in diseased tissue.In certain embodiments, the subject is determined to have both CLDN18.2 expression and PD-L1 expression in diseased tissue.

[0122] In certain embodiments, the subject is further determined to have low or no PD-L1 expression in the affected tissue. In certain embodiments, the subject is determined to have CLDN18.2 expression and low or no PD-L1 expression in the affected tissue.

[0123] PD-1 / PD-L1 axis inhibitors (e.g., PD-1 inhibitors, PD-L1 inhibitors) are a group of immune checkpoint inhibitors that lead to activation, proliferation and / or augmentation of T cell signaling and are used as frontline treatments for multiple types of cancer with remarkable curative effects. However, immune checkpoint inhibitors are only beneficial in a small percentage of patients and have been shown to be less active in some cancers, especially those with low PD-L1 expression (see Darvin et al., 2018, Immune checkpoint inhibitors: recent progress and potential biomarkers. Exp Mol Med 50(12):165). For example, only approximately 20% of eligible patients achieve any durable benefit, and many of these patients will eventually relapse with drug-resistant disease and subsequently experience disease progression (see DEMeyers et al., Targeting the PD-1 / PD-L1 axis for the treatment of non-small-cell lung cancer. Curr Oncol. 2018 Aug;25(4):e324-e334). Studies have investigated the correlation between tumor PD-L1 expression and treatment efficacy, and have shown that PD-L1 overexpression is associated with a significantly higher objective response rate (ORR) (Gettinger et al., Overall survival and long-term safety of nivolumab (anti-programmed death 1 antibody, BMS-936558, ONO-4538) in patients with previously treated advanced non-small-cell lung cancer. J Clin Oncol. 2015;33:2004-12.; Garon et al., Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372:2018-28. doi:10.1056 / NEJMoa1501824).Publications and reports at the 2021 American Society of Clinical Oncology (ASCO) Annual Meeting also showed that patients with tumors with low PD-L1 expression may not benefit much from treatment with PD-1 / PD-L1 inhibitors.

[0124] The present disclosure has surprisingly discovered that in tumor tissue that expresses CLDN18.2 and, optionally, has low or no expression of PD-L1, binding of a CLDN18.2 antagonist (e.g., an anti-CLDN18.2 antibody) to CLDN18.2 expressed on the surface of cells in the tumor tissue can significantly upregulate PD-L1 expression in the tumor cells.

[0125] Based at least in part on the discoveries described above, the present disclosure provides a method of treating a CLDN18.2-associated disease or condition in a subject in need thereof, where the subject is determined, or has been determined, to have a) CLDN18.2 expression in diseased tissue (e.g., cells of a cancerous or tumor tissue), and b) low or absent expression of PD-L1 in the diseased tissue.

[0126] The disclosure further provides a method of sensitizing a subject to a PD-1 / PD-L1 axis inhibitor in a subject determined to have low or no expression of PD-L1 in diseased tissue by administering a therapeutically effective amount of a CLDN18.2 antagonist, optionally in combination with a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor.

[0127] The disclosure further provides a method of increasing tumor responsiveness to treatment with a PD-1 / PD-L1 inhibitor in a subject having a tumor that is resistant or refractory to treatment with a PD-1 / PD-L1 inhibitor by administering a therapeutically effective amount of a CLDN18.2 antagonist, optionally in combination with a therapeutically effective amount of a PD-1 / PD-L1 inhibitor.

[0128] A) Combination therapy methods In one aspect, the present disclosure provides a method for treating a CLDN18.2-related disease or condition in a subject in need thereof. In certain embodiments, the method provided herein comprises administering to the subject a therapeutically effective amount of a CLDN18.2 antagonist in combination with a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor.

[0129] In certain embodiments, the subject is determined to have CLDN18.2 expression in diseased tissue.In certain embodiments, the subject is determined to have both CLDN18.2 expression and PD-1 expression in diseased tissue.In certain embodiments, the subject is determined to have no or low PD-L1 expression in diseased tissue.

[0130] In certain embodiments, the methods provided herein further comprise selecting a subgroup of patients who have expression of CLDN18.2 in diseased tissue but low or absent PD-L1 expression. In certain embodiments, the selected subgroup of patients is eligible for combination therapy of CLDN18.2 and PD-1 / PD-L1 inhibitors to treat the disease or condition. In certain embodiments, the disease or condition is cancer, the patient is a cancer patient, and the diseased tissue is cancerous tissue.

[0131] B) Methods of sensitizing a disease to PD-1 / PD-L1 inhibitors In another aspect, the disclosure further provides a method of sensitizing a disease or condition (e.g., cancer) in a subject in need thereof to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the disease or condition is characterized by low or absent expression of PD-L1 in diseased tissue (e.g., cancerous or tumor tissue) obtained from the subject.

[0132] In certain embodiments, the method comprises determining the presence or expression level of CLDN18.2 in diseased tissue obtained from the subject. In certain embodiments, the subject is determined to have CLDN18.2 expression in diseased tissue.

[0133] In certain embodiments, when the presence of CLDN18.2 is determined in the previous determination step or when the expression level of CLDN18.2 reaches a threshold level, then a therapeutically effective amount of a CLDN18.2 antagonist is administered to such a subject, thereby sensitizing the disease or condition in such a subject to treatment with a PD-1 / PD-L1 inhibitor. In certain embodiments, the method further comprises administering a therapeutically effective amount of a PD-1 / PD-L1 inhibitor after the subject is sensitized to treatment with a PD-1 / PD-L1 inhibitor, to achieve significantly improved therapeutic efficacy. The PD-1 / PD-L1 inhibitor can also be administered simultaneously with, before, or after the CLDN18.2 antagonist (e.g., anti-CLDN18.2 antibody).

[0134] As used herein, the term "threshold level" in relation to CLDN18.2 expression refers to the minimum expression level of CLDN18.2 that is detectable using conventional techniques, such as immunohistochemistry (IHC) and other suitable methods.

[0135] C) Methods for reducing or overcoming disease resistance to PD-1 / PD-L1 inhibitors In another aspect, the disclosure also provides a method of increasing the responsiveness of a tumor to treatment with a PD-1 / PD-L1 system inhibitor in a subject, where the subject is determined to have, or has been determined to have, a tumor that is resistant or refractory to treatment with a PD-1 / PD-L1 system inhibitor.

[0136] In certain embodiments, the methods include determining the presence or expression level of CLDN18.2 in a tumor sample obtained from the subject.

[0137] In certain embodiments, if the presence of CLDN18.2 is determined for a subject in the previous determination step or if the expression level of CLDN18.2 reaches a threshold level, a therapeutically effective amount of a CLDN18.2 antagonist is administered to such subject, optionally in combination with a therapeutically effective amount of a PD-1 / PD-L1 system inhibitor, thereby increasing the responsiveness of tumors in the subject to treatment with a PD-1 / PD-L1 system inhibitor.

[0138] Resistance to a PD-1 / PD-L1 inhibitor may be de novo resistance or acquired resistance. As used herein, the term "de novo" with respect to resistance to treatment with a PD-1 / PD-L1 inhibitor refers to resistance that arises with the first PD-1 / PD-L1 inhibitor treatment, i.e., a subject with de novo resistance is not responsive the first time they receive a PD-1 / PD-L1 inhibitor treatment. The term "acquired" with respect to resistance to treatment with a PD-1 / PD-L1 inhibitor refers to resistance that arises during treatment with a PD-1 / PD-L1 inhibitor that is not present with the first PD-1 / PD-L1 inhibitor treatment, i.e., a subject with acquired resistance can respond to treatment with a PD-1 / PD-L1 inhibitor and later becomes resistant or non-responsive to treatment with a PD-1 / PD-L1 inhibitor.

[0139] Resistance to treatment with a PD-1 / PD-L1 inhibitor can be detected by various methods known in the art, for example, by measuring the reduction in tumor volume following treatment with a PD-1 / PD-L1 inhibitor.

[0140] Selection of appropriate patient subgroups in targeted therapy can greatly maximize efficacy, reduce costs, and avoid missed treatment opportunities. For example, as described above, cancer patients with low or no PD-L1 expression are often not eligible for treatment with PD-1 / PD-L1 inhibitors. However, by using a process of detecting CLDN18.2 expression in such patients and selecting patients with CLDN18.2 expression, the selected patients who are otherwise not responsive to treatment with PD-1 / PD-L1 inhibitors become eligible for and responsive to treatment with PD-1 / PD-L1 inhibitors when combined with CLDN18.2 antagonists. On the other hand, by using the CLDN18.2 detection and selection process described above, combination therapy will not be wasted in patients with no or undetectable CLDN18.2 expression, so that these patients will have enough time to search for more suitable therapies and avoid missing optimal treatment opportunities.

[0141] In certain embodiments, the disease or condition is further resistant or refractory to a second therapy selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, and combinations thereof.

[0142] In certain embodiments, the disease or condition is resistant or refractory to combination therapy with a PD-1 / PD-L1 axis inhibitor (e.g., a standard of care anti-PD-1 antibody or anti-PD-L1 antibody) and chemotherapy (e.g., a standard of care chemotherapeutic agent, e.g., oxaliplatin and fluorouracil (5FU)).

[0143] The term "chemotherapy", as used herein, refers to the treatment of cancer (cancerous or tumor cells) with one or more cytotoxic anti-tumor, anti-neoplastic drugs, sometimes referred to as "chemotherapeutic agents" or "chemotherapeutic drugs", as part of a standardized regimen. Exemplary chemotherapeutic agents include, but are not limited to, methotrexate and 5-fluorouracil (5-FU), oxaliplatin, alkylating agents (e.g., thiotepa and cyclophosphamide (Cytoxan™)), alkyl sulfonates (e.g., busulfan, improsulfan and piposulfan), aziridines (e.g., benzodopa, carboquone, meturedopa and uredopa), emylermine and memilamelamelamine (e.g., altrexate, cyclosulf ... acetogenins, camptothecins (e.g., the synthetic analog topotecan), bryostatins, kallistatins, CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin), cryptophycins (especially cryptophycin and cryptophycin), dolastatins, duocarmycins (synthetic analogs, KW-202, KW-2 ...189 and CBI-TMI), erytherobin, pancratistatin, sarcodictin, spongiostatin, cisplatin, nitrogen mustards (e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard), nitrosoureas (e.g., carmustine, chlorozotocin, foremustine, lomustine, nimustine, ranimustine), folic acid analogs (e.g., demopterin, methotrexate ... Rexate, pteropterin, trimetrexate, purine analogues (e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine), androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone), adrenal antidrugs (e.g., aminoglutethimide, mitotane, trilostane), folic acid supplements (e.g., frolinic acid, acid), aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, hestravucil, bisantrene, edatrexate, defofamine, demecolcine, diaziquone, elformucine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids (e.g., maytansine and ansamitocin), mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PThese include SKTM, razoxane, rhizoxin, schizofiran, spirogermanium, tenuazonic acid, triazicon, 2,2,2"-trichlorotriemylamine, trichothecenes, urethanes, vindesine, dacarbazine, mannomustine, mitobronitol, and mitolactol. In certain embodiments, the chemotherapy or chemotherapeutic agent is oxaliplatin and / or 5FU.

[0144] Methods for determining likelihood of eligibility for or responsiveness to treatment with a CLDN18.2 antagonist Based at least in part on the findings of the present disclosure as described above, CLDN18.2 can be considered as a biomarker whose presence predicts the subject's responsiveness to the combination therapy of CLDN18.2 antagonist and PD-1 / PD-L1 inhibitor treatment.In certain embodiments, the subject has a tumor that is resistant or refractory to treatment with PD-1 / PD-L1 inhibitor and, if necessary, standard of care chemotherapy.The resistance can be de novo resistance or acquired resistance.

[0145] In another aspect, the present disclosure also provides a method for determining the eligibility of a subject with low or no expression of PD-L1 for combination therapy with a CLDN18.2 antagonist and a PD-1 / PD-L1 system inhibitor, or the likelihood of a subject's responsiveness to combination therapy with a CLDN18.2 antagonist and a PD-1 / PD-L1 system inhibitor.

[0146] In certain embodiments, the method includes contacting a sample obtained from a subject with a CLDN18.2 diagnostic agent (e.g., anti-CLDN18.2 diagnostic antibody) under conditions that allow detection of CLDN18.2 expression. If the sample has positive expression of CLDN18.2 in the cells of the sample, the subject is determined to be suitable for or likely to respond to combination therapy with a CLDN18.2 antagonist and a PD-1 / PD-L1 inhibitor, and if no expression of CLDN18.2 is detected in the sample, the subject is determined to be not suitable for or likely to respond to treatment with a CLDN18.2 antagonist.

[0147] In certain embodiments, a subject is identified as likely to respond to combination therapy with a CLDN18.2 antagonist and a PD-1 / PD-L1 inhibitor based on having expression of CLDN18.2 in the diseased tissue, and optionally, low or no PD-L1 expression in the diseased tissue.

[0148] Determine PD-L1 and / or CLDN18.2 expression The method of use provided herein comprises determining the expression of CLDN18.2 and / or PD-L1.In certain embodiments, the subject is determined to have CLDN18.2 expression in diseased tissue.In certain embodiments, the subject is determined to have both CLDN18.2 expression and PD-1 expression in diseased tissue.In certain embodiments, the subject is determined to have no or low PD-L1 expression in diseased tissue.

[0149] Any suitable method known in the art may be used and is described in detail below.

[0150] i. Sample preparation In certain embodiments, the subject is a human. In certain embodiments, the methods provided herein further include providing a biological sample from the subject, the biological sample comprising diseased tissue (e.g., cancerous tissue or tumor tissue).

[0151] Any biological sample suitable for carrying out the method provided herein can be obtained from a subject.As used herein, "biological sample" refers to a biological specimen obtained by sampling from a subject, optionally with additional processing.The collection of sample from a subject is carried out according to the standard protocol generally taken by a hospital or clinic, such as during biopsy.

[0152] In certain embodiments, the sample may be a biological sample that contains cancer cells or non-cancer cells. For example, the non-cancer cells may be derived from the same tissue or organ in which the cancer cells are also found. In certain embodiments, a biological sample that contains or is suspected to contain cancer cells may be obtained from a subject. In some embodiments, the biological sample may be derived from cancer cells or cancer tissue or tumor-infiltrating immune cells. In certain embodiments, the biological sample is tumor tissue.

[0153] In some embodiments, the biological sample is a fresh or preserved sample obtained from tumor tissue, for example, by tumor biopsy or fine needle aspiration. In some embodiments, the sample can be any biological fluid that contains cancer cells or non-cancerous cells (e.g., peripheral blood mononuclear cells (PBMCs)).

[0154] Examples of biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal washings, pleural fluid, peritoneal fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, and the like, and tissues such as biopsied tissues (e.g., biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tissue, lung tissue, colon tissue, liver tissue, prostate tissue, brain tissue, neural tissue, meningeal tissue, colon tissue, kidney tissue, endometrial tissue, cervical tissue, lymph node tissue, muscle tissue, or skin tissue), paraffin-embedded tissue. In further embodiments, the biological sample comprises cells, tissues, blood, plasma, serum, urine, mouthwash, stool, saliva, and any combination thereof.

[0155] In certain embodiments, the sample may be further processed by a desired method to determine the expression level of at least one biomarker, such as CLDN18.2 and / or PD-L1.

[0156] ii. Determination of CLDN18.2 expression In certain embodiments, CLDN18.2 expression is determined from a diseased tissue (eg, from a biological sample).

[0157] In some embodiments, CLDN18.2 expression in diseased tissue (eg, cancerous or tumor tissue) is higher than or equivalent to expression in healthy or non-cancerous gastric cells or tissue.

[0158] In some embodiments, CLDN18.2 expression in diseased tissue (e.g., cancerous or tumor tissue) is lower than expression in healthy or non-cancerous gastric cells or tissue, but higher than expression in healthy or non-cancerous tissues or organs other than the stomach.

[0159] In some embodiments, CLDN18.2 expression in diseased tissue (e.g., cancerous or tumor tissue) is comparable to expression in healthy or non-cancerous tissues or organs other than the stomach and is detectable by an anti-CLDN18.2 diagnostic antibody.

[0160] In some embodiments, expression of CLDN18.2 is on the cell surface or membrane-associated.

[0161] The presence and / or expression level of CLDN18.2 in diseased tissue (e.g., cancerous tissue or tumor tissue) can be determined by various methods known in the art. In certain embodiments, the biological sample can be further processed to isolate analytes such as, for example, nucleic acids or proteins. The presence and / or expression level of CLDN18.2 can be determined, for example, by quantitative fluorescence cytometry, immunohistochemistry (IHC) or nucleic acid-based methods. For example, the biological sample from the subject can be exposed to an anti-CLDN18.2 antibody or antigen-binding fragment that binds and detects expressed CLDN18.2 protein.

[0162] In certain embodiments, the expression of CLDN18.2 in diseased tissue (e.g., cancerous tissue or tumor tissue) is determined and measured by IHC. In certain embodiments, the expression level of human CLDN18.2 protein in cancerous tissue or tumor tissue from a subject can be determined according to the method described in Example 6 provided herein.

[0163] In certain embodiments, a subject is determined or has been determined to have high CLDN18.2 expression in diseased tissue (e.g., cancerous or tumor tissue) from the subject. In certain embodiments, the diseased tissue is determined or has been determined to have CLDN18.2 expression that is higher than or equivalent to expression in healthy or non-cancerous gastric cells or tissue. High CLDN18.2 expression in a biological sample, e.g., diseased tissue (e.g., cancerous or tumor tissue), refers to expression at an intensity of at least 2+ as measured by IHC, and at least 40% (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 10 ... "positive staining" refers to expression of CLDN18.2 at a level that stains positively in at least 85%, at least 90%, at least 95%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-80%, 50-70%, 50-60%, 60-80%, 60-70% or 70-80%).

[0164] In certain embodiments, the subject is determined or determined to have moderate CLDN18.2 expression in diseased tissue (e.g., cancerous tissue or tumor tissue) from the subject.In certain embodiments, the diseased tissue is determined or determined to have CLDN18.2 expression that is lower than that in healthy or non-cancerous gastric cells or gastric tissue, but higher than that in healthy or non-cancerous tissue or organ other than stomach.Moderate CLDN18.2 expression in biological sample, for example, diseased tissue (e.g., cancerous tissue or tumor tissue), refers to the expression of CLDN18.2 at an intensity of at least 1+ and less than 2+ as measured by IHC, and at a level where at least 30% (or at least 35%) of cells, but less than 40%, stain positive in IHC.

[0165] In certain embodiments, the subject is determined or has been determined to have low CLDN18.2 expression in diseased tissue (e.g., cancerous tissue or tumor tissue) from the subject.In certain embodiments, the diseased tissue is determined or has been determined to have CLDN18.2 expression that is equivalent to that in healthy or non-cancerous tissue or organ other than stomach, and is detectable by anti-CLDN18.2 diagnostic antibody. Low CLDN18.2 expression in a biological sample, e.g., diseased tissue (e.g., cancerous or tumor tissue), refers to expression of CLDN18.2 at an intensity above 0 but less than 1+ as measured by IHC, and at a level where above 0 but less than 30% (e.g., 5%, 10%, 15%, 20%, 25%, 5-25%, 10-25%, 15-25%, 20-25%, 5-20%, 5-15%, 5-10%, 10-20% or 10-15%) of cells stain positive in IHC.

[0166] The anti-CLDN18.2 diagnostic antibody can be any anti-CLDN18.2 antibody that can sensitively detect the expression of CLDN18.2 in diseased cells or diseased tissues. For example, the anti-CLDN18.2 diagnostic antibody can be one described in PCT / CN2021 / 095411. In a specific embodiment, the anti-CLDN18.2 diagnostic antibody is 14G11, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0167] iii. Determination of PD-L1 expression In certain embodiments, PD-L1 expression is determined from the diseased tissue (e.g., from a biological sample).

[0168] The same biological sample (e.g., diseased tissue, e.g., cancerous or tumor tissue) for determining expression of CLDN18.2 can also be measured, or simultaneously, to determine whether there is expression of PD-L1 in the same sample.

[0169] Similar to determining CLDN18.2 expression, PD-L1 expression in a biological sample, e.g., a diseased tissue (e.g., a cancerous or tumor tissue), can be determined by various methods known in the art. A biological sample containing or suspected to contain cancer cells can be obtained from a subject. The biological sample can contain cancer cells, cancer tissue, and immune cells (e.g., tumor-associated immune cells, e.g., tumor-infiltrating immune cells). The biological sample may be further processed to isolate analytes, e.g., nucleic acids (e.g., mRNA) or proteins. The presence and / or expression level of PD-L1 can be determined, for example, by quantitative fluorescence cytometry, immunohistochemistry (IHC), or nucleic acid-based methods (e.g., RNA sequencing). For example, a biological sample from a subject can be exposed to an anti-PD-L1 antibody or antigen-binding fragment that binds to and detects expressed PD-L1 protein. Alternatively, PD-L1 can be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, and the like.

[0170] In certain embodiments, PD-L1 expression is determined by IHC. Specific PD-L1 staining by IHC requires appropriate patient material, a sensitive primary anti-PD-L1 antibody, and a suitable staining protocol. There are three main scoring algorithms for PD-L1 staining that are specific to tumor type and clinical decision: tumor proportion score (TPS), immune proportion score (IPS), and combined positive score (CPS) (see Schildhaus et al., Predictive value of PD-L1 diagnostics.Pathologe.2018 Nov;39(6):498-519. doi:10.1007 / s00292-018-0507-x).

[0171] TPS can be defined by the percentage of tumor cells positive for PD-L1 staining in relation to the total tumor cells in a biological sample, a detailed description of which can be found, for example, in Piper et al.,Can PD-L1 tumor proportion score be used as the key to unlocking the KEYNOTE studies of pembrolizumab in advanced lung cancer? Transl Lung Cancer Res 2019;8(5):715-722.

[0172] IPS can be defined by the percentage of tumor-associated immune cells positive for PD-L1 expression in relation to total tumor-associated immune cells, a detailed description of which can be found, for example, in Yang et al., PD-L1 expression on tumor cells and tumor infiltrating immune cells in Chinese colorectal cancer patients. Journal of Clinical Oncology, Volume 38, Issue 15_suppl.

[0173] CPS can be defined by the number of PD-L1 stained cells (including but not limited to tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells and multiplied by 100; detailed descriptions can be found, for example, in Yamashita et al., Prognostic impacts of the combined positive score and the tumor proportion score for programmed death ligand-1 expression by double immunohistochemical staining in patients with advanced gastric cancer. Gastric Cancer. 2020 Jan;23(1):95-104; and Dako et al., Development of the combined positive score(CPS) for the evaluation of PD-L1 in solid tumors with the immunohistochemistry assay PD-L1 IHC 22C3 pharmDx. Journal of Clinical Oncology, Volume 35, Issue 15_suppl, 2017. CPS can also be defined by the percentage of PD-L1 staining cells (including but not limited to tumor cells, lymphocytes, macrophages) in relation to the total number of viable tumor cells, as described in Example 6 of the present disclosure.

[0174] A CPS for PD-L1 expression in a sample can be obtained using PD-L1 IHC 22C3 pharmDx or PD-L1 IHC 28-8 pharmDx. The PD-L1 IHC 28-8 pharmDx assay (Agilent Technologies, Santa Clara, CA, USA), a kit for detecting PD-L1 expression, is approved as a complementary diagnostic method for use with nivolumab in cancers such as nonsquamous non-small cell lung cancer (NSQNSCL) and squamous cell carcinoma of the head and neck (SCCHN). PD-L1 IHC 28-8 pharmDx contains the optimized reagents and protocols required to complete IHC staining of formalin-fixed paraffin-embedded (FFPE) samples using the Autostainer Link 48 and Dako PT Link pretreatment modules (Phillips T, Simmons P, Inzunza HD, et al. Development of an automated PD-L1 mmunohistochemistry(IHC)assay for non-small cell lung cancer. Appl Immuno Molec Morph 2015;23(8):541-9). Briefly, FFPE samples are first incubated with a primary monoclonal antibody against PD-L1 or a negative control reagent (NCR), followed by a linker antibody specific for the host species of the primary antibody, and then with a ready-to-use visualization reagent containing a secondary antibody molecule coupled to a dextran polymer backbone and a horseradish peroxidase molecule, and then subjected to light microscopy for visualization of PD-L1 staining.

[0175] PD-L1 IHC 28-8 pharmDx was used in CheckMate-649, a randomized, multicenter, open-label study in patients (n=1581) with previously untreated advanced or metastatic gastric, esophagogastric junction, and esophageal adenocarcinoma. The study enrolled patients regardless of PD-L1 expression level, and tumor samples from patients were evaluated in a central laboratory using the PD-L1 IHC 28-8 pharmDx assay. The clinical trial demonstrated a statistically significant improvement in overall survival (OS) and progression-free survival (PFS) for patients with tumors with PD-L1 expression of CPS ≥ 5, which was not observed for patients with tumors with PD-L1 expression of CPS < 5 (information obtained from https: / / www.fda.gov / media / 124784 / download).

[0176] Different algorithms may be used for different tumor types. For example, TPS can be applied to determine PD-L1 expression in lung cancer, head and neck cancer and melanoma. CPS and IPS are standard measurements of PD-L1 expression in urothelial cancer. For TPS, only PD-L1 staining of tumor cells is considered PD-L1 positive, and staining of cells other than tumor cells is not considered PD-L1 positive, while CPS includes PD-L1 expression in tumor-associated immune cells and IPS is limited to PD-L1 expression in certain immune cells (e.g., tumor-associated immune cells) (see Schildhaus et al., Predictive value of PD-L1 diagnostics.Pathologe.2018 Nov;39(6):498-519).

[0177] In certain embodiments, PD-L1 expression is determined by CPS using IHC. As mentioned above, CPS can be determined based on the number of PD-L1 positive cells (including but not limited to tumor, lymphocytes and macrophages) in relation to the total tumor cells in the biological sample, thus allowing the capture of PD-L1 expression in tumor and immune cells in a single reading. For example, a PD-L1 expression level of 5% (or a CPS of 5) in a biological sample, such as a diseased tissue (e.g., cancerous tissue or tumor tissue), means that 5% of cells (including but not limited to tumor, lymphocytes and macrophages) in relation to the total tumor cells in the biological sample are positive for PD-L1 staining.

[0178] In certain embodiments, in the methods provided herein, the subject is determined to have low or no PD-L1 expression in cells or in the diseased tissue.

[0179] As used herein, the term "low expression of PD-L1" may refer to a PD-L1 expression level that is lower or below a reference level.

[0180] In certain embodiments, the term "reference level" with respect to PD-L1 expression refers to a threshold (e.g., minimum) expression level of PD-L1 in a biological sample, e.g., diseased tissue (e.g., cancerous tissue or tumor tissue), derived from a subject that is responsive to treatment with a PD-1 / PD-L1 system inhibitor. The expression of PD-L1 can be measured by the methods provided herein. For different tumor types and / or when different PD-L1 detection assays are used, the threshold expression level of PD-L1 expression may be different. For example, the threshold expression level for gastric cancer may be 5% (or CPS of 5) as measured by PD-L1 IHC 28-8 pharmDx assay, i.e., the methods provided herein are for treating gastric cancer that has PD-L1 expression in gastric tumor tissue lower than 5% (or CPS of 5) as measured by PD-L1 IHC 28-8 pharmDx assay and has expression of CLDN18.2 in gastric tumor tissue. The threshold expression level for gastric cancer may be, in some embodiments, 4% (or a CPS of 4), 3% (or a CPS of 3), 2% (or a CPS of 2) or 1% (or a CPS of 1) as measured by a PD-L1 IHC 28-8 pharmDx assay.In other embodiments, the threshold level for gastric cancer may be 10% (or 10 CPS), 9% (or 9 CPS), 8% (or 8 CPS), 7% (or 7 CPS), 6% (or 6 CPS), 5% (or 5 CPS), 4% (or 4 CPS), 3% (or 3 CPS), 2% (or 2 CPS) or 1% (or 1 CPS) as measured by the PD-L1 IHC 22C3 pharmDx assay, a detailed description of which can be found, for example, in Dako et al., Development of the combined positive score (CPS) for the evaluation of PD-L1 in solid tumors with the immunohistochemistry assay PD-L1 IHC 22C3 pharmDx. Journal of Clinical Oncology, Volume 35, Issue 15_suppl, 2017.

[0181] In some embodiments of the methods provided herein, the subject is determined to have PD-L1 expression in diseased tissue (e.g., cancerous or tumor tissue) that is less than or equal to 20% (or 20 CPS), 15% (or 15 CPS), 10% (or 10 CPS), 9% (or 9 CPS), 8% (or 8 CPS), 7% (or 7 CPS), 6% (or 6 CPS), 5% (or 5 CPS), 4% (or 4 CPS), 3% (or 3 CPS3), 2% (or 2 CPS) or 1% (or 1 CPS).

[0182] In certain embodiments, the subject is determined to have 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of cells in the diseased tissue that are positive for PD-L1 expression, but still have PD-L1 expression that is detectable by an anti-PD-L1 diagnostic antibody.

[0183] In some embodiments, a subject is determined to have PD-L1 expression in diseased tissue (e.g., cancerous or tumor tissue) that is equivalent to that in healthy or non-cancerous tissue.

[0184] As used herein, the term "no PD-L1 expression" refers to the absence of any detectable PD-L1 signal in a biological sample using various techniques such as IHC with an anti-PD-L1 diagnostic antibody. In some embodiments, PD-L1 expression in diseased tissue is low or not detectable by an anti-PD-L1 diagnostic antibody.

[0185] The reagents used to detect PD-L1 expression throughout the specification may be anti-PD-L1 diagnostic antibodies, such as 22C3 as described in US20170285037A1, the disclosure of which is incorporated herein by reference in its entirety, and the commercially available clone 28-8, which is a monoclonal rabbit anti-PD-L1.

[0186] In some other embodiments of the methods provided herein, PD-L1 expression in the diseased tissue (e.g., cancerous or tumor tissue) reaches, is equal to, or is even higher than a reference level.

[0187] Treatment In certain embodiments, the methods provided herein further comprise administering to the subject a CLDN18.2 antagonist in combination with a PD-1 / PD-L1 inhibitor. In certain embodiments, the CLDN18.2 antagonist and the PD-1 / PD-L1 inhibitor are each administered to the subject in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" of the CLDN18.2 antagonist or the PD-1 / PD-L1 inhibitor used in the methods provided herein varies depending on various factors known in the art, such as body weight, age, past medical history, current drug therapy, the subject's health status and cross-reactivity, allergies, sensitivities and potential for adverse side effects, as well as the route of administration and the extent of disease development. Dosage amounts can be proportionally reduced or increased by those skilled in the art (e.g., physicians or veterinarians) as indicated by these and other circumstances or requirements.

[0188] In certain embodiments, the CLDN18.2 antagonist and / or PD-1 / PD-L1 system inhibitor can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 3 mg / kg, about 3 mg / kg to about 30 mg / kg, about 3 mg / kg to about 20 mg / kg, about 6 mg / kg to about 20 mg / kg, about 3 mg / kg to about 10 mg / kg, or about 6 mg / kg to about 10 mg / kg. In certain embodiments, the dosage can be varied during the course of treatment. In certain embodiments, the dosage can be varied during the course of treatment depending on the subject's response. The dosing regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose can be administered, or multiple divided doses can be administered over time.

[0189] In certain embodiments, the present disclosure provides a method for treating a CLDN18.2-related disease or condition in a subject identified as likely to respond to a combination therapy treatment of a CLDIN18.2 antagonist and a PD-1 / PD-L1 inhibitor. In certain embodiments, the treating step comprises administering a therapeutically effective amount of a CLDIN18.2 antagonist and a therapeutically effective amount of a PD-1 / PD-L1 inhibitor to the subject identified as likely to respond to a combination therapy treatment of a CLDIN18.2 antagonist and a PD-1 / PD-L1 inhibitor.

[0190] The CLDN18.2 antagonist used in the methods provided herein may comprise an anti-CLDN18.2 antibody or an antigen-binding fragment. The anti-CLDN18.2 antibody may be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a labeled antibody, a bivalent antibody, or an anti-idiotypic antibody.

[0191] In certain embodiments, the anti-CLDN18.2 antibody has high binding affinity to a biological sample having high expression of CLDN18.2.

[0192] In certain embodiments, the anti-CLDN18.2 antibody has high binding affinity to a biological sample having moderate expression of CLDN18.2.

[0193] In certain embodiments, the anti-CLDN18.2 antibody has high binding affinity for a biological sample having low expression of CLDN18.2.

[0194] High, moderate or low expression of CLDN18.2 in a biological sample can be defined in the same manner as above by comparing healthy or non-cancerous gastric cancer tissue other than healthy or non-cancerous gastric cancer tissue, or by scoring using the results obtained from IHC.

[0195] In certain embodiments, an anti-CLDN18.2 antibody comprises heavy chain HCDR1, HCDR2, and HCDR3, and / or light chain LCDR1, LCDR2, and LCDR3 sequences, The HCDR1 sequence comprises GYNMN (SEQ ID NO:1) or a homologous sequence thereof with at least 80% sequence identity; The HCDR2 sequence comprises NIDPYYGGTSYNQKFKG (SEQ ID NO: 2) or a homologous sequence thereof of at least 80% sequence identity; the HCDR3 sequence comprises MYHGNAFDY (SEQ ID NO:3) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence comprises KSSQSLLNSGNLKNYLT (SEQ ID NO: 4) or a homologous sequence thereof with at least 80% sequence identity; the LCDR2 sequence comprises WASTRKS (SEQ ID NO:5) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence includes QNDYSYPLT (SEQ ID NO:6) or a homologous sequence thereof with at least 80% sequence identity.

[0196] Although CDRs are known to be responsible for antigen binding, it has been found that not all six CDRs are essential or invariant. In other words, it is possible to replace, change or modify one, two or three CDRs in an anti-CLDN18.2 antibody while substantially maintaining the specific binding affinity to CLDN18.2.

[0197] In certain embodiments, the anti-CLDN18.2 antibody comprises the heavy chain CDR3 sequence of MYHGNAFDY (SEQ ID NO: 21). The heavy chain CDR3 region is located at the center of the antigen-binding site, and therefore is believed to make the most contact with the antigen and provide the most free energy to the affinity of the antibody to the antigen. The heavy chain CDR3 is also believed to be the most diverse CDR of the antigen-binding site in terms of length, amino acid composition, and conformation due to multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81). The diversity of the heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45) and desirable antigen-binding affinities (Schier R, etc. J Mol Biol. 263: 551-67).

[0198] In some embodiments, anti-CLDN18.2 antibody comprises all or part of heavy chain variable domain and / or all or part of light chain variable domain.In one embodiment, anti-CLDN18.2 antibody is a single domain antibody consisting of all or part of heavy chain variable domain provided herein.More information about such single domain antibody is available in the art (see, for example, US Patent No. 6,248,516).

[0199] In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:8.

[0200] In certain embodiments, the anti-CLDN18.2 antibody further comprises an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region. In certain embodiments, the anti-CLDN18.2 antibody further comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0201] In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises a Cκ or Cλ.

[0202] In certain embodiments, the anti-CLDN18.2 antibodies and antigen-binding fragments thereof provided herein comprise a constant region of an IgG1 isotype. In certain embodiments, the constant region of human IgG1 comprises SEQ ID NO: 9 or a homologous sequence thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0203] The constant region of IgG1 isotype can induce effector functions such as ADCC or CDC. The effector function of anti-CLDN18.2 antibody can cause cytotoxicity against cells expressing CLDN18.2. Effector function can be evaluated using various assays, such as Fc receptor binding assay, C1q binding assay, and cytolysis assay, and any of the above-mentioned assays for determining ADCC or CDC.

[0204] In certain embodiments, the constant region contains one or more amino acid residue substitutions or modifications that confer increased CDC or ADCC as compared to the wild-type constant region.

[0205] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. The lysis of the target cells is extracellular, requires direct cell-cell contact, and does not involve complement. ADCC can be viewed as a mechanism that directly induces various degrees of immediate tumor destruction, leading to antigen presentation and induction of tumor-specific T-cell responses. In vivo induction of ADCC is believed to trigger tumor-specific T-cell responses and host-derived antibody responses. In certain embodiments, the constant region comprises one or more amino acid residue substitutions relative to SEQ ID NO:9 selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof. In certain embodiments, the constant region comprises the sequence of SEQ ID NO:11, and optionally further comprises the sequence of SEQ ID NO:10.

[0206] In certain embodiments, the anti-CLDN18.2 antibody is humanized. Humanized antibodies or antigen-binding fragments are desirable because they are less immunogenic in humans. Humanized antibodies have chimeric variable regions when non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can essentially be performed by substituting non-human (e.g., mouse) CDR genes for the corresponding human CDR genes in human immunoglobulin genes (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536). In certain embodiments, the humanized light and heavy chains of the present disclosure are substantially non-immunogenic in humans and maintain substantially the same or much higher affinity for CLDN18.2 than the parent antibody.

[0207] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment provided herein comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity but retaining specific binding affinity to CLDN18.2, particularly human CLDN18.2.

[0208] In certain embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment provided herein comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, or homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity but retaining specific binding affinity to CLDN18.2, particularly human CLDN18.2.

[0209] In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14; The light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:16.

[0210] In certain embodiments, a humanized anti-CLDN18.2 antibody may comprise a heavy chain variable region fused to the constant region of a human IgG1 isotype and a light chain variable region fused to the constant region of a human kappa chain.

[0211] The humanized anti-CLDN18.2 antibodies provided herein retain specific binding affinity to biological samples expressing CLDN18.2, and in this embodiment, at least equal to or even better than the parent antibody.The humanized antibodies provided herein also retain their functionality in that all antibodies can mediate cell killing by ADCC, CDC and the induction of apoptosis induced by crosslinking of targets on the tumor cell surface, and by direct inhibition of proliferation.

[0212] In certain embodiments, the anti-CLDN18.2 antibody comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO:39; The light chain comprises the amino acid sequence of SEQ ID NO:40.

[0213] In certain embodiments, the anti-CLDN18.2 antibody is capable of inducing the expression of PD-L1 in affected tissues of a subject.

[0214] The anti-CLDN18.2 antibodies used in the methods provided herein can also include various types of variants of the antibody sequences provided herein.

[0215] In certain embodiments, the variant comprises one or more modifications or substitutions in one, two or three of the CDR sequences mentioned above, one or more FR sequences, the heavy or light chain variable region sequences provided herein, and / or the constant region (e.g., Fc region). Such antibody variants maintain the specific binding affinity of their parent antibody to CLDN18.2, but have one or more desirable properties imparted by the modification or substitution. For example, the antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or improved effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues), suitable for naming several types. In certain embodiments, the anti-CLDN18.2 antibodies used in the methods provided herein also encompass glycosylation variants with improved effector function, such as ADCC or CDC.

[0216] In certain embodiments, the anti-CLDN18.2 antibody used in the methods provided herein is non-fucosylated. The term "afucosylation" or "afucosylated" refers to the reduction or removal of the core fucose of the N-glycan attached to the antibody. The majority of glycans of human IgG antibodies are known as G0, G1, and G2, which are complex biantennary molecules with 0, 1, or 2 terminal galactose core fucose residues.

[0217] Afucosylated antibody variants are described, for example, in U.S. Patent Application Publication No. 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, U.S. Patent Application Publication No. 2003 / 0115614, U.S. Patent Application Publication No. 2002 / 0164328, U.S. Patent Application Publication No. 2004 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, U.S. Patent Application Publication No. 2004 / 0132140, U.S. Patent Application Publication No. 2004 / 0132141, U.S. Patent Application Publication No. 2004 / 0132142, U.S. Patent Application Publication No. 2004 / 0132143, U.S. Patent Application Publication No. 2004 / 0132144, U.S. Patent Application Publication No. 2004 / 0132145, U.S. Patent Application Publication No. 2004 / 0132146 ... They can be made using methods known in the art, such as those described in U.S. Patent Application Publication No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO 2005 / 053742, WO 2002 / 031140, Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

[0218] In certain embodiments, the antibody glycosylation variant is afucosylated at Asn297 site in the CH2 region of the Fc of the antibody. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues), but may be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody.

[0219] In certain embodiments, the anti-CLDN18.2 antibodies used in the methods provided herein also include cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues. A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful for conjugation, for example, via maleimide or haloacetyl, with cytotoxic and / or imaging compounds, labels, or radioisotopes, among others, at the site of the engineered cysteine. Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine ​​residues are known in the art, see, for example, WO 2006 / 034488.

[0220] In certain embodiments, the constant region of an anti-CLDN18.2 antibody or antigen-binding fragment thereof used in the methods provided herein comprises one or more amino acid residue substitutions compared to SEQ ID NO:9 (i.e., the wild-type sequence) selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof. In certain embodiments, the constant region comprises the sequence of SEQ ID NO:11.

[0221] In certain embodiments, the anti-CLDN18.2 antibodies used in the methods provided herein also include anti-CLDN18.2 antigen-binding fragments, such as diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies or bivalent domain antibodies.

[0222] In certain embodiments, the anti-CLDN18.2 antibody used in the methods provided herein is bivalent, tetravalent, hexavalent or multivalent. The term "valency" as used herein refers to the presence of a specified number of antigen-binding sites in a given molecule. Thus, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two binding sites, four binding sites and six binding sites in an antigen-binding molecule, respectively. Any molecule that is more than bivalent is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0223] A bivalent molecule can be monospecific when both binding sites are specific for binding to the same antigen or epitope. This, in certain embodiments, achieves stronger binding to the antigen or epitope than its monovalent counterpart. Similarly, a multivalent molecule can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences, despite having the same specificity).

[0224] Bivalent can also be bispecific, when the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific, when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope). Bispecific antibodies that can be used in the methods provided herein can target both CLDN18.2 and a checkpoint molecule, such as PD-1, PD-L1, PD-L2, CLTA-4, SIRPα, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, or CD83. Exemplary bispecific antibodies that can be used in the methods provided herein include, but are not limited to, a bispecific antibody targeting CLDN18.2 and CD3, a bispecific antibody targeting CLDN18.2 and 4-1BB, a bispecific antibody targeting CLDN and TGFβ, a bispecific antibody targeting CLDN18.2 and SIRPα, and a bispecific antibody targeting CLDN18.2 and IL-15.

[0225] The anti-CLDN18.2 antibodies used in the methods provided herein can also be antibody-drug conjugates (ADCs) comprising any of the above-described anti-CLDN18.2 antibodies conjugated to a cytotoxic agent.

[0226] In certain embodiments, the cytotoxic agent can be any agent that is harmful to cells or can damage or kill cells.In certain embodiments, the cytotoxic agent is optionally a toxin, a chemotherapeutic agent (e.g., a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a growth inhibitor, or other anticancer agent), or a radioisotope.

[0227] Examples of toxins include bacterial and plant toxins, such as diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin, alvin, modeccin, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PARI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, restrictocin, phenomycin, enomycin, and the trichothecenes (see, e.g., WO 93 / 21232). Such large molecule toxins can be conjugated to the antibodies or antigen-binding fragments provided herein using methods known in the art, for example, as described in Vitetta et al (1987) Science, 238:1098.

[0228] Cytotoxic agents also include small molecule toxins and chemotherapeutic drugs such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansine and maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623; U.S. Pat. No. 5,208,020), calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.53:3336-3342), taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vindesine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thioepa They may also be chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine), calicheamicins, maytansinoids, dolastatins, uristatins such as MMAE and MMAF (U.S. Pat. Nos. 5,635,483 and 5,780,588), dolostatin, trichothecenes, and CC1065, and derivatives thereof having cytotoxic activity. .

[0229] The cytotoxic agent can also be a highly radioactive isotope. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Sm 153 , Bi 212 , P 32 , Pb 212, and radioisotopes of Lu. Methods for conjugating radioisotopes to antibodies, for example via suitable ligand reagents, are known in the art (see, for example, WO 94 / 11026, Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, NY, Pubs. (1991)). The ligand reagent has a chelating ligand that can bind, chelate, or otherwise complex with the radioisotope metal, and also has a functional group that is reactive with the thiol of cysteine ​​of the antibody or antigen-binding fragment. Exemplary chelating ligands include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Tex.).

[0230] In certain embodiments, in the ADCs used in the methods provided herein, the antibodies (or antigen-binding fragments thereof) are conjugated to one or more cytotoxic agents at an antibody:drug ratio of about 1 to about 20, about 1 to about 6, about 2 to about 6, about 3 to about 6, about 2 to about 5, about 2 to about 4, or about 3 to about 4.

[0231] The anti-CLDN18.2 antibody used in the methods provided herein may also be replaced by immune cells expressing chimeric antigen receptors (CARs) or genetically modified TCRs, which include anti-CLDN18.2 antigen-binding domains and T cell activation domains as described above. Chimeric antigen receptors (CARs) are engineered chimeric receptors that combine the antigen-binding domains of antibodies with one or more signaling domains for T cell activation. Immune cells, such as T cells and natural killer (NK) cells, can be genetically engineered to express CARs. T cells expressing CARs are called CAR-T cells. T cells expressing genetically modified TCRs are called TCR-T cells. CARs and genetically modified TCRs can mediate antigen-specific cellular immune activity in T cells, allowing CAR-T / TCR-T cells to eliminate cells expressing the targeted antigen (e.g., tumor cells). In one embodiment, binding of a CAR-T / TCR-T cell provided herein to CLDN18.2 expressed on a cell, such as a cancer cell, results in proliferation and / or activation of the CAR-T / TCR-T cell, and the activated CAR-T / TCR-T cell can release cytotoxic factors, such as perforin, granzymes, and granulysin, which can initiate cytolysis and / or apoptosis of the cancer cell.

[0232] In some embodiments, the T cell activation domain of a CAR comprises a costimulatory signaling domain and a TCR signaling domain, which can be linked to each other in a random or specified order, optionally by a short peptide linker (e.g., a glycine-serine doublet linker) having, for example, between 2 and 10 amino acids in length.

[0233] In some embodiments, the CAR further comprises a transmembrane domain. When expressed in a cell, the anti-CLDN18.2 antigen-binding domain is extracellular and the T cell activation domain is intracellular.

[0234] In certain embodiments, the CAR comprises an anti-CLDN18.2 antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to CLDN18.2 and comprises an antigen-binding fragment of an antibody provided herein.

[0235] In certain embodiments, CAR or genetically modified TCR is bispecific.In certain embodiments, the bispecific CAR or TCR used in the methods provided herein can specifically bind to the first and second epitopes of CLDN18.2, or can specifically bind to CLDN18.2 and a second antigen, such as CD3, 4-1BB, TGFβ, SIRPa and IL-15.

[0236] The term "PD-1 / PD-L1 axis inhibitor" refers to a molecule (e.g., a small molecule, an antibody, etc.) that inhibits a PD-1 / PD-L1 axis binding partner, e.g., the interaction between PD-1 and PD-L1, and eliminates the inhibitory effects on T cell function (e.g., proliferation, cytokine production, and target cell killing) that result from signaling through the PD-1 / PD-L1 signaling axis. A PD-1 / PD-L1 axis inhibitor may include a PD-1 inhibitor or a PD-L1 inhibitor.

[0237] The term "PD-1 inhibitor" as used herein refers to a molecule that reduces, abrogates, inhibits, blocks, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, e.g., PD-L1, PD-L2. In certain embodiments, a PD-1 inhibitor is a molecule that blocks the binding of PD-1 to its binding partners, e.g., PD-L1, PD-L2. For example, a PD-1 inhibitor can be an anti-PD-1 antibody or antigen-binding fragment thereof, a fusion protein, an oligopeptide, an immunoadhesin, and other molecules that reduce, abrogate, inhibits, blocks, or interferes with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In some embodiments, a PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the PD-1 inhibitor is nivolumab (OPDIVO; BMS-936558), dostallimab (TSR-042), pembrolizumab (KEYTRUDA; MK-3475), MEDI0680 (AMP-514), MEDI4736, BI 754091, pidilizumab (CT-011), cemiplimab (LIBTAYO, REGN2810), spartalizumab (PDR001), cetrelimab (JNJ 63723283), toripalimab (JS001), PF-06801591, tislelizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, lambrolizumab or camrelizumab (SHR-1210), sintilimab (Tyvyt, IBI308), penprimab (AK105).

[0238] In some embodiments, the PD-1 inhibitor is nivolumab (OPDIVO; BMS-936558).

[0239] In some embodiments, the PD-1 inhibitor used in the methods provided herein comprises an anti-PD-1 antibody comprising a heavy chain HCDR1, HCDR2, and HCDR3 and / or a light chain LCDR1, LCDR2, and LCDR3 sequence, The HCDR1 sequence comprises NSGMH (SEQ ID NO:55) or a homologous sequence thereof of at least 80% sequence identity; The HCDR2 sequence comprises VIWYDGSKRYYADSVKG (SEQ ID NO:56) or a homologous sequence thereof of at least 80% sequence identity; The HCDR3 sequence comprises NDDY (SEQ ID NO:57) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence comprises RASQSVSSYLA (SEQ ID NO:58) or a homologous sequence thereof of at least 80% sequence identity; The LCDR2 sequence comprises DASNRAT (SEQ ID NO:59) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence includes QQSSNWPRT (SEQ ID NO:60) or a homologous sequence thereof with at least 80% sequence identity.

[0240] In certain embodiments, the PD-1 inhibitor used in the methods provided herein comprises an anti-PD-1 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:61; The light chain variable region comprises the amino acid sequence of SEQ ID NO:62.

[0241] In certain embodiments, the PD-1 inhibitor used in the methods provided herein comprises an anti-PD-1 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO:63; The light chain comprises the amino acid sequence of SEQ ID NO:64.

[0242] The term "PD-L1 inhibitor" refers to a molecule that reduces, abrogates, inhibits, blocks, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1, B7-1. For example, a PD-L1 inhibitor can be an anti-PD-L1 antibody or antigen-binding fragment thereof, a fusion protein, an immunoadhesin, an oligopeptide, and other molecules that reduce, abrogate, inhibits, blocks, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1, B7-1. In some embodiments, the PD-L1 inhibitor is an anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll antibody is selected from the group consisting of atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446), BMS-936559, avelumab (bavencio), lodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105, STI-1040, CS1001, adebulerimab (SHR-1316), and SHR-1701.

[0243] In some embodiments, the PD-L1 inhibitor used in the methods provided herein is a bispecific antibody that targets both PD-L1 and another checkpoint molecule, such as PD-1, PD-L1, PD-L2, CLTA-4, SIRPα, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, or CD83. In certain embodiments, the PD-L1 inhibitors used in the methods provided herein are bispecific antibodies that target both PD-L1 and another checkpoint molecule, such as TGFβ, 4-1BB, CTLA4, LAG3, or TIGIT.

[0244] In certain embodiments, the PD-L1 inhibitors used in the methods provided herein comprise an anti-PD-L1 antibody that comprises a heavy chain HCDR1, HCDR2, and HCDR3, and / or a light chain LCDR1, LCDR2, and LCDR3 sequence, The HCDR1 sequence comprises DYYMN (SEQ ID NO: 22) or a homologous sequence thereof of at least 80% sequence identity; The HCDR2 sequence comprises DINPNNAETLYNHKFKG (SEQ ID NO: 23) or a homologous sequence thereof of at least 80% sequence identity; The HCDR3 sequence comprises WGDGPFAY (SEQ ID NO: 24) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence comprises KASQNVGAAVA (SEQ ID NO:25) or a homologous sequence thereof of at least 80% sequence identity; The LCDR2 sequence comprises SVSDRYT (SEQ ID NO:26) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence includes QQYSNYPT (SEQ ID NO:27) or a homologous sequence thereof with at least 80% sequence identity.

[0245] In certain embodiments, the PD-L1 inhibitor used in the methods provided herein comprises an anti-PD-L1 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17; The light chain variable region comprises the amino acid sequence of SEQ ID NO:18.

[0246] In certain embodiments, the PD-L1 inhibitors used in the methods provided herein comprise an anti-PD-L1 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20; The light chain comprises the amino acid sequence of SEQ ID NO:21.

[0247] In certain embodiments, the PD-L1 inhibitors used in the methods provided herein comprise an anti-PD-L1 antibody that comprises a heavy chain HCDR1, HCDR2, and HCDR3, and / or a light chain LCDR1, LCDR2, and LCDR3 sequence, The HCDR1 sequence comprises TYWMH (SEQ ID NO: 32) or a homologous sequence thereof of at least 80% sequence identity; The HCDR2 sequence comprises MIQPNSGGTKYNEKFKK (SEQ ID NO: 33) or a homologous sequence thereof of at least 80% sequence identity; The HCDR3 sequence comprises GAGTVDYFDY (SEQ ID NO: 34) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence comprises RASESVDIYGNSFMH (SEQ ID NO: 35) or a homologous sequence thereof of at least 80% sequence identity; The LCDR2 sequence comprises RASNLES (SEQ ID NO:36) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence includes QQSTEDPYT (SEQ ID NO: 37) or a homologous sequence thereof with at least 80% sequence identity.

[0248] In certain embodiments, the PD-L1 inhibitor used in the methods provided herein comprises an anti-PD-L1 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:28; The light chain variable region comprises the amino acid sequence of SEQ ID NO:29.

[0249] In certain embodiments, the PD-L1 inhibitors used in the methods provided herein comprise an anti-PD-L1 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 30; The light chain comprises the amino acid sequence of SEQ ID NO:31.

[0250] In certain embodiments, the PD-L1 inhibitors used in the methods provided herein comprise an anti-PD-L1 antibody that comprises a heavy chain HCDR1, HCDR2, and HCDR3, and / or a light chain LCDR1, LCDR2, and LCDR3 sequence, The HCDR1 sequence comprises DSWIH (SEQ ID NO: 45) or a homologous sequence thereof of at least 80% sequence identity; The HCDR2 sequence comprises WISPYGGSTYYADSVKG (SEQ ID NO: 46) or a homologous sequence thereof of at least 80% sequence identity; The HCDR3 sequence comprises RHWPGGFDY (SEQ ID NO: 47) or a homologous sequence thereof of at least 80% sequence identity; The LCDR1 sequence comprises RASQDVSTAVA (SEQ ID NO: 48) or a homologous sequence thereof of at least 80% sequence identity; The LCDR2 sequence comprises SASFLYS (SEQ ID NO:49) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence includes QQYLYHPAT (SEQ ID NO:50) or a homologous sequence thereof with at least 80% sequence identity.

[0251] In certain embodiments, the PD-L1 inhibitor used in the methods provided herein comprises an anti-PD-L1 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:51; The light chain variable region comprises the amino acid sequence of SEQ ID NO:52.

[0252] In certain embodiments, the PD-L1 inhibitors used in the methods provided herein comprise an anti-PD-L1 antibody comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO:53; The light chain comprises the amino acid sequence of SEQ ID NO:54.

[0253] The CLDN18.2 antagonists and PD-1 / PD-L1 axis inhibitors used in the methods provided herein can be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, e.g., intravenous infusion, intramuscular, or intradermal injection), or parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes. In certain embodiments, administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0254] In certain embodiments, administration of a composition comprising an anti-CLDN18.2 antibody precedes, is concurrent with, or follows administration of a composition comprising a PD-1 / PD-L1 axis inhibitor.

[0255] In certain of these embodiments, the CLDN18.2 antagonist administered in combination with one or more PD-1 / PD-L1 inhibitors in the methods provided herein may be administered simultaneously with the one or more PD-1 / PD-L1 inhibitors, and in certain of these embodiments, the CLDN18.2 antagonist and one or more PD-1 / PD-L1 inhibitors may be administered as part of the same pharmaceutical composition. However, the CLDN18.2 antagonist administered "in combination" with the PD-1 / PD-L1 inhibitor need not be administered at the same time as the agent or in the same composition. A CLDN18.2 antagonist administered before or after a PD-1 / PD-L1 inhibitor is considered to be administered "in combination" with the PD-1 / PD-L1 inhibitor, as this phrase is used herein, even if the CLDN18.2 antagonist and the PD-1 / PD-L1 inhibitor are administered by different routes. When possible, a PD-1 / PD-L1 axis inhibitor administered in combination with a CLDN18.2 antagonist will be administered according to the schedule described in the product information sheet of the PD-1 / PD-L1 axis inhibitor, or according to protocols described in the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)), or known in the art.

[0256] In another aspect, the disclosure provides for the use of a pharmaceutical composition comprising a therapeutically effective amount of a) a CLDN18.2 antagonist, b) a PD-1 / PD-L1 axis inhibitor, or c) both, and one or more pharma- ceutically acceptable carriers, in the manufacture of a medicament for treating a disease or condition in a subject in need thereof. In some embodiments, the disease or condition is characterized by a) having CLDN18.2 expression in the affected tissue, and b) having low or no expression of PD-L1 in the affected tissue.

[0257] II. Kit In another aspect, the present disclosure provides a kit useful for treating a disease or condition in a subject in need thereof, comprising a first container comprising a CLDN18.2 antagonist, a second container comprising a PD-1 / PD-L1 system inhibitor, and optionally instructions for use of the kit. In some embodiments, the disease or condition is characterized by a) having CLDN18.2 expression in the diseased tissue, and b) having low or no expression of PD-L1 in the diseased tissue. Suitable containers include, for example, vials (e.g., dual-chamber vials), bottles, syringes (e.g., single or dual-chamber syringes), and test tubes. The containers can be formed from a variety of materials, such as glass or plastic.

[0258] In another aspect, the disclosure provides a kit comprising a CLDN18.2 antagonist and a package insert containing instructions for using the CLDN18.2 antagonist in combination with a PD-1 / PD-L1 axis inhibitor to treat a disease or condition in a subject in need thereof. In some embodiments, the disease or condition is characterized by a) having CLDN18.2 expression in the diseased tissue, and b) having low or no expression of PD-L1 in the diseased tissue.

[0259] In another aspect, the disclosure provides a kit comprising a PD-1 / PD-L1 inhibitor and a package insert containing instructions for using the PD-1 / PD-L1 inhibitor in combination with a CLDN18.2 antagonist to treat a disease or condition in a subject in need thereof. In some embodiments, the disease or condition is characterized by a) having CLDN18.2 expression in the diseased tissue, and b) having low or no expression of PD-L1 in the diseased tissue.

[0260] In another aspect, the disclosure provides a kit comprising an anti-CLDN18.2 antibody or antigen-binding fragment and a package insert containing instructions for use of the anti-CLDN18.2 antibody or antigen-binding fragment in combination with a PD-1 / PD-L1 axis inhibitor to treat a disease or condition in a subject having low expression of CLDN18.2 and / or PD-L1. Any of the PD-1 / PD-L1 axis inhibitors described herein or shown to be effective in inhibiting PD-1 / PD-L1 axis signaling can be included in the kit.

[0261] As used herein, the term "package insert" refers to instructions included in a commercial package of a pharmaceutical that contain information regarding, for example, indications, dosage, directions for use, administration, contraindications, other medications that should be combined with the packaged product, and / or warnings regarding the use of such medications.

[0262] In another aspect, the present disclosure also provides a kit for predicting the responsiveness of a subject to treatment with a CLDN18.2 antagonist in combination with a PD-1 / PD-L1 inhibitor, the kit comprising one or more reagents for detecting the presence of CLDN18.2 and / or PD-L1 in a biological sample obtained from a subject, or one or more reagents for measuring the expression level of CLDN18.2 and / or PD-L1 in a biological sample obtained from a subject. In certain embodiments, the biological sample is a tumor tissue. In certain embodiments, the kit further comprises instructions for predicting the responsiveness of a subject to treatment with a CLDN18.2 antagonist in combination with a PD-1 / PD-L1 inhibitor. The instructions may include the contents as described above.

[0263] The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0264] III. Disease or Condition The disease or condition to be diagnosed, sensitized or treated by the method or kit provided herein can be cancer.In certain embodiments, the cancer is selected from the group consisting of gastric cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, digestive cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma and adenocarcinoma.

[0265] Examples of cancer include, but are not limited to, non-small cell lung cancer (squamous / non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (e.g., basal breast carcinoma, ductal carcinoma, and lobular breast carcinoma), and carcinoma), pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, myeloma, mycosis fungoides, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancies, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma carcinoma), papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatocellular carcinoma, bile ductal carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumors, EBV-positive and -negative PTLD, and diffuse large B-cell lymphoma. These include: DLBCL, plasmablastic lymphoma, extranodal NK / T cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary central nervous system lymphoma, spinal axis tumor, brain stem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinal germ cell tumor.

[0266] In certain embodiments, the cancer is gastric cancer, lung cancer, colon cancer, or a combination thereof.

[0267] In certain embodiments, the cancer is a CLDN18.2-expressing cancer. "CLDN18.2-expressing cancer," as used herein, refers to any cancer or tumor involving cancer cells that express CLDN18.2.

[0268] Examples of cancers that express CLDN18.2 include, but are not limited to, gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), ovarian cancer, colon cancer, colorectal cancer, gastrointestinal mesenchymal tumor (GIST), gastrointestinal carcinoid tumor, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, appendix cancer; prostate cancer, kidney cancer (e.g., renal cell carcinoma), liver cancer, head and neck cancer, and gallbladder cancer and metastases thereof, e.g., gastric cancer metastases, e.g., Krukenberg's tumor, peritoneal dissemination metastases, and lymph node metastases.

[0269] In certain embodiments, the cancer that expresses CLDN18.2 can be adenocarcinoma, for example advanced adenocarcinoma.In certain embodiments, the cancer is selected from intragastric, esophageal, pancreatic, biliary, lung and ovarian adenocarcinoma.In certain embodiments, the cancer that expresses CLDN18.2 includes gastric cancer, esophageal cancer, particularly lower esophageal cancer, esophageal-gastric junction cancer and gastroesophageal cancer. EXAMPLES

[0270] While the present disclosure has been particularly shown and described with reference to specific embodiments, some of which are preferred, it should be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the disclosure as disclosed herein.

[0271] [Example 1] Upregulation of PD-L1 expression on tumor cells by anti-claudin 18.2 (CLDN18.2) antibody in the presence of PBMCs In this assay, three different CLDN18.2-expressing gastric tumor cell lines, NUGC-4 (JCRB, Cat. No. JCRBB0834), KATOIII (ATCC, Cat. No. HTB-103) and SNU-620 (Cobioer, Cat. No. CBP60508), were used as target cells and added to cell culture plates. No antibody, 30 μg / ml of anti-CLDN18.2 antibody 18B10, 30 μg / ml of isotype control hIgG1 or 5 ng / ml of IFN-γ were added into the corresponding wells with target cells. After co-culture of the plates at 37°C for 30 min, human PBMCs were added as effector cells at an E / T ratio of 40:1. The plates were incubated at 37°C for 72 h. At the end of the incubation, 100 μl of cell culture supernatant from each well was transferred to another cell culture plate seeded with the same target cells. The second plate was incubated at 37°C for 72 hours, after which target cells were harvested and analyzed for PD-L1 expression by flow cytometry using a mouse anti-human PD-L1 antibody (BD Pharmingen, catalog no. 557924). S1 represents supernatant of target cells alone, S2 represents supernatant of target cells and human PBMCs with isotype control hIgG1, S3 represents supernatant of target cells and human PBMCs with anti-CLDN18.2 antibody 18B10, and S4 represents supernatant of target cells with IFN-γ.

[0272] As shown in Figure 1A-1C, compared with the S1 group, which expressed a basal level of PD-L1 in target cells, the S3 and S4 groups showed increased PD-L1 expression, while the S2 group only underwent a slight change. This result indicated that the anti-CLDN18.2 antibody could upregulate the PD-L1 expression in target tumor cells. This raises the possibility of a potential synergistic effect when both anti-CLDN18.2 antibody and PD-1 / PD-L1 inhibitors are combined for cancer treatment.

[0273] [Example 2] Combination of anti-CLDN18.2 and anti-PD-L1 antibodies AM4B6 in tumor models To evaluate the potential synergistic effects of combining claudin 18.2 antibodies with checkpoint inhibitors, such as anti-PDL1 antibodies, the following experiments were performed using both the NCI-H460-CLDN18.2 xenograft model (as a lung cancer model), the CT-26-CLDN18.2 syngeneic model (as a colorectal cancer model) and the MC38-CLDN18.2 syngeneic model (as a colon adenocarcinoma model).

[0274] 1. Antitumor efficacy of anti-CLDN18.2 and anti-PD-L1 antibodies in the NCI-H460-CLDN18.2 xenograft tumor model NCI-H460-CLDN18.2 was purchased from Kyinno (catalog number, KC-1450) and was stably transfected with human CLDN18.2 expression. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment (Hyclone). Logarithmic-phase growing cells were harvested and counted for tumor inoculation. Female SPF-grade NOD-SCID mice were inoculated with 3×10 6 NCI-H460-CLDN18.2 cells and 1.5 × 10 6 Human PBMCs were inoculated with 50% Matrigel. Approximately 4 hours after inoculation, animals were selected and randomized into 4 groups (n=10). Animals were then treated with 10mg / kg isotype control, 10mg / kg anti-CLDN18.2 antibody 18B10, 10mg / kg anti-PD-L1 antibody AM4B6, 10mg / kg anti-CLDN18.2 antibody 18B10 and 10mg / kg anti-PD-L1 antibody AM4B6 by ip injection twice a week for 5 weeks. Tumor size was measured in two dimensions using calipers (INSIZE) two or three times a week, and volume was calculated according to the formula: V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively, in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test with p < 0.05. * <0.05 and ** Differences were considered significant when <0.01.

[0275] As shown in Figure 2, compared with the isotype control, the anti-CLDN18.2 antibody 18B10 was able to slightly (but statistically significantly) inhibit tumor growth, while the anti-PD-L1 antibody AM4B6 alone had no significant effect on tumor growth. Specifically, the AM4B6 group only had very slight inhibition, with a TGI of 17.6% (Table 1). In contrast, the combination of the two antibodies had a significantly improved effect on tumor growth, with the TGI increasing to 53.6% (Table 1).

[0276] [Table 1]

[0277] 2. Antitumor efficacy of anti-CLDN18.2 and anti-PD-L1 antibodies in the CT26-CLDN18.2 syngeneic tumor model CT26 is a syngeneic mouse tumor model. CT26-CLDN18.2 was purchased from Kyinno (catalog number, KC-1195) and was stably transfected with human CLDN18.2 expression. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment (Hyclone). Logarithmic growing cells were harvested and counted for tumor inoculation. Female SPF grade BALB / c mice were inoculated with 2 × 10 6 CT26-CLDN18.2 cells were inoculated into each mouse. 3 Once tumors had grown to 100 mm Hg, animals were selected and randomized into 4 groups (n=8). Animals were then treated with either 10 mg / kg isotype control, 10 mg / kg anti-CLDN18.2 antibody 18B10, 3 mg / kg anti-PD-L1 antibody atezolizumab, or 10 mg / kg anti-CLDN18.2 antibody 18B10 and 3 mg / kg anti-PD-L1 antibody atezolizumab by ip injection twice weekly for 3 weeks. Tumor size was measured twice weekly in two dimensions using calipers (INSIZE), and volume was calculated according to the formula: V=0.5a×b 2where a and b are the long and short diameters of the tumor, respectively, in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test with p < 0.05. * <0.05 and ** Differences were considered significant when <0.01.

[0278] As shown in Figure 3, after administration, the tumor volume of mice in the 10 mg / kg isotype control group continued to increase. Treatment with 10 mg / kg 18B10, 3 mg / kg atezolizumab, and the combination of 10 mg / kg 18B10 and 3 mg / kg atezolizumab all showed better antitumor activity compared to treatment with the isotype control, as shown in Figure 3. In addition, the tumor inhibition activity of the combination group continued for about 2 weeks after the last dosing day (day 19), indicating that there may be some immune memory effect of the combination treatment. The TGI of the combination of the two antibodies also increased to 88.44% (Table 2).

[0279] [Table 2]

[0280] 3. Antitumor efficacy of anti-CLDN18.2 and anti-PD-L1 antibodies in the MC38-CLDN18.2 syngeneic tumor model MC38-CLDN18.2 was purchased from Kyinno (catalog number, KC-1449) and was stably transfected with human CLDN18.2 expression. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment (Hyclone). Logarithmic-phase growing cells were harvested and counted for tumor inoculation. Female SPF-grade C57BL / 6 mice were inoculated with 2 × 10 6 MC38-CLDN18.2 cells were inoculated into each tumor. 3Once tumors had grown to 1000 mg / kg, animals were selected and randomized into 4 groups (n=6). Animals were then treated with 13 mg / kg isotype control, 10 mg / kg anti-CLDN18.2 antibody 18B10, 3 mg / kg anti-PD-L1 antibody atezolizumab, 10 mg / kg anti-CLDN18.2 antibody 18B10 and 3 mg / kg anti-PD-L1 antibody atezolizumab by ip injection twice weekly for 4 weeks. Tumor size was measured twice weekly in two dimensions using calipers (INSIZE) and volume was calculated according to the formula: V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively, in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test with p < 0.05. * <0.05 and ** Differences were considered significant when <0.01.

[0281] As shown in Figure 4, after dosing, treatment with either 10 mg / kg 18B10, 3 mg / kg atezolizumab, or the combination of 10 mg / kg 18B10 and 3 mg / kg atezolizumab all showed better antitumor activity compared to treatment with the isotype control, as shown in Figure 4. In addition, the tumor inhibitory activity of the combination group persisted for about 3 weeks after the last dosing day (day 28), which is similar to the efficacy data of the CT26-CLDN18.2 model. The combination of 18B10 and PD-L1 Ab may have added some immune memory effect to maintain antitumor activity for a long time. The TGI of the combination of the two antibodies was also increased to 94.34% (Table 3).

[0282] [Table 3]

[0283] [Example 3] Combination of anti-CLDN18.2 and anti-PD-1 antibodies RMP1-14 in tumor models The anti-tumor efficacy of anti-CLDN18.2 and anti-PD-1 antibodies was also evaluated in the CT26-CLDN18.2 syngeneic tumor model.

[0284] CT26-CLDN18.2 cells were routinely subcultured twice a week by trypsin-EDTA treatment (Hyclone). Cells in logarithmic growth phase were harvested and counted for tumor inoculation. Female SPF grade BALB / c mice were inoculated with 2 × 10 6 CT26-CLDN18.2 cells were inoculated into each tumor. The tumor size was approximately 80 mm. 3 When the tumors had grown to 100 mm HgG1, animals were selected and randomized into 4 groups (n=10). The animals were then treated twice weekly for 4 weeks by ip injection with 10 mg / kg hIgG1 control and 5 mg / kg 2A3 (rat IgG2a control), 10 mg / kg anti-CLDN18.2 antibody 18B10 and 5 mg / kg 2A3, 5 mg / kg rat anti-mouse PD-1 antibody RMP1-14 (purchased from BioXcell, USA) and 10 mg / kg hIgG1 control and 10 mg / kg anti-CLDN18.2 antibody 18B10 in combination with 5 mg / kg RMP1-14 at Rgb1-14. Tumor size was measured twice weekly in two dimensions using calipers (INSIZE), and volume was calculated according to the formula: V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively, in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test with p < 0.05. * <0.05 and ** Differences were considered significant when <0.01.

[0285] As shown in Figure 5A, after administration, the tumor volume of the mice in the 10 mg / kg isotype control group continued to increase. All treatment groups with either 10 mg / kg 18B10 + 5 mg / kg 2A3, 10 mg / kg isotype control hIgG1 + 5 mg / kg RMP1-14, or a combination of 10 mg / kg 18B10 and 5 mg / kg RMP1-14 showed better antitumor activity compared to treatment with the isotype control, as shown in Figure 5A. Furthermore, all tumors in the combination group (18B10 + RMP1-14) had regressed by day 29 (TGI = 100%), while there was no tumor regression in the 18B10 + 2A3 group and only half of the tumors had regressed in the isotype control + RMP1-14 group (Table 4).

[0286] [Table 4]

[0287] [Example 4] Efficacy of 18B10 combination with anti-PD-1 and chemotherapeutic agents against the CT26-hCLDN18.2 tumor model The mouse colon cancer cell line CT26 was transfected with the CLDN18.2 gene and screened for stable expression of CLDN18.2, designated CT26-hCLDN18.2. CT26-hCLDN18.2 cells were maintained in vitro as monolayer cultures in RPMI1640 medium (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100 U / ml penicillin, 100 μg / ml streptomycin (Hyclone), and 1 μg / mL puromycin (Gibco) at 37 °C in an atmosphere containing 5% CO2 in air. Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment (Hyclone). Logarithmic-phase growing cells were harvested and counted for tumor inoculation. Female SPF-grade BABL / c mice were inoculated with 2 × 10 6 CT26-hCLDN18.2 cells were inoculated with 50% Matrigel. The tumor size was approximately 80 mm 3At time point, tumor-bearing mice were selected and randomized into 4 groups (n=10). Animals were treated with 10 mg / kg hIgG1 control and 1 mg / kg 2A3 (anti-rat IgG2a control), 10 mg / kg 18B10, 1 mg / kg RMP1-14 (anti-mouse PD1) and 1 mg / kg oxaliplatin and 5 mg / kg 5FU, and 10 mg / kg 18B10 in combination with 1 mg / kg RMP1-14 (anti-mouse PD1) and 1 mg / kg oxaliplatin and 5 mg / kg 5FU, for antibodies by ip injection twice a week for 3 weeks, and for chemotherapy by iv injection once a week for 3 weeks. Tumor size was measured in two dimensions using calipers (INSIZE) two or three times a week, and volume was calculated according to the formula: V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively, in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test with p < 0.05. * <0.05 and ** Differences were considered significant when <0.01. The triple combination therapy had enhanced antitumor activity over 18B10 alone and the combination of anti-PD-1 and chemotherapy drugs, as shown in Table 5 and Figure 5B.

[0288] [Table 5]

[0289] [Example 5] IHC-based assessment of CLDN18.2 and PD-L1 expression in clinical and patient-derived xenograft (PDX) samples across various tumor types CLDN18.2 and PD-L1 co-expression status in clinical cancer samples To investigate the expression levels and overlap status of CLDN18.2 and PD-L1 in gastric and cholangiocarcinoma, immunohistochemistry (IHC) was performed on these 4% neutral buffered formalin-fixed paraffin-embedded (FFPE) tumor sections using in-house developed and validated recombinant anti-CLDN18.2 (14G11) and commercially available anti-PD-L1 (22C3) monoclonal antibodies. After deparaffinization and rehydration, all sections proceeded to antigen retrieval by boiling at 97-99°C for 25 min in EnVision™ FLEX target retrieval solution (Dako, K8002), then quenched and blocked with EnVision™ FLEX peroxidase blocking reagent (Dako, K8002) and incubated with appropriately diluted 14G11 (0.6 μg / mL) and 22C3 (1 μg / mL) antibodies, respectively. Antibody binding was visualized using EnVision™ FLEX+ Mouse (LINKER), followed by EnVision™ FLEX / HRP and EnVision™ FLEX substrate working solution (Dako, K8002). Sections were finally counterstained with hematoxylin and mounted with permanent mounting medium.

[0290] All samples were scored by the relative percentage of positively stained tumor cells to all viable tumor cells for CLDN18.2, or by the combined positive score (CPS) of total stained immune and tumor cells to all viable tumor cells for PD-L1, with membrane staining of different intensities (negative (0), weak (1+), moderate (2+), strong (3+)) as presented in Table 6. Only membrane staining with an intensity of ≥2+ was considered positive. Weak to strong membrane signals of CLDN18.2 and PD-L1 were generated by 14G11 and 22C3 in gastric and cholangiocarcinoma cancer tissues, respectively (see Figure 6A). CLDN18.2 and PD-L1 expression levels and overlap across gastric and cholangiocarcinoma cancer tissues are summarized in Table 7. The results show that most samples have CLDN18.2 expression in gastric and cholangiocarcinoma cancers. Furthermore, the rate of no or low PD-L1 expression among CLDN18.2-expressing (IHC score ≥ 1+) tumor samples in gastric cancer was 93.75%, and the rate of no or low PD-L1 expression among CLDN18.2-expressing (IHC score ≥ 1+) tumor samples in cholangiocarcinoma was 100%.

[0291] [Table 6]

[0292] [Table 7]

[0293] CLDN18.2 and PD-L1 co-expression status in PDX samples Biotinylated 14G11 (14G11 biotin) and 22C3 (22C3 biotin) were prepared. Briefly, biotin amidocaproate NHS ester (Sigma, B2643-10MG) was prepared in anhydrous DMF at 20 mg / mL as a stock solution. To each 1 mg of 14G11 antibody to be labeled, 10 μl of the stock solution was added and mixed gently at room temperature for 1 hour. Low molecular weight reaction products were removed by desalting on a Zeba™ Spin desalting column (ThermoFisher, 89890) according to the manufacturer's instructions.

[0294] Immunohistochemistry (IHC) was performed on slides of 4% neutral buffered formalin fixed paraffin embedded PDX samples, including gastric and pancreatic cancer. After deparaffinization and rehydration, all slides proceeded to antigen retrieval by boiling in EnVision™ FLEX target retrieval solution (Dako, K8002) at 97-99°C for 25 min, then quenched and blocked with IHC Biotin Block Kit (MaiXin, BLK-0001) according to the manufacturer's instructions, and incubated with 10 μg / mL in-house biotinylated monoclonal mouse anti-claudin 18.2 (14G11 biotin) and anti-PD-L1 (22C3 biotin) antibodies, respectively, for 30 min at 37°C. Antibody binding was visualized using horseradish peroxidase-labeled streptavidin (MaiXin, SP KIT-D1) and EnVision™ FLEX substrate working solution (Dako, K8002). Finally, sections were counterstained with hematoxylin and mounted with permanent mounting medium. IHC results were scored for staining intensity, pattern, and percent positive (see Table 6).

[0295] CLDN18.2 and PD-L1 expression levels and overlap across gastric and pancreatic PDX cancer tissues are summarized in Table 8. The results show that most samples have CLDN18.2 expression in gastric and pancreatic cancer. Furthermore, CLDN18.2 expression (IHC scoring ≧1+, see FIG. 6B) in both gastric and pancreatic cancer) is 100% with no or low PD-L1 expression among tumor samples.

[0296] [Table 8]

[0297] [Example 6] Efficacy of 18B10 combination with PD-1 antibody against GC-02-0007 PDX tumor model in HSC-NSG-hIL-15 mice GC-02-0007 gastric tumor tissue was obtained from Beijing Cancer Hospital and passaged in nude mice to establish a PDX bank. The expression levels of CLDN18.2 and PD-L1 in the GC-02-0007 PDX tumor model were measured as described in the section "CLDN18.2 and PD-L1 co-expression status in PDX samples" in Example 5, and the results were shown in Table 8 "Gastric cancer". HSC-NSG-hIL-15 mice were human HSC-reconstituted human IL-15 mice that could maintain a certain human NK cell ratio purchased from JAX lab. Small tumor tissue blocks with a diameter of about 3 mm excised from tumor-bearing mice by integrated tumor dissection were subcutaneously inoculated into each mouse. A few days after inoculation, tumors of about 50 mm in diameter were observed. 3 Animals with tumor sizes of 0.01 to 0.01 mm were selected and randomized into 4 groups, each group consisting of 5 mice. Mice were then treated with 30 mg / kg isotype control, 20 mg / kg 18B10, 10 mg / kg nivolumab, and a combination of 18B10 and nivolumab by ip injection twice a week for 4 weeks. Animals were sacrificed by CO2 inhalation at the end of the study. Tumor size was measured in two dimensions using a vernier caliper (INSIZE) two or three times a week, and volume was calculated according to the formula: V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively, in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test with p < 0.05. * <0.05 and ** Differences were considered significant when <0.01.

[0298] Treatment with the combination of 18B10 and nivolumab reduces tumor volume more effectively than treatment with nivolumab alone.

[0299] [Example 7] Efficacy of 18B10-HaLa in combination with anti-PD-1 and chemotherapeutic drugs against NUGC4-hCLDN18.2 and human PBMC co-inoculated tumor models The human gastric cancer cell line NUGC4 was transfected with the CLDN18.2 gene and screened for stable expression of CLDN18.2, designated NUGC4-hCLDN18.2. NUGC4-hCLDN18.2 cells were maintained in vitro as monolayer cultures in RPMI1640 medium (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100 U / ml penicillin, 100 μg / ml streptomycin (Hyclone) and 1 μg / mL puromycin (Gibco) at 37 °C in an atmosphere containing 5% CO2 in air. Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment (Hyclone). Logarithmic-phase growing cells were harvested and counted for tumor inoculation. Female SPF-grade NOD-SCID mice were injected with 5 × 10 mixed 100-μg / ml nucleoside-containing 1 ... 6 NUGC4-hCLDN18.2 cells and 5 × 10 6 Human PBMCs were inoculated with 50% Matrigel. Approximately 4 hours after inoculation, mice were selected and randomized into 4 groups (n=10). Animals were treated with isotype control and vehicle, 10 mg / kg 18B10-HaLa, 5 mg / kg nivolumab and 3 mg / kg oxaliplatin / 10 mg / kg 5-FU, and a combination of 10 mg / kg 18B10-HaLa, 5 mg / kg nivolumab and 3 mg / kg oxaliplatin / 10 mg / kg 5-FU, for antibodies by ip injection twice a week for 6 weeks, and chemotherapy by iv injection once a week for 6 weeks. Tumor size was measured in two dimensions using calipers (INSIZE) two or three times a week, and volume was calculated according to the formula: V=0.5a×b 2where a and b are the long and short diameters of the tumor, respectively, in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test with p < 0.05. * <0.05 and ** Differences were considered significant when p<0.01. The triple combination therapy had significantly enhanced antitumor activity over 18B10-HaLa alone (p<0.01) and the combination of anti-PD-1 and chemotherapy drugs (p<0.01), as shown in Table 9 and Figure 7.

[0300] [Table 9]

[0301] [Example 8] Efficacy of 18B10 in combination with anti-PD-1 and chemotherapeutic drugs against MFC / hCLDN18.2 tumor model The mouse gastric cancer cell line MFC was transfected with the CLDN18.2 gene and screened for stable expression of CLDN18.2, designated MFC / CLDN18.2. MFC / CLDN18.2 cells were maintained in vitro as monolayer cultures in DMEM medium (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100 U / ml penicillin, 100 μg / ml streptomycin (Hyclone) and 1 μg / mL puromycin (Gibco) at 37 °C in an atmosphere containing 5% CO2 in air. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment (Hyclone). Growing cells in the logarithmic growth phase were harvested and counted for tumor inoculation. Female SPF grade 615 mice were inoculated with 2 × 10 6 Approximately 5 days after inoculation, approximately 90 mm 3Tumor-bearing mice were selected and randomized into 4 groups (n=9) with tumor sizes of 10 mg / kg 18B10-HaLa, 1 mg / kg RMP1-14 and 1 mg / kg oxaliplatin / 5 mg / kg 5-FU, and a combination of 10 mg / kg 18B10-HaLa, 1 mg / kg RMP1-14 and 1 mg / kg oxaliplatin / 5 mg / kg 5-FU. The antibodies were treated twice weekly for 3 weeks by ip injection, and the chemotherapy drugs were treated once weekly for 3 weeks by iv injection. Tumor size was measured twice or thrice weekly in two dimensions using calipers (INSIZE), and volume was calculated according to the formula: V=0.5a×b 2 where a and b are the long and short diameters of the tumor, respectively, in mm 3 Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test with p < 0.05. * <0.05 and ** Differences were considered significant when <0.01. The triple combination therapy had enhanced antitumor activity (TGI at day 19: 72.22% vs. 62.02%) over 18B10-HaLa alone (p<0.01) and over the combination of anti-PD-1 and chemotherapy drugs, as shown in Table 10 and Figure 8.

[0302] [Table 10]

[0303] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5]

Table 11-6

Table 11-7

Table 11-8

Claims

1. A pharmaceutical composition for use in a method for treating a CLDN18.2-associated disease or condition in a subject in need thereof, comprising: The pharmaceutical composition comprises a therapeutically effective amount of a) a CLDN18.2 antagonist, b) a PD-1 / PD-L1 system inhibitor, or c) both, and one or more pharmaceutically acceptable carriers; The method includes administering to the subject a therapeutically effective amount of a CLDN18.2 antagonist in combination with a therapeutically effective amount of a PD-1 / PD-L1 system inhibitor; The subject is determined to have CLDN18.2 expression in affected tissue.

2. The pharmaceutical composition described in claim 1, wherein the method further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent.

3. The pharmaceutical composition described in claim 1, wherein CLDN18.2 expression in the diseased tissue is higher than or equivalent to expression in healthy or non-cancerous gastric cells or gastric tissue.

4. The pharmaceutical composition described in claim 1, wherein CLDN18.2 expression in the diseased tissue is lower than expression in healthy or non-cancerous gastric cells or gastric tissue, but higher than expression in healthy or non-cancerous tissue or organ other than the stomach.

5. The pharmaceutical composition described in claim 1, wherein CLDN18.2 expression in the diseased tissue is equivalent to expression in healthy or non-cancerous tissues or organs other than the stomach and is detectable by an anti-CLDN18.2 diagnostic antibody.

6. The pharmaceutical composition described in claim 1, wherein the subject is determined to have PD-L1 expression in the affected tissue.

7. The pharmaceutical composition of claim 1, wherein the subject is determined to have low or no PD-L1 expression in the affected tissue.

8. The pharmaceutical composition described in claim 7, wherein PD-L1 expression in the affected tissue is lower than a reference level.

9. The pharmaceutical composition described in claim 7, wherein 20% or less of the cells in the affected tissue are positive for PD-L1 expression.

10. The pharmaceutical composition described in claim 9, wherein the cells of the affected tissue include disease cells and immune cells in the affected tissue.

11. The pharmaceutical composition described in claim 7, wherein PD-L1 expression in the diseased tissue is equivalent to that in healthy or non-cancerous tissue, or wherein PD-L1 expression in the diseased tissue is low or not detectable by an anti-PD-L1 diagnostic antibody.

12. 1. A pharmaceutical composition for use in a method of sensitizing a disease or condition to treatment with a PD-1 / PD-L1 axis inhibitor in a subject in need thereof, comprising: The pharmaceutical composition comprises a therapeutically effective amount of a) a CLDN18.2 antagonist, b) a PD-1 / PD-L1 system inhibitor, or c) both, and one or more pharmaceutically acceptable carriers; the subject is determined to have low or absent expression of PD-L1 in affected tissue; a) determining the presence or expression level of CLDN18.2 in diseased tissue obtained from the subject; and b) when the presence of CLDN18.2 is determined in step a) or when the expression level of CLDN18.2 reaches a threshold level in step a), administering to the subject a therapeutically effective amount of a CLDN18.2 antagonist, optionally in combination with a therapeutically effective amount of a PD-1 / PD-L1 system inhibitor; thereby sensitizing the disease or condition to treatment with a PD-1 / PD-L1 system inhibitor. A pharmaceutical composition comprising:

13. The pharmaceutical composition of claim 12, wherein step b) further comprises administering a chemotherapeutic agent to the subject.

14. 1. A pharmaceutical composition for use in a method for increasing tumor responsiveness to treatment with a PD-1 / PD-L1 system inhibitor in a subject, comprising: The pharmaceutical composition comprises a therapeutically effective amount of a) a CLDN18.2 antagonist, b) a PD-1 / PD-L1 system inhibitor, or c) both, and one or more pharmaceutically acceptable carriers; the subject is determined to have a tumor that is resistant or refractory to treatment with a PD-1 / PD-L1 system inhibitor; The method includes: a) determining the presence or expression level of CLDN18.2 in a tumor sample obtained from the subject; and b) when the presence of CLDN18.2 is determined in step a) or when the expression level of CLDN18.2 reaches a threshold level in step a), administering to the subject a therapeutically effective amount of a CLDN18.2 antagonist, optionally in combination with a therapeutically effective amount of a PD-1 / PD-L1 system inhibitor; thereby increasing the responsiveness of the tumor to treatment with a PD-1 / PD-L1 system inhibitor in the subject. A pharmaceutical composition comprising:

15. The pharmaceutical composition of claim 14, wherein step b) further comprises administering a chemotherapeutic agent to the subject.

16. The pharmaceutical composition of claim 1, 12, or 14, wherein the CLDN18.2 antagonist comprises an anti-CLDN18.2 antibody, e.g., a monoclonal anti-CLDN18.2 antibody, a bispecific antibody targeting CLDN18.2 and a second antigen (e.g., CD3, 4-1BB, TGFβ, SIRPα, and IL15), or an immune cell expressing a chimeric antigen receptor (CAR) or genetically modified TCR comprising an anti-CLDN18.2 antigen-binding domain.

17. The anti-CLDN18.2 antibody comprises heavy chain HCDR1, HCDR2, and HCDR3 and / or light chain LCDR1, LCDR2, and LCDR3 sequences, the HCDR1 sequence comprises GYNMN (SEQ ID NO: 1) or a homologous sequence thereof with at least 80% sequence identity; the HCDR2 sequence comprises NIDPYYGGTSYNQKFKG (SEQ ID NO: 2) or a homologous sequence thereof with at least 80% sequence identity; the HCDR3 sequence comprises MYHGNAFDY (SEQ ID NO: 3) or a homologous sequence thereof with at least 80% sequence identity; the LCDR1 sequence comprises KSSQSLLNSGNLKNYLT (SEQ ID NO: 4) or a homologous sequence thereof with at least 80% sequence identity; the LCDR2 sequence comprises WASTRKS (SEQ ID NO: 5) or a homologous sequence thereof of at least 80% sequence identity; 17. The pharmaceutical composition of claim 16, wherein the LCDR3 sequence comprises QNDYSYPLT (SEQ ID NO: 6) or a homologous sequence thereof of at least 80% sequence identity.

18. The anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, 18. The pharmaceutical composition of claim 17, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

8.

19. 17. The pharmaceutical composition of claim 16, wherein the constant region comprises one or more amino acid residue substitutions relative to SEQ ID NO: 9 selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, or any combination thereof.

20. The pharmaceutical composition described in claim 19, wherein the constant region comprises the sequence of SEQ ID NO: 11, and optionally, the constant region further comprises the sequence of SEQ ID NO:

10.

21. The anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14; 20. The pharmaceutical composition of claim 19, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO:

16.

22. The anti-CLDN18.2 antibody, comprising a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 39; 17. The pharmaceutical composition of claim 16, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

40.

23. The pharmaceutical composition of claim 16, wherein the anti-CLDN18.2 antibody is capable of inducing expression of PD-L1 in the affected tissue of the subject.

24. The pharmaceutical composition of claim 16, wherein the PD-1 / PD-L1 system inhibitor comprises a PD-1 inhibitor selected from the group consisting of antibodies, small molecules, and combinations thereof; or wherein the PD-1 / PD-L1 system inhibitor comprises a PD-L1 inhibitor selected from the group consisting of antibodies, small molecules, and combinations thereof.

25. (i) The PD-1 inhibitor is selected from the group consisting of nivolumab (OPDIVO; BMS-936558), dostallimab (TSR-042), pembrolizumab (KEYTRUDA; MK-3475), MEDI0680 (AMP-514), MEDI4736, BI 754091, pidilizumab (CT-011), cemiplimab (LIBTAYO, REGN2810), spartalizumab (PDR001), cetrelimab (JNJ 63723283), toripalimab (JS001), PF-06801591, tislelizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, lambrolizumab or camrelizumab (SHR-1210), sintilimab (Tyvyt, IBI308), penprimab (AK105), di an anti-PD-1 antibody selected from the group consisting of mbelelimab, retifanlimab, selplulimab, balstilimab, geptanolimab, prorugolimab, ezabenlimab, sasanlimab, pimivalimab, budigalimab, nofazinelimab, sindelizumab, MGA404, Sym021, BAT1306, and HX008; or (ii) The PD-L1 inhibitor is atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446), BMS-936559, avelumab (bavencio), rodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, embafolimab (KN035), MDX-1105, STI-1040, CS1001, adeburelimab (SHR-131 6), SHR-1701, TOB2450, vintrafusp, LP002, STI-3031, cosibelimab, pakmilirumab, NM01, LDP, AMP-224, galibrimab (BGB-A333), A167, SCD-135, opucolimab, GR1405, and optionally, the PD-L1 inhibitor is atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446); or the PD-L1 inhibitor comprises a bispecific antibody that targets both PD-L1 and another checkpoint molecule selected from the group consisting of PD-1, PD-L1, PD-L2, CLTA-4, SIRP, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, or CD83; 25. The pharmaceutical composition of claim 24.

26. The PD-L1 inhibitor comprising an anti-PD-L1 antibody comprising heavy chain HCDR1, HCDR2, and HCDR3 and / or light chain LCDR1, LCDR2, and LCDR3 sequences; (a) the HCDR1 sequence comprises DYYMN (SEQ ID NO: 22) or a homologous sequence thereof with at least 80% sequence identity; the HCDR2 sequence comprises DINPNNAETLYNHKFKG (SEQ ID NO: 23) or a homologous sequence thereof with at least 80% sequence identity; the HCDR3 sequence comprises WGDGPFAY (SEQ ID NO: 24) or a homologous sequence thereof of at least 80% sequence identity; the LCDR1 sequence comprises KASQNVGAAVA (SEQ ID NO: 25) or a homologous sequence thereof of at least 80% sequence identity; the LCDR2 sequence comprises SVSDRYT (SEQ ID NO: 26) or a homologous sequence thereof of at least 80% sequence identity; the LCDR3 sequence comprises QQYSNYPT (SEQ ID NO: 27) or a homologous sequence thereof of at least 80% sequence identity; or (b) the HCDR1 sequence comprises TYWMH (SEQ ID NO: 32) or a homologous sequence thereof with at least 80% sequence identity; the HCDR2 sequence comprises MIQPNSGGTKYNEKFKK (SEQ ID NO: 33) or a homologous sequence thereof with at least 80% sequence identity; the HCDR3 sequence comprises GAGTVDYFDY (SEQ ID NO: 34) or a homologous sequence thereof of at least 80% sequence identity; the LCDR1 sequence comprises RASESVDIYGNSFMH (SEQ ID NO: 35) or a homologous sequence thereof of at least 80% sequence identity; the LCDR2 sequence comprises RASNLES (SEQ ID NO: 36) or a homologous sequence thereof of at least 80% sequence identity; The LCDR3 sequence comprises QQSTEDPYT (SEQ ID NO: 37) or a homologous sequence thereof with at least 80% sequence identity; 17. The pharmaceutical composition of claim 16.

27. The PD-L1 inhibitor comprises an anti-PD-L1 antibody comprising a heavy chain variable region and a light chain variable region; (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 18; or (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28; the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29; 17. The pharmaceutical composition of claim 16.

28. The PD-L1 inhibitor, comprising an anti-PD-L1 antibody comprising a heavy chain and a light chain; (a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20; the light chain comprises the amino acid sequence of SEQ ID NO: 21; or (b) the heavy chain comprises the amino acid sequence of SEQ ID NO: 30; the light chain comprises the amino acid sequence of SEQ ID NO:

31.

28. The pharmaceutical composition of claim 27.

29. The pharmaceutical composition of claim 16, wherein the disease or condition is cancer or the affected tissue contains cancer cells.

30. The pharmaceutical composition of claim 29, wherein the cancer is selected from the group consisting of gastric cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain tumor, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, digestive cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, and adenocarcinoma.

31. The pharmaceutical composition of claim 16, wherein the disease or condition is resistant or refractory to treatment with a PD-1 / PD-L1 system inhibitor, and optionally the resistance is de novo resistance or acquired resistance.

32. The pharmaceutical composition of claim 31, wherein the disease or condition is resistant or refractory to combination therapy with a PD-1 / PD-L1 system inhibitor and a chemotherapeutic agent.

33. The chemotherapeutic agent may be an antimetabolite, such as methotrexate and 5-fluorouracil (5-FU), oxaliplatin, alkylating agents (e.g., thiotepa and cyclophosphamide (Cytoxan™)), alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan), aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa), emylermines and memylamelamines (e.g., altretamine, triemylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemirolomelamine), acetogenins, camptothecins (e.g., the synthetic analog topotecan), bryostatin, kallistatin, CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin), cryptophycins (e.g., cryptophycin and cryptophycin), dolastatin, duocarmycin (synthetic analogs, KW-2, 189 and CBI-TMI), eleutherobin, pancratistatin, sarcodictine, spongiostatin, cisplatin, nitrogen mustards (e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicine, fenesterine, prednimustine, trofosfamide, uracil mustard), nitrosoureas (e.g., carmustine, chlorozotocin, foremustine, lomustine, nimustine, ranimustine), folic acid analogs (e.g., demopterin, methotrexate, Rexate, pteropterin, trimetrexate, purine analogues (e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine), pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine), androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone), antiadrenal agents (e.g., aminoglutethimide, mitotane, trilostane), folic acid supplements (e.g., florinic acidacid)]), aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, hestravcil, bisantrene, edatrexate, defofamine, demecolcine, diaziquone, elformucine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids (e.g., maytansine and ansamitocin), mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, P 33. The pharmaceutical composition of claim 32, wherein the active ingredient is selected from the group consisting of SK™, razoxane, rhizoxin, schizofiran, spirogermanium, tenuazonic acid, triazicon, 2,2,2"-trichlorotriemylamine, trichothecene, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, and mitolactol.

34. A kit for use in treating a disease or condition in a subject in need thereof, comprising: a first container containing a CLDN18.2 antagonist; a second container containing a PD-1 / PD-L1 system inhibitor; and, optionally, instructions for use of the kit; The disease or condition is characterized by a) CLDN18.2 expression in the affected tissue, and / or b) low or no expression of PD-L1 in the affected tissue.

35. A kit comprising a CLDN18.2 antagonist and a package insert containing instructions for using the CLDN18.2 antagonist in combination with a PD-1 / PD-L1 system inhibitor to treat a disease or condition in a subject in need thereof, The disease or condition is characterized by a) CLDN18.2 expression in the affected tissue, and / or b) low or no expression of PD-L1 in the affected tissue.

36. The kit described in claim 34, wherein the CLDN18.2 antagonist and the PD-1 / PD-L1 system inhibitor are contained in a pharmaceutical composition described in any one of claims 1 to 33.

37. The kit described in claim 35, wherein the CLDN18.2 antagonist is contained in a pharmaceutical composition described in any one of claims 1 to 33.

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