Cancer treatment with a PD-1 inhibitor or a PD-L1 inhibitor and an antibody-drug conjugate targeting Claudin 18.2

Abstract

There is provided a combination therapy comprising an inhibitor of PD-1 or an inhibitor of PD-L1 and an antibody-drug conjugate targeting Claudin 18.2. The present invention is, for example, a method for treating cancer in a patient in need of treatment for cancer, the method comprising administering to the patient an inhibitor of PD-1 or an inhibitor of PD-L1, and an antibody-drug conjugate (ADC) comprising a drug moiety covalently attached to an anti-Claudin 18.2 antibody comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of SEQ ID NOs: 3-8.

Description

Background Art

[0001] Background Claudin is a family of proteins that form important components of tight cell-cell junctions. They establish paracellular barriers that control the flow of molecules between cells. The proteins have an N-terminus and a C-terminus in the cytoplasm. Different claudins are expressed in different tissues, and changes in their functions have been associated with the formation of cancer in their respective tissues. Claudin-1 is expressed in colorectal cancer, claudin-18 is expressed in gastric cancer, and claudin-10 is expressed in hepatocellular carcinoma.

[0002] Claudin-18 has two isoforms, isoform 1 and isoform 2. Isoform 2 (claudin 18.2 or CLDN18.2) is a very selective cell lineage marker. The expression of claudin 18.2 in normal tissues is strictly restricted to differentiated epithelial cells of the gastric mucosa and was not present in the gastric stem cell zone. Claudin 18.2 was retained in malignant transformation and was expressed in a significant proportion of primary gastric cancers and their metastases. Ectopic activation of claudin 18.2 was also seen at high frequency in tumors of the pancreas, esophagus, ovary, and lung. These data suggest that CLDN18.2 has a very restricted expression pattern in normal tissues, with frequent ectopic activation in diverse human cancers.

Summary of the Invention

Means for Solving the Problems

[0003] Abstract It has been discovered herein that an antibody-drug conjugate based on LM-001 was able to synergistically inhibit anti-PD-1 antibody and tumor growth. This is thought to be at least due to the excellent ability of LM-001 to induce receptor-mediated antibody internalization. Amino acid residues on the claudin 18.2 protein that are important for binding to LM-001 include amino acid residues (e.g., W30, L49, W50, C53, C63, and R80) that are important for stabilizing the conformation of the extracellular loop. More importantly, residues involved in binding to the antibody are contemplated to include N45, Y46, G48, V54, R55, E56, S58, F60, and E62, which are located between the β3 and β4 strands of the first extracellular loop, and Y169 and G172, which are in the β5 of the second extracellular loop. In contrast, known anti-claudin 18.2 antibodies are thought to bind to only one of the extracellular loops.

[0004] According to one embodiment of the present disclosure, there is provided a method for treating cancer in a patient in need thereof, the method comprising administering to the patient an antibody-drug conjugate (ADC) comprising an inhibitor of PD-1 or an inhibitor of PD-L1, and a drug moiety covalently attached to an anti-claudin 18.2 antibody comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of SEQ ID NOs: 3-8. In some embodiments, the anti-claudin 18.2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 1 and a VL comprising the amino acid of SEQ ID NO: 2.

[0005] In some embodiments, the anti-claudin 18.2 antibody is attached to 2-10 drug moieties, preferably 2-6 drug moieties.

[0006] In some embodiments, the drug moiety is a cytotoxic agent or a cell division inhibitor. In some embodiments, the drug moiety is a maytansinoid or an auristatin. In some embodiments, the drug moiety comprises DM1 or DM4. In some embodiments, the drug moiety comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0007] In some embodiments, the drug moiety is attached to an antibody or a fragment thereof via a linker. In some embodiments, the linker is hydrolysable under acidic conditions. In some embodiments, the linker comprises valine-citrulline.

[0008] In some embodiments, the ADC comprises 2 - 6 MMAE or MMAF conjugated to an anti-Claudin 18.2 antibody via valine-citrulline.

[0009] In some embodiments, the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170 and BMS-986189. In some embodiments, the inhibitor of PD-1 is selected from the group consisting of pembrolizumab, nivolumab, semaprilumab, dostarlimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012, AMP-224, and AMP-514. In some embodiments, the inhibitor is toripalimab.

[0010] In some embodiments, the inhibitor and the ADC are each administered once every 1 - 6 weeks. In some embodiments, the ADC is administered at a dose of 0.1 mg / kg - 10 mg / kg. In some embodiments, the ADC is administered at a dose of 0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg, 1.6 mg / kg, 2.0 mg / kg or 2.4 mg / kg.

[0011] In some embodiments, the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

[0012] In certain embodiments, a method for treating cancer in a patient in need of cancer treatment, comprising administering to the patient an inhibitor of PD-1 or an inhibitor of PD-L1 and a vcMMAE-conjugated anti-Claudin 18.2 antibody (ADC), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 1 and a VL comprising the amino acid of SEQ ID NO: 2; each antibody is conjugated to 2 to 6 units of vcMMAE; the ADC is administered at a dose of 1.2 mg / kg to 1.8 mg / kg; and the cancer is gastrointestinal cancer. A method is provided.

[0013] In some embodiments, the gastrointestinal cancer is gastric cancer (GC) or gastroesophageal junction cancer (GJC). In some embodiments, the inhibitor of PD-1 or the inhibitor of PD-L1 is toripalimab. In some embodiments, the inhibitor of PD-1 or the inhibitor of PD-L1 is nivolumab.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0018] Detailed Description Definition It should be noted that the term "one (a)" or "one (an)" entity refers to one or more of that entity. For example, "an antibody" is understood to represent one or more antibodies. Thus, the terms "one (a)" (or "one (an)"), "one or more", and "at least one" can be used interchangeably herein.

[0019] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a whole antibody and any antigen-binding fragment, or a single chain thereof. Thus, the term "antibody" includes any protein or peptide containing a molecule that contains at least a portion of an immunoglobulin molecule having the biological activity of binding to an antigen. Such examples include, but are not limited to, the heavy or light chain of a complementarity-determining region (CDR) or the ligand-binding portion thereof, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0020] The terms "antibody fragment" or "antigen-binding fragment", as used herein, are part of an antibody such as F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv, etc. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, Spiegelmers and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0021] The term antibody encompasses a wide variety of biochemically distinguishable classes of polypeptides. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with several subclasses among them (e.g., γ1-γ4). It is this chain type that determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Subclasses (isotypes) of immunoglobulins, e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and are known to confer functional differentiation. Each modified version of those classes and isotypes is readily recognized by those skilled in the art from the perspective of the present disclosure and is thus within the scope of the present disclosure. All classes of immunoglobulins are clearly within the scope of the present disclosure, and the following discussion generally targets the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons, and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds to form a "Y" shaped configuration, where the light chains begin at the mouth of the "Y" and together with the continuous heavy chains pass through the variable regions.

[0022] Antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab’ and F(ab’)2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing either the VK or VH domain, fragments generated from a Fab expression library, and anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulins or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of the immunoglobulin molecule.

[0023] The light chain is classified as either kappa or lambda (κ, λ). The class of each heavy chain can bind to either a kappa or lambda light chain. Generally, the light chain and the heavy chain are covalently bound to each other, and the "tail" portions of the two heavy chains are bound to each other by a covalent disulfide linkage or a non-covalent linkage when the immunoglobulin is produced by any of a hybridoma, a B cell or a genetically engineered host cell. In the heavy chain, the amino acid sequence continues from the N-terminus of the branched end of the Y-shaped structure to the C-terminus at the bottom of each chain.

[0024] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light chain (VK) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases such that they are more distal from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxy termini of the heavy and light chains, respectively.

[0025] As indicated above, the variable region enables the antibody to selectively recognize and specifically bind to an epitope on the antigen. That is, the VK and VH domains of the antibody, or a subset of the complementarity-determining regions (CDRs), combine to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites present at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) in each of the VH and VK chains. In some examples, such as certain immunoglobulin molecules derived from Camelidae species or engineered based on Camelidae immunoglobulins, the complete immunoglobulin molecule may consist of only heavy chains without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0026] In naturally occurring antibodies, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous sequences of amino acids that specifically position to form the antigen-binding domain because the antibody assumes its three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain are referred to as the "framework" regions and exhibit little intermolecular variation. The framework regions mainly assume the form of a β-sheet higher-order structure, and the CDRs form connected loops and, in some cases, form parts of the β-sheet structure. Thus, the framework regions act to form a scaffold provided for the positioning of the CDRs in the correct orientation by non-covalent intermolecular interactions. The antigen-binding domain formed by the correctly positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its associated epitope. The amino acids that make up the CDRs and the framework regions can each be readily identified by one of ordinary skill in the art for any given heavy or light chain variable region because they have been previously defined (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)).

[0027] "Specifically binds" or "has specificity for" generally means that an antibody binds to an epitope via its antigen-binding domain and that the binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope more readily than it binds to a random, unrelated epitope via its antigen-binding domain. The term "specificity" is used herein to quantify the relative affinity by which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B", or it may be said that antibody "A" binds to epitope "C" with higher specificity than to the related epitope "D".

[0028] As used herein, the terms "treating" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (alleviate) undesirable physiological changes or disorders such as the progression of cancer. Beneficial clinical outcomes or desired clinical outcomes include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, remission or temporary suppression of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to survival that would be predicted in the absence of treatment. Those in need of treatment include those already having a condition or disorder, those having a tendency to have a condition or disorder, or those in whom a condition or disorder should be prevented.

[0029] As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal" mean any subject, particularly a mammalian subject, for which diagnosis, prognosis prediction or treatment is desired. Mammalian subjects include humans, domestic animals, agricultural animals and zoo, sports or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, beef cattle, dairy cows, etc.

[0030] As used herein, phrases such as "in a patient in need of treatment" or "a subject in need of treatment" include subjects such as mammalian subjects that would benefit from administration of an antibody or composition of the present disclosure used, for example, for detection, for diagnostic procedures, and / or for treatment.

[0031] Combined treatment It has been discovered herein that an antibody-drug conjugate based on LM-001 was able to synergistically inhibit tumor growth with an anti-PD-1 antibody. For example, 18 days after treatment, the anti-PD-1 antibody (LM-PD1) alone at 5 mg / kg achieved a tumor growth inhibition (TGI) rate of 10.52% (Table 3); ADC LM-002 alone was able to achieve TGI rates of 10.30% (2.5 mg / kg) and 24.45% (5 mg / kg) at two different doses, respectively.

[0032] When these two agents were used in combination, their TGI rates were 48.65% (2.5 mg / kg of LM-PD1 and 2.5 mg / kg of LM-002) and 42.63% (2.5 mg / kg of LM-PD1 and 5 mg / kg of LM-002). Both of these TGI rates were higher than the sum of their individual TGI rates, demonstrating synergy. Also importantly, the two different doses of LM-002 produced a significant anti-tumor effect as single agents (10.30% vs. 24.45%), but there was no such difference for the combination. Therefore, this result demonstrates that the combination with the anti-PD-1 antibody reduced the requirements for LM-002.

[0033] Interestingly, the combination was also tested in human patients with gastric cancer (GC) or gastroesophageal junction cancer (GJC). Again, the combination therapy showed significant efficacy, resulting in complete responses in at least one patient and partial responses in the rest. Surprisingly, when combined with the same dose (240 mg) of the anti-human PD-1 antibody toripalimab, the combination with the lower dose of LM-002 (1.6 mg / kg) showed better efficacy than the combination with the higher dose (2.0 mg / kg).

[0034] Accordingly, one embodiment of the present disclosure provides a method for treating cancer in a patient in need of cancer treatment. In some embodiments, the method involves administering to the patient an antibody-drug conjugate (ADC) comprising an inhibitor of PD-1 or an inhibitor of PD-L1, and a drug moiety covalently attached to an anti-Claudin 18.2 antibody.

[0035] In one embodiment, there is also provided an inhibitor of PD-1 or an inhibitor of PD-L1 for use in treating cancer in a patient, wherein the patient is further treated with an ADC comprising a drug moiety covalently attached to an anti-Claudin 18.2 antibody. There is also provided an ADC comprising a drug moiety covalently attached to an anti-Claudin 18.2 antibody for use in treating cancer in a patient, wherein the patient is further treated with an inhibitor of PD-1 or an inhibitor of PD-L1. Yet another embodiment provides an inhibitor of PD-1 or an inhibitor of PD-L1, and an ADC comprising a drug moiety covalently attached to an anti-Claudin 18.2 antibody for use in treating cancer.

[0036] In some embodiments, the anti-Claudin 18.2 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of SEQ ID NOs: 3-8. In some embodiments, the anti-Claudin 18.2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 1 and a VL comprising the amino acid of SEQ ID NO: 2.

[0037] As demonstrated, the anti-Claudin 18.2 antibody LM-001 has an excellent ability to induce receptor-mediated antibody internalization, even when compared to the clinical candidate IMAB362 (claudiximab). The significant increase (which is intended) in the ability of the antibodies of the present disclosure to induce receptor-mediated antibody internalization may be due to how these antibodies bind to the Claudin 18.2 protein. Amino acid residues on the Claudin 18.2 protein that are important for binding to LM-001 include amino acid residues (e.g., W30, L49, W50, C53, C63, and R80) that are important for stabilizing the conformation of the extracellular loop. W30, L49, and W50 are part of the W-LW-C-C consensus motif that helps to stabilize the conformation of loop 1. C53 and C63 form a disulfide bond between beta-strands. R80 may be important for maintaining the interaction between parallel Claudin 18.2 molecules on the cell surface or for stabilizing the conformation of loop 1.

[0038] Also important for antibody binding are the residues N45, Y46, G48, V54, R55, E56, S58, F60, E62, Y169, and G172. Of these, N45, Y46, G48, V54, R55, E56, S58, F60, and E62 are located within the β3 strand or through C63 to the β4 strand. This region consisting of residues 45-63 (NYQGLWRSCVRESSGFTEC) of SEQ ID NO: 9 is referred to herein as the "β3-β4 loop", which is part of the first extracellular loop (loop 1) of Claudin 18.2. In contrast, Y169 and G172 are part of the β5 strand of the second extracellular loop (loop 2) (residues 169-172 of SEQ ID NO: 3; YTFG).

[0039] A large increase in activity by the antibodies of the present disclosure to induce receptor-mediated antibody internalization (which is intended) is due to their ability to bind to residues in both the β3-β4 loop and the β5 strand. In this context, known anti-Claudin 18.2 antibodies are thought to bind to only one of the loops. [Table 5]

[0040] In one embodiment, the ADC comprises LM-001 covalently attached to a drug moiety. The drug moiety may be a reactive group that reacts with a conjugation point in the antibody or may be modified to include it. For example, the drug moiety can be attached by alkylation (e.g., at the epsilon amino group of lysine or at the N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification between hydroxyl and carboxyl groups, amidation at amino or carboxyl groups, and conjugation to thiols.

[0041] In some embodiments, the number p of conjugated drug moieties per antibody molecule ranges from 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 on average. In some embodiments, p ranges from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3 on average. In other embodiments, p is 1, 2, 3, 4, 5, 6, 7 or 8 on average. In some embodiments, p ranges from about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2 on average. In some embodiments, p ranges from about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4 or about 2 to about 3 on average. In some embodiments, p ranges from 1 to 6, 2 to 5, or 3 to 4.

[0042] For example, if the chemical activation of a protein results in the formation of a free thiol group, the protein may be conjugated with a sulfhydryl-reactive agent. In one aspect, the agent is substantially specific for the free thiol group. Examples of such agents include maleimide, haloacetamide (e.g., iodo, bromo or chloro), haloester (e.g., iodo, bromo or chloro), halomethyl ketone (e.g., iodo, bromo or chloro), benzyl halide (e.g., iodide, bromide or chloride), vinyl sulfone and pyridylthio.

[0043] The drug can be linked to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. Cleavable linkers are typically sensitive to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases such as lysosomal proteases or endosomal proteases. In an exemplary embodiment, the linker can be a dipeptide linker, such as valine-citrulline (val-cit or VC), phenylalanine-lysine (phe-lys) linker, or maleimidocaproic acid-valine-citrulline (citruline)-p-aminobenzyl oxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (smcc). Sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (smcc) conjugation occurs via a maleimide group that reacts with sulfhydryl (thiol, -SH), and its sulfosuccinimidyl ester is reactive towards primary amines (as seen at lysine and the protein or peptide N-terminus). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Additional suitable cleavable linkers include disulfide linkers. For example, a linker such as the mc linker may be covalently bound to the antibody to such an extent that the antibody has to be degraded intracellularly to release the drug.

[0044] The linker can contain groups for conjugation to the antibody. For example, the linker can contain amino, hydroxyl, carboxyl, or sulfhydryl reactive groups (such as maleimide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethyl ketone (e.g., iodo, bromo, or chloro), benzyl halide (e.g., iodide, bromide, or chloride), vinyl sulfone, and pyridylthio).

[0045] In some embodiments, the drug moiety is a cytotoxic agent or a cell division inhibitor, an immunosuppressive agent, a radioisotope, a toxin, etc. The conjugate can be used to inhibit the growth of tumor cells or cancer cells, to cause apoptosis in tumor or cancer cells, or to treat cancer in a patient. Thus, the conjugate can be used in various situations for the treatment of cancer in animals. The conjugate can be used to deliver the drug to tumor cells or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds to or associates with cancer cells that express Claudin 18.2, and the conjugate and / or the drug can be taken up into the interior of the tumor cells or cancer cells through receptor-mediated endocytosis.

[0046] Once inside the cell, one or more specific peptide sequences (e.g., in the linker) within the conjugate are hydrolytically cleaved by one or more tumor cell or cancer cell-associated proteases, releasing the drug. The released drug then freely moves within the cell, inducing cytotoxic or cell division inhibitory activity, or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and then the drug either penetrates the cell or acts on the cell surface.

[0047] Examples of drug moieties or payloads include DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide), and are selected from the group consisting of. DM1 is a derivative of the tubulin inhibitor maytansine, while MMAD, MMAE, and MMAF are auristatin derivatives. In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE.In some embodiments, the drug moiety is a maytansinoid or an auristatin.

[0048] In some embodiments, antibody LM001 is conjugated to MMAE via a Val-Cit linker. In some embodiments, antibody LM001 is conjugated to MMAF via a Val-Cit linker. In some embodiments, each antibody is conjugated to 1-20, 2-20, 2-8, 2-6, 2-5, or 3-4 Val-Cit-MMAE or Val-Cit-MMAF.

[0049] An inhibitor of PD-1 or an inhibitor of PD-L1 refers to any therapeutic agent, small molecule or antibody that can inhibit the activity of the PD-1 and PDL1 immune checkpoint proteins present on the surface of cells. PD-1 and PD-L1 inhibitors act to inhibit the association of programmed death-ligand 1 (PD-L1) with its receptor, programmed cell death protein 1 (PD-1). The interaction of these cell surface proteins is involved in the suppression of the immune system, occurs after infection, limits the death of bystander host cells, and prevents autoimmune diseases.

[0050] In some embodiments, the PD-1 inhibitor or the PD-L1 inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. Examples of PD-1 inhibitors include, but are not limited to, atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189. Examples of PD-L1 inhibitors include, but are not limited to, pembrolizumab, nivolumab, semipramab, dostarlimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012, AMP-224, and AMP-514.

[0051] Pembrolizumab (previously MK-3475 or lambrolizumab, Keytruda) was developed by Merck and was first approved by the Food and Drug Administration in 2014 for the treatment of melanoma. It has subsequently been approved for metastatic non-small cell lung cancer and squamous cell carcinoma of the head and neck. In 2017, it became the first immunotherapy drug approved for use based on tumor genetic mutations rather than tumor location.

[0052] Nivolumab (Opdivo) was developed by Bristol-Myers Squibb and was first approved by the FDA in 2014 for the treatment of melanoma. It has subsequently been approved for squamous cell carcinoma of the lung, renal cell carcinoma, and Hodgkin lymphoma.

[0053] Cemiplimab (Libtayo) was developed by Regeneron Pharmaceuticals and was first approved by the FDA in 2018 for the treatment of cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC that is not a candidate for curative surgery or curative radiation.

[0054] Dostarlimab (Jemperli) was developed by GlaxoSmithKline and was first approved by the FDA in April 2021 for the treatment of mismatch repair deficient (dMMR) recurrent or advanced endometrial cancer. On August 17, 2021, the FDA granted priority approval for the treatment of mismatch repair deficient (dMMR) recurrent or advanced solid tumors.

[0055] JTX-4014 is being developed by Jounce Therapeutics and as of 2020, it has entered Phase I clinical trials. Spartalizumab (PDR001) is a PD-1 inhibitor developed by Novartis to treat both solid tumors and lymphomas, which as of 2018 has entered Phase III clinical trials. Camrelizumab (SHR1210) is an anti-PD-1 monoclonal antibody introduced by Jiangsu HengRui Medicine Co., Ltd, which has currently received conditional approval in China for the treatment of relapsed or refractory classical Hodgkin lymphoma.

[0056] Sintilimab (IBI308) is a human anti-PD-1 antibody developed by Innovent and Eli Lilly for patients with non-small cell lung cancer (NSCLC).Tislelizumab (BGB-A317) is a humanized IgG4 anti-PD-1 monoclonal antibody in important Phase 3 and Phase 2 clinical trials in solid tumors and blood cancers.

[0057] Toripalimab (JS 001) is a humanized IgG4 monoclonal antibody against PD-1 under clinical trials. INCMGA00012 (MGA012) is a humanized IgG4 monoclonal antibody developed by Incyte and MacroGenics. AMP-224 is being developed by AstraZeneca / MedImmune and GlaxoSmithKline. AMP-514 (MEDI0680) is being developed by AstraZeneca.

[0058] Atezolizumab (Tecentriq) is a fully humanized IgG1 (immunoglobulin 1) antibody developed by Roche Genentech. In 2016, the FDA approved atezolizumab for urothelial cancer and non-small cell lung cancer.

[0059] Avelumab (Bavencio) is a fully human IgG1 antibody developed by Merck Serono and Pfizer. Avelumab has been approved by the FDA for the treatment of metastatic Merkel cell carcinoma. It failed in a Phase III clinical trial for gastric cancer.

[0060] Durvalumab (Imfinzi) is a fully human IgG1 antibody developed by AstraZeneca. Durvalumab has been approved by the FDA for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer after chemoradiation.

[0061] Furthermore, several PD-L1 inhibitors are in the experimental stage of development. KN035 is a PD-L1 antibody with a subcutaneous formulation currently under clinical evaluation in the United States, China, and Japan. CK-301 by Checkpoint Therapeutics is a PD-L1 inhibitor developed by Dana Farber and is currently in a Phase 3 trial for NSCLC.

[0062] AUNP12 is a 29-mer peptide as the first peptidic PD-1 / PD-L1 inhibitor developed by Aurigene and Laboratoires Pierre Fabre, which is being evaluated in clinical trials following promising in vitro results.

[0063] CA-170, discovered by Aurigene / Curis as a PD-L1 and VISTA antagonist, has been shown as a potent small molecule inhibitor in vitro. This compound is currently under a Phase I clinical trial for mesothelioma patients.

[0064] BMS-986189 is a macrocyclic peptide discovered by Bristol-Myers Squibb, and its pharmacokinetics, safety, and tolerability are currently being studied in healthy subjects.

[0065] In certain embodiments, the PD-1 inhibitor is toripalimab. In another embodiment, the PD-1 inhibitor is nivolumab.

[0066] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of cancers of the stomach, pancreas, esophagus, ovary, and lung.

[0067] Additional diseases or disorders related to increased cell survival that can be treated, prevented, diagnosed, and / or prognosed using the antibodies or their variants or derivatives of the present disclosure include, but are not limited to, progression and / or metastasis of malignant tumors, including leukemia (acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, and, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma, and other solid tumors such as sarcomas and carcinomas related to disorders.

[0068] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors including the specific antibody, their variants or derivatives being used, the patient's age, weight, general health, sex and diet, as well as the time of administration, rate of excretion, drug combinations, and the severity of the particular disease being treated. The determination of such factors by a healthcare provider is within the scope of ordinary skill in the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the properties of the compound being used, the severity of the disease and the desired effect. The amount used can be determined by the principles of pharmacology and pharmacokinetics well known in the art.

[0069] The methods of administration of the antibody, fragment, or antibody-drug conjugate include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transnasal, epidural, and oral routes. The antigen-binding polypeptide or composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelial or inner layer of the skin mucosa (such as oral mucosa, rectal and intestinal mucosa, etc.), and may also be administered together with other bioactive agents. Accordingly, the pharmaceutical composition containing the antigen-binding polypeptide of the present disclosure may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (such as by powder, ointment, droplet or transdermal patch), intraorally, or as an oral or nasal spray.

[0070] As used herein, the term "parenteral" refers to a mode of administration that includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injections and infusions.

[0071] In some embodiments, the PD-L1 inhibitor or PD-1 inhibitor is administered once every week, every two weeks, or once every 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In some embodiments, the ADC is administered once every week, every two weeks, or once every 3, 4, 5, 6, 7, 8, 9 or 10 weeks.

[0072] In some embodiments, the PD-L1 inhibitor or PD-1 inhibitor and the ADC are administered on the same day. In some embodiments, the PD-L1 inhibitor or PD-1 inhibitor and the ADC are administered during the same medical visit. In some embodiments, the PD-L1 inhibitor or PD-1 inhibitor and the ADC are administered separately. In some embodiments, the PD-L1 inhibitor or PD-1 inhibitor and the ADC are administered in a combination formulation.

[0073] In some embodiments, the PD-L1 inhibitor or PD-1 inhibitor (e.g., toripalimab) is administered at a dosage of about 0.1 mg / kg to 10 mg / kg. In some embodiments, each dosage is at least 0.1 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, 1.2 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.8 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, or 5 mg / kg. In some embodiments, each dosage is 0.2 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, 1.2 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.8 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 5 mg / kg or 10 mg / kg or less. In some embodiments, each dosage is 0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg, 1.6 mg / kg, 2.0 mg / kg or 2.4 mg / kg.

[0074] In some embodiments, the ADC is administered at a dosage of about 0.1 mg / kg to 10 mg / kg. In some embodiments, each dosage is at least 0.1 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, 1.2 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.8 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, or 5 mg / kg. In some embodiments, each dosage is 0.2 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, 1.2 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.8 mg / kg, 2 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.8 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 5 mg / kg or 10 mg / kg or less. In some embodiments, each dosage is 0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg, 1.6 mg / kg, 2.0 mg / kg or 2.4 mg / kg.

[0075] As observed in Example 4 and Figure 4, higher dosages of the ADC do not necessarily result in higher efficacy. In fact, among the GC and GEJ patients tested, a lower dosage of the ADC (1.6 mg / kg) resulted in a higher response (even a complete response) than a higher dosage (2.0 mg / kg). In some embodiments, therefore, each dosage of the ADC is 1.0 mg / kg to 2.0 mg / kg. In some embodiments, each dosage of the ADC is 1.2 mg / kg to 1.9 mg / kg. In some embodiments, each dosage of the ADC is 1.2 mg / kg to 1.8 mg / kg. In some embodiments, each dosage of the ADC is 1.4 mg / kg to 1.8 mg / kg. In some embodiments, each dosage of the ADC is 1.5 mg / kg to 1.7 mg / kg. In some embodiments, each dosage of the ADC is 1.55 mg / kg to 1.65 mg / kg. In some embodiments, the cancer being treated is a gastrointestinal cancer, such as GC or GEJ.

[0076] In certain embodiments, a method for treating cancer in a patient in need of cancer treatment, the method comprising administering to the patient an inhibitor of PD-1 or an inhibitor of PD-L1 and a vcMMAE-conjugated anti-Claudin 18.2 antibody (ADC), wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 1 and a VL comprising the amino acid of SEQ ID NO: 2; each antibody is conjugated to 2 to 6 units of vcMMAE; the ADC is administered at a dose of 1.2 mg / kg to 1.8 mg / kg; and the cancer is a gastrointestinal cancer.

[0077] In some embodiments, the gastrointestinal cancer is gastric cancer (GC) or gastroesophageal junction cancer (GJC). In some embodiments, the inhibitor of PD-1 or the inhibitor of PD-L1 is toripalimab. In some embodiments, the inhibitor of PD-1 or the inhibitor of PD-L1 is nivolumab.

[0078] Composition The present disclosure also provides a pharmaceutical composition. Such a composition comprises an effective amount of an antibody, fragment or antibody-drug conjugate, and a pharmaceutically acceptable carrier.

[0079] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory authority of the federal or state government or listed for use in animals, more specifically in humans, in the United States Pharmacopeia or other generally recognized pharmacopeias. Further, a "pharmaceutically acceptable carrier" is generally a non-toxic, solid, semi-solid or liquid diluent, excipient, encapsulating material, or any type of formulation adjuvant.

[0080] The term "carrier" refers to a diluent, adjuvant, additive, or vehicle that is administered together with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids such as water and oils of petroleum, animal, vegetable or synthetic origin, including peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline, as well as aqueous solutions of dextrose and glycerol, can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical additives include starch, glucose, lactose, sucrose, gelatin, malt, corn, wheat flour, talc, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Optionally, the composition can also contain trace amounts of a wetting or emulsifying agent, or a pH buffer such as acetate, citrate or phosphate. Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic pressure such as sodium chloride or dextrose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. The composition can be formulated as a suppository using traditional binders and carriers such as triglycerides. Oral formulations can contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E. W. Martin, which is incorporated herein by reference. Such compositions preferably contain a therapeutically effective amount of the antigen-binding polypeptide in purified form, together with a suitable amount of carrier, to provide a form for proper administration to a patient. The formulation must be suitable for the mode of administration. Parenteral preparations can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[0081] In certain embodiments, the composition is formulated according to defined procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile, isotonic, aqueous buffer. If necessary, the composition may also contain solubilizing agents and local anesthetics such as lignocaine to relieve pain at the injection site. Generally, the components are supplied either separately or mixed together in unit dosage forms, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of the active agent. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided to mix the components prior to administration.

Example

[0082] (Example 1) Preparation and testing of an anti-Claudin 18.2 antibody-drug conjugate This example prepared an antibody-drug conjugate (ADC) having the antibody 4F11E2 HC N55E-LC S32A ("LM-001") disclosed in International Publication No. WO 2019 / 219089. LM-001 has the following sequences.

Table 1

[0083] The residues W30, N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62, C63, R80, Y169, and G172 of the Claudin 18.2 protein were determined to be involved in the binding to this antibody. W30 is part of a cluster of residues in the first half of the first extracellular domain of the Claudin 18.2 protein. N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62, and C63 are in a second cluster of residues within the same extracellular domain. On the other hand, Y169 and G172 are located in or near the second extracellular domain. The Claudin 18.2 protein has a short intracellular N-terminus, a large first extracellular loop (Loop 1, or ECS1) containing a consensus W-LW-C-C motif, a shorter second extracellular loop (Loop 2, or ECS2), and four transmembrane segments in the intracellular C-terminal tail. Loop 1 contains four β-strands β1, β2, β3, and β4, and the loop contains one β-strand β5.

[0084] To prepare the ADC (designated LM-002), the antibody was mixed with approximately 3-fold TCEP and stirred at 37 °C for 2 hours. The reaction system was quickly added dropwise in an amount more than 8-fold with respect to VC-MMAE, incubated on ice for 1 hour, and 20-fold excess cysteine was added with respect to the drug linker to terminate the reaction. Finally, the ADC product was purified by elution through Sephadex G-25 equilibrated in PBS and concentrated by centrifugal ultrafiltration. The conjugate was filtered through a 0.2 μm filter under aseptic conditions and stored at -80 °C for analysis and testing. The drug-antibody ratio was analyzed by UV spectrophotometry, the monomer content was analyzed by SEC-HPLC, and the free drug content was analyzed by RP-HPLC. The drug-antibody ratio (DAR) of vcMMAE-conjugated LM-001 was 3.76.

[0085] (Example 2) Combination of Antibody-Drug Conjugate and PD-1 Antibody In this example, the antitumor efficacy of individual LM-002 and anti-PD1 antibody (LM-PD1) as single agents, and LM-002 in combination with anti-PD1 antibody, was evaluated in a C57BL / 6 mouse model with LLC1 (Lewis lung carcinoma) cells expressing human CLDN18.2.

[0086] Table 2 below shows the experimental design.

Table 2

[0087] Cell culture LLC1 / H_CLDN18.2 tumor cells were maintained in vitro in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C in an atmosphere of 5% CO2 in air. The tumor cells were passaged regularly, twice a week. Growing cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0088] Tumor inoculation Each mouse was subcutaneously inoculated in the right axilla (lateral) with 0.1 mL of LLC1 / H_CLDN18.2 tumor cells (0.3×10 6 cells) in PBS for tumor development. When the tumor volume reached approximately 50 mm 3 (which is 10 days after cell inoculation), the animals were randomly grouped according to tumor volume. LM-002 and LM-PD1 dosing was initiated for the efficacy study. The administration of the test substances and the number of animals in each group are shown in Table 2.

[0089] Observation All procedures regarding the handling, care and treatment of animals in the study were performed in accordance with the guidelines approved by B&K's Institutional Animal Care and Use Committee (IACUC) and Medsyin, which follows the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). At the time of routine monitoring, animals were checked daily for tumor growth and any effect of treatment on normal behavior such as mobility, food and water consumption (by visual inspection only), weight gain / loss (weight was measured every 2 days or twice a week), eye / hair matting, and any other abnormal effects listed in the protocol. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

[0090] Tumor Measurements and Endpoints The primary endpoint is to see if tumor growth can be delayed or if the mice can be cured. The experimental endpoints are: 1) the mean tumor volume in the control group reaches 2000 mm 3 or 2) mice were treated for 3 weeks. The experiment was terminated when one of the conditions was met.

[0091] Tumor size was measured three times a week in two dimensions using calipers (number 105688, sylvac Dantsin) and the volume was calculated according to the formula: V = 0.5a × b 2 where a and b were the long and short diameters of the tumor, respectively, to calculate the tumor size in mm 3 The tumor size was then used to calculate the T / C(%) value. T / C(%) was calculated using the formula: T / C%=(Ti-T0) / (Vi-V0)×100 (Ti is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the first day of treatment, Vi is the mean tumor volume of the vehicle control group on the same day as Ti, and V0 is the mean tumor volume of the vehicle group on the first day of treatment).

[0092] TGI (Tumor Growth Inhibition) was calculated for each group using the formula: TGI (%) = [100 - T / C]%. According to the guiding principles of anti-tumor drugs in the Drug Testing Center, t / C% ≤ 40% is considered that the drug is effective. According to the NIH Guidelines, TGI% ≥ 58% is considered that the drug is effective.

[0093] Statistical Analysis Summary statistics including the mean and standard error of the mean (SEM) were provided for the tumor volume of each group at each time point (detailed in Table 6 of Section 10.1). Statistical analysis of the differences in tumor volume between groups was performed on the data obtained at the treatment time points. The P-value was calculated by Microsoft Excel 2013.

[0094] A t-test was performed to compare the tumor volumes between groups. A value of p < 0.05 was considered statistically significant.

[0095] Results The study was completed on the 18th day according to the experimental requirements. Detailed information on the tumor volumes in different groups is shown below.

[0096] Body Weight The body weights of LLC1 / H_CLDN18.2 tumor-bearing mice were regularly monitored as an indirect measure of toxicity. After treatment, the body weights of the mice in each group did not decrease significantly. The detailed changes in the body weights and the relative changes in body weights of LLC1 / H_CLDN18.2 tumor-bearing mice after administration are shown in Figures 1 and 2. The tumor growth curves of LLC1 / H_CLDN18.2 tumor-bearing mice after administration are shown in Figure 3. The data are also shown in Tables 3 - 4. [Table 3] [Table 4]

[0097] This example was initiated to test the antitumor efficacy of antibodies (LM-002 and LM-PD1) in the LLC1 / H_CLDN18.2 model. The changes in the body weight and relative body weight of mice on the 18th day after administration are shown in Figures 1-2. During the administration period, mice in all groups had no significant weight loss, and mice in the administration groups had good tolerance.

[0098] After administration, the average tumor volumes of the PBS group, LM-PD1 (5 mg / kg) group, LM-002 (2.5 mg / kg) group, LM-002 (5 mg / kg) group, LM-002 (2.5 mg / kg) + LM-PD1 (5 mg / kg) group, and LM-002 (5 mg / kg) + LM-PD1 (5 mg / kg) group were 2399.71 mm 3 、2153.09 mm 3 、2158.26 mm 3 、1826.44 mm 3 、1259.28 mm 3 、1400.26 mm 3 respectively (Figure 3). The T / C values of the LM-PD1 (5 mg / kg) group, LM-002 (2.5 mg / kg) group, LM-002 (5 mg / kg) group, LM-002 (2.5 mg / kg) + LM-PD1 (5 mg / kg) group, and LM-002 (5 mg / kg) + LM-PD1 (5 mg / kg) group were 89.48%, 89.70%, 75.55%, 51.35%, and 57.37% respectively. The TGI values were 10.52%, 10.3%, 25.45%, 48.65%, and 42.63% respectively (Table 3). T / C% < 40% and TGI% > 58% indicate that the treatment is effective.

[0099] The p-values in the treatment group compared with the vehicle group were shown in Table 4. At the end of the experiment (day 18), compared with the PBS group, the 5 mg / kg LM-002 and combination groups showed significant antitumor activity, and compared with the LM-PD1 monotherapy group, the combination of LM-002 (2.5 mg / kg) + LM-PD1 (5 mg / kg) showed significantly higher antitumor activity (P = 0.0277). Interestingly, the lower dose LM-002 (2.5 mg / kg) + LM-PD1 (5 mg / kg) group had an antitumor effect no inferior to that of the higher dose LM-002 (5 mg / kg) + LM-PD1 (5 mg / kg) group.

[0100] (Example 3) Clinical trial protocol A combination of the anti-Claudin 18.2 antibody-drug conjugate LM-002 and the anti-PD-1 antibody tripalimab is tested in a clinical trial.

[0101] Tripalimab is administered IV at 240 mg every three weeks. LM-002 is administered intravenously on day 1 every three weeks. For Phase I, there are two pre-specified dose levels of LM-002 (0.2 mg / kg, 0.4 mg / kg). For Phase Ib, there are six pre-specified dose levels of LM-002 (0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg, 1.6 mg / kg, 2.0 mg / kg and 2.4 mg / kg) in combination with a fixed dose of tripalimab (240 mg).

[0102] Patients with advanced solid tumors are enrolled in this trial.

[0103] (Example 4) Clinical trial results A combination of the anti-Claudin 18.2 antibody-drug conjugate LM-002 (1.6 mg / kg and 2.0 mg / kg) and the anti-PD-1 antibody tripalimab (240 mg) was tested in a pilot human study. The patients treated had gastric cancer (GC) or gastroesophageal junction cancer (GEJ).

[0104] Eight patients were enrolled in this trial, and three patients were evaluable for baseline tumor assessment with at least one post-baseline assessment at the target lesion. Their responses are presented in the chart of Figure 4, which was very positive. One patient had a complete response (CR), and two patients had a partial response (PR). Interestingly, the combination with the lower dose (1.6 mg / kg) of LM-002 was significantly more effective than the combination with the higher LM-002 dose (2.0 mg / kg).

[0105] The present disclosure is not intended to be limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any composition or method that is a functional equivalent is within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and changes can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they come within the scope of the appended claims and their equivalents.

[0106] All publications and patent applications mentioned in this specification are hereby incorporated by reference in the same manner as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A composition for treating cancer in patients requiring cancer treatment, wherein the composition comprises a PD-1 inhibitor or a PD-L1 inhibitor, and an antibody-drug conjugate (ADC) comprising a drug portion covalently attached to an anti-claudin 18.2 antibody containing the amino acid sequences of SEQ ID NOs: VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively.

2. The composition according to claim 1, wherein the anti-claudin 18.2 antibody comprises VH containing the amino acid sequence of SEQ ID NO: 1 and VL containing the amino acids of SEQ ID NO:

2.

3. The composition according to claim 1 or 2, wherein the anti-claudin 18.2 antibody is attached to 2 to 10 of the drug portions, preferably 2 to 6 of the drug portions, and more preferably 3 to 4 of the drug portions.

4. The composition according to claim 1, wherein the drug portion is a cytotoxic agent or a cell division inhibitor.

5. The composition according to claim 4, wherein the drug portion is a mytansinoid or auristatin.

6. The composition according to claim 5, wherein the drug portion comprises DM1 or DM4.

7. The composition according to claim 6, wherein the drug portion comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

8. The composition according to claim 1, wherein the drug portion is attached to the antibody or a fragment thereof via a linker.

9. The composition according to claim 8, wherein the linker is hydrolyzable under acidic conditions.

10. The composition according to claim 8, wherein the linker comprises valine-citrulline.

11. The composition according to claim 1, wherein the ADC comprises 2 to 6 MMAEs or MMAFs conjugated to the anti-claudin 18.2 antibody via valine-citrulline.

12. The composition according to claim 1, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189.

13. The composition according to claim 1, wherein the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, dostallimab, JTX-4014, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, INCMGA00012, AMP-224, and AMP-514.

14. The composition according to claim 1, wherein the inhibitor is tripalimab.

15. The composition according to claim 1, wherein the inhibitor and the ADC are each administered once every 1 to 6 weeks.

16. The composition according to claim 1, wherein the ADC is administered in a dose of 0.1 mg / kg to 10 mg / kg.

17. The composition according to claim 16, wherein the ADC is administered in doses of 0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg, 1.6 mg / kg, 2.0 mg / kg, or 2.4 mg / kg.

18. The composition according to claim 16, wherein the ADC is administered in a dose of 1.2 mg / kg to 1.8 mg / kg.

19. The composition according to claim 1, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, digestive tract cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

20. A composition for treating cancer in patients requiring cancer treatment, wherein the composition comprises a PD-1 inhibitor or a PD-L1 inhibitor and a vcMMAE-conjugated anti-claudin 18.2 antibody (ADC), The antibody comprises VH containing the amino acid sequence of SEQ ID NO: 1 and VL containing the amino acids of SEQ ID NO: 2; Each antibody is conjugated with 2 to 6 units of the aforementioned vcMMAE; The aforementioned ADC is administered at a dose of 1.2 mg / kg to 1.8 mg / kg; The aforementioned cancer is a gastrointestinal cancer. composition.

21. The composition according to claim 20, wherein the digestive tract cancer is gastric cancer (GC) or gastroesophageal junction cancer (GJC).

22. The composition according to claim 20 or 21, wherein the PD-1 inhibitor or the PD-L1 inhibitor is tripalimab.

23. The composition according to claim 20 or 21, wherein the PD-1 inhibitor or the PD-L1 inhibitor is nivolumab.

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