Cancer treatment using PD-1 or PD-l1 inhibitors and cladin 18.2-targeting antibody drug conjugates

Abstract

In one embodiment, a combination therapy is provided that includes an inhibitor of PD-1 or PD-L1 and an antibody drug conjugate that targets claudin 18.2.

Description

Background Art

[0001] Tight junction proteins (claudins) are a class of proteins that are important components of tight cell junctions. They establish a paracellular barrier that controls the flow of molecules between cells. These proteins have an N-terminus and a C-terminus in the cytoplasm. Different claudins are expressed in different tissues, and changes in their functions are associated with the formation of cancer in the corresponding tissues. Claudin-1 is expressed in colon 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 highly selective cell lineage marker. The expression of Claudin 18.2 in normal tissues is strictly restricted to differentiated epithelial cells of the gastric mucosa, but is absent in the gastric stem cell zone. Claudin 18.2 is retained in malignant transformation and is expressed in a significant proportion of primary gastric cancers and their metastases. Frequent ectopic activation of claudin 18.2 has also been found in pancreatic, esophageal, ovarian, and lung tumors. These data suggest that CLDN18.2 has a highly restricted expression pattern in normal tissues and is frequently ectopically activated in a variety of human cancers. Summary of the invention

[0003] It is found herein that LM-001-based antibody-drug conjugates are able to synergistically inhibit tumor growth with anti-PD-1 antibodies. This is believed 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 those that are important for stabilizing the conformation of the extracellular loop (e.g., W30, L49, W50, C53, C63, and R80). More importantly, residues speculated to be involved in binding antibodies 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 located in β5 of the second extracellular loop. In contrast, known anti-claudin 18.2 antibodies are believed to bind only to 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, comprising administering to the patient an inhibitor of PD-1 or PD-L1 and an antibody drug conjugate (ADC), the antibody drug conjugate comprising a drug moiety covalently linked to an anti-claudin 18.2 antibody, the anti-claudin 18.2 antibody comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise 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 linked to 2 to 10 drug moieties, preferably 2 to 6 drug moieties.

[0006] In some embodiments, the drug moiety is a cytotoxic agent or a cytostatic agent. 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 connected to the antibody or fragment thereof via a linker. In some embodiments, the linker is hydrolyzable under acidic conditions. In some embodiments, the linker comprises valine-citrulline.

[0008] In some embodiments, the ADC comprises 2 to 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 PD-1 inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cemiplimab, 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 to 6 weeks. In some embodiments, the ADC is administered at a dose of 0.1 mg / kg to 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, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer.

[0012] In a specific embodiment, a method for treating cancer in a patient in need thereof is provided, comprising administering to the patient an inhibitor of PD-1 or 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 2-6 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.

[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 PD-L1 is toripalimab. In some embodiments, the inhibitor of PD-1 or PD-L1 is nivolumab. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The weight changes of tumor-bearing mice in different groups are shown. The data points represent the mean weight of the groups. The error bars represent the standard error of the mean (SEM).

[0015] Figure 2 The relative changes in body weight of tumor-bearing mice in different groups are shown. The data points represent the mean relative changes in body weight of the groups. The error bars represent the standard error of the mean (SEM).

[0016] Figure 3 Tumor growth curves after administration of test substances to LLC1 / H_CLDN18.2 tumor-bearing mice are shown. Data points represent group means, and error bars represent standard error of the mean (SEM).

[0017] Figure 4 Responses of three human patients treated with LM-002 (1.6 mg / kg or 2.0 mg / kg) and toripalimab (240 mg) are shown. DETAILED DESCRIPTION

[0018] definition

[0019] It should be noted that the term "a" or "an" entity refers to one or more (one or more) of the entity; for example, "an antibody" should be understood to mean one or more antibodies. Thus, the terms "a" (or "a"), "one (kind) or more (kind)" and "at least one (kind)" can be used interchangeably herein.

[0020] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein or peptide that contains a molecule that contains at least a portion of an immunoglobulin molecule that has biological activity that binds to an antigen. Such examples include, but are not limited to, a heavy or light chain complementary determining region (CDR) or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein.

[0021] As used herein, the term "antibody fragment" or "antigen binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isomers, 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.

[0022] The term antibody encompasses a wide variety of biochemically distinguishable polypeptides. It will be appreciated by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γl-γ4). The nature of the chain determines the "class" of the antibody, IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to confer functional specificity. In view of the present disclosure, modified versions of each of these classes and isotypes are easily discernible to those skilled in the art, and are therefore within the scope of the present disclosure. All immunoglobulin classes are obviously within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises 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 connected by disulfide bonds in a "Y" configuration, with the light chains bracketing the heavy chains, starting at the mouth of the "Y" and extending across the variable region.

[0023] The antibodies, antigen-binding polypeptides, variants or derivatives of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized 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 comprising VK or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules of the present disclosure can be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0024] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be combined with either a κ or λ light chain. Typically, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, and the "tails" of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0025] The light chain and the heavy chain are divided into structural homology regions and functional homology regions. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains (VK) of the light chain part and the variable domains (VH) of the heavy chain part determine the recognition and specificity of the antigen. On the contrary, the constant domains (CK) of the light chain and the constant domains (CH1, CH2 or CH3) of the heavy chain confer important biological properties, such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more away from the antigen binding site or amino terminus of the antibody. The N-terminal part is the variable region, and the C-terminal part is the constant region; the CH3 and CK domains actually contain the carboxyl termini of the heavy chain and the light chain, respectively.

[0026] As mentioned above, variable region allows antibody selective recognition and specific binding to epitopes on antigens. That is, the subset combination of the VK domain and VH domain or complementary determining region (CDR) of the antibody forms a variable region that defines a three-dimensional antigen binding site. This four-level antibody structure forms an antigen binding site present at each arm terminal of 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) on each of the VH and VK chains. In some cases, such as some immunoglobulin molecules derived from camelid species or based on camelid immunoglobulin engineering, a complete immunoglobulin molecule can be composed only of heavy chains, without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0027] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form an antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain (called "framework" regions) show less intermolecular variability. The framework regions primarily adopt a β-pleated structure, and the CDRs form loops that connect the β-pleated structure and, in some cases, form part of the β-pleated structure. Thus, the framework regions serve to form a scaffold that positions the CDRs in the correct orientation through interchain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface promotes non-covalent binding of an antibody to its cognate epitope. One of ordinary skill in the art can readily identify the amino acids comprising the CDRs and framework regions, respectively, for any given heavy or light chain variable region because they have been precisely defined (see "Sequences of Proteins of Immunological Interest", Kabat, E. et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)).

[0028] "Specific binding" or "specific for..." generally refers to the binding of an antibody to an epitope through its antigen binding domain, and the binding requires a certain complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope through its antigen binding domain more easily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody binds to a certain epitope. For example, antibody "A" can be said to have a higher specificity for a given epitope than antibody "B", or it can be said that antibody "A" binds to epitope "C" with a higher specificity than to a related epitope "D".

[0029] As used herein, the term "treatment" refers to therapeutic treatment and preventive or defensive measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or disorders, such as the progress of cancer. Beneficial or desired clinical results include but are not limited to detectable or undetectable symptom relief, disease degree mitigation, stable disease state (i.e., no deterioration), disease progression delay or slowing, disease state improvement or mitigation, and relief (whether partial or complete). "Treatment" can also mean that the survival period is extended compared to the expected survival period if not receiving treatment. Those subjects who need to be treated include those subjects who have already suffered from a disease or disorder and those subjects who are prone to a disease or disorder or those subjects who want to prevent a disease or disorder.

[0030] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo animals, sports animals or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0031] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects, who would benefit by administering an antibody or composition of the disclosure, such as for detection, for diagnostic procedures, and / or for therapy.

[0032] Combination therapy

[0033] This paper found that the antibody drug conjugate based on LM-001 can synergistically inhibit tumor growth with anti-PD-1 antibody. For example, 18 days after treatment, only anti-PD-1 antibody (LM-PD1) achieved a tumor growth inhibition (TGI) rate of 10.52% at 5 mg / kg (Table 3); only ADC LM-002 was able to achieve TGI rates of 10.30% (2.5 mg / kg) and 24.45% (5 mg / kg) at two different doses.

[0034] When these two agents were used in combination, their TGI rates were 48.65% (2.5 mg / kg LM-PD1 and 2.5 mg / kg LM-002) and 42.63% (2.5 mg / kg LM-PD1 and 5 mg / kg LM-002), respectively. Both TGI rates were greater than the sum of their individual TGI rates, indicating a synergistic effect. It is also important to note that while two different doses of LM-002 resulted in significant anti-tumor effects as single agents (10.30% vs. 24.45%), there was no such difference when combined. Therefore, this result suggests that the combination with anti-PD-1 antibodies reduces the need for LM-002.

[0035] Also interestingly, the combination was tested in human patients with gastric cancer (GC) or gastroesophageal junction cancer (GJC). Again, the combination therapy showed significant efficacy, resulting in a complete remission in at least one patient and partial remission in the remaining patients. Surprisingly, when combined with the same dose (240 mg) of toripalimab (an anti-human PD-1 antibody), the combination with a lower dose of LM-002 (1.6 mg / kg) showed better efficacy than the combination with a higher dose (2.0 mg / kg).

[0036] Therefore, one embodiment of the present disclosure provides a method for treating cancer in a patient in need thereof. In some embodiments, the method requires administering to the patient an inhibitor of PD-1 or PD-L1 and an antibody drug conjugate (ADC) comprising a drug moiety covalently linked to an anti-claudin 18.2 antibody.

[0037] In one embodiment, an inhibitor of PD-1 or PD-L1 for treating cancer in a patient is also provided, wherein the patient is further treated with an ADC comprising a drug moiety covalently linked to an anti-claudin 18.2 antibody. An ADC for treating cancer in a patient is also provided, wherein the ADC comprises a drug moiety covalently linked to an anti-claudin 18.2 antibody, wherein the patient is further treated with an inhibitor of PD-1 or PD-L1. Another embodiment provides an inhibitor of PD-1 or PD-L1 for treating cancer and an ADC comprising a drug moiety covalently linked to an anti-claudin 18.2 antibody.

[0038] In some embodiments, the anti-claudin 18.2 antibody comprises VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and VL CDR3, which respectively comprise the amino acid sequences of SEQ ID NO: 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 sequence of SEQ ID NO: 2.

[0039] As demonstrated, even compared to the clinical candidate drug IMAB362 (claudiximab), the anti-claudin18.2 antibody LM-001 has an excellent ability to induce receptor-mediated antibody internalization. It is speculated that the ability of the antibodies disclosed herein to induce receptor-mediated antibody internalization is greatly increased, which can be attributed to how these antibodies bind to the claudin18.2 protein. The amino acid residues on the claudin 18.2 protein that are important for binding to LM-001 include those that are important for stabilizing the conformation of the extracellular loop (e.g., W30, L49, W50, C53, C63, and R80). W30, L49, and W50 are part of the W-LW-CC consensus motif and help stabilize the conformation of loop 1. C53 and C63 form a disulfide bond between β chains. R80 may be important for maintaining interactions between parallel claudin 18.2 molecules on the cell surface or stabilizing the conformation of loop 1.

[0040] Residues N45, Y46, G48, V54, R55, E56, S58, F60, E62, Y169 and G172 are also important for antibody binding. Among them, N45, Y46, G48, V54, R55, E56, S58, F60 and E62 are located in the β3 strand or positioned by C63 in the β4 strand. This region consists of residues 45-63 of SEQ ID NO:9 (NYQGLWRSCVRESSGFTEC), referred to herein as the "β3 to 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 (residues 169-172 of SEQ ID NO:3; YTFG) of the second extracellular loop (loop 2).

[0041] It is speculated that the antibodies disclosed herein have greatly increased activity in inducing receptor-mediated antibody internalization due to their ability to bind to residues in the β3 to β4 loops and the β5 strand. In this context, it is thought that known anti-claudin 18.2 antibodies bind to only one of the loops.

[0042] Human claudin 18.2 sequence

[0043]

[0044] In one embodiment, the ADC includes LM-001 covalently linked to a drug moiety. The drug moiety can be a group that reacts with a coupling point on an antibody or is modified to include a group that reacts with a coupling point on an antibody. For example, the drug moiety can be linked by alkylation (e.g., at ε-aminolysine or the N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification between hydroxyl and carboxyl groups, amidation of amino or carboxyl groups, and conjugation to sulfhydryl groups.

[0045] In some embodiments, the number of drug moieties conjugated per antibody molecule, p, ranges from 1 to 8 on average; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. 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 ranges from 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. 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. In some embodiments, p ranges from 1 to 6, 2 to 5, or 3 to 4.

[0046] For example, when chemical activation of a protein results in the formation of a free sulfhydryl group, the protein can be coupled with a sulfhydryl reactive agent. On the one hand, the reagent is a reagent that is substantially specific for free sulfhydryl groups. Such reagents include, for example, maleimide, haloacetamide (e.g., iodo, bromo or chloro), halogenated esters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g., iodo, bromo or chloro), benzyl halides (e.g., iodide, bromide or chloride), vinyl sulfones and pyridine sulfhydryls.

[0047] The drug can be connected to the antibody or fragment through a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are generally easy to cleave 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 maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl, mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate, smcc). Sulfo-smcc coupling occurs through a maleimide group that reacts with sulfhydryl (thiol, -SH), while its Sulfo-NHS ester is reactive with primary amines (such as those found at lysine and the N-terminus of proteins or peptides). Another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Other suitable cleavable linkers include disulfide linkers. The linker can be covalently bound to the antibody to the extent that the antibody must be degraded intracellularly to release the drug, such as mc linkers, etc.

[0048] The linker can include a group for connection to the antibody. For example, the linker can include an amino, hydroxyl, carboxyl or sulfhydryl reactive group (e.g., maleimide, haloacetamide (e.g., iodo, bromo or chloro), haloester (e.g., iodo, bromo or chloro), halomethylketone (e.g., iodo, bromo or chloro), benzyl halide (e.g., iodide, bromide or chloride), vinyl sulfone and pyridinethio).

[0049] In some embodiments, the drug moiety is a cytotoxic agent or a cytostatic agent, an immunosuppressant, a radioisotope, a toxin, etc. The conjugate can be used to inhibit the proliferation of tumor cells or cancer cells, cause apoptosis of tumors or cancer cells, or be used to treat cancer in patients. The conjugate can be used in various environments to treat animal cancer accordingly. The conjugate can be used to deliver drugs to tumor cells or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds or associates with cancer cells expressing claudin 18.2, and the conjugate and / or the drug can be absorbed into tumor cells or cancer cells through receptor-mediated endocytosis.

[0050] Once inside the cell, one or more specific peptide sequences in the conjugate (e.g., in the linker) are hydrolyzed and cleaved by one or more tumor cell or cancer cell-related proteases, resulting in drug release. The released drug then migrates freely within the cell and induces cytotoxicity or cell inhibition or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug subsequently penetrates the cell or acts on the cell surface.

[0051] Examples of drug moieties or payloads are selected from the group consisting of: DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethyl auristatin-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-monomethyl auristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valinyl-L-valinyl-(3R,4 S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropionyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethyl auristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L- Valyl-N5-(aminocarbonyl)-L-ornithine]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). DM1 is a derivative of the microtubule 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.

[0052] In some embodiments, antibody LM001 is coupled to MMAE via a Val-Cit linker. In some embodiments, antibody LM001 is coupled to MMAF via a Val-Cit linker. In some embodiments, each antibody is coupled to 1-20, 2-20, 2-8, 2-6, 2-5, or 3-4 Val-Cit-MMAE or Val-Cit-MMAF.

[0053] Inhibitors of PD-1 or PD-L1 are any therapeutic agent, small molecule, or antibody that is able to inhibit the activity of the PD-1 and PDL1 immune checkpoint proteins present on the cell surface. PD-1 and PD-L1 inhibitors work by inhibiting the binding of programmed death ligand 1 (PD-L1) to 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 and occurs after infection to limit bystander host cell killing and prevent autoimmune disease.

[0054] In some embodiments, the PD-1 or PD-L1 inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. Example PD-1 inhibitors include, but are not limited to, atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189. Example PD-L1 inhibitors include, but are not limited to, pembrolizumab, nivolumab, cemiplizumab, dostalimumab, JTX-4014, spartalizumab, carrelizumab, sindilimab, tislelizumab, toripalizumab, INCMGA00012, AMP-224, and AMP-514.

[0055] Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda) was developed by Merck and first approved by the U.S. Food and Drug Administration in 2014 for the treatment of melanoma. It was later approved for metastatic non-small cell lung cancer and head and neck squamous cell carcinoma. In 2017, it became the first immunotherapy drug approved for use based on tumor genetic mutations rather than tumor location.

[0056] Nivolumab (Opdivo) was developed by Bristol-Myers Squibb and first received FDA approval in 2014 for the treatment of melanoma. It was later approved for squamous cell lung cancer, renal cell carcinoma, and Hodgkin lymphoma.

[0057] Cemiplimab (Libtayo), developed by Regeneron Pharmaceuticals, was first approved by the FDA in 2018 for the treatment of cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC that is not amenable to curative surgery or curative radiation therapy.

[0058] Dostarlimab (Jemperli), developed by GlaxoSmithKline, was first approved by the FDA in April 2021 for the treatment of recurrent or advanced endometrial cancer with mismatch repair deficiency (dMMR). On August 17, 2021, the FDA granted accelerated approval for the treatment of recurrent or advanced solid tumors with mismatch repair deficiency (dMMR).

[0059] JTX-4014 is being developed by Jounce Therapeutics and is in Phase I trials as of 2020. Spartalizumab (PDR001) is a PD-1 inhibitor developed by Novartis for the treatment of solid tumors and lymphomas and has entered Phase III trials as of 2018. Camrelizumab (SHR1210) is an anti-PD-1 monoclonal antibody launched by Jiangsu Hengrui Medicine Co., Ltd., which recently received conditional approval in China for the treatment of relapsed or refractory classical Hodgkin lymphoma.

[0060] 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 pivotal Phase 3 and Phase 2 clinical trials for solid tumors and hematological cancers.

[0061] Toripalimab (JS 001) is a humanized IgG4 monoclonal antibody against PD-1 in clinical development. INCMGA00012 (MGA012) is a humanized IgG4 monoclonal antibody being developed by Incyte and MacroGenics. AMP-224 is being developed by AstraZeneca / MedImmune and GlaxoSmithKline. AMP-514 (MEDI0680) is being developed by AstraZeneca.

[0062] Atezolizumab (Tecentriq) is a fully humanized IgG1 (immunoglobulin 1) antibody developed by Roche Genentech. In 2016, the FDA approved atezolizumab for urothelial carcinoma and non-small cell lung cancer.

[0063] Avelumab (Bavencio) is a fully human IgG1 antibody developed by Merck Serono and Pfizer. Avelumab is approved by the FDA for the treatment of metastatic Merkel cell carcinoma. It failed in a Phase III clinical trial in gastric cancer.

[0064] Durvalumab (Imfinzi) is a fully human IgG1 antibody developed by AstraZeneca. Durvalumab is approved by the FDA for the treatment of urothelial carcinoma after chemoradiation and unresectable non-small cell lung cancer.

[0065] There are also some PD-L1 inhibitors in the experimental stage of development. KN035 is a PD-L1 antibody in the form of a subcutaneous formulation and is currently undergoing clinical evaluation in the United States, China and Japan. Checkpoint Therapeutics' CK-301 is a PD-L1 inhibitor developed by Dana Farber and is currently undergoing a phase 3 trial for NSCLC.

[0066] AUNP12 is a 29-mer peptide that is the first peptic PD-1 / PD-L1 inhibitor developed by Aurigene and Laboratoires Pierre Fabre and is currently being evaluated in clinical trials after promising in vitro results.

[0067] CA-170, discovered by Aurigene / Curis, is a PD-L1 and VISTA antagonist that has been shown to be a potent in vitro small molecule inhibitor. The compound is currently in Phase I clinical trials for mesothelioma patients.

[0068] BMS-986189 is a macrocyclic peptide discovered by Bristol-Myers Squibb and its pharmacokinetics, safety and tolerability are currently being studied in healthy subjects.

[0069] In a specific embodiment, the PD-1 inhibitor is toripalimab. In another embodiment, the PD-1 inhibitor is nivolumab.

[0070] 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 gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer.

[0071] Other diseases or conditions associated with improved cell survival that can be treated, prevented, diagnosed and / or prognosed with the antibodies of the present disclosure or variants or derivatives thereof include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythrocytic leukemias)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic The invention relates to polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovialoma, mesothelioma, Ewing's tumor, and polycythemia vera. 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, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0072] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative thereof used, the patient's age, weight, general health, sex, diet, and the time of administration, excretion rate, drug combination, and severity of the specific disease being treated. The judgment of these factors by the medical caregiver is within the ordinary skill of the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles known in the art.

[0073] The method of administration of antibody, fragment or antibody drug conjugate includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptide or composition can be applied by any convenient approach, such as by infusion or bolus injection, by absorption through epithelium or mucocutaneous lining (such as oral mucosa, rectum and intestinal mucosa, etc.), and can be applied together with other bioactive agents. Therefore, the pharmaceutical composition containing antigen-binding polypeptide of the present disclosure can be oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, local (such as by powder, ointment, drops or transdermal patch), oral administration, or as oral or nasal spray.

[0074] The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0075] In some embodiments, the PD-L1 or PD-1 inhibitor is administered once every 1 week, every 2 weeks, or every 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, the ADC is administered once every 1 week, every 2 weeks, or every 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks.

[0076] In some embodiments, the PD-L1 or PD-1 inhibitor and the ADC are administered on the same day. In some embodiments, the PD-L1 or PD-1 inhibitor and the ADC are administered during the same medical visit. In some embodiments, the PD-L1 or PD-1 inhibitor and the ADC are administered separately. In some embodiments, the PD-L1 or PD-1 inhibitor and the ADC are administered as a combined formulation.

[0077] In some embodiments, the PD-L1 or PD-1 inhibitor (e.g., Teplizumab) is administered at a dose of about 0.1 mg / kg to 10 mg / kg. In some embodiments, each dose 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 dose is no more than 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. In some embodiments, each dose 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.

[0078] In some embodiments, the ADC is administered at a dose of about 0.1 mg / kg to 10 mg / kg. In some embodiments, each dose 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 dose is no more than 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. In some embodiments, each dose 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.

[0079] As in Example 4 and Figure 4As shown, higher doses of ADC do not necessarily lead to higher efficacy. In fact, in the GC and GEJ patients tested, a lower dose of ADC (1.6 mg / kg) produced a higher (even complete) response than a higher dose (2.0 mg / kg). Therefore, in some embodiments, each dose of ADC is 1.0 mg / kg to 2.0 mg / kg. In some embodiments, each dose of ADC is 1.2 mg / kg to 1.9 mg / kg. In some embodiments, each dose of ADC is 1.2 mg / kg to 1.8 mg / kg. In some embodiments, each dose of ADC is 1.4 mg / kg to 1.8 mg / kg. In some embodiments, each dose of ADC is 1.5 mg / kg to 1.7 mg / kg. In some embodiments, each dose of ADC is 1.55 mg / kg to 1.65 mg / kg. In some embodiments, the cancer being treated is gastrointestinal cancer, such as GC or GEJ.

[0080] In a specific embodiment, a method for treating cancer in a patient in need thereof is provided, comprising administering to the patient an inhibitor of PD-1 or 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 2-6 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.

[0081] In some embodiments, the gastrointestinal cancer is gastric cancer (GC) or gastroesophageal junction cancer (GJC). In some embodiments, the inhibitor of PD-1 or PD-L1 is toripalimab. In some embodiments, the inhibitor of PD-1 or PD-L1 is nivolumab.

[0082] Composition

[0083] The present disclosure also provides pharmaceutical compositions, which contain an effective amount of an antibody, fragment or antibody-drug conjugate, and an acceptable carrier.

[0084] In a specific embodiment, the term "pharmaceutically acceptable" refers to approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, especially humans. Further, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0085] The term "carrier" refers to a diluent, adjuvant, auxiliary material or excipient used together with the therapeutic agent. Such a pharmaceutical carrier can be a sterile liquid, such as water and oil, including those oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer, such as acetate, citrate or phosphate. Antimicrobial agents such as benzyl alcohol or methyl parabens are also contemplated; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity such as sodium chloride or dextrose. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated into a suppository together with conventional binders and carriers (e.g., triglycerides). Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such a composition will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in a purified form, together with an appropriate amount of a carrier to provide a form for appropriate administration to a patient. The formulation should be compatible with the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0086] In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to the human being according to conventional procedures. Usually, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to alleviate the pain at the injection site. Usually, the composition is provided separately or mixed with a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate provided in a closed container, such as an ampoule or a sachet indicating the amount of the active agent. In the case where the composition is used by infusion, it can be distributed with an infusion bottle equipped with sterile pharmaceutical grade water or saline. In the case where the composition is used by injection, an ampoule of sterile water for injection or saline can be provided so that the composition can be mixed before use.

[0087] Example

[0088] Example 1: Preparation and testing of anti-claudin 18.2 antibody drug conjugates

[0089] In this example, an antibody drug conjugate (ADC) was prepared, wherein the antibody was the antibody 4F11E2 HC N55E-LC S32A ("LM-001") disclosed in WO2019219089. LM-001 has the following sequence.

[0090] Table 1 LM-001 sequence

[0091]

[0092]

[0093] Residues W30, N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62, C63, R80, Y169 and G172 of claudin 18.2 protein have been identified as being involved in binding to the antibody. W30 is part of a cluster of residues in the first half of the first extracellular domain of claudin 18.2 protein. N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62 and C63 are located 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. Claudin 18.2 has four transmembrane segments, a short intracellular N-terminus, a large first extracellular loop (loop 1 or ECS1) containing a consensus W-LW-CC motif, a shorter second extracellular loop (loop 2 or ECS2), and an intracellular C-terminal tail. Loop 1 includes four β strands β1, β2, β3, and β4, and the loop includes one β strand β5.

[0094] To prepare the ADC (called LM-002), the antibody was mixed with about three times of TCEP and stirred at 37°C for 2 hours. Eight times of VC-MMAE was quickly added to the reaction system and incubated on ice for 1 hour, and 20 times excess cysteine ​​was added relative to the drug linker to quench the reaction. Finally, the ADC product was purified by elution with Sephadex G-25 balanced with PBS and concentrated by centrifugal ultrafiltration. The conjugate was filtered through a 0.2 μm filter under sterile conditions and stored at -80°C for analysis and testing. The drug-antibody ratio was analyzed by UV spectroscopy, 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-coupled LM-001 was 3.76.

[0095] Example 2: Combination of Antibody Drug Conjugate and PD-1 Antibody

[0096] This example evaluates the anti-tumor efficacy of LM-002 and anti-PD1 antibody (LM-PD1) alone as single agents and in combination with anti-PD1 antibody in a C57BL / 6 mouse model with LLC1 (Lewis lung carcinoma) cells expressing human CLDN18.2.

[0097] Table 2 below shows the experimental design.

[0098] Table 2 Groups and treatments in efficacy studies

[0099]

[0100] a. Number of animals in each group

[0101] b. Dosing volume: Adjust the dosing volume based on body weight, 5 μL / g or 10 μL / g. If the body weight loss is >15%, the treatment regimen can be adjusted.

[0102] c.QW: once a week; biw: twice a week

[0103] Cell culture

[0104] 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 and 5% CO2 in air. Tumor cells were routinely subcultured twice a week. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0105] Tumor inoculation

[0106] Each mouse was subcutaneously inoculated with LLC1 / H_CLDN18.2 tumor cells (0.3×10 6 ) for tumor development. When the tumor volume reaches about 50mm 3 At 10 days after cell inoculation, the animals were randomly divided into groups according to the tumor volume. LM-002 and LM-PD1 were started to be administered for efficacy study. The test substance administration and the number of animals in each group are shown in Table 2.

[0107] Observations

[0108] All procedures related to animal handling, care, and treatment in the study were performed in accordance with guidelines approved by the B&K and Medsyin Institutional Animal Care and Use Committees (IACUC) and followed the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). During routine monitoring, animals were examined daily for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption (by observation only), weight gain / loss (body weight was measured every two days or twice a week), eye / hair matting, and any other abnormal effects specified in the protocol. Deaths and observed clinical signs were recorded based on the number of animals in each subset.

[0109] Tumor Measurements and End Points

[0110] The primary endpoint was to observe whether tumor growth could be delayed or whether the mice could be cured. 3 The experiment was terminated when one of the conditions was met.

[0111] Tumor size was measured three times a week in two dimensions using a caliper (No. 105688, sylvac Dantsin) and expressed in mm using the following formula: 3 The unit represents volume: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively. The tumor size is then used to calculate the T / C (%) value. T / C (%) is calculated using the following formula: T / C% = (Ti-T0) / (Vi-V0) × 100, Ti is the average tumor volume of the treatment group on a given day, T0 is the average tumor volume of the treatment group on the first day of treatment, Vi is the average tumor volume of the vehicle control group on the same day as Ti, and V0 is the average tumor volume of the vehicle group on the first day of treatment.

[0112] The TGI (tumor growth inhibition) of each group was calculated using the following formula: TGI (%) = [100-T / C]%. According to the guidelines of the Center for Drug Review for Anti-tumor Drugs, t / C% ≤ 40% is considered effective. According to the NIH guidelines, TGI% ≥ 58% is considered effective.

[0113] Statistical analysis

[0114] Summary statistics of tumor volume at each time point for each group are provided, including the mean and standard error of the mean (SEM) (see Table 6 in Section 10.1 for details). Statistical analysis of differences in tumor volume between groups was performed on the data obtained at the treatment time point. P values ​​were calculated by Microsoft Excel 2013.

[0115] T-test was performed to compare tumor volumes among the groups. A p value < 0.05 was considered statistically significant.

[0116] result

[0117] According to the experimental requirements, the study was completed on day 18. Detailed information on tumor volumes in different groups is shown below.

[0118] weight

[0119] The body weight of LLC1 / H_CLDN18.2 tumor-bearing mice was monitored regularly as an indirect measure of toxicity. After treatment, the body weight of mice in each group did not decrease significantly. The detailed changes in body weight and relative changes in body weight of LLC1 / H_CLDN18.2 tumor-bearing mice after administration are shown in Figure 2. Figure 1 and Figure 2 The tumor growth curve of LLC1 / H_CLDN18.2 tumor-bearing mice after administration is shown in Figure 3 The data are shown in Table 3-4.

[0120] Table 3 T / C (%), TGI (%) after treatment (N=8)

[0121]

[0122] Table 4 P values ​​of different groups at different time points

[0123]

[0124]

[0125] This example aims to test the anti-tumor effect of antibodies (LM-002 and LM-PD1) in LLC1 / H_CLDN18.2 model. The weight change and relative weight change of mice on day 18 after administration are shown in Figure 2. Figure 1-2 During the administration period, mice in all groups did not show significant weight loss, and the mice in the administration groups were well tolerated.

[0126] 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 、2153.09mm 3 、2158.26mm 3 、1826.44mm 3 、1259.28mm 3 、1400.26mm3 ( Figure 3 ). The T / C of 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 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.

[0127] The p values ​​of the treatment groups compared to the vehicle group are shown in Table 4. At the end of the experiment (day 18), the 5 mg / kg LM-002 group and the combination group showed significant anti-tumor activity compared with the PBS group, and the combination of LM-002 (2.5 mg / kg) + LM-PD1 (5 mg / kg) showed significantly higher anti-tumor activity compared with the LM-PD1 monotherapy group (P = 0.0277). Interestingly, the lower dose LM-002 (2.5 mg / kg) + LM-PD1 (5 mg / kg) group had a comparable anti-tumor effect compared with the higher dose LM-002 (5 mg / kg) + LM-PD1 (5 mg / kg) group.

[0128] Example 3 Clinical trial plan

[0129] The combination of the anti-claudin 18.2 antibody-drug conjugate LM-002 and the anti-PD-1 antibody toripalimab will be tested in clinical trials.

[0130] Teplizumab will be administered as 240 mg intravenous injection once every 3 weeks. LM-002 will be administered intravenously on day 1 once every 3 weeks. For Phase I, there are 2 predefined dosing levels of LM-002 (0.2 mg / kg, 0.4 mg / kg). For Phase Ib, 6 predefined dosing 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) are combined with a fixed dose of Teplizumab (240 mg / kg).

[0131] Patients with advanced solid tumors were enrolled in this trial.

[0132] Example 4 Clinical trial results

[0133] The 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 toripalimab (240 mg) was tested in a pilot human trial. Patients treated had gastric cancer (GC) or gastroesophageal junction cancer (GEJ).

[0134] Eight patients were enrolled in this trial, of whom three had baseline tumor assessments and at least one post-baseline target lesion assessment. Figure 4 The results are 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).

[0135] ***

[0136] The scope of the present disclosure is not limited by the specific embodiments described, which are intended to be a single illustration of the various aspects of the present disclosure, and any functionally equivalent compositions or methods are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and changes may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and changes of the present disclosure as long as these modifications and changes fall within the scope of the appended claims and their equivalents.

[0137] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for treating cancer in a patient in need thereof, comprising administering to the patient an inhibitor of PD-1 or PD-L1 and an antibody drug conjugate (ADC), wherein the antibody drug conjugate comprises a drug portion covalently linked to an anti-claudin 18.2 antibody, wherein the anti-claudin 18.2 antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise the amino acid sequences of SEQ ID NOs: 3-8. 2 . The method of claim 1 , wherein 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 sequence of SEQ ID NO:

2. 3 .

3. The method according to claim 1 or 2, wherein the anti-claudin 18.2 antibody is linked to 2 to 10 drug moieties, preferably 2-6 drug moieties, more preferably 3-4 drug moieties.

4. The method of any preceding claim, wherein the drug moiety is a cytotoxic or cytostatic agent.

5. The method of claim 4, wherein the drug moiety is a maytansine or an auristatin.

6. The method of claim 5, wherein the drug moiety comprises DM1 or DM4.

7. The method of claim 6, wherein the drug moiety comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

8. The method according to any one of the preceding claims, wherein the drug moiety is linked to the antibody or fragment thereof via a linker.

9. The method of claim 8, wherein the linker is hydrolyzable under acidic conditions.

10. The method of claim 8, wherein the linker comprises valine-citrulline.

11. The method according to any one of the preceding claims, wherein the ADC comprises 2 to 6 MMAE or MMAF conjugated to an anti-claudin 18.2 antibody via valine-citrulline.

12. The method according to any one of the preceding claims, wherein the inhibitor of PD-L1 is selected from the group consisting of: atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170 and BMS-986189.

13. The method according to any of the preceding claims, wherein the inhibitor of PD-1 is selected from the group consisting of: pembrolizumab, nivolumab, cemiplizumab, dostalimab, JTX-4014, spartalizumab, carrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012, AMP-224 and AMP-514.

14. The method according to any one of the preceding claims, wherein the inhibitor is toripalimab.

15. The method according to any one of the preceding claims, wherein the inhibitor and the ADC are each administered once every 1 to 6 weeks.

16. The method according to any one of the preceding claims, wherein the ADC is administered at a dose of 0.1 mg / kg to 10 mg / kg.

17. The method of claim 16, wherein 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.

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

19. The method according to any of the preceding claims, 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, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer and thyroid cancer.

20. A method for treating a cancer patient in need thereof, comprising administering to the patient an inhibitor of PD-1 or 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 sequence of SEQ ID NO: 2; Each antibody was conjugated with 2–6 units of vcMMAE; The ADC is administered at a dose of 1.2 mg / kg to 1.8 mg / kg; The cancer is a gastrointestinal cancer.

21. The method of claim 20, wherein the gastrointestinal cancer is gastric cancer (GC) or gastroesophageal junction cancer (GJC).

22. The method of claim 20 or 21, wherein the inhibitor of PD-1 or PD-L1 is toripalimab.

23. The method of claim 20 or 21, wherein the inhibitor of PD-1 or PD-L1 is nivolumab.

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