Methods and compositions for predicting therapeutic efficacy of cancer therapy and cancer prognosis

Abstract

The present invention relates generally to methods and compositions for predicting therapeutic efficacy of cancer therapy and prognosis of cancer. Disclosed are markers associated with favorable and adverse outcomes in certain cancer treatments, respectively, and useful as prognostic markers for cancer. Methods relating to these markers are disclosed for predicting the effectiveness of cancer treatments and prognosing the clinical outcome of cancer patients.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201680021996.1 and invention name “Methods and compositions for predicting therapeutic efficacy and cancer prognosis of cancer treatment”. The original application is the PCT international application PCT / EP2016 / 058061 filed on April 13, 2016, which entered the Chinese national phase on October 13, 2017. Technical Field

[0002] The present invention generally relates to methods and compositions for predicting the therapeutic efficacy of cancer treatments and for prognosing cancer. The present invention discloses markers that are associated with favorable and unfavorable outcomes in certain cancer treatments, respectively, and can be used as prognostic markers for cancer. Methods involving these markers are disclosed for predicting the efficacy of cancer treatments and for prognosing the clinical outcomes of cancer patients. Background Art

[0003] Cancer of the stomach and esophagus (gastroesophageal; GE) has the highest unmet medical need among malignancies. Gastric cancer is the second leading cause of death worldwide. Despite the aggressiveness of established standard treatments associated with significant side effects, the incidence of esophageal cancer has risen in recent decades, and the overall five-year survival rate for GE cancer is 20% to 25%. There is a high medical need for patients with this cancer type, and innovative drugs are needed.

[0004] The tight junction molecule claudin 18 isotype 2 (CLDN18.2) is a cancer-associated splice variant of claudin 18 [Niimi, T., et al., Mol Cell Biol, 2001. 21(21): 7380-90; Tureci, O., et al., Gene, 2011. 481(2): 83-92]. CLDN18.2 is a 27.8 kDa transmembrane protein containing four transmembrane domains and two small extracellular loops (loop 1 is surrounded by hydrophobic regions 1 and 2; loop 2 is surrounded by hydrophobic regions 3 and 4). CLDN18.2 is a highly selective gastric lineage antigen that is expressed only on short-lived differentiated gastric epithelial cells and cannot be detected in any other normal human tissues. This antigen is ectopically expressed at significant levels in a variety of human cancers including gastroesophageal cancer and pancreatic cancer [Sahin, U., et al., Clin Cancer Res, 2008.14 (23): p. 7624-34]. CLDN18.2 protein is also frequently detected in lymph node metastasis and distant metastasis of gastric cancer. Since target downregulation by siRNA technology leads to inhibition of gastric cancer cell proliferation, CLDN18.2 appears to be involved in the proliferation of CLDN18.2-positive tumor cells.

[0005] IMAB362 is a chimeric monoclonal antibody of the IgG1 subtype against CLDN18.2. IMAB362 recognizes the first extracellular domain of CLDN18.2 with high affinity and specificity and does not bind to any other claudin family members including the closely related claudin 18 splice variant 1 (CLDN18.1).

[0006] In human xenografts expressing CLDN18.2, survival benefits and tumor regression have been observed in mice after administration of IMAB362. When administered intravenously in relevant animal species, toxicity in gastric tissue was not observed due to inaccessibility of the target epitope. However, the tumor target becomes accessible to IMAB362 during malignant transformation. IMAB362 has four independent and highly effective mechanisms of action: (i) antibody-dependent cellular cytotoxicity (ADCC); (ii) complement-dependent cytotoxicity (CDC); (iii) apoptosis induction induced by cross-linking of the target on the tumor surface; and (iv) direct inhibition of proliferation.

[0007] A previous Phase I trial has evaluated IMAB362 as a monotherapy at a single dose in patients with advanced gastroesophageal cancer. In this trial, five IMAB362 doses (33 mg / m 2 , 100mg / m 2 , 300mg / m 2 , 600mg / m 2 and 1000 mg / m 2 ) as a monotherapy. The study showed that a single administration of this antibody at up to 1000 mg / m 2 The drug was safe and well tolerated at doses of 300 mg / m 2 Group and 600mg / m 2 The best results regarding antitumor activity were obtained in the 300 mg / m 2 In two patients of the 2-group, the disease was controlled and they were assessed as non-CR, non-PD (CD=complete response; PD=progressive disease) because they had only non-target lesions. The duration of non-CR and non-PD was about two months and six weeks, respectively. The tumor marker levels of these three patients remained stable. 2 One patient in the group presented with stable disease (SD). The duration of SD was about 2 months.

[0008] Based on the highly potent mechanism of action of IMAB362 inducing cell killing, the survival benefit of mice bearing CLDN18.2-positive tumors treated with IMAB362, the absence of any indication of IMAB362-related toxicity, and the promising results of the Phase I trial, a Phase IIa study was initiated. This Phase IIa clinical trial was conducted to determine the safety, tolerability, and antitumor activity of repeated doses of IMAB362 in patients with metastatic, refractory, or recurrent disease of advanced gastric or lower esophagus adenocarcinoma demonstrated by histology.

[0009] In this Phase IIa trial, the investigational drug was administered to three cohorts that were recruited sequentially. The first cohort of three patients was given a lower dose level (300 mg / m 2 Body surface area) received repeated doses of IMAB362. The antibody was given as a 2-hour intravenous infusion. Since no indication of IMAB362-related toxicity was detected in the first cohort, the IMAB362 dose was increased to 600 mg / m in the second cohort (three patients). 2 In the third cohort, 19 patients were assigned the same dose (repeated administration of 600 mg / m 2 Body surface area). Patient samples from this group were analyzed for several accompanying analytes (i.e., ADCC, CDC, immunophenotyping, and genetic immune polymorphisms). All patients in all groups received repeated doses of IMAB362 (5 administrations) every two weeks at visits 2, 5, 6, 7, and 8.

[0010] The differences in responsiveness of antigen-positive tumors (overexpressing the target antigen to a similar degree) to intervention with therapeutic monoclonal antibodies (e.g., IMAB362) suggest that there are additional factors associated with treatment outcome. This requires careful selection of patients who may benefit from antibody therapy.

[0011] Therefore, it is necessary to develop a test to measure the suitability of a patient for antibody therapy. The present invention addresses this need by providing markers associated with favorable and unfavorable outcomes in antibody therapy, respectively. In addition, the present invention shows that these markers can be used as markers for prognosticating the clinical outcome of cancer patients.

[0012] The findings presented herein can be used to select appropriate treatments for cancer patients, and in particular to decide whether antibody therapy should be administered to cancer patients. Summary of the invention

[0013] The present invention provides methods for SNP (single-nucleotide polymorphism) genotyping, which are used, for example, to assess the likelihood that an individual will respond to therapeutic treatment for cancer, to select a treatment or prevention regimen (e.g., to decide whether to administer a particular therapeutic agent to an individual who has cancer or is at increased risk of developing cancer in the future), or to assess an individual's prognosis regarding disease severity and recovery.

[0014] The present invention is based on the following discovery: certain genotypes of SNPs are associated with the sensitivity / insensitivity of cancer to antibody therapy (e.g., CLDN18.2-positive cancer, in particular, IMAB362 therapy of CLDN18.2-positive gastroesophageal cancer). The present invention is also based on the following discovery: certain genotypes of SNPs are associated with the clinical outcome of cancer patients and can therefore be used to prognose cancer.

[0015] In one aspect, the invention relates to methods of evaluating:

[0016] (i) whether a cancer patient with a tumor that is positive for a tumor antigen is a responder to treatment with an antibody directed against said tumor antigen, and / or

[0017] (ii) whether the cancer patient (preferably a cancer patient with a tumor antigen-positive tumor) will experience progression-free survival,

[0018] The method comprises determining the genotype of one or more single nucleotide polymorphisms selected from the group consisting of FCGR2A rs1801274, MUC1 rs4072037, IL-10 rs1800896, DNMT3Ars1550117, SMAD4 rs12456284, EGF rs4444903, CDH1 rs16260, ERCC1 rs11615, and FCGR3Ars396991 in a sample obtained from the patient.

[0019] In one embodiment, the presence of the heterozygous FCGR2A rs1801274[CT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0020] In one embodiment, the presence of the homozygous FCGR2A rs1801274[TT] genotype and / or the homozygous FCGR2Ars1801274[CC] genotype indicates an increased risk that the cancer patient is not a responder to treatment with the antibody and / or an increased risk that the cancer patient does not experience progression-free survival.

[0021] In one embodiment, the presence of the homozygous MUCl rs4072037[AA] genotype indicates a reduced risk that the cancer patient will not be a responder to treatment with the antibody and / or a reduced risk that the cancer patient will not experience progression-free survival.

[0022] In one embodiment, the presence of the homozygous MUCl rs4072037[GG] genotype indicates an increased risk that the cancer patient will not be a responder to treatment with the antibody and / or an increased risk that the cancer patient will not experience progression-free survival.

[0023] In one embodiment, the presence of the homozygous IL-10 rs1800896[GG] genotype indicates a reduced risk that the cancer patient will not be a responder to treatment with the antibody and / or a reduced risk that the cancer patient will not experience progression-free survival.

[0024] In one embodiment, the presence of the heterozygous DNMT3A rs1550117[GA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0025] In one embodiment, the presence of the heterozygous SMAD4 rs12456284[GA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0026] In one embodiment, the presence of the homozygous EGF rs4444903[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0027] In one embodiment, the presence of the homozygous CDH1 rs16260[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0028] In one embodiment, the presence of the homozygous ERCC1 rs11615[TT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0029] In one embodiment, the presence of a heterozygous FCGR3A rs396991 [TG] genotype and / or a homozygous FCGR3Ars396991 [TT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0030] In one embodiment, the presence of the homozygous FCGR3A rs396991[GG] genotype indicates an increased risk that the cancer patient is not a responder to treatment with the antibody and / or an increased risk that the cancer patient does not experience progression-free survival.

[0031] In one embodiment, the tumor antigen is CLDN18.2 protein.

[0032] In one aspect, the invention relates to methods of evaluating:

[0033] (i) whether a cancer patient with a CLDN18.2-positive tumor is a responder to treatment with an antibody directed against the CLDN18.2 protein, and / or

[0034] (ii) whether the cancer patient (preferably a cancer patient with a CLDN18.2-positive tumor) will experience progression-free survival,

[0035] The method comprises determining the genotype of one or more single nucleotide polymorphisms selected from the group consisting of FCGR2A rs1801274, MUC1 rs4072037, IL-10 rs1800896, DNMT3Ars1550117, SMAD4 rs12456284, EGF rs4444903, CDH1 rs16260, ERCC1 rs11615, and FCGR3Ars396991 in a sample obtained from the patient.

[0036] In one embodiment, the presence of the heterozygous FCGR2A rs1801274[CT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0037] In one embodiment, the presence of the homozygous FCGR2A rs1801274[TT] genotype and / or the homozygous FCGR2Ars1801274[CC] genotype indicates an increased risk that the cancer patient is not a responder to treatment with the antibody and / or an increased risk that the cancer patient does not experience progression-free survival.

[0038] In one embodiment, the presence of the homozygous MUCl rs4072037[AA] genotype indicates a reduced risk that the cancer patient will not be a responder to treatment with the antibody and / or a reduced risk that the cancer patient will not experience progression-free survival.

[0039] In one embodiment, the presence of the homozygous MUCl rs4072037[GG] genotype indicates an increased risk that the cancer patient will not be a responder to treatment with the antibody and / or an increased risk that the cancer patient will not experience progression-free survival.

[0040] In one embodiment, the presence of the homozygous IL-10 rs1800896[GG] genotype indicates a reduced risk that the cancer patient will not be a responder to treatment with the antibody and / or a reduced risk that the cancer patient will not experience progression-free survival.

[0041] In one embodiment, the presence of the heterozygous DNMT3A rs1550117[GA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0042] In one embodiment, the presence of the heterozygous SMAD4 rs12456284[GA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0043] In one embodiment, the presence of the homozygous EGF rs4444903[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0044] In one embodiment, the presence of the homozygous CDH1 rs16260[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0045] In one embodiment, the presence of the homozygous ERCC1 rs11615[TT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0046] In one embodiment, the presence of a heterozygous FCGR3A rs396991 [TG] genotype and / or a homozygous FCGR3Ars396991 [TT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

[0047] In one embodiment, the presence of the homozygous FCGR3A rs396991[GG] genotype indicates an increased risk that the cancer patient is not a responder to treatment with the antibody and / or an increased risk that the cancer patient does not experience progression-free survival.

[0048] In one embodiment of all aspects of the invention, the antibody acts by recruiting the patient's immune system to destroy tumor cells. In one embodiment, the antibody acts by antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). In one embodiment, the antibody is a monoclonal antibody. In one embodiment of all aspects of the invention, the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 17 or 51 or a fragment thereof, and the light chain comprising the amino acid sequence shown in SEQ ID NO: 24 or a fragment thereof.

[0049] In one embodiment of all aspects of the invention non-responsiveness to treatment with said antibody comprises a relative decrease in one or more of: survival, progression-free survival, relapse-free survival, distant relapse-free survival and stable disease.

[0050] In one aspect, the invention relates to a method of treating a cancer patient, the method comprising:

[0051] a. assessing whether a cancer patient is a responder to treatment with an antibody by the method of the invention, and

[0052] b. (i) treating the cancer patient with the antibody if the risk of the patient not being a responder to treatment with the antibody is reduced; or (ii) not treating the cancer patient with the antibody and / or treating the cancer patient with a treatment regimen that includes a treatment different from treatment with the antibody if the risk of the patient not being a responder to treatment with the antibody is increased.

[0053] In one embodiment, the treatment regimen includes treatment that is independent of the patient's immune system. In one embodiment, the treatment regimen does not include treatment with antibodies that work by recruiting the patient's immune system to destroy tumor cells. In one embodiment, the treatment regimen includes surgery, chemotherapy, and / or radiation. In one embodiment, the treatment regimen includes treatment with a small molecule inhibitor of the tumor antigen and / or an antibody-drug conjugate in which the antibody is directed to the tumor antigen. In one embodiment, the antibody-drug conjugate is an antibody coupled to a radioactive portion, a chemotherapeutic portion, or a toxin portion. In one embodiment, the antibody-drug conjugate is an antibody coupled to a cell inhibitory or cytotoxic compound.

[0054] In one aspect, the present invention relates to a method for evaluating the clinical outcome of a cancer patient, the method comprising determining the genotype of one or more single nucleotide polymorphisms selected from the group consisting of FCGR2Ars1801274, MUC1 rs4072037, IL-10 rs1800896, DNMT3A rs1550117, SMAD4 rs12456284, EGF rs4444903, CDH1 rs16260, ERCC1 rs11615, and FCGR3A rs396991 in a sample obtained from the patient.

[0055] In one embodiment, the presence of the heterozygous FCGR2A rs1801274[CT] genotype indicates a reduced risk of a poor clinical outcome.

[0056] In one embodiment, the presence of a homozygous FCGR2A rs1801274[TT] genotype and / or a homozygous FCGR2Ars1801274[CC] genotype indicates an increased risk of a poor clinical outcome.

[0057] In one embodiment, the presence of the homozygous MUCl rs4072037[AA] genotype indicates a reduced risk of a poor clinical outcome.

[0058] In one embodiment, the presence of the homozygous MUCl rs4072037[GG] genotype indicates an increased risk of a poor clinical outcome.

[0059] In one embodiment, the presence of the homozygous IL-10 rs1800896[GG] genotype indicates a reduced risk of a poor clinical outcome.

[0060] In one embodiment, the presence of the heterozygous DNMT3A rs1550117[GA] genotype indicates a reduced risk of a poor clinical outcome.

[0061] In one embodiment, the presence of the heterozygous SMAD4 rs12456284[GA] genotype indicates a reduced risk of a poor clinical outcome.

[0062] In one embodiment, the presence of the homozygous EGF rs4444903[AA] genotype indicates a reduced risk of a poor clinical outcome.

[0063] In one embodiment, the presence of the homozygous CDH1 rs16260[AA] genotype indicates a reduced risk of a poor clinical outcome.

[0064] In one embodiment, the presence of the homozygous ERCC1 rs11615[TT] genotype indicates a reduced risk of a poor clinical outcome.

[0065] In one embodiment, the presence of a heterozygous FCGR3A rs396991[TG] genotype and / or a homozygous FCGR3Ars396991[TT] genotype indicates a reduced risk of a poor clinical outcome.

[0066] In one embodiment, the presence of the homozygous FCGR3A rs396991[GG] genotype indicates an increased risk of a poor clinical outcome.

[0067] In one embodiment, the clinical outcome of the cancer patient is assessed including the possibility of predicting one or more of the following: survival, progression-free survival, recurrence-free survival, distant recurrence-free survival, and stable disease. In one embodiment, a poor clinical outcome includes a relative reduction in one or more of the following: survival, progression-free survival, recurrence-free survival, distant recurrence-free survival, and stable disease.

[0068] In one embodiment, the patient has a tumor that is positive for a tumor antigen and is receiving treatment with an antibody directed against said tumor antigen.

[0069] In one embodiment of all aspects of the invention, the sample is a sample comprising DNA. In one embodiment, the DNA has been extracted from a body sample of a patient. In one embodiment, the DNA has been extracted from blood.

[0070] In one embodiment of all aspects of the invention, the tumor is a solid tumor. In one embodiment, the tumor is a gastroesophageal tumor. In one embodiment, the tumor is advanced gastric or lower esophageal adenocarcinoma. In one embodiment, the cancer is gastroesophageal cancer. In one embodiment, the cancer is advanced gastric or lower esophageal adenocarcinoma.

[0071] In another aspect, the present invention relates to a kit comprising means for determining the genotype of one or more single nucleotide polymorphisms selected from the following in a sample obtained from a patient: FCGR2A rs1801274, MUC1rs4072037, IL-10 rs1800896, DNMT3A rs1550117, SMAD4 rs12456284, EGF rs4444903, CDH1 rs16260, ERCC1 rs11615 and FCGR3A rs396991. In one embodiment, the kit can be used to perform the method of all aspects of the present invention. In one embodiment, the kit also comprises a data carrier. In a preferred embodiment, the data carrier is an electronic or non-electronic data carrier. In one embodiment, the data carrier comprises instructions for how to perform the method of all aspects of the present invention.

[0072] Other objects, advantages and features of the present invention will become apparent from the following detailed description when considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 : Single nucleotide polymorphisms with statistically significant genotype frequency shift between patients and controls (χ 2 Test, p<0.05).

[0074] The assignment of SNP-specific genotypes to the bar segments is shown. Pat: patient population, Co: control population.

[0075] Figure 2 : The relative frequency of the homozygous risk genotype for each patient relative to the number of SNP risk factors studied for each patient. Patients are sorted by increasing frequency of cumulative homozygous risk factors.

[0076] Figure 3 : Progression-free survival of PP patients differentiated by rs1801274 (FCGR2A) genotype (Kaplan-Meier curve).

[0077] Figure 4 : Progression-free survival of FAS patients differentiated by rs1801274 (FCGR2A) genotype (Kaplan-Meier curve).

[0078] Figure 5 : Progression-free survival of PP patients differentiated by rs1800896 (IL-10) genotype (Kaplan-Meier curve).

[0079] Figure 6 : Progression-free survival of FAS patients differentiated by rs1800896 (IL-10) genotype (Kaplan-Meier curve).

[0080] Figure 7 : Progression-free survival of FAS patients differentiated by rs1550117 (DNMT3A) genotype (Kaplan-Meier curve).

[0081] Figure 8 : Progression-free survival of PP patients differentiated by rs12456284 (SMAD4) genotype (Kaplan-Meier curve).

[0082] Fig. 9 : Progression-free survival of PP patients differentiated by rs4072037 (MUC1) genotype (Kaplan-Meier curve).

[0083] Fig.10: Progression-free survival of FAS patients differentiated by rs4072037 (MUC1) genotype (Kaplan-Meier curve).

[0084] Fig.11 : Progression-free survival of FAS patients differentiated by rs4444903 (EGF) genotype (Kaplan-Meier curve).

[0085] Fig.12 : Progression-free survival of FAS patients differentiated by rs16260 (CDH1) genotype (Kaplan-Meier curve).

[0086] Fig.13 : Progression-free survival of PP patients differentiated by rs11615 (ERCC1) genotype (Kaplan-Meier curve).

[0087] Fig.14 : Progression-free survival of PP patients differentiated by rs396991 (FCGR3A) genotype (Kaplan-Meier curve). DETAILED DESCRIPTION

[0088] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art are generally understood.

[0089] Hereinafter, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The embodiments and preferred embodiments described differently should not be construed as limiting the present invention to only the embodiments clearly described. This specification should be understood to support and include embodiments that combine the embodiments clearly described with any number of disclosed and / or preferred elements. In addition, unless otherwise indicated by the context, any arrangement and combination of all described elements in this application should be considered to be disclosed by the specification of the application.

[0090] Preferably, the terms used herein are as in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel; and H. ed., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0091] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques as explained in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd edition, J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0092] Throughout this specification and the appended claims, unless otherwise required by the context, the word "include / comprise" and variations should be understood to mean including the member, integer or step or member, integer or step group, but not excluding any other member, integer or step or member, integer or step group, although in some embodiments, such other members, integers or steps or members, integers or steps The group can be excluded, that is, the subject is to include the member, integer or step or member, integer or step group. Unless otherwise specified herein or obviously contradictory to the context, the nouns used in the context of describing the present invention (especially in the context of the claims) without quantifiers should be interpreted as covering one / kind and / or more / kind. The description of the value range herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise specified herein, each individual value is incorporated into this specification as if it were recorded separately herein. Unless otherwise specified herein or obviously contradictory to the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as" or "such as") provided herein is intended only to better illustrate the present invention and does not limit the scope of the present invention otherwise claimed. Language in this specification should not be construed as indicating any non-claimed element essential to the practice of the present invention.

[0093] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, guidelines, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention.

[0094] The inventors provide tests for measuring the suitability of patients for certain cancer treatments (particularly antibody treatments) and drawing conclusions about the prognosis of cancer patients. The results obtained using these tests enable physicians to decide on the appropriate treatment for cancer patients, and in particular decide whether antibody treatment should be administered to a particular cancer patient.

[0095] The term "single nucleotide polymorphism" or "SNP" refers to a DNA sequence variation that usually occurs within a population, in which a single nucleotide in a genome (or other consensus sequence) is different between members of a biological species or between paired chromosomes. SNPs can occur in the coding sequence of a gene, in the non-coding region of a gene, or in the intergenic region (the region between genes). Due to the degeneracy of the genetic code, SNPs in the coding sequence can but do not necessarily change the amino acid sequence of the protein produced. Therefore, there are two types of SNPs in the coding region, synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not in the protein coding region can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by this type of SNP is called eSNP (expression SNP), and can be upstream or downstream of the gene.

[0096] The genotype of a SNP can be determined using a variety of methods known in the art. Analytical methods for discovering new SNPs and detecting known SNPs include, for example, DNA sequencing, capillary electrophoresis, mass spectrometry, single-strand conformation polymorphism (SSCP), electrochemical analysis, denaturing HPLC and gel electrophoresis, restriction fragment length polymorphism, and hybridization analysis.

[0097] Methods for determining which nucleotide is present at a particular SNP position described herein (for either or both alleles) may be referred to by phrases such as "determining the genotype of a SNP" or "SNP genotyping." Thus, these phrases may refer to detecting a single allele (nucleotide) at a SNP position, or may encompass detecting two alleles (nucleotides) at a SNP position (e.g., to determine the homozygous or heterozygous state of a SNP position). In addition, these phrases may also refer to detecting an amino acid residue encoded by a SNP (e.g., an alternative amino acid residue encoded by a different codon generated by an alternative nucleotide at a SNP position).

[0098] Reagents that specifically detect the specific target SNP positions disclosed herein and are preferably specific for the specific nucleotides (alleles) of the target SNP positions (i.e., the reagents preferably can distinguish between different alternative nucleotides of the target SNP positions, thereby allowing the identity of the nucleotides present at the target SNP positions to be determined) can be used for SNP detection. Generally speaking, such detection reagents hybridize with nucleic acid molecules containing the target SNP by complementary base pairing in a sequence-specific manner, and distinguish the target variant sequence from other nucleic acid sequences (e.g., forms known in the art) in the test sample. An example of a detection reagent is a non-naturally occurring nucleic acid primer or probe that hybridizes with a target nucleic acid containing the SNP disclosed herein. In a preferred embodiment, such primers or probes can distinguish nucleic acids having specific nucleotides (alleles) at the target SNP position from other nucleic acids having different nucleotides at the same target SNP position. In addition, the detection reagent can hybridize with specific regions at the SNP position 5' and / or 3'. It is obvious to those skilled in the art that such detection reagents (e.g., such primers and probes) can be used directly as reagents for genotyping one or more SNPs disclosed herein, and can be incorporated into any kit form.

[0099] For analyzing SNPs, it may be appropriate to use oligonucleotides specific for alternative SNP alleles. Such oligonucleotides for detecting single nucleotide variations in a target sequence may be referred to by terms such as "allele-specific oligonucleotides," "allele-specific probes," or "allele-specific primers."

[0100] The SNP detection reagent can be labeled with a reporter (e.g., a fluorescent reporter dye) that emits a detectable signal. Although the preferred reporter dye is a fluorescent dye, any reporter dye that can be connected to a detection reagent such as an oligonucleotide probe or primer is suitable according to the present invention. In another embodiment, the detection reagent can also be labeled with a quencher dye, especially when the reagent is used as a self-quenching probe (e.g., a TaqMan probe). The SNP detection reagent disclosed herein may also include other labels, including but not limited to biotin for streptavidin binding, haptens for antibody binding, and oligonucleotides for binding to other complementary oligonucleotides.

[0101] According to the present invention, it is also considered that the SNP nucleotide to be identified is not included (or is not complementary to it), but is used to measure the reagent of one or more SNPs disclosed in this article. For example, primers that flank the target SNP position but do not directly hybridize with it can be used for primer extension reactions in which the primer is hybridized to the region adjacent to the target SNP position (that is, within one or more nucleotides from the target SNP site). During the primer extension reaction, if a specific nucleotide (allele) is present in the target SNP site, the primer is usually not able to extend through the target SNP site, and the primer extension product can be detected to determine which SNP allele is present in the target SNP site. For example, specific ddNTPs are usually used in primer extension reactions to terminate primer extension once the ddNTP is incorporated into the extension product. Therefore, according to the present invention, even if the sequence bound does not necessarily include the SNP site itself, the nucleic acid molecule in the region adjacent to the SNP site is bound and used to measure the SNP site.

[0102] The term "FCGR2A" relates to the human FCGR2A gene. This gene encodes the low affinity immunoglobulin gamma Fc region receptor II-a (CD32) and is a member of the immunoglobulin Fc receptor gene family. The protein encoded by this gene is a cell surface receptor found on phagocytic cells (such as macrophages and neutrophils) and is involved in the process of phagocytosis and immune complex clearance. Alternative splicing produces multiple transcript variants.

[0103] Preferably, the term "FCGR2A" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 61 of the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 62 of the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0104] rs1801274 is a SNP in the FCGR2A gene. rs1801274 (C) encodes the arginine (R) allele, and the (T) allele encodes the variant histidine (H). This SNP is an intragenic conversion substitution with the following codon changes: CAT, CGT, and results in a missense mutation. This SNP is referred to by many names in the literature, including A519C and R131H. The background sequence is as follows:

[0105]

[0106] The term "MUC1" refers to the human MUC1 gene. The gene encodes mucin 1, cell surface associated (MUC1) or polymorphic epithelial mucin (PEM), which is a member of the mucin family and a membrane-bound glycosylated phosphoprotein. The protein is anchored to the apical surface of many epithelia via a transmembrane domain. Outside the transmembrane domain is a SEA domain containing a cleavage site for releasing a large extracellular domain. The protein plays a protective role by binding to pathogens and also functions in cell signaling capabilities.

[0107] Preferably, the term "MUC1" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 63 of the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 64 of the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0108] rs4072037 is a SNP in the MUC1 gene. This SNP is an intragenic conversion substitution with the following codon changes: ACA, ACG, and results in a silent mutation. The background sequence is as follows:

[0109]

[0110] The term "IL-10" refers to the human IL-10 gene. This gene encodes interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), which is an anti-inflammatory cytokine.

[0111] Preferably, the term "IL-10" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 65 of the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 66 of the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0112] rs1800896 is a SNP in the IL-10 gene. This SNP is an intergenic / unknown intragenic conversion substitution. The background sequence is as follows:

[0113]

[0114] The term "DNMT3A" relates to the human DNMT3A gene. This gene encodes DNA (cytosine-5)-methyltransferase 3A. The protein encoded by this gene is an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA.

[0115] Preferably, the term "DNMT3A" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 67 of the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 68 of the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0116] rs1550117 is a SNP in the DNMT3A gene. This SNP is an intragenic conversion substitution within the DNMT3A promoter region. The background sequence is as follows:

[0117]

[0118] The term "SMAD4" refers to the human SMAD4 gene. This gene encodes mother DPP homolog 4 (Mothers againstdecapentaplegic homolog 4). The protein encoded by this gene is involved in cell signaling and belongs to the Darfwin protein family that regulates members of the TGFβ protein superfamily. It binds to receptor-regulated SMADs, such as SMAD1 and SMAD2, and forms a complex that binds to DNA and acts as a transcription factor. It is the only known mammalian coSMAD.

[0119] Preferably, the term "SMAD4" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 69 of the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 70 of the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0120] rs12456284 is a SNP in the SMAD4 gene. This SNP is an intragenic conversion substitution in the 3′-UTR. The background sequence is as follows:

[0121]

[0122] The term "EGF" refers to the human EGF gene. This gene encodes epidermal growth factor. EGF is a growth factor that stimulates cell growth, proliferation and differentiation by binding to its receptor EGFR.

[0123] Preferably, the term "EGF" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 71 in the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 72 in the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0124] rs4444903 is a SNP in the EGF gene. This SNP is an intragenic conversion substitution in the 5′-UTR. The background sequence is as follows:

[0125]

[0126] The term "CDH1" refers to the human CDH1 gene. This gene encodes Cadherin-1, also known as CAM 120 / 80 or E-cadherin (E-cadherin) or uvomorulin. The protein is a classical member of the Cadherin superfamily. It is a calcium-dependent cell-cell adhesion glycoprotein consisting of five extracellular Cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail. Loss of function is believed to promote the progression of cancer by increasing proliferation, invasion, and / or metastasis.

[0127] Preferably, the term "CDH1" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 73 of the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 74 of the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0128] rs16260 is a SNP in the CDH1 gene. This SNP is an intragenic transversion substitution located in the promoter region of the CDH1 gene. The background sequence is as follows:

[0129]

[0130] The term "ERCC1" refers to the human ERCC1 gene. This gene encodes the DNA excision repair protein ERCC-1. The function of the ERCC1 protein is mainly in the nucleotide excision repair of damaged DNA.

[0131] Preferably, the term "ERCC1" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 75 of the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 76 of the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0132] rs11615 is a SNP in the ERCC1 gene. This SNP is a silent intragenic conversion substitution. The background sequence is as follows:

[0133]

[0134] The term "FCGR3A" relates to the human FCGR3A gene. This gene encodes the low affinity immunoglobulin gamma Fc region receptor III-A. The protein encoded by this gene is part of a cluster of molecules on the surface of differentiated cells.

[0135] Preferably, the term "FCGR3A" relates to a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 77 of the sequence listing or a variant of the nucleic acid sequence, preferably consisting of the same, and a protein encoded by the nucleic acid, preferably a protein comprising the amino acid sequence of SEQ ID NO: 78 of the sequence listing or a variant of the amino acid sequence, preferably consisting of the same.

[0136] rs396991 is a SNP in the FCGR3A gene. This SNP is an intragenic transversion substitution with the following codon changes: GTT, TTT, and causes a missense mutation. rs396991 (T) encodes the phenylalanine (F) allele, and the (G) allele encodes the variant valine (V). The background sequence is as follows:

[0137]

[0138] Sealantin is a family of proteins that are the most important components in tight junctions, in which they establish a paracellular barrier that controls the flow of molecules in the intercellular spaces between epithelial cells. Sealantin is a transmembrane protein that spans the membrane 4 times, with an N-terminus and a C-terminus both located in the cytoplasm. The first extracellular loop or domain consists of an average of 53 amino acids, and the second extracellular loop or domain consists of about 24 amino acids. The cell surface proteins of the sealant family (e.g., CLDN18.2) are expressed in tumors of different origins, and are particularly suitable as target structures associated with antibody-mediated cancer immunotherapy due to their selective expression (not expressed in toxicity-related normal tissues) and localization to the plasma membrane.

[0139] The term "CLDN" as used herein means claudin and includes CLDN18.2. Preferably, claudin is human claudin.

[0140] The term "CLDN18" relates to claudin 18 and includes any variants including claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

[0141] The term "CLDN18.2" preferably relates to human CLDN18.2, and in particular to a protein comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 according to the sequence listing or a variant of said amino acid sequence. The first extracellular loop or domain of CLDN18.2 preferably comprises amino acids 27 to 81, more preferably amino acids 29 to 78 of the amino acid sequence shown in SEQ ID NO: 1. The second extracellular loop or domain of CLDN18.2 preferably comprises amino acids 140 to 180 of the amino acid sequence shown in SEQ ID NO: 1. The first and second extracellular loops or domains preferably form the extracellular part of CLDN18.2.

[0142] CLDN18.2 is selectively expressed in normal tissues of differentiated epithelial cells of the gastric mucosa. CLDN18.2 is expressed in cancers of different origins, such as pancreatic cancer, esophageal cancer, gastric cancer, bronchial cancer, breast cancer, and ENT tumors. CLDN18.2 is a valuable target for preventing and / or treating primary tumors (e.g., gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer) and their metastases (particularly gastric cancer metastasis (e.g., Krukenberg tumor), peritoneal metastasis, and lymph node metastasis).

[0143] The term "CLDN18.1" preferably relates to human CLDN18.1 and in particular to a protein comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 2 according to the sequence listing or a variant of said amino acid sequence.

[0144] "Prognosis" as used herein refers to the prediction of an outcome, particularly the likelihood of progression-free survival (PFS) or disease-free survival (DFS). Survival is usually calculated as the average number of months (or years) in which 50% of patients survive, or the percentage of patients alive after 1, 5, 15, and 20 years. Prognosis is important for treatment decisions because patients with a good prognosis are usually offered less invasive treatments, while patients with a poor prognosis are usually offered more aggressive treatments, such as a wider range of chemotherapeutic drugs.

[0145] As used herein, "predicting" means providing information about the likely response of a disease to different therapeutic treatments.

[0146] The phrase "indicative of a risk" refers to an indication of a certain degree of likelihood or probability. The phrase "indicative of a reduced risk" refers to a low degree of likelihood or probability. The phrase "indicative of an increased risk" refers to a certain higher or high degree of likelihood or probability.

[0147] If an event "indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody", then the event indicates that the cancer patient is a responder to treatment with the antibody, i.e. the patient is likely to be a responder to treatment with the antibody, and optionally, the patient is more likely to be a responder to treatment with the antibody than if the patient was not a responder to treatment with the antibody.

[0148] If an event "indicates an increased risk that the cancer patient is not a responder to treatment with the antibody", then the event indicates that the cancer patient is not a responder to treatment with the antibody, i.e. the patient is likely to be a non-responder to treatment with the antibody, and optionally, the patient is more likely to be a non-responder to treatment with the antibody than if the patient was a responder to treatment with the antibody.

[0149] If an event "indicates a reduced risk of a poor clinical outcome," then the event indicates a good clinical outcome, ie, it is likely that a good clinical outcome will occur, and optionally, it is more likely that a good clinical outcome will occur than a poor clinical outcome.

[0150] If an event "indicates an increased risk of a poor clinical outcome," then the event indicates a poor clinical outcome, ie, it is likely that a poor clinical outcome will occur, and optionally, it is more likely that a poor clinical outcome will occur than a good clinical outcome.

[0151] If an event "indicates a reduced risk that a cancer patient does not experience progression-free survival", then the event indicates that a cancer patient experiences progression-free survival, i.e., the patient is likely to experience progression-free survival, and optionally, the patient is more likely to experience progression-free survival than if the patient does not experience progression-free survival.

[0152] If an event "indicates an increased risk that a cancer patient will not experience progression-free survival," then the event indicates that the cancer patient will not experience progression-free survival, i.e., the patient is likely not to experience progression-free survival, and optionally, the patient is more likely not to experience progression-free survival than if the patient experienced progression-free survival.

[0153] The term "sample" used herein refers to any material obtained from an object and can be used for analytical purposes (particularly for determining the genotype of one or more single nucleotide polymorphisms). In certain embodiments, the sample described herein can be or can be derived from any tissue, cell and / or biological fluid from, for example, a mammal or person to be tested. The sample can be separated from a patient (for example, from the human body). The sample can be a graded and / or purified sample. For example, the sample encompassed by the present invention can be or can be derived from a tissue (for example, a section or explant) sample, a single cell sample, a cell colony sample, a cell culture sample, a blood (for example, whole blood or blood fraction, such as a blood cell fraction, serum or plasma) sample, a urine sample or a sample from other peripheral sources. In a particularly preferred embodiment, the sample is a tissue sample (for example, from a biopsy of an object having or suspected of having cancerous tissue). For example, the sample can be a biopsy of a tumor. Samples can be obtained from a patient before therapeutic treatment begins, during therapeutic treatment and / or after therapeutic treatment, for example, before, during or after the administration of cancer treatment.

[0154] The sample material can be used to produce nucleic acid extracts (including DNA and / or RNA), protein or membrane extracts from any body fluid (e.g., blood, serum, plasma, urine, saliva, sputum, gastric juice, semen, tears, sweat, etc.), skin, hair, cells (especially nucleated cells), biopsy, buccal swab, or tissue or tumor sample.

[0155] The invention further relates to a test kit, which comprises for example the following components: a reagent for determining the genotype of one or more SNPs as described herein. In the context of the present invention, the term "kit of parts" (abbreviated as: test kit) is to be understood as any combination of at least some components identified herein, which are combined into functional units in spatial coexistence, and may comprise other components. For example, the test kit may comprise a preselected primer or probe specific to a nucleic acid sequence, the nucleic acid sequence comprising one or more SNPs whose genotypes are to be determined. The test kit may also comprise an enzyme (for example, a polymerase such as Taq) suitable for amplifying nucleic acids and deoxynucleotides and buffer required for the reaction mixture of amplification. The test kit may also comprise a probe specific to one or more SNPs. In certain embodiments, the components are detectably labeled.

[0156] The kit of the present invention may comprise (i) a container and / or (ii) a data carrier. The container may be filled with one or more of the above-mentioned components or reagents. The data carrier may be a non-electronic data carrier, such as a graphic data carrier, such as an information leaflet, an information sheet, a bar code or an access code; or an electronic data carrier, such as a floppy disk, a compact disc (CD), a digital versatile disk (DVD), a microchip or other semiconductor-based electronic data carrier. The access code may allow access to a database, such as an Internet database, a centralized or decentralized database. The data carrier may contain instructions for allowing analysis of the results obtained with the kit, and in particular for using the kit in the method of the present invention.

[0157] Alternatively or additionally, the kit may include materials desirable from a commercial and user perspective, including buffers, reagents and / or diluents.

[0158] Based on the results obtained (ie, based on the genotype of one or more single nucleotide polymorphisms), the physician can select a cancer treatment, in particular an antibody treatment, to which the patient is predicted to be responsive. Preferably, a cancer treatment to which the patient is predicted not to be responsive is not administered to the patient.

[0159] Based on the prediction that the patient will not respond to antibody therapy (particularly antibody therapy that works by recruiting the patient's immune system to destroy tumor cells), the physician may choose to administer a cancer therapy other than antibody therapy (particularly antibody therapy that works by recruiting the patient's immune system to destroy tumor cells). In particular, the physician may choose to administer chemotherapy.

[0160] Based on the results predicting that the patient will be responsive to antibody therapy (particularly antibody therapy that works by recruiting the patient's immune system to destroy tumor cells), the physician may choose to administer the antibody therapy (particularly antibody therapy that works by recruiting the patient's immune system to destroy tumor cells), optionally in combination with chemotherapy.

[0161] The term "(therapeutic) treatment" as used herein in particular for cancer treatment relates to any treatment intended to improve the patient's health and / or prolong (increase) the patient's lifespan. The treatment can eliminate cancer, reduce the size or number of tumors in patients, prevent or slow the occurrence of cancer in patients, inhibit or slow the occurrence of new cancers in patients, reduce the frequency or severity of symptoms in patients, and / or reduce recurrence in patients who currently have or have previously had cancer. The (therapeutic) treatment of cancer can be selected from surgery, chemotherapy, radiotherapy and targeted therapy. A particularly preferred treatment according to the present invention is a cancer treatment involving therapeutic monoclonal antibodies directed against tumor antigens (e.g. CLDN18.2 expressed on target cells).

[0162] Adjuvant therapy is treatment given in addition to primary, primary, or initial treatment. Surgery and combined treatment regimens used to treat cancer have made this term primarily used to describe adjuvant cancer treatment. An example of adjuvant therapy is additional treatment usually given after surgery where all detectable disease has been removed but where there is still a statistical risk of recurrence due to occult disease.

[0163] Terms such as "responsiveness" or "responder" in the context of treatment refer to the fact that a patient has a therapeutic benefit from a given treatment modality, and in particular refer to the observation of a reduction, prevention, or elimination of a disease, including shortening the duration of a disease, arresting or slowing the progression or worsening of a disease, inhibiting or slowing new disease occurrence and / or recurrence, preventing or delaying the onset of a disease or its symptoms, reducing the frequency or severity of symptoms, and / or prolonging the life of a patient in a patient currently suffering from or previously suffering from a disease. In particular, it refers to the observation of a reduction in tumor mass or a prolongation of tumor-free time, recurrence-free time, or overall survival time.

[0164] Terms such as "non-responsiveness" or "non-responder" in the context of treatment refer to the fact that the patient has no therapeutic benefit from a given treatment modality, particularly no reduction, prevention or elimination of the disease is observed, i.e., the patient is resistant to the treatment.

[0165] A complete response is defined as the absence of any residual disease (e.g., cancer) and is typically assessed by pathological analysis of the tissue samples obtained. In this case, the term "pathological complete response" (pCR) is often used. In particular, pCR is defined as the absence of any residual invasive tumor cells in the original tumor bed. However, the definition of pCR can vary between different grading systems. Pathological complete response has been shown to be a prognostic factor for overall better survival, but also a prognostic factor for disease-free survival and recurrence-free survival.

[0166] Relapse-free survival was defined as the time from randomization to first relapse or recurrence, second cancer, or death.

[0167] Progression-free survival (PFS) is a survival rate that measures the length of time during which the disease being treated (usually cancer) does not get worse, both during and after drug administration or treatment. It is sometimes used as a measure to study the health of people with a disease to try to determine how a new treatment works, and it is often used as a clinical endpoint in randomized controlled trials of cancer treatments.

[0168] According to the present invention, the term "cancer patient experiences progression-free survival" relates to a cancer patient having a prolonged period without disease progression, in particular when compared to an average for the patient and / or when compared to patients who are non-responders to a given treatment modality. Preferably, the prolonged period is at least 4, preferably at least 5, more preferably at least 6 months, such as at least 7 months or at least 8 months, the period starting from, for example, the time of the first administration of treatment.

[0169] The term "clinical outcome" is defined as the clinical result of a disease, such as a reduction or improvement in symptoms, particularly following treatment.

[0170] The term "recurrence" with respect to cancer includes the appearance of tumor cells at the same site and organ of primary disease, distant metastases that may occur even many years after the initial diagnosis and treatment of the cancer, or local events such as infiltration of tumor cells into regional lymph nodes.

[0171] The terms "individual" and "object" are used interchangeably herein. It refers to a human, non-human primate or other mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse or primate), which may be affected by a disease or condition (e.g., cancer) or be susceptible to a disease or condition (e.g., cancer), but may or may not suffer from the disease or condition. In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "object" do not represent a specific age, and therefore encompass adults, the elderly, children and newborns. In some preferred embodiments of the present invention, "individual" or "object" is a "patient". The term "patient" means a treatment subject, particularly a sick subject, according to the present invention.

[0172] In a particularly preferred embodiment, the method of the invention is performed on a patient who has been diagnosed with cancer.

[0173] "Target cell" shall mean any undesirable cell, such as a cancer cell. In some preferred embodiments, the target cell expresses CLDN18.2.

[0174] In the context of the present invention, terms such as "protection", "prevention" or "preventative" relate to preventing the occurrence and / or spread of a disease in a subject, and in particular minimizing the chance that a subject will develop a disease or delaying the onset of a disease. For example, a subject at risk for cancer would be a candidate for treatment to prevent cancer.

[0175] "At risk" means a subject identified as having a higher than normal chance of developing a disease, particularly cancer, compared to the general population. In addition, a subject who has had or currently has a disease, particularly cancer, is a subject at increased risk of developing the disease because the subject may go on to develop the disease. A subject who currently has or has had cancer also has an increased risk of cancer metastasis.

[0176] As used herein, the term "combination" refers to the use of more than one treatment or therapeutic agent in the context of administering a treatment. The use of the term "combination" does not limit the order in which the treatment or therapeutic agents are administered to a subject. The treatment or therapeutic agent can be administered before, concomitantly with, or after the second treatment or therapeutic agent is administered to the subject. Preferably, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or within a certain time interval so that the treatment or therapeutic agent can act together. In a specific embodiment, the treatment or therapeutic agent is administered to the subject in a certain order, amount, and / or within a certain time interval so that it provides an improved benefit compared to if it is administered in other ways (particularly independently of each other). Preferably, the improved benefit is a synergistic effect.

[0177] The term "disease" refers to an abnormal condition that affects the body of an individual. Generally speaking, a disease is interpreted as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originating from an external source, such as an infectious disease, or it may be caused by an internal dysfunction, such as an autoimmune disease. For humans, "disease" is often more broadly used to refer to any condition that causes pain, dysfunction, suffering, social problems or death in a sick individual or similar problems in those in contact with the individual. In this broader sense, diseases sometimes include injuries, disabilities, disorders, syndromes, infections, isolated symptoms, abnormal behaviors, and atypical changes in structure and function, which may be considered distinguishable categories in other contexts and for other purposes. Diseases often affect individuals not only physically but also emotionally, because contracting and coexisting with many diseases can change an individual's view of life and an individual's personality. According to the present invention, the term "disease" includes cancer, particularly those cancer forms described herein. Any reference to cancer or a specific cancer form herein also includes its cancer metastasis. In a preferred embodiment, the disease to be treated according to the present application involves cells expressing tumor antigens (e.g., CLDN18.2).

[0178] According to the present invention, "diseases involving cells expressing tumor antigens" means that tumor antigens (e.g., CLDN18.2) are expressed in cells of diseased tissues or organs. In one embodiment, the expression of tumor antigens in cells of diseased tissues or organs is increased compared to the state in healthy tissues or organs. Increase means an increase of at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In one embodiment, expression is only found in diseased tissues, while expression in corresponding healthy tissues is suppressed. According to the present invention, diseases involving cells expressing tumor antigens include cancer diseases. In addition, according to the present invention, cancer diseases are preferably those in which cancer cells express tumor antigens.

[0179] The term "cancer disease" or "cancer" refers to or describes a physiological condition characterized by unregulated cell growth in an individual. Examples of cancer include, but are not limited to, epithelial cancer, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancer include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, sex and reproductive organ cancer, Hodgkin's Disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas. According to the present invention, the term "cancer" also includes cancer metastasis. Preferably, a "cancer disease" is characterized by cells expressing a tumor antigen (eg, CLDN18.2), and cancer cells express such a tumor antigen. Cells expressing a tumor antigen (eg, CLDN18.2) are preferably cancer cells, preferably cancer cells of a cancer described herein.

[0180] According to the present invention, the term "tumor" or "neoplastic disease" refers to an abnormal growth of cells (referred to as neoplastic cells, tumorigenic cells or tumor cells), preferably forming a swelling or lesion. "Tumor cell" means an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow after the stimulus that triggered the new growth has ceased. Tumors show a partial or complete loss of structural organization and functional coordination with normal tissues, and usually form a distinct mass of tissue that can be benign, pre-malignant or malignant.

[0181] In one embodiment, the cancer according to the present invention relates to cancer cells expressing tumor antigens (eg, CLDN18.2). In one embodiment, the cancer is tumor antigen positive, for example, CLDN18.2 positive. In one embodiment, the expression of tumor antigens (eg, CLDN18.2) is located on the surface of the cell. In one embodiment, at least 50%, preferably 60%, 70%, 80% or 90% of the cancer cells are tumor antigen positive (eg, CLDN18.2 positive), and / or at least 40%, preferably at least 50% of the cancer cells are positive for surface expression of tumor antigens (eg, CLDN18.2). In one embodiment, at least 95% or at least 98% of the cancer cells are tumor antigen positive, for example, CLDN18.2 positive. In one embodiment, at least 60%, at least 70%, at least 80% or at least 90% of the cancer cells are positive for surface expression of tumor antigens (eg, CLDN18.2).

[0182] In one embodiment, the cancer involving cancer cells expressing CLDN18.2 or CLDN18.2-positive cancer is selected from the group consisting of: gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer, and metastases thereof, in particular gastric cancer metastases (e.g., Krukenberg tumors), peritoneal metastases, and lymph node metastases. In one embodiment, the cancer is adenocarcinoma, in particular advanced adenocarcinoma. Particularly preferred cancer diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung, and ovary. In one embodiment, the cancer is selected from the group consisting of: gastric cancer, esophageal cancer (in particular, lower esophageal cancer), cancer of the eso-gastric junction, and gastroesophageal cancer. In a particularly preferred embodiment, the cancer is gastroesophageal cancer, such as metastatic, refractory, or recurrent advanced gastroesophageal cancer. In one embodiment, the CLDN18.2-positive tumor is a tumor of the above-mentioned cancer types.

[0183] Embodiments involving CLDN18.2-positive tumors or cancer cells expressing CLDN18.2 preferably involve the use of antibodies that have the ability to bind to CLDN18.2. In one embodiment, the antibody that has the ability to bind to CLDN18.2 is a monoclonal antibody, a chimeric antibody or a humanized antibody, or an antibody fragment.

[0184] According to the invention, "carcinoma" is a malignant tumor originating from epithelial cells. This group represents the most common cancers and includes common forms of breast cancer, prostate cancer, lung cancer and colon cancer.

[0185] "Adenocarcinoma" is a cancer that originates from glandular tissue. This tissue is also part of a larger tissue category called epithelial tissue. Epithelial tissue includes skin, glands, and a variety of other tissues that line the cavities and organs of the body. Epithelium is derived from the ectoderm, endoderm, and mesoderm embryologically. To be classified as an adenocarcinoma, the cell does not necessarily need to be part of a gland, as long as it has secretory properties. This form of cancer can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated adenocarcinomas may not be the case. By staining cells from a biopsy, a pathologist will determine whether the tumor is an adenocarcinoma or some other type of cancer. Due to the ubiquity of glands in the body, adenocarcinomas can occur in many tissues of the body. Although each gland may not secrete the same substance, as long as the cell has exocrine function, it is considered glandular, and therefore its malignant form is called adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize (if there is enough time to do so). Ovarian adenocarcinoma is the most common type of ovarian cancer. These include serous and mucinous adenocarcinomas, clear cell adenocarcinomas, and endometrioid adenocarcinomas.

[0186] "Metastasis" means that cancer cells spread to other parts of the body from its original position. The formation of metastasis is a very complicated process, and depends on the malignant cells to break away from the primary tumor, invade the extracellular matrix, penetrate the endothelial basement membrane to enter the body cavity and blood vessels and then infiltrate the target organ after being transported by the blood. Finally, the growth of new tumors at the target site depends on angiogenesis. Even after removing the primary tumor, tumor metastasis often occurs, because tumor cells or components can retain and produce metastasis potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis", which relates to the metastasis away from the primary tumor and the local lymph node system. In one embodiment, the term "metastasis" according to the present invention relates to lymph node metastasis. The metastasis of a specific form of treatment using the present invention is derived from the metastasis of the gastric cancer as the primary site. In some preferred embodiments, such gastric cancer metastasis is Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0187] A refractory cancer is a malignancy for which a particular treatment is refractory, either initially unresponsive to treatment or becomes unresponsive over time. The terms "refractory," "unresponsive," or "resistant" are used interchangeably herein.

[0188] Krukenberg tumor is an uncommon metastatic tumor of the ovary, accounting for 1% to 2% of all ovarian tumors. The prognosis of Krukenberg tumor remains poor, and treatment of Krukenberg tumor has not yet been established. Krukenberg tumor is a metastatic signet ring cell adenocarcinoma of the ovary. The stomach is the primary site of most Krukenberg tumor cases (70%). Colon cancer, appendix cancer, and breast cancer (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumors have been reported from gallbladder cancer, bile duct cancer, pancreatic cancer, small intestine cancer, ampulla of Vater cancer, cervical cancer, and bladder / urachal cancer.

[0189] The term "surgery" as used herein includes the removal of a tumor during surgery. Surgery is a common treatment for cancer. A surgeon may use local excision to remove a tumor.

[0190] The term "chemotherapy" as used herein refers to the use of a chemotherapeutic agent or combination of chemotherapeutic agents, preferably to stop cancer cell growth by killing cells or by stopping cell division. When chemotherapy is taken orally or by injection into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy). When chemotherapy is placed directly into the cerebrospinal fluid, an organ, or a body cavity (e.g., the abdomen), the drugs primarily affect cancer cells in those areas (regional chemotherapy).

[0191] Chemotherapeutic agents according to the present invention include cytostatic compounds and cytotoxic compounds. Traditional chemotherapeutic agents work by killing cells that divide rapidly (one of the main characteristics of most cancer cells). This means that chemotherapy also harms cells that divide rapidly under normal circumstances, such as cells in bone marrow, digestive tract and hair follicles. This causes the most common side effects of chemotherapy. According to the present invention, the term "chemotherapy" preferably does not include antibodies that target proteins (tumor antigens) that are abnormally expressed in cancer cells and that work by raising the patient's immune system to destroy tumor cells. However, antibodies that target proteins (tumor antigens) that are abnormally expressed in cancer cells and work by the therapeutic part or therapeutic agent conjugated thereto can be regarded as a form of chemotherapy. However, in the strictest sense, the term "chemotherapy" according to the present invention does not include targeted therapy.

[0192] According to the present invention, the term "chemotherapeutic agent" includes taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, etoposide, bleomycin, vinorelbine, cyclophosphamide, and combinations thereof. According to the present invention, reference to a chemotherapeutic agent is to include any prodrug of the agent, such as an ester, salt or derivative (e.g., conjugate). An example is a conjugate of the agent with a carrier substance, such as protein-bound paclitaxel, such as albumin-bound paclitaxel. Preferably, the salt of the agent is pharmaceutically acceptable.

[0193] Taxanes are a class of diterpenoid compounds that were originally obtained from natural sources such as plants of the genus Taxus, but some have been synthesized artificially. The main mechanism of action of taxane drugs is to disrupt microtubule function, thereby inhibiting the cell division process. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0194] According to the present invention, the term "docetaxel" refers to a compound having the following formula:

[0195]

[0196] In particular, the term "docetaxel" refers to the compound 1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytaxane-11-ene-2α,4,13α-triyl 4-acetate 2-benzoate 13-{(2R,3S)-3-[(tert-butoxycarbonyl)-amino]-2-hydroxy-3-phenylpropanoate}.

[0197] According to the present invention, the term "paclitaxel" refers to a compound having the following formula:

[0198]

[0199] In particular, the term "paclitaxel" refers to the compound (2α,4α,5β,7β,10β,13α)-4,10-bis-(acetyloxy)-13-{[(2R,3S)3-(benzoylamino)-2-hydroxy-3-phenylpropionyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytaxanthin-11-en2-yl benzoate.

[0200] According to the present invention, the term "platinum compound" refers to a compound containing platinum in its structure, such as a platinum complex, and includes compounds such as cisplatin, carboplatin and oxaliplatin.

[0201] The term "cisplatin" or "cisplatinum" refers to the compound cis-diamminedichloroplatinum (II) (CDDP) of the formula:

[0202]

[0203] The term "carboplatin" refers to the compound cis-diamino(1,1-cyclobutanedicarboxylate)platinum(II) of the formula:

[0204]

[0205] The term "oxaliplatin" refers to a platinum compound complexed with a diaminocyclohexane carrier ligand of the following formula:

[0206]

[0207] In particular, the term "oxaliplatin" refers to the compound [(1R,2R)-cyclohexane-1,2-diamine](oxalate-O,O')platinum(II). Oxaliplatin for injection is also commercially available under the trade name Eloxatine.

[0208] The term "nucleoside analog" refers to structural analogs of nucleosides, a class that includes both purine analogs and pyrimidine analogs.

[0209] The term "gemcitabine" is a compound that is a nucleoside analog of the formula:

[0210]

[0211] In particular, the term refers to the compound 4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)pyrimidin-2(1H)-one or 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,2-dihydropyrimidin-2-one.

[0212] The term "nucleoside analogs" includes fluoropyrimidine derivatives, such as fluorouracil and its prodrugs. The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (sold under the trade names Adrucil, Carac, Efudix, Efudex and Fluoroplex) is a compound that is a pyrimidine analog of the formula:

[0213]

[0214] In particular, the term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.

[0215] The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent that is a prodrug that is converted to 5-FU in tissues. Orally administrable capecitabine has the following formula:

[0216]

[0217] In particular, the term refers to the compound [1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]carbamic acid pentyl ester.

[0218] The term "folinic acid" or "leucovorin" refers to a compound that can be used in synergistic combination with the chemotherapeutic agent 5-fluorouracil. Thus, if reference is made herein to the administration of 5-fluorouracil or a prodrug thereof, in one embodiment the administration may include administration in combination with folinic acid. Folinic acid has the following formula:

[0219]

[0220] In particular, the term refers to the compound (2S)-2-{[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridin-6-yl)methylamino]benzoyl]amino}glutaric acid.

[0221] According to the present invention, the term "camptothecin analogue" refers to a derivative of the compound camptothecin (CPT; (S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione). Preferably, the term "camptothecin analogue" refers to a compound comprising the following structure:

[0222]

[0223] According to the present invention, preferred camptothecin analogs are inhibitors of the DNA enzyme topoisomerase I (topoisomerase I, topo I). Preferred camptothecin analogs according to the present invention are irinotecan and topotecan.

[0224] Irinotecan is a drug that prevents DNA unwinding by inhibiting topoisomerase I. In chemical terms, it is a semisynthetic analog of the natural alkaloid camptothecin with the following formula:

[0225]

[0226] In particular, the term "irinotecan" refers to the compound (S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxol-1H-pyrano[3',4':6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4'-bipiperidine]-1'-carboxylate.

[0227] Topotecan is a topoisomerase inhibitor of the formula:

[0228]

[0229] In particular, the term "topotecan" refers to the compound (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride.

[0230] Anthracyclines are a class of drugs commonly used in cancer chemotherapy that are also antibiotics. Structurally, all anthracyclines share a common tetracyclic 7,8,9,10-tetrahydrotetracene-5,12-quinone structure and generally require glycosylation at specific sites.

[0231] Anthracyclines preferably cause one or more of the following mechanisms of action: 1. Inhibit DNA and RNA synthesis by intercalating between base pairs of DNA / RNA chains, thereby preventing the replication of rapidly growing cancer cells. 2. Inhibit the topoisomerase II enzyme, preventing the relaxation of supercoiled DNA and thus blocking DNA transcription and replication. 3. Generate iron-mediated free oxygen radicals, which damage DNA and cell membranes.

[0232] According to the present invention, the term "anthracycline" preferably relates to an agent, preferably an anticancer agent for inducing apoptosis, preferably by inhibiting the rebinding of DNA in topoisomerase II.

[0233] Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodoxorubicin, pyrarubicin, valrubicin, N-trifluoro-acetyl doxorubicin-14-valerate, aclarubicin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyano-morpholino-DOX), 2-pyrrolo-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone, and aclarubicin A (aclarubicin). Mitoxantrone is a member of the anthracenedione class of compounds, which are anthracycline analogs that lack the sugar moiety of anthracyclines but retain a planar polycyclic aromatic ring structure that allows for intercalation into DNA.

[0234] Epirubicin is an anthracycline of the formula:

[0235]

[0236] and is marketed under the trade name Ellence in the United States and under the trade name Pharmorubicin or Epirubicin Ebewe elsewhere. In particular, the term "epirubicin" refers to the compound (8R, 10S)-10-[(2S, 4S, 5R, 6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracene-5,12-dione. In some chemotherapy regimens, epirubicin is preferred over doxorubicin (the most popular anthracycline) because it appears to cause fewer side effects.

[0237] The term "etoposide" refers to a semisynthetic derivative of podophyllotoxin that exhibits antitumor activity. Etoposide inhibits DNA synthesis by forming a complex with topoisomerase II and DNA. The complex induces double-stranded DNA breaks and prevents repair by binding to topoisomerase II. Accumulated breaks in DNA prevent entry into the mitotic phase of cell division and lead to cell death. Etoposide has the following formula:

[0238]

[0239] In particular, the term refers to the compound 4'-demethyl-epipodophyllotoxin 9-[4,6-O-(R)-ethylidene-β-D-pyranoglucopyranoside], 4'-(dihydrogen phosphate).

[0240] The term "bleomycin" refers to a glycopeptide antibiotic produced by the bacterium Streptomyces verticillus. When used as an anticancer agent, it acts by causing DNA breaks. Bleomycin preferably comprises a compound having the formula:

[0241]

[0242] The term "vinorelbine" refers to an antimitotic chemotherapeutic drug that is a semisynthetic vinca alkaloid and is administered as a treatment for certain types of cancer, including breast cancer and non-small cell lung cancer. Vinorelbine preferably comprises a compound having the formula:

[0243]

[0244] Cyclophosphamide is a nitrogen mustard alkylating agent from the oxazophorine group. The main use of cyclophosphamide is in the treatment of some forms of cancer with other chemotherapeutic agents. Cyclophosphamide preferably comprises a compound having the formula:

[0245]

[0246] In the context of this invention, the term "radiation therapy" refers to the use of high-energy x-rays or other types of radiation to kill cancer cells or keep them from growing. There are two types of radiation therapy. External radiation therapy uses a machine outside the body to send radiation to the cancer. Internal radiation therapy uses radioactive materials sealed in needles, seeds, wires, or catheters that are placed directly in or near the cancer. The way radiation therapy is given depends on the type and stage of the cancer being treated.

[0247] According to the present invention, the term "targeted therapy" relates to any treatment that can be used to preferentially target diseased cells (e.g., cancer cells) without targeting or to a lesser extent non-disease cells. Targeting of diseased cells preferably results in killing of diseased cells and / or impairing their proliferation or viability. Such treatments include: i) naked or therapeutically conjugated antibodies, antibody fragments and proteins (e.g., antibodies or antibody conjugates against CLDN18.2 described herein) that target certain cell surface targets on diseased cells (e.g., tumor antigens, such as CLDN18.2); or ii) small molecules that impair the proliferation or viability of diseased cells. In a specific embodiment, the agent binds to an antigen that is expressed at a higher level on diseased stem cells than on normal stem cells. In a specific embodiment, the agent specifically binds to a tumor antigen. Traditional chemotherapy or radiotherapy is not considered a "targeted therapy", although it is often directed against tumors. In addition, the term "antibody therapy" according to the present invention preferably does not include treatment with an antibody, fragment or derivative thereof conjugated to a therapeutic moiety, but only involves treatment with an antibody, fragment or derivative thereof that acts by recruiting the patient's immune system to destroy tumor cells.

[0248] The term "antigen" refers to a substance comprising an epitope to which an immune response is generated and / or directed. The term "antigen" particularly includes proteins, peptides, polysaccharides, nucleic acids (especially RNA and DNA) and nucleotides. The term "antigen" also includes substances that become antigenic and sensitizing only by conversion (e.g., in a molecule, intermediate or by completion with body proteins). The antigen or its processed product is preferably recognized by a T cell or B cell receptor or by an immunoglobulin molecule (e.g., an antibody). In a preferred embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, such as CLDN18.2.

[0249] In the context of the present invention, the term "tumor antigen" or "tumor-associated antigen" relates to an antigen present in a tumor cell. The antigen is preferably present on a tumor cell, for example on the surface of a tumor cell. Preferably, a "tumor antigen" is expressed by a tumor cell. In one embodiment, the term "tumor antigen" relates to a protein that is abnormally expressed in a tumor cell when compared to a normal (ie, non-tumor) cell. For example, expression may be found only in tumor cells and not in normal (ie, non-tumor) cells, or the expression level may be higher in tumor cells compared to normal (ie, non-tumor) cells. In one embodiment, the term "tumor antigen" relates to a protein that is specifically expressed in a limited number of tissues and / or organs or a specific developmental stage under normal conditions and is expressed or abnormally expressed in one or more tumors or cancerous tissues. In the context of the present invention, a tumor antigen is preferably associated with the cell surface of a cancer cell, and is preferably not expressed, only rarely expressed, or expressed at a lower level in normal tissues and cells. Preferably, according to the present invention, if the expression level is below the detection limit and / or if the expression level is too low to allow the tumor antigen-specific antibody added to the cell to bind, the tumor antigen is not expressed in the cell. A particularly preferred tumor antigen according to the present invention is CLDN18.2.

[0250] According to the present invention, the term "tumor antigen-positive cancer" or "tumor antigen-positive tumor" or similar terms means a cancer or tumor comprising cancer cells or tumor cells that express a tumor antigen, preferably expressed on the surface of the cancer cells or tumor cells. If the tumor antigen is located on the surface of the cell and can be approached to bind to a tumor antigen-specific antibody added to the cell, the tumor antigen is expressed on the surface of the cell.

[0251] In a preferred embodiment of the present invention, "tumor antigen-positive cancer" or "tumor antigen-positive tumor" is a "CLDN18.2-positive cancer" or "CLDN18.2-positive tumor". According to the present invention, the term "CLDN18.2-positive cancer" or "CLDN18.2-positive tumor" means a cancer or tumor comprising cancer cells or tumor cells expressing CLDN18.2 (preferably expressed on the surface of the cancer cells or tumor cells).

[0252] "Cell surface" is used according to its normal meaning in the art, and thus includes the exterior of the cell that is accessible to proteins and other molecules for binding.

[0253] In the context of the present invention, the term "extracellular portion" refers to a portion of a molecule (e.g., a protein) that faces the extracellular space of a cell and is preferably accessible from the outside of the cell, e.g., by an antigen binding molecule (e.g., an antibody) located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0254] According to the present invention, CLDN18.2 is not substantially expressed in the cell if the expression level is low compared to the expression in gastric cells or gastric tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% or even lower than that expressed in gastric cells or gastric tissue. Preferably, CLDN18.2 is not substantially expressed in the cell if the expression level exceeds the expression level in non-cancerous tissue other than the stomach by no more than 2 times, preferably 1.5 times, and preferably does not exceed the expression level in the non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in the cell if the expression level is below the detection limit and / or if the expression level is too low to allow binding of a CLDN18.2-specific antibody added to the cell.

[0255] According to the present invention, CLDN18.2 is expressed in a cell if the expression level exceeds the expression level in non-cancerous tissues other than the stomach by preferably more than 2 times, preferably 10 times, 100 times, 1000 times or 10000 times. Preferably, CLDN18.2 is expressed in a cell if the expression level is above the detection limit and / or if the expression level is high enough to allow binding of a CLDN18.2-specific antibody added to the cell. Preferably, CLDN18.2 expressed in a cell is expressed or exposed on the surface of the cell.

[0256] The term "epitope" refers to an antigenic determinant in a molecule, i.e., a portion of a molecule that is recognized by the immune system (e.g., recognized by an antibody). For example, an epitope is a discrete three-dimensional site on an antigen that is recognized by the immune system. An epitope is usually composed of a chemically active surface group of a molecule such as an amino acid or a sugar side chain, and usually has a specific three-dimensional structural feature and a specific charge feature. The difference between a conformational and a non-conformational epitope is that, in the presence of a denaturing solvent, the binding to the former is lost while the binding to the latter is not lost. The epitope of a protein preferably comprises a continuous or discontinuous portion of the protein, and the length is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids, for example, the length of the epitope can be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids.

[0257] The term "antibody" includes glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and any molecule comprising an antigen-binding portion of such a glycoprotein. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, antibody fragments or derivatives (including but not limited to single-chain antibodies (e.g., scFv) and antigen-binding antibody fragments (e.g., Fab and Fab' fragments)), and also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described herein. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0258] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity. In one embodiment, the monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a non-human animal (e.g., a mouse) fused with an immortalized cell.

[0259] As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from animals that are transgenic or transchromosomal with respect to immunoglobulin genes (e.g., mice) or hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express the antibody (e.g., from transfectomas); (c) antibodies isolated from recombinant combinatorial antibody libraries; and (d) antibodies prepared, expressed, created or isolated by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences.

[0260] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0261] The term "humanized antibody" refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only a complementarity determining region (CDR) grafted to a suitable framework region in a variable domain. The antigen binding site may be wild-type or modified by one or more amino acid substitutions, such as being modified to be closer to being similar to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences (e.g., humanized mouse antibodies comprising all six CDRs from a mouse antibody). Other forms have one or more CDRs that are changed relative to the original antibody.

[0262] The term "chimeric antibody" refers to the following antibodies, wherein a portion of each of the amino acid sequences of the heavy and light chains is homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to a corresponding sequence in another. Generally speaking, the variable regions of both the light and heavy chains simulate the variable regions of an antibody derived from a mammal, while the constant portion is homologous to the sequence of an antibody derived from another. An obvious advantage of this chimeric form is that, in combination with a constant region derived from, for example, a human cell preparation, the variable region can be conveniently obtained from currently known sources using readily available B cells or hybridomas from non-human host organisms. Although the variable region has the advantage of being easy to prepare and the specificity is not affected by the source, compared with the constant region from a non-human source, when the antibody is injected, the constant region is human and is less likely to induce an immune response from a human subject. However, this definition is not limited to this specific example.

[0263] Antibodies can be derived from different species including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0264] The antibodies described herein include IgA (e.g., IgA1 or IgA2), IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more particularly an IgG1κ or IgG1λ isotype (i.e., IgG1, κ, λ); an IgG2a antibody (e.g., IgG2a, κ, λ); an IgG2b antibody (e.g., IgG2b, κ, λ); an IgG3 antibody (e.g., IgG3, κ, λ); or an IgG4 antibody (e.g., IgG4, κ, λ).

[0265] As used herein, "heterologous antibodies" are defined relative to the transgenic organism that produces such antibodies. The term refers to antibodies having amino acid sequences or encoding nucleic acid sequences corresponding to those found in organisms that do not consist of the transgenic organism and are typically derived from species other than the transgenic organism.

[0266] As used herein, "heterohybrid antibody" refers to an antibody having light and heavy chains of different biological origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.

[0267] The antibodies described herein are preferably separated. "Separated antibodies" used herein are intended to refer to antibodies that are substantially free of other antibodies with different antigenic specificities (e.g., antibodies that are specifically bound to tumor antigens are substantially free of antibodies that specifically bind to antigens that are different from tumor antigens). However, antibodies that are specifically bound to epitopes, isotypes or variants of human tumor antigens can have cross-reactivity with other related antigens (e.g., other related antigens from other species (e.g., tumor antigen species homologs)). In addition, the separated antibodies can be substantially free of other cellular materials and / or chemicals. In one embodiment of the invention, the combination of "separated" monoclonal antibodies relates to antibodies with different specificities and with clearly determined compositions or mixtures.

[0268] The term "antigen-binding portion" of an antibody (or simply "binding portion") or "antigen-binding fragment" of an antibody (or simply "binding fragment") or similar terms refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; (vi) isolated complementarity determining regions (CDRs); and (vii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected using recombinant methods through synthetic linkers, so that they can be made into a single protein chain, in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Another example is a binding domain immunoglobulin fusion protein, which comprises (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in US 2003 / 0118592 and US 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0269] The term "binding domain" in conjunction with the present invention characterizes the structure (of an antibody for example) which binds / interacts with a given target structure / antigen / epitope. Thus, a binding domain according to the present invention represents an "antigen interaction site".

[0270] All antibodies and antibody derivatives (eg, antibody fragments) described herein for the purposes of the present invention are encompassed within the term "antibody." The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of an antibody with other substances or antibodies, or an antibody fragment.

[0271] Naturally occurring antibodies are usually monospecific, i.e., they bind to a single antigen. The present invention includes antibodies that bind to a target cell (by engaging a tumor antigen) and a second entity such as a cytotoxic cell (e.g., by engaging a CD3 receptor). The antibodies of the present invention may be bispecific or multispecific - e.g., trispecific, tetraspecific, etc.

[0272] The term "bispecific molecule" is intended to include substances with two different binding specificities. For example, the molecule can bind to or interact with (a) a cell surface antigen and (b) a receptor on the surface of an effector cell (e.g., an Fc receptor). The term "multispecific molecule" is intended to include substances with more than two different binding specificities. For example, the molecule can bind to or interact with (a) a cell surface antigen, (b) a receptor on the surface of an effector cell (e.g., an Fc receptor), and (c) at least one other component. Therefore, the term "antibody against a tumor antigen" includes, but is not limited to, bispecific, trispecific, tetraspecific and other multispecific molecules against tumor antigens as well as other targets (e.g., Fc receptors on effector cells). The term "bispecific antibody" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0273] According to the present invention, the antibody can exert its therapeutic effect by recruiting the patient's immune system to destroy tumor cells and / or through the therapeutic moiety or therapeutic agent coupled to the antibody. For the purposes of the present invention, such antibody conjugates can be considered to be covered by the term "chemotherapeutic agent", while antibodies that exert their therapeutic effect by recruiting the patient's immune system to destroy tumor cells are not.

[0274] In the context of the present invention, the antibody is preferably capable of acting by recruiting the patient's immune system to destroy tumor cells, i.e. the antibody, in particular when bound to its target (e.g. a tumor antigen on a diseased cell), elicits immune effector functions as described herein. Preferably, the immune effector functions are directed against cells, such as cancer cells carrying a tumor antigen (e.g. CLDN18.2) on their surface.

[0275] In the context of the present invention, the term "immune effector function" includes any function mediated by a component of the immune system that results in, for example, inhibition of tumor growth and / or inhibition of tumorigenesis, including inhibition of tumor dissemination and metastasis. Preferably, the immune effector function results in killing cancer cells. Such functions include complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells carrying tumor antigens, cell lysis of cells carrying tumor antigens, and / or inhibition of proliferation of cells carrying tumor antigens. Binding agents can also simply act by binding to tumor antigens on the surface of cancer cells. For example, antibodies can block the function of tumor antigens or induce apoptosis simply by binding to tumor antigens on the surface of cancer cells.

[0276] Antibody-dependent cell-mediated cytotoxicity

[0277] ADCC describes the cell-killing ability of effector cells, particularly lymphocytes, which preferably requires that the target cells be labeled with antibodies.

[0278] ADCC preferably occurs when the antibody binds to the antigen on the tumor cell and the antibody Fc domain engages with the Fc receptor (FcR) on the surface of the immune effector cell. Several Fc receptor families have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be considered as a mechanism for direct induction of variable degrees of immediate tumor destruction leading to antigen presentation and induction of tumor-directed T cell responses. Preferably, the in vivo induction of ADCC will result in tumor-directed T cell responses and host-derived antibody responses.

[0279] Complement-dependent cytotoxicity

[0280] CDC is another cell killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are also very effective in directing CDC through the classical complement activation pathway. Preferably, in this cascade reaction, the formation of antigen-antibody complexes leads to the exposure of multiple C1q binding sites in close proximity on the CH2 domain of the antibody molecule (e.g., IgG molecule) (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low-affinity C1q-IgG interaction into a high-affinity interaction, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of effector cell chemotaxis / activators C3a and C5a. Preferably, the complement cascade ends with the formation of a membrane attack complex, which produces holes in the cell membrane that are conducive to the free entry and exit of water and solutes into and out of the cell.

[0281] To inhibit tumor growth and / or tumorigenesis, according to the present invention, the antibodies may be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radioisotope. Cytotoxins or cytotoxic agents include any agent that is harmful to cells and specifically kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, amanitin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Suitable therapeutic agents for forming antibody conjugates include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin, (formerly daunomycin and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A.

[0282] Antibodies can also be conjugated to radioactive isotopes (eg, iodine-131, yttrium-90, or indium-111) to produce cytotoxic radiopharmaceuticals.

[0283] The antibody conjugates of the present invention can be used to modulate a given biological response, and the drug moiety should not be construed as being limited to typical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. Such proteins can include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

[0284] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Ahalysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).

[0285] The term "antibodies against tumor antigens" or similar terms relate to antibodies against tumor antigens or having the ability to bind to tumor antigens. The term "binding" according to the present invention preferably relates to specific binding.

[0286] According to the present invention, an antibody is capable of binding to a predetermined target if it has significant affinity for the predetermined target and binds to it in a standard assay. This is usually measured by the equilibrium dissociation constant (K D) to measure "affinity" or "binding affinity". Preferably, the term "significant affinity" refers to a dissociation constant (K D ) binds to the intended target: 10 -5 M or smaller, 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, or 10 -12 M or smaller.

[0287] An antibody is (substantially) not capable of binding to a target if it has no significant affinity for the target and does not bind significantly, in particular undetectably, to the target in a standard assay. Preferably, an antibody does not detectably bind to the target if present in a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 μg / ml or more. Preferably, an antibody does not detectably bind to the target if its K of binding to the target is less than 10%. D is the K of the antibody binding to the intended target. D At least 10 times, 100 times, 10 3 times, 10 4 times, 10 5 times or 10 6 times higher, then it does not have significant affinity for the target. For example, if the K of an antibody binding to a target that the antibody is capable of binding to is D For 10 -7 M, then the K for binding to a target for which the antibody has no significant affinity D For at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 M.

[0288] If an antibody is able to bind to a predetermined target and it is not able to bind to other targets, i.e. it does not have a significant affinity for other targets and does not bind significantly thereto in standard assays, then the antibody is specific for the predetermined target. According to the invention, if an antibody is able to bind to a tumor antigen but is (substantially) not able to bind to other targets, then the antibody is specific for the tumor antigen. Preferably, the antibody is specific for a tumor antigen if the affinity and binding to such other targets do not significantly exceed the affinity or binding to a tumor antigen-unrelated protein, such as bovine serum albumin (BSA), casein, human serum albumin (HSA) or a non-tumor antigen transmembrane protein, such as an MHC molecule or transferrin receptor, or any other specified polypeptide. Preferably - if the K of the antibody binding to the predetermined target is D is the K that binds to its nonspecific target D At least 10 times, 100 times, 10 3 times, 10 4 times, 10 5 times or 10 6 fold lower, then the antibody is specific for the target. For example, if the K of an antibody binding to its specific target is D For 10 -7 M, then the K of its nonspecific target binding D For at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 M

[0289] The binding of an antibody to a target can be determined experimentally using any suitable method; see, e.g., Berzofsky et al., "Antibody-Antigen Interactions" in Fundamental Immunology, Paul, WE, ed., Raven Press New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992); and the methods described herein. Affinity can be readily determined using conventional techniques, such as by equilibrium dialysis; by using a BIAcore 2000 instrument using the general procedures outlined by the manufacturer; by radioimmunoassay using a radiolabeled target antigen; or by other methods known to the skilled artisan. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NY Acad. ScL, 51: 660 (1949). The measured affinity of a particular antibody-antigen interaction may vary if measured under different conditions (e.g., salt concentration, pH). Therefore, affinity and other antigen binding parameters (e.g., K D 、IC 50 ) is preferably measured using standardized solutions of antibodies and antigens and a standardized buffer.

[0290] As used herein, "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

[0291] As used herein, "isotype switching" refers to a phenomenon in which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

[0292] As used herein, the term "naturally occurring" when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man in the laboratory is naturally occurring.

[0293] As used herein, the term "rearranged" refers to the configuration of a heavy or light chain immunoglobulin locus in which a V segment is positioned adjacent to a DJ or J segment in a conformation that encodes essentially a complete VH or VL domain, respectively. Rearranged immunoglobulin (antibody) loci can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer / nonamer homology element.

[0294]

[00136] The term "unrearranged" or "germline configuration" as used herein with respect to a V segment refers to a configuration in which the V segment has not recombined to be immediately adjacent to a D or J segment.

[0295] Preferably, antibodies against tumor antigens bind to cells expressing tumor antigens to induce or mediate killing of cells expressing tumor antigens. Cells expressing tumor antigens are preferably cancer cells, and in particular cells of cancer diseases described herein. Preferably, antibodies induce or mediate cell killing by inducing one or more of the following: complement dependent cytotoxicity (CDC)-mediated lysis, antibody dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, and inhibition of proliferation of cells expressing tumor antigens. Preferably, ADCC-mediated cell lysis occurs in the presence of effector cells, which are selected from monocytes, mononuclear cells, NK cells, and PMNs in some specific embodiments. Inhibition of cell proliferation can be measured in vitro by determining cell proliferation in an assay using bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU). BrdU is a synthetic nucleoside that is an analog of thymidine and can be incorporated into newly synthesized DNA in replicating cells (during the S phase of the cell cycle), thereby replacing thymidine during DNA replication. Detection of the incorporated chemical using, for example, an antibody specific for BrdU indicates cells that are actively replicating their DNA.

[0296] In some preferred embodiments, the antibodies described herein may be characterized by one or more of the following properties:

[0297] a) Specificity for tumor antigens;

[0298] b) a binding affinity to a tumor antigen of about 100 nM or less, preferably about 5 nM to 10 nM or less, more preferably about 1 nM to 3 nM or less;

[0299] c) the ability to induce or mediate CDC on tumor antigen-positive cells;

[0300] d) the ability to induce or mediate ADCC on tumor antigen-positive cells;

[0301] e) the ability to inhibit the growth of tumor antigen-positive cells;

[0302] f) Ability to induce apoptosis of tumor antigen-positive cells.

[0303] In one embodiment, the antibody to a tumor antigen has the ability to bind to an epitope present in the tumor antigen (preferably an epitope located within the extracellular domain of the tumor antigen). Preferably, the antibody to a tumor antigen is specific to the tumor antigen. Preferably, the antibody to a tumor antigen binds to a tumor antigen expressed on the surface of a cell. In some particularly preferred embodiments, the antibody to a tumor antigen binds to a natural epitope of a tumor antigen present on the surface of a living cell.

[0304] According to the present invention, an antibody having the ability to bind to CLDN18.2 or an antibody against CLDN18.2 is an antibody capable of binding to an epitope present in CLDN18.2 (preferably an epitope located in the extracellular domain of CLDN18.2, in particular the first extracellular domain of CLDN18.2, preferably amino acids 29 to 78). In some particular embodiments, the antibody having the ability to bind to CLDN18.2 is an antibody capable of binding to the following epitopes: (i) an epitope on CLDN18.2 that is not present on CLDN18.1, preferably SEQ ID NOs: 3, 4 and 5; (ii) an epitope located on CLDN18.2-loop 1, preferably SEQ ID NO: 8; (iii) an epitope located on CLDN18.2-loop 2, preferably SEQ ID NO: 10; (iv) an epitope located on CLDN18.2-loop D3, preferably SEQ ID NO: 11; (v) an epitope comprising CLDN18.2-loop 1 and CLDN18.2-loop D3; or (vi) a non-glycosylated epitope located on CLDN18.2-loop D3, preferably SEQ ID NO: 9.

[0305] According to the present invention, the antibody having the ability to bind to CLDN18.2 is preferably an antibody having the ability to bind to CLDN18.2 but not to CLDN18.1. Preferably, the antibody having the ability to bind to CLDN18.2 is specific for CLDN18.2. Preferably-the antibody having the ability to bind to CLDN18.2 is an antibody having the ability to bind to CLDN18.2 expressed on the surface of a cell. In some particularly preferred embodiments, the antibody having the ability to bind to CLDN18.2 binds to a natural epitope of CLDN18.2 present on the surface of a living cell. Preferably, the antibody having the ability to bind to CLDN18.2 binds to one or more peptides selected from SEQ ID NOs: 1, 3 to 11, 44, 46 and 48 to 50. Preferably, the antibody having the ability to bind to CLDN18.2 is specific for the above-mentioned protein, peptide or immunogenic fragment or derivative thereof. The antibody having the ability to bind to CLDN18.2 can be obtained by a method comprising the following steps: immunizing an animal with a protein or peptide comprising an amino acid sequence selected from SEQ ID NOs: 1, 3 to 11, 44, 46 and 48 to 50, or a nucleic acid or host cell expressing the protein or peptide. Preferably, the antibody binds to cancer cells (particularly cells of the above-mentioned cancer types), and preferably does not substantially bind to non-cancer cells.

[0306] Preferably, an antibody having the ability to bind to CLDN18.2 binds to cells expressing CLDN18.2 to induce or mediate killing of cells expressing CLDN18.2. Cells expressing CLDN18.2 are preferably cancer cells, and are particularly selected from tumorigenic gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer cells. Preferably, the antibody induces or mediates cell killing by inducing one or more of the following: complement dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, and inhibition of proliferation of cells expressing CLDN18.2. Preferably, ADCC-mediated cell lysis occurs in the presence of effector cells, which are selected from monocytes, mononuclear cells, NK cells, and PMNs in some particular embodiments.

[0307] In some preferred embodiments, antibodies having the ability to bind to CLDN18.2 can be characterized by one or more of the following properties:

[0308] a) Specificity for CLDN18.2;

[0309] b) has a binding affinity to CLDN18.2 of about 100 nM or less, preferably about 5 nM to 10 nM or less, more preferably about 1 nM to 3 nM or less;

[0310] c) the ability to induce or mediate CDC on CLDN18.2-positive cells;

[0311] d) the ability to induce or mediate ADCC on CLDN18.2-positive cells;

[0312] e) ability to inhibit the growth of CLDN18.2-positive cells;

[0313] f) Ability to induce apoptosis of CLDN18.2 positive cells.

[0314] In a particularly preferred embodiment, the antibody having the ability to bind to CLDN18.2 is produced by a hybridoma deposited at DSMZ (Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) and having the following name and accession number:

[0315] a.182-D1106-055, accession number DSM ACC2737, deposited on October 19, 2005;

[0316] b.182-D1106-056, accession number DSM ACC2738, deposited on October 19, 2005;

[0317] c.182-D1106-057, accession number DSM ACC2739, deposited on October 19, 2005;

[0318] d.182-D1106-058, accession number DSM ACC2740, deposited on October 19, 2005;

[0319] e.182-D1106-059, accession number DSM ACC2741, deposited on October 19, 2005;

[0320] f.182-D1106-062, accession number DSM ACC2742, deposited on October 19, 2005;

[0321] g.182-D1106-067, accession number DSM ACC2743, deposited on October 19, 2005;

[0322] h.182-D758-035, accession number DSM ACC2745, deposited on November 17, 2005;

[0323] i.182-D758-036, accession number DSM ACC2746, deposited on November 17, 2005;

[0324] j.182-D758-040, accession number DSM ACC2747, deposited on November 17, 2005;

[0325] k.182-D1106-061, accession number DSM ACC2748, deposited on November 17, 2005;

[0326] l.182-D1106-279, accession number DSM ACC2808, deposited on October 26, 2006;

[0327] m.182-D1106-294, accession number DSM ACC2809, deposited on October 26, 2006;

[0328] n.182-D1106-362, accession number DSM ACC2810, deposited on October 26, 2006.

[0329] Some preferred antibodies according to the present invention are those produced by and obtainable from the above hybridomas: namely, 37G11 in the case of 182-D1106-055, 37H8 in the case of 182-D1106-056, 38G5 in the case of 182-D1106-057, 38H3 in the case of 182-D1106-058, 39F11 in the case of 182-D1106-059, 43A11 in the case of 182-D1106-062, 43A12 in the case of 182-D1106-067. 61C2 in the case of 182-D758-035, 26B5 in the case of 182-D758-036, 26D12 in the case of 182-D758-040, 42E12 in the case of 182-D1106-061, 125E1 in the case of 182-D1106-279, 163E12 in the case of 182-D1106-294, 175D10 in the case of 182-D1106-362, and chimeric and humanized forms thereof.

[0330] In one embodiment, the antibody having the ability to bind to CLDN18.2 is an antibody selected from the following: (i) an antibody produced by and / or obtainable from a clone deposited under the following accession numbers: DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809 or DSM ACC2810; (ii) an antibody that is a chimeric or humanized form of the antibody in (i); (iii) an antibody having the specificity of the antibody in (i); and (iv) an antibody comprising an antigen binding portion or antigen binding site (particularly a variable region) of the antibody in (i), and preferably having the specificity of the antibody in (i).

[0331] Preferred chimeric antibodies and their sequences are shown in the table below.

[0332]

[0333] In some preferred embodiments, the antibodies according to the present invention (particularly antibodies in chimeric form) include antibodies comprising a heavy chain constant region (CH), the heavy chain constant region comprising an amino acid sequence derived from a human heavy chain constant region, such as the amino acid sequence shown in SEQ ID NO: 13 or a fragment thereof. In other preferred embodiments, the antibodies according to the present invention (particularly antibodies in chimeric form) include antibodies comprising a light chain constant region (CL), the light chain constant region comprising an amino acid sequence derived from a human light chain constant region, such as the amino acid sequence shown in SEQ ID NO: 12 or a fragment thereof. In a particularly preferred embodiment, the antibodies according to the present invention (particularly antibodies in chimeric form) include antibodies comprising CH and comprising CL, the CH comprising an amino acid sequence derived from human CH, such as the amino acid sequence shown in SEQ ID NO: 13 or a fragment thereof, and the CL comprising an amino acid sequence derived from human CL, such as the amino acid sequence shown in SEQ ID NO: 12 or a fragment thereof.

[0334] In one embodiment, the antibody capable of binding to CLDN18.2 is a chimeric mouse / human IgG1 monoclonal antibody comprising a κ murine variable light chain, a human κ light chain constant region allotype Km(3), a murine heavy chain variable region, a human IgG1 constant region, allotype G1m(3).

[0335] In certain preferred embodiments, the chimeric form of the antibody includes an antibody comprising a heavy chain and / or a light chain, wherein the heavy chain comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 16, 17, 18, 19, 51 and fragments thereof, and the light chain comprises an amino acid sequence selected from SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 28 and fragments thereof.

[0336] In certain preferred embodiments, chimeric forms of antibodies include antibodies comprising a combination of heavy and light chains selected from the following possibilities (i) to (ix):

[0337] (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 14 or a fragment thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 21 or a fragment thereof;

[0338] (ii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 15 or a fragment thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 20 or a fragment thereof;

[0339] (iii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 16 or a fragment thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 22 or a fragment thereof;

[0340] (iv) the heavy chain comprises the amino acid sequence of SEQ ID NO: 18 or a fragment thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 25 or a fragment thereof;

[0341] (v) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 24 or a fragment thereof;

[0342] (vi) the heavy chain comprises the amino acid sequence of SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 23 or a fragment thereof;

[0343] (vii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 26 or a fragment thereof;

[0344] (viii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 19 or a fragment thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 27 or a fragment thereof;

[0345] (ix) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 19 or a fragment thereof and the light chain comprises the amino acid sequence shown in SEQ ID NO: 28 or a fragment thereof; and

[0346] (x) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 51 or a fragment thereof, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 24 or a fragment thereof.

[0347] Antibodies according to (v) or (x) are particularly preferred.

[0348] As used above, "fragment" or "fragment of an amino acid sequence" relates to a part of an antibody sequence, i.e., refers to a sequence of an antibody sequence shortened at the N-terminus and / or C-terminus, which when replacing said antibody sequence in an antibody retains the binding of said antibody to CLDN18.2, and preferably retains the function of said antibody described herein, such as CDC-mediated cleavage or ADCC-mediated cleavage. Preferably, a fragment of an amino acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98% or 99% of the amino acid residues from said amino acid sequence. A fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 16, 17, 18, 19, 51, 20, 21, 22, 23, 24, 25, 26, 27 and 28 preferably relates to said sequence in which 17, 18, 19, 20, 21, 22 or 23 amino acids at the N-terminus are removed.

[0349] In a preferred embodiment, the antibody capable of binding to CLDN18.2 comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NO: 29, 30, 31, 32, 33, 34 and fragments thereof.

[0350] In a preferred embodiment, the antibody capable of binding to CLDN18.2 comprises a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43 and fragments thereof.

[0351] In certain preferred embodiments, the antibody having the ability to bind to CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possibilities (i) to (ix):

[0352] (i) VH comprises the amino acid sequence of SEQ ID NO: 29 or a fragment thereof, and VL comprises the amino acid sequence of SEQ ID NO: 36 or a fragment thereof;

[0353] (ii) VH comprises the amino acid sequence set forth in SEQ ID NO: 30 or a fragment thereof, and VL comprises the amino acid sequence set forth in SEQ ID NO: 35 or a fragment thereof;

[0354] (iii) VH comprises the amino acid sequence set forth in SEQ ID NO: 31 or a fragment thereof, and VL comprises the amino acid sequence set forth in SEQ ID NO: 37 or a fragment thereof;

[0355] (iv) VH comprises the amino acid sequence set forth in SEQ ID NO:33 or a fragment thereof, and vL comprises the amino acid sequence set forth in SEQ ID NO:40 or a fragment thereof;

[0356] (v) VH comprises the amino acid sequence set forth in SEQ ID NO: 32 or a fragment thereof, and VL comprises the amino acid sequence set forth in SEQ ID NO: 39 or a fragment thereof;

[0357] (vi) VH comprises the amino acid sequence of SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence of SEQ ID NO: 38 or a fragment thereof;

[0358] (vii) VH comprises the amino acid sequence set forth in SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence set forth in SEQ ID NO: 41 or a fragment thereof;

[0359] (viii) VH comprises the amino acid sequence of SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence of SEQ ID NO: 42 or a fragment thereof;

[0360] (ix) VH comprises the amino acid sequence shown in SEQ ID NO: 34 or a fragment thereof, and VL comprises the amino acid sequence shown in SEQ ID NO: 43 or a fragment thereof.

[0361] The antibody according to (v) is particularly preferred.

[0362] According to the present invention, the term "fragment" refers in particular to one or more complementarity determining regions (CDRs), preferably at least CDR3 variable regions, of a heavy chain variable region (VH) and / or a light chain variable region (VL). In one embodiment, the one or more complementarity determining regions (CDRs) are selected from the group of complementarity determining regions CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the term "fragment" refers to complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0363] In a preferred embodiment, the antibody having the ability to bind to CLDN18.2 comprises a VH comprising a group of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (vi):

[0364] (i) CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14;

[0365] (ii) CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15;

[0366] (iii) CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16;

[0367] (iv) CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17;

[0368] (v) CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18; and

[0369] (vi) CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, and CDR3: positions 117 to 128 of SEQ ID NO: 19.

[0370] In a preferred embodiment, the antibody having the ability to bind to CLDN18.2 comprises a VL comprising a group of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix):

[0371] (i) CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20;

[0372] (ii) CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21;

[0373] (iii) CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22;

[0374] (iv) CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23;

[0375] (v) CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24;

[0376] (vi) CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25;

[0377] (vii) CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26;

[0378] (viii) CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27; and

[0379] (ix) CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0380] In a preferred embodiment, the antibody having the ability to bind to CLDN18.2 comprises a combination of VH and VL, each of which comprises a group of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix):

[0381] (i) VH: CDR1: positions 45 to 52 of SEQ ID NO: 14, CDR2: positions 70 to 77 of SEQ ID NO: 14, CDR3: positions 116 to 125 of SEQ ID NO: 14, VL: CDR1: positions 49 to 53 of SEQ ID NO: 21, CDR2: positions 71 to 73 of SEQ ID NO: 21, CDR3: positions 110 to 118 of SEQ ID NO: 21;

[0382] (ii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 15, CDR2: positions 70 to 77 of SEQ ID NO: 15, CDR3: positions 116 to 126 of SEQ ID NO: 15, VL: CDR1: positions 47 to 58 of SEQ ID NO: 20, CDR2: positions 76 to 78 of SEQ ID NO: 20, CDR3: positions 115 to 123 of SEQ ID NO: 20;

[0383] (iii) VH: CDR1: positions 45 to 52 of SEQ ID NO: 16, CDR2: positions 70 to 77 of SEQ ID NO: 16, CDR3: positions 116 to 124 of SEQ ID NO: 16, VL: CDR1: positions 47 to 52 of SEQ ID NO: 22, CDR2: positions 70 to 72 of SEQ ID NO: 22, CDR3: positions 109 to 117 of SEQ ID NO: 22;

[0384] (iv) VH: CDR1: positions 44 to 51 of SEQ ID NO: 18, CDR2: positions 69 to 76 of SEQ ID NO: 18, CDR3: positions 115 to 125 of SEQ ID NO: 18, VL: CDR1: positions 47 to 58 of SEQ ID NO: 25, CDR2: positions 76 to 78 of SEQ ID NO: 25, CDR3: positions 115 to 122 of SEQ ID NO: 25;

[0385] (v) VH: CDR1: positions 45 to 52 of SEQ ID NO: 17, CDR2: positions 70 to 77 of SEQ ID NO: 17, CDR3: positions 116 to 126 of SEQ ID NO: 17, VL: CDR1: positions 47 to 58 of SEQ ID NO: 24, CDR2: positions 76 to 78 of SEQ ID NO: 24, CDR3: positions 115 to 123 of SEQ ID NO: 24;

[0386] (vi) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 23, CDR2: positions 76 to 78 of SEQ ID NO: 23, CDR3: positions 115 to 123 of SEQ ID NO: 23;

[0387] (vii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, VL: CDR1: positions 47 to 58 of SEQ ID NO: 26, CDR2: positions 76 to 78 of SEQ ID NO: 26, CDR3: positions 115 to 123 of SEQ ID NO: 26;

[0388] (viii) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19 - VL: CDR1: positions 47 to 58 of SEQ ID NO: 27, CDR2: positions 76 to 78 of SEQ ID NO: 27, CDR3: positions 115 to 123 of SEQ ID NO: 27; and

[0389] (ix) VH: CDR1: positions 45 to 53 of SEQ ID NO: 19, CDR2: positions 71 to 78 of SEQ ID NO: 19, CDR3: positions 117 to 128 of SEQ ID NO: 19, vL: CDR1: positions 47 to 52 of SEQ ID NO: 28, CDR2: positions 70 to 72 of SEQ ID NO: 28, CDR3: positions 109 to 117 of SEQ ID NO: 28.

[0390] In other preferred embodiments, the antibody having the ability to bind to CLDN18.2 preferably comprises one or more complementarity determining regions (CDRs) of the heavy chain variable region (VH) and / or the light chain variable region (VL) of a monoclonal antibody against CLDN18.2 (preferably a monoclonal antibody against CLDN18.2 described herein), preferably at least the CDR3 variable region, and preferably comprises one or more complementarity determining regions (CDRs) of the heavy chain variable region (VH) and / or the light chain variable region (VL) described herein, preferably at least the CDR3 variable region. In one embodiment, the one or more complementarity determining regions (CDRs) are selected from the group of complementarity determining regions CDR1, CDR2 and CDR3 described herein. In a particularly preferred embodiment, the antibody having the ability to bind to CLDN18.2 preferably comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) of a monoclonal antibody against CLDN18.2 (preferably the monoclonal antibody against CLDN18.2 described herein), and preferably comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or light chain variable region (VL) described herein.

[0391] In one embodiment, an antibody comprising one or more CDRs, CDR groups or combinations of CDR groups as described herein comprises said CDRs and intervening framework regions. Preferably - the portion will also include at least about 50% of either or both of the first and fourth framework regions - the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Antibody construction by recombinant DNA techniques may result in the introduction of residues encoded by linkers introduced to facilitate cloning or other manipulation steps at the N-terminus or C-terminus of the variable region, including the introduction of linkers connecting the variable region of the invention to additional protein sequences, including immunoglobulin heavy chains, other variable regions (e.g., in the production of double-chain antibodies) or protein tags.

[0392] In one embodiment, an antibody comprising one or more CDRs, sets of CDRs or combinations of sets of CDRs described herein comprises said CDRs in a human antibody framework.

[0393] Mention herein that an antibody comprises a specific chain or a specific region or sequence in relation to its heavy chain preferably relates to the situation wherein all heavy chains of said antibody comprise said specific chain, region or sequence. This applies correspondingly to the light chain of the antibody.

[0394] It should be understood that the antibodies described herein can be delivered to the patient by administering nucleic acid (e.g., RNA) encoding the antibody and / or by administering a host cell comprising nucleic acid (e.g., RNA) encoding the antibody. Therefore, when administered to the patient, the nucleic acid encoding the antibody can be in a naked form or in a suitable delivery vehicle (e.g., in the form of liposomes or viral particles), or present in a host cell. The nucleic acid provided can produce antibodies in a continuous manner over a long period of time-thereby at least partially alleviating the instability observed for therapeutic antibodies. The nucleic acid to be delivered to the patient can be produced by recombinant means. If the nucleic acid is administered to the patient when not present in the host cell, it is preferably taken up by the patient's cells for expressing the antibody encoded by the nucleic acid. If the nucleic acid is administered to the patient while being present in the host cell, it is preferably expressed by the host cell in the patient, thereby producing the antibody encoded by the nucleic acid.

[0395] As used herein, the term "nucleic acid" is intended to include DNA and RNA - e.g., genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0396] Nucleic acid can be contained in a vector. The term "vector" used herein includes any vector known to the technician, including a plasmid vector, a cosmid vector, a phage vector (e.g., lambda phage), a viral vector (e.g., an adenovirus or a baculovirus vector) or an artificial chromosome vector (e.g., a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC) or a P1 artificial chromosome (PAC)). The vector includes an expression vector and a cloning vector. The expression vector comprises a plasmid and a viral vector, and generally contains a desired coding sequence and a suitable DNA sequence necessary for expressing an operably connected coding sequence in a specific host organism (e.g., a bacterium, a yeast, a plant, an insect or a mammal) or in an in vitro expression system. Cloning vectors are generally used for transforming and amplifying a certain desired DNA fragment, and may lack the functional sequence required for expressing the desired DNA fragment.

[0397] In the context of the present invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA, substantially pure RNA), synthetic RNA, recombinantly produced RNA, and modified RNA that is different from naturally occurring RNA by adding, deleting, replacing, and / or changing one or more nucleotides. Such changes may include the addition of non-nucleotide substances, such as addition to the end or interior of the RNA, such as at one or more nucleotides of the RNA. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0398] According to the present invention, the term "RNA" includes and preferably relates to "mRNA", which means "messenger RNA" and relates to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. mRNA generally comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to technicians. For example, there are a variety of commercially available in vitro transcription kits.

[0399] In order to improve the expression and / or stability of the RNA used according to the invention, it can be modified, preferably without changing the sequence of the expressed peptide or protein.

[0400] In the case of RNA used according to the invention, the term "modification" includes any RNA modification that is not naturally present in the RNA. Such modified RNA is encompassed by the term "RNA" herein.

[0401] For example, the RNA according to the invention may have modified naturally occurring or synthetic ribonucleotides to improve its stability and / or reduce cytotoxicity. For example, in one embodiment, in the RNA used according to the invention, 5-methylcytidine partially or completely (preferably completely) replaces cytidine. As an alternative or supplement, in one embodiment, in the RNA used according to the invention, pseudouridine partially or completely (preferably completely) replaces uridine.

[0402] In one embodiment, the term "modification" refers to providing a 5'-cap or a 5'-cap analog to RNA. The term "5'-cap" refers to a cap structure found on the 5' end of an mRNA molecule, and is generally composed of a guanosine nucleotide connected to the mRNA by an uncommon 5'-5' triphosphate connection. In one embodiment, the guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap (m7G). In the context of the present invention, the term "5'-cap" includes 5'-cap analogs that are similar to RNA cap structures and are modified to have the ability to stabilize RNA (if attached to RNA, preferably in vivo and / or in cells).

[0403] Preferably, the RNA expresses its encoded protein or peptide if delivered to (ie, transfected into) a cell, particularly a cell present in vivo.

[0404] The term "transfection" refers to the introduction of nucleic acid (particularly RNA) into a cell. For the purposes of the present invention, the term "transfection" also includes the introduction of nucleic acid into a cell or the uptake of nucleic acid by such a cell, wherein the cell may be present in a subject (e.g., a patient). Therefore, according to the present invention, the cell used to transfect the nucleic acid described herein may be present in vitro or in vivo, for example, the cell may form a part of an organ, tissue and / or organism of the patient. According to the present invention, transfection may be transient or stable. For some applications of transfection, it is sufficient as long as the transfected genetic material is only transiently expressed. Since the nucleic acid introduced during the transfection process is usually not integrated into the nuclear genome, the exogenous nucleic acid will be diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acid greatly reduce the dilution rate. If the desired transfected nucleic acid is actually retained in the genome of the cell and its daughter cells, a stable transfection must be performed. RNA can be transfected into cells to transiently express its encoded protein.

[0405] The term "stability" of RNA relates to the "half-life" of the RNA. "Half-life" relates to the period of time required to eliminate half of the activity, amount or number of a molecule. In the context of the present invention, the half-life of an RNA indicates the stability of the RNA. The half-life of an RNA can affect the "duration of expression" of the RNA. It can be expected that an RNA with a long half-life will be expressed for a long time.

[0406] In the context of the present invention, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", wherein the term "in vitro transcription" refers to a process in which RNA (particularly mRNA) is synthesized in vitro in a cell-free system (preferably using a suitable cell extract). Preferably, a cloning vector is used to produce the transcript. These cloning vectors are generally referred to as transcription vectors - and are encompassed within the term "vector" according to the present invention.

[0407] According to the present invention, the term "translation" relates to the process by which a chain of messenger RNA directs the assembly of an amino acid sequence in a cell's ribosomes to produce a peptide or protein.

[0408] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and / or peptides or proteins, for example, by transcription and / or translation. With respect to RNA, the term "expression" or "translation" particularly relates to the production of peptides or proteins. It also includes partial expression of nucleic acids. In addition, expression can be transient or stable. According to the present invention, the term expression also includes "abnormal expression" or "abnormal expression".

[0409] According to the present invention, "abnormal expression" or "abnormal expression" means that the expression is changed (preferably increased) compared to a reference (e.g., a state in a subject not suffering from a disease associated with abnormal or abnormal expression of a certain protein (e.g., a tumor antigen). Increased expression means an increase of at least 10%, particularly at least 20%, at least 50% or at least 100%, or more. In one embodiment, expression is present only in diseased tissues, while expression in healthy tissues is suppressed.

[0410] The term "specific expression" refers to that a protein is essentially expressed only in a specific tissue or organ. For example, a tumor antigen specifically expressed in gastric mucosa means that the protein is mainly expressed in gastric mucosa, and is not expressed in other tissues, or is not expressed to a significant degree in other tissues or organ types. Therefore, a protein that is only expressed in cells of gastric mucosa and expressed at a significantly lower degree in any other tissue (e.g., testis) is specifically expressed in cells of gastric mucosa. In some embodiments, tumor antigens may also be specifically expressed in more than one tissue type or organ (e.g., in 2 or 3 tissue types or organs, but preferably in no more than 3 different tissues or organ types) under normal conditions. In this case, the tumor antigen is specifically expressed in these organs. For example, if a tumor antigen is preferably expressed to an approximately equal degree in the lungs and stomach under normal conditions, the tumor antigen is specifically expressed in the lungs and stomach.

[0411] According to the present invention, the term "RNA encoding" means that RNA, if present in a suitable environment (preferably within a cell), can be expressed to produce the protein or peptide it encodes.

[0412] Some aspects of the invention rely on adoptive transfer of host cells: host cells are transfected in vitro with nucleic acids (e.g., RNA) encoding the antibodies described herein and transferred (preferably after ex vivo expansion from low precursor frequencies to clinically relevant cell numbers) to a recipient (e.g., a patient). Host cells used for treatment according to the invention may be autologous, allogeneic, or syngeneic relative to the recipient being treated.

[0413] The term "autologous" is used to describe any material that comes from the same subject. For example, an "autologous transplant" refers to a transplant of tissue or organ from the same subject. Such a procedure is advantageous because it overcomes immune barriers that would otherwise lead to rejection.

[0414] The term "allogeneic" is used to describe any material from different individuals of the same species. Two or more individuals are considered to be allogeneic to each other when the genes at one or more loci are not identical.

[0415] The term "isogenic" is used to describe any material from individuals or tissues having the same genotype, ie, identical twins or animals or tissues thereof from the same inbred strain.

[0416] The term "heterologous" is used to describe something that is composed of multiple different elements. As an example, transferring the bone marrow of one individual into a different individual constitutes a heterologous transplant. Heterologous genes are genes that come from a source other than the subject.

[0417] According to the present invention, the term "peptide" comprises oligopeptides and polypeptides and refers to a substance comprising 2 or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more, and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids covalently linked by peptide bonds. The term "protein" refers to large peptides, preferably peptides having more than 100 amino acid residues, but in general the terms "peptide" and "protein" are synonymous and are used interchangeably herein.

[0418] The teachings given herein regarding specific amino acid sequences (such as those shown in the sequence listing) should be interpreted as also relating to variants of said specific sequences, which produce sequences that are functionally equivalent to said specific sequences, such as amino acid sequences that exhibit properties that are the same or similar to those of the specific amino acid sequence.

[0419] An important property is to retain the binding of the antibody to its target or maintain the effector function of the antibody. Preferably, a sequence that is a variant relative to a specific sequence retains the binding of the antibody to its target when it replaces the specific sequence in the antibody, and preferably retains the function of the antibody described herein, such as CDC-mediated cleavage or ADCC-mediated cleavage.

[0420] Those skilled in the art will appreciate that, in particular, the sequences of the CDRs, hypervariable regions and variable regions may be modified without losing the ability of the antibody to bind to its target. For example, the CDR regions are identical or highly homologous to the regions of the antibodies specified herein. "Highly homologous" means that 1 to 5, preferably 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in the CDRs. In addition, the hypervariable regions and variable regions may be modified such that they show significant homology to the regions of the antibodies specifically disclosed herein.

[0421] The term "variant" according to the present invention refers in particular to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologues, in particular those occurring naturally. Allelic variants relate to changes in the normal sequence of a gene, the meaning of which is often unclear. Whole gene sequencing generally identifies a variety of allelic variants of a given gene. Species homologues are nucleic acids or amino acid sequences that have a different species origin from a given nucleic acid or amino acid sequence. The term "variant" should encompass any post-translationally modified variants and conformational variants.

[0422] For the purposes of the present invention, "variants" of amino acid sequences include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. Amino acid deletion variants comprising deletions at the N-terminus and / or C-terminus of a protein are also referred to as N-terminal and / or C-terminal truncated variants.

[0423] Amino acid insertion variants include the insertion of a single or two or more amino acids in a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site of the amino acid sequence, but random insertion and appropriate screening of the resulting products are also possible.

[0424] Amino acid addition variants comprise amino-terminal and / or carboxyl-terminal fusions of one or more amino acids (eg, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids).

[0425] Amino acid deletion variants are characterized by removing one or more amino acids from the sequence, for example, by removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position of the protein.

[0426] The amino acid substitution variant is characterized in that at least one residue in the sequence is removed and another residue is inserted in its place. It is preferred to modify the position that is not conserved between homologous proteins or peptides in the amino acid sequence and / or replace the amino acid with other amino acids with similar properties. Preferably, the amino acid changes in the protein variant are conservative amino acid changes, that is, the replacement of similar charged or uncharged amino acids. Conservative amino acid changes involve the replacement of one of the families of amino acids related to its side chain. Generally speaking, naturally occurring amino acids are divided into the following four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Sometimes, phenylalanine, tryptophan and tyrosine are collectively classified as aromatic amino acids.

[0427] Preferably, the degree of similarity (preferably identity) between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Preferably - the degree of similarity or identity is given to an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, then preferably the similarity or identity degree is given to at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably contiguous amino acids. In some preferred embodiments, the similarity or identity degree is given to the total length of the reference amino acid sequence. Comparisons for determining sequence similarity (preferably sequence identity) can be performed using tools known in the art, preferably using optimal sequence alignment, such as using Align, using standard settings (preferably EMBOSS::needle, matrix: Blosum62, gap opening 10.0, gap extension 0.5).

[0428] "Sequence similarity" refers to the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0429] The term "identity percentage" is intended to indicate the percentage of identical amino acid residues between two sequences to be compared obtained after optimal alignment, which percentage is purely statistical, the differences between the two sequences being randomly distributed and over their entire length. The sequence comparison between two amino acid sequences is conventionally performed by comparing these sequences after optimal alignment, the comparison being performed by segments or by a "comparison window" to identify and compare local regions with sequence similarity. Optimal alignments of sequences for comparison can be generated, in addition to manual work, by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482; by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444; or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0430] The percent identity is calculated by determining the number of identical positions between the two compared sequences, dividing that number by the number of compared positions, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0431] The term "cell" or "host cell" preferably relates to intact cells, i.e. cells with intact membranes that have not released their normal intracellular components (e.g. enzymes, organelles or genetic material). Intact cells are preferably living cells, i.e. living cells that can perform their normal metabolic functions. Preferably, according to the present invention, the term relates to any cell that can be transfected with exogenous nucleic acids. Preferably, the cell can express the nucleic acid in the recipient when it is transfected with exogenous nucleic acids and transferred into the recipient. The term "cell" includes bacterial cells; other available cells are yeast cells, fungal cells or mammalian cells. Suitable bacterial cells include cells from: Gram-negative bacterial strains, such as strains of Escherichia coli, Proteus and Pseudomonas; and Gram-positive bacterial strains, such as strains of Bacillus, Streptomyces, Staphylococcus and Lactococcus. Suitable fungal cells include cells from Trichoderma, Neurospora and Aspergillus species. Suitable yeast cells include cells from Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica) and Hansenula species. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK, etc. However, amphibian cells, insect cells, plant cells, and any other cells in the art for expressing heterologous proteins may also be used. Mammalian cells are particularly preferred for adoptive transfer, such as cells from humans, mice, hamsters, pigs, goats, and primates. Cells can be derived from a large number of tissue types, and include primary cells and cell lines, such as cells of the immune system (particularly antigen presenting cells, such as dendritic cells and T cells), stem cells (such as hematopoietic stem cells and mesenchymal stem cells), and other cell types. Antigen presenting cells are cells that display antigens on their surface in the context of a major histocompatibility complex. T cells can recognize this complex using their T cell receptors (TCRs).

[0432] The term "transgenic animal" refers to an animal having a genome comprising one or more transgenes (preferably heavy and / or light chain transgenes) or a transchromosome (integrated or not integrated into the natural genomic DNA of the animal) and preferably capable of expressing the transgene. For example, a transgenic mouse may have a human light chain transgene and a human heavy chain transgene or a human heavy chain transchromosome, so that when immunized with a tumor antigen and / or a cell expressing a tumor antigen, the mouse produces human anti-tumor antigen antibodies. The human heavy chain transgene may be integrated into the chromosomal DNA of the mouse, as in the case of a transgenic mouse (e.g., HuMAb mouse-such as HCo7 or HCo12 mouse), or the human heavy chain transgene may be maintained outside the chromosome, as in the case of a transchromosome (e.g., KM) mouse described in WO 02 / 43478. Such a transgenic and transchromosome mouse may be capable of producing a variety of isotypes (e.g., IgG, IgA and / or IgE) of human monoclonal antibodies against tumor antigens by performing VDJ recombination and isotype conversion.

[0433] As used herein, "reduce," "lower," or "inhibit" means an overall decrease or the ability to cause an overall decrease in a level (e.g., expression level or cell proliferation level) of preferably 5% or greater, 10% or greater, 20% or greater, more preferably 50% or greater, and most preferably 75% or greater.

[0434] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even higher.

[0435] Antibodies described herein can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although the somatic cell hybridization method is preferred, in principle, other techniques for producing monoclonal antibodies can be adopted, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries.

[0436] The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

[0437] Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (described, for example, in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995); see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0438] In another preferred embodiment, transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system can be used to produce human monoclonal antibodies. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice". The production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in WO2004035607.

[0439] Another strategy for generating monoclonal antibodies is to directly isolate the gene encoding the antibody from lymphocytes producing antibodies with defined specificities, for example, see Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For details on recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

[0440] In order to produce antibodies, mice can be immunized with carrier-conjugated peptides from antigenic sequences (i.e., sequences to which antibodies are directed), enriched preparations of recombinantly expressed antigens or fragments thereof, and / or cells expressing antigens as described. Alternatively, mice can be immunized with DNA encoding antigens or fragments thereof. If immunization with purified preparations or enriched preparations of antigens does not produce antibodies, mice can also be immunized with cells expressing antigens (e.g., cell lines) to promote immune responses.

[0441] Plasma and serum samples obtained by tail vein or retro-orbital bleeding can be used to monitor immune responses during the course of the immunization protocol. Mice with adequate immunoglobulin titers can be used for fusions. Three days before sacrifice and spleen removal, mice can be boosted intraperitoneally or intravenously with antigen-expressing cells to increase the proportion of specific antibody-secreting hybridomas.

[0442] In order to produce hybridomas producing monoclonal antibodies, splenocytes and lymph node cells can be isolated from mice immunized and fused with suitable immortalized cell lines (e.g., mouse myeloma cell lines). Then, the hybridomas obtained can be screened for the production of antigen-specific antibodies. Then, single wells can be screened by ELISA to obtain hybridomas that secrete antibodies. Antigen-expressing cells can be used to identify antibodies that are specific to antigens by immunofluorescence and FACS analysis. Hybridomas that secrete antibodies can be re-plated, screened again, and if still positive for monoclonal antibodies, subclones can be performed by limiting dilution. Then, stable subclones can be cultured in vitro in tissue culture medium to produce antibodies for characterization.

[0443] Antibodies may also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods, as is known in the art (Morrison, S. (1985) Science 229: 1202).

[0444] For example, in one embodiment, the target gene (e.g., antibody gene) can be connected to an expression vector (e.g., eukaryotic expression plasmid), such as the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP338841, or other expression systems known in the art. The purified plasmid with the cloned antibody gene can be introduced into a eukaryotic host cell (e.g., CHO cell, NS / 0 cell, HEK293T cell or HEK293 cell or other eukaryotic cells (e.g., cells from plants, fungi or yeast cells) as an alternative). The method for introducing these genes can be the method described in the art, such as electroporation, lipofectine, lipofectamine, etc. After these antibody genes are introduced into host cells, cells expressing antibodies can be identified and selected. These cell representatives can then be amplified and scaled up to produce antibodies according to their expression levels. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0445] Alternatively, cloned antibody genes may be expressed in other expression systems, including prokaryotic cells, e.g., microorganisms such as E. coli. In addition, antibodies may be produced in non-human transgenic animals, e.g., in milk from sheep and rabbits or in eggs from hens, or in transgenic plants; see, e.g., Verma, R., et al. (1998) J. Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R, et al. (1999) Biol. Chem. 380: 825-839.

[0446] Chimerism

[0447] When repeatedly applied, mouse antibodies are highly immunogenic in people, resulting in reduced therapeutic effects. The main immunogenicity is mediated by the heavy chain constant region. If the corresponding antibody is chimeric or humanized, the immunogenicity of mouse antibodies in people can be reduced or completely avoided. Chimeric antibodies are antibodies whose different parts come from different animal species, such as those with variable regions from mouse antibodies and human immunoglobulin constant regions. The chimeric nature of antibodies is achieved by connecting the variable regions of mouse antibody heavy and light chains with the constant regions of human heavy and light chains (e.g., as described in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, chimeric antibodies are produced by connecting human κ-light chain constant regions to mouse light chain variable regions. In an equally preferred embodiment, chimeric antibodies can be produced by connecting human λ-light chain constant regions to mouse light chain variable regions. The preferred heavy chain constant regions for producing chimeric antibodies are IgG1, IgG3 and IgG4. Other preferred heavy chain constant regions for generating chimeric antibodies are IgG2, IgA, IgD and IgM.

[0448] Humanization

[0449] Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). Therefore, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Since the CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific naturally occurring antibody can be expressed by constructing an expression vector comprising CDR sequences from a specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of high affinity secondary library antibodies that are evenly distributed in the variable regions alone.

[0450] The ability of an antibody to bind antigen can be determined using standard binding assays (eg, ELISA, Western blot, immunofluorescence, and flow cytometric analysis).

[0451] In order to purify the antibody, the selected hybridoma can be cultured in a two-liter spinner bottle to purify the monoclonal antibody. Alternatively, the antibody can be produced in a dialysis-based bioreactor. The supernatant can be filtered and (if necessary) concentrated, followed by affinity chromatography with protein G agarose or protein A agarose. The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be replaced with PBS, and the concentration can be determined by OD280 using a 1.43 extinction coefficient. The monoclonal antibody can be divided into aliquots and stored at -80 ° C.

[0452] To determine whether a selected monoclonal antibody binds to a unique epitope, site-directed mutagenesis or multiple site-directed mutagenesis can be used.

[0453] In order to determine the isotype of the antibody, isotype ELISA can be performed with a variety of commercial kits (e.g., Zymed, Roche Diagnostics). The wells of the microtiter plate can be coated with anti-mouse Ig. After blocking, the plate is reacted with a monoclonal antibody or purified isotype control at ambient temperature for 2 hours. The wells can then be reacted with a mouse IgG1, IgG2a, IgG2b or IgG3, IgA or mouse IgM specific peroxidase conjugated probe. After washing, the plate can be developed with ABTS substrate (1 mg / ml) and analyzed at an OD of 405 to 650. Alternatively, the IsoStrip mouse monoclonal antibody isotype typing kit (Roche, catalog number 1493027) can be used as described by the manufacturer.

[0454] In order to prove the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to living cells expressing antigens, flow cytometry can be used. Cell lines expressing antigens naturally or after transfection and negative controls lacking antigen expression (cultured under standard culture conditions) can be mixed with different concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS, and can be incubated at 4°C for 30 minutes. After washing, anti-IgG antibodies labeled with APC or Alexa647 can bind to monoclonal antibodies bound to antigens under the same conditions as primary antibody staining. Samples can be analyzed by flow cytometry using a FACS instrument using light and side scatter properties to set gates for single living cells. In order to distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, a co-transfection method can be used. Cells transiently transfected with plasmids encoding antigens and fluorescent markers can be stained as described above. Transfected cells can be detected in a fluorescent channel different from cells stained with antibodies. Since most transfected cells express both transgenes, antigen-specific monoclonal antibodies preferentially bind to cells expressing the fluorescent marker, whereas non-specific antibodies bind to non-transfected cells in comparable proportions. In addition to or in lieu of flow cytometry assays, alternative assays utilizing fluorescence microscopy can be used. Cells can be accurately stained exactly as described above and examined by fluorescence microscopy.

[0455] To demonstrate the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to live cells expressing the antigen, immunofluorescence microscopy analysis can be used. For example, cell lines expressing the antigen spontaneously or after transfection and negative controls lacking antigen expression are cultured in chamber slides under standard culture conditions in DMEM / F12 medium supplemented with 10% fetal calf serum (FCS), 2mM L-glutamine, 100IU / mL penicillin and 100μg / mL streptomycin. The cells are then fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with monoclonal antibodies against the antigen at 25°C for 30 minutes. After washing, the cells are reacted with an anti-mouse IgG secondary antibody (Molecular Probes) labeled with Alexa555 under the same conditions. The cells are then examined by fluorescence microscopy.

[0456] Cell extracts from cells expressing the antigen and suitable negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. Anti-mouse IgG peroxidase can be used to detect IgG binding and visualized with ECL substrate.

[0457] The reactivity of the antibody to the antigen can also be tested by immunohistochemistry in a manner well known to the skilled artisan, for example, using paraformaldehyde- or acetone-fixed frozen sections or paraformaldehyde-fixed paraffin-embedded tissue sections from non-cancerous or cancerous tissue samples obtained from patients during routine surgical procedures or from mice bearing xenograft tumors inoculated with cell lines expressing the antigen either spontaneously or after transfection. For immunostaining, antibodies reactive with the antigen can be incubated followed by incubation with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the supplier's instructions.

[0458] The antibodies can be tested for their ability to mediate phagocytosis and killing of cells expressing tumor antigens. In vitro testing of monoclonal antibody activity will provide an initial screen prior to testing in vivo models.

[0459] Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0460] Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear cells, or other effector cells from healthy donors can be purified by Ficoll Hypaque density centrifugation followed by lysis of contaminating erythrocytes. Washed effector cells can be suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum or, alternatively, 5% heat-inactivated human serum and incubated with the treated cells at various effector:target ratios. 51 Cr-labeled target cells expressing tumor antigens are mixed. Alternatively, target cells can be labeled with a fluorescence enhancing ligand (BATDA). The highly fluorescent chelate of europium to the enhancing ligand released from dead cells can be measured by a fluorimeter. Another alternative technique can utilize transfection of target cells with luciferase. Only living cells can then oxidize the added lucifer yellow. Purified anti-tumor antigen IgG can then be added at different concentrations. An irrelevant human IgG can be used as a negative control. Depending on the type of effector cells used, the assay can be performed at 37°C for 4 to 20 hours. The fluorescence can be measured by measuring the presence of luciferase in the culture supernatant. 51 Cell lysis of a sample can be determined by the presence of Cr release or EuTDA chelates. Alternatively, luminescence resulting from oxidation of Lucifer Yellow can be a measure of viable cells.

[0461] Anti-tumor antigen monoclonal antibodies can also be tested in various combinations to determine whether cell lysis is enhanced with multiple monoclonal antibodies.

[0462] Complement-dependent cytotoxicity (CDC)

[0463] The ability of monoclonal anti-tumor antigen antibodies to mediate CDC can be tested using a variety of known techniques. For example, complement serum can be obtained from blood in a manner known to the skilled artisan. In order to determine the CDC activity of mAbs, different methods can be used. For example,51 Cr release or increased membrane permeability can be assessed using a propidium iodide (PI) exclusion assay. Briefly, target cells can be washed and 5 × 10 5 / mL and incubate with different concentrations of mAb at room temperature or at 37°C for 10 to 30 minutes. Serum or plasma can then be added to a final concentration of 20% (v / v) and the cells can be incubated at 37°C for 20 to 30 minutes. All cells from each sample can be added to the PI solution in the FACS tube. The mixture can then be immediately analyzed by flow cytometry analysis using FACSArray.

[0464] In an alternative assay, the induction of CDC can be determined on adherent cells. In one embodiment of this assay, 24 hours prior to the assay, 3×10 4 Cells were inoculated in tissue culture flat-bottom microtiter plates at a density of 1 / well. The next day, growth medium was removed and cells were incubated with antibodies in triplicate. Control cells were incubated with growth medium or a growth medium comprising 0.2% saponin for determining background lysis and maximum lysis, respectively. After incubation at room temperature for 20 minutes, supernatant was removed, and 20% (v / v) human plasma or serum (pre-warmed to 37°C) in DMEM was added to cells and incubated for another 20 minutes at 37°C. All cells from each sample were added to propidium iodide solution (10 μg / mL). Then, supernatant was replaced with PBS containing 2.5 μg / ml ethidium bromide, and the fluorescence emission after excitation at 520 nm was measured using Tecan Satire at 600 nm. Specific lysis percentage was calculated as follows: specific lysis %=(sample fluorescence-background fluorescence) / (maximum lysis fluorescence-background fluorescence)×100.

[0465] Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies

[0466] Monoclonal anti-tumor antigen antibodies can be incubated, for example, with tumor antigen-positive tumor cells or tumor cells transfected with tumor antigens at 37°C for about 20 hours to test the ability to induce apoptosis. Cells can be harvested, washed in annexin-V binding buffer (BD biosciences), and incubated in the dark for 15 minutes with annexin-V (BD biosciences) conjugated to FITC or APC. All cells from each sample can be added to the PI solution (10 μg / ml in PBS) in the FACS tube and immediately evaluated by flow cytometry (as above). Alternatively, the general inhibition of cell proliferation by monoclonal antibodies can be detected with commercially available kits. The DELFIA cell proliferation kit (Perkin-Elmer, catalog number AD0200) is a non-isotopic immunoassay based on measuring 5-bromo-2'-deoxyuridine (BrdU) incorporated during DNA synthesis of proliferating cells in a microplate. Monoclonal antibodies labeled with europium are used to detect incorporated BrdU. Cells are fixed using a fixative solution and DNA is denatured to allow antibody detection. Unbound antibody is washed away and DELFIA Inducer is added to dissociate the europium ions from the labeled antibody into solution where they form highly fluorescent chelates with components of the DELFIA Inducer. The fluorescence measured in the assay using time-resolved fluorimetry is proportional to DNA synthesis in the cells of each well.

[0467] Preclinical research

[0468] The antibodies described herein can also be tested in in vivo models (eg, in immunodeficient mice bearing xenograft tumors inoculated with a cell line expressing the tumor antigen) to determine their efficacy in controlling the growth of tumor cells expressing the tumor antigen.

[0469] After the tumor cells expressing tumor antigens are xenotransplanted into immunocompromised mice or other animals, the antibodies described herein can be used for in vivo studies. Antibodies can be administered to tumor-free mice, followed by injection of tumor cells to measure the effect of the antibodies in preventing tumor formation or tumor-related symptoms. Antibodies can be administered to mice carrying tumors to determine the therapeutic efficacy of the corresponding antibodies in reducing tumor growth, metastasis or tumor-related symptoms. Antibody administration can be combined with the administration of other substances (such as cytostatic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors or other antibodies) to determine the synergistic efficacy and potential toxicity of the combination. In order to analyze the toxic side effects mediated by antibodies, animals can be inoculated with antibodies or control agents, and symptoms that may be associated with tumor antigen-antibody therapy can be thoroughly studied. Possible side effects of in vivo administration of tumor antigen antibodies particularly include toxicity at tissues expressing tumor antigens. Antibodies that recognize tumor antigens in humans and other species (e.g., mice) are particularly useful for predicting potential side effects mediated by monoclonal tumor antigen antibodies administered in humans.

[0470] Mapping of epitopes recognized by antibodies can be performed as described in detail in "Epitope Mapping Protocols (Methods in Molecular Biology), Glenn E. Morris ISBN-089603-375-9 and in "Epitope Mapping: A Practical Approach" Practical Approach Series, 248, Olwyn MR Westwood, Frank C. Hay.

[0471] The compounds and agents described herein may be administered in the form of any suitable pharmaceutical composition.

[0472] The pharmaceutical compositions are preferably sterile and contain an effective amount of an antibody described herein and optionally other agents discussed herein to produce a desired response or desired effect.

[0473] The pharmaceutical composition is generally provided in a uniform dosage form and can be prepared in a manner known per se. The pharmaceutical composition can, for example, be in the form of a solution or a suspension.

[0474] Pharmaceutical compositions may contain salts, buffer substances, preservatives, carriers, diluents and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to the non-toxicity of materials that do not interact with the action of the active ingredients of the pharmaceutical composition.

[0475] Non-pharmaceutically acceptable salts can be used to prepare pharmaceutically acceptable salts and are included in the present invention. Such pharmaceutically acceptable salts include those prepared from the following acids in a non-limiting manner: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

[0476] Suitable buffer substances for use in pharmaceutical compositions include acetic acid in salt form, citric acid in salt form, boric acid in salt form, and phosphoric acid in salt form.

[0477] Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0478] Injectable preparations may contain pharmaceutically acceptable excipients, such as Ringer lactate.

[0479] The term "carrier" refers to an organic or inorganic composition of natural or synthetic nature, in which the active ingredients are combined to promote, enhance or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a patient.

[0480] Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers.

[0481] The term "excipient" as used herein is intended to mean all substances that may be present in a pharmaceutical composition but are not active ingredients, such as, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents or coloring agents.

[0482] The agents and compositions described herein can be administered by any conventional route, for example, by parenteral administration, including by injection or infusion. Administration is preferably carried out parenterally (eg, intravenously, intraarterially, subcutaneously, intradermally or intramuscularly).

[0483] Compositions suitable for parenteral administration generally contain a sterile aqueous or nonaqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solution or suspension media.

[0484] The agents and compositions described herein are administered in an effective amount. An "effective amount" refers to an amount that achieves a desired response or desired effect alone or in combination with another dosage. In the case of treating a specific disease or a specific condition, the desired response preferably refers to inhibiting the progression of the disease. This includes slowing the progression of the disease, and in particular interrupting or reversing the progression of the disease. The desired response in treating a disease or condition may also be to delay or prevent the onset of the disease or condition. In particular, the term "effective amount" refers to a therapeutic amount that is sufficient to prevent the occurrence, recurrence or onset of cancer and one or more symptoms thereof, reduce the severity and duration of cancer, improve one or more symptoms of cancer, prevent the progression of cancer, cause the regression of cancer and / or prevent the metastasis of cancer. In one embodiment of the invention, the amount of treatment is effective for achieving stabilization, reduction or elimination and / or eradication, removal or control of primary cancer, metastatic cancer and / or recurrent cancer of a cancer stem cell population.

[0485] The effective amount of the medicament or composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient (including age, physiological condition, body size and weight), the duration of treatment, the type of concomitant treatment (if any), the specific route of administration and similar factors. Therefore, the dosage of the medicament described herein can depend on a number of such parameters. In the event that the response in the patient is insufficient at the initial dose, a higher dose can be used (or an effectively higher dose can be achieved by a different, more localized route of administration).

[0486] The agents and compositions provided herein can be used alone or in combination with conventional treatment regimens such as surgery, irradiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated).

[0487] The treatment of cancer represents an area where combination strategies are particularly desirable, because the combined effects of two, three, four or even more cancer drugs / treatments often produce significantly stronger synergies than the effects of a single treatment method. Therefore, in another embodiment of the invention, cancer treatment can be effectively combined with a variety of other drugs. Among them are, for example, combinations with conventional tumor treatments, multi-epitope strategies, additional immunotherapies, and targeted angiogenesis or apoptosis treatment methods (for review, see, for example, Andersen et al. 2008: Cancer treatment: the combination of vaccination with other therapies. Cancer Immunology Immunotherapy, 57 (11): 1735-1743). The sequential administration of different agents can inhibit the growth of cancer cells at different checkpoints, while other agents can, for example, inhibit the survival or metastasis of new blood vessels, malignant cells, potentially converting cancer into a chronic disease.

[0488] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0489] Example

[0490] Example 1: Descriptive analysis of genetic immune polymorphisms

[0491] Individual patterns of single nucleotide polymorphisms (SNPs) in a patient's genome can predict the response rate to the therapeutic antibody IMAB362. To investigate such SNP patterns, all patients were genotyped for multiple SNPs with known or putative roles in immune response and gastric cancer susceptibility.

[0492] Specifically, the following issues are addressed:

[0493] a. SNP genotypes of each patient for the polymorphisms studied.

[0494] b. Frequency of SNP genotypes in the patient population.

[0495] c. Identification of patients with polymorphisms that may directly interfere with the mode of action of IMAB362 (Fc receptor and complement system polymorphisms).

[0496] d. Accumulation of SNP genotypes described as risk factors for gastric cancer susceptibility, cancer progression, or cancer treatment for each patient.

[0497] e. Correlation between SNP genotypes and clinical outcomes.

[0498] f. Correlation between SNP genotype and progression-free survival (PFS).

[0499] Genetic polymorphism analysis was performed on all patients in Cohorts 1, 2 and 3. Patient blood samples were collected on day 1 (V2a, before infusion).

[0500] Whole blood samples (9 ml, EDTA-Monovette) were collected from all patients. EDTA blood was stored in 1 ml aliquots at -20°C at the research center immediately after sample collection. EDTA blood samples were transported on dry ice (-70°C) and stored at -20°C. Upon arrival, blood samples were immediately stored at -20°C until DNA isolation.

[0501] The SNPs of interest were selected by focusing on SNPs known to affect immune system function through literature research, and in particular SNPs that have been described to affect the mode of action of therapeutic antibodies (such as Fc receptor and complement system polymorphisms). SNPs that have been described to affect gastric cancer patient survival, (gastric) cancer susceptibility or gastric cancer progression were also selected and studied.

[0502] In Fluidigm Biomark TM TaqMan PCR-based SNP genotyping TM Genetic polymorphisms were analyzed using the PCR assay (46 standard, 5 custom; Life Technologies). DNA isolation was performed according to standard protocols for isolating genomic DNA from whole blood. Fluidigm Biomarl TMThe real-time PCR analysis platform allows genotyping of up to 96 patient samples with 96 SNPs in one measurement because a lab chip with 96 channels for patient DNA samples and 96 orthogonal channels for SNP determination is applied to the patient samples and specific SNP primers. Patient genomic DNA is pre-amplified by specific target amplification (STA). In Fluidigm Biomark rM TaqMan real-time PCR analysis platform was used to perform TaqMan real-time PCR on preamplified DNA under standard conditions. TM Real-time PCR analysis. Allele determination of SNPs was performed for each patient and each assay was performed using proprietary Fluidigm software and statistical analysis software "R". Subgroups of SNPs were determined by classical Sanger sequencing due to equivocal Fluidigm results.

[0503] The genetic polymorphism of 51 single nucleotide polymorphisms (SNPs) was determined for 53 patients. The blood sample from 1 patient did not allow sufficient DNA extraction to analyze SNPs. Only 6 SNP genotypes were determined for a subgroup of 20 patients. Due to technical problems, the genotype of MDM2 SNP rs2279744 was not determined in 9 patients. The PTGS2 rs20417 genotyping result of 1 patient was unclear and was not further studied.

[0504] The matrix of SNP genotypes of the tested patients is determined to allow statistical testing of the genotype frequency shift of the patient population compared to the genotype frequency in the Caucasian control population. The SNP genotype frequencies in the Caucasian control population are based on the data collected by the international SNP genotyping project (HapMap-CEU, PGA-EUROPEAN-PANEL, CAUC1, pilot_1_CEU_low_coverage_panel, CEU_GENO_PANEL, PDR-90) stored in the public database dbSNP (National Center for Biotechnology Information, Bethesda (MD, USA). The number of patients of each genotype of a given SNP is compared with the number of patients of each genotype in the Caucasian control population. The number of patients of each genotype of the control population is calculated by multiplying the relative SNP genotype frequency in the population provided by the number of research samples reported. This allows direct chi-square test (Chi square test) to determine the statistically significant difference between the patient population and the corresponding control population.

[0505] Chi-square tests were performed for 48 of the 51 studied SNPs. No data on SNP genotype frequencies are available in public databases for SNPs C1QA (rs1044378), FCGR2C (Q57X (C->T)) and MDM2 (rs2279744). SNPs with statistically significant changes in genotype frequency between patient and control groups (5 of 48 SNPs, p<0.05) are shown in Figure 1 middle.

[0506] Four of these five SNPs have been shown to play a role in cancer / gastric cancer susceptibility. All four cancer / gastric cancer susceptibility SNPs did show overrepresentation of the respective cancer-associated genotypes in the patient population, as expected for gastric cancer patients (Table 1).

[0507] To date, one of these five SNPs (rs12146727 (C1S)) has not been shown to be a risk / susceptibility factor for cancer or gastric cancer. To date, this SNP has only been described as a putative risk factor for cardiovascular disease.

[0508] Table 1: Gastric cancer susceptibility-associated SNPs with statistically significant differences in genotype frequencies between patients and control groups.

[0509]

[0510] 49 of the 51 research SNPs in the patient population show variant allele patterns in the patient population studied. This allows the frequency shift of the SNP alleles between the test patient subgroups, which ideally can help identify the putative responder population. Only 2 SNPs, C1QA (rs1044378) and FCGR2C (AHN1ME8) show constant SNP genotypes in all patients, thereby preventing any type of differential analysis. For 5 SNPs, statistically significant allele frequency shifts can be determined in this study compared with the control population, which provides a proof of principle that the SNP allele frequency depends on the composition of a given population. Therefore, the tested SNP selection is very suitable for future identification of SNP biomarker candidates.

[0511] Fc receptor and complement system polymorphisms may directly interfere with the mode of action of IMAB362. Patients were genotyped for SNP alleles in genes that may affect the efficacy of antibody-based treatments, such as FCGR3A (F176V [T→G], rs396991), FCGR2A (H131R [T→C], rs1801274), and C1QA ([276A→G], rs172378) (Table 2).

[0512] Patients were also genotyped for published SNP alleles of the FCGR2C gene (Q57X [C→T], no rs number) and complement system factors C1S (R119H [G→A], rs12146727) and C1QA (rs292001, rs1044378). These SNPs have not been shown to affect antibody therapy but are included as candidate SNPs of interest.

[0513] Table 2: Patients with Fc receptor and complement system polymorphisms. SNP genotypes of patients with well-documented Fc receptor and complement system polymorphisms are listed. The FCGR3A Val / Val polymorphism with a putative positive effect on antibody therapy is in bold and underlined. The FCGR2A (Arg / Arg) and C1QA [G / G] polymorphisms with a putative adverse effect on antibody therapy are shaded grey and highlighted in bold.

[0514]

[0515] A total of 23 patients show at least one fully documented Fc receptor and complement system polymorphism. 4 patients (100411, 101120, 200336 and 400109) are homozygous for FCGR3A allele (F176V [T → G]), which is reported to improve response rate and progression-free survival in antibody therapy. 12 patients are homozygous for FCGR2A allele (H131R [T → C]), and other 10 patients are homozygous for C1QA allele ([276A → G]). It has been shown that these two SNPs can have an adverse effect on antibody therapy. In total, 21 patients are homozygous for FCGR2A allele (H131R [T → C]) or C1QA allele ([276A → G]) (patient 100511 is homozygous for two SNP alleles).

[0516] The correlation of the above findings with patient disease progression can provide insight into the effects of Fc receptor and complement system polymorphisms on IMAB362 treatment.

[0517] The progression of the disease in patients and the efficacy of antibody therapy may be affected by the accumulation of SNPs described as risk factors for gastric cancer susceptibility, cancer progression or cancer therapy. Of the 51 SNPs studied, up to 43 SNPs allow classification of the respective SNP genotypes as "risk" and "non-risk" genotypes. The number of homozygous SNP risk factor genotypes for each patient was counted, as these are generally described as the most relevant risk alleles. Figure 2 The relative frequency of the number of homozygous risk genotypes per patient relative to the number of studied SNP risk factors per patient is shown in .

[0518] The observed accumulation of the investigated risk genotypes per patient ranged from 14% to 46%. This wide distribution allowed the investigation of whether the accumulation of SNP risk genotypes per patient was associated with the patient's clinical outcome.

[0519] In summary, 53 of 54 patients were successfully genotyped for 51 SNPs. 49 of 51 SNPs showed variant SNP allele patterns, allowing significant changes in the SNP genotype frequencies of patient subgroups to be analyzed. Homozygous Fc receptor and complement system polymorphisms described as antibody therapy modulators were found in 23 of 53 patients. The accumulation of the risk genotype studied for each patient was observed to be 14% to 46%.

[0520] Example 2: Correlation between SNP genotyping and clinical outcomes

[0521] The purpose of associating clinical outcomes with genetic polymorphism genotypes is to identify putative SNP biomarker candidates that predict clinical outcomes in patients. Putative biomarker candidates identified in this analysis will be validated in subsequent Phase IIb and Phase III studies. Validating putative biomarker candidates in Phase IIb will allow for differentiation of putative prognostic and predictive SNP candidates.

[0522] The association analysis of each SNP with clinical outcomes was performed independently on two defined Phase IIa clinical trial patient populations: the "full analysis set" (FAS) population with 40 patients and the "per protocol set" population with 21 patients.

[0523] The absolute frequency of the genotype of each SNP in each clinical outcome group ("responder", "non-responder") of the patient population was quantified by SAS Enterprise Guide 6.1. The absolute genotype frequencies were organized in contingency tables (3×2 or 2×2) structured by clinical outcomes and SNP genotypes. The standard statistical test used was Pearson's Chi square test. If the numerical structure of the data set prohibited the use of the Pearson Chi square test, Fisher's exact test was applied to the 2×2 contingency table in some cases. The statistical significance level applied was p < 0.05. Correlation analysis was performed using the statistical analysis software SAS Enterprise Guide 6.1.

[0524] To investigate the effect of SNP genotype on progression-free survival, Kaplan-Meier curves were calculated for each group and then formally compared using the statistical log-rank test. The statistical significance level applied was p < 0.05. Log-rank statistics were implemented using the statistical analysis software SAS Enterprise Guide 6.1.

[0525] Correlation of clinical outcomes with SNP genotyping was performed to identify putative predictive or prognostic SNP biomarker candidates. Correlation was studied in two patient populations (FAS population and PP population).

[0526] The FAS population included 40 patients, 12 of whom were defined as "responders" (clinical outcome "partial remission" or "stable disease") and 28 as "non-responders" (clinical outcome "progressive disease"). One patient sample (100801, non-responder) in the FAS population was not available for SNP analysis as described above, so the maximum number of FAS patients analyzed for association was reduced to 39. The PP population included 21 patients, of which 10 were responders and 11 were non-responders.

[0527] The number of patients studied per SNP varied from 20 to 39 (in the FAS population) and from 20 to 21 (in the PP population).

[0528] Correlation analysis was performed as described above. In total, of the 51 SNPs studied, 2 showed statistically significant associations with clinical outcomes in both the FAS and PP populations.

[0529] The two SNPs that showed statistical association between clinical outcome and respective SNP genotype in both populations were FCGR2A rs1801274 (p = 0.0004 [PP]; p = 0.008 [FAS]) and IL-10 rs1800896 (p = 0.042 [PP], p = 0.022 [FAS]) (Table 3). For each of these two SNPs, the number of patients statistically tested for each SNP was 21 (PP) and 39 (FAS).

[0530] Table 3: SNPs showing statistical association between clinical outcomes and SNP genotypes in PP and FAS populations.

[0531]

[0532] (Chi-square test, statistical significance: p < 0.05)

[0533] Five SNPs showed association with clinical outcomes in one patient population (FAS or PP), as can be shown for DNMT3 Ars1550117 [PP, p = 0.035], SMAD4 rs12456284 [FAS, p = 0.02], MUC1 rs4072037 (FAS, p = 0.03), EGF rs4444903 [FAS, p = 0.049], and CDH1 rs16260 [FAS p = 0.049]) (Table 4).

[0534] Table 4: In PP or SNPs that showed statistical association between clinical outcomes and SNP genotypes in the FAS population.

[0535]

[0536] (Chi-square test, statistical significance: p < 0.05)

[0537] Examining over- or under-representation of SNP genotypes in responder / non-responder patients may allow for providing scientific explanations for statistically significant frequency differences.

[0538] The genotypes of two SNPs, rs11615 (ERCC1) and rs396991 (FCGR3A) were associated with prolonged progression-free survival (PFS) in the PP population (Table 5).

[0539] Table 5: SNPs showing statistical association between prolonged PFS and SNP genotype in the PP population.

[0540]

[0541] For each of the 9 SNPs listed, the number of patients statistically tested per SNP was 21 (PP) and 39 (FAS).

[0542] FCGR2A rs1801274 [C / T]: In PP, all patients carrying the heterozygous rs1801274 [CT] genotype were indeed responders (8), which is reflected in the high significant p-value of the statistical test (0.0004). All PR patients (4 out of 4) showed this genotype. Most non-responders (73%, 8 out of 11) showed the homozygous [TT] genotype (Table 6). This genotype distribution pattern can also be seen in the FAS population, but it is not as obvious as in the PP population (Table 7). Multiple non-responder patients in the FAS population also had the [CT] genotype (30%), which resulted in a less obvious but still highly statistically significant p-value.

[0543] Table 6: List of rs1801274 (FCGR2A) genotypes in PP patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0544]

[0545] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0546] Table 7: List of rs1801274 (FCGR2A) genotypes in FAS patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0547]

[0548] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0549] Survival analysis FCGR2A rs1801274[C / T]: The high significance overrepresentation of rs1801274 genotype [CT] in the expected responder group is also reflected in the association with prolonged progression-free survival (PFS). Figure 3 ) and FAS( Figure 4 ), the [CT] genotype was also highly significantly associated with prolonged PFS (PP p=0.0007, FAS p=0.03). However, it is interesting that during the first 60 treatment days, FAS patients with the [TT] genotype showed a trend towards a higher PFS rate compared to patients with the [CC] or [CT] genotypes. Therefore, survival analysis confirms that rs1801274 (FCGR2A) is a very interesting putative biomarker candidate with predictive or prognostic properties.

[0550] IL-10 rs1800896[A / G]: In PP, none of the non-responder patients carried the homozygous rs1800896[GG] genotype (Table 8). This genotype was found at an elevated frequency (40%) in responder patients (4 out of 10). Only 1 out of 10 responders (10%) showed the [AA] genotype, with the remaining responders showing the heterozygous [GA] genotype. In FAS, a comparable genotype frequency distribution could be observed (Table 9), but the [GG] genotype could be observed at a low frequency in non-responder patients in this population (11%, 3 out of 27).

[0551] Table 8: List of rs1800896 (IL-10) genotypes in PP patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0552]

[0553] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0554] Table 9: List of rs1800896 (IL-10) genotypes in FAS patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0555]

[0556] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0557] Survival analysis rs1800896 (IL-10) [A / G]: The rs1800896 [GG] genotype was significantly over-represented in responder patients. The statistical correlation between [GG] genotype and PFS showed that in the PP and FAS populations, [GG] genotype was not significantly associated with PFS (PP p = 0.27 ( Figure 5 ));FAS p=0.08,( Figure 6 )). However, the p-value for the FAS survival association was close to significance, which may indicate that in a larger population with reduced statistical noise, significance may be well achieved. Overall, rs1800896 (IL-10) is an interesting putative biomarker candidate.

[0558] DNMT3A rs1550117 [G / A]: In PP, 4 responders (40%) showed the [GA] genotype, while all non-responders showed the [GG] genotype (p=0.03, Table 10).

[0559] Table 10: List of rs1550117 (DNMT3A) genotypes in PP patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0560]

[0561] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0562] Survival analysis rs1550117 (DNMT3A) [G / A]: In the responder patients in the PP population, the rs1550117 [GA] genotype was significantly over-represented. In the FAS population, the PFS difference between [GA] and [GG] carriers was borderline significant (FAS p = 0.058) ( Figure 7 ).

[0563] In the FAS population, only one patient was a carrier of the [AA] genotype.

[0564] SMAD4 rs12456284 [G / A]: In FAS, the [GA] genotype was found to be statistically significantly overrepresented relative to the [AA] and [GG] genotypes in the responder population (7 of 12 patients, 58%) (p=0.023, Table 11). In the FAS non-responder population, the frequency of the [GA] genotype can be found at a frequency of 19% (5 of 27 non-responders). In the PP population, this association was indicated by trend significance (p=0.081, data not shown).

[0565] Table 11: List of rs12456284 (SMAD4) genotypes in FAS patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0566]

[0567] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0568] Survival analysis rs12456284 (SMAD4) [G / A]: The rs12456284 [GA] genotype was significantly over-represented in FAS responders and showed the same trend in PP responders. The statistical correlation between rs12456284 genotype and PFS showed that in the PP population, using the Gehan-Brelow-Wilcoxon test, the [GA] genotype was significantly associated with PFS (PP p = 0.048) ( Figure 8), while the significance using the log-rank test was p=0.35. The Gehan-Brelow-Wilcoxon test gave higher weight to PFS events at early time points compared to the log-rank test, and indeed the difference between [GA] and [AA] carriers was most significant during the first 100 days of this Phase IIa clinical trial. In the FAS population, the [GA] genotype was not significantly associated with PFS (p=0.20 (log-rank), p=0.23 (Gehan-Brelow-Wilcoxon)), but visual inspection indicated that [GA] carriers had a trend toward prolonged PFS.

[0569] MUC1 rs4072037 [A / G]: In FAS, the rs4072037 genotype with the highest frequency of 67% was found in the responder population to be [AA] (8 of 12), while non-responders showed this genotype in only 26% of patients (7 of 27). None of the responder patients showed the homozygous [GG] genotype (Table 12), while non-responders showed the [GG] genotype at a ratio of 22% (6 of 27). This differential genotype distribution in responder and non-responder FAS patients was statistically significant (p=0.03). A comparable genotype distribution pattern was found in the PP population (data not shown), where responders showed almost the same relative [AA] genotype frequency of 70% (7 of 10) as in the FAS population (trend significance p=0.11).

[0570] Table 12: List of rs4072037 (MUC1) genotypes in FAS patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0571]

[0572] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0573] Survival analysis rs4072037 (MUC1) [A / G]: The significant overrepresentation of the rs4072037 genotype [AA] in responder patients may indicate the association of this genotype with PFS. Statistical tests showed that the [AA] genotype was significantly associated with PFS in both the PP and FAS populations (PP p=0.001, ( Fig. 9 );FAS p=0.02,( Fig.10 )). This survival analysis confirmed that rs4072037 (MUC1) is a very interesting putative predictive or prognostic biomarker candidate.

[0574] EGF rs4444903 [G / A]: In FAS, the rs4444903 genotype [AA] was significantly over-represented in the responder population (5 of 12; 42%) compared to the non-responder population (3 of 27; 11%) (Table 13). In the PP population, this asymmetric distribution was not statistically significant (p=0.32, data not shown).

[0575] Table 13: List of rs4444903 (EGF) genotypes in FAS patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0576]

[0577] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0578] Survival analysis rs4444903 (EGF) [G / A]: There was no statistically significant association between the rs4444903 [AA] genotype and PFS in either the PP or FAS populations (FAS p = 0.1; PP p = 0.16). However, a trend toward prolonged PFS was observed in both the PP and FAS populations ( Fig.11 ).

[0579] CDH1 rs16260 [C / A]: In FAS, the rs16260 genotype [AA] was present at a significantly higher frequency in responders (5 of 12, 42%) compared to the non-responder group (3 of 27, 11%) (p=0.049, Table 14). In PP, this asymmetric distribution between the two patient groups was not significant (p=0.72, data not shown).

[0580] Table 14: List of rs16260 (CDH1) genotypes in FAS patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0581]

[0582] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0583] Survival analysis rs16260 (CDH1) [C / A]: In the FAS population, the association between rs16260 (CDH1) genotype [AA] and PFS approached statistical significance (log-rank test p = 0.065, Gehan-Brelow-Wilcoxon test p = 0.032) ( Fig.12 ).

[0584] ERCC1 rs11615 [C / T]: In PP, a trend was found with a higher frequency of rs11615 genotype [TT] in the responder group (3 out of 10; 30%) (p = 0.068; non-responder group (0%)). In contrast, the homozygous [CC] genotype was only present in the non-responder group (2 patients) (Table 15).

[0585] Table 15: List of rs11615 (ERCC1) genotypes in PP patients and their respective frequencies in responder (PR and SD) and non-responder patients (PD).

[0586]

[0587] RESP: responder, NONRESP: non-responder, PFS: progression-free survival, abs freq.: absolute frequency, relfreq.: relative frequency

[0588] Survival analysisrs11615(ERCC1)[C / T]:rs11615[TT] genotype was present only in the responder group in the PP group. Statistical correlation between rs11615 genotype and PFS showed that rs11615 genotype in PP was highly significantly associated with PFS, with [CT] and [TT] carriers showing prolonged survival compared to [CC] carriers (PP p=0.0001)( Fig.13 ). Despite this outstanding significance value, it should be noted that there were only 2 patients with the [CC] genotype and 3 patients with the [TT] genotype in the PP. However, in the FAS population, the same effect could be observed as a trend (FAS p=0.13, data not shown), suggesting that this effect is also valid in a larger patient population.

[0589] Survival analysis FCGR3A rs396991 [T / G]: In neither PP nor FAS was the genotype of SNP rs396991 associated with clinical outcome (FAS p=0.49; PP p=0.29, data not shown). However, survival analysis in the PP population showed with high statistical significance that patients with genotypes [TG] and [TT] showed improved PFS compared with [GG] (p=0.0007, Fig.14). This effect was also observed in the FAS population (p=0.25; data not shown). Although the PP population reached significance, it should be noted that only 3 PP patients were [GG] carriers.

[0590] Example 3: Discussion accompanying immune polymorphism analysis

[0591] The primary objective of this Phase IIa trial is to evaluate the safety and efficacy of the therapeutic anti-CLDN18.2 mononuclear antibody IMAB362 in patients with gastroesophageal adenocarcinoma. In addition, a concomitant analysis of genetic immune response polymorphisms was performed to evaluate parameters that could be used as potential predictive or prognostic biomarkers associated with IMAB362 treatment.

[0592] Discussion on Descriptive Immune Polymorphism Analysis

[0593] Genetic polymorphisms in the patient genome have been shown to alter the response rate to therapeutic antibodies. To investigate the effect of individual genetic variations on response rate, the genotypes of 51 single nucleotide polymorphisms (SNPs) with known or putative roles in immune response and gastric cancer susceptibility or progression were determined in patients.

[0594] In this study, 51 SNPs of 53 of the 54 study patients were successfully genotyped. For 5 SNPs, statistically significant changes in genotype frequency in the patient population compared to the control population could be detected. It has been previously shown that 4 of these SNPs are associated with cancer / gastric cancer susceptibility. In the study population, the cancer / gastric cancer-related genotypes of each of these 4 SNPs are overrepresented, as expected in the patient population with advanced GC. The overrepresentation of each homozygous genotype can indicate a recessive mode of action, suggesting impaired gene function relative to enhanced gene activity. This is emphasized by published data, for example, it has been reported that the gastric cancer-related AA genotype of SNP rs16260 in CDH1 causes a downregulation of CDH1 expression, because its position in the promoter of CDH1 is at -160.

[0595] Polymorphisms in genes involved in immune signaling were studied, even though these polymorphisms have not been described as gastric cancer risk factors before. Genetic polymorphisms in genes encoding immune signaling factors have been shown to significantly modulate the risk of developing gastric cancer. Therefore, the response rate of antibody-based cancer treatments may also be affected by these SNPs.

[0596] The IL-2 genotype GG (SNP rs2069762), which is overrepresented in the patient population, is associated with an increased risk of gastric atrophy induced by Helicobacter pylori (H. pylori) infection and may predispose to gastric cancer. The CTLA4 SNP rs231775 and rs2274223 (PLCE1) genotypes have been described as GC susceptibility risk factors. However, since the published studies on rs231775 are contradictory in the sequence of the genotype, no conclusions can be drawn here. Fcγ receptor and complement system polymorphisms were studied in this study. The potentially beneficial FCGR3A genotype encoding Val / Val[GG] was detected in 4 APT patients, and the FCGR2A genotype (Arg / Arg)[CC] with potential adverse effects could be detected in 12 APT patients.

[0597] It has been demonstrated that CDC as a secondary effector mechanism is also affected by SNP polymorphisms: Carriers of the A allele of the polymorphism ([276A->G], rs172378) in the complement component C1qA showed a prolonged response after rituximab treatment of follicular lymphoma. A complement system polymorphism in C1QA with the genotype 'GG' was detected in 10 patients and may adversely affect the response. However, the SNP polymorphism rs12146727 in the complement component C1S has so far only been described in screenings not related to antibody therapy or cancer.

[0598] The identification of significant genotype frequency shifts between patient and control populations suggests that SNP genotype frequency shifts can be used as predictive and prognostic markers in clinical studies.

[0599] The accumulation of SNP risk alleles can also have an impact on the clinical outcome of patients. In order to allow such analysis, the number of homozygous SNP risk genotypes is counted for each patient. The correlation of these numbers with treatment response can provide a deep understanding of the role of SNP risk factor accumulation.

[0600] Discussion on the correlation between SNP genotyping and clinical outcomes

[0601] FCGR2A rS1801274:

[0602] Examination of over- or under-represented FCGR2A genotypes showed that in the PP population, all patients with the heterozygous rs1801274 genotype [CT] were responder patients, and patients with a partial response (PR) had only this genotype. The over-represented homozygous genotype in the non-responder population was [TT]. Observation of these frequency distributions alone does not allow conclusions to be drawn whether the [CT] genotype is beneficial or whether [TT] is detrimental. In most studies investigating the effects of SNP genotypes, the respective homozygous genotype showed the strongest biological effect, indicating a generally recessive mode of action, reflecting impaired gene function versus enhanced gene activity of both alleles. In cases where SNP alleles result in increased genetic activity, a stepwise effect of the biological effect is often observed: one allele (i.e., heterozygous) increases gene activity, and two alleles (i.e., homozygous) increase gene activity even more. In both gain- or loss-of-function cases, the strongest biological / clinical effect is usually observed in patients with the homozygous genotype. Under this assumption, overrepresentation of the homozygous [TT] genotype in the non-responder populations in both the PP and FAS populations would result in adverse effects.

[0603] However, this was unexpected because the rs1801274 FCGR2A [TT] genotype has been described as a factor with a prolonging effect on PFS in many clinical studies. In our phase IIa clinical trial, a more rigorous examination of the association between genotype and PFS in FAS non-responder patients showed that during the first 60 days of treatment, FAS PD patients with the [TT] genotype did show a trend toward higher PFS time, as compared with FAS PD patients with the [CT] genotype (compare Tables 7 and Figure 4 ). The explanation for this observation consistent with the underrepresentation of [TT] in responders with extended PFS may be the superposition of two different molecular mechanisms: first, the rs1801274[CT] genotype may be a marker for responder patients. This is a new observation that has not been described in the literature so far, and this may indicate that this genotype is a predictive marker for IMAB362 treatment. The molecular mechanism underlying this new observation has not yet been resolved.

[0604] A second observation, already described in the literature for other therapeutic anticancer antibodies, would be the prolonged PFS in patients with the FCGR2A[TT] genotype. In our Phase IIa study, this effect was due to the superposition of the presumed first mechanism that could only be observed as a trend in non-responder patients. Mechanistically, the second observation could be explained by the improved binding affinity of IgG1 antibodies to the FCGR2A 131 His / His receptor allele (encoded by the [TT] genotype) as opposed to the weaker binding affinity to the homozygous FCGR2A 131 Arg / Arg receptor allele (encoded by the [CC] genotype): In a study systematically investigating the effects of Fcγ receptor polymorphisms, it has recently been shown that antibodies of the IgG1 isotype indeed bind to the two allelic forms of the Fcγ receptor IIA with different affinities, with H131 having a higher affinity compared to R131. It is generally assumed that the differential affinity of IgG antibodies to FCGR2A receptor alleles affects the triggering rate of effector mechanisms and, therefore, affects prolonged PFS in patients carrying high-affinity receptor alleles. The report provides data supporting this hypothesis, showing that the Fcγ receptor polymorphisms FCGR2A H131R and FCGR3A F176V (Phe>Val, rs396991) may have an impact on the clinical efficacy of trastuzumab-based IgG1 antibody therapy in patients with metastatic breast cancer. Patients with the genotypes FCGR3A176Val / Val and FCGR2A 131 His / His showed significantly better response rates and progression-free survival. The same polymorphisms were also associated with the response rate of B-cell lymphoma patients treated with rituximab (IgG1). In another study, prolonged PFS after cetuximab (IgG1) treatment was associated with the FCGR3A 176 Val / Val genotype. Controversially, recently there have been authoritative studies reporting no association between Fcγ receptor polymorphisms and survival, response rate, or progression-free survival discussed with respect to antibodies. In the BCIRG-006 trial of the Breast Cancer International Research Group (BCIRG), 1218 patients were treated in a randomized study with two groups containing trastuzumab and a control group without trastuzumab. The association between the Fcγ receptor polymorphisms and trastuzumab efficacy reported above could not be confirmed. A long-term study of 460 patients using rituximab in combination with chemotherapy in follicular lymphoma reported no association between Fcγ receptor polymorphisms and progression-free survival. In the REACH trial with 419 patients in which patients received fludarabine and cyclophosphamide (FC) or rituximab plus FC, FCGR2A and FCGR3A polymorphisms did not significantly affect the results.Recent cetuximab trials have also yielded inconsistent results, not recommending Fcγ receptor polymorphisms as a useful biomarker. This may reflect intrinsic population factors or differences in concurrent chemotherapy regimens.

[0605] MUC1 rs4072037:

[0606] MUC1 is a transmembrane glycoprotein of the mucin family. Mucin is a high molecular weight protein that is extensively O-glycosylated by oligosaccharides and n-glycan chains in the N-terminal extracellular domain. Mucin is expressed on the apical surface of the epithelium lining the respiratory and gastrointestinal tracts and tubes in the liver, pancreas and kidney. Transmembrane mucins span the membrane with an α-helix and provide a protective lining to the extracellular space with their sugar chains. Mucins secreted into the extracellular space build a mucus gel layer, which serves as an additional physical protection for the epithelium. Transmembrane MUC1 and secreted mucins MUC5C and MUC6 are the main mucins expressed in the stomach. MUC1 is translated into a single polypeptide chain, which is automatically cut. The N-terminal extracellular domain (MUC1-N) initially remains non-covalently linked to the transmembrane / cytoplasmic intracellular domain (MUC1-C). This intracytoplasmic intracellular domain is used as a signaling domain, which can enter the nucleus and associate with many transcription factors to directly activate gene expression. Cellular stress can lead to proteolytic cleavage of the MUCl-N and MUCl-C domains through a second proteolytic site. This can also be observed in cancer cells, where MUCl is no longer expressed in an orderly manner at the apical membrane of the cell, but can be found overexpressed and localized throughout the cell. The consequence is shedding of the extracellular domain (also known as CA15-3) to the extracellular space and intracellular localization of MUCl-C. The intracytoplasmic signaling domain acts as an oncogene (e.g., by activating Wnt / β-catenin signaling and blocking apoptotic pathways).

[0607] However, the extracellular domain of MUC1 is not only a static structural component, but also plays an important role during signaling events at the cell membrane. It has been demonstrated that the glycosylation and expression status of the extracellular domain of MUC1 regulate the interaction between membrane signaling molecules and the extracellular matrix. It has been reported that under-glycosylated MUC1-N in tumor cells enhances signaling between membrane molecules (such as ICAM-1 or E-selectin) and the MUC1 core protein. In addition, mucin expression and glycosylation status appear to mask membrane-associated molecules. In cancer cells, masking of HER2 protein by mucin expression has been described as a possible resistance mechanism for trastuzumab treatment.

[0608] The MUC1 polymorphism rs4072037 'A' allele has been described as a risk factor for gastric cancer susceptibility. This polymorphism is a G->A exchange in exon 2, which results in alternative splicing of MUC1 exactly in the predicted signal peptide cleavage site of MUC1. Defective cleavage of the signal peptide can lead to abnormal MUC1 protein localization or glycosylation pattern and, therefore, defective protein function.

[0609] In this Phase IIa clinical trial, the rs4072037[AA] genotype has been found to be statistically associated with the responder population. It can be inferred that the under-glycosylated or under-expressed [AA] allele form of MUC1 allows IMAB362 to better approach the membrane target molecule CLDN18.2 expressed on cancer cells, thereby improving the therapeutic efficacy. This makes rs4072037 a predictive biomarker.

[0610] IL-10 rs1800896:

[0611] IL-10 is a key regulator of the immune system with pleiotropic functions. It is known that IL-10 is used as an anti-inflammatory immunosuppressive cytokine by inhibiting macrophage-dependent antigen-specific T cell proliferation and macrophage-dependent production of cytokines by T cells. However, IL-10 is also described as an immunostimulatory cytokine that enhances B cell, granulocyte and mast cell differentiation and growth as well as NK cell and CD8+T cell activation. The pleiotropic potential of IL-10 is also reflected by the extensive expression of IL-10 in many immune cell types (including Th2 cells, Treg cells, Th3 cells, NK T cells, B cells, macrophages and dendritic cells). This dual role of IL-10 is reflected in tumor promotion and tumor suppression potential: IL-10 secreted by tumor cells or tumor-infiltrating immune cells (such as macrophages) allows tumor cells to escape immune surveillance by mechanisms that have only been partially elucidated. One mechanism described involves Treg cells that contribute to the induction of peripheral tolerance through the expression of immunoregulatory cytokines (such as IL-10). Another mechanism reported is the inhibition of cross-presentation of tumor-associated antigens by dendritic cells and thus preventing T cells from initiating an effective immune response against tumor cells. On the other hand, the display of IL-10 by malignant cells leads to the downregulation of HLA class I proteins, resulting in increased sensitivity to NK cell cytotoxicity.

[0612] The IL-10 promoter polymorphism rs1800896 at position (-1082) is of interest because the 'G' allele has been reported as a risk factor for gastric cancer and renal cancer. The 'G' allele of this polymorphism has been reported to be associated with reduced IL-10 expression in vitro compared to the 'A' allele. The [GG] genotype of rs1800896 was overrepresented in responder patients of this Phase IIa clinical trial, which may indicate a lower relative expression level of IL-10. It can be inferred that patients carrying the [GG] genotype have lower IL-10 expression, which in turn may make it more difficult for tumor cells to escape immune surveillance through one of the above mechanisms. In fact, none of the 12 FAS responder patients showed elevated IL-10 serum levels, as opposed to 22% of FAS non-responder patients (6 of the 27 measured). However, other authors have pointed out that the 'A' allele is associated with reduced IL-10 expression.

[0613] It should also be noted that IL-10 signals through the intracellular mediator Stat3, and Stat3 activation is dependent on MUC1-C. Therefore, the functional interaction of MUC1 with IL-10 may be the reason why both molecules were proven to be statistically significant biomarker candidates in this Phase IIa clinical trial. Finally, FCGR2A is expressed on macrophages, which are usually the main source of IL-10 in the tumor microenvironment. If these putative biomarker candidates prevail in ongoing and future studies, the study of the functional interaction of these factors may be of considerable interest.

[0614] rs1550117 (DNMT3A):

[0615] Rs1550117 is a SNP in the DNMT3A gene, which encodes an enzyme DNA (cytosine-5)-methyltransferase 3A that catalyzes the transfer of methyl groups to specific CpG structures in DNA, thereby inducing epigenetic modifications. It has been shown that the genotype [AA] confers an increased risk of gastric cancer compared to [GG] or [GA]. In this study, [AA] was found in only one patient in the FAS population and in no patients in the PP population. This may indicate that [AA] also confers a survival risk, preventing third- and fourth-line treatment for [AA] carriers in this Phase IIa clinical trial. The finding that [GA] was significantly associated with clinical outcomes in PP suggests that this marker has the potential to serve as a predictive biomarker for IMAB362 treatment.

[0616] rs12456284 (SMAD4):

[0617] rs12456284 is a SNP in the SMAD4 gene, which encodes the intracellular TGFβ / BMP signaling co-transducer "maternal DPP homolog 4". It has been published that the [GG] genotype significantly reduces the risk of gastric cancer. The statistically significant over-representation of the heterozygous [GA] genotype relative to the [AA] genotype in the FAS responder population suggests that this genotype can be used as a predictive biomarker. The prolonged PFS of the PP population patients carrying [GA] is a supportive fact.

[0618] rs4444903(EGF):

[0619] The functional polymorphism rs4444903 in the promoter region of the EGF gene was observed to regulate the level of EGF protein, and higher amounts of EGF factor were detected in the serum of [GG] carriers. The G allele of this polymorphism and the [GG] genotype showed a significant association with an increased risk of gastrointestinal cancer in a meta-analysis.

[0620] In this phase IIa clinical trial, the genotype [AA] was significantly over-represented in FAS responders, and patients with this genotype showed a trend toward prolonged PFS in both the FAS and PP populations. This may indicate that the rs4444903 [AA] genotype is a predictive or prognostic biomarker.

[0621] rs16260(CDH1):

[0622] The cell adhesion protein E-cadherin 1 (E-cadherin) is a member of the calcium-dependent cadherin superfamily. Loss of function has been associated with the progression of cancer. The rs16260[A] allele in the CDH1 promoter has been shown to reduce the transcription efficiency of E-cadherin 1. In addition, the -160A allele of CDH1 has been described as a susceptibility factor for gastric carcinogenesis.

[0623] In this study, rs16260[AA] genotype carriers were statistically overrepresented among FAS responders. This may indicate that the [AA] genotype is a putative predictive biomarker.

[0624] rs11615 (ERCC1) and rs396991 (FCGR3A):

[0625] Two SNPs, rs11615 (ERCC1, DNA repair protein "excision repair cross-complementation group 1") and rs396991 (FCGR3A, low affinity immunoglobulin gamma Fc region receptor III-A), both showed genotype (ERCC1 [TT], FCGR3A [TG] and [TT]) associated with prolonged PFS. This may indicate that these SNPs are predictive or prognostic biomarkers.

[0626]

[0627] New international patent applications

[0628] Ganymed Pharmaceuticals AG, etc.

[0629] "Methods and compositions for predicting therapeutic efficacy of cancer treatment and cancer prognosis"

[0630] My proposal number: 342-85 PCT

[0631] _______________________________________________

[0632] Additional page for biological materials

[0633] Identification of other deposits

[0634] 1) Name and address of the depositary of the deposits (DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748):

[0635] DSMZ - German Collection of Microorganisms and Cell Cultures

[0636] Mascheroder Weg 1b

[0637] 38124 Braunschweig

[0638] DE

[0639] 2) Name and address of the depository of the deposit (DSM ACC2808, DSM ACC2809, DSM ACC2810):

[0640] DSMZ - German Collection of Microorganisms and Cell Cultures

[0641] Inhoffenstr.7 B

[0642] 38124 Braunschweig

[0643] DE

[0644]

[0645]

[0646] Additional notes on all the above collections:

[0647] -Mus musculus myeloma P3X63Ag8U.1 fused with mouse spleen cells

[0648] -Hybridoma secreting antibodies against human claudin-18A2

[0649] 3) Depositor:

[0650] All the above deposits are made by the following depositors:

[0651] Ganimede Pharmaceuticals

[0652] Freiligrathstraβe 12

[0653] 55131 Mainz

[0654] DE

Claims

1. Evaluate the following methods: (i) whether a cancer patient with a tumor that is positive for a tumor antigen is a responder to treatment with an antibody directed against said tumor antigen, and / or (ii) whether the cancer patient, preferably a cancer patient having a tumor antigen-positive tumor, will experience progression-free survival, The method comprises determining the genotype of one or more single nucleotide polymorphisms selected from the group consisting of FCGR2A rs1801274, MUC1 rs4072037, IL-10 rs1800896, DNMT3A rs1550117, SMAD4 rs12456284, EGF rs4444903, CDH1 rs16260, ERCC1 rs11615, and FCGR3A rs396991 in a sample obtained from the patient.

2. The method of claim 1, wherein the presence of the heterozygous FCGR2A rs1801274[CT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

3. The method of claim 1, wherein the presence of a homozygous FCGR2A rs1801274[TT] genotype and / or a homozygous FCGR2Ars1801274[CC] genotype indicates an increased risk that the cancer patient is not a responder to treatment with the antibody and / or an increased risk that the cancer patient does not experience progression-free survival.

4. The method of claim 1, wherein the presence of the homozygous MUC1 rs4072037[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

5. The method of claim 1, wherein the presence of the homozygous MUC1 rs4072037[GG] genotype indicates an increased risk that the cancer patient is not a responder to treatment with the antibody and / or an increased risk that the cancer patient does not experience progression-free survival.

6. The method of claim 1, wherein the presence of the homozygous IL-10 rs1800896[GG] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

7. The method of claim 1, wherein the presence of a heterozygous DNMT3A rs1550117[GA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

8. The method of claim 1, wherein the presence of a heterozygous SMAD4 rs12456284[GA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

9. The method of claim 1, wherein the presence of the homozygous EGF rs4444903[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

10. The method of claim 1, wherein the presence of the homozygous CDH1 rs16260[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

11. The method of claim 1, wherein the presence of the homozygous ERCC1 rs11615[TT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

12. The method of claim 1, wherein the presence of a heterozygous FCGR3A rs396991 [TG] genotype and / or a homozygous FCGR3Ars396991 [TT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

13. The method of any one of claims 1 to 12, wherein the tumor antigen is CLDN18.2 protein.

14. Evaluate the following methods: (i) whether a cancer patient with a CLDN18.2-positive tumor is a responder to treatment with an antibody directed against the CLDN18.2 protein, and / or (ii) whether the cancer patient, preferably a cancer patient with a CLDN18.2-positive tumor, will experience progression-free survival, The method comprises determining the genotype of one or more single nucleotide polymorphisms selected from the group consisting of FCGR2A rs1801274, MUC1 rs4072037, IL-10 rs1800896, DNMT3A rs1550117, SMAD4 rs12456284, EGF rs4444903, CDH1 rs16260, ERCC1 rs11615, and FCGR3A rs396991 in a sample obtained from the patient.

15. The method of claim 14, wherein the presence of the heterozygous FCGR2A rs1801274[CT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

16. The method of claim 14, wherein the presence of a homozygous FCGR2A rs1801274[TT] genotype and / or a homozygous FCGR2Ars1801274[CC] genotype indicates an increased risk that the cancer patient is not a responder to treatment with the antibody and / or an increased risk that the cancer patient does not experience progression-free survival.

17. The method of claim 14, wherein the presence of the homozygous MUC1 rs4072037[AA] genotype indicates a reduced risk that the cancer patient will not be a responder to treatment with the antibody and / or a reduced risk that the cancer patient will not experience progression-free survival.

18. The method of claim 14, wherein the presence of the homozygous MUC1 rs4072037[GG] genotype indicates an increased risk that the cancer patient will not be a responder to treatment with the antibody and / or an increased risk that the cancer patient will not experience progression-free survival.

19. The method of claim 14, wherein the presence of the homozygous IL-10 rs1800896[GG] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

20. The method of claim 14, wherein the presence of the heterozygous DNMT3A rs1550117[GA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

21. The method of claim 14, wherein the presence of the heterozygous SMAD4 rs12456284[GA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

22. The method of claim 14, wherein the presence of the homozygous EGF rs4444903[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

23. The method of claim 14, wherein the presence of the homozygous CDH1 rs16260[AA] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

24. The method of claim 14, wherein the presence of the homozygous ERCC1 rs11615[TT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

25. The method of claim 14, wherein the presence of a heterozygous FCGR3A rs396991 [TG] genotype and / or a homozygous FCGR3Ars396991 [TT] genotype indicates a reduced risk that the cancer patient is not a responder to treatment with the antibody and / or a reduced risk that the cancer patient does not experience progression-free survival.

26. The method of any one of claims 1 to 25, wherein the antibody acts by recruiting the patient's immune system to destroy tumor cells.

27. The method of any one of claims 1 to 26, wherein the antibody acts via antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

28. The method of any one of claims 1 to 27, wherein the antibody is a monoclonal antibody.

29. The method of any one of claims 1 to 28, wherein the antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 17 or 51 or a fragment thereof, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 24 or a fragment thereof.

30. The method of any one of claims 1 to 29, wherein non-responsiveness to treatment with the antibody comprises a relative decrease in one or more of: survival, progression-free survival, relapse-free survival, distant relapse-free survival, and stable disease.

31. A method of treating a cancer patient, the method comprising: a. assessing whether the cancer patient is a responder to treatment with an antibody by the method of any one of claims 1 to 30, and b. (i) if the risk of the patient not being a responder to treatment with the antibody is reduced, treating the cancer patient with the antibody; or (ii) if the risk of the patient not being a responder to treatment with the antibody is increased, not treating the cancer patient with the antibody and / or treating the cancer patient with a treatment regimen that includes a treatment different from treatment with the antibody.

32. The method of claim 31, wherein the treatment regimen comprises a treatment that is independent of the patient's immune system.

33. The method of claim 31 or 32, wherein the treatment regimen does not comprise treatment with an antibody that acts by recruiting the patient's immune system to destroy tumor cells.

34. The method of any one of claims 31 to 33, wherein the treatment regimen comprises surgery, chemotherapy, and / or radiation.

35. The method of any one of claims 31 to 34, wherein the treatment regimen comprises treatment with a small molecule inhibitor of the tumor antigen and / or an antibody-drug conjugate in which the antibody is directed against the tumor antigen.

36. The method of claim 35, wherein the antibody-drug conjugate is an antibody coupled to a radioactive moiety, a chemotherapeutic moiety, or a toxin moiety.

37. The method of claim 35 or 36, wherein the antibody-drug conjugate is an antibody coupled to a cytostatic or cytotoxic compound.

38. A method for evaluating the clinical outcome of a cancer patient, the method comprising determining the genotype of one or more single nucleotide polymorphisms selected from the group consisting of FCGR2A rs1801274, MUC1 rs4072037, IL-10 rs1800896, DNMT3A rs1550117, SMAD4 rs12456284, EGF rs4444903, CDH1 rs16260, ERCC1 rs11615, and FCGR3A rs396991 in a sample obtained from the patient.

39. The method of claim 38, wherein the presence of the heterozygous FCGR2A rs1801274[CT] genotype indicates a reduced risk of a poor clinical outcome.

40. The method of claim 38, wherein the presence of a homozygous FCGR2A rs1801274[TT] genotype and / or a homozygous FCGR2Ars1801274[CC] genotype indicates an increased risk of a poor clinical outcome.

41. The method of claim 38, wherein the presence of a homozygous MUCl rs4072037[AA] genotype indicates a reduced risk of an adverse clinical outcome.

42. The method of claim 38, wherein the presence of a homozygous MUCl rs4072037[GG] genotype indicates an increased risk of a poor clinical outcome.

43. The method of claim 38, wherein the presence of the homozygous IL-10 rs1800896[GG] genotype indicates a reduced risk of a poor clinical outcome.

44. The method of claim 38, wherein the presence of a heterozygous DNMT3A rs1550117[GA] genotype indicates a reduced risk of a poor clinical outcome.

45. The method of claim 38, wherein the presence of a heterozygous SMAD4 rs12456284[GA] genotype indicates a reduced risk of an adverse clinical outcome.

46. ​​The method of claim 38, wherein the presence of the homozygous EGF rs4444903[AA] genotype indicates a reduced risk of an adverse clinical outcome.

47. The method of claim 38, wherein the presence of a homozygous CDH1 rs16260[AA] genotype indicates a reduced risk of a poor clinical outcome.

48. The method of claim 38, wherein the presence of the homozygous ERCC1 rs11615[TT] genotype indicates a reduced risk of a poor clinical outcome.

49. The method of claim 38, wherein the presence of a heterozygous FCGR3A rs396991[TG] genotype and / or a homozygous FCGR3Ars396991[TT] genotype indicates a reduced risk of a poor clinical outcome.

50. The method of any one of claims 38 to 49, wherein assessing a cancer patient's clinical outcome comprises predicting the likelihood of one or more of: survival, progression-free survival, recurrence-free survival, distant recurrence-free survival, and stable disease.

51. The method of any one of claims 39 to 49, wherein an adverse clinical outcome comprises a relative decrease in one or more of: survival, progression-free survival, relapse-free survival, distant relapse-free survival, and stable disease.

52. The method of any one of claims 38 to 51, wherein the patient has a tumor that is positive for a tumor antigen and is receiving treatment with an antibody directed against the tumor antigen.

53. The method of any one of claims 1 to 52, wherein the sample is a sample comprising DNA.

54. The method of claim 53, wherein the DNA has been extracted from a body sample of the patient.

55. The method of claim 53 or 54, wherein the DNA has been extracted from blood.

56. The method of any one of claims 1 to 55, wherein the tumor is a solid tumor.

57. The method of any one of claims 1 to 56, wherein the tumor is a gastroesophageal tumor.

58. The method of any one of claims 1 to 57, wherein the tumor is advanced gastric or lower esophageal adenocarcinoma.

59. The method of any one of claims 1 to 58, wherein the cancer is gastroesophageal cancer.

60. The method of any one of claims 1 to 59, wherein the cancer is advanced gastric or lower esophageal adenocarcinoma.

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