combination of claudin 18.2 antibody therapy for cancer treatment

专利摘要:
Patent Summary for: "Combination of therapy involving claudin 18.2 antibodies for cancer treatment". The present invention provides a combination therapy for the effective treatment and / or prevention of diseases associated with cldn18.2 expressing cells, including cancerous diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer and metastasis
公开号:BR112014028941B1
申请号:R112014028941-7
申请日:2013-05-21
公开日:2019-11-12
发明作者:Regina Stadler Christiane；Adriana Maria Heinz Cornelia；Jadwiga Utsch Magdalena；Mitnacht-Kraus Rita；Denis Jacobs Stefan；Sahin Ugur；Türeci Özlen
申请人:Ganymed Pharmaceuticals Ag；Tron Translationale Onkologie An Der Johannes Gutenbert Univ Mainz Gemeinnuetzige Gmbh；
IPC主号:

专利说明:

Invention Patent Descriptive Report: “COMBINATION OF THERAPY INVOLVING ANTIBODIES AGAINST CLAUDIN 18.2 FOR CANCER TREATMENT.
FIELD OF THE INVENTION [001] Cancer of the stomach and esophagus (gastroesophageal; GE) are among the malignant neoplasms of greatest unmet medical need. Gastric cancer is the second leading cause of cancer death worldwide. The incidence of esophageal cancer has increased in recent decades, coinciding with a change in the histological type and location of the primary tumor. Esophageal adenocarcinoma is now more prevalent than squamous cell carcinoma in the United States and Western Europe, with most tumors located in the distal esophagus. The overall five-year survival rate for GE cancer is 20-25%, despite the aggressiveness of the established standard treatment associated with significant side effects.
[002] Most patients have metastatic or locally advanced disease, and must undergo first-line chemotherapy. Treatment regimens are based on a structure derived mainly from platinum and fluoropyrimidine combined with a third compound (for example, taxane or anthracyclines). Still, median progression-free survival of 5 - 7 months and survival
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2/159 overall median of 9 to 11 months is the best that can be expected.
[003] The lack of one of the main benefits of the most recent combination chemotherapy regimens for these types of cancer has stimulated research on the use of targeted agents. Recently, for positive gastroesophageal HER2 / neu-cancer Trastuzumab was approved. However, as only about 20% of patients express the target and are eligible for this treatment, the medical need is still high.
[004] The Claudin 18 tight junction molecule processing variant 2 (Claudin 18.2 (CLDN18.2)) is a member of the tight claudin junction protein family. CLDN18.2 is a 27.8 kDa transmembrane protein, comprising four membrane domains that it measures with two small extracellular loops.
[005] In normal tissues, there is no detectable expression of CLDN18.2 by RT-PCR with the exception of the stomach. Immunohistochemistry with specific antibodies CLDN18.2 reveals the stomach as the only positive tissue.
[006] CLDN18.2 is a highly selective gastric lineage antigen exclusively expressed on short-lived differentiated gastric epithelial cells. CLDN18.2 is maintained in the course of the malignant transformation and thus appears
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3/159 often on the surface of human gastric cancer cells. In addition, this pan-tumor antigen is activated ectopically at significant levels in esophageal, pancreatic and pulmonary adenocarcinomas. The CLDN18.2 protein is also located in lymph node metastases from gastric cancer adenocarcinomas and distant metastases, especially to the ovary (the so-called Krukenberg tumors).
[007] The chimeric IgGl antibody IMAB362, which is directed against CLDN18.2 was developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes the first extracellular domain (ECD1) of CLDN18.2 with high affinity and specificity. IMAB362 do not bind to any other member of the claudine family including the closely related amendment variant of Claudin 18.1 (CLDN18.1). IMAB362 shows precise tumor cell specificity and includes four highly potent mechanisms independent of action. After binding to target IMAB362 mediates cell death by ADCC, CDC and induction of cross-linked apoptosis of the target on the surface of the tumor cell and direct inhibition of proliferation. Thus, IMAB362 efficiently lyses CLDN18.2-positive cells, including human gastric cancer cell lines in vitro and in vivo. Mice with CLDN18.2-positive cancer cell lines have a
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4/159 survival benefit and up to 40% of mice show tumor regression when treated with IMAB362.
[008] IMAB362 toxicity and PK profile / TK of IMAB362 has been thoroughly examined in mice and cynomolgus monkeys including 28-day dose range studies, repeated-dose toxicity studies in a cinomologist and a three-month repeated dose toxicity study in mice. In both mice (the longest duration of treatment was weekly administration for 3 months, the highest dose levels were 400 mg / kg) and monomolgus monkeys (up to 5 weekly applications of up to 100 mg / kg) in repeated doses of IMAB362 iv are well tolerated. No signs of systemic or local toxicity are induced. Specifically, no gastric toxicity has been observed in any toxicity study. IMAB362 does not induce immune activation and release of cytokines. There were no adverse effects on male or female reproductive organs. IMAB362 does not bind to tissues that do not have the target. Biodistribution studies in mice indicate that the reason for the lack of gastric toxicity is more likely to compartmentalize tight junctions at the site in the normal gastric luminal epithelium, which appears to severely impair the accessibility of the IMAB362 epitope. This compartmentalization is lost after the malignant transformation
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5/159 making the drugable epitope by IMAB362.
[009] IMAB362 is in early clinical trials. Phase I of the clinical study was carried out in humans. 5 dose cohorts (33 mg / m ² , 100 mg / m ² , 300 mg / m ² , 600 mg / m ² , 1000 mg / m ² ) from 3 patients each received a single intravenous administration of IMAB362 and were observed for 28 days. IMAB362 was very well tolerated, with no relevant safety observation in patients. In one patient, all measured tumor markers decreased significantly within 4 weeks after treatment. In an ongoing phase Ila IMAB362 clinical study is given in a repetitive manner.
The data presented here indicates that bisphosphonates such as zoledronic acid (ZA), particularly when administered in conjunction with recombinant interleukin-2 (IL-2), increase the activity of an anti-CLDN18.2 antibody such as IMAB362. The underlying mechanism is the activation and expansion of a highly cytotoxic immune cell population (γ9δ2 T cells).
[0011] In addition, data are presented demonstrating that chemotherapeutic agents can stabilize or increase the expression of CLDN18.2 on the surface of cancer cells in an 8.2ut increased drugability CLDN18.2 resutling by an anti-CLDNl antibody, such as
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6/159
IMAB362. A synergistic effect of an anti-CLDN1.8 antibody such as IMAB362 has been observed with certain chemotherapy regimens, in specific chemotherapy regimens used for the treatment of gastric cancer or treatment of solid human tumors. Human cancer cells pretreated with chemotherapy are more susceptible to specific antibody-induced target death. In mouse tumor models, tumor control with an anti-8.2 CLDNl antibody plus chemotherapy is superior to that performed with 8.2 l of anti-CLD antibody as a single agent.
SUMMARY OF THE INVENTION [0012] The present invention, in general, provides a combination therapy for the effective treatment and / or prevention of diseases associated with cells expressing CLDN18.2, including cancerous diseases, such as gastric cancer, cancer of the esophagus, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head-neck cancer and gallbladder cancer and metastases from work, notably metastasis of gastric cancer, such as Krukenberg tumors, peritoneal metastasis and ganglion metastasis. Particularly preferred cancerous diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile ducts,
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7/159 lung and ovary.
[0013] In one aspect, the present invention provides a method of treating or preventing a cancerous disease comprising administering to a patient an antibody that has the ability to bind CLDN18.2 in combination with a γδ T cell stimulating agent. The γδ T-cell stimulating agent can be administered before, simultaneously with or after administration of the antibody that has the ability to bind to CLDN18.2, or a combination thereof.
[0014] In one embodiment, the cells are γδ Vy9V62 T cells. In one embodiment, the γδ T cell stimulating agent is a bisphosphonate, such as a nitrogen-containing bisphosphonate (aminobiphosphonate). In one embodiment, the γδ T-cell stimulating agent is selected from the group consisting of zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid, incadronic acid and its salts . In one embodiment, the γδ T-cell stimulating agent is administered in combination with interleukin-2.
[0015] In one embodiment, the method of the invention further comprises administering a stabilizing agent or increasing the expression of CLDN18.2. Expression
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8/159 of CLDN18.2 is preferably on the cell surface of a cancer cell.
[0016] The expression of the stabilizing or increasing agent of CLDN18.2 can be a cytotoxic agent and / or cytostatic agent. In one embodiment, the agent for stabilizing or increasing the expression of CLDN18.2 comprises an agent that induces a cell cycle arrest or an accumulation of cells in one or more phases of the cell cycle, preferably in one or more phases of the cycle cell other than the Gl-phase. The expression of the stabilizing or enhancing agent of CLDN18.2 may comprise an agent selected from the group consisting of anthracyclines, platinum compounds, taxanes, nucleoside analogs and camptothecin analogs, or prodrugs thereof, and combinations thereof . The term stabilizing or increasing agent of 18.2 CLD may comprise an agent selected from the group consisting of epirubicin, oxaliplatin, cisplatin, 5 fluorouracil or its prodrugs, such as capecitabine, docetaxel, irinotecan, and combinations thereof. The stabilizing or enhancing agent of CLDN18.2 may comprise a combination of oxaliplatin and 5-fluorouracil or its prodrugs, a combination of cisplatin and 5fluorouracil or its prodrugs, a combination of at least one anthracycline and oxaliplatin, a combination of fur
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9/159 minus an anthracycline and cisplatin, a combination of at least one anthracycline and 5-fluorouracil or its prodrugs, a combination of at least one taxane and oxaliplatin, a combination of at least one taxane and cisplatin, a combination of hair minus one taxane and 5-fluorouracil or its prodrugs, or a combination of at least one camptothecin analog and 5-fluorouracil or prodrugs thereof. The expression of the stabilizing or increasing agent of CLDN18.2 may be an agent that induces immunogenic cell death. The immunogenic cell death inducing agent can comprise an agent selected from the group consisting of anthracyclines, oxaliplatin and their combinations. The expression stabilizing or increasing agent of CLDN18.2 may comprise a combination of epirubicin and oxaliplatin. In one embodiment, the method of the invention comprises administering at least one anthracycline, at least one platinum compound and at least one of 5-fluorouracil and its prodrugs. Anthracycline can be selected from the group consisting of epirubicin, doxorubicin, daunorubicin, idarubicin and valrubicin. Preferably, anthracycline is epirubicin. The platinum compound can be selected from the group consisting of cisplatin and oxaliplatin. The nucleoside analog can be selected from the group consisting of 5-fluorouracil
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10/159 and its prodrugs. Taxane can be selected from the group consisting of paclitaxel and docetaxel. The camptothecin analog can be selected from the group consisting of irinotecan and topotecan. In one embodiment, the method of the invention comprises the administration of (i) epirubicin, oxaliplatin and 5-fluorouracil, (ii) epirubicin, oxaliplatin and capecitabine, (iii) epirubicin, cisplatin and 5-fluorouracil, (iv) epirubicin, cisplatin and capecitabine or (v) folinic acid, and oxaliplatin 5-fluorouracil.
[0017] The method of the invention can further comprise the administration of at least one other chemotherapeutic agent which can be a cytotoxic agent.
[0018] The antibody that has the ability to bind to CLDN18.2 can bind to native CLDN18.2 epitopes present on the surface of living cells. In one embodiment, the antibody that has the ability to bind to 18.2 CLD binds to the first extracellular loop of CLDN18.2. In one embodiment, the antibody that has the ability to bind to CLDN18.2 mediates cell death by one or more complement-dependent cytotoxicity (CDC) mediated lysis, antibody-dependent cell cytotoxicity (ADCC) mediated by lysis, induction of apoptosis and inhibition of proliferation. In one embodiment, the antibody that has the ability to bind CLDN18.2 is a monoclonal antibody,
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11/159 a chimeric or humanized antibody, or a fragment of an antibody. In one embodiment, the antibody that has the ability to bind to a CLDN18.2 antibody is selected from the group consisting of (i) an antibody produced by and / or obtainable from a clone deposited under the Accession number No. DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2742, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2289, or DSM ACC2809, or which is a chimerized or humanized form of the antibody in (i), (iii) an antibody having the specificity of the antibody under (i) and (iv) an antibody comprising the antigen-binding or antigen-binding portion, in particular the variable region, of the antibody under (i) and, preferably, with the specificity of the antibody under (i). In one embodiment, the antibody is coupled to a therapeutic agent, such as a toxin, a radioisotope, a drug or a cytotoxic agent.
[0019] In one embodiment, the method of the invention comprises administering the antibody which has the ability to bind CLDN18.2 at a dose of up to 1000 mg / m ² In one embodiment, the method of the invention comprises administering the antibody which has the ability to bind to CLDN18.2 repeatedly at a dose of 300 to 600 mg / m ² .
[0020] In one embodiment, the cancer is CLDN18.2
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12/159 positive. In one embodiment, the cancer disease is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head-neck cancer, gallbladder cancer and metastasis. The cancer disease can be a Krukenberg tumor, peritoneal metastasis and / or lymph node metastasis. In one embodiment, cancer is an adenocarcinoma, in particular, an advanced adenocarcinoma. In one embodiment, the cancer is selected from the group consisting of cancer of the stomach, cancer of the esophagus, in particular, of the lower esophagus, cancer of the esogastric junction and gastro-oesophageal cancer. The patient may be an HER2 / neu negative patient or a patient with an HER2 / neu positive status, but not eligible for trastuzumab therapy.
[0021] According to the invention, CLDN18.2 preferably has the amino acid sequence according to SEQ ID NO: 1.
[0022] In another aspect, the present invention provides a medicinal preparation comprising an antibody that has the ability to bind CLDN18.2 and a γδ T cell stimulating agent. The medicinal preparation of the present invention may further comprise an
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13/159 stabilization or increased expression of CLDN18.2. The antibody that has the ability to bind to CLDN18.2 and the T cell stimulating agent γδ, and, optionally, the stabilizing or increasing agent of CLDN18.2 expression, may be present in the medical preparation in a mixture or separated each other.
[0023] The medical preparation can be a kit comprising a first container that includes the antibody that has the ability to bind CLDN18.2 and a container that includes the γδ T-cell stimulating agent, and, optionally, a container, including the stabilizing agent or increased expression of CLDN18.2. The medical preparation may further include printed instructions for the use of the preparation for the treatment of cancer, in particular, for the use of the preparation in a method of the invention. Different embodiments of the medical preparation, and in particular of the γδ T-cell stimulating agent and the stabilizing or enhancing agent of CLDN18.2 expression are as described above for the method of the invention.
[0024] The present invention also provides the agents described herein, such as the antibody that has the ability to bind CLDN18.2 for use in the methods described herein, for example, for administration in combination with a γδ T cell stimulating agent, and, optionally, a
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14/159 stabilizing agent or increased expression of CLDN18.2.
[0025] Other features and advantages of the present invention will be evident from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS [0026] Figure 1 shows the effect of chemotherapy on gastric cancer cells. Cultivation of atoIII cells for 96 hours leads to a stop of the cell cycle in phases G0 / G1 and negative regulation of CLDN18.2. Cytostatic compounds, resulting in a cell cycle arrest at different stages of the cell cycle (S-phase (5-FU) or G2-phase (epirubicin)) stabilizes CLDN18.2-expression.
[0027] Figure 2 shows the effect of chemotherapy on gastric cancer cells, a / b: Effect of chemotherapy on the levels of transcription and proteins of CLDN18.2 in gastric cancer cells, c: Flow cytometry of extracellular IMAB362 of binding in gastric cancer cells treated with chemotherapeutic agents.
[0028] Figure 3 shows the effect of chemotherapy on gastric cancer cells. Cytostatic compounds, resulting in a cell cycle stop at different phases of the cell cycle (-S / G2 phase (irinotecan) or in G2 phase (docetaxel)).
[0029] Figure 4 shows IMAB362 ADCC mediated death
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15/159 induced by gastric cancer cells after pretreatment with chemotherapy.
[0030] Figure 5 shows the effect of chemotherapy on gastric cancer cells, a: Cells treated with irinotecan, docetaxel or Cisplatin exhibit a lower level of viable cells compared to target medium cultured cells, b: CLDN18.2 expression in cells treated with irinotecan, docetaxel or Cisplatin is increased compared to cultured cells medium, c / d: Treatment of cells with irinotecan, docetaxel or cisplatin increases the potency of IMAB362 to induce ADCC.
[0031] Figure 6 shows the effects of chemotherapy on IMAB362-induced CDC [0032] Figure 7 shows the effects of chemotherapy on effector cells [0033] Figure 8 shows PBMC expansion cultures in supplemented ZA / IL-2 [0034] Figure 9 shows T cell enrichment in Vy9V52 ZA / IL-2 supplemented PBMC cultures [0035] Figure 10 shows enrichment of Vy9V52 T cells in medium supplemented with ZA and an increased dose of IL-2 [0036] Figure 11 shows the expansion and cytotoxic activity of Vy9V52 T cells over co-incubation with
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16/159 ZA-pulsed monocytes and human cancer cells [0037] Figure 12 shows the ZAdependent development of different cell types in PBMC cultures [0038] Figure 13 shows the display of surface markers on Vy9V52 T cells after ZA / IL-2 treatment [0039] Figure 14 shows ADCC activity of T cells with Vy9V62 IMAB362 in NUGC of 4 gastric cancer cells positive CLDN18.2 [0040] Figure 15 shows ADCC of EVIAB362 using Vy9V52 T cells as effector cells [0041 ] Figure 16 shows the effects of ZA on location on the surface of target cells in CLDN18.2 [0042] Figure 17 shows the effects of chemotherapy and ZA / IL-2 in the treatment of effector cells [0043] Figure 18 shows the biodistribution studies with conjugated antibodies in mice [0044] Figure 19 shows the beginning of treatment of HEK293-CLDN xenografts 18.2 tumor [0045] Figure 20 shows the treatment of advanced HEK293-CLDN18.2 xenoenxe tumor symptoms [0046] Figure 21 shows the effect of IMAB362 on the growth of subcutaneous tumor from gastric cancer xenografts [0047] Figure 22 shows the effects of immunotherapy
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17/159 with IMAB362 in NCI-N87-CLDN18.2 gastric carcinoma xenografts [0048] Figure 23 shows the effects of combination therapy with IMAB362 and EOF regimen in NCI-N87-CLDN18.2 xenografts [0049] Figure 24 shows the effects of combination therapy with IMAB362 and EOF regimen in UGC-4-CLDN18.2 xenotransplants [0050] Figure 25 shows the effects of T-Vy9V62 cell IL-2 ZA induced in the control of macroscopic tumors by IMAB362 in NSG mice [0051] Figure 26 shows the effects of combination therapy with IMAB362 and EOF regimen on CLS 103 ~ allograft cldnl8.2 tumors
DETAILED DESCRIPTION OF THE INVENTION [0052] Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein, as these may vary. It is also to be understood that the terminology used here is for the purpose of describing only particular embodiments, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless otherwise defined, all terms
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18/159 technicians and scientists used here have the same meanings as commonly understood by a technician versed in the subject.
[0053] In the following, 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 way and in any number to create additional embodiments. The variously described examples and preferred embodiments are not to be construed to limit the present invention to only those embodiments explicitly described. This description must be understood to support and encompass embodiments that combine the embodiments explicitly described, with any number of the elements described and / or preferred. In addition, any changes and combinations of all elements described in this application should be considered disclosed by the description of this application, unless the context indicates otherwise.
[0054] Preferably, the terms used here are defined as described in A multilingual glossary of biotechnological terms: (IUPAC Recommendations), HGW Leuenberger, B. Nagel, and H. Kolbl, Eds, Helvetica Chimica Acta, CH-4010 Basel , Switzerland., (1995).
[0055] The practice of the present invention will employ,
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19/159 unless otherwise specified, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques that are explained in the literature in the field (cf., for example, Molecular Cloning: A Labocamundongory Manual, 2 ^nd Edition, J. Sambrook et al. Eds., Cold Spring Harbor Labocamundongory Press, Cold Spring Harbor, 1989).
[0056] Throughout this specification and the claims that follow, unless the context otherwise requires, the word understand, and variations such as understand and understand, will be understood to imply the inclusion of a declared element, whole or step or group of members, integers or steps, but not the exclusion of any other member, integer or step or group of partners, integers or steps, although in some modalities, such another member, integer or step or group of partners, integers or steps they can be excluded, that is, the object consists of the inclusion of an indicated member, whole or step or group of partners, whole or steps. The terms one and a reference area similar to that used in the context of the description of the invention (especially in the context of the claims) should be understood to cover both the singular and the plural, unless otherwise indicated herein or in clear contradiction with the context. Value range quote here is intended only to serve as a
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20/159 abbreviated method of referring individually to each separate value that belongs within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were described herein individually. All of the methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (for example, such as), provided herein is intended merely to better illustrate the invention and does not represent a limitation on the scope of the invention otherwise claimed. No language in the specification should be interpreted as indicating any unclaimed elements essential to the practice of the invention.
[0057] Several documents are cited throughout the text of this specification. Each of the documents cited here (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether above or below, are hereby incorporated by reference in their entirety. Nothing here should be construed as an admission that the invention is not authorized to anticipate this disclosure by virtue of the state of the art.
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21/159 [0058] The term CLDN18 relates to Claudina 18 and includes variants, including the claudin 18 processing variant 1 (Claudin 18.1 (CLDN18.1)) and the claudin 18 processing variant 2 (claudin 18.2 ( CLDN18.2)).
[0059] The term CLDN18.2 refers to CLDN18.2 preferably, human, and in particular to a protein
which comprises, preferably, what It consists at sequence in amino acids from according to SEQ ID NO: 1 gives listing in strings or a variant gives referred sequence in amino acids. [0060] The term CLDN18 ! .1 refers to if the CLDN18 .1 preferably human, and in particular, The a protein which comprises, preferably, what It consists at sequence in amino acids from according to SEQ ID NO: 2 gives listing in
sequences or a variant of said amino acid sequence.
[0061] The term variant according to the invention refers, in particular, to mutants, splicing variants, isoforms, conformations, allelic variants, species variants and homologous species, in particular, those that are naturally present. An allelic variant refers to a change in the normal sequence of a gene, the meaning of which is often unclear. Complete genetic sequencing often identifies
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22/159 numerous allelic variants of a given gene. A species homolog is a sequence of nucleic acid amino acids or with a different species of origin from which a given sequence of nucleic acid or amino acids. The term variant covers all post-translationally modified variants and conformation variants.
[0062] According to the invention, the term cancer positive for CLDN18.2 means cancer involving cancer cells that express CLDN18.2, preferably on the surface of said cancer cells.
[0063] Cell surface is used according to its normal meaning in the state of the art, and thus includes the outer part of the cell that is accessible for binding by proteins and other molecules.
[0064] CLDN18.2 is expressed on the surface of cells that are located on the surface of said cells and is accessible for binding the specific antibodies of CLDN18.2 added to the cells.
[0065] According to the invention, CLDN18.2 is not substantially expressed in a cell, if the level of expression is lower compared to the expression in cells of the stomach or stomach tissue. Preferably, the level of expression is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of the expression in cells of the
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23/159
stomach or fabric of stomach or same bottom. In preferably, CLDN18.2 no is substantially expressed in an cell if The level in expression higher at the level in
expression in non-cancerous tissue except stomach by no more than 2 times, preferably 1.5 times, and preferably, does not exceed the level of expression in which said non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in a cell, if the expression level is below the detection limit and / or if the expression level is too low to allow the binding of CLDN18.2 specific antibodies added to the cells .
[0066] According to the invention, CLDN18.2 is expressed in a cell, if the level of expression is higher than the level of expression in non-cancerous tissue other than stomach, preferably more than 2 times, preferably 10 times, 100 times 1000- double, or 10,000 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 the binding of CLDN18.2-specific antibodies added to the cells. Preferably, CLDN18.2 expressed in a cell is expressed or exposed on the surface of said cell.
[0067] According to the invention, the term disease refers to any pathological condition, including cancer,
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24/159 in particular, those forms of cancer described here. Any reference in this document to cancer or particular forms of cancer also includes cancer metastases. In a preferred embodiment, a disease to be treated in accordance with the present application involves cells that express CLDN18.2.
[0068] Diseases associated with cells expressing CLDN18.2 or similar expressions mean according to the invention that CLDN18.2 is expressed in cells of a diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in cells of a diseased tissue or organ is increased compared to the state of a healthy tissue or organ. An increase refers to 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 1,000%, at least 10000 % or even more. In one embodiment, the expression is found only in diseased tissue, while the expression in healthy tissue is suppressed. According to the invention, diseases associated with cells that express CLDN18.2 include cancerous diseases. In addition, according to the invention, cancerous diseases are preferably those in which the cancer cells express CLDN18.2.
[0069] As used herein, a cancer disease or cancer includes a disease characterized by a
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25/159 regulated aberrant cell growth, proliferation, differentiation, adhesion and / or migration. Cancer cell means an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow after the stimuli that started the new growth to cease. Preferably, a cancer disease is characterized by cells that express CLDN18.2 and a cancer cell that expresses CLDN18.2. A cell that expresses CLDN18.2 is preferably a cancer cell, preferably a cancer cell described herein.
[0070] Adenocarcinoma is a cancer that originates in the glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands and a variety of other tissues that line the body's cavities and organs. Epithelium is embryologically derived from ectoderm, endoderm and mesoderm. To be classified as adenocarcinoma, cells do not necessarily have to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to look like the glandular tissue that they are derived from, while little differentiated cannot. By staining the cells from a biopsy, a pathologist will
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26/159 determine whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinomas can arise in many tissues in the body, due to the ubiquitous nature of the glands within the body. While each gland cannot be the same substance that secretes, as long as there is an exocrine function for the cell, it is considered a malignant gland and its shape is therefore called adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize given enough time to do so. Ovarian adenocarcinoma is the most common type of ovarian cancer. It includes serous and mucinous adenocarcinomas, clear cell adenocarcinoma and endometrioid adenocarcinoma.
[0071] By metastasis is meant the spread of cancer cells from their original site to another part of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, the penetration of the basal endothelial membranes to enter the body cavity and vessels, and then after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor because the tumor cells or components can remain and develop the potential
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27/159 metastatic. In one embodiment, the term metastases, according to the invention which is from the primary tumor and the regional lymph node system. In one embodiment, the term metastasis according to the invention refers to ganglionic metastasis. A particular form of metastasis that is treatable using the therapy of the invention originates from gastric cancer metastases as the primary site. In preferred embodiments such gastric cancer metastasis is Krukenberg tumors, peritoneal metastases and / or lymphatic metastasis.
[0072] Krukenberg's tumor is an unusual metastatic tumor in the ovary accounting for 1% to 2% of all ovarian tumors. The prognosis of the Krukenberg tumor is still very poor and there is no established treatment for Krukenberg tumors. Krukenberg tumor is a metastatic adenocarcinoma signet ring of ovarian cells. Stomach is the main site, in most cases of Krukenberg tumor (70%). Colon, appendix, and breast carcinomas (mainly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg's tumor arising from carcinomas of the gallbladder, bile ducts, pancreas, small intestine, Vater's ampoule, cervix and urinary bladder / urachus have been reported. The interval between the diagnosis of a primary carcinoma and the subsequent discovery of ovarian involvement is usually
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28/159 months or less, but longer periods have been reported. In many cases, the primary tumor is very small and can escape detection. A previous history of a carcinoma of the stomach or other organ can be obtained in only 20% to 30% of cases.
[0073] Krukenberg's tumor is an example of the selective spread of cancers, more generally in the stomachovarian axis. This axis of tumor dissemination has historically attracted the attention of many pathologists, especially when gastric neoplasms were found to have selective metastasis to the ovaries, without involvement of other tissues. The gastric carcinoma metastasis pathway to the ovaries has been a mystery for a long time, but it is now evident that retrograde lymphatic spread is the most likely route of metastasis.
[0074] Women with Krukenberg tumors tend to be exceptionally young for patients with metastatic carcinoma as they are typically in the fifth decade of life, with an average age of 45 years. This young age of distribution may be related, in part, to the increased frequency of signet ring of gastric carcinoma in young women. Common symptoms are usually related to involvement of the ovaries, the most common of which
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29/159 what are abdominal pain and bloating (mainly, because of ovarian tumors usually bilateral and often large). The remaining patients have nonspecific gastrointestinal symptoms or are asymptomatic. In addition, the Krukenberg tumor is supposedly associated with virilization resulting from the production of hormones by the ovarian stroma. Ascites is present in 50% of cases and usually reveals malignant cells.
[0075] Krukenberg's tumors are bilateral in more than 80% of reported cases. The ovaries are usually enlarged asymmetrical, with a contoured contour. The sectioned surfaces are yellow or white; they are usually solid, although they are occasionally cystic. It is important to note that the capsular surface of the ovaries with Krukenberg tumors is usually smooth and free of adhesions or peritoneal deposits. Of note, other metastatic tumors to the ovary tend to be associated with surface implants. This may explain why Krukenberg's tumor morphology may deceptively appear as a primary ovarian tumor. However, bilateralism in Krukenberg's tumor is consistent with its metastatic nature.
[0076] Patients with Krukenberg tumors have an overall mortality rate that is significantly high. THE
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Most patients die within two years (median survival, 14 months). Several studies show that the prognosis is poor when the primary tumor is identified after metastasis to the ovary is discovered, and the prognosis becomes worse, if the primary tumor remains covered.
[0077] No ideal treatment strategy for Krukenberg tumors has been clearly established in the literature. Whether a surgical resection should be performed has not been treated properly. Chemotherapy or radiation therapy has no significant effect on the prognosis of patients with Krukenberg tumors.
[0078] By treating is meant administering a compound or composition or a combination of compounds or compositions to an individual in order to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in one subject; stop or delay a disease in a subject; inhibit or delay the development of a new disease in a subject; decrease the frequency or severity of symptoms and / or recurrences in a subject who currently has or has had a disease; and / or prolong, that is, increase the subject's useful life.
[0079] In particular, the term treatment of a disease includes cure, shortening the duration, improving, preventing, slowing or inhibiting the progression or worsening of,
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31/159 or to prevent or delay the onset of a disease or its symptoms.
[0080] The term patient means, according to the invention for the treatment of a subject, in particular, a sick subject, including humans, non-human primates or other animals, in particular mammals, such as cows, horses, pigs, sheep, goats, dogs, cats or rodents, such as mice and mice. In a particularly preferred embodiment, a patient is a human being.
[0081] γδ T cells (delta gamma T cells) represent a small subset of T cells that have a distinct T cell receptor (TCR) on their surface. Most T cells have a TCR composed of two glycoprotein chains called a- and β-TCR chains. In contrast, in γδ T cells, the TCR is composed of a γ chain and a δ chain. This group of T cells is generally much less common than αβ T cells. Human γδ T cells play an important role in responding to surveillance stress, such as infectious diseases and autoimmunity. Changes induced by transformation into tumors are also suggested to trigger γδ T-cell-mediated stress responses and increase anti-tumor immunity. Importantly, after coupling the antigen, γδ T cells activated in lesion sites
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32/159 provide cytokines (for example, INFY, TNFa) and / or chemokines that mediate the recruitment of other effector cells and show effector functions, such as immediate cytotoxicity (death receptor pathway and cytolytic granule pathway) and ADCC.
[0082] Most γδ T cells in peripheral blood express the T cell receptor Vy9V52 (TCRy6). Vy9V52 T cells are unique to humans and primates and are assumed to play an essential role in initiating and detecting danger by invading pathogens as they expand dramatically in many acute infections and may be superior to all other lymphocytes within a few days, for example, in tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis and malaria.
[0083] γδ T cells respond to small non-peptide phosphorylated antigens (phosphoantigens), such as pyrophosphates synthesized in bacteria and isopentenyl pyrophosphate (IPP) produced in mammalian cells via the mevalonate pathway. Considering that the production of IPP in normal cells is not sufficient for the activation of γδ T cells, the deregulation of the mevalonate pathway in tumor cells leads to the accumulation of IPP and activation of γδ T cells. IPP can also be increased by therapeutically aminobiphosphonates, which inhibit the enzyme mevalonate pathway
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33/159 fRNAesyl pyrophosphate synthase (FPPS). Among others, zoledronic acid (ZA, zoledronate, Zometa ™, Novartis) represents such an aminobiphosphonate, which is already clinically administered to patients for the treatment of osteoporosis and metastatic bone diseases. After treatment of PBMC in vitro, ZA is taken, especially, by monocytes. ΓΡΡ accumulates in monocytes and differentiate into antigen presenting cells that stimulate the development of γδ T cells. In this configuration, the addition of interleukin-2 (IL-2) is preferred as the growth and survival factor for activated γδ T cells. Finally, certain alkylated amines have been described to activate Vy9V62 T cells in vitro, however, only in millimolar concentrations.
[0084] According to the invention, the term γδ T cell stimulating agent refers to compounds that stimulate the development of γδ T cells, in particular Vy9V62 T cells, in vitro and / or in vivo, in particular, through of induction of γδ T cell activation and expansion. Preferably, the term refers to compounds that, in vitro and / or in vivo increase in isopentenyl pyrophosphate (ΓΡΡ) produced in mammalian cells, preferably via the mevalonate inhibit the enzyme fRNAesyl pyrophosphate (FPP).
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34/159 [0085] A particular group of compounds that stimulate γδ T cells are bisphosphonates, in particular nitrogen-containing bisphosphonates (N-bisphosphonates; aminobiphosphonates).
[0086] For example, bisphosphonates suitable for use in the invention may include one or more of the following compounds including analogs and derivatives, pharmaceutical salts, hydrates, esters, and conjugated prodrugs:
- [1-hydroxy-2- (1H-imidazol-l-yl) ethane-1,1-di-yl] bis (phosphonic acid), zoledronic acid, for example, zoledronate;
- Phosphonic acid (dichloro-phosphono-methyl), for example, clodronate
- {1-hydroxy-3- [methyl (pentyl) amino] propane-1,1-diyl} bis (phosphonic acid), ibandronic acid, eg ibandronate
- (3-amino-1-hydroxypropane-1,1-di-yl) bis (phosphonic acid), pamidronic acid, for example, pamidronate;
- (1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl) phosphonic, risedronic acid, for example, risedronate;
- Phosphonic acid (1-2-hydroxy-imidazo [1,2-a] pyridin-3-yl-1-phosphonoethyl), minodronic acid;
- [3 - (dimethylamino) - 1-hydroxypropane- 1,1-diyl] bis (phosphonic acid), olpadronic acid.
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- [4-Amino-1-hydroxy-1 (hydroxy-oxido-phosphoryl) butyl] phosphonic acid, alendronic acid, for example, alendronate;
- [(cycloheptylamino) methylene] bis (phosphonic acid), incadronic acid;
- (1-hydroxyethan-1,1-di-yl) bis (phosphonic acid), etidronic acid, for example, etidronate; and
- {[(4-chlorophenyl) thio]} methylene} bis (phosphonic acid), tiludronic acid.
[0087] According to the invention, zoledronic acid (INN) or zoledronate (marketed by Novartis under the trade names Zometa, Zomera, Aclasta and Reclast) is a particularly preferred bisphosphonate. Zometa is used to prevent bone fractures in cancer patients, such as multiple myeloma and prostate cancer, as well as to treat osteoporosis. It can also be used to treat malignant hypercalcemia and can be useful for treating pain from bone metastases.
[0088] In a particularly preferred embodiment, a γδ T cell stimulating agent according to the invention is administered in combination with IL-2. Such a combination proved to be particularly effective in mediating expansion and activation of γ9δ2 T cells.
[0089] Interleukin-2 (IL-2) is an interleukin, a
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36/159 type of cytokine signaling molecule in the immune system. It is a protein that attracts lymphocytes and is part of the body's natural response to microbial infection, and in discrimination between foreigners (non-self) and self. IL-2 mediates its effects by binding to IL-2 receptors, which are expressed by lymphocytes.
[0090] The IL-2 used, according to the invention can be any IL-2 or support allowing the stimulation of γδ T cells and can be derived from any species, preferably human. 11-02 may be isolated, produced recombinantly or synthetically IL-2 and can occur naturally or be modified from IL-2 naturally.
[0091] The term stabilizing agent or increased expression of CLDN18.2 refers to an agent or a combination of agents, the starting supply to cells results in increased RNA and / or protein levels of CLDN18.2 preferably in an increase in the levels of CLDN18.2 protein on the cell surface, compared to the situation where the cells are not supplied with the agent or combination of agents. Preferably, the cell is a cancer cell, in particular a cancer cell that expresses CLDN18.2, as a cell of the types of cancer is described here again. The term stabilizing or increasing expression of CLDN18.2 refers, in particular, to
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37/159 an agent or combination of agents to which the disposition of cells results in a higher density of CLDN18.2 on the surface of said cells compared to the situation where the cells are not supplied with the agent or combination of agents . Stabilization The expression of CLDN18.2 includes, in particular, the situation where the agent or combination of agents prevents a decrease or reduces a decrease in the expression of CLDN18.2, for example, expression of CLDN18.2 would decrease without delivery of the agent or the combination of agents and disposition of the agent or combination of agents prevents said decrease or reduced said decrease in expression CLDN18.2. Increasing the expression of CLDN18.2 includes, in particular, the situation where the agent or combination of agents increases the expression of CLDN18.2, for example, expression of CLDN18.2 would decrease, or remain essentially constant increase without supply of the agent or combination of agents and disposition of agent or combination of agents CLDN18.2 increases expression compared to the situation without the supply of agent or combination of agents so that the resulting expression is higher compared to the situation where the expression of CLDN18.2 would decrease, remain essentially constant or increase without provision of the agent or combination of agents.
[0092] According to the invention, the term
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38/159 stabilization or increased expression of CLDN18.2 includes chemotherapeutic agents or combinations of chemotherapeutic agents such as cytostatic agents. Chemotherapeutic agents can affect cells in one of the following ways: (1) Damage to cell DNA, so that it can no longer reproduce, (2) inhibit the synthesis of new DNA strands so that cell replication is not possible , (3) Stop the cell mitosis processes, so that the cells cannot divide into two cells.
[0093] According to the invention, the term stabilizing or increasing expression of CLDN18.2 refers preferably to an agent or a combination of a cytostatic compound such agents or a combination of cytostatic compounds the provision of which cells, namely in cancer cells, results in cells being trapped in or accumulating in one or more phases of the cell cycle, preferably in one or more phases of the cell cycle other than the gl- and GO-phases, preferably others of the than the G-phase, preferably in one or more of the G2- or S-phase of the cell cycle, such as the G1 / G2, S / G2, G2 or S-phase of the cell cycle. The term cells of being trapped in or accumulating in one or more phases of the cell cycle means that the percentage of cells that are in which said one or more phases of the cell cycle increases.
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Each cell goes through a cycle that comprises four phases in order to replicate itself. The first phase is called Gl when the cell is preparing to duplicate its chromosomes. The second phase is called S, and in this phase of DNA synthesis occurs and the DNA is duplicated. The next phase is the G2 phase, when the RNA and protein duplicate. The final step is the M phase, which is the actual cell division phase. In this final step, the DNA and RNA divide and duplicate and move to the separate ends of the cell, and, in fact, the cell divides into two identical, functional cells. Chemotherapeutic agents that are DNA damaging agents usually lead to an accumulation of cells in the G1 and / or G2 phase. Chemotherapeutic agents that block cell growth by interfering with DNA synthesis, such as antimetabolites normally lead to an accumulation of cells in the S phase. Examples of these drugs are 6-mercaptopurine and 5-fluorouracil.
[0094] According to the invention, the term stabilizing or increasing expression of CLDN18.2 includes anthracyclines, such as epirubicin, platinum compounds, such as cisplatin and oxaliplatin, nucleoside analogs, such as 5-fluorouracil or its precursors, taxanes such as docetaxel, and camptothecin analogues, such as irinotecan and topotecan, and
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40/159 drug combinations, such as drug combinations comprising one or more of the anthracyclines, such as epirubicin, oxaliplatin and 5-fluorouracil, such as a drug combination comprising oxaliplatin and 5 fluorouracil or other drug combinations described herein.
[0095] In a preferred embodiment, a stabilizing or enhancing agent of CLDN18.2 expression is an immunogenic cell death inducing agent.
[0096] In specific circumstances, cancer cells can introduce a lethal stress pathway associated with the emission of a combination of space-timed signals that is decoded by the immune system to activate tumor-specific immune responses (Zitvogel L. et al (2010 ) Cell 140: 798 - 804). In such a scenario, cancer cells are triggered to emit signals that are detected by innate immune effectors, such as dendritic cells to trigger a cognate immune response involving CD8 + T cells and IFN-γ signaling, so that death tumor cells can induce a productive anti-cancer immune response. These signs include pre-apoptotic exposure of the accompanying endoplasmic reticulum (ER) calreticulin (CRT) on the cell surface, pre-apoptotic ATP secretion, and post release
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41/159 apoptotic of the nuclear HMGB1 protein. Together, these processes constitute the molecular determinants of immunogenic cell death (ICD). Anthracyclines, oxaliplatin, and γ irradiation are able to induce all the signals that define ICD, while cisplatin, for example, which is deficient in CRT induction translocation from ER to the surface of dying cells - a process that requires stress ER requires supplementation by tapsigargina, an ER stress inducer.
[0097] According to the invention, the term immunogenic cell death-inducing agent refers to an agent or combination of agents that, when supplied to cells, in particular cancer cells, is able to induce cells to introduce a pathway of lethal stress that ultimately results in tumor-specific immune responses. In particular, an immunogenic cell death-inducing agent when delivered to cells induces cells to emit a space-time-defined combination of signals, including, in particular, the pre-apoptotic exposure of the accompanying endoplasmic reticulum (ER) calreticulin (CRT) in surface cell, pre-apoptotic ATP secretion, and post-apoptotic release of the nuclear HMGB1 protein.
[0098] According to the invention, the term immunogenic cell death-inducing agent includes anthracyclines and
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42/159 oxaliplatin.
[0099] Anthracyclines are a class of drugs commonly used in cancer chemotherapy, which are also antibiotics. Structurally, all anthracyclines share a common 7,8,9,10-tetrahydrotetracene5,12-quinone four ring structure and normally require glycosylation at specific sites.
[00100] Anthracyclines preferably take about one or more of the following mechanisms of action: 1. Inhibition of DNA and RNA synthesis by intercalation between base pairs of the DNA / RNA chain, thus preventing rapid replication the growth of cancer cells. 2. The inhibition of the topoisomerase II enzyme, preventing the relaxation of supercoiled DNA, thus preventing DNA transcription and replication. 3. Creation of free oxygen radicals
iron-mediated that damage DNA and membranes cell phones. [00101] From a deal with The invention, the term anthracycline, preferably, if refers to an agent,
preferably, an anti-cancer agent to induce apoptosis, preferably by inhibiting the rewiring of DNA into topoisomerase II.
[00102] Preferably, according to the invention, the term anthracycline refers, generally to a class of compounds that have the following ring structure
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including analogs and derivatives, pharmaceutical salts, hydrates, esters, and conjugated prodrugs.
[00103] Examples of anthracycline and anthracycline analogs include, but are not limited to, daunorubicin (daunomycin), doxorubicin (adriamycin), epirubicin, idarubicin, rhodomycin, pyrarubicin, valrubicin, N-trifluoro-acetyl doxorubin, 14-valecammonium holinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrroline-doxorubicin (2-PDOX), 5iminodaunomycin, mitoxantrone and aclacinomycin A (aclarrubicin). Mitoxantrone is a member of the class of anthracendione compounds, which has anthracycline analogues that lack the anthracycline sugar molecule, but maintain the structure of the polycyclic planar aromatic ring that allows intercalation in DNA.
[00104] Particularly preferred as anthracillin according to the invention is a compound of the following formula:
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where [00105] Ri is selected from the group consisting of H and OH, R2 is selected from the group consisting of H and OMe, R3 is selected from the group consisting of H and OH, and R4 is selected from the group consisting of H and OH.
[00106] In one embodiment, R1 is H, R2 represents OMe, R3 represents H, and R4 is OH. In another embodiment, R1 is OH, R2 represents OMe, R3 represents H, and R4 is OH. In another embodiment, R1 is OH, R2 represents OMe, R3 is OH, and R4 is H. According to another embodiment, R1 is H, R2 is H, R3 represents H, and R4 is OH.
[00107] Specifically contemplated as anthracycline in the context of the present invention is epirubicin. Epirubicin is an anthracycline medication that has the following formula:
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The OH
The HO
O O OH O
H3C ^O
CH3
OH
NH ₂ and is marketed under the trade name Ellence in the USA and farmorubicin 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-Oxan2-yl] oxy -6, 1 1-dihydroxy-8- (2-hydroxyacetyl) -l-methoxy8-methyl-9,10-dihydro-7H-tetracen-5,12-dion. Epirubicin is favored over doxorubicin, the most popular anthracycline, in some chemotherapy regimens, as it appears to cause fewer side effects.
[00108] According to the invention, the term platinum compound refers to compounds containing platinum in their structure, such as platinum complexes and includes compounds such as cisplatin, carboplatin and oxaliplatin.
[00109] The term cisplatin or cisplatin refers to the compound cz ^' s-diaminodichloroplatin (II) (CDDP) of the following formula:
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Ο / ,,, ρ ^ ΝΗ, CK> JH.
[00110] The term carboplatin refers to the compound cis-diamine (1,1-cyclobutanedicarboxylate) platinum (II) with the following formula:
[00111] The term oxaliplatin refers to a compound that is a platinum compound that is complexed with a diaminocyclohexane carrier binder the following formula:
[00112] In particular, the term oxaliplatin refers to the compound [(IR, 2R) -cyclohexane-1,2-diamine] (ethanedioate-O, 0 ') platinum (II). Injectable oxaliplatin is also marketed under the brand name Eloxatine.
[00113] The term nucleoside analog refers to a structural analog of a nucleoside, a category that includes both purine analogs and pyrimidine analogs.
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In particular, the term nucleoside analog refers to derivatives of fluoropyrimidine which includes fluorouracil and its prodrugs.
[00114] The term fluorouracil or 5-fluorouracil (5FU or f5U) (sold under the brand names Adrucil, Carac, Efudix, Efudex and Fluoroplex) is a compound that is a pyrimidine analog with the following formula:
[00115] In particular, the term refers to the compound 5-fluoro-1H-pyrimidine-2,4-dione.
[00116] The term capecitabine (Xeloda, Roche) refers to a chemotherapeutic agent that is a prodrug that is converted to 5-FU in tissues. Capecitabine, which can be administered orally has the following formula:
[00117] In particular, the term refers to the pentyl compound [1- (3,4-dihydroxy-5-methyl-2-yl) -5-fluor-2-oxo-1H
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48/159 pyrimidin-4-yl] carbamate.
[00118] Taxanes are a class of diterpene compounds that were derived from primary natural sources, such as plants of the Taxus genus, but some have been artificially synthesized. The main mechanism of action of the drug taxane class is the disruption of microtubule function, thereby inhibiting the cell division process. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).
[00119]
According to the invention, the term docetaxel refers to a formula:
following compound with the
[00120]
According to the invention, the term paclitaxel refers to a compound with the following formula:
O ^ NH O ^H 3 ^C
O''
OH
O
O ^ CH3
O
CH3
CH
OH £ O ^ O _H - o ^H O ^ CH3
O
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49/159 [00121] In accordance with the invention, the term camptothecin analog refers to derivatives of the compound camptothecin (CPT; (S) -4-ethyl-4-hydroxy-1H-pyran [3 ', 4': 6.7] indolizine [1,2-b] quinoline-3,14- (4H, 12H) -dione). Preferably, the term camptothecin analog refers to compounds that comprise the following structure:
OH [00122] According to the invention, preferred camptothecin analogs are inhibitors of the DNA enzyme topoisomerase I (Topo I). Preferred camptothecin analogs according to the invention are irinotecan and topotecan.
[00123] Irinotecan is a drug preventing from unwinding DNA by inhibiting topoisomerase I. In chemical terms, it is a semi-synthetic analogue of the natural alkaloid camptothecin having the following formula:
[00124] In particular, the term irinotecan refers to the compound (S) -4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxyPetition 870160018043, 05/05/2016, p. 58/206
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3, 14-dioxo-1H-pyran [3,476,7] -indolizine [1,2,2-b] quinolin-9-yl- [1,4'bipiperidine] - 1'-carboxylate.
[00125] Topotecan is a topoisomerase inhibitor of the formula:
H3C
N
H3C
HO [00126] In particular, the term topotecan refers to the compound (S) -10 - [(dimethylamino) methyl] -4-ethyl-4,9-dihydroxy-1 H-pyran [3 ', 4': 6 , 7] indolizine [1,2-b] quinoline-3, 14 (4H, 12H) -dione monochloride mouse.
[00127] According to the invention, a stabilizing or increasing expression agent of CLDN18.2 may be a chemotherapeutic agent, in particular a chemotherapeutic agent in the treatment of established cancer and may be part of a combination of drugs, such as a combination of drugs established for use in the treatment of cancer. This drug combination can be a combination of drugs used in chemotherapy, and it can be a combination of drugs as used in a chemotherapy regimen selected from the group consisting of EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, FOLFOX chemotherapy,
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Folfiri chemotherapy, chemotherapy and chemotherapy FLOT DCF.
[00128] The combination of drugs used in EOX chemotherapy has epirubicin, oxaliplatin and capecitabine. The combination of drugs used in ECF chemotherapy comprises epirubicin, cisplatin and 5-fluorouracil. The combination of drugs used in ECX chemotherapy has epirubicin, cisplatin and capecitabine. The combination of drugs used in EOF chemotherapy comprises epirubicin, oxaliplatin and 5-fluorouracil.
[00129] Epirubicin is usually administered at a dose of 50 mg / m2, cisplatin 60 mg / m2, oxaliplatin 130 mg / m2, prolonged intravenous infusion of 5-fluorouracil, 200 mg / m2 / day orally and capecitabine 625 mg / m2 twice daily for a total of eight 3-week cycles.
[00130] The combination of drugs used in FLO chemotherapy comprises of 5-fluorouracil, folinic acid and oxaliplatin (normally 5-fluorouracil 2,600 mg / m2 for 24 hours of infusion, folinic acid 200 mg / m2 and oxaliplatin 85 mg / m2, at every 2 weeks).
[00131] FOLFOX is a chemotherapy regimen, made of folinic acid (leucovorin), 5-fluorouracil and oxaliplatin. The recommended dose regimen given every two weeks is the
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52/159 next: Day 1: Oxaliplatin 85 mg / m ² IV infusion and leucovorin 200 mg / m ² IV infusion, followed by 5-FU 400 mg / m ² IV bolus, followed by 5- FU 600 mg / m ² IV infusion as a 22-hour continuous infusion;
[00132] Day 2: leucovorin 200 mg / m ² IV infusion over 120 minutes, followed by 5-FU 400 mg / m ² IV bolus given over 2-4 minutes, followed by 5-FU 600 mg / m ² IV as a 22- hour continuous infusion.
[00133] The combination of drugs used in Folfiri chemotherapy comprises of 5-fluorouracil, leucovorin, and irinotecan.
[00134] The combination of drugs used in DCF chemotherapy comprises the administration of docetaxel, cisplatin and 5-fluorouracil.
[00135] The combination of drugs used in FLOT chemotherapy comprises the administration of docetaxel, oxaliplatin, 5-fluorouracil and folinic acid.
[00136] The term folinic acid or leucovorin refers to a compound useful in synergistic combination with the chemotherapy agent 5-fluorouracil. Folinic acid has the following formula:
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[00137] In particular, the term refers to the compound (2S) -2 - {[4 - [(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydrolH-pteridin -6- yl) methylamino] benzoyl] amino] of pentanedioic acid.
[00138] For the administration of a stabilizing agent or increased expression of CLDN18.2, in one embodiment, the chemotherapy pattern according to the EOX regimen in combination with an antibody that has the ability to bind CLDN18.2 , in particular IMAB362 is administered for max. 8 cycles. Doses and schedules can be as follows:
[00139] 50 mg / m2 of epirubicin will be administered how infusion iv 15 minutes nc i day 1 of each cycle during The phase of EOX. [00140] 130 mg / m2 oxaliplatin will be administered iv how infusion 2 h day 1 of each cycle during the phase in EOX.
[00141] 625 mg / m2 Capecitabine are taken twice a day for 21 days, morning and evening, beginning
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54/159 with the night of day 1 of each cycle during the EOX phase.
• 1000 mg of antibody / m2 will be administered iv as a 2 h infusion on day 1 of cycle 1. Thereafter, 600 mg / m2 of antibody iv will be administered as a 2 h infusion on day 1 of each cycle of another. after the infusion of oxaliplatin is complete.
[00142] After the end of chemotherapy, the patient will continue with 600 mg / m2 of antibody as an infusion of 2 h every 3 or 4 weeks.
[00143] In one embodiment of the present invention, standard chemotherapy, according to the EOX regimen in combination with ZA / IL-2 and an antibody that has the ability to bind to CLDN18.2, in particular, IMAB362 is administered for up to 8 cycles (24 weeks).
[00144] The term antigen refers to an agent, such as a protein or peptide that comprises an epitope against which an immune response is directed and / or is being directed. In a preferred embodiment, an antigen is an antigen associated with a tumor, such as CLDN18.2, that is, a constituent of cancer cells that can be derived from the cytoplasm, the cell surface and the cell nucleus, in particular , antigens that are produced, preferably, in large quantities, intracellular or as surface antigens in cancer cells.
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55/159 [00145] In the context of the present invention, the term tumor-associated antigen preferably refers to proteins that are under normal conditions specifically expressed in a limited number of tissues and / or organs or at specific stages of development and they are expressed or expressed aberrantly in one or more tumors or tissue cancer. In the context of the present invention, the tumor-associated antigen is preferably associated with the cell surface of a cancer cell and, preferably, is not or only rarely expressed in normal tissues.
[00146] The term epitope refers to an antigenic determinant in a molecule, that is, the part of a molecule that is recognized by the immune system, for example, that is recognized by an antibody. For example, epitopes are discrete, three-dimensional sites on an antigen, which are recognized by the immune system. Epitopes typically consist of chemically active surface clusters of molecules such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that the bond to the former, but not the latter, is lost in the presence of denaturing solvents. An epitope of a
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56/159 protein, such as CLDN18.2, preferably comprises a continuous or batch portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, more preferably between 10 and 25 amino acids in length, for example, the epitope
can be, of preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length. [00147] O antibody term if refers The an
glycoprotein that comprises at least two (H) heavy chains and two light (L) inter-chains linked by disulfide bonds, and includes any molecule that comprises an antigen-binding portion thereof. The term antibody includes monoclonal antibodies and antibody fragments or derivatives, including, without limitation, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, for example, scFv and antigen binding antibody fragments such as Fab fragments and Fab 'e also includes all recombinant forms of antibodies, for example, antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein. Each heavy chain is comprised of a variable heavy chain region (here abbreviated as VH) and a variable
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57/159 heavy chain constant region. Each light chain is comprised of a variable region of the light chain (here abbreviated as VL) and a constant region of the light chain. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called structural regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classical complement system.
[00148] The antibodies described herein can be human antibodies. The term human antibody, as used herein, is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin
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58/159 (eg, mutations introduced by random mutagenesis, or site specific in vitro or by somatic mutation in vivo).
[00149] The term humanized antibody refers to a molecule containing an antigen-binding site that is substantially derived from an immunoglobulin of a non-human species, in which the remaining immunoglobulin structure of the molecule is based on the structure and / or the following a human immunoglobulin. The antigen-binding site can include either the complete variable domains fused to the constant domains or just the complementarity determining regions (CDR) grafted into suitable structural regions in the variable domains. Antigen binding sites can be wild-type or modified by one or more amino acid substitutions, for example, modified to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody that contains all six CDRs of the mouse antibody). Other forms have one or more CDRs that are altered with respect to the original antibody.
[00150] The term chimeric antibody refers to these antibodies, where a portion of each of the
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59/159 amino acid sequences of light and heavy chains is homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a certain corresponding class, while the rest of the chain is homologous to the corresponding sequences in another corresponding one. Typically, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammal, while the constant portions are homologous to the sequences of antibodies derived from another. A clear advantage for such chimeric forms is that the variable region can be conveniently derived from presently known sources using B cells or hybridomas readily available from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and specificity is unaffected by the source, the constant region being human, they are less likely to elicit an immune response from a human individual when antibodies are injected than the constant region from from a non-human source. However, the definition is not limited to this particular example.
[00151] The terms of an antibody (or simply binding moiety) similar terms or fragment of
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60/159 antigen binding to an antibody (or simply binding fragment) or antigen binding portion 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 within the term of an antibody antigen binding portion include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH domains; (ii) F (ab ') 2, divalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al, (1989) Nature 341: 544 - 546), which consists of a VH domain; (vi) the isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs that can optionally be joined by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked, using recombinant methods, by a synthetic linker that allows them to be made as a single protein chain, in which the VL and VH regions match
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61/159 to form monovalent molecules (known as 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 encompassed by the term antigen binding fragment of an antibody. Another example is immunoglobulin fusion proteins that bind to the domain comprising (i) a polypeptide binding domain that is fused to a polypeptide from the immunoglobulin chRNA region, (ii) a CH2 immunoglobulin heavy chain from the constant region fused to the chRNAeira region, and (iii) a CH3 immunoglobulin from the constant chain heavy chain fused to the CH2 constant region. The polypeptide binding domain can be a variable region of the heavy chain or a variable region of the light chain. The immunoglobulin fusion proteins that bind to the domain are further described 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 way as intact antibodies are.
[00152] The term bispecific molecule is intended to include any agent, for example, a protein, peptide or protein or complex peptide, which has two
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62/159 different connection specifics. For example, the molecule can bind to, or interact with (a) a cell surface antigen, and (b) an Fc receptor on the surface of an effector cell. The term multispecific molecule or heterospecific molecule is intended to include any agent, for example, a protein, peptide, or protein or peptide complex, which has more than two different binding specificities. For example, the molecule can bind to, or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, triespecific, tetraspecific, and other multispecific molecules that are directed towards CLDN18.2, and for other purposes, such as Fc receptors on effector cells. The term bispecific antibodies also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with domains complementary to another chain and creating two antigen-binding sites (see, for example, Holliger, P., et al (1993) Proc Natl Acad Sci USA 90: 6444-6448; Poljak, RJ, et al
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63/159 (1994) Structure 2: 1121-1123).
[00153] An antibody can be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a medication (for example, an immunosuppressant) or a radioisotope. A cytotoxin or cytotoxic agent includes any agent that is harmful to and, in particular, kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitramicin, actinomycin D, 1 , tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues thereof. Suitable therapeutic agents for the formation of antibody conjugates include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil decarbazine), alkylating agents (p eg, mecloretamine, thiope chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cis-dichlorodiamine-platinum (II) cisplatin) eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (for example,
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64/159 dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC), and anti-mitotic agents (eg, 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 yet 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.
[00154] Antibodies can also be conjugated to a radioisotope, for example, iodine-131, yttrium-90 or iidium111, to generate cytotoxic radiopharmaceuticals.
[00155] The antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be interpreted as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or its active fragment, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or γ-interferon; or, biological response modifiers such as, for example,
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65/159 lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), colony stimulating factor granulocyte (G-CSF), or other growth factors.
[00156] Techniques for conjugating such a therapeutic antibody portion are well known, see, for example, RNAon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (Eds.), 243-56 (Alan R. Liss, Inc., 1985) pp .; Hellstrom et al., Antibodies for drug delivery, in Controlled Drug Delivery (2nd ^to Ed.), Robinson et al. (Eds.), 623-53 (Marcel Dekker, Inc., 1987) pp .; Thorpe, Antibody with cytotoxic agents Agents in Cancer Therapy: A Review, on monoclonal antibodies '84: Biological and clinical applications, Pincheraet al. (Eds.), Pp 475-506 (1985); The analysis, results and future perspectives of the therapeutic use of Radiolabeled Antibody in cancer therapy, in monoclonal antibodies in cancer detection and therapy, Baldwin et al. (Eds.), Pp. 30316 (Academic Press 1985), and Thorpe et al., A Cytotoxic Preparation and Properties of Antibody-Toxin Conjugates, Immunol. Rev., 62: 119-58 (1982).
[00157] As used herein, an antibody is
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66/159 derived from a germline sequence, in particular, whether the antibody is obtained from a system by immunizing an animal or by screening a library of immunoglobulin genes, and in which the selected antibody is at least 90%, more preferably at least 95%, even more preferably, at least 96%, 97%, 98%, or 99% identical in the amino acid sequence with the amino acid sequence encoded by the germline immunoglobulin gene. Typically, an antibody derived from a germline sequence will in particular exhibit no more than 10 amino acid differences, more preferably, no more than 5, or even more preferably, no more than 4, 3, 2, or one difference in amino acids from the amino acid sequence encoded by the germline immunoglobulin gene.
[00158] As used herein, the term heteroantibodies refers to two or more antibodies, their derivatives, or antigen-binding regions linked together, at least two of which have different specificities. These different specificities include a binding specificity for an Fc receptor on an effector cell, and a binding specificity for an antigen or epitope on a target cell, for example, a tumor cell.
[00159] The antibodies described here can be
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67/159 monoclonal antibodies. The term monoclonal antibody as used herein refers to a preparation of antibody molecules of the single molecular composition. A monoclonal antibody has a unique binding and affinity specificity. In one embodiment, monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a non-human animal, for example, a mouse, fused to an immortalized cell.
[00160] The antibodies described herein can be recombinant antibodies. The term recombinant antibody, as used herein, includes all antibodies that are prepared, expressed, raised or isolated by recombinant means, such as: (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transcromosomal with respect to immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library; and (d) antibodies prepared, expressed, raised or isolated by any other means that involve splicing the immunoglobulin gene sequences to other DNA sequences.
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68/159 [00161] The antibodies described herein can be derived from different species, including, but not limited to, mouse, mouse, rabbit, guinea pig and human.
[00162] The antibodies described herein include polyclonal and monoclonal antibodies and include IgA, such as iGal or IgA2, IgGl, IgG2, IgG3, IgG4, IgE, IgM, IgD and antibodies. In various embodiments, the antibody is an IgGl antibody, more particularly an IgGl, kappa or IgGl, lambda isotype (i.e. IgGl, κ, λ), an IgG2a antibody (for example, IgG2a, κ, λ), an antibody IgG2b (for example, IgG2b, K, λ), an IgG3 antibody (for example, IgG3, κ, λ) or an IgG4 antibody (for example, IgG4, κ, λ).
The term transfectoma, as used herein, includes recombinant eukaryotic host cells that express an antibody, such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant or fungal cells, including yeast cells.
[00164] As used herein, a heterologous antibody is defined in relation to a transgenic organism producing such an antibody. This term refers to an antibody having an amino acid sequence or a coding nucleic acid sequence corresponding to that found in an organism that is not constituted, the transgenic organism, and being generally derived from an exception, the
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69/159 organism of transgenic species.
[00165] As used herein, a hetero antibody refers to an antibody having light and heavy chains from different origins in the organisms. For example, an antibody having a human heavy chain associated with a murine light chain is a hetero-antibody.
[00166] The invention includes all antibodies and antibody derivatives, as described herein, which for the purposes of the present invention are encompassed by the term antibody. The term antibody derivatives refers to any modified form of an antibody, for example, an antibody conjugate and the other agent or antibody, or an antibody fragment.
[00167] The antibodies described herein are preferably isolated. An isolated antibody, as used herein, is intended to refer to an antibody that is substantially free of other antibodies that have different antigen specificities (for example, an isolated antibody that specifically binds to CLDN18.2 is substantially free of antibodies that bind specifically to antigens other than CLDN18.2). An isolated antibody that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may, however, cross-react with other related antigens, for example.
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70/159 example, from other species (for example, CLDN18.2 homologous species). In addition, an isolated antibody can be substantially free of other cellular material and / or chemicals. In one embodiment of the invention, an isolated monoclonal antibody combination refers to antibodies having different specificities and being combined into a well-defined composition or mixture.
[00168] The term bond according to the invention preferably refers to a specific bond.
[00169] According to the present invention, an antibody is able to bind to a predetermined target, if it has significant affinity for said predetermined target and binds to said predetermined target in conventional assays. Affinity or binding affinity is often measured by the dissociation equilibrium constant (KD). Preferably, the term significant affinity refers to binding to a predetermined target, with a dissociation constant (KD) of 10 ^-5 M or less, 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, or 10 ^-12 M or less.
[00170] An antibody is (substantially) unable to bind to a target that has no significant affinity for that target and does not bind
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significant, in particular, no if turns on so detectable by said target, in essay pattern. In preferably the antibody is not turn on in form detectable
for said target if it is present in a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 µg / ml or higher. Preferably, an antibody has no significant affinity for a target that binds to that target with a Kd that is at least 10 times, 100 times, 103 times, 104 times, 10 ⁵ times, or 106 times more higher than KD for binding to the predetermined target to which the antibody is capable of binding. For example, if the KD for binding an antibody to the target to which the antibody is capable of binding is 10 ^-7 M, the KD for binding to a target to which the antibody has no significant affinity would be is at least 10 -6 M, 10 - ⁵ M, 10 -4 M, 10 -3 M, 10 - ² M or 10 ^-1 M.
[00171] An antibody is specific to a predetermined target, if it is able to bind to that predetermined target, while it is not able to bind to other targets, that is, it has no significant affinity for the other objectives and is not binds significantly to other targets in standard assays. According to the invention, an antibody is specific for CLDN18.2 if it is able to bind to
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CLDN18.2, but is not (substantially) able to bind to other targets. Preferably, an antibody is specific for CLDN18.2 if the affinity for binding and such other targets does not significantly exceed the affinity for protein or CLDN18.2-related binding, such as bovine serum albumin (BSA), casein, human serum albumin (HSA) or non-claudin transmembrane proteins, such as MHC molecules or transferrin receptor or any other specified polypeptide. Preferably, an antibody is specific to a predetermined target, binds to that target with a D that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 106 times lower than the KD for binding to a target for which it is not specific. For example, if the KD for binding an antibody to the target for which it is specific is 10 ^-7 M, the KD for binding to a target for which it is not specific would be at least 10 ^{- 6} M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M, or 10 ^-1 M.
[00172] The binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., Antigen-antibody 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 here
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73/159 described. Affinities can be easily determined using conventional techniques, such as by equilibrium dialysis; using the BIAcore 2000 instrument, through general procedures described by the manufacturer; by radioimmunoassay using the radiolabeled target antigen; or by another method known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NY Acad. Sci, 51: 660 (1949). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, for example, salt concentration, pH. Thus, measurements of affinity and other antigen binding parameters, for example, KD, IC50, are preferably made with standardized antibody and antigen solutions, and a standardized buffer.
[00173] As used herein, isotype refers to the class of antibodies (for example, IgM or IgGl) that is encoded by genes in the heavy chain constant regions.
[00174] As used herein, isotype switching refers to the phenomenon by which the class, or isotype, of an antibody changes from an Ig class to one of the other Ig classes.
[00175] The term naturally occurring as used herein, as applied to an object refers to the
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74/159 the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that was not intentionally modified by humans in the laboratory is naturally occurring.
[00176] The term rearranged as used herein refers to a configuration of an immunoglobulin heavy chain or light chain in which the locus of a V segment is positioned immediately adjacent to a J segment or in a conformation essentially encoding a complete VH or VL domain, respectively. The locus of the rearranged immunoglobulin (antibody) gene can be identified by comparison with germline DNA; a rearranged locus will have at least one recombined heptamer / nonomer homology element.
[00177] The term germline configuration or not rearranged, as used here in reference to a segment V refers to the configuration in which the segment V is not recombined so as to be immediately adjacent to a segment D or J.
[00178] According to the invention, an antibody that has the ability to bind to CLDN18.2 is an antibody capable of binding to an epitope present in CLDN18.2, preferably
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75/159 an epitope located within the extracellular domains of CLDN18.2, in particular the first extracellular domain, preferably the amino acids at positions 29-78 of CLDN18.2. In particular embodiments, an antibody that has the ability to bind to CLDN18.2 is an antibody capable of binding to (i) an epitope on CLDN18.2 that is not present on CLDN18.1, preferably SEQ ID NO : 3, 4, and 5, (ii) an epitope located on CLDN18.2-loopl, preferably SEQ ID NO: 8, (iii) an epitope located on CLDN18.2-Loop2, preferably SEQ ID NO : 10, (iv) an epitope located on CLDN18.2-loopD3, preferably SEQ ID NO: 11, (v) an epitope, comprising CLDN18.2-loopl and CLDN18.2-loopD3, or (vi) an non-glycosylated epitope located at CLDN18.2 loopD3, preferably SEQ ID NO: 9.
[00179] According to the invention, an antibody that has the ability to bind to CLDN18.2, preferably, is an antibody that has the ability to bind to CLDN18.2, but not to CLDN18.1. Preferably, an antibody that has the ability to bind to CLDN18.2 is specific to CLDN18.2. Preferably, an antibody that has the ability to bind to CLDN18.2, preferably, is an antibody that has the ability to bind to CLDN18.2 expressed on the cell surface. In particular preferred embodiments, an antibody that has the ability to bind to CLDN18.2 binds
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76/159 to native CLDN18.2 epitopes present on the surface of living cells. Preferably, an antibody that has the ability to bind to CLDN18.2 binds to one or more peptides selected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50. Preferably, an antibody that has the ability to bind to CLDN18.2 is specific for the aforementioned proteins, or fragments of immunogenic peptides or derivatives thereof. An antibody that has the ability to bind to CLDN18.2 can be obtained by a method that comprises the step of immunizing an animal with a protein or peptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 - 11, 44, 46, and 48 - 50, or a host cell for nucleic acid or expressing said protein or peptide. Preferably, the antibody binds to cancer cells, in particular cancer cells of the types mentioned above, and preferably does not bind substantially to non-cancer cells.
[00180] Preferably, the binding of an antibody that has the ability to bind to cells that express CLDN18.2 CLDN18.2 induces or mediates killing cells that express CLDN18.2. Cells expressing CLDN18.2 are preferably cancer cells and are, in particular, selected from the group consisting of cells
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77/159 tumorigenic gastric, esophagus, pancreas, lung, ovary,
colon, liver, head-neck, and cells in cancer gives gallbladder. Preferably, the antibodies induce or mediate the death of cells through gives induction in one or more
lysis-mediated complement-dependent cytotoxicity (CDC), lysis-mediated antibody-dependent cell cytotoxicity (ADCC), apoptosis, and inhibition of proliferation of cells expressing CLDN18.2. Preferably, cell lysis-mediated ADCC occurs in the presence of effector cells, which in particular embodiments are selected from the group consisting of monocytes, mononuclear cells, N cells and PMN. 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 analogue of thymidine and can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle), replacing with thymidine during DNA replication. Detecting the incorporated chemistry using, for example, antibodies specific for BrdU indicates cells that have been actively replicating their DNA.
[00181] In preferred embodiments, the antibodies described herein can be characterized by one or more of the
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78/159 following properties:
a) specificity for CLDN18.2;
b) a binding affinity for CLDN18.2 of about 100 nM or less, preferably about 5-10 nM or less and more preferably about 1 -3 nM or less,
c) the ability to induce or mediate CDC on CLDN18.2 positive cells;
d) the ability to induce or mediate ADCC in CLDN18.2 positive cells;
e) the ability to inhibit the growth of CLDN18.2 positive cells;
f) the ability to induce apoptosis of CLDN18.2 positive cells.
[00182] In a particularly preferred embodiment, an antibody that has the ability to bind to CLDN18.2 is produced by a hybridoma deposited with DSMZ (Mascheroder Weg lb, 31824 Braunschweig, Germany; new address: Inhoffenstr 7B, 31824 Braunschweig, Germany ), and having the following name and access number:
The. 182-D1106-055, number of access DSM ACC2737, filed on October 19, 2005; B. 182-D1106-056, number of access DSM ACC2738, filed on October 19, 2005 ç. 182-D1106-057, number of access DSM ACC2739,
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filed on October 19, 2005,d. 182-D1106-058, DSM accession number ACC2740, filed on October 19, 2005;and. 182-D1106-059, DSM access number deposited on October 19, 2005; ACC2741, f. 182-D1106-062, DSM access number deposited on October 19, 2005; ACC2742, g. 182-D1106-067, DSM accession number deposited on October 19, 2005; ACC2743, H. 182-D758-035, DSM accession number ACC2745, on November 17, 2005; deposited i. 182-D758-036, accession number DSM ACC2746,on November 17, 2005 deposited j. 182-D758-040, accession number DSM ACC2747,on November 17, 2005 deposited
k. 182-D1106-061, DSM accession number ACC2748, filed on November 17, 2005;
l. 182-D1106-279, DSM accession number ACC2808, filed on October 26, 2006;
m. 182-D1106-294, DSM accession number ACC2809, filed on October 26, 2006
n. 182-D1106-362, DSM accession number ACC2810, filed on October 26, 2006.
[00183] Preferred antibodies, according to
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80/159 invention are those produced by, and obtained from, the hybridomas described above; that is, 37G11, in the case of Dl-182 106-055, 37H8, in the case of Dl-182 106-056, 38G5, in the case of Dl-182 106-057, 38H3, in the case of Dl-182 106-058 , 39F11 in the case of 182-D 1106-059, 43A11, in the case of D-182 1106-062, 61C2, in the case of 182-D1106-067, 26B5, in the case of 182-D758035, 26D12, in the case of 182 -D758-036, 28D10, in the case of 182D758-040, 42E12 in the case of 182-Dl 106-061, 125E1 in the case of 182-Dl 106-279, 163E12, in the case of Dl-182 106 -294, and 175D10 in the case of Dl-182 106-362; and the chimerized and humanized forms of these.
[00184] Preferred chimerized antibodies and their sequences are shown in the table below
clone mAb Isotype Regionvariable Chimerized antibody Jailheavy 43A11 182-D1106-062 IgG2a SEQ ID NO: 29 SEQ ID NO: 14163E12 182-D1106-294 IgG3 SEQ ID NO: 30 SEQ ID NO: 15125E1 182-D1106-279 IgG2a SEQ ID NO: 31 SEQ ID NO: 16166E2 182-D1106-308 IgG3 SEQ ID NO: 33 SEQ ID NO: 18175D10 182-D1106-362 IgG1 SEQ ID NO: 32 SEQ ID NO: 1745C1 182-D758-187 IgG2a SEQ ID NO: 34 SEQ ID NO: 19 JailLight 43A11 182-D1106-062 IgK SEQ ID NO: 36 SEQ ID NO: 21163E12 182-D1106-294 IgK SEQ ID NO: 35 SEQ ID NO: 20125E1 182-D1106-279 IgK SEQ ID NO: 37 SEQ ID NO: 22
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81/159
166E2 182-D1106-308 IgK SEQ ID NO: 40 SEQ ID NO: 25 175D10 182-D1106-362 IgK SEQ ID NO: 39 SEQ ID NO: 24 45C1 182-D758-187 IgK SEQ ID NO: 38 SEQ ID NO: 23 45C1 182-D758-187 IgK SEQ ID NO: 41 SEQ ID NO: 26 45C1 182-D758-187 IgK SEQ ID NO: 42 SEQ ID NO: 27 45C1 182-D758-187 IgK SEQ ID NO: 43 SEQ ID NO: 28
[00185] In preferred embodiments, the antibodies, in particular, chimerized forms of the antibodies, according to the invention include antibodies comprising a heavy chain constant region (CH) comprising an amino acid sequence derived from a heavy chain of the human constant region , such as the amino acid sequence represented by SEQ ID NO: 13, or a fragment thereof. In other preferred embodiments, the antibodies, in particular chimerized forms of antibodies, according to the invention include antibodies comprising a light chain constant region (CL), which comprises an amino acid sequence derived from a human light chain constant region, such as such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof. In a particularly preferred embodiment, antibodies, in particular chimerized forms of antibodies, according to the invention include antibodies comprising a CH comprising an amino acid sequence derived from a human CH, such as the amino acid sequence represented
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82/159 by SEQ ID NO: 13 or a fragment thereof and comprising a CL comprising an amino acid sequence derived from a human CL such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof.
[00186] In one embodiment, an antibody that has the ability to bind to CLDN18.2 is a mouse / human IgGl chimeric monoclonal antibody comprising kappa, murine variable light chain, human kappa light chain, allotype km constant region (3) , murine heavy chain variable region, IgGl human constant region, Glm allotype (3).
[00187] On certain preferred embodiments, forms
chimerized antibodies include antibodies comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, and a fragment thereof and / or comprising a light chain comprising an amino acid sequence selected from the group consisting of SEQ
ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, and one of yours
fragment.
[00188] On certain preferred embodiments, forms
chimerized antibodies include antibodies comprising
a combination of heavy chains and light chains
selected from the following possibilities: (i) to (ix):
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83/159
(i) the heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 14 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID AT THE: 21 or one your fragment, (ii) the heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 15 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID AT THE: 2 0 or one your fragment, (iii) the heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 16 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID AT THE: 22 or one your fragment, (iv) the heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 18 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID AT THE: 25 or one your fragment, (go heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 17 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID AT THE: 24 or one your
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84/159 fragment,
(via heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 19 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID AT THE: 23 or one your fragment, (vii) < the heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 19 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID AT THE: 2 6 or one your fragment, (viii) the heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 19 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID NO: 27 or a fragment of it, and (ix) a heavy chain comprises an sequence in amino acids represented by SEQ ID AT THE: 19 or one your fragment and the light chain comprises an sequence in amino acids represented by SEQ ID AT THE: 28 or one your fragment. [00189] Fragment or fragment of a sequence in amino acids as used above, refers to to a part in an antibody sequence, that is, an sequence what
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85/159 represents the shortened antibody sequence at the N-terminal and / or C-terminal, which when replacing said antibody in the sequence of an antibody retains the binding of said antibody to CLDN18.2 and preferably functions of said antibody, as described herein, for example, CDC or ADCC mediated lysis mediated lysis. 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 NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and preferably refers to said sequence in which 17, 18, 19, 20, 21, 22 or 23 amino acids at the N-terminal are removed.
[00190] In a preferred embodiment, an antibody that has the ability to bind to CLDN18.2 comprises a heavy chain variable region (VH) that comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, and one yours
fragment.
[00191] In a preferred embodiment, an antibody
that has the ability to bind to CLDN18.2 comprises a light chain variable region (VL) that comprises an amino acid sequence selected from the group that
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86/159 consists of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, and a fragment.
[00192] In certain preferred embodiments, an antibody that has the ability to bind to CLDN18.2 comprises a combination of the heavy chain variable region (VH) and the light chain variable region (VL) selected from the following possibilities: (i) to (ix):
(i) VH comprises an amino acid sequence represented by SEQ ID NO: 29 or a fragment thereof and VL comprises an amino acid sequence represented by SEQ ID NO: 36 or a fragment thereof, (ii) VH comprises a sequence of amino acids represented by SEQ ID NO: 30 or a fragment thereof and VL comprises an amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof, (iii) VH comprises an amino acid sequence represented by SEQ ID NO: 31 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof, (iv) the VH comprises an amino acid sequence represented by SEQ ID NO: 33 or a fragment thereof and the VL comprises a sequence amino acid represented by SEQ ID NO: 40 or a fragment thereof, (v) VH comprises an amino acid sequence
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87/159 represented by SEQ ID NO: 32 or a fragment thereof and VL comprises an amino acid sequence represented by SEQ ID NO: 39 or a fragment thereof, (vi) VH comprises an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof, (vii) the VH comprises an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 41 or a fragment thereof, (viii) VH comprises an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO : 42 or a fragment thereof, and (ix) the VH comprises an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 43 or a fragment thereof.
[00193] In a preferred embodiment, an antibody that has the ability to bind to CLDN18.2 comprises a VH comprising a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (vi ):
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88/159 (i): CDR1 positions 45-52 of SEQ ID NO: 14, CDR2: 70-77 positions of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14, (ii) CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: 70-77 of SEQ ID NO: positions 15, CDR3: 116-126 positions of SEQ ID NO: 15, (iii): CDR1 positions 45-52 of SEQ ID NO : 16, CDR2: 70-77
gives SEQ ID NO positions: 16 CDR3: SEQ ID AT THE positions 116-124 in: 16,(iv): CDR1 positions 45 -52 of SEQ ID AT THE : 17, CDR2 : 70-77 gives SEQ ID NO positions: 17 CDR3: SEQ ID AT THE positions 116-126 in: 17,(v): CDR1 positions 44- 51 from SEQ ID AT THE: 18, CDR2 : 69-76
SEQ ID NO: positions 18, CDR3 SEQ ID NO: 115-125 positions: 18, and (vi): CDR1 positions 45-53 of SEQ ID NO: 19, CDR2: 71-78 positions of SEQ ID NO: 19 , CDR3: positions 117-128 of SEQ ID NO: 19.
[00194] In a preferred embodiment, an antibody that has the ability to bind to CLDN18.2 comprises a VL comprising a set of complementarity determining regions CDR1, CDR2 and CDR3 selected from the following embodiments (i) to (ix ):
(i): CDR1 positions 47-58 of SEQ ID NO: 20, CDR2:
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89/159 positions 76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of
SEQ ID NO: 20,
(ii) CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID NO : 21, CDR3 : 110 positions - 118: SEQ ID NO 21,(iii): CDR1 positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO : 22 CDR3: positions 109-117 SEQ ID NO of: 22,(iv): CDR1 positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 of SEQ ID NO : 23 CDR3: positions 115-123 SEQ ID NO of: 23,(v): CDR1 positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID NO : 24 CDR3: positions 115-123 SEQ ID NO: 24,(vi): CDR1 positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID NO : 25 CDR3: positions 115-122 SEQ ID NO: 25,(vii): CDR1 positions 47-58 of SEQ ID NO: 26, CDR2:
positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 SEQ ID NO of: 26, (viii): CDR1 positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID NO : 27, CDR3: positions 115-123 SEQ ID NO of: 27, and (ix): CDR1 positions 47-52 of SEQ ID NO: 28, CDR2:
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90/159 positions 70-72 positions of SEQ ID NO: 28, CDR3: positions 109117 of SEQ ID NO: 28.
[00195] In a preferred embodiment, an antibody that has the ability to bind to CLDN18.2 comprises a combination of VH and VL each comprising a set of complementarity determining regions CDRL, CDR2 and CDR3 selected from the following embodiments (i) to (ix):
(i): VH: CDR1 positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO 14, VL: CDR1: positions 49 - 53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID NO: 21 CDR3: positions 110-118 of SEQ ID NO: 21, (ii) VH: CDR1: positions positions 70-77 of SEQ ID NO: SEQ ID NO: 15, VL: CDR1: 47 positions 76-78 of SEQ ID NO: SEQ ID NO: 20, (iii) VH: CDR1: positions 70-77 of SEQ ID NO:
45-52 of SEQ ID NO: 15, CDR2:
15, CDR3: positions 116-126 of
-58 of SEQ ID NO: 20, CDR2:
20, CDR3: positions 115-123 of
45-52 of SEQ ID NO: 16, CDR2:
16, CDR3: positions 116-124 of
SEQ ID NO: 16, VL: CDRe: positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO: 22 CDR3: positions 109-117 of SEQ ID NO: 22, (iv) VH : CDR1: positions 44-51 of SEQ ID NO: 18, CDR2:
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91/159
positions 69-76 of SEQ ID NO: 18 CDR3: positions 115-125 from SEQ
ID NO: 18, VL: CDR1: positions 47 -58 of SEQ ID NO: 25, CDR2:
positions 76-78 of SEQ ID NO: 25 CDR3: positions 115-122 from SEQ
ID NO: 25,
(v): VH: CDR1 positions 45-52 of SEQ ID NO: 17, CDR2:
positions 70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17, VL: CDR1: positions 47 -58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO: 24, (vi) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19 CDR3: positions 117- 128 of SEQ ID NO: 19, VL: CDR1: positions 47 -58 of SEQ ID NO: 23, CDR2: positions 76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO: 23, ( vii): VH: CDR1 positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19 CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO: 26, (viii) VH: CDR1: positions 45-53 of SEQ ID NO: 19 , CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID NO: 27 CDR3: positions 115-123 of SEQ ID NO: 27, and
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92/159 (ix) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1 : positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 of SEQ ID NO: 28 CDR3: positions 109-117 of SEQ ID NO: 28.
[00196] In other preferred embodiments, an antibody that has the ability to bind to CLDN18.2 preferably comprises one or more of the complementarity determining regions (CDR), preferably at least the variable region of CDR3, of the heavy chain variable region (VH) and / or variable light chain region (VL) of a monoclonal antibody against CLDN18.2, preferably a monoclonal antibody against CLDN18.2 described herein, and preferably comprises one or more of the complementarity determining regions (CDR), preferably at least the variable CDR3 region, the variable heavy chain (VH) regions and / or variable light chain (VL) regions described herein. In said embodiment, one or more of the complementarity determining regions (CDRs) are selected from a set of complementarity determining regions CDR1, CDR2 and CDR3 described herein. In a particularly preferred embodiment, an antibody that has the ability to bind to CLDN18.2 preferably comprises the regions determining
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93/159 complementarity CDR1, CDR2 and CDR3 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, and preferably, comprises the complementarity determining region of CDR1, CDR2 and CDR3 of the variable regions of the heavy chain (VH) and / or variable regions of the light chain (VL) described herein.
[00197] In one embodiment, an antibody comprising one or more CDRs, a set of CDRs or a combination of CDR sets as described herein comprises said CDR, together with its intervening structural regions. Preferably, the portion will also include at least about 50% of one or both of the first and fourth structure regions, at 50%, with the C-terminal being 50% of the first structure region and the N-terminal being 50% % of the fourth structure region. Antibody construction using recombinant DNA techniques can result in the introduction of N- or C-terminal residues into the variable regions encoded by ligands introduced to facilitate cloning or other manipulation steps, including the introduction of ligands to join the variable regions of the invention other protein sequences including immunoglobulin heavy chains, other variable domains (e.g.
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94/159 (eg, in the production of diabody) or protein markers.
[00198] In one embodiment of an antibody comprising one or more CDRs, a set of CDRs or a combination of CDR sets as described herein comprises said CDR in a human antibody structure.
[00199] Reference made in this document to an antibody comprising with respect to its heavy chain a specific chain or a particular region or sequence preferably refers to the situation in which all heavy chains of said antibody comprise the said chain in particular in the region or sequence. This applies correspondingly to the light chain of an antibody.
[00200] The term nucleic acid, as used herein, is intended to include DNA and RNA. A nucleic acid can be single-stranded or double-stranded, but is preferably double-stranded DNA.
[00201] According to the invention, the term expression is used in its most general meaning and comprises the production of RNA or RNA and protein / peptide. It also comprises partial expression of nucleic acids. In addition, the expression can be performed transiently or stably.
[00202] The teaching is given here with respect to
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95/159 specific amino acid sequences, for example, those shown in the sequence listing, are to be interpreted so as to also relate to the variants of said specific sequences that result in sequences that are functionally equivalent to said specific sequences, for example amino acid sequences exhibiting properties identical or similar to those of specific amino acid sequences. An important property is to retain the binding of an antibody to its target or to support the effector functions of an antibody. Preferably, a sequence that is a variant that relates to a specific sequence, when replacing the specific sequence of an antibody retains the binding of said antibody to CLD 18.2 and preferably functions of said antibody, as described herein , for example, CDC or ADCC mediated lysis mediated lysis.
[00203] It will be appreciated by those skilled in the art that, in particular, the CDR sequences, hypervariable and variable regions can be modified without losing the ability to bind CLDN18.2. For example, CDR regions will be identical or highly homologous to the antibody regions specified herein. Highly homologous, it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2, substitutions can be made in the CDR. Beyond
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In addition, the hypervariable and variable regions can be modified so that they show substantial homology to the antibody regions specifically described herein.
[00204] For the purposes of the present invention, variants of an amino acid sequence comprise insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. Amino acid deletion variants that comprise the deletion at the N-terminal and / or the C-terminal end of the protein are also called N-terminal and / or C-terminal truncation variants.
[00205] amino acid insertion variants comprise single or double insertions or more of amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insert, one or more amino acid residues are inserted at a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.
[00206] Variants of addition of amino acids comprise
amino fusions and / or carboxy-terminal one or more amino acids, such like 1 , 2, 3, 5, 10, 20, 30, 50, or more amino acids. [00207] Variants in deletion in amino acid are
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97/159 characterized by the removal of one or more amino acids from the sequence, such as removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can be in any position of the protein.
[00208] Amino acid substitution variants are characterized by at least one residue in the sequence to be removed and another residue to be inserted in its place. Preference is given to modifications being in positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or to replace amino acids with others having similar properties. Preferably, amino acid changes in protein variants are conservative amino acid changes, i.e., similarly charged or unloaded amino acid substitutions. A conservative amino acid change involves replacing one of a family of amino acids that are related in its side chains. Naturally occurring amino acids are generally divided into four families: acid (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and not polar charged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes
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98/159 classified together as aromatic amino acids.
[00209] Preferably, the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence that is a variant
gives said given sequence of amino acids it will be of hair any less cer where is it 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%, 97%, 98%, or 99%. The degree of similarity or identity is given
preferably for 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, the degree of similarity or identity is preferred for 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 the continuous amino acids. In preferred embodiments, the degree of similarity or identity is given over the entire length of the reference amino acid sequence.
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99/159
Alignment to determine sequence similarity, preferably sequence identity can be done with the tools of the known art, preferably using the best sequence alignment, for example, using Align, using the default settings, preferably EMBOSS: needle , Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
[00210] Similarity sequence indicates the percentage of amino acids that are either identical or that represent conservative amino acid substitutions. Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.
[00211] The term percent identity is intended to denote a percentage of amino acid residues that are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and along its entire length. Sequence comparisons between two amino acid sequences are conventionally carried out by comparing these sequences, after having aligned them optimally, said comparison being made by the segment or by the comparison window to identify and compare local regions of sequence similarity. O
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100/159 optimal alignment of sequences for comparison can be produced, in addition manually, using the local homology algorithm of Smith and Waterman, 1981, App Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using Pearson and Lipman's similarity search method, 1988, Proc. Natl Acad. Sci. EUA 85, 2444, or through computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis) .
[00212] The percentage of identity is calculated by determining the number of identical positions between the two sequences to be compared, dividing this value by the number of positions in comparison and multiplying the result obtained by 100, in order to obtain the percentage of identity between these two strings.
[00213] The term transgenic animal refers to an animal with a genome that comprises one or more transgenes, preferably heavy and / or light chain transgenes, or transchromosomes (whether integrated or not integrated into the animals' natural genomic DNA) and which it is preferably capable of expressing the transgenes. For example, a transgenic mouse may have a human light chain transgene and a human
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101/159 human transgene, either human heavy chain or transchromosome heavy chain, such that the mouse produces human anti-CLDN18.2 antibodies when immunized with CLDN18.2 antigen and / or cells that express CLDN18.2. The human heavy chain transgene can be integrated into the mouse chromosomal DNA, as is the case with transgenic mice, for example HuMAb mice, such as HCo7 or HCol2 mice, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal (e.g., KM) mice, as described in WO 02/43478. Such transgenic and trans-chromosomal mice may be able to produce various isotypes of human monoclonal antibodies to CLDN18.2 (for example, IgG, IgA and / or IgE) by undergoing VDJ recombination and isotype exchange.
[00214] Reduction, decrease or inhibit, as used herein, means an overall decrease or the ability to cause an overall decrease, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or greater, and, more preferably, 75% or greater, at the level, for example, at the level of expression or the level of cell proliferation.
[00215] Terms such as increasing or improving refer, preferably, to an increase or improvement of about
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102/159 of 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 1,000%, at least 10000%) or even more.
Mechanisms of action mAb [00216] While the following provides considerations about the mechanism underlying the therapeutic efficacy of the antibodies of the invention it is not to be considered as limiting the invention in any way.
[00217] The antibodies described here preferably interact with components of the immune system, preferably through ADCC or CDC. The antibodies described here can also be used to target payloads (for example, radioisotopes, drugs or toxins) to kill tumor cells directly or can be used in synergy with traditional chemotherapeutic agents, attacking tumors through complementary mechanisms of action, which may include the anti-tumor immune responses that may have been compromised due to cytotoxic side effects of chemotherapy on T lymphocytes. However, the antibodies described here may also have an effect simply by binding to CLDN18.2
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103/159 on the cell surface, thus, for example, preventing cell proliferation.
Antibody-dependent cell mediated cytotoxicity [00218] ADCC describes the ability to kill cells from effector cells, as described herein, in specific lymphocytes, which preferably requires the target cell to be labeled by an antibody.
[00219] ADCC preferably occurs when antibodies bind to antigens on tumor cells and the Fc antibody domains involve Fc receptors (FcR) on the surface of immune effector cells. Several families of Fe receptors have been identified, and specific cell populations characteristically expressed defined Fc receptors. ADCC can be seen as a mechanism to directly induce a variable degree of immediate tumor destruction that leads to antigen presentation and the induction of T cell responses directed to the tumor. Preferably, in vivo ADCC induction will lead to tumor-directed T cell responses and host-derived antibody responses.
Complement-dependent cytotoxicity [00220] CDC is another method of killing cells that can be driven by antibodies. IgM isotype is the most effective for complement activation. IgGl and IgG3 are also very
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104/159 effective, both in directing CDC through the complement pathway, classic activation. Preferably, in this cascade, the formation of antigen-antibody complexes results in the unveiling of CLQ multiple binding sites in close proximity in the CH2 domains of participating antibody molecules, such as IgG molecules (Clq is one of the three subcomponents of the Cl complement) . Preferably, these uncovered Clq binding sites convert the previously low affinity Clq-IgG interaction to one of high avidity, which triggers a cascade of events involving a number of other complement proteins and leads to the proteolytic release of cell chemotactics effector / activating agents C3a and C5a. Preferably, the complement cascade ends in the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.
[00221] The antibodies described herein can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example, the Kohler and Milstein standard somatic cell hybridization technique, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques can be used
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105/159 to produce monoclonal antibodies, for example, oncogenic viral or B-lymphocyte transformation, or phage display techniques using libraries of antibody genes.
[00222] The preferred animal system for the preparation of hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well-established process. Immunization protocols and techniques for the isolation of immunized splenites for fusion are known in the art. Fusion partners (for example, murine myeloma cells) and fusion procedures are also known.
[00223] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the mouse and rabbit system (for example, described 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)).
[00224] In yet another preferred embodiment, human monoclonal antibodies can be generated using transgenic mice that carry transchromosomal or parts of the human immune system instead of the mouse system. These transgenic and trans-chromosomal mice include mice, known as HuMAb KM mice and mice, respectively, and are
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106/159 collectively referred to here as transgenic mice. The production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in WO 2004 035607.
[00225] Yet another strategy for the generation of monoclonal antibodies is to directly isolate genes that encode lymphocyte antibodies that produce antibodies of the specificity defined for example, see Babcock et al., 1996; An innovative strategy for the production of monoclonal antibodies from lymphocytes, isolated individuals that produce antibodies of defined specificities. For details of recombinant antibody engineering see also Welschof and Kraus, recombinant antibodies for cancer therapy ISBN-089603-918-8 and Benny KC Lo Antibody Engineering ISBN 1-58829092-1.
[00226] To generate antibodies, mice can be immunized with peptides conjugated to carriers derived from the antigen sequence, that is, the sequence against which the antibodies are being directed, an enriched preparation of the antigen or its fragments and / or recombinantly expresses the cells that express the antigen, as described. Alternatively, mice can be immunized with DNA that encodes the antigen or its fragments. In the case where immunizations using a
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107/159 purified or enriched antigen preparation do not result in antibodies, mice can also be immunized with cells that express the antigen, for example, a cell line, to promote immune responses.
[00227] The immune response can be monitored throughout the course of the immunization protocol with plasma and serum samples to be obtained by tail vein or retroorbital hemorrhages. Mice with sufficient immunoglobulin titers can be used for fusions. Mice can be boosted intraperitoneally or intravenously with cells that express the antigen 3 days before sacrifice and removal of the spleen to increase the rate of specific antibody-secreting hybridomas.
[00228] To generate hybridomas that produce monoclonal antibodies, splenocytes and lymph node cells from immunized mice can be isolated and fused with an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antibodies specific for the antigen. The individual wells can then be screened by ELISA for antibodies that secrete hybridomas. Immunofluorescence and by FACS analysis using cells that express the antigen, antibodies with specificity for the antigen can
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108/159 be identified. The antibody can be replated secretory hybridomas, sieved again, and if still positive for monoclonal antibodies they can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to generate the antibody in tissue culture medium for characterization.
[00229] Antibodies can also be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods, as are well known in the art (Morrison, S. (1985) Science 229: 1202).
[00230] For example, in one embodiment, the gene (s) of interest, for example, antibody genes, can be linked in an expression vector, such as a eukaryotic expression plasmid as used by the gene expression system of GS disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expression systems well known in the art. The plasmid purified with the cloned antibody genes can be introduced into eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293T cells or HEK293 cells or, alternatively, other eukaryotic cells, such as cells derived from plants, fungi or plants. yeast cells. The method used to introduce these genes can be the methods
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109/159 described in the state of the art, such as electroporation, lipofectin, lipofectamine or others. After introducing these antibody genes into host cells, cells that express the antibody can be identified and selected. These cells represent transfectomas that can then be amplified for their level of expression and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.
[00231] Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, for example, E. coli. In addition, antibodies can be produced in transgenic non-human animals, such as in sheep and rabbit milk or in chicken eggs, or in transgenic plants; for example, see 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.
Chimerization [00232] Murine monoclonal antibodies can be used as therapeutic antibodies in humans when labeled with radioactive isotopes or toxins. Unlabeled murine antibodies are highly immunogenic in humans
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110/159 when applied repeatedly leading to a reduction in the therapeutic effect. The main immunogenicity is mediated by the heavy chain constant regions. The immunogenicity of murine antibodies in humans can be reduced or completely avoided if the respective antibodies are chimerized or humanized. Chimeric antibodies are antibodies, the different portions of which are derived from different animal species, such as those that have a variable region derived from a murine antibody and a human immunoglobulin constant region. Chimerization of antibodies is achieved by the union of the variable regions of the murine heavy and light chain of antibodies with the constant region of the human heavy and light chain (for example, as described by Kraus et al., In Methods in Molecular Biology series, antibodies recombinants for cancer ISBN 089603-918-8-therapy). In a preferred embodiment, chimeric antibodies are generated by joining human light chain kappa from the constant region to the variable region of the murine light chain. In one embodiment, also preferred chimeric antibodies can be generated by joining the human lambda-light chain from the constant region to the variable region of the murine light chain. The preferred heavy chain constant regions for generating chimeric antibodies are IgG1, IgG3 and IgG4. Other constant regions
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Preferred heavy chain 111/159 for the generation of chimeric antibodies are IgG2, IgA, IgD and IgM.
Humanization [00233] Antibodies interact with target antigens predominantly through amino acid residues that are located in the complementarity determining regions of six heavy and light chain (CDR). For this reason, the amino acid sequences within CDRs are more diverse among individual antibodies than sequences outside CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of naturally occurring specific antibodies by constructing expression vectors that include CDR sequences from the antibody that occurs naturally grafted specifically into structural sequences of a different antibody with different properties (see, for example, Riechmann, L. et al (1998) Nature 332: 323-327; Jones, P. et al (1986) Nature 321: 522525; and Queen, C. et al. (1989) Proc Natl Acad Sci USA 86: 10029-10033). Such structure sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from sequences of
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112/159 mature antibody genes because they do not include completely assembled variable genes, which are formed by V (D) J during the maturation of adherent B cells. Germline gene sequences will also be different from the sequences of an antibody high affinity secondary repertoire in the individual uniformly throughout the variable region.
[00234] The ability of antibodies to bind an antigen can be determined using standard binding assays (eg, ELISA, Western Blot, immunofluorescence and flow cytometric analysis).
[00235] To purify antibodies, the selected hybridomas can be grown in two-liter spinner flasks for the purification of monoclonal antibody. Alternatively, antibodies can be produced based on dialysis bioreactors. Supernatants can be filtered and, if necessary, concentrated before affinity chromatography with protein G-Sepharose or protein A-Sepharose. The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged for PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be divided into aliquots and
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113/159 stored at -80 ° C.
[00236] To determine whether the selected monoclonal antibodies bind to single epitopes, site-directed mutagenesis or multiple site-directed mutations can be used.
[00237] To determine the antibody isotype, ELISAs isotype with various commercial kits (for example, Zymed, Roche Diagnostics) can be performed. Microtiter plate wells can be coated with anti-mouse Ig. After blocking, the plates are reacted with monoclonal antibodies or purified isotype controls, at room temperature for two hours. The wells can then be reacted with either a mouse IgGl, IgG2a, IgG2b or IgG3, IgA or mouse specific IgM peroxidase conjugated. After washing, the plates can be developed with the sub-mouse ABTS (1 mg / ml) and analyzed at OD 405-650. Alternatively, the IsoStrip mouse kit isotyping Monoclonal Antibody (Roche, Cat. No. 1493027) can be used as described by the manufacturer.
[00238] In order to demonstrate the presence of antibodies in the serum of immunized mice or binding of monoclonal antibodies to cells that express live antigen, flow cytometry can be used. Cell lines that express naturally or after transfection of antigens
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114/159 and negative controls without antigen expression (grown under normal growing conditions) can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS, and can be incubated at 4 ° C for 30 min. After washing, the anti-Alexa647 or APC-labeled IgG antibody can bind to the monoclonal antibody bound to the antigen under the same conditions as the staining of the primary antibody. Samples can be analyzed by flow cytometry with a FACS instrument using light and side-to-door dispersion properties in individual living cells. In order to distinguish monoclonal antibodies specific for the antigen from non-specific ligands in a single measurement, the co-transfection method can be employed. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained, as described above. The transfected cells can be detected in a different fluorescence channel than cells labeled with the antibody. Since most transfected cells express both transgenes, antigen-specific monoclonal antibodies bind
preferably to highlighter fluorescence of cells what express, While that the antibodies no specific if call in an proportion comparable at cells no
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115/159 transfected. An alternative assay using fluorescence microscopy can be used in addition to or instead of the flow cytometry assay. The cells can be stained exactly as described above and examined by fluorescence microscopy.
[00239] In order to demonstrate the presence of antibodies in the serum of immunized mice or binding of monoclonal antibodies to live cells that express the antigen, immunofluorescence microscopy analysis can be used. For example, cell lines that express either spontaneously or after missing antigen transfection and negative antigen controls are grown on growth chamber slides under standard conditions in DMEM / F12 medium, supplemented with 10% fetal calf serum (FCS ), 2 mM L-glutamine, 100 IU / ml penicillin and 100 ug / ml streptomycin. The cells can then be fixed with methanol or paraformaldehyde or left untreated. The cells can then be made to react with monoclonal antibodies against the antigen for 30 min. at 25 ° C. After washing, cells can be reacted with an Alexa555-secondary IgG-labeled anti-mouse antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.
[00240] Cell extracts from cells that
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116/159 express antigens and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) electrophoresis in polyacrylamide gel. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. Binding IgG can be detected using anti-mouse IgG peroxidase and developed with substl mouse ECL.
[00241] Antibodies can be further tested for antigen reactivity by immunohistochemistry in a manner well known to a person skilled in the art, for example, using acetone or fixed paraformaldehyde, cryosections or tissue sections embedded in paraffin fixed with paraformaldehyde to from non-cancer tissue or cancer tissue samples obtained from patients during routine surgical procedures or from mice bearing xenografted tumors inoculated with cell lines that express spontaneously or after transfection of antigens. For immunostaining, antibodies reactive to the antigen can be incubated followed by anti-mouse goat horseradish-conjugated horseradish peroxidase or DAKO antibodies according to the manufacturer's instructions.
[00242] Antibodies can be tested for their
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117/159 ability to mediate phagocytosis and cell death expressing CLDN18.2. The in vitro monoclonal antibody activity assay will provide an initial screening before testing in in vivo models.
Antibody-dependent cell-mediated cytotoxicity (ADCC):
[00243] Briefly, polymorphonuclear cells (PMN), NK cells, monocytes, mononuclear cells or other effector cells, from healthy donors can be purified by Ficoll Hypaque density, followed by lysis of contaminating erythrocytes. The washed effector cells can be suspended in RPMI supplemented with 10% heat-inactivated fetal calf serum or, alternatively, with 5% heat-inactivated human serum and mixed with ⁵¹ Cr-labeled target cells that express CLDN18.2, in various ratios from effector cells to target cells. Alternatively, the target cells can be labeled with a fluorescence enhancing ligand (BATDA). A highly fluorescent europium chelate with the reinforcement ligand that is released from dead cells can be measured by a fluorometer. Another alternative technique can use transfection of target cells with luciferase. Added lucifer yellow can then be oxidized by only viable cells. Purified anti-CLDN18.2 IgG can then
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118/159 be added in various concentrations. Irrelevant human IgG can be used as a negative control. The tests can be carried out for 4 to 20 hours at 37 ° C, depending on the type of effector cell used. The samples can be tested for cytolysis by measuring ⁵¹ Cr release or the presence of EuTDA chelate in the culture supernatant. Alternatively, the luminescence resulting from the oxidation of lucifer yellow can be a measure of viable cells.
[00244] Anti-CLDN18.2 monoclonal antibodies can also be tested in various combinations to determine whether cytolysis is enhanced with several monoclonal antibodies. Complement-dependent cytotoxicity (CDC):
[00245] Anti-CLDN18.2 monoclonal antibodies can be tested for their ability to mediate CDC, using a variety of known techniques. For example, for the serum supplement they can be obtained from the blood in a manner known to the qualified person. To determine the CDC activity of mAbs, different methods can be used. ^5I Release can, for example, be measured or high membrane permeability can be assessed using a propidium iodide (PI) exclusion assay. Briefly, the target cells can be washed and 5 x 10 ⁵ / ml can be incubated with several
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119/159 mAb concentrations for 10-30 min. at room temperature or at 37 ° C. The serum or plasma can then be added to a final concentration of 20% (v / v) and the cells incubated at 37 ° C for 20-30 min. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be analyzed immediately by flow cytometry analysis using FACSArray.
[00246] In an alternative assay, CDC induction can be determined in adherent cells. In one embodiment of this assay, cells are seeded 24 hours before the assay with a density of 3 x 10 ⁴ / well in flat-bottomed tissue culture microtiter plates. The next day of growth medium is removed and the cells are incubated in triplicate with the antibodies. Control cells are incubated with growth medium or growth medium containing 0.2% saponin to determine background and maximum lysis, respectively. After incubation for 20 min. at room temperature, the supernatant is removed and 20% (v / v) of human plasma or serum in DMEM (preheated to 37 ° C) is added to the cells and incubated for another 20 min. at 37 ° C. All cells from each sample are added to the propidium iodide solution (10 pm). Then, the supernatants are
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120/159 replaced by ml of PBS containing fluorescence emitting bromide and 2.5 µg / ethidium after excitation at 520 nm is measured at 600 nm using a Tecan satire. The percentage of specific lysis is calculated as follows:% specific lysis = (sample fluorescence-background fluorescence) / (maximum fluorescence background lysis by fluorescence) x 100.
Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies:
[00247] To test the ability to initiate apoptosis, anti-CLDN18.2 monoclonal antibodies can, for example, be incubated with CLDN18.2 positive tumor cells, for example, SNU-16, DAN-G, KATO-III or CLDN18.2 tumor cells transfected at 37 ° C for about 20 hours. The cells can be harvested, washed in annexin-V (BD) binding biological sciences, and incubated with annexin V conjugated with (BD biosciences) or APC FITC for 15 min. in the dark. All cells from each sample can be added to the PI solution (10 µg / ml in PBS) in a FACS tube and evaluated immediately by flow cytometry (as above). Alternatively, a general inhibition of cell proliferation by monoclonal antibodies can be detected with commercially available kits. The DELFIA Cell Proliferation Kit (Perkin
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Elmer, Cat. No. AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis of proliferating cells in microplates. Embedded BrdU is detected through a europium-labeled monoclonal antibody. To allow the detection of antibodies, cells are fixed and DNA is denatured using Fix solution. Unbound antibody is removed by DELFIA wash and inducer is added to dissociate europium ions from the labeled antibody in solution, where they form highly fluorescent chelates, with components of the DELFIA inducer. The measured fluorescence - using time-resolved fluorometry in detection - is proportional to the DNA synthesis in the cell of each well.
Pre-clinical studies [00248] Monoclonal antibodies that bind to CLDN18.2 can also be tested in an in vivo model (for example in immunodeficient mice with xenografted tumors inoculated with cell lines that express CLDN18.2, for example DAN -G, SNU-16, or KATO-III, or after transfection, for example HEK293) to determine its effectiveness in controlling the growth of tumor cells that express CLDN18.2.
[00249] In in vivo studies, after xenogen CLDN18.2 expressing tumor cells in mice
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122/159 immunocompromised or in other animals can be performed using the antibodies described herein. The antibodies can be administered to tumor-free mice, followed by injection of the tumor cells to measure the effects of the antibodies to prevent the formation of tumors or tumor-related symptoms. The antibodies can be administered to tumor-bearing mice to determine the therapeutic effectiveness of the respective antibodies to reduce tumor growth, tumor metastasis or related symptoms. Antibody application can be combined with the application of other substances such as cystostatic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors or other antibodies to determine the synergistic effectiveness and potential toxicity of combinations. To analyze toxic side effects mediated by animal antibodies, they can be inoculated with antibodies or control reagents and thoroughly investigated for symptoms possibly related to CLDN18.2antihody therapy. Possible side effects of in vivo application of CLDN18.2 antibodies include particularly toxicity in CLDN18.2 expressing tissues, including the stomach. Antibodies that recognize CLDN18.2 in humans and other species, for example, mice, are
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123/159 particularly useful for predicting potential side effects mediated by application of CLDN18.2-monoclonal antibodies in humans.
[00250] Mapping of epitopes recognized by antibodies can be performed as described in detail in Epitope Mapping Protocols (Methods in Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and in Epitope Mapping: A Practical Approach Practical Approach Series , 248 by Olwyn MR Westwood, Frank C. Hay.
[00251] The compounds and agents described herein can be administered in the form of any suitable pharmaceutical composition.
[00252] Pharmaceutical compositions are generally supplied in a uniform dosage form and can be prepared in a manner known per se. A pharmaceutical composition can, for example, be in the form of a solution or suspension.
[00253] A pharmaceutical composition can comprise salts, buffer substances, preservatives, carriers, diluents and / or excipients which are preferably pharmaceutically acceptable. The term pharmaceutically acceptable refers to the lack of toxicity of a material that does not interact with the action of the active component of the pharmaceutical composition.
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124/159 [00254] Salts that are not pharmaceutically acceptable can be used for the preparation of pharmaceutically acceptable salts and are included in the invention. Pharmaceutically acceptable salts of this type include, but are not limited to, those prepared from the following hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic hydrochloric acids, and the like. 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.
[00255] Buffer substances suitable for use in a pharmaceutical composition include acetic acid in a salt, citric acid in a salt, boric acid in a salt of phosphoric acid and a salt.
[00256] Preservatives suitable for use in a pharmaceutical composition include benzalkonium chloride, chlorobutanol, thimerosal and paraben.
[00257] An injectable formulation can comprise a pharmaceutically acceptable excipient, such as RL.
[00258] The term vehicle refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined to facilitate, improve or allow application. According to the invention, the term
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125/159 carrier also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient.
[00259] Possible carrier substances for parenteral administration are, for example, sterile water, Ringer's, lactated Ringer's, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy- propylene copolymers.
[00260] The term excipient, when used here, is intended to indicate all substances that may be present in a pharmaceutical composition and that are not active ingredients, such as, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring or coloring agents.
[00261] The agents and compositions described herein can be administered via any conventional route, such as parenteral administration, including injection or infusion. Administration is preferably parenteral, for example by intravenous, intra-arterial, subcutaneous, intradermal or intramuscular route.
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126/159 [00262] Compositions suitable for parenteral administration usually comprise a sterile aqueous or non-aqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of vehicles and solvents are compatible with Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as a solution or suspending medium.
[00263] The agents and compositions described herein are administered in effective amounts. An effective amount refers to the amount that achieves a desired reaction or a desired effect alone or in conjunction with other doses. In the case of treating a particular disease or condition, the desired reaction preferably relates to inhibiting the course of the disease. This comprises slowing down the progress of the disease and, in particular, halting or reversing the progress of the disease. The desired reaction in treating a disease or condition can also be delayed onset or prevent the onset of said disease or condition.
[00264] An effective amount of an agent described herein or the composition will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, size and
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127/159 weight, duration of treatment, type of follow-up therapy (if present), specific route of administration and similar factors. Therefore, the doses administered of the agents described herein can depend on several of such parameters. In the event that a patient's reaction is insufficient with an initial dose, higher doses (or higher doses effectively achieved by a different, more localized route) of administration can be used.
[00265] The agents and compositions described herein can be administered to patients, for example, in vivo, to treat or prevent a variety of disorders, such as those described herein. Preferred patients include human patients with diseases that can be corrected or improved by administering the agents and compositions described herein. This includes disorders involving the cells
characterized per a pattern of changed expression in CLDN18.2. [00266] Per example, in an achievement, the antibodies here described can be used to treat one
patient with a cancerous disease, for example, a cancerous disease as described herein is characterized by the presence of cancer cells that express CLDN18.2.
[00267] Compositions and treatment methods
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128/159 described according to the invention pharmaceuticals can also be used for immunization or vaccination to prevent a disease described herein.
[00268] The present invention is further illustrated by the following examples which are not to be construed as limiting the scope of the invention.
EXAMPLES
Example 1: CLDN18.2 expression of human gastric cancer cell lines is stabilized by in vitro treatment with chemotherapeutic agents [00269] KatoIII cells, a human gastric tumor cell line was cultured in RPMI 1640 medium (Invitrogen) containing 20% of FCS (Perbio) and 2 mM Glutamax (Invitrogen) at 37 ° C and 5% CO2, with or without cytostatic compounds. Epirubicin (Pfizer) was tested at a concentration of 10 or 100 ng / ml, 5-FU (Neofluor from NeoCorp AG) was tested at a concentration of 10 or 100 ng / ml, and oxaliplatin (Hospira) was tested at a concentration of 50 or 500 ng / ml. It was also used; the combination of all three epirubicin compounds (10 ng / ml, oxaliplatin 500 ng / ml, 5-FU 10 ng / ml EOF). 8 x 10 ⁵ KatoIII cells were cultured for 96 hours without changing the medium, or for 72 hours followed by 24 hours of culture in standard medium to release the cells from stopping the cell cycle
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129/159 in a 6-well tissue culture plate at 37 ° C, 5% CO2. The cells were harvested with EDTA / trypsin, washed and analyzed.
[00270] For the extracellular detection of CLDN18.2 cells, they were stained with the monoclonal anti-CLDN18.2 IMAB362 antibody (Ganymed) or an isotyp-matched control antibody (Ganymed). As a secondary goat antiHuIgG-APC reagent from Dianova it was used.
[00271] Phases of the cell cycle were determined based on the measurement of cellular DNA content. This makes it possible to discriminate between cells in the G1-, S or G2 -phase of the cell cycle. In the duplication of DNA in the S phase occurs while in the G2 -phase cells grow and prepare for mitosis. Cell cycle analysis was performed using the reagent kit from DNA CycleTEST PLUS BD Biosciences following the manufacturer's protocol. Flow cytometry acquisition and analysis were performed using BD FACS CantoII (BD Biosciences) and Fluxo Jo software (Tree Star).
[00272] The columns in Figure Ia and b show the respective percentage of cells in G1, S or in the G2 phase of the cell cycle. Medium cultured KatoIII cells show a cell cycle arrest predominantly at the G-phase. Cells treated with 5-FU are blocked predominantly at the S-phase. Epirubicin or KatoIII cells treated with EOF
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130/159 show a cell cycle arrest predominantly in the G2 phase. KatoIII cells treated with oxaliplatin show enrichment of cells predominantly in the Gl- and G2 phases. As can be seen in Figure 1c, a stop of the cell cycle in the S or G2 phase results in the stabilization or positive regulation of CLDN18.2. As soon as the cells are released from any phase of the cell cycle (Figure 1b), the expression of CLDN18.2 on the cell surface of KatoIII cells is upregulated (Figure Id).
[00273] NUGC-4 and KATO III cells were treated with 5-FU + OX (10 ng / ml 5-FU and 500 ng / ml oxaliplatin), EOF (10 ng / ml epirubicin, 500 ng oxaliplatin / ml and 10 ng / ml 5 -FU) or FLO (10 ng / ml 5-FU, 50 ng / ml folinic acid and 500 ng / ml oxaliplatin) for 96 hours. RNA from chemotherapy pretreated cells NUGC-4 and KATO III was isolated and converted to cDNA. Level of transcription CLDN18.2 was analyzed in quantitative real-time PCR. The results are shown in Figure 2a as relative expression, compared to the transcription level of the HPRT gene. Figure 2b shows a Western blot of CLDN18.2 and untreated and treated cell loading control actin from NUGC-4. The intensity of the luminescence signal is shown in relation to actin in percent.
[00274] Pre-treatment of NUGC-4 and KATO III cells
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131/159 with EOF, FLO, as well as 5-FU + OX chemotherapeutic combination results in an increase in RNA and protein levels of CLDN18.2 as shown by quantitative real-time PCR (Figure 2a) and Western blot (Figure 2b ).
[00275] Binding IMAB362 in gastric cancer cells NUGC-4 and KATO III treated with EOF (10 ng / ml epirubicin, 500 ng ml of oxaliplatin / and 10 ng / ml 5FU) or FLO (10 ng / ml of 5-FU, 50 ng / ml folinic acid and 500 ng / ml oxaliplatin) were analyzed for 96 hours by flow cytometry. The amount of CLDN18.2 protein marked by IMAB362 on the surface of gastric cancer cell lines is increased, as shown in Figure 2c. This effect was more prominent in cells pretreated with EOF or FLO.
[00276] KatoIII cells were pretreated for 4 days with Irinotecan or docetaxel and analyzed for CLDN18.2 expression and cell cycle arrest. Treatment of cells with irinotecan resulted in a dose-dependent inhibition of cell growth and a cell cycle arrest in the / S-phase G2 (Figure 3). The treatment of cells with Docetaxel resulted in a dose-dependent inhibition of cell growth and a stop of the cell cycle in the G2 phase (Figure 3).
Example 2: Pretreatment of gastric cancer cells
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132/159 human with chemotherapy results in increased efficiency of IMAB362-mediated ADCC [00277] IMAB362-mediated ADCC was investigated using gastric cancer NUGC-4 cells as a target, which either were pretreated with 10 ng / ml of 5-FU and 500 ng oxaliplatin ml / (5-FU + OX), 10 ng / ml epirubicin, 500 ng oxaliplatin ml / and 10 ng / ml 5-FU (EOF) or 10 ng / ml 5-FU, 50 ng / folinic acid ml and 500 ng / ml oxaliplatin (FLO) for 96 hours (40 effectontarget ratios: 1) or untreated. EC50 values were obtained from 7 healthy donors for untreated and EOF, FLO or 5-FU + OX pretreated NUGC-4 cells.
[00278] As shown in Figure 4a, the dose / response curves in pretreated cells shifted up and to the left relative to untreated target cells. This resulted in a higher maximum lysis and a decrease in the EC50 values for one third of the untreated cells (Figure 4b).
[00279] Peripheral blood mononuclear cells (PBMCs), including NK cells, monocytes, mononuclear cells or other effector cells from healthy human donors were purified by Ficoll-Hypaque density. The washed effector cells were seeded in X-Vivo medium. KatoIII cells that express endogenously CLDN18.2
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133/159 and are of gastric origin were used as target cells in this scenario. The target cells stably express luciferase, yellow lucifer, which is oxidized by only viable cells. Purified anti-CLDN18.2 IMAB362 antibody was added in various concentrations and as an isotype control antibody to an irrelevant chim hulgGI antibody was used. The samples were tested for cytolysis by measuring the luminescence resulting from the oxidation of yellow lucifer which is a value for the amount of viable cells remaining after IMAB362 induced cytotoxicity. KatoIII pretreated for 3 days with Irinotecan (1000 ng / ml), docetaxel (5 ng / ml) or cisplatin (2000 ng / ml) were cultured compared to medium and untreated IMAB362 ADCC-induced target cells was quantified.
[00280] KatoIII cells pretreated for 3 days with irinotecan, docetaxel or Cisplatin exhibited a lower level of viable cells compared to target medium cultured cells (Figure 5a) and claudinl8.2 expression in cells pretreated with irinotecan, docetaxel or cisplatin was increased compared to medium cultured cells (Figure 5b).
[00281] Furthermore, pretreatment of KatoIII cells with irinotecan, docetaxel or Cisplatin increased the potency of IMAB362 to induce ADCC (Figure 5c, d).
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Example 3: Chemotherapy results in increased efficiency of CDAB-induced IMAB362 [00282] Effects of chemotherapeutic agents on IMAB362-induced CDC were analyzed by pretreatment of KATO III gastric cancer cells with 10 ng / ml of 5-FU and 500 ng oxaliplatin mL / (5-FU + OX) for 48 hours. Dose response curves induced by pre-treated chemotherapeutic IMAB362 CDC representative using KATO III cells are shown in Figure 6. Pretreatment of tumor cells for 48 hours increased the potency of IMAB362 to induce CDC, resulting in greater lysis maximum cell count of pretreated tumor cells compared to untreated cells.
Example 4: Ability of immune effector cells to perform ADCC-mediated IMAB362 is not compromised by treatment with chemotherapeutic agents [00283] The chemotherapeutic agents used in EOF or FLO regimen are highly potent in inhibiting target cell proliferation. To investigate the adverse effects of chemotherapy on effector cells, PBMCs from healthy donors were treated with 10 ng / ml epirubicin, 500 ng ml oxaliplatin / and 10 ng / ml 5-FU (EOF) or 10 ng / ml 5-FU, 50 ng / ml folinic acid and 500 ng oxaliplatin ml / (FLO) for 72 hours before
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135/159 application in CCDA tests. Figure 7a shows EC 50 values from 4 healthy donors and Figure 7b shows dose / response curves representative of ADCC-induced IMAB362 using EOF or FLO pretreated in effector cells. IMAB362-induced ADCC of NUGC-4 gastric carcinoma cells is not compromised due to EOF or FLO chemotherapies.
Example 5: A combination of ZA / IL-2 results in the expansion of optimal treatment of cultures of peripheral blood mononuclear cells (PBMC) [00284] The effect of ZA / IL-2 on the proliferation of PBMC cultures was evaluated in vitro. PBMCs were harvested from healthy human donors and the cultures were treated with a single dose of ZA. IL-2 was added every 3-4 days. Specifically, PBMCs derived from 3 different healthy human donors (# 1, # 2, # 3) were cultured in RPMI medium (1x10 ⁶ cells / ml) for 14 days at 1 μΜ higher ZA (300 U / ml) or low (25 U / ml), doses of IL-2; cf. Figure 8a. PBMC from the same donors were grown in RPMI medium additionally for 14 days with 300 U / ml IL-2 plus ZA or without ZA; cf. Figure 8b. Increase in the number of cells was determined by counting live cells on days 6, 8, 11 and 14.
[00285] In medium supplied with a high dose of IL-2 of about 2 - 5 times more expanded cells, compared
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136/159 with cultures supplied with a low dose of IL-2 (Figure 8a). Cell expansion in medium without ZA was approximately 2 times less compared to cells grown in medium with ZA (Figure 8b). These data demonstrate the need to apply both ZA and IL-2 compounds in an appropriate combination to ensure cell expansion.
[00286] Example 6: ZA / IL-2 results in treatment expansion of large amounts of Vy9V52 T cells in PBMC cultures [00287] PBMC were grown for 14 days in RPMI medium supplemented with 300 U / ml IL-2 and with or without 1 μΜ ZA. The percentage of Vy9V52 T cells within the CD3 + lymphocyte population (Figure 9a) and the percentage of CD 16+ cells within the CD3 + Vy9 + V62 + T cell population (Figure 9b) was determined by multicolor FACS on day 0 and day 14. The results were plotted for each donor on the scatter plot. Figure 9c shows a scatterplot showing the increase (enrichment) in the number of CD3 + Vy9 + V52 + and CD3 + CD 16+ Vy9 + V52 + T cells within the lymphocyte population over time. The number of cells seeded on day 0 and the number of cells harvested on day 14 were taken into account.
[00288] IL-2 in addition to PBMC cultures is necessary for lymphocyte survival and growth.
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They expand efficiently in cultures supplied with 300 U / ml of IL-2. FACS analysis using specific antibodies Vy9 and V52 reveals that the addition of ZA / IL-2 specifically induces the accumulation of Vy9V62 T cells (Figure 9a). After 14 days, the CD3 + lymphocyte population can comprise up to 80% of Vy9V52 T cells. A portion of T cells express Vy9V62 CD 16, while the enrichment of these cells within the CD3 + lymphocyte population is 10-700fold, donor dependent (Figures 9b and 9c). The enrichment of CD16 + Vy9 + V62 + T cells in 10-600fold cultures is greater compared to cultures grown without ZA (Figure 9c). We conclude that ZA / IL-2 treatment of PBMCs in vitro results in over-regulation of the CD-Fcylll receptor mediating ADCC 16 in a significant proportion of γδ T cells.
Example 7: IL-2 affects the expansion of Vy9V52 T cells in a dose-dependent manner [00289] The addition of ZA in cultures is the most important factor in inducing the development of Vy9V52 T cells. It is well known that IL-2 is necessary for the growth and survival of T cells.
[00290] PBMC were cultured for 14 days in RPMI medium supplemented with 1 μΜ ZA and increasing IL-2 concentrations. IL-2 was added on day 0 and on day
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4. The enrichment of CD 16 + Vy9 + V62 + T cells within the CD3 + lymphocyte population was determined by multicolor FACS stained on day 0 and day 14. To compare the different donors, the amount of CD16 + Vy9 + V52 + T cells harvested after culturing with 600 U / ml IL-2 was defined as 100%; cf.
[00291] Figure 10, on the left. In addition, the ADCC activity of isolated cultures grown for 14 days at increasing concentrations of IL-2 was tested; cf. Figure 10, right.
[00292] IL-2 dose response analysis was confirmed which also stimulates the growth and survival of the VY9V62 subset of T cells. By adding low concentrations of IL-2 in the medium, a correlation was found between the dose of IL-2 and the percentage of CD16 + Vy9V62 T cells within the CD3 + lymphocyte population (Figure 10, left). ADCC activity of cells cultured at higher concentrations of IL-2 (150-600U / ml) was improved compared to cells cultured at low concentrations of IL-2 (Figure 10, right).
Example 8: ZA induces the production of IPP in monocytes and cancer cells stimulate either the expansion of νγ9νδ2 T cells [00293] fresh PBMC (Exp. # 1) or 14 days ZA / IL-2 stimulated cultures of Vy9V52 T cells that were incubated
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139/159 or without monocytes (Exp # 2-5.) (Effector ratio: monocyte 1: 0), with 0.2fold (4: 1) or 5fold (ratio 1: 4) the amount of monocytes + 1 μΜ ZA. The enrichment of Vy9V62 T cells in co-cultures after 14 days was determined by multicolor FACS, while the expansion of the culture was considered in the calculation. The T cell enrichment factor cultured with Vy9V62 monocytes in a 1: 4 ratio was defined as 100% for each experiment. The increase of monocytes in culture resulted in an enrichment of Vy9V62 T cells of more than 10fold. This effect was clearly ZAdependent; cf. Figure 11a.
[00294] In addition, human stomach cancer cells (NUGC-4-luciferase) and murine stomach cancer cells (CLS103 - Spotted Calcein) were pretreated with or without 5 μΜ ZA for two days. Purified human Vy9V52 T cells were MACS (day 14) and co-cultured with cancer cells for 24 h. The cytotoxicity of T cells against untreated and treated Vy9V52 Z Target cells were determined by measuring the remaining activity of luciferase or fluorescent calcein; cf. Figure 1 £ target cells (NUGC-4 and CLS103) were pretreated with or without 5 μΜ ZA for 2 days and subsequently incubated for 4 h with mitomycin-C (50 MI) to prevent proliferation. 14d purified MACS resting old
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Vy9V52 human T cells and ³ H-thymidine were added to the target cells and co-cultures were incubated for 48 h at 37 ° C. Proliferation was determined by measuring ³ H thymidine incorporation in DNA, using a MicroBeta scintillation counter. The proliferation of non-target cells treated with ZA and W / o Vy9V52 T cells was defined as 100%; cf. Figure 1 lc.
[00295] As shown in Figures 1 £ and 1 lc the pulsed activated human cancer cells ZA-Vy9V52 T cells in terms of cytotoxicity (5-10fold) and proliferation (1.4-1.8 times), whereas the cell line of murine cancer CLS103 failed to cause these effects on Vy9V62 T cells.
Example 9: ZA / IL-2 affects the treatment composition of PBMC cultures [00296] The growth and differentiation of specific cell types in PBMC cultures depends on the presence of cytokines. These components are added either to (for example, growth factors present in serum, IL-2) or medium secreted by the immune cells themselves. Which type of cells evolves also depends on the initial composition of PBMC and on genetic characteristics. To analyze the overall increase in effector cells (NK cells and Vy9V52 T cells) PBMCs of 10
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141/159 different donors were cultured in the presence of 300 U / ml IL-2 and with or without 1 μΜ ZA for 14 days. The number of effector cells within the lymphocyte population was identified by multicolor FACS staining using CD3, CD 16, CD56, Vy9 and V62 antibodies. CD3-CD56 + CD16 + cells represent NK cells and CD3 + Vy9 + V52 + represent Vy9V52 T cells.
[00297] FACS analysis revealed that multicolored on top of IL-2 treatment mainly develop NK cells, whereas in ZA / IL-2 cells treated Vy9V52 T cultures are predominantly expanded (Figure 12). Example 10: ZA IL-2 generates the treatment of effector memory cells + Vy9V52 [00298] The subpopulations of T lymphocytes can be delineated with the aid of two surface markers, the high mw isoform of the common lymphocyte antigen CD45RA and the receptor chemokine CCR7. CCR7 + naive and central memory cells (CM) T are characterized by the ability to circulate repeatedly in lymph nodes and antigen encounter. In contrast, memory effector (EM) and effector T RA + (Temra) lymphocytes for low-regular CCR.7 and seem to specialize in migrating to peripheral non-lymphoid tissues for example, to infected or tumor sites. EM cells can be subdivided based on CD27
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142/159 differential and CD28 expression. The progressive loss of CD28 and CD27 surface expression is concomitant with increased regulation of the cytolytic capacity of cells. In addition, the level of CD57 correlates with the expression of granzymes and perforins and thus represents a third marker that indicates cytotoxicity / cell maturation.
[00299] PBMC were grown with or w / o 1 μΜ ZA and 300 U / ml of IL-2 for 14 days. The expression of the different surface markers was determined by multicolor FACS analysis on day 0 (PBMC) and day 14. Naive cells are CD45RA + CCR7 +, central memory cells (CM) are CD45RA- CCR7 +, Temra are CD45RA + CCR7- and effector memory cells (EM) are negative for both markers; cf. Figure 13a. In addition, the cytolytic activity of Vy9V52 T cells was determined by staining for CD27 and CD57 markers; cf. Figure 13b, c. In addition, the development of N cell-like characteristics important for ADCC activity was analyzed by staining with CD3 + CD 16 (antibody binding) and CD56 (adhesion) cells; cf. Figure 13d.
[00300] Multicolor FACS analysis of Vy9V52 T cells revealed that treatment ZA / IL-2 clearly stimulated the development of EM Vy9V62 T cells that are CD27 and CD57 + (Figure 13b-c). In addition to increased activity
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143/159 cytolytic, an increase in the level of CD 16 and CD56, which are known from NK cells has been observed (CD3-CD16 + CD56 +) to be involved in ADCC in the CD3 + cell population (Figure 13d).
[00301] Taken together, these data imply that the treatment of PBMC ZA results in the development of CD16 + Vy9 + V52 + effector memory T cells, which are able to migrate to peripheral non-lymphoid tissues and that high activity markers display cytolytics. In combination with the tumor antibody IMAB362 target these cells are extremely well used to migrate to, target and kill tumor cells.
Example 11: ZA / IL-2 expanded VY9V52 effector T cells are potent for IMAB362 mediated CLDN18.2 dependent ADCC [00302] Similar to NK cells, the ZA / IL-2 expanded Vy9V52 T cells are positive for CD 16 (see Figure 9 and 13), the FcyRIII receptor, through which an antibody bound to the cell triggers ADCC. To assess whether Vy9V62 T cells are capable of inducing potent ADCC in conjunction with IMAB362 a series of experiments were performed.
[00303] PBMCs derived from two different donors (# 1 and # 2) were grown in medium with 300 U / ml IL-2 and with or without 1 μΜ ZA. After 14 days the cells were harvested and added with increasing concentrations (0.26 ng / ml - 200
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144/159 ug / ml) from IMAB362 to NUGC-4 expressing CLDN18.2 cells.
[00304] Specific death was determined in luciferase assays; cf. Figure 14a. Figure 14b, c gives an overview of the tests carried out ADCC with 27 donors grown on 300 U / ml of IL-2 and with or w / o ZA. NUGC-4 served as target cells. For each donor, the EC50 values (b), calculated from the dose-response curves and the maximum specific death rate at the dose of IMAB362 (c) 200 μ were plotted on the scatter plots.
[00305] Strong IMAB362-dependent ADCC activity was observed against CLDN18.2-positive NUGC-4 cells using PBMC cultured for 14 days with ZA / IL-2 (Figure 14a). Using ZA / cultures of PBMC treated with IL-2, ADCC depends on the presence of Vy9V62 T cells (Figures 12 and 15). If cells are grown without ZA, ADCC activity is reduced for most donors. In these cultures, residual ADCC activity is N-cell dependent (Figures 11 and 14). When testing more than 20 donors, CCDA trials reveal that ZA / IL-2 from PBMC treatment improves EC 50 and maximum specific death rates, compared to PBMC in culture with IL-2 alone.
[00306] In addition, PBMCs from two different donors (# 1 + # 2) were grown with 1 μΜ ZA and 300 U / ml IL
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2. These effector cell cultures were used in CCDA assays with CLDN18.2-positive (NUGC-4, KATO III) and (SK-BR-3), target human negative cell lines (E: T ratio 40: 1 ). IMAB362 antibody (200 µg / ml 0.26 ng / ml) was added in increasing amounts. ADCC was measured in luciferase assays; cf. Figure 15a. The same experiment, as described in (a) was performed with NUGC-4 target cells and effector cells harvested from cultures treated with ZA / IL-2 at different time points; cf. Figure 15b. The same experiment, as described in (a), was performed using -4 NUGC as target cells; cf. Figure 15c. The ZA / IL-2 cells were either expanded or used directly, or Vy9V52 T cells were purified from the cultures using MACS TCRy5 screening (Miltenyi Biotech). Degree of purity of more than 97.0% of T cells in Vy9V52 lymphocytes was obtained.
[00307] Strong ADCC activity against CLDN18.2 negative CLDN18.2 negative human tumor cell lines, but was not observed (Figure 15a). In addition, no ADCC activity is obtained with isotype control antibodies (not shown). In the course of ZA / IL-2 the treatment of lytic ADCC activity increases over time for a fraction of the donors (Figure 15b). The dose / effect curve of IMAB362 shifts up and to the left
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146/159 showing improved EC50 values and maximum rates of lysis over time. Compared to the unconditioned PBMC, Vy9V52 effector T cells enriched by ZA / IL-2 treatment are able to achieve a higher maximum murder rate of CLDN18.2-positive target cells, plus they require lower concentrations of IMAB362 for the same rate of murder.
[00308] To confirm that T cells are Vy9V62 from the reservoir for lytic activity, these cells were isolated with> 97% purity by ZA / IL-2 magnetic screening cell in culture from PBMC populations on day 14. ADCC activity in together with IMAB362 is retained and partially improved due to the higher purity. These data confirm that Vy9V52 T cells are primarily responsible for the ADCC activity seen with 14 day old PBMC cultures (Figure 15c).
Example 12: Treatment of target cell lines with ZA / IL-2 does not affect the surface expression of CLDN18.2 [00309] IMAB362 triggered modes of action are strictly dependent on the presence and amount of detectable extracellular CLDN18.2. Therefore, the influence of ZA / IL-2 on CLDN18.2 surface density treatment was analyzed by flow cytometry using CLDN18.2 endogenous cell lines expressing NUGC-4 and KATO III.
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Specifically, the flow cytometry analysis of the link
IMAB362 in NUGC-4 unpermeabilized gastric cancer cells
pretreated with ZA / IL-2 or ZA / IL-2 + EOF or ZA / IL-2 + 5-FU / OX during 72 hours it was made. [00310] ZA / IL-2 in treatment vitro reveals no changes at amount of CLDN18.2 surface of
location; cf. Figure 16.
Example 13: IMCC362-ZA / IL-2-mediated ADCC increase in PBMC treatment is not compromised by EOF pretreatment [00311] Commitment cell proliferation chemotherapeutic agents. In contrast ZA / IL-2 treatment triggers the expansion of Vy9V52 T cells. To analyze the influence of these opposite interactions on effector cells, PBMC from six healthy donors were cultured with IL-2 ZA ZA or IL-2 + EOF for 8 days prior to application in CCDA assays (E: T ratio 15: 1) . IMAB362 concentrations resulting in 50% ADCC-mediated lysis of untreated NUGC-4 target cells (EC50 were determined).
[00312] Increase of IMAB362 ADCC-induced NUGC-4 cells due to treatment with ZA PBMC IL-2 is not significantly altered by combined treatment of PBMC with EOF (Figure 17).
Example 14: Targeting in vivo tumors of IMAB362 CLDN18.2-positive and antitumor effects of IMAB362 in
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148/159 human tumor cell xenografts in nude mice [00313] To investigate in IMAB362 targeting tumor cells in vivo, 8 ( ^Λ g Dyelight ® 680-labeled antibody was administered intravenously to nude mice that were xenografted via subcutaneous with the human gastric cancer cell line NUGC-4. NUGC-4 cells exhibit superficial expression of CLDN18 0.2, as well as HER2 / neu (trastuzumab target), but are negative for CD20. Control studies were conducted by injecting NUGC-4 groups of mice grafted with either trastuzumab-labeled Dyelight 680 (positive control group) or labeled Dyelight ^® 680-rituximab (negative control) .IMAB362 accumulates strongly and exclusively in tumor xenografts, as demonstrated by the live image of mice, using a Xenogen ® fluorescence imaging system 24 hours after intravenous injection of antibodies (Figure 18). IMAB362 is effective retained in the detectable positive target of the tumor and in comparable intensity, even after 120 hours (Figure 18). Trastuzumab is also detected exclusively in xenografts 24 hours after injection. The trastuzumab signal is flushed out quickly within 120 hours after injection. No signal is detected with rituximab.
[00314] In addition, IMAB362 was used to treat
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149/159 nude mice with CLDN18.2-positive xenograft tumors. Studies of early treatment models (with administrations of IMAB362 as early as three days after tumor cell inoculation) were performed. In addition, advanced tumor treatment experiments were started up to 9 days after tumor cell inoculation when the tumors had reached volumes of about 60 120 mm ³ .
[00315] The nude mice were inoculated subcutaneously with ¹ x 10 ⁷ transfectants HEK293-CLDN18.2. The treatment of 10 mice per group started 3 days after tumor inoculation. The mice were treated with 200 µg IMAB362, infliximab as isotype control and PBS twice a week for 6 weeks alternating intravenous and intraperitoneal routes of application. Whereas all mice in the groups treated with PBS or isotype control died within 70-80 days, the animals treated with IMAB362 had a survival benefit (Figure 19). Not only was the time to death prolonged, but 4 out of 10 mice survived the entire 210-day observation period.
[00316] The treatment of 9 to 10 mice per group was started when the average tumor volume reached 88 mm (62-126 mm). Before treatment the mice were
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150/159 stratified in test groups to ensure comparable tumor sizes in all groups. The mice were treated with 200 µg IMAB362, isotype control or PBS twice a week for 6 weeks alternating intravenous and intraperitoneal routes of application. All mice in the groups treated with PBS or isotype control died within 50-100 days. The animals treated with IMAB362 had a survival benefit, with almost twice the median survival time (47 versus 25 days). Three of these mice survived the entire observation period (figure 20).
[00317] It is important to note that the anti-tumor efficacy in vivo depends on the presence of the target in the tumor cells. No IMAB362 treatment antitumor effects were observed in mice grafted with HEK293 CLDN18.2-negative tumor cells.
[00318] The gastric tumor model NUGC-4 was used to investigate the efficacy of IMAB362 against cancer cells with endogenous expression of CLDN18.2. NUGC-4 cells grow aggressively in nude mice.
[00319] 1 x 10 ⁷ NUGC-4 gastric cancer cells were injected subcutaneously into the left flank of nude athymic mice (n = 9 for IMAB362 group; n = 8 for control groups). IMAB362 (200 mg per injection) and
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151/159 the controls were applied iv alternating twice a week and ip, starting 6 days after the tumor inoculation with iv injection. Tumor sizes were monitored twice a week. The data shown in Figure 21a are means with SEM. Tumor growth in mice treated with IMAB362 was significantly inhibited compared to mice treated with controls (* p <0.05). Figure 21b shows tumor volumes on day 21 after tumor inoculation. The volumes of tumors in IMAB362 treated mice were smaller than the significant tumors in control mice (* p <0.05).
[00320] When LXL 0 ⁷ tumor cells are inoculated into mice, the average survival time of untreated mice is no more than 25 days. Treatment with IMAB362, cetuximab, trastuzumab or isotype and buffer controls was started when the tumor volume reached an average size of about 109 mm (63-135 mm). The mice were stratified in size depending on treatment groups (Figure 21). IMAB362 has been shown to significantly reduce the rate of tumor growth. There was no significant reduction in tumor growth compared to saline or antibody controls for this tumor model that grows aggressively. The delay in tumor growth was associated with a lack of
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152/159 significantly increased the mean survival time of mice treated IMAB362 (31 days versus 25 days).
[00321] The antitumor activity of IMAB362 was examined with two models of human gastric carcinoma xenotransplant using NCI-N87 or NUGC-4 cells with IMAB362 CLDN18.2 target lentivirus transduction (NCI-N87CLDN18.2 and NUGC-4- CLDN18.2).
[00322] NCI-N87-CLDN18.2 xenograft tumors were inoculated subcutaneously by injection of 1x10 NCI-N87-CLDN18.2 cells into the flank of 8 nude mice (female, 6 weeks old) per treatment group. The treatment was started 5 days after the tumor inoculation by intravenous injection of 800 ug IMAB362 or with 200 μΐ NaCl 0.9% for the saline control group. Intravenous administration was continued weekly for the duration of the observation period. Tumor size and animal health were monitored semi-weekly. Figure 22a shows the effects of treatment on the growth of IMAB362 tumors. The size of the tumors was measured sc twice a week (mean + EPM, *** p <0.001). Figure 22b shows Kaplan-Meier survival. The mice were sacrificed when the tumor reached a volume of 1400 mm.
[00323] Thus, the continuous treatment ΓΜΑΒ362 inhibited (p <0.001) the highly significant growth of NCI-N87
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CLDN18.2 gastric carcinoma xenografts (Figure 22a) of the tumor. The delay in tumor growth was associated with a significantly longer (p <0.05) longer survival of IMAB362 treated mice (Figure 22b).
[00324] IMAB362 rapid growth immunotherapy of NUGC-4-CLDN18.2 xenografts resulted in significantly (p <0.05) smaller tumor sizes on day 14 of treatment. After the first two weeks of treatment IMAB362 tumor progression of NUGC-4-CLDN18.2 was very aggressive. However, the inhibition of the growth of the NUGC-4-CLDN18.2 tumor until day 14 of treatment resulted in a significant (p <0.05) greater survival of the IMAB362 treated mice.
[00325] In summary, IMAB362 was highly effective in treating gastric carcinoma xenografts showing significant delay in tumor progression and prolongs survival in endogenous CLDN18.2-positive tumor models. In very aggressive tumor model systems, these anti-tumor effects of IMAB362 are less prominent, but still significant, highlighting the strong anti-tumor capacity of IMAB362.
Example 15: Anti-tumor effects of IMAB362 combined with chemotherapy in mouse tumor models [00326] In vitro, IMAB362-mediated ADCC is more efficient in pretreated human gastric cancer cells
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154/159 with combinations of chemotherapeutic agents including EOF and 5-FU + OX. Therefore, the antitumor impact of combining these compounds with IMAB362 was investigated in vivo in mouse tumor models.
[00327] NCI-N87-CLDN18.2 xenograft tumors were inoculated by injecting ¹ x 10 7 subcutaneous NCI-N87-CLDN18.2 cells into the flank of 9 mice for each treatment group. Tumor-bearing mice were treated according to the EOF regimen with 1.25 mg / kg epirubicin, 3.25 mg / kg oxaliplatin and 56.25 mg / kg intraperitoneal 5-fluorouracil on days 4, 11, 18 and 25 after tumor inoculation, followed by intravenous injection of 800 ug IMAB362 24 hours after chemotherapy administration. IMAB362 treatment was continued weekly. Tumor size and animal health were monitored semi-weekly. Figure 23 shows one of the effects of combined treatment on tumor growth.
[00328] The size of the tumors was measured sc twice a week (mean + SEM; * p <0.05). Figure 23b shows Kaplan-Meier survival. The mice were sacrificed when the tumor reached a volume of 1400 mm ³ .
[00329] Tumor NCI-N87-CLDN18.2 bearing nude mice treated with IMAB362 or EOF regimen showed highly significant suppressed tumor growth compared to
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155/159 control mice. Additional IMAB362 treatment in combination with EOF chemotherapy resulted in significantly (p <0.05) greater inhibition of tumor growth than treatment with the EOF regimen alone (Figure 23a). The average survival of mice in the saline control group was 59 days. IMAB362 weekly treatment of mice prolonged significantly median survival of 76 days similar to mouse survival in the EOF group with an average survival of 76 days, too. But combined treatment with IMAB362 and EOF increased the median survival by 81 days (Figure 23b).
[00330] Xenograft tumors were inoculated by injection of 1x10 'NUGC-4-CLDN 18.2 subcutaneous cells, in the flank, of 10 nude mice (female, six weeks old) per treatment group. The mice were treated on day 3, 10, 17 and 24 with chemotherapeutic agents. IMAB362 treatment was continued weekly. Figure 24a shows the tumor growth curves sc NUGC-4 ~ xenotransplants CLDN18.2 (mean + SEM). Figure 24b shows Kaplan-Meier survival (Log-rank (Mantel-Cox) Test, ** p <0.01).
[00331] Subcutaneous NUGC-4-CLDN18.2 xenotransplant tumors grow very aggressively. Despite IMAB362 treatment of tumor carriers nude mice significantly inhibited tumor growth compared to
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156/159 to the control group treated with saline. In combination therapy with EOF, IMAB362 effects on tumor growth NUGC-4-CLDN18.2 was masked by growth inhibition due to EOF treatment, showing no increased inhibition of tumor growth compared to treatment with EOF alone (Figure 24a). However, the median survival of mice treated with EVIAB362 and EOF regimen was highly significant (p <0.01) prolonged, compared to the survival of mice treated with EOF alone (Figure 24b).
Example 16: ZA / IL-2 expanded VY9V52 T cells improve IMAB362-mediated control of advanced tumors in vivo [00332] To investigate the combined activity of IMAB362 and ZA / IL-2 generated γδ T cells in mouse systems, we used NSG mice. NSG mice lack mature T cells, B cells, natural killer cells (NK), multiple cytokine signaling pathways, and they have many defects in innate immunity, whereas niches in primary and secondary immune tissues are permissive for colonization by human immune cells.
[00333] NSG mice were inoculated subcutaneously with an XLO ⁷ HEK293 transfectedCLDN18.2 cells. On the same day, the mice received 8 x 10 ⁶ human PBMC enriched in Vy9V52 T cells, which were
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157/159 grown for 14 days in supplemented medium-ZA. In addition, the mice were injected with 5 µg / kg and ZA 5000 U of IL-2 (Proleukin). To keep human T cells functional, IL-2 was administered weekly and semi-ZA weekly. When the tumors became HEK293-CLDN18.2 macroscopically visible treatment, semi-weekly with 200 µg of IMAB362 was started. In addition to 9 mice treated as described, two groups of control mice were established. One group did not receive human γδ T cells, the other group was treated with an isotype control antibody instead of IMAB362. Consequence of CLDN18.2-positive tumors in mice treated with ΓΜΑΒ362 in the presence of human γδ T cells and ZA was significantly inhibited and almost nulled, whereas in mice treated with either an isotype control antibody or human T effectors lacking cell, the tumors grew aggressively and mice had to be terminated prematurely (Figure 25).
Example 17: Antitumor effects of IMAB362 combined with chemotherapy in mouse tumor models [00334] Antitumor activity of IMAB362 in combination with chemotherapy was examined in subcutaneous allograft gastric carcinoma in immunocompetent mice outbred NMRI using CLS 103 cells with transduction of NMRI
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158/159 murine cldnl8.2 lentivirus (cldnl8.2 CLS-103 ~).
[00335] CLS-103 ~ allograft tumors inoculated cldnl8.2 by injecting lxlO ^6-CLS-103 cells subcutaneous cldnl8.2 in NMRI mice flank 10 for each treatment group. Tumor-bearing mice were treated with 1.25 mg / kg epirubicin, 3.25 mg kg oxaliplatin and 56.25 mg / kg 5-fluorouracil (EOF) intraperitoneally on days 3, 10, 17 and 24 after tumor inoculation, followed by intravenous injection of 800 ug IMAB362 24 hours after each chemotherapy administration. IL-2 was administered semi-weekly by subcutaneous injection of
3000 IE. After the end of chemotherapy, IMAB362 and IL-2
treatment was continued throughout the observation period.
[00336] Tumor size and animal health were monitored semi-weekly. The mice were sacrificed when the tumor reached a volume of 1400 mm ³ or tumors became ulcerative.
[00337] How can be seen in Figure 26, CLS-103 ~
8.2 cldnl tumor-bearing NMRI mice treated with IMAB362 or EOF alone did not show significant tumor growth inhibition compared to the saline control group. In contrast, the combination of EOF chemotherapy and treatment resulted in EV1AB362 significantly greater than
Petition 870160018043, of 05/05/2016, p. 167/206
159/159 inhibition of tumor growth and prolonging the survival of mice with tumors. These observations indicate the existence of additive or synergistic therapeutic effects even by combining EOF chemotherapy and IMAB362 immunotherapy. IL-2 treatment showed no effect on tumor growth.

权利要求:
Claims (13)
[1]
1. Use of an antibody characterized by the fact that it is for the preparation of a medicine for the treatment or prevention of a cancerous disease, in combination with bisphosphonate and interleukin-2, in which the antibody is selected from the group consisting of (1) an antibody that binds to CLDN18.2 and comprises an antibody heavy chain comprising the amino acid sequence represented by SEQ ID NO: 32 and an antibody light chain comprising the amino acid sequence represented by SEQ ID NO: 39, and (2) an antibody produced by and / or obtained by a clone deposited under accession number DSM ACC2810.
[2]
2. Use, according to claim 1, characterized by the fact that bisphosphonate is selected from the group consisting of zoledronic acid, clodronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, olpadronic acid, alendronic acid , incadronic acid and its salts.
[3]
3. Use according to claim 1 or 2, characterized by the fact that the antibody mediates cell death by one or more lysis-mediated complement-dependent cytotoxicity (CDC), lysis-mediated antibody-dependent cell cytotoxicity (ADCC) , the induction of apoptosis and inhibition of proliferation.
[4]
4. Use according to any one of claims 1

[5]
Use according to any one of claims 1 to 4, characterized in that it comprises using the antibody at a dose of 300 to 600 mg / m ² .
[6]
6. Use according to any one of claims 1 to 5, characterized by the fact that the cancer is CLDN18.2 positive.
[7]
Use according to any one of claims 1 to 6, characterized by the fact that the cancer is an adenocarcinoma, in particular, an advanced adenocarcinoma.
[8]
8. Use according to any one of claims 1 to 7, characterized by the fact that the cancer is selected from the group consisting of cancer of the stomach, cancer of the esophagus, in particular, of the lower esophagus, cancer of the esophageal junction gastric and gastroesophageal cancer.
[9]
Use according to any one of claims 1 to 8, characterized by the fact that the patient is a HER2 / neu negative patient or in the status of a HER2 / neu positive patient, but not eligible for trastuzumab therapy.
[10]
10. Use according to any one of claims 1 to 9, characterized by the fact that CLDN18.2 has the amino acid sequence defined by SEQ ID NO: 1.
[11]
11. Use according to any one of claims 1 to 10, characterized by the fact that the heavy chain of the
3/3

[12]
12. Kit characterized by the fact that it comprises an antibody and an agent, in which the agent is a bisphosphonate which is used in combination with interleukin-2, and in which the antibody is selected from the group consisting of (1) a antibody that binds to CLDN18.2 and comprises an antibody heavy chain comprising the amino acid sequence represented by SEQ ID NO: 32 and an antibody light chain comprising the amino acid sequence represented by SEQ ID NO: 39, and (2) an antibody produced by and / or obtained by a clone deposited under DSM accession number ACC2810.
[13]
13. Kit according to claim 12, characterized in that the antibody heavy chain comprises the amino acid sequence represented by SEQ ID NO: 17 and the antibody light chain comprises the amino acid sequence represented by SEQ ID NO: 24.

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法律状态:
2017-01-03| B65X| Notification of requirement for priority examination of patent application|

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2017-02-07| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|

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2017-02-21| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2018-08-21| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2019-03-06| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2019-08-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2019-11-12| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 21/05/2013, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 21/05/2013, OBSERVADAS AS CONDICOES LEGAIS |

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2020-03-17| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REF. RPI 2549 DE 12/11/2019 QUANTO AO TITULAR E AO INVENTOR. |

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2021-06-08| B25D| Requested change of name of applicant approved|Owner name: TRON - TRANSLATIONALE ONKOLOGIE AN DER JOHANNES GUTENBERG-UNIVERSITAET MAINZ GEMEINNUETZIGE GMBH (DE) ; GANYMED PHARMACEUTICALS GMBH (DE) |

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2021-06-29| B25D| Requested change of name of applicant approved|Owner name: GANYMED PHARMACEUTICALS GMBH (DE) ; TRON - TRANSLATIONALE ONKOLOGIE AN DER UNIVERSITAETSMEDIZIN DER JOHANNES GUTENBERG UNIVERSITAET MAINZ GEMEINNUETZIGE GMBH (DE) |

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2021-07-20| B25A| Requested transfer of rights approved|Owner name: TRON - TRANSLATIONALE ONKOLOGIE AN DER UNIVERSITAETSMEDIZIN DER JOHANNES GUTENBERG UNIVERSITAET MAINZ GEMEINNUETZIGE GMBH (DE) ; ASTELLAS PHARMA INC. (JP) |

优先权:

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申请号 | 申请日 | 专利标题

---

PCT/EP2012/002211|WO2013174404A1|2012-05-23|2012-05-23|Combination therapy involving antibodies against claudin 18.2 for treatment of cancer|

---

PCT/EP2013/001503|WO2013174509A1|2012-05-23|2013-05-21|Combination therapy involving antibodies against claudin 18.2 for treatment of cancer|

---