claudin 18.2 antibody therapy for cancer treatment

专利摘要:
Patent Summary for: "therapy involving claudin 18.2 antibodies for cancer treatment". The present invention generally provides effective therapy for the treatment and / or prevention of diseases associated with cldn-expressing cells 18,2, in particular cancerous diseases such as gastro-oesophageal cancer. Data are presented demonstrating that administration of an anti-cldn18.2 antibody to human patients with gastroesophageal cancer is safe and well tolerated to a dose of at least 1,000 mg / m2. In addition, data are presented demonstrating that the antibody is fully functional in these patients to effect effects on anti-tumor cells and evidence of anti-tumor activity was obtained.
公开号:BR112015023910B1
申请号:R112015023910
申请日:2014-03-17
公开日:2019-09-10
发明作者:Sahin Ugur；Türeci Özlem
申请人:Ganymed Pharmaceuticals Ag；Tron Translationale Onkologie An Der Univ Der Johannes Gutenberg Univ Mainz Gemeinnuetzige Gmbh；
IPC主号:

专利说明:

Invention Patent Descriptive Report for: “THERAPY INVOLVING ANTIBODIES AGAINST CLAUDIN 18.2 FOR CANCER TREATMENT.
[001] Cancers 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 present with locally advanced or metastatic disease have to undergo first-line chemotherapy. Treatment regimens are based on a structure of platinum and fluoropyrimidine derivatives, mainly combined with a third compound (for example, taxane or anthracyclines). Still, the average free survival of 5 to 7 months and the average global survival of 9 to 11 months are the best that can be expected.
2/217 [003]
The lack of a major benefit from the latest generation of various chemotherapy combination regimens for these types of cancer has spurred research on the use of targeted agents.
Recently for FFer2 / neupositive gastroesophageal cancers, Trasruzumab has been approved. However, as only ~
20% of patients who express the target and are eligible for this treatment, the medical need remains high.
[004] The tight junction molecules claudin 18 variant of splice 2 (2 claudin 18.2 (CLDN18.2)) is a member of the tight junction claudin 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 except for 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 malignant transformation and thus appears frequently on the surface of human gastric cancer cells. In addition, this pan-tumor antigen is ectopically activated at significant levels in the esophagus,
3/217 pancreas and pulmonary adenocarcinomas. The CLDN18.2 protein is also located in lymphatic metastases from gastric cancer adenocarcinomas and, especially, in distant metastases to the ovary (so-called Krukenberg tumors).
[007] The chimeric IgGl antibody IMAB362 that 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 does not bind to any other member of the claudine family including Claudine 18's closely related processing variant 1 (CLDN18.1). IMAB362 shows precise tumor cell specificity and includes four highly potent independent mechanisms of action. After binding to the target, IMAB362 mediates cell death by ADCC, CDC and induction of cross-linked apoptosis induced by the target on the surface of the tumor cell and direct inhibition of proliferation. Thus, IMAB362 cells efficiently lyse CLDN1.8-positive, including human gastric cancer cell lines in vitro and in vivo. Mice with CLDN18.2-positive cancer cell lines have a survival benefit and up to 40% of mice show tumor regression when treated with IMAB362.
[008] The toxicity and PK / TK profile of IMAB362 has been thoroughly examined in mice and cynomolgus monkeys,
4/217 including studies to determine the dose range, 28 days of repeated dose toxicity studies in cinomolgos and a 3-month repeated dose toxicity study in mice in both mice (the longest the duration of treatment at weekly administration for 3 months, higher dose levels of 400 mg / kg) and cynomolgus monkeys (up to 5 weekly applications of up to 100 mg / kg) 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 healthy gastric luminal epithelium, which appears to severely impair the accessibility of the IMAB362 epitope. This compartmentalization is lost upon the malignant transformation making drug epitopes covered by IMAB362.
[009] Results are presented demonstrating that administration of an anti-CLDN18.2 antibody, such as IMAB362 to human patients with gastroesophageal cancer is
5/217 safe and well tolerated up to a dose of at least 1000 mg / m ² · In addition, the data presented here demonstrate that the antibody is fully functional in these patients to perform effects on anti-tumor cells and evidence for anti-tumor activity. tumor was obtained ·
SUMMARY OF THE INVENTION [0010] The present invention, in general, provides a therapy for the treatment and / or prevention of diseases associated with cells that express CLDN18.2, including cancerous diseases, such as gastric cancer, esophageal cancer, 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 metastasis, including metastasis 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, lung and ovary · [0011] In a first aspect, the present invention provides a method of treating or preventing a cancerous disease, comprising administering to a patient a antibody that has the ability to bind to CLDN 18.2, wherein the antibody is administered to provide a serum level of at least 40 pg / ml. In different
In 6/217 embodiments, the antibody is administered so as to provide a serum level of at least 50 μg / ml, at least 150 μg / ml, at least 300 μg / ml, at least 400 μg / ml or at least , 500 μg / ml. In different embodiments, the antibody is administered in order to provide a serum level of no more than 800 μg / ml, 700 μg / ml, 600 μg / ml, 550 μg / ml or 500 μg / ml. In one embodiment, the serum level provided is between 40 μg / ml and 700 μg / ml, preferably 40 μg / ml and 600 μg / ml, preferably 50 μg / ml and 500 μg / ml, such as between 150 μg / ml and 500 μg / ml or 300 μg / ml and 500 μg / ml. The term serum level, as used in this specification, means a concentration of the substance in question in blood serum. In one embodiment, the serum level is provided for at least 7 days, or at least 14 days. In one embodiment, the method comprises administering a dose of antibody doses / doses of at least 300 mg / m ² , such as at least 600 mg / m ² , preferably up to 1500 mg / m ^2, up to 1200 mg / m ² m ² up to 1000 mg / m ² .
[0012] In a second 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 to CLDN18.2, wherein the antibody is administered at a dose of at least
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0 mg / m2, such as at least 600 mg / m ² and preferably up to 1500 mg / m ² , up to 1200 mg / m ² up to 1000 mg / m ² · [0013] In a third aspect, this The invention provides a method of treating or preventing a cancerous disease, comprising administering to a patient an antibody that has the ability to bind to CLDN18.2, wherein at least 50%, preferably 60%, 70%, 80 % or 90% of the patient's cancer cells are CLDN18.2 positive and / or at least 40%, preferably 50% or 60% of the patient's cancer cells are positive for the surface expression of CLDN18.2. In this regard, the present invention also provides a method of treating or preventing a cancerous disease, said method comprising: a. identification of a patient who exhibits at least 50%, preferably 60%, 70%, 80% or 90% of CLDN18.2 positive cancer cells and / or at least 40%, preferably 50% or 60% cancer cells that are positive for CLDN18.2 surface expression; and b. administering to said patient an antibody that has the ability to bind to CLDN 18.2. In one embodiment, at least 95% or at least 98% of the patient's cancer cells are CLDN18.2 positive. In one embodiment, at least 70%, at least 80% or at least 90% of the patient's cancer cells are positive for the surface expression of
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CLDN18.2.
[0014] In an embodiment of the method of any of the aspects described here, the treatment of cancer of the disease results in obtaining the disease stable. In one embodiment, stable disease is achieved for at least 2 months, at least 3 months, or at least 6 months.
[0015] In a fourth aspect, the present invention provides a method of achieving stable disease in a cancer patient that comprises administering to the patient an antibody that has the ability to bind to CLDN18.2. In one embodiment, stable disease is achieved for at least 2 months, at least 3 months, or at least 6 months.
[0016] In one embodiment of the method of any of the aspects described here, the antibody is administered in a single dose or in multiple doses.
[0017] In a fifth 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 to CLDN18.2, wherein the antibody is administered in multiple doses.
[0018] According to the invention, the antibody is administered in multiple doses, the antibody is preferably administered in at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7
9/217 doses, at least 8 doses, at least 9 doses or at least 10 doses and, preferably, up to 30, 25, 20, 15 or 10 doses. The doses of the antibody are preferably administered at intervals of at least 7 days, at least 10 days, at least 14 days, or at least 20 days. The doses of the antibody are preferably administered at intervals of between 7 and 30 days, 10 days and 20, and preferably about 14 days.
[0019] In an embodiment of the third, fourth or fifth aspect method, the antibody is administered so as to provide a serum level of at least 40 pg / ml. In different embodiments, the antibody is administered to provide a serum level of at least 50 pg / ml, at least 150 pg / ml, at least 300 pg / ml, at least 400 pg / ml, or at least at least 500 pg / ml. In different embodiments, the antibody is administered to provide a serum level of no more than 800 pg / ml, 700 pg / ml, 600 pg / ml, 550 pg / ml or 500 pg / ml. In one embodiment, the serum level provided is between 40 pg / ml and 700 pg / ml, preferably 40 pg / ml and 600 pg / ml, preferably 50 pg / ml and 500 pg / ml, such as between 150 pg / ml and 50 0 pg / ml or 30 0 pg / ml and 50 0 pg / ml. In one embodiment, the serum level is provided for at least 7 days, or at least 14 days. In one embodiment, the method comprises administering a dose / doses of antibody
10/217 of at least 300 mg / m ² such as at least 600 mg / m ² and preferably up to 1500 mg / m ² , up to 1200 mg / m ² or up to 10 0 0 mg / m ² · [0020 ] In an embodiment of the method of any of the aspects mentioned above, the method further comprises the administration of one or more selected from the group consisting of anti-emetics, anti-spasmodics, parasympatholytics and agents that protect the gastric mucosa.
[0021] In a sixth 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 to CLDN 18.2 and one or more selected from the group that it consists of antiemetics, anti-spasmodics, parasympatholytics and agents that protect the gastric mucosa.
[0022] If the method of the invention comprises the administration of one or more selected from the group consisting of anti-emetics, anti-spasmodics, parasympatholytics and agents that protect the gastric mucosa, the method in different embodiments comprises administering: (I) one antiemetic and an antispasmodic; (ii) antispasmodics and an agent that protects the gastric mucosa, (iii) an anti-emetic and an agent that protects the gastric mucosa or (iv) an antiemetic, an antispasmodic and an
11/217 agent that protects the gastric mucosa.
[0023] In one embodiment, an antiemetic is administered as antiemetic prophylaxis prior to administration of the antibody. In one embodiment, an antiemetic is administered as an intervention antiemetic simultaneously with and / or after administration of the antibody. In one embodiment, the antiemetic comprises a 5-HT 3 receptor antagonist and / or a neurokinin 1 (NK1) receptor antagonist. Preferably, the NK1 receptor antagonist comprises aprepitant (for example, Splice) and the 5-HT3 receptor antagonist comprises ondansetron (for example, Zofran), granisetron (for example, Kytril, Sancuso) or Palonosetron (for example, Aloxi), or a combination of two or more of these.
[0024] In one embodiment, the antispasmodic comprises butylscopolamine (Buscopan).
[0025] In one embodiment, the agent that protects the gastric mucosa comprises an agent that reduces the production of gastric acid. In one embodiment, the agent that protects the gastric mucosa comprises an agent selected from the group consisting of proton pump inhibitors, misoprostol and omeprazole. In one embodiment, the agent that protects the gastric mucosa comprises a combination of a proton pump inhibitor and misoprostol. In one embodiment, the proton pump inhibitor comprises
12/217 pantoprazole (e.g., Pantozole).
[0026] In one embodiment, the method of the invention comprises administering to the patient an NK1 receptor antagonist, such as aprepitant (e.g., Amendment), a 5-HT3 receptor antagonist, such as ondansetron (e.g., Zofran) , granisetron (eg Kytril, Sancuso) or Palonosetron (eg Aloxi), or a combination of two or more of these, such as an antispasmodic butylscopolamine (eg Buscopan) and a proton pump inhibitor, such as pantoprazole (e.g., Pantozole).
[0027] In one embodiment, the method of any of the above, the antibody is administered by iv infusion In one embodiment, the intravenous infusion is over a period of time between 1 and 4 hours, preferably about 2 hours.
[0028] In a sixth aspect, the present invention provides a method of determining the responsiveness of a cancer patient for treating or preventing a cancer disease comprising administering an antibody that has the ability to bind to CLDN18. 2, said method comprising the step of determining the blood level of one or more markers in the patient, wherein the one or more markers are selected from the group consisting of CA 125, CA 15-3, CA 19-9 , CEA, IL-2, IL-15,
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IL-6, IFNy, and TNFα. In this regard, before and after administration of an antibody that has the ability to bind to CLDN18.2, such as after administration of a single dose of the antibody, biological samples such as blood can be taken from the patient to establish the level of one or more labels. Several samples can be taken from the same tissue to determine average levels and to account for possible fluctuations in those levels. The level of one or more markers following administration of the antibody is compared to the level determined before administration. The effect of the antibody on the patient can therefore be identified by a desired change in the marker level followed by administration of an antibody that has the ability to bind to CLDN18.2. If the patient experiences a desired change in the marker level followed by administration of an antibody that has the ability to bind to treatment with the CLDN18.2 antibody having the ability to bind to CLDN18.2 it can be initiated.
[0029] In one embodiment, the level is determined in the blood, plasma or serum.
[0030] In one embodiment, the one or more markers are selected from the group consisting of CA 125, CA 15-3, CA 19-9, CEA, IL-2, IL-15, IFNy, and TNFa and a decrease at the level of at least one of the markers following antibody administration indicates that the patient responds
14/217 to the treatment or prevention of a cancer disease.
[0031] In one embodiment, the marker is 1L-6 and an increase in the level of the marker following administration of the antibody indicates that the patient responds to the treatment or prevention of a cancer disease.
[0032] In an eighth aspect, the present invention provides a method for determining whether a cancer patient is favorable to the treatment or prevention of a cancer disease comprising administering an antibody that has the ability to bind CLDN18.2, said method comprising the step of determining the percentage of cancer cells positive CLDN18.2.
[0033] In this embodiment, prior to administration of an antibody that has the ability to bind to CLDN18.2, a biological sample, such as a tumor sample (e.g., a tumor biopsy) can be taken from the patient to establish the level of cancer cells positive for CLDN18.2. Several samples can be taken to determine an average level and to take into account any fluctuations in those levels. If a patient has the desired level of CLDN18.2 positive cancer cells, an antibody that has the ability to bind CLDN18.2 can be administered.
[0034] In one embodiment, a level of at least 50%, preferably 60%, 70%, 80% or 90%, at least 95% or at least 98% of positive cancer cells
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CLDN18.2 indicates that the patient is susceptible to treatment or prevention of a cancer disease. In one embodiment, a level of at least 40%, preferably at least 50%, at least 60%, at least 70%, at least 80% or at least 90% cancer cells that are positive for the surface expression of CLDN18.2 indicates that the patient is susceptible to treatment or prevention of a cancer disease.
[0035] 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 CLDN18.2 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 apoptosis and proliferation inhibition. In one embodiment, the antibody that has the ability to bind to a CLDN18.2 monoclonal antibody is either chimeric or humanized antibodies, or an antibody fragment. 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 The. DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM
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ACC2740, DSM ACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2747, DSM ACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) an antibody that is a chimerized or humanized form of the antibody (humanized or i) of the antibody ), (iii) an antibody having the specificity of the antibody under (i), and (iv) an antibody comprising the antigen binding antigen binding portion or site, in particular of the variable region, of the antibody under (i) and preferably having 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.
[0036 ] In a concretization, cancer is CLDN18.2 positive. In an concretization, the cells of cancer express in mode The CLDN18.2. In one embodiment, the expression in CLDN18 .2 it's on the surface of cells In a
embodiment, at least 50%, preferably 60%, 70%, 80% or 90% of the cancer cells are CLDN18.2 positive and / or at least 40%, preferably at least 50% of the cancer cells are positive for the surface expression of CLDN18.2. In one embodiment, at least 95% or at least 98% of the cancer cells are CLDN18.2 positive. In one embodiment, at least 60%, at least 70%, at least 80% or at least 90% of the cancer cells are positive for surface expression of CLDN18,2.
[0037] In one embodiment, the disease is cancer
17/217 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 same. 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, 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. In a particularly preferred embodiment, the cancer is gastroesophageal cancer, such as metastatic, advanced refractory or recurrent gastroesophageal cancer. The patient may be a HER2 / neu negative patient or a patient with an HER2 / neu positive status, but not eligible for trastuzumab therapy. In one embodiment, the patient has had previous therapy with at least one drug selected from the group consisting of pyrimidine analogs (for example, fluorouracil and / or capecitabine), platinum compounds (for example cisplatin and / or oxaliplatin ), epirubicin, docetaxel and detoxifying agents for antineoplastic treatment (eg calcium folinate and / or folinic acid). In one embodiment, the patient has a state of
18/217 between 0 and 1 and / or a KRNAofsky index of between 70 and 100% of ECOG performance. In a particularly preferred embodiment, the patient is a human patient.
[0038] According to the invention, preferably CLDN18.2 has the amino acid sequence according to SEQ ID NO: 1.
The present invention also provides the agents described herein, such as the antibody that has the ability to bind to CLDN 18.2 for use in the methods described herein.
[0040] Other features and advantages of the present invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS [0041] Figure 1 shows the mean blood concentration of IMAB362 during the study.
[0042] Figure 2 shows the ADCC activity of patients' PBMCs. (A) PBMC were purified from 6 patient blood samples 7 days (open squares) or 14 days (black squares) after IMAB362 administration. Specific rates of lysis of stomach cancer target NUGC-4, cells expressing CLDN18.2, obtained after adding 31.63 μ / IMAB362 πι1 and PBMC from a healthy donor or PBMC from patients (E: T = 20 : 1) for 24 h. (B) dependent on IMAB362 concentration specific cell lysis
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NUGC-4 obtained 24 hours after adding PBMC from different patients (graphical display ± standard deviation, p value was calculated using the unpaired t test); (C) ADCC response curves from healthy control PBMC after addition of increasing concentrations IMAB362. The assays were performed in parallel for each ADCC analysis with the patient's PBMC. (D) ADCC response curve of patients' PBMCs after the addition of increasing IMAB362 concentrations (for 0202 non-PBMC patients were obtained sufficient to generate a curve). (E) Maximum death rates for all patients and maximum healthy donors were calculated with the GraphPad Prism software using the non-linear build-in regression analysis tool.
[0043] Figure 3 shows the ability of patient complement components to induce CDC-mediated IMAB362. The assays were performed with CLDN 18.2 and CHO-Kl luciferase positive target cells. The cells, serum (20% v / v) and antibodies were incubated for 80 min at 37 ° C. Patient samples were prepared by adding 0.5 g / ml IMAB362 fresh to pre-infusion serum samples ( gray bars). HSC: Healthy control pool of human serum spiked with 0.3-10 µg / ml IMAB362 (positive control). Hi: inactivated by the combined heat of human serum enriched with 10 ug / ml IMAB362 (negative control). The number of patients is indicated. Error bars: ± standard deviation
20/217 [0044] Figure 4 shows the ability of patient complement components to interact with IV IMAB362 administered over time. Standardized CDC assays were pertbrmed by adjusting the IMAB362 concentration in each 0.5 µg ml sample, using each patient's serum pre-infusion (dilution factor 10-680fold). The (A / B) CDC assays were performed as described in Figure 3. (C) Each point represents a patient measurement. Open square: 0.5 ug ml IMAB362 in human serum. The P values obtained with the paired t test. Error bars: mean ± standard deviation.
[0045] Figure 5 shows the kinetics of cytotoxicity induced by circulating IMAB362 administered i.v.
[0046] Target NUGC-4 cells, PBMC from a healthy donor (E: T = 40: 1) and patient serum samples (25% v / v) as an antibody and complement source were used in an assay cytotoxicity to measure total integrated cytotoxic activity. From each patient, serum samples were collected 1, 7, 14 and 28-32 days after IMAB362 administration. Patients were treated with increasing doses of IMAB362 (33-1000 mg / m ² ) ^. The concentration of antibody present in the assay is indicated under each bar. HSC: control of human serum spiked with 200.0 pg / ml of fresh IMAB362 (EC80-100). PSC: pre-infusion serum control Patient spiked with fresh 200.0 pg / ml (IMAB362). na: not available.
21/217 [0047] Figure 6 shows the kinetics of the ADCC activity of IMAB362 in the heat-inactivated patient's serum. The test was performed as described in the previous figure, except here the patient's complement was white bar inactivated by heat (56 ° C, 30 minutes) to highlight the ADCC activity (parts of black and gray bars) and to calculate the additive effects of the components of serum (parts).
[0048] Figure 7 shows the CDC activity induced by IMAB362 present in the patient's serum. The assays were performed with CDC CLDN18.2 and CHO-Kl luciferase positive target cells. They were incubated for 80 min with 20% patient serum obtained 1, 7, 14 and 28-32 days after the infusion (v / v) of antibodies. Patients were treated with a dose of IMAB362 33 at 1000 mg / m ² . The concentration of antibody present in each assay is indicated below each bar. HSC: Healthy control of human serum pool enriched with decreasing concentrations of IMAB362 as indicated. PC: positive control (patient pre-infusion of serum enriched with 10 pg / ml IMAB362).
[0049] Figure 8 shows pharmacokinetic results of repeated infusions of IMAB362 in patients. Mean ± SD of concentration in pg / ml) of 1MAB362 in the serum of 4 patients treated with repeated doses of 300 mg / m2 (cohort 1, left Figure) and up to 30 patients (first infusion of 30 patients, fifth infusion 12 patients) treated with doses
22/217 repeated at 600 mg / m ² (cohort 2 and 3 cohort together, figure on the right). Arrows indicate 1MAB362 infusions. First
infusion was administered on day 0. [0050] The figure 9 shows progression free in survival of s patients in the analysis set complete (FAS). [0051] The figure 10 shows progression in
free survival of patients in the set per protocol (PP) (n = 20).
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 here since these may vary. It is also to be understood that the terminology used herein 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 technical and scientific terms used herein have the same meanings as are commonly understood by a person skilled in the art.
[0053] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it must be understood
23/217 that they can be combined in any form and in any number of embodiments to create additional ones. The variously described embodiments of preferred examples are not to be interpreted to limit the present invention to only explicitly described embodiments. This description should be understood to support and cover embodiments that combine the embodiments explicitly described with any number of the described and / or preferred elements. 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 The multilingual glossary of biotechnological terms: (IUPAC Recommendations), Leuenberger HGW, B. Nagel, and H. Kolbl, Eds, Helvética Chimica Acta, CH-4010 Basel, Switzerland., (1995).
[0055] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques, which are explained in the literature in the field (cf., for example, Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, J. Sambrook et al., Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).
[0056] Throughout this descriptive report and the
24/217 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 member, whole or step or group of members, whole or steps, but not the exclusion of any other member, integer or step or group of members, integers or steps, although in some embodiments, such another member, integer or step or group of members, integers or steps may be excluded, ie , the object consists of the inclusion of a declared member, whole or step or group of members, integers 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) are to be understood to cover both the singular and the plural, unless otherwise indicated here or clearly contradicted by the context . Recitation of ranges of values here is intended only to serve as an abbreviated method of referring individually to each separate value that falls within the range. Unless otherwise indicated here, each individual value is incorporated into the specification as if it were described here 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 example language
25/217 (for example, as), provided herein is intended only to better illustrate the invention and is not a limitation on the scope of the otherwise claimed invention. No language in the specification should be understood to indicate any unclaimed element 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 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 a previous invention.
[0058] The term CLDN18 refers to claudin 18 and includes variants, including splice 1 variant claudin 18 (claudin 18.1 (CLDN18.1)) and splice 2 variant claudin 18 (claudin 18.2 (CLDN18.2)).
[0059] The term CLDN18.2 refers, preferably, to human CLDN 18.2, and in particular to a protein which preferably comprises the amino acid sequence according to SEQ ID NO: 1 of the listing sequences or a variant of said amino acid sequence.
26/217 [0060] The term CLDN18.1 refers to CLDN18.1 preferably, human, and, in particular, to a protein that preferably comprises the amino acid sequence according to SEQ ID NO : 2 of the sequence listing or a variant of that amino acid sequence.
[0061] The term variant, according to the invention refers, in particular, to mutants, processing variants, conformations, isoforms, 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 numerous allelic variants of a given gene. A homologous species is an amino acid or nucleic acid sequence with a different species of origin than a given nucleic acid or amino acid sequence. 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 a cancer involving cancer cells expressing CLDN18.2, preferably on the surface of said cancer cells.
[0063] Cell surface is used, according to the
27/217 its normal meaning in the state of the art, and thus includes the outer part of the cell that is accessible for binding proteins and other molecules.
[0064] CLDN18.2 is expressed on the cell surface that is 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 stomach or stomach tissue or even lower. Preferably, CLDN18.2 is not substantially expressed in a cell, if the level of expression is higher than the level of expression in non-cancerous tissue other than stomach by not more than 2 times, preferably 1.5 times, and preferably , do not exceed the level of expression at 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 binding of specific CLDN18.2 antibodies added to the cells.
[0066] According to the invention, CLDN 18.2 is expressed
28/217 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 times or 10,000 times. Preferably, CLDN18.2 is expressed in a cell, if the expression level is higher than 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, in particular the forms of cancer described herein. Any reference in this document to cancer or particular forms of cancer also includes cancer metastasis thereof. In a preferred embodiment, a disease to be treated according to 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 healthy state in a tissue or organ. An increase refers to an increase of at least 10%,
29/217 in particular, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In one embodiment, expression is found only in diseased tissue, whereas 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, preferably cancerous diseases are those in which the cancer cells express CLDN18.2.
[0069] As used herein, a cancer or cancer disease includes a disease characterized by regulated aberrant cell growth, proliferation, differentiation, adhesion and / or migration. The three malignant properties of cancer (uncontrolled growth (division beyond normal limits), invasion (intrusion and destruction of adjacent tissues), and sometimes metastasis (spread to other sites in the body via lymph or blood)) differentiate cancers from tumors benign, which are self-limiting, and do not invade or metastasize. Most cancers form a tumor, but some, like leukemia, do not. Cancerous cell is understood as an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow after the stimuli that initiated new growth cease. Preferably, a cancer disease is characterized by cells that express CLDN18.2 and a cell
30/217 cancerous expressed CLDN18.2. A cell that expresses CLDN 18.2 preferably is a cancer cell, preferably a cancer cell described herein.
[0070] According to the invention, the term tumor or tumor disorder refers to an abnormal growth of cells (called neoplastic cells, tumor cells or tumor cells), preferably forming a swelling or injury. Tumor cells are understood as an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow after the stimuli that initiated new growth cease. Tumors show a partial or complete lack of structural organization and functional coordination with normal tissue, and generally form a precipitate with the tissue, which can be benign, pre-malignant or malignant.
[0071] According to the invention, a tumor is preferably a malignant tumor. Malignant tumor is used for cancer.
[0072] Adenocarcinoma is a cancer that originates in the glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. The skin's epithelial tissue includes glands and a variety of other tissue that lines the body's cavities and organs. Epithelium is embryologically derived from the ectoderm, endoderm and mesoderm. To be classified as adenocarcinoma, the
31/217 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 resemble glandular tissue that are derived from, while poorly differentiated cannot. By staining the cells from a biopsy, a pathologist will 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 secreting the same substance, while 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.
[0073] By metastasis is meant the spread of cancer cells from their original location 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 cavity and
32/217 vessels in the body, and then, after being transported by blood, the target organ infiltration. 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 metastatic potential. In one embodiment, the term metastasis according to the invention refers to metastasis refers to a remote metastasis that 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, which is treatable with the therapy of the invention originates from metastasis of gastric cancer as the primary site. In preferred embodiments, such gastric cancer metastases are Krukenberg tumors, peritoneal metastases and / or lymphatic metastasis.
[0074] 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 an adenocarcinoma signet ring of metastatic cells of the ovary. Stomach is the primary site, in most cases of Krukenberg tumor (70%). Colon, appendix, and breast carcinomas (carcinoma
33/217 mainly lobular invasive) are the next most common primary sites. Rare cases of Krukenberg tumor arising from carcinomas of the gallbladder, biliary tract, pancreas, small intestine, Vater's ampoule, cervix, urinary bladder and / urachus have been reported.
[0075] 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 gastric signet ring carcinomas in young women. Common symptoms are usually related to ovarian involvement, the most common of which are abdominal pain and bloating (mainly, because of the usually bilateral and often large ovarian tumors). The remaining patients have nonspecific gastrointestinal symptoms or are asymptomatic. In addition, Krukenberg's tumor is supposedly associated with virilization resulting from hormone production by ovarian stroma. Ascites is present in 50% of cases and usually reveals malignant cells.
[0076] Krukenberg's tumors are bilateral in more than 80% of reported cases. The ovaries are usually asymmetrically enlarged, with a contoured shape. The surfaces are sectioned yellow or white; they are
34/217 normally solid, although they are occasionally cystic. It is important to note that the capsular surface of the ovaries with Krukenberg tumors is typically smooth and free of adhesions or peritoneal deposits. Note that other metastatic ovarian tumors 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.
[0077] Patients with Krukenberg tumors have an overall mortality rate that is significantly high. Most patients die within 2 years (median survival, 14 months). Several studies show that the prognosis is poor, when the primary tumor is identified after ovarian metastasis is discovered, the prognosis and becomes worse when the primary tumor remains secret.
[0078] By treating is meant administering a compound or composition or a combination of compounds or compositions to a patient in order to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in a 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 has
35/217 currently or who already had an illness; and / or prolong, that is, increase the patient's useful life.
[0079] In particular, the term treatment of a disease includes healing, shortening the duration, improving, preventing, delaying or inhibiting the progression or worsening of, or preventing or delaying the onset of a disease or its symptoms.
[0080] The term patient means, according to the invention, a subject for treatment, in particular, a sick subject, including humans, non-human primates or other animals, namely mammals, such as cows, horses, pigs, sheep, goats, dogs, cats or rodents, such as mice and rats. In a particularly preferred embodiment, a patient is a human being.
[0081] According to the invention, an antibody having the ability to bind to CLDN 18.2 can be administered in combination with, that is, simultaneously with, followed by and / or thereafter, a stabilizing agent or increased expression of the CLDN 18.2.
[0082] The term stabilizing or increasing expression of CLDN 18.2 refers to an agent or combination of agents whose cell supply results in increased RNA and / or CLDN 18.2 protein levels, from preferably in an increase in the levels of CLDN 18.2 protein on the cell surface compared to the situation in
36/217 that 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 CLDN 18.2, 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 an agent or combination of agents whose supply of cells results in a higher density of 18.2 CLDN on the surface of said cells compared to situation where the cells are not supplied with the agent or combination of agents. Stabilization of CLDN 18.2 expression includes, in particular, the situation where the agent or combination of agents prevents a decrease or reduces a decrease in CLDN 18.2 expression, for example, CLDN 18.2 expression would decrease without agent delivery either the combination of agents and disposition of the agent or the combination of agents prevents said decrease or reduces said decrease of 18.2 CLDN expression. Increasing CLDN 18.2 expression includes, in particular, the situation where the agent or combination of agents increases CLDN 18.2 expression, for example, CLDN 18.2 expression would decrease, remain essentially constant, or increase without agent delivery or the combination of agents and agent disposition or combination of agents CLDN18.2 increases expression compared to the situation without the supply of agent or the
37/217 combination of agents so that the resulting expression is greater 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.
[0083] According to the invention, the term stabilizing or increasing 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) DNA damage to cells, so that they can no longer reproduce, (2) inhibit the synthesis of new DNA strands, so that no replication of cells is possible, (3) Cease the cell mitosis processes, so that the cells cannot divide into two cells.
[0084] According to the invention, the term stabilizing or increasing expression agent of CLDN18.2 preferably refers to an agent or a combination of cytostatic agents, such compounds one or a combination of cytostatic compounds to provide that cells, in particular 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 other than the Gl phase, preferably in one or more of the G2 or S phase of the
38/217 cell cycle, such as phases G1 / G2, S / G2, G2 or S of the cell cycle. The term cells 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 of the phases of the cell cycle increases. Each cell goes through a four-phase cycle in order to replicate itself. The first phase called Gl is when the cell prepares to replicate 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 split DNA and RNA duplicates and moves to the separate ends of the cell, the cell, and actually divides into two identical, functional cells. Chemotherapeutic agents that are DNA damaging agents normally 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.
[0085] According to the invention, the term stabilizing or increasing expression agent of CLDN18.2 includes anthracyclines, such as epirubicin, platinum compounds,
39/217 such as oxaliplatin and cisplatin, nucleosides such as 5-fluorouracil or its such as taxanes, docetaxel, and analogs of prodrug analogues, camptothecin, such as irinotecan and topotecan, and 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 5fluorouracil or other drug combinations described herein.
In a preferred embodiment, a stabilizing or increasing expression agent of CLDN18.2 is an immunogenic agent that induces cell death.
In specific circumstances, cancer cells can introduce a lethal stress path 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 that involves so that the CD8 + T cells and IFN-γ signaling of tumor cell death can induce a productive anti-cancer immune response. These signs include
40/217 the pre-apoptotic exposure of the accompanying endoplasmic reticulum (ER) calreticulin (CRT) on the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGBl. 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 ER stress - requires supplementation by tapsigargina, an ER stress inducer.
[0088] According to the invention, the term cell death-inducing immunogenic agent refers to an agent or combination of agents that, when supplied to cells, in particular cancer cells, is capable of inducing cells to introduce a lethal stress pathway that ultimately results in tumor-specific immune responses. In particular, a cell death-inducing immunogenic agent when supplied 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 HMGB protein 1.
[0089] According to the invention, the term cell death-inducing immunogenic agent includes anthracyclines and oxaliplatin.
[0090] 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.
[0091] Anthracyclines preferably bring one or more of the following mechanisms of action: inhibition of the synthesis of I. DNA and RNA by intercalation between base pairs of the DNA / RNA chain, thus preventing the rapid growth replication of cancer cells. 2. Inhibition of the topoisomerase II enzyme, preventing the supercoiled DNA from relaxing and thereby blocking transcription and replication. 3. Creation of free oxygen radicals mediated by iron that damage DNA and cell membranes.
[0092] According to the invention, the term anthracycline preferably refers to an agent, preferably an anti-cancer agent to induce apoptosis, preferably inhibiting the rewiring of DNA into topoisomerase II.
[0093] Preferably, according to the invention, the term anthracycline generally refers to a
42/217 class of compounds having the following ring structure
including analogs and derivatives pharmaceutical salts, hydrates, esters, and pr [0094] daunorubicin epirubicin, valrubicin, o-drugs
Examples include, (daunomycin), idarubicin,
N-trifluor-acetyl conjugates.
analogues but are not doxorubicin rhodomycin, doxorubicin anthracycline and limited to, (adriamycin), pyrarubicin,
14-valerate, aclacinomycin, morpholynorthorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (DOX cyanomorpholine-), 2 pyrroline-doxorubicin (2-PDOX), 5-iminodaunomycin, mitoxantrone and aclacinomycin A (aclaracinomycin). Mitoxantrone is a member of the class of anthracendion compounds that are anthracycline analogues that lack the sugar portion of anthracyclines, but retain the planar polycyclic aromatic ring structure that allows intercalation in DNA.
[0095] Particularly preferred as anthracillin, according to the invention is a compound with the following formula:
43/217
on what
Ri is selected from consists of
H
R2 o
group that and OH, is
selected from between the group what It consists in H and OMe, R3 is selected starting of the group what It consists in H and OH, and R4 is selected starting of the group what It consists in H and OH. [0096] In a embodiment, R1 is H, R2 represents OMe, R3 is H, and R4 is OH . In another embodiment, R1 represents an OH group. R2 is OMe, R3 is H, and R4 is OH. In another
R3
R1 represents an embodiment, represents OMe, is
OH, R2 group OH, and R4 is H.
According to another embodiment, R1 is H, R2 is H, R3 is H, and R4 is OH.
Specifically contemplated as anthracycline in the context of the present invention epirubicin.
epirubicin is an anthracycline medication that has the following formula:
and is commercialized
under the trade name Ellence in the U.S.
44/217 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) -1-methoxy8-methyl-9,10-dihydro-7H-tetracen-5,12-dione. Epirubicin is favored over doxorubicin, the most popular anthracycline, in some chemotherapy regimens, as it appears to cause fewer side effects.
[0098] 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.
[0099] The term cisplatin or cisplatinum refers to the compound cis-diaminodichloroplatin (II) (CDDP) with the following formula:
7, _ρι , .λνη ₃ c <-nh ₃ [00100] The term carboplatin refers to the compound cis-diamine (1,1-cyclobutanedicarboxylate) platinum (II) with the following formula:
^Η, “Χ <>
h ₃ n ^x
45/217 [00101] The term oxaliplatin refers to a compound that is a platinum compound that is complexed with a diaminocyclohexane carrier linker the following formula:
[00102] In particular, the term oxaliplatin refers to the compound [(IR, 2R) -cyclohexane-1,2-diamine] (ethanedioate-0,0 ') platinum (II). Injectable oxaliplatin is also marketed under the brand name Eloxatine.
[00103] The term nucleoside analog refers to a structural analog of a nucleoside, a category that includes both purine analogs and pyrimidine analogs. In particular, the term nucleoside analog refers to derivatives of fluoropyrimidine which includes fluorouracil and its prodrugs.
[00104] The term fluorouracil or 5-fluorouracil (5FU or f5U) (sold under the brand names Adrucil, Carac, Efudix, Eftidex and Fluoroplex) is a compound that is a pyrimidine analog with the following formula:
[00105] In particular, the term refers to the compound
46/217
5-fluoro-1 H-pyrimidine-2,4-dione.
[00106] The term capecitabine (Xeloda, Roche) refers to a chemotherapeutic agent that is a prodrug that is converted to 5-FU in tissues. Capecitabine that can be administered orally has the following formula:
[00107] In particular, the term refers to the compound pentyl [1- 3,4-dihydroxy-5-methyl-2-yl) -5-fluoro-2-oxo-1 Hpirimidin-4-yl] carbamate.
[00108] Taxanes constitute a class of diterpene compounds, which were derived from natural sources in the first place, such as plants of the genus Taxus, but some were 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).
[00109] According to the invention, the term docetaxel refers to a compound having the following formula:
47/217 refers to a compound camptothecin (CPT;
indolizine [l
According to the
CH3 invention, the term paclitaxel compound having the following formula:
According to (S) according to the invention, the term camptothecin derivative analog of
-4-ethyl-4-hydroxy-1H-pyran [3 ', 4': 6,7], 2-b] quinoline-3, 14- (4H, 12H) -dione).
Preferably, the term camptothecin analog refers to compounds that comprise the following structure:
, preferred camptothecin analogs are inhibitors of the enzyme of
DNA topoisomerase
I (top I).
Preferred camptothecin analogs
48/217 according to the invention are irinotecan and topotecan.
[00113] 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:
Ha [00114] In particular, the term irinotecan refers to the compound (S) -4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy3,14-dioxolH-pyran [3 ', 4': 6.7] -indolizino [1,2-b] quinolin-9-yl- [1,4'bipiperidine] -''-carboxylate.
[00115] Topotecan is a topoisomerase inhibitor of the formula:
HaC
N
H3C
HO [00116] 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 monohydrochloride.
[00117] According to the invention, a stabilizing or increasing agent of CLDN18.2 expression can be a chemotherapeutic agent, in particular, a chemotherapeutic agent in the treatment of established cancer and can
49/217 be part of a combination of drugs, such as a combination of drugs established for use in cancer treatment. 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, chemotherapy FOLFOX, chemotherapy Folfiri, chemotherapy FLOT and chemotherapy DCF.
[00118] The combination of drugs used in EOX chemotherapy comprises 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 comprises epirubicin, cisplatin and capecitabine. The combination of drugs used in EOF chemotherapy comprises epirubicin, oxaliplatin and 5-fluorouracil.
[00119] Epirubicin is usually administered at a dose of 50 mg / m ² , cisplatin 60 mg / m ² , oxaliplatin 130 mg / m ² , prolonged venous infusion of 5-fluorouracil, 200 mg / m ² / day and for oral capecitabine 625 mg / m ² twice daily for a total of eight cycles of 3 weeks.
[00120] The combination of drugs used in the
50/217 FLO chemotherapy comprises 5-fluorouracil, folinic acid and oxaliplatin (usually 5-fluorouracil of 2,600 mg / m ² for 24 hours of infusion, folinic acid 200 mg / m ² and oxaliplatin of 85 mg / m ² , each 2 weeks).
[00121] FOLFOX is a chemotherapy regimen composed of folinic acid (leucovorin), 5-fluorouracil and oxaliplatin. The recommended dose regimen given every two weeks is as follows: Day 1: Oxaliplatin 85 mg / m ² IV infusion and leucovorin 200 mg / m ² IV infusion, followed by 5-FU 400 tng / m ² IV bolus, followed by 5-FU 600 mg / m ² IV infusion as a 22-hour continuous infusion; 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.
[00122] The combination of drugs used in Folfiri chemotherapy comprises of 5-fluorouracil, leucovorin and irinotecan.
[00123] THE combination in drugs used at chemotherapy DCF comprises in docetaxel, cisplatin and 5- fluorouracil. [00124] THE combination in drugs used at chemotherapy FLOT comprises in docetaxel, oxaliplatin, 5-
fluorouracil and folinic acid.
[00125] The term folinic acid or leucovorin is used
51/217 refers to a compound useful in synergistic combination with the chemotherapy agent 5-fluorouracil folinic acid has the following formula:
[00126]
In particular
the term refers to the acid compound (2S) -2 - {[4 - [(2-amino-5-formyl-4-oxo-5,6,7,8 tetrahydro-1H-pteridine-6-yl) methylamino] benzoyl] amino} pentanedioic.
[00127] According to the invention, an antibody having the ability to bind to CLDN18.2 can be administered in combination with, that is, simultaneously with, followed by and / or thereafter, a γδ T cell stimulating agent.
[00128] 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 compound with two glycoprotein chains called A- and β-TCR chains. In contrast, in γδ T cells, the TCR consists of a γ chain and a δ chain. This group of T cells is generally much less common than T cells. Γδ human T cells play
52/217 an important role in the response to stress-surveillance, such as infectious diseases and autoimmunity. Changes induced by transformation into tumors are also suggested to elicit γδ T-mediated stress-response responses and increase anti-tumor immunity. Importantly, after coupling the antigen, γδ-activated T cells at lesional sites provide cytokines (eg INFY, TNFa) and / or chemokines that mediate the recruitment of other effector cells and show immediate effector functions such as cytotoxicity (via receptor death and cytolytic granules pathways) and ADCC.
[00129] Most γδ T cells in peripheral blood express the T cell receptor Vy9V82 (TCRy0). Vy9V52 T cells are unique to humans and primates and are assumed to play an early and essential role in detecting danger by invading pathogens as they expand dramatically in many acute infections and can be superior to all other lymphocytes within a few days , for example, in tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis and malaria. 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
53/217 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 aminobiphosphonates therapeutically, which inhibit the mevalonate enzyme fRNAesyl pyrophosphate synthase (FPP) pathway. 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. Through treatment of PBMC in vitro, ZA is taken especially by monocytes. IPP accumulates in monocytes and they differentiate for antigen presenting cells to 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 γδ activated T cells. Finally, certain alkylated amines have been described to activate Vγ9Vδ2 T cells in vitro, however, only in millimolar concentrations.
[00130] According to the invention, the term stimulating agent γδ T cells refers to compounds that stimulate the development of γδ T cells, in particular Vγ9Vδ2 T cells, in vitro and / or in vivo, in particular, through induction activation and expansion of γδ T cells. Preferably, the term refers to compounds that, in
54/217
vitro and / or in vivo, increased isopentenyl pyrophosphate
(PIP) produced in mammalian cells, preferably by inhibiting the fevnaesyl pyrophosphate synthase (FPP) mevalonate pathway.
[00131] A particular group of compounds that stimulate the cells T γδ are bisphosphonates, in particular, bisphosphonates containing nitrogen (N-bisphosphonates;
aminobiphosphonates).
[00132] 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:
[00133] [l-hydroxy-2- (l-yl-lH-imidazole) ethane-l, l-
diyl] bis (phosphonic acid), zoledronic acid, for example, zoledronate;
[00134] Phosphonic acid (dichloro-phosphono-methyl), for example, clodronate (1-hydroxy-3- [methyl (pentyl) amino] propane-1,1-di-yl} bis (phosphonic acid), ibandronic acid, for example, ibandronate (3-amino-lhydroxypropane-l, l-diyl) bis (phosphonic acid), pamidronic acid, for example, pamidronate: (1-hydroxy-l-phosphono-
2-pyridin-3-yl-ethyl) phosphonic acid, risedronic acid, for example, risedronate; (L-Hydroxy-2-imide2o [1,2-a] pyridin-
3-yl-1-phosphonoethyl) phosphonic, minodronic acid; [3 (dimethylamino) -1-hydroxypropane-1,1-di-yl] bis (acid
55/217 phosphonic), olpadronic acid.
[00135] [4-amino-1-hydroxy-1- (hydroxy-oxido-phosphoryl) butyl] phosphonic, alendronic acid, for example alendronate;
[00136] [(Cycloheptylamino) methylene] bis (phosphonic acid), incadronic acid;
[00137] (1-hydroxyethane-1,1-di-yl) bis (phosphonic acid), etidronic acid, for example etidronate; and [00138] [(4-chlorophenyl) thio] methylene} bis (phosphonic acid), tiludronic acid.
[00139] 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 patients with cancers such as multiple myeloma and prostate cancer, as well as for the treatment of osteoporosis. It can also be used to treat malignant hypercalcemia and can be useful for treating pain from bone metastases.
[00140] 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
56/217 to treat malignant hypercalcemia and may be useful for the treatment of bone metastasis pain.
[00141] In a particularly preferred embodiment, a γδ T cell stimulating agent according to the invention is administered in combination with IL-2. This combination proved to be particularly effective in mediating expansion and activation of γ9δ2 T cells.
[00142] Interleukin-2 (IL-2) is an interleukin, a 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 the discrimination between external (non-self) and self. IL-2 mediates its effects by binding to IL-2 receptors, which are expressed by lymphocytes.
[00143] 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 natural IL-2.
[00144] According to the invention, the term antiemetic refers to a compound, composition or medication that is effective against vomiting and / or nausea. In one embodiment, the anti-emetic includes a 5-HT3 receptor antagonist and / or a
57/217 neuroquinin 1 (NK1).
[00145] 5-HT3 receptor antagonists block serotonin receptors in the central nervous system and gastrointestinal tract. Examples of these include, but are not limited to, ondansetron (Zofran) which can be administered in an oral tablet form, oral dissolution tablet form, or an injection, Dolasetron (Anzemet) which can be administered in tablet form or as an an injection, Granisetron (Kytril, Sancuso) which can be administered in the form of a tablet (Kytril), oral solution (Kytril), injection (Kytril), or in a transdermal patch for the upper arm (Sancuso), Tropisetron (Navoban ) that can be administered in capsules orally or as an injection, Palonosetron (Aloxi) that can be administered as an injection or in oral capsules and mirtazapine (Remeron).
[00146] NKL receptor antagonists include, but are not limited to aprepitant (Amendment).
[00147] A preferred combination of a 5-HT3 receptor antagonist and an NKL receptor antagonist is a combination of ondansetron (Zofran) and aprepitant (Amendment).
[00148] Additional antiemetics that can be used according to the invention, in particular in combination with a 5-HT 3 receptor antagonist and / or an NKL receptor antagonist include, but are not limited to, metoclopramide (Reglan) acting about the GI tract as a
58/217 prokinetic, Lorazepam, Atropine, alizapride (Litican, Plitican, Superan, Vergentan) and dimenhydrinate (Dramamine, Driminate, Gravol, Gravamin, Vomex, Vertirosan).
[00149] According to the invention, an antispasmodic (synonym: spasmolytic) can be administered. According to the invention, the term antispasmodic refers to a compound, composition or medication that suppresses muscle spasms. Preferably, an antispasmodic is useful for contraction of smooth muscle. Preferred according to the invention are antispasmodics that are effective in treating spasmodic activity in the digestive system. Thus, preferred antispasmodics are effective in relieving gastrointestinal spasms.
[00150] Antispasmodics include, but are not limited to, scopolamine butylbromide which is also known as scopolamine butylbromide, butylhyoscine and butylbromide hyoscine. It is marketed under the trade name Buscopan by Boehringer Ingelheim GmbH, Germany.
[00151] According to the invention, a parasympatholytic can be administered. According to the invention, the term parasympatholytic refers to a compound, composition or medication that reduces the activity of the parasympathetic nervous system. Parasympatholytics include, but are not limited to, atropine.
[00152] According to the invention, the term inhibitor of
59/217 proton pump refers to a compound, composition or medication whose main action is a pronounced and prolonged reduction in the production of gastric acid.
[00153] Proton pump inhibitors include benzimidazole derivatives and imidazopyridine derivatives. Examples of proton pump inhibitors include, but are not limited to, Omeprazole (brand names: Gasec, Losec, Prilosec, Zegerid, ocid, Lomac, Omepral, omez,), lansoprazole (brand names: Prevacid, Zoton, Monolifum , Inhibitol, Levant, Lupizole), dexlansoprazole (trade name: apidex, Dexilant), Esomeprazole (brand names: Nexium, Esotrex, esso), pantoprazole (brand names: Protonix, Somac, Pantoloc, Pantozole, Zentro, ZENTRO, Pan , Control, TECTA), Rabeprazole (brand names: ACIPHEX, Pariet, Erraz, Zechin, Rabecid, Nzole-D, Rabeloc, Razo) and Haprazol (brand names: Ilapro, Lupilla, Adiza).
[00154] According to the invention, other compounds, compositions or medicaments can be administered, which have a protective effect on the gastric mucosa, in particular, if a non-steroidal anti-inflammatory drug (NSAID) is administered.
[00155] For example, other compounds, compositions or medications can be administered to avoid the common adverse effect of NSAIDs gastric ulceration, in particular to prevent NSAID-induced gastric ulcers. In a
60/217 embodiment, misoprostol can be administered, which is a synthetic analogue of prostaglandin E1 (PGE1) that is used for the prevention of NSAID-induced gastric ulcers. Misoprostol acts on gastric parietal cells, inhibiting gastric acid secretion coupled to G protein receptors mediated by inhibition of adenylathocyclase, which leads to a decrease in levels of intracellular cyclic AMP and decreased activity of the proton pump on the surface apical of the parietal cell.
[00156] In addition, omeprazole has proven to be at least as effective as misoprostol in treating NSAID-induced ulcers, but significantly better tolerated.
[00157] Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of drugs that provide analgesic and antipyretic effects (fever reduction), and, in higher doses, anti-inflammatory effects. The term non-steroid distinguishes these drugs from steroids. The most prominent members of this group of drugs are aspirin, ibuprofen and naproxen.
[00158] One of the main adverse reactions (ADRs) associated with NSAIDS relates to the effect of the gastrointestinal tract (GI) of NSAIDS. These effects are in many cases severe enough to represent the risk of ulcer perforation and upper gastrointestinal bleeding. NSAID patients experience dyspepsia, adverse events
61/217 upper gastrointestinal associated with NSAIDS, irritation of gastrointestinal ulceration (GI) and GI. NSAIDs cause a double attack on the GI tract: the acid molecules directly irritate the gastric mucosa, and the inhibition of COX-1 and COX-2 reduces levels of protective prostaglandins. The inhibition of prostaglandin synthesis in the GI tract causes increased gastric acid secretion, decreased bicarbonate secretion, reduced mucus secretion and decreased trophic effects on the epithelial mucosa. Thus, NSAIDs are preferably not administered according to the invention. Paracetamol or acetaminophen, which is not classified as an NSAID as it only has weak anti-inflammatory effects can be administered as analgesic agents according to the invention, however, it may not be effective for pain management and, thus, the administration of a NSAIDs may become necessary, in particular to avoid administration of opioids.
[00159] Commonly, adverse gastric effects (but not necessarily intestinal) can be reduced by means of suppressing acid production, by concomitant use of a proton pump inhibitor, for example, omeprazole, esomeprazole; or the prostaglandin analogue misoprostol.
[00160] 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
62/217 addressed. In a preferred embodiment, an antigen is a tumor-associated antigen, 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 preferably produced in large quantities, intracellularly or as cancer cell surface antigens.
[00161] 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 are expressed or expressed aberrantly in one or more of the tumor 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.
[00162] The term epitope refers to an antigenic determinant of a molecule, that is, to 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 chains
63/217 sides of sugars, and generally have specific three-dimensional structural characteristics, as well as specific load characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former, but not the latter, is lost in the presence of denaturing solvents. An epitope of a 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 preference 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24 or 25 amino acid gone on length. [00163] O term antibody if refers to a
glycoprotein comprising at least two (H) and two light heavy chains (L) inter-chains linked by disulfide bridges, and includes any molecule comprising an antigen-binding portion thereof. The term antibody includes monoclonal antibodies and their 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 fragments Fab and Fab 'and also includes all recombinant forms of antibodies,
64/217 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
constant region in heavy chain. Each chain Light is consisting of an variable region light chain ( on here abbreviated as VL ) and a chain constant region Light . At
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 VP 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 antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
[00164] 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 immunoglobulin sequences of the
65/217 human germline. The human antibodies described herein can include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random mutagenesis, either site specific in vitro or by somatic mutation in vivo).
[00165] 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 may include either the complete variable domains fused to the constant domains or only the complementarity determining regions (CDR) grafted into appropriate 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 from the mouse antibody). Other forms have one or more CDRs that are altered with respect to the original antibody.
66/217 [00166] The term chimeric antibody refers to these antibodies, where a portion of each of the amino acid sequences of the light and heavy chains is homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class corresponding, while the rest of the chain is homologous to the corresponding sequences in another corresponding. 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 of such chimeric forms is that the variable region can conveniently be derived from presently known sources using readily available B cells or hybridomas 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 not affected 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.
67/217 [00167] The terms antigen binding portion of an antibody or simply 9 ”binding portion”) or antigen binding fragment of an antibody (or simply binding fragment) or antigen binding portion refers to one or more fragments of an antibody retaining 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 fragments covered by the term antigen-binding portion of an antibody include (i) Fab fragments, monovalent fragments consisting of the 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) isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs that can optionally be joined by a synthetic linker. Furthermore, 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
68/217 single protein chain, where the VL and VH regions pair 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 a domain comprising (i) a polypeptide from the binding domain that is fused to a polypeptide from the immunoglobulin chRNA region, (ii) from the CH2 heavy immunoglobulin chain from the constant region fused to the chRNAeira region, and (iii) a CH3 immunoglobulin from the heavy chain from the constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. The immunoglobulin fusion proteins that bind to the domain are further disclosed in US 2003/01 18592 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.
[00168] The term bispecific molecule is intended to include any agent, for example, a protein, a peptide or a complex protein or peptide, which has
69/217 two different connection specificities. 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, tetraspecitic, 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 (1994) Structure 2 : 1121 - 1123).
70/217 [00169] An antibody can be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., 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, mitramycin, actinomycin D, 1, dehydrochloride, 1H , procaine, 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 (e.g., methotrexate, 6mercaptopurine, 6-thioguanine, cytarabine, fludarabin, decarbazine 5-fluorouracil), alkylating agents (e.g. mecloretamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin (formerly, anthracyclines) ) and doxorubicin), antibiotics (for example, dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC), and anti-mitotic agents (for example,
71/217 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 the preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A.
[00170] Antibodies can also be conjugated to a radioisotope, for example, iodine-131, yttrium-90 or indium111 to generate cytotoxic radiopharmaceuticals.
[00171] 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 DAIG portion can be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or an active fragment thereof, 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, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), colonies of stimulating factor granulocyte macrophages (GM- Colony stimulating factor
72/217 granulocytes (G-CSF), or other growth factors.
[00172] Techniques for conjugating such therapeutic portion of antibodies 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 (Maree Dekker, Inc., 1987) pp .; Thorpe, Antibody Carriers of cytotoxic agents in cancer therapy: a review of monoclonal antibodies '84: Clinical and biological applications, Pincheraet al. (eds.), pp 475-506 (1985); The analysis, results and foreseeable future of the therapeutic use of Radiolabeled Antibody in cancer therapy, in monoclonal antibodies for Cancer Detection and therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., The preparation and properties of the antibody-toxin conjugated cytotoxic, Immunol. Rev., 62: 11-19-58 (1982).
[00173] As used herein, an antibody is derived from a germline sequence in particular if 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
73/217 minus 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 particular germline sequence will 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 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
[00174] As used herein, the term heteroantibodies refers to two or more antibodies, their derivatives, or the 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.
[00175] The antibodies described herein can be monoclonal antibodies. The term monoclonal antibody, as used herein, refers to a preparation of antibody molecules of simple 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
74/217 obtained from a non-human animal, for example, mouse, fused with an immortalized cell.
[00176] 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 (e.g., a mouse) that is transgenic or transchromosomal with respect to for immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectome, (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 from other DNA sequences.
[00177] The antibodies described herein can be derived from different species, including, but not limited to, mouse, rat, rabbit, guinea pig and human.
[00178] The antibodies described herein include monoclonal and polyclonal antibodies and include, such as IgA1 or igAl antibodies, IgGl, IgG2, IgG3, IgG4, IgE, IgM and IgD. In several embodiments, the antibody is an antibody of
75/217
IgGl, more particularly, an IgGl, kappa isotype or IgGl, lambda isotype (i.e., IgGl, κ, λ), an IgG2a antibody (for example, IgG2a, κ, λ), an IgG2b antibody (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.
[00180] 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 does not consist of the transgenic organism, and is generally derived from species other than the transgenic organism.
[00181] As used herein, a heterohybrid 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 heterohybrid antibody.
[00182] The invention includes all antibodies and
76/217 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.
[00183] 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 are specifically bind 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, from other species (for example, homologous CLDN18.2 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 that have different specificities and be combined into a well-defined composition or mixture.
[00184] The term connection according to the invention, of
77/217 preference, refers to a specific connection.
[00185] 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.
[00186] An antibody is (substantially) unable to bind to a target that does not have significant affinity for said target and does not bind significantly, in particular, it cannot detectably bind to said target in standard tests. Preferably, the antibody will not detectably bind to 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 greater. 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, 10 ³ times of, 10 ⁴ times of, 10 ⁵ times of, or 10 ⁶ times more
78/217 higher than the 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.
[00187] An antibody is specific for a predetermined target, if it is able to bind to that predetermined target, although it is not able to bind to other targets, that is, it has no significant affinity for the other objectives and does not bind significantly to other targets in conventional assays. According to the invention, an antibody is specific to CLDN18.2 if it is able to bind to 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 binding to unrelated proteins or CLDN18.2, such as bovine serum albumin (BSA), casein, human serum albumin (HSA) or non-membrane transmembrane proteins, such as transferrin or receptor MHC molecules or any other specified polypeptide. Preferably, an antibody is specific to a predetermined target, it binds to that target with a
79/217
Kd which is at least 10 times, 100 times, 10 ³ times of, 10 ⁴ times of, 10 ⁵ times of, or 10 ⁶ times of less than 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 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M, or 10 ^-11 M.
[00188] The binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., Antibody Antigen Interactions In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the methods described here. Affinities can be easily determined using conventional techniques, such as by equilibrium dialysis; using the BIAcore 2000 instrument, using general procedures defined by the manufacturer; by radioimmunoassay using the radiolabeled target antigen; or by any other 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. ScL, 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, affinity measurements and other parameters
80/217 antigen binding, for example, KD, IC50, are preferably made with standardized antibody and antigen solutions, and a standardized buffer.
[00189] 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.
[00190] 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.
[00191] The term naturally occurring, as used here, as applied to an object refers to 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.
[00192] The term rearranged as used herein refers to an immunoglobulin heavy or light chain configuration in which a segment V chain locus is positioned immediately adjacent to a D or J coding segment or segment essentially a full VH or VL domain , respectively. A rearranged immunoglobulin (antibody) locus of genes can be identified by comparison
81/217 with the germline DNA; a rearranged locus will have at least one recombined heptamer / nonomer homology element.
[00193] The term or configuration of the non-rearranged germline, 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.
[00194] 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 an epitope located within the extracellular domains of CLDN18.2, in particular the first extracellular domain, preferably amino acid 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) a non-glycosylated epitope located on CLDN18. 2loopD3, preferably SEQ ID NO: 9.
82/217 [00195] According to the invention an antibody that has the ability to bind CLDN18.2, preferably is an antibody that has the ability to bind 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 is preferably 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 CLDN18.2 binds 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 comprising 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 to express said protein or peptide. Preferably, the antibody binds to cells
83/217 of cancer, in particular cells of the types of cancer mentioned above, and preferably does not bind substantially to non-cancer cells.
[00196] Preferably, binding of an antibody that has the ability to bind to cells that express CLDN18.2 CLDN18.2 induces or mediates the death of cells that express CLDN18.2. The cells that express CLDN18.2 are preferably cancer cells and are, in particular, selected from the group consisting of gastric, oesophageal, pancreatic, lung, ovary, colon, liver, head-neck, and head cells, of gallbladder cancer. Preferably, antibodies induce or mediate cell death by inducing one or more complement-dependent cytotoxicity-mediated lysis (CDC), antibody-dependent cell cytotoxicity-mediated lysis (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, NK and PMN cells. The inhibition of cell proliferation can be measured in vitro by determining the proliferation of cells in an assay using bromodeoxyuridine (5-bromo-2-deoxyuridine, from BrdU). BrdU is a synthetic nucleoside that is an analogue of thymidine and can
84/217 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.
[00197] In preferred embodiments, the antibodies described herein can be characterized by one or more of the 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.
[00198] In a particularly preferred embodiment, an antibody that has the ability to bind to CLDN18.2 is produced by a hybridoma deposited in the DSMZ (Mascheroder Weg lb, 31824 Braunschweig, Germany; new
85/217 address: Inhoffenstr. 7B, 31824 Braunschweig, Germany) and
with the following designation number and access: ACC2737 The. 182-D1 106-055, number of access DSM filed on 19 October 2005 B. 182-D1 106-056, number of access DSM ACC2738 filed on 19 October 2005 ç. 182-D 1 106-057, number of access DSM ACC2739 filed on 19 October 2005 d. 182-D1 106-058, number of access DSM ACC2740 filed on 19 October 2005 and. 182-D1 106-059, number of access DSM ACC2741 filed on 19 October 2005 f. 182-D1 106-062, number of access DSM ACC2742 filed on 19 October 2005, g. 182-D 1106-067, number of access DSM ACC2743 filed on 19 October 2005 H. 182-D75 8-035, number of access DSM ACC2745 deposited on 17 November 2005 I. 182-D75 8-036, number of access DSM ACC2746 deposited on 17 November 2005 j. 182-D75 8-040, number of access DSM ACC2747 deposited on 17 November 2005 k. 182-D 1 106-061, number of access DSM ACC2748 deposited on 17 November 2005 1. 182-D1 106-279, number of access DSM ACC2808
86/217
filed on 26 October 2006 m. 182-D1 106-294, DSM accession number ACC2809, filed on 26 October 2006, n. 182-D1 106-362, DSM accession number ACC2810, filed on 26 October 2006.
Preferred antibodies according to the invention are those produced by, and obtained from, the hybridomas described above; that is, 37G1 1 in the case of 182-D1 106-055, 37H8 in the case of 182-D1106-056, 38G5 in the case of
182-D1 106-057, 38H3, in the case of 182-D1 106-058, 39F1 1 in the case of 182-D1 106-059, 1 1 43 A, in the case of D-182 1 106-062, 61C2, in the case of 182-D1 106-067, 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 D-182 1 106-061 , 125E1 in the case of D-182 1 106-279, 163E12 in the case of D-1 182 106-294, and 175D10 in the case of 182-dl 106-362; and the chimerized and humanized forms of these.
[00200] Preferred Chimerized Antibodies and their sequences are shown in the following table:
clone mAb Isotype Variable region 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: 17
87/217
45C1 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: 22166E2 182-D1106-308 IgK SEQ ID NO: 40 SEQ ID NO: 25175D10 182-D1106-362 IgK SEQ ID NO: 39 SEQ ID NO: 2445C1 182-D758-187 IgK SEQ ID NO: 38 SEQ ID NO: 2345C1 182-D758-187 IgK SEQ ID NO: 41 SEQ ID NO: 2645C1 182-D758-187 IgK SEQ ID NO: 42 SEQ ID NO: 2745C1 182-D758-187 IgK SEQ ID NO: 43 SEQ ID NO: 28
[00201] In preferred embodiments, antibodies, in particular, chimerized forms of 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, 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 the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof. In a particularly preferred embodiment, the antibodies, in particular, chimerized forms of antibodies according to the
The invention includes antibodies comprising a CH comprising an amino acid sequence derived from a human CH, such as the amino acid sequence represented by SEQ ID NO: 13 or a fragment thereof and comprising a CL comprising a sequence of amino acids derived from a human CL, such as the amino acid sequence represented by SEQ ID NO: 12 or a fragment thereof.
[00202] 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, human IgGl constant region, Glm allotype (3).
[00203] In certain preferred embodiments, chimerized forms of 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 which comprise 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 a fragment thereof.
[00204] In certain preferred embodiments, chimerized forms of antibodies include antibodies comprising
89/217 a combination of heavy chains and light chains selected from the following possibilities: (i) to (ix):
(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
90/217
amino acids represented by SEQ ID AT THE: 2 4 or one your 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: 26 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 AT THE: 27 or one your fragment, 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: 2 8 or one your
fragment.
[00205] The antibody according to (v) is particularly preferred.
[00206]
Fragment or fragment of a sequence of
91/217 amino acids as used above, refers to a part of an antibody sequence, that is, a sequence that represents the antibody sequence shortened at the Ne / or C- terminus, which when replacing said antibody an antibody sequence 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 of said sequence amino acids. 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, and 28, of a preferred mode relates to wherein said amino acid sequence 17, 18, 19, 20, 21, 22 or 23 at the N-terminus is removed.
[00207] 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 a fragment thereof.
[00208] In a preferred embodiment, an antibody that has the ability to bind to CLDN18.2 comprises a
92/217 light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, and one thereof fragment.
[00209] 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):
[001] 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, [002] VH comprises a sequence of amino acids represented by SEQ ID NO: 30 or a fragment thereof and the VL comprises an amino acid sequence represented by SEQ ID NO: 35 or a fragment thereof, [003] 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, [004] the VH comprises an amino acid sequence represented by SEQ ID NO: 33 or a fragment thereof and the VL comprises a sequence of amino acids represented by SEQ ID NO: 40 or a fragment thereof,
93/217 [005] VH comprises an amino acid sequence 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, [006] 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, [007] 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, [008] 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: 42 or a fragment thereof, [009] VH comprises an amino acid sequence represented by SEQ ID NO: 34 or a fragment thereof and VL comprises an amino acid sequence represented a by SEQ ID NO: 43 or a fragment thereof.
[00210] 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 ):
94/217
(i): CDR1: positions 45 - 52 of SEQ ID NO: 14, CDR2: positions 70 - 77 of SEQ ID AT THE: 14, CDR3: positions 116 - 125 of SEQ ID NO: 14 ^, (ii) CDR1: positions 45 - 52 of SEQ ID NO: 15, CDR2: positions 70 - 77 of SEQ ID AT THE: 15, CDR3: positions 116 - 126 of SEQ ID NO: 15 ^, (iii ) CDR1: positions 45 - 52 of SEQ ID NO: 16, CDR2: positions 70 - 77 of SEQ ID AT THE: 16, CDR3: positions 116 - 124 of SEQ ID NO: 16 ^, (iv) CDR1: positions 45 - 52 of SEQ ID NO: 17, CDR2: positions 70 - 77 of SEQ ID AT THE: 17, CDR3: positions 116 - 126 of SEQ ID NO: 17 ^, (v): CDR1 positions 4 4 - 51 of SEQ ID NO: 18, CDR2: positions 69 - 76 of SEQ ID AT THE: 18, CDR3: positions 115 - 125 of SEQ ID NO: 18 , and (saw) : CDR1 positions 45 - 53 of SEQ ID NO: 19, CDR2: positions 71 - 78 of SEQ ID AT THE: 19, CDR3: positions 117 - 128
SEQ ID NO: 19.
[00211] 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:
positions 76 - 78 of SEQ ID NO: 20, CDR3: positions 115 - 123
95/217 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: positions 110 - 118 of 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 of SEQ ID NO: 22, (iv) 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, (v): 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): CDR1 positions 47 - 58 of SEQ ID NO: 25, CDR2: positions 76 - 78 of SEQ ID NO: 25, CDR3: positions 115 - 122 of 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 1 15-123 of SEQ ID NO: 26, (viii) 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 (ix) CDR1: positions es 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.
96/217 [00212] 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 CDR1, CDR2 and CDR3 selected from of 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 45 - 52 of SEQ ID NO: 15, CDR2: positions 70 - 77 of SEQ ID NO: 15, CDR3: positions 116 - 126 of SEQ ID NO: 15, VL: CDR1: positions 47 - 58 of SEQ ID NO: 20, CDR2: positions 76-78 of SEQ ID NO: 20, CDR3: positions 115
- 123 of SEQ ID NO: 20, (iii) VH: CDR1: positions 45 - 52 of SEQ ID NO: 16, CDR2: positions 70 - 77 of SEQ ID NO: 16, CDR3: positions 116 - 124 of SEQ ID NO : 16, VL: CDR1: 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: positions 69-76 of SEQ ID NO: 18, CDR3: positions 115-125 of 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
97/217 of 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 115123 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 115123 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: posi tions 76 - 78 from SEQ ID NO: 27, CDR3: positions 115 - 123 from SEQ ID NO: 27, and [00213] (ix) VH: CDR1: positions 45 - 53 from 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 of SEQ ID NO: 28, CDR3:
98/217 positions 109-117 of SEQ ID NO: 28.
[00214] 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 the CDR3 region heavy chain variable (VH) and / or variable light chain (VL) region 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 the light chain variable regions (VL) described herein. In one embodiment, said one or more of the complementarity determining regions (CDRs) are selected from a set of CDR1, CDR2 and CDR3 complementarity determining regions described herein. In a particularly preferred embodiment, an antibody that has the ability to bind to CLDN18.2 preferably comprises the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or the light chain variable region (VL) of a monoclonal antibody against CLDN18.2, preferably of a monoclonal antibody against CLDN18.2 described herein, and preferably
99/217 comprises the complementarity determining regions
CDR1, CDR2 and CDR3 of variable regions of the chain heavy (VH) and / or the regions light chain variables (VL) on here descr ito.[00215] In a realization of a antibody
comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs, together with their intervening structural regions. Preferably, the portion will also include at least about 50% of any one or both of the first and fourth structural regions, at 50%, with the C-terminal being 50% of the first structural region and the N-terminal being 50% fourth region. Construction of antibodies prepared by recombinant DNA techniques can result in the introduction of N- or C-terminal residues to 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., in the production of diabodies) or protein markers.
[00216] In one embodiment of an antibody comprising one or more CDR, a set of CDRs or a combination of CDR sets, as described herein comprises said CDR in an antibody structure
100/217 human.
[001] Reference made in this document to an antibody comprising with respect to the heavy chain of it of a specific chain or of a particular region or sequence preferably refers to the situation where all heavy chains of said specific antibody of the chain comprise said, region or sequence. This applies correspondingly to the light chain of an antibody. Reference made in this specification to an antibody comprising with respect to the heavy chain of a specific chain or a particular region or sequence preferably refers to the situation where all heavy chains of said specific chain antibody comprise said, region or sequence. This applies correspondingly to the light chain of an antibody.
[002] 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.
[003] 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
101/217 stably.
[004] The teaching given here with respect to specific amino acid sequences, for example, those shown in the SEQuências list, is to be interpreted so as to also refer to variants of said specific sequences that result in sequences that are functionally equivalent to said specific sequences, for example, amino acid sequences that exhibit 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 maintain 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 CLDN18.2 and preferably functions of said antibody, as described herein , for example, CDC or ADCC mediated lysis mediated lysis.
[005] It will be appreciated by a technician versed in the subject that, in particular, the sequences of the CDR, hypervariable and variable regions can be modified without losing the ability to link CLDN18.2. For example, CDR regions will be identical or highly homologous to the antibody regions specified herein. By highly homologous it is considered 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
Furthermore, the hypervariable and variable regions can be modified so that they show substantial homology to the antibody regions specifically described herein.
[006] For the purposes of the present invention, variants of an amino acid sequence comprise amino acid 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 variants of C-terminal and / or N-terminal truncation.
[007] Amino acid insertion variants comprise single or two or more amino acid insertions in a particular amino acid sequence. In the case of variants of the SEQ of amino acids having an insertion, one or more amino acid residues are inserted in a particular location in an amino acid sequence, although the insertion at random, with appropriate screening of the resulting product is also possible.
[008] Amino acid addition variants 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. [009] Variants in deletion of amino acids are
characterized by the removal of one or more amino acids from the
103/217 sequence, such as by removing 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can be in any position of the protein.
[0010] 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 the replacement of amino acids with others that have similar properties. Preferably, the amino acid changes in variant proteins are conservative amino acid changes, i.e., similar loaded 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), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.
104/217 [00217] Preferably, the degree of similarity, preferably of identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for a region of amino acids 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 determined by the entire length of the reference amino acid sequence. Alignment to determine the similarity of the Sequence, preferably the identity of the
105/217
Sequence can be performed with the tools known from the state of the art, preferably using the best alignment of SEQuências, for example, using ALIGN, using the standard configurations, preferably EMBOSS :: needle, Matrix: Blosum62, open slits 10, 0, Gap Extend 0.5.
[00218] Sequence similarity 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.
[00219] The term percentage 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 to be distributed randomly and along its entire length. Sequence comparisons between two amino acid sequences are conventionally carried out by comparing these sequences, after having optimally aligned them, said comparison being made by the segment or by the comparison window to identify and compare local regions of Sequence similarity. Optimal alignment of sequences for
106/217 comparison can be produced, in addition manually, using the local homology algorithm of Smith and Waterman, 1981, App ads. 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, N and BLAST TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. ).
[00220] 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 comparison positions and multiplying the result obtained by 100, in order to obtain the percentage of identity between these two sequences.
[00221] 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 (integrated or not integrated into the animals' natural genomic DNA) and which is preferably capable of expressing the transgenes. For example, a transgenic mouse can have a human light chain transgene and a human transgene, either human heavy chain or transchromosome heavy chain, such that the mouse
107/217 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 transcromosomal mice (for example, KM), as described in WO 02/43478. Such transgenic and transcromosomal 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 change.
[00222] 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 even more preferably 75% or greater, at the level, for example, at the level of expression or the level of cell proliferation.
[00223] Terms such as increasing or improving refer, preferably, to an increase or improvement of about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more
108/217 preferably, at least 80%, and more preferably at least 100%, at least 200%, at least 500%, at least 1,000%, at least 10000% or even more.
Mechanisms of action mAb [00224] Although the following provides considerations about the mechanism underlying the therapeutic effectiveness of the antibodies of the invention, it is not to be considered as limiting the invention in any way.
[00225] The antibodies described herein 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 directly kill tumor cells or can be used in synergy with traditional chemotherapeutic agents, to attack tumors through complementary mechanisms of action , which may include the anti-tumor immune responses that may have been compromised as a result of cytotoxic side effects of a chemotherapeutic T lymphocyte. However, the antibodies described here may also have an effect simply by binding to CLDN18.2 on the cell surface, in this way, for example, preventing cell proliferation.
Antibody-dependent cell-mediated cytotoxicity
109/217
Antibody-dependent cell mediated cytotoxicity [00226] ADCC describes the ability to kill cells from effector cells, as described herein, particularly lymphocytes, which preferably requires the target cell to be labeled by an antibody.
[00227] ADCC preferably occurs when antibodies bind to antigens on tumor cells and the antibody Fe domains involve Fc receptors (FcR) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express 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, induction of ADCC in vivo will lead to tumor-directed T cell responses and host-derived antibody responses.
Complement-dependent cytotoxicity [00228] CDC is another method of killing cells that can be driven by antibodies. The IgM isotype is most effective for complement activation. IgGl and IgG3 are also very effective, both in directing CDC through the complement pathway,
110/217 classic activation. Preferably, in this cascade, the formation of antigen-antibody complexes results in the unveiling of Clq binding sites in multiple proximity in the CH2 domains of participating antibody molecules, such as IgG molecules (Clq is one of the three subcomponents of the complement
Cl). Preferably, these uncovered Clq binding sites convert the interaction
Clq-IgG previously had low affinity for one with high avidity, which triggers a cascade of events that involve a series of other complement proteins and leads to the proteolytic release of effector cell chemotactics / activation 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.
Antibody Production and Testing [00229] The antibodies described here 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 for the production of monoclonal antibodies, for example
111/217 viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of antibody genes.
[00230] 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 procedure. Immunization protocols and techniques for the isolation of immunized splenocytes for fusion are known in the art. Fusion partners (for example, murine myeloma cells) and fusion procedures are also known.
[00231] Other preferred animal systems for the preparation of hybridomas that secrete monoclonal antibodies are the mouse and the 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)).
[00232] In yet another preferred embodiment, human monoclonal antibodies can be produced using transgenic mice that carry transcromosomal or parts of the human immune system instead of the mouse system. These transgenic and transchromosomal mice include mice, known as Km mice and HuMAb mice, respectively, and are collectively referred to here as transgenic mice.
112/217
The production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in WO 2004035607 [00233] Yet another strategy for the generation of monoclonal antibodies is to directly isolate genes encoding lymphocyte antibodies that produce antibodies of the specificity defined by for example, see Babcock et al., 1996; An innovative strategy to generate monoclonal antibodies from isolated lymphocytes, individual producers of 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.
[00234] To generate antibodies, mice can be immunized with vehicle-conjugated peptides 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 expressed cells expressing the antigen, as described. Alternatively, mice can be immunized with DNA that encodes the antigen or its fragments. In the event that immunizations using a purified or enriched antigen preparation do not result in antibodies, mice can be immunized with
113/217 cells that express the antigen, for example, a cell line, to promote immune responses.
[00235] The immune response can be monitored throughout the immunization protocol with samples of plasma and serum to be obtained through the tail vein or retro-orbital 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 antibodies that secrete specific hybridomas.
[00236] To generate hybridomas that produce monoclonal antibodies, splenocytes and lymph node cells from immunized mice can be isolated and fused with a suitable 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. By immunofluorescence and FACS analysis using cells that express the antigen, antibodies with specificity for the antigen can be identified. The antibody can be replated secretory hybridomas, sieved again and if still positive for monoclonal antibodies they can be subcloned via
114/217 limiting dilution. The stable subclones can then be cultured in vitro to generate the antibody in tissue culture medium for characterization.
[00237] 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).
[00238] 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 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 those described in the prior art, such as electroporation, lipofectin, lipofectamine or others. After the introduction of these antibody genes into host cells, cells that express the antibody can be
115/217 identified and selected.
[00239] These cells represent the transfectomas that can then be amplified to their level of expression and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.
[00240] 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 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 [00241] Murine monoclonal antibodies can be used as therapeutic antibodies in humans when labeled with radioactive toxins or isotopes. Unmarked murine antibodies are highly immunogenic in humans 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
116/217 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 joining the variable regions of the murine heavy and light chain of antibodies with the human heavy and light chain constant region (for example, as described by Kraus et al., In Methods in Molecular Biology, series of recombinant cancer antibodies ISBN 0-89603-918-8-therapy). In a preferred embodiment, chimeric antibodies are generated by joining the human kappa light chain from the constant region to the murine light chain variable region. 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 preferred heavy chain constant regions for generating chimeric antibodies are IgG2, IgA, IgD and IgM.
Humanization [00242] Antibodies interact with target antigens predominantly through amino acid residues that
117/217 are located in the complementarity determining regions of six heavy and light chains (CDR). For this reason, the amino acid sequences within CDRs are more diverse between 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 antibodies by constructing specific expression vectors that include CDR sequences from the naturally occurring antibody grafted into specific sequences structural effects 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: 522 - 525; 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 vary from the mature antibody gene sequences because they do not include fully assembled variable genes, which are formed by V (D) J during maturation of cells joining B. Germline gene sequences, too will be different from the sequences of an antibody of high affinity secondary repertoire in the individual uniformly throughout the region
118/217 variable.
[00243] The ability of antibodies to bind an antigen can be determined using standard binding assays (eg, ELISA, Western Blot, immunofluorescence and flow cytometric analysis).
[00244] To purify antibodies, selected hybridomas can be grown in two-liter spinner flasks for monoclonal antibody purification. 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 fractionated and stored at -80 ° C.
[00245] To determine whether the selected monoclonal antibodies bind to single epitopes, site-directed mutagenesis or multiple site-directed mutations can be used.
[00246] To determine the isotype of antibodies, ELISA of isotypes with various commercial kits (for example, Zymed, Roche Diagnostics) can be performed. Plate wells
119/217 microtiter can be coated with mouse anti-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 mouse IgGl, IgG2a, IgG2b or IgG3, IgA or mouse IgM specific to peroxidase conjugated probes. After washing, the plates can be developed with ABTS substrate (1 mg / ml) and analyzed at OD 405-650. Alternatively, the isotyping mouse monoclonal antibody IsoStrip kit (Roche, Cat. No. 1,493,027) can be used as described by the manufacturer.
[00247] 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 antigen and negative controls lacking antigen expression (grown under normal growth 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
120/217 analyzed by flow cytometry with a FACS instrument using light and lateral dispersion properties, the door 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. The cells transiently transfected with plasmids encoding 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 preferentially bind to cells that express fluorescence markers, while non-specific antibodies bind in a comparable proportion of untransfected cells. 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.
[00248] In order to demonstrate the presence of antibodies in the serum of immunized mice or binding of monoclonal antibodies to living cells that express the antigens, immunofluorescence microscopy analysis can be used. For example, cell lines that express either
121/217 spontaneously or after transfection and negative antigen controls lacking antigen expression are grown in 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, the cells can react with an Alexa555 labeled with secondary antibody anti-mouse IgG (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.
[00249] Cell extracts from cells that express the 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 ECL substrate.
[00250] The antibodies can be further tested for reactivity with the antigen by immunohistochemistry in a manner well known to the person skilled in the art, for example.
122/217 example, using acetone or paraformaldehyde fixed cryo cuts or paraffin-embedded tissue sections fixed with paraformaldehyde 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 expressing spontaneously or after antigen transfection. For immunostaining, antibodies reactive to the antigen can be incubated followed by conjugated goat anti-mouse horseradish peroxidase or goat anti-rabbit antibodies (DAKO) according to the manufacturer's instructions.
[00251] Antibodies can be tested for their 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 cytotoxicity (ADCC): [00252] 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. Washed effector cells can be suspended in RPMI medium supplemented with 10% thermally inactivated fetal calf serum or, alternatively, with 5% heat-inactivated human serum and mixed with ^{5 eu}
123/217 Cr-labeled target cells expressing 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 be added at various concentrations. Human IgG can be irrelevant 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. 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 may be a measure of viable cells.
[00253] 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) [00254] Anti-CLDN18.2 monoclonal antibodies can be tested for their ability to mediate CDC, using a
124/217 variety of known techniques. For example, the serum supplement can be obtained from the blood in a manner known to a person skilled in the art. To determine the CDC activity of the mAbs, different methods can be used. ⁵¹ Cr can release, 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 various concentrations of mAb for 1030 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.
[00255] 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 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 for
125/217 determination of 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 µg / ml). Then, the supernatants are replaced with 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 Safire. The percentage of specific lysis is calculated as follows:% specific lysis = (sample fluorescence-background fluorescence) / (maximum fluorescence of lysis by background fluorescence) x 100.
Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies [00256] 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 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 Biosciences buffer and incubated with annexin V conjugated with (Biosciences BD) conjugated with FITC or APC for 15 min. in the dark. All cells from
126/217 of 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 means of monoclonal antibodies can be detected with commercially available kits. The DELFIA Cell Proliferation Kit (PerkinElmer, 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 using the europium-labeled monoclonal antibody. To allow the detection of antibodies, the cells are fixed and the DNA is denatured using correction solution. The 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 inducer DELFIA. The measured fluorescence - using time-resolved fluorometry in detection - is proportional to the DNA synthesis in the cell of each well.
Preclinical studies [00257] 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,
127/217 or after transfection, for example HEK293) to determine its effectiveness in controlling the growth of tumor cells that express CLDN18.2.
[00258] In in vivo studies, after CLDN18.2 xenograft expressing tumor cells in immunocompromised mice or in other animals can be performed using the antibodies described here. 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 tumor-related tumors. The antibodies administered to mice with or symptoms may be tumor 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 efficacy 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 CLDN18.2-antibody treatment. Possible side effects of
In vivo application of CLDN18.2 antibodies includes toxicity, particularly in the expression of CLDN18.2 in tissues, including the stomach. Antibodies that recognize CLDN18.2 in humans and other species, for example, mice, are particularly useful for predicting potential side effects mediated by application of CLDN18.2-monoclonal antibodies in humans.
[00259] 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 Epitope Mapping: A Practical Approach Practical Approach Series, 248 by Olwyn MR Westwood, Frank C. Hay.
[00260] The compounds and agents described herein can be administered in the form of any suitable pharmaceutical composition.
[00261] Pharmaceutical compositions are usually 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.
[00262] A pharmaceutical composition can comprise its salts, buffer substances, preservatives, carriers, diluents and / or excipients which are preferably pharmaceutically acceptable. The term
129/217 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.
[00263] 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 acids: hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, hydrochloric, 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.
[00264] Buffer substances suitable for use in a pharmaceutical composition include acetic acid, in a salt thereof, citric acid in a salt, boric acid in a salt of phosphoric acid and a salt thereof.
[00265] Preservatives suitable for use in a pharmaceutical composition include benzalkonium chloride, chlorobutanol, parabens and thimerosal.
[00266] An injectable formulation can comprise a pharmaceutically acceptable excipient, such as Ringer Lactate.
[00267] The term vehicle refers to a component
130/217 organic or inorganic, natural or synthetic in nature, in which the active component is combined to facilitate, improve or allow application. According to the invention, the term carrier also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient.
[00268] Possible support 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 / polymers polyoxyethylene / propylene polyoxy glycolide or copolymers.
[00269] 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.
[00270] The agents and compositions described herein can be administered by any conventional route, such as parenteral administration, including by injection or infusion. Administration is preferably parenteral, for example,
131/217 intravenously, intra-arterially, subcutaneously, intradermally or intramuscularly.
[00271] 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. The 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.
[00272] 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.
[00273] An effective amount of an agent or composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient,
132/217 including age, physiological condition, size and 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 not sufficient with an initial dose, higher doses (or higher doses effectively achieved by a different, more localized route) of administration can be used.
[00274] 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 cells characterized by an altered expression pattern of CLDN18.2.
[00275] For example, in one embodiment, the antibodies described herein can be used to treat a 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 .
[00276] Compositions and methods of treatment
Pharmaceutical 133/217 described in accordance with the invention can also be used for immunization or vaccination to prevent a disease described herein.
[00277] 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: First single-dose, multi-center, escalation-in-human clinical trial, phase I, open, iv infusion to assess the safety and tolerability of IMAB362 in hospitalized patients with advanced gastroesophageal cancer [00278] A first clinical study of scaling of IV infusion in human, single-dose, phase I, open, multi-center humans in IMAB362 was performed to determine the maximum tolerated dose or applicable single dose (BAT) of IMAB362, examine for safety, tolerability and profile of adverse effects of IMAB362, determine the pharmacokinetic profile of single increasing doses of IMAB362, determine the immunogenicity of a single dose application of IMAB362, and determine the potential for anti-tumor activity of IMAB362 in patients with advanced gastroesophageal cancer (GE).
[00279] This study was designed as a first phase I human, multicenter, inter-patient single dose escalation non-randomized, open clinical study with a
134/217 single intravenous infusion of IMAB362 and one to a period of 4 weeks following treatment-free.
[00280] To be included in the study, patients had to meet all of the following inclusion criteria:
• Metastatic, refractory or recurrent disease of advanced gastroesophageal cancer confirmed by histology • Expression CLDN18.2 confirmed by immunohistochemistry or availability of a tumor tissue sample suitable for determining the expression CLDN18.2 • Standard chemotherapy before containing a fluoropyrimidine, a compound platinum and / or epirubicin, and - if clinically appropriate - docetaxel • At least one measurable site of the disease, according to the RECIST criteria (computed tomography (CT) scans or magnetic resonance tomography (MRT) no more than 6 weeks before study entry) • 18 years of age or older • Written informed consent after being informed about the study • Performance status ECOG (PS) 0-1 OR Kamofsky 70
- 100% • Life expectancy> 3 months • Platelet count> 100,000 / mm ³ • Hemoglobin> 10 g / dl
135/217 • 1NR <1.5 • Normal bilirubin • AST and ALT <2.5 times the normal limit (ULN) (5 times ULN if liver metastases are present) • Creatinine <1.5 x ULN • For women with potential (last menstruation less than 2 years before admission) to become pregnant: Negative pregnancy test (β-HCG) at baseline and using two highly effective methods for contraception 8 weeks after the study drug infusion • Male patients must use a contraceptive method accepted for 8 weeks after the study drug is infused.
[00281] Patients who have one or more of the following criteria were not included in the study:
• Pregnancy or breast-feeding • Allergic reaction or intolerance before a monoclonal antibody, including humanized and chimeric antibodies • Before inclusion in the present study • Less than 3 weeks after antitumor chemotherapy or previous radiation therapy • Other agents or experimental devices simultaneously or within 4 weeks before this study
136/217 • Other anti-cancer agents or concomitant therapies • History of positive test for human immunodeficiency virus (HIV) antibody • Known hepatitis • Serious or uncontrolled diseases, including, but not limited to any of the following:
> Ongoing or active infection requiring parenteral antibiotics> Symptomatic congestive heart failure> Unstable angina pectoris> Uncontrolled hypertension> Clinically significant cardiac arrhythmia> Myocardial infarction in the last 6 months> Gastric bleeding within the last four weeks> Peptic ulcer symptomatic> Clinical symptoms of brain metastasis or documented metastases> Psychiatric illness or social situations that would prevent compliance Study> Concomitant administration of anticoagulation agents with vitamin K antagonists (eg Coumadina); and
137/217> Concomitant administration of therapeutic doses of heparin (prophylactic doses are acceptable).
[00282] Of a total of 29 patients, 15 patients received the study medication and were assigned to one of the dose groups (33, 100, 300, 600 or 1000 mg IMAB362 / m ²⁾ ·
These patients formed the safety population (SP). Since no dose-limiting toxicities potentially occurred in any of the dosing groups, no other patients had to be tested to confirm potential dose-limiting toxicities. Therefore, no more than 3 patients in each dose group, that is, 15 patients in general, received the study medication.
[00283] Assignment of patients to the different dose cohorts 1MAB362 is given in Table 1, below.
Table 1: Patient allocation.
Dose cut IMAB362 Patient No. 33 mg / m ² 0201 0103 0104 100 mg / m ² 0202 0105 0203 300 mg / m ² 1101 0403 1201
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600 mg / m ² 0302 0204 1202 1000 mg / m ² 0205 0106 0112
EVALUATION A. SECURITY
[00284] IMAB362 was found to be safe is well tolerated. [00285] Only 25 AEs (Adverse events), what occurred in 8 of patients were classified how related how treatment. Related AEs how
treatment were similar between dose groups. More than half of these AEs were gastrointestinal disorders (mainly nausea, vomiting). Only one of these related adverse events was classified as severe (vomiting), while all the others were mild or moderate. All related adverse events recovered, with the exception of one case of dysgeusia (grade 1 CTC (mild)) with unknown result and one case of increased GGT (grade 2 CTC (moderate)), which did not recover.
[00286] No dose-limiting toxicity (DLT), defined as an AE related to treatment that occurred during or within four weeks after the study drug infusion and era or Grade 3 toxicity (except
139/217 for nausea, vomiting and alopecia) or toxicity grade 4 or 5 (according to version 3.0 of the CTC), was observed in any of the dose groups. Therefore, the maximum tolerated or single applicable dose (BAT) of IMAB362 determined in the present study is 1000 mg / m ^2. [00287] No related SAE and no unexpected serious suspicion of adverse reaction (SUSAR) occurred in the present study.
[00288] Only 7 patients had at least one laboratory value outside the reference range assessed as grade 3 (severe). There was no dose-effect relationship and no clear relationship to the study drug. No grade 4 CTC (life-threatening) or 5 (death) laboratory values have been reported.
[00289] In conclusion, there are no significant differences in the profile of AE and other safety parameters between the dose groups could be seen. Generally speaking, IMAB362 given in a single dose has been observed to be safe and well tolerated with nausea and vomiting being the most common related adverse event.
B. EVALUATION OF PHARMACOKINETICS AND IMMUNOGENICITY [00290] For the determination of drug concentration in serum levels IMAB362, all patients were measured immediately before the infusion of study medication, at the end of the infusion, at 3, 8, 12 and 24 hours after the end of
140/217 infusion and on days 3 (V3), 5 (V4), 8 (V5), 15 (V6) and 29 (V7).
[00291] An overview of the serum levels of IMAB362 in the course of the study for each patient is given in Table 2. For reasons unknown to a patient (No. 1201) in 300 mg / m ² group of the dose of a low level of serum IMAB362 (12,633 ug / mL) It was measured before the study drug was infused (V2, day 0).
Table 2: Time course of serum level IMAB362 [g / ml] per patient
Pat. Dose groupIMAB362 V20h post infusion V23h post infusion V28h post infusion V212:00post infusion V224h post infusion V3 day 2 V4 day 5(± 1) V5 day 8(± 1) V6 day 15(± 2) V7 day 29(± 7) 0103 33 mg / m ² 14.9 14.6 12.1 13.7 11.7 8.0 5.3 5.4 3.8 1.9 0104 33 mg / m ² 12.5 15.1 12.3 10.1 9.7 11.3 7.0 3.6 2.3 1.3 0201 33 mg / m ² 15.4 12.6 12.7 10.6 11.2 9.5 9.5 6.6 5.3 2.3 0105 100 mg / m ² 75.6 65.8 61.6 63.4 48.8 37.5 24.4 19.0 15.8 11.3 0202 100 mg / m ² 59.3 49.4 47.4 41.2 41.4 35.6 18.5 17.1 12.8 6.4 0203 100 mg / m ² 38.5 36.8 41.1 35.5 36.6 25.4 19.6 18.2 12.6 6.3 0403 300 mg / m ² - 164.2 161.6 125.0 114.0 90.2 59.8 43.9 20.9 11.4 1101 300 mg / m ² 176.1 171.5 145.5 150.5 146.5 86.3 65.9 65.3 42.7 36.8 1201 300 mg / m ² 153.1 169.5 147.7 124.6 97.8 135.1 124.3 63.7 47.7 33.3 0204 600 mg / m ² 315.7 298.2 285.8 284.3 340.4 218.0 136.7 - 87.5 52.9 0302 600 mg / m ² 361.6 335.0 342.6 285.1 239.1 140.9 82.2 28.4 11.0 2.0 1202 600 mg / m ² 242.2 290.1 281.0 237.6 207.9 170.2 131.6 58.1 37.0 23.6 0106 1000 mg / m ² 493.7 606.1 488.9 465.9 452.7 367.8 259.9 158.8 79.3 34.7 0112 1000 mg / m ² 359.0 465.9 375.9 356.5 311.0 273.1 220.6 192.8 154.1 84.4 0205 1000 mg / m ² 479.7 435.9 366.7 331.6 343.9 279.7 193.9 155.3 105.9 38.9
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Peak concentration (Cma x) of each patient is printed in bold type [00292] The average peak concentrations observed (C max) per dose group are shown in Table 3. Increased average values for C max correspond to the increasing doses of infusion of IMAB362.
Table 3: Maximum concentrations (C max) of IMAB362 during study - Summary of descriptive statistics.
Dose groupIMAB362 Cmax [g / mL] N average SD Min Average Max 33 mg / m ² 3 15.1 0.3 14.9 15.1 15.4 100 mg / m ² 3 58.7 17.3 41.1 59.3 75.6 300 mg / m ² 3 169.9 5.9 164.2 169.5 176.1 600 mg / m ² 3 330.7 36.7 290.1 340.4 361.6 1000 mg / m ² 3 517.3 77.3 465.9 479.7 606.1
The graphical representation of the mean blood concentrations of IMAB362 during the study is given in Figure 1.
[00293] The highest levels were measured from IMAB362 directly at the end of the infusion up to 8 hours after the end of the infusion. At 3 hours after the end of the infusion, the mean concentration 1MAB362 was 14.1 µg ml in the 33 mg / m ^{2 group} , 50.7 pg / ml in doses in the 100 mg / m ^{2 group} , 164.2 pg / mL in the 300 mg / m ^{2 group} , 307.8 pg / mL in the 600 mg / m ^{2 group} , and 50 2.6 pg / mL in the 1000 mg / m ^{2 group} .
[00294] Pharmacokinetics of IMAB362 is dose-dependent.
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The highest dose levels were observed during the first 8 hours after the 2-hour infusion. The average half-life of IMAB362 was 8.5 days in general, ranging from about 5 to about 12 days in the different dose groups.
[00295] It was determined from in vitro studies of the mode of action that concentrations of IMAB362 of 50 pg / ml of robust execution of effects on anti-tumor cells through ADCC, CDC and inhibition of proliferation can be expected and that EC50 values of ADCC and CDC, which are considered as the main mode of actions, are still covered with half this level of concentration. Based on this knowledge, doses of 300 mg / m ² and 600 mg / m ² have been identified for closer evaluation in multiple dose studies with IMAB362. Patients receiving IMAB362 300 mg / m ² and 600 mg / m ² were clearly above these levels on day 8 (V5) and close to 50 pg / ml on day 15 (V6).
[00296] There was no evidence of anti-drug antibodies in patients after this single dose of IMAB362. C. EVALUATION OF ANTITUMORAL ACTIVITY [00297] The main measure for assessing the potential for antitumor activity was the tumor state according to RECIST (version 1.0) classification at 2 to 5 weeks after the IMAB362 infusion (V6 / V7). As all patients completed the study according to the protocol, assessments were performed exclusively at V7, that is, 4 to 5 weeks
143/217 after the infusion of drugs. All patients were assessed by CT.
[00298] Three patients had no measurable disease (patients 1101 and 1201 had no target lesion, for patient 0302 the respective data were not available), but were included in the population for the analysis of antitumor activity, as this was not an evaluation formal effectiveness.
[00299] In general, for none of the patients a complete or partial response could be evaluated. Stable disease was observed for one of 15 patients in the 600 mg / m ² dose group. While in the treated patients the percentage of tumor cells stained positively for CLDN18.2 ranged from 1% to 80% (up to 50% of tumor cells with membrane staining), 90% or more of the tumor cells of this patient stained positive for CLDN18 0.2 with a large fraction of the tumor cells exhibit membrane staining. Two patients in the 300 mg / m ^{2 group} also did not progress and did not have a target lesion that were not assessed for objective tumor response and were classified as non-CR, non-PD. The duration of PD was about 2 months. The duration of non-CR, non-PD was about two months and 6 months, respectively.
[00300] An overview of the overall response by the patient is given in Table 4.
Table 4: Tumor status (general response) at V7 per patient
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Cutting doseIMAB362 Patient No. V7 Tumor Status 33 mg / m ² 0103 Progressive disease 0104 Progressive disease 0201 Progressive disease 100 mg / m ² 0105 Progressive disease 0202 Progressive disease 0203 Progressive disease 300 mg / m ² 0403 Progressive disease 1101 * Non-CR, non-PD 1201 * Non-CR, non-PD 600 mg / m ² 0204 Stable disease 0302 * Progressive disease 1202 Progressive disease 1000 mg / m ² 0106 Progressive disease 0112 Progressive disease 0205 Progressive disease
Patients without measurable disease (patients 1101 and
1201 had no target lesion, for patient 0302 the respective data were not available).
[00301] The different parameters that contribute to the assessment of the state of the tumor (global response) are described below.
[00302] Regarding the change in the sum of the largest diameters (target lesion), state of non-target lesions after IMAB362
145/217 treatment, occurrence of new injuries, an overview of the results of the evaluation after IMAB362 treatment (evaluated in V7) is shown in Table 5.
[00303] Table 5: Evaluation of parameters for evaluation of tumor status in V7 per patient
Group doseIMAB362 No. of patient and Percentage change in the sum of the longest diameter in the target lesion Unequivocal progression from a non-target injury New injuries 33 mg / m ² 0103 + 30.2% Yes No 0104 + 10.0% Yes Yes 0201 + 33.3% Yes Yes 100 mg / m ² 0105 + 25.9% No Yes 0202 + 35.9% Yes Yes 0203 + 35.1% No non-target injury no 300 mg / m ² 0403 -37.8% Yes Yes 1101 No non-target injury Diceunavailable No 1201 No non-target injury no no
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600 mg / m ² 0204 + 7.4% no no 0302 Diceunavailable Diceunavailable Yes 1202 + 66.7% no Yes 1000 mg / m ² 0106 + 36.5% no Yes 0112 + 40.0% no no 0205 -2.8% Without injurynon-target Yes
[00304] The percentage change in sum of the longest target lesion diameters from VI to V7 did not show any clear difference for different treatment doses.
[00305] For non-target lesions an unequivocal progression (from VI to V7) has been reported more frequently in patients at the lower doses, but not at the doses of 600 mg / m ² and 1000 mg / m ² .
[00306] In a patient in the 300 mg / m ^{2 group} (0403) an unequivocal progression was observed in non-target lesion and a decrease in the longer diameter in a target lymph node lesion.
[00307] Regarding new lesions, no preference was observed for one of the dose groups.
[00308] In the case of patients 0302 (600 mg / m ² of dose group) and 0205 (1000 mg / m ² of dose group) the occurrence of new injuries was the reason for the assessment of
147/217 global response as a progressive disease.
[00309] For the assessment of the state of the non-target lesions according to RECIST, the level of serum tumor antigens CA 125, CA 15-3, CA 19-9, and CEA was determined by the central laboratory at V2 (day 1, before infusion), V6 and V7.
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Dose groupPatient ID Tumor marker Time point Level Outside the reference range 300 mg / m ² 1101 CA 125 V2 (before 21.2 U / mL inf ..) 20.6 U / mL V6 27.7 U / mL CA 15-3 V7 21.3 U / mL V2 (before 21.0 U / mL inf ..) 21.3 U / mL CA 19-9 V6 <0.6 U / mL V7 <0.6 U / mL V2 (before <0.6 U / mL CEA inf ..) 1.7 ng / mL V6 2.0 ng / mL V7 2.1 ng / mL 1201 CA 125 V2 (before 13.5 U / mL inf ..) 13.7 U / mL V6 11.5 U / mL CA 15-3 V7 11.6 U / mL V2 (before 11.3 U / mL inf ..) 11.3 U / mL CA 19-9 V6 68.0 U / mL Yes V7 68.8 U / mL Yes V2 (before 63.0 U / mL Yes CEA inf ..) 3.2 ng / mL V6 2.6 ng / mL V7 3.0 ng / mL V2 (before inf ..)V6V7V2 (before inf ..)V6V7 600 mg / m ² 0204 CA 125 V2 (before 59.2 U / mL Yes inf ..) 50.2 U / mL Yes V6 35.1 U / mL Yes CA 15-3 V7 477.5 U / mL Yes V2 (before 372.3 U / mL Yes inf ..) 310.4 U / mL Yes CA 19-9 V6 > 10,000 U / mL Yes V7 5667 U / mL Yes V2 (before 3979 U / mL Yes CEA inf ..) 40.3 ng / mL Yes V6 25.2 ng / mL Yes V7 19.4 ng / mL Yes V2 (before inf ..)V6V7
149/217 [00310] Of the 3 patients with stable disease or non-CR non-PD according to the imaging, two patients had stable tumor marker levels during the observation period. One patient (0204) showed a profound decrease in all 4 tumor markers after treatment. Most patients with progressive disease, in contrast, had increased levels of tumor markers.
[00311] Tumor status (according to the RECIST classification) at 4 to 5 weeks after IMAB362 infusion (V6 / V7) was compared to baseline. In general, none of the patients could be assessed as a complete or partial response. One of the patients (15 in a 600 mg / m ² dose group) had a stable disease at the end of the study. Two patients in the 300 mg / m ² dose group with the disease not measurable did not show CR / non-PD. In line with this, the levels of tumor markers in these three patients either remained stable (2 patients) or even decreased profoundly (1 patient). Most patients with progressive disease showed increased levels of tumor markers over time.
[00312] Regarding the parameters that contribute to the evaluation of the tumor state (total response), a decrease in an injury was observed in the 300 mg / m ² dose group ^. In the screening (VI), 13 of the 15 patients had a total of 32 non-target lesions. After IMAB362 treatment
150/217 (assessed at V7) an unambiguous progression from a non-target lesion was reported for a total of 5 patients, 3 in the 33 mg / m ² dose group, 1 in the 100 mg / m ² dose group and 1 in the dose group 300 mg / m ² . For none of these 5 patients, the overall response was assessed as only progressive disease due to the progression of their non-target lesions. A total of 17 new lesions were observed over the course of the study, evenly distributed across the dose groups. In the case of 2 patients (in the 600 mg / m ² and 1000 mg / m ² dose groups), the occurrence of new lesions was the reason for assessing the overall response as a progressive disease.
[00313] In addition, auxiliary data were collected from selected patients, which shows that patients and the components of patients' PBMC serum are fully functional and potent in mediating the main IMAB362 modes of action of CDC and ADCC, respectively.
[00314] In conclusion, for anti-tumor activity suggests (stable disease, tumor marker decrease) were observed in the dose groups of 300 mg / m ² and / m 600 mg / m ² . Due to the small sample size of the dose groups, it is difficult to conclude on trends for effectiveness.
C. GLOBAL CONCLUSIONS [00315] This study was designed as a phase I, inhuman, multicenter, single-dose, inter-patient escalation, non-randomized, open clinical study with a
151/217 single intravenous infusion of IMAB362 and a free treatment for a period of 4 weeks.
[00316] A total of 15 patients received study medication and were assigned to one of the dose groups (33, 100, 300, 600 or 1000 mg I AB362 / m ² ) · The dose groups can be considered as comparable. There are no relevant imbalances regarding the baseline and demographic characteristics that could be observed.
[00317] With regard to the primary study objective, no dose limiting toxicity (DLT) was observed in any of the dose groups. Therefore, the single dose of IMAB362 applicable in the present study was 1000 mg / m ^2. IMAB362 was safe and well tolerated, with nausea and vomiting being the most common related adverse events.
[00318] The profile of AE and the incidence of AEs was found to be similar across different dosing groups. No apparent differences between dose groups can be seen in numbers of individual patients with clinically significant deterioration in all hematological, biochemical or coagulation parameters.
[00319] Regarding the potential anti-tumor activity of IMAB362, according to the RECIST criteria, a complete or partial response could not be observed for any of the patients. One of the patients (15 in the
152/217 dose of 600 mg / m ² ), presented stable disease at the end of the study. Two patients in the 300 mg / m ² dose group with the disease not measurable did not show CR / non-PD. Of these 3 patients with stable disease, according to the imaging, they had two levels of stable tumor markers during the observation period. One patient showed a profound decrease in all 4 tumor markers after treatment.
[00320] This and the pharmacokinetic studies, showing that the serum levels directed to IMAB362 are reached at a dose level of 600 mg / m ² , the support that this dose has yet to be evaluated.
[00321] Furthermore, auxiliary data confirms that patients' immune effectors are fully functional and potent in mediating the main modes of action of IMAB362 from CDC and ADCC, respectively.
Example 2: Potency of the Drug [00322] The objectives of in vitro analyzes performed for this Phase I clinical study included an analysis of whether (i) effector cells present in the patient's blood are capable of inducing IMAB362-dependent ADCC, (ii) the The patient's complement system is capable of inducing IMAB362 dependent on CDC and (iii) the ability to induce IMAB362 ADCC and CDC is altered after administration to patients.
[00323] Different types of assays were performed to study the cytolytic activity induced by IMAB362 after
153/217 administration to patients in detail. The assays were performed with either the patient's serum or patient isolated from PBMC from blood samples (Table 7). For comparison and to verify the functionality of serum CDC and ADCC, assays from a pool of human serum (generated from healthy human subjects), in which fresh IMAB362 was serially diluted in parallel, was included in each assay. To test the functionality of CCDA assays with PBMC, blood cells isolated from a healthy donor were used as a positive control in the same assay for each patient.
A. Materials and Methods [00324] For the different in vitro assays of patient serum samples were collected before the infusion of IMAB362 and 1, 7, 14 and 28 - 32 days after the administration of IMAB362 antibody (Table 7). They were used as an antibody source and complement IMAB362 in CDC or as a source of antibodies in serum CCDA assays. Pre-infusion of patient serum was used as non-negative control IMAB362 and for dilution of patient serum samples to adjust the 1MAB362 concentration to 0.5 ug / ml. Fresh blood samples were collected 14 days after the infusion (7 days for patient 0203) and were used as a source of effector cells for CCDA assays.
[00325] Table 7: Summary of serum and blood samples collected for each patient, no: serum sample not
154/217 was obtained from the clinical study site
Subject No. IMAB362 dose [mg / m ² ] Pre-infusion(y / n) serum obtained infusion in after day blood obtained on the day 0201 33 s 1 7 14 30 14 0202 100 s 1 7 14 28 14 0203 100 s 1 7 14 28 14 0403 300 s 1 7 14 28 7 0204 600 s 1 at the. 14 32 14 0205 1000 s 1 7 14 30 14
[00326] Blood samples were collected from patients (Table 7), serum was collected and serum aliquots were prepared and immediately stored at -80 ° C. Analysis of all these samples was performed in a single experiment after collection of all serum samples 24.
[00327] For ADCC, fresh blood samples (15 ml of Na 2 EDTA) were used to isolate the PBMC and CCDA assays were performed the next day.
[00328] The ability of patients' PBMCs to induce ADCC in conjunction with IMAB362 was tested ex vivo using fresh 15 ml of Na 2 -EDTA anticoagulated blood samples obtained from patients (14 days 7 days for patient 0203) after IMAB362 administration. PBMC from blood samples were isolated on arrival using Ficoll density gradient centrifugation. PBMCs were cultured for 24 h and ADCC assays were performed the next day with NUGC4 cancer cells transfected with CLDN18.2 luciferase human gastric device as targets in conjunction with several
155/217 concentrations of IMAB362 added exogenously. PBMCs were added in an E: T ratio of 20: 1 and the assays were incubated for 24 h at 37 ° C, 5% C0 2 PBMCs obtained from a healthy donor were tested in the same configuration in parallel to analyze assay validity (positive assay control). This PBMC stock was stored in liquid N 2 and for each ADCC assay with patient PBMCs, an aliquot of this PBMC stock was thawed and analyzed in parallel.
[00329] Characterized materials used were:
• CLDN18.2 positive target cells: NUGC4-10CHl luci eGFP # 2 stomach cancer cells stable and transfected;
• positive control effector cells: PBMC obtained from a healthy donor (frozen N very -stock ID: 276-SMS09-00706, 4e7c / vial, MNZ, 08.07.07.SJA) • functional control antibody: IMAB362 in serial dilutions (0.4 ng ml -126.5 g / ml) • Negative control antibody assay: Istotype control (Rituximab, 126.5 μ / πι1) [00330] The ability of the patient's serum components to induce cytotoxicity complement dependent (CDC) in conjunction with IMAB362 was analyzed ex vivo over time. Serum samples were collected and stored at -80 ° C and all patient samples were
156/217 tested in parallel in the same experiment, with the addition of pre-infusion of serum to which a fixed amount of 0.5 g / ml IMAB362 (representing the in vitro CE 5 S concentration) was added exogenously, samples were also collected 1, 7, 14 and 28-32 days after IMAB362 administration were tested, in which 1MAB362 circulates had to be adjusted to 0.5 µg / ml (CDC) with normalization. The final serum concentration for each assay was adjusted to 20%. The transfected CHO-Kl-luciferase cells stably transfected with 18.2 CLDN were used as targets. For comparison a mixture of serum from healthy human donors spiked with IMAB362 was tested.
[00331] Characterized materials used were:
• CLDN18.2 positive target cells: stably transfected cells CHO-K1 P740 2A5 # luci.
• positive assay control: IMAB362 serial dilutions (1: 3.16), prepared in the human serum pool from healthy donors, resulting in final concentrations in the range of 31.6 ng / ml and 10.0 ug / ml.
• Functional antibody control: IMAB362 adjusted to 0.5 g / ml of final assay concentration in each patient's preintusion serum sample.
• Negative control antibody assay: Isotype control antibody diluted in human pooled serum (Rituximab).
[00332] The kinetics of cytotoxicity mediated by general IMAB362 in human circulation, which integrates its
157/217 ability to induce ADCC and CDC, was analyzed in a one-tube assay.
[00333] The serum from each patient collected 7, 14 and 2832 days after iv administration of IMAB362, and thus comprising the patient's complement factors plus circulating IMAB362, was tested in this assay. The serum was applied in each test at a final serum concentration of 25% (v / v). Healthy control PBMCs were added as effector cells, to the step that NUGC-4 cells served as target cells with an E: T of40: l ratio.
[00334] In an additional configuration, the serum was to destroy the complement activity inactivated by heat. This second assay therefore reflects only the ADCC activity induced by IMAB362 present in the patient's serum.
[00335] During Phase I of the study, serum samples were collected and stored at -80 ° C. All patient samples were tested in parallel in the same experiment.
[00336] Characterized materials used were:
• CLDN18.2 positive target cells: stably luciferase-transf NUGC-4 10CH1 1 luci eGFP # 2 stomach cancer cells.
• Effector cells: PBMCs from a healthy donor (fresh buffy coat).
• Functional control antibody: IMAB362 dilutions
158/217 series (0.26 ng / ml - 200.0 g / ml) was added to the human serum mixture.
• positive control Sample: Patient pre-infusion serum sample enriched with IMAB362 (200.0 µg ml) (representing ECs -ioo for IMAB362 in this scenario).
• Negative control antibody assay: isotype control antibody in the human serum pool (Rituximab).
[00337] The ability of IMAB362 to interact and activate the complement present in the patient's serum and to induce complement-dependent cytotoxicity (CDC) after prolonged circulation in the patient's blood was analyzed 1 ex vivo, 7, 14 and 28-32 days after IMAB362 administration. The assay was performed directly using serum samples from patients in the assay (CDC without standardization). As a positive control pre-infusion serum to which a fixed amount of 10 µg / ml IMAB362 (representing in vitro in ECQ S -I o 3rd concentration) was added exogenously. The final serum concentration for each assay was adjusted to 20%. The CHO-K.1-luciferase cells transfected stably transfected with CLDN18.2 were used as targets. For comparison a mixture of serum from healthy human donors spiked with IMAB362 was tested.
[00338] During the Phase I study, serum samples were collected and stored at -80 ° C. All patient samples were tested in parallel in the same experiment.
159/217 [00339] Characterized materials used were:
• CLDN18.2 positive target cells: stably transfected CHO-K1 P740 2A5 # luci cells.
• Functional control antibody: IMAB362 serial dilutions (1: 3.16), prepared in the human serum pool from healthy donors, resulting in final concentrations in the range of 31.6 ng / ml to 10.0 g / ml.
• positive sample control: serum samples from pre-infusion patients each spiked with IMAB362 (10.0 µg / ml) in (in vitro CDC-CE concentration 0-100).
• Negative control antibody assay: Isotype control antibody diluted in human pooled serum.
B. RESULTS [00340] PBMC ability of patients to mediate ADCC [00341] In order to analyze the patient's immune cell ability to lyse cells that express tumor CLDN18.2, NUGC-4-gastric cancer cells, endogenously expressing CLDN18. 2, were incubated with increasing concentrations of IMAB362 and with patients' PBMCs. PBMC trials from a healthy donor were included as a functional control.
[00342] The PBMCs of the patients show dose-dependent IMAB362 lysis rates, with a maximum of 27 to 77% at a concentration of -30 ng / ml. This is not
160/217 significantly different (unpaired t test) from the maximum lysis rates of 14 to 56% obtained with healthy control PBMCs tested in the same assays (Figure 2). ADCC activity was more profound for patient 0204.
[00343] These data show, that the PBMCs of gastric cancer patients are not inferior in ADCC induction of CLDN18.2 positive gastric cancer human cells in conjunction with IMAB362, compared to PBMCs obtained from healthy donors.
[00344] Ability of the patient's complement system to induce CDC [00345] The ability of the patient's complement to interact with IMAB362 present in the serum and induce CDC has been tested. The pre-infusion serum samples were added with fresh 0.5 µg / ml of CDC IMAB362 activity and compared with the same concentration of antibody spiked in the human serum pool. Serum / antibody samples were incubated with CHO-K1 P740 2A5 # luci cells and lysis was determined after 80 min by measuring luciferase activity.
[00346] All patients were able to induce significant CDC within 80 minutes (Figure 3). For 5 out of 6 patients, maximum lysis rates ranging from 50 to 71% were observed. This is comparable to that of the data obtained by means of tests in parallel with a mixture of
161/217 healthy controls (64.5%). Notable, patient 0204 showed greater CDC activity with fresh IMAB362 (93.9%).
[00347] Ability of soluble effectors in the patient's serum to induce cell death intravenously with circulating IMAB362 [00348] Next, the ability of patients' serum to interact with IV IMAB362 administered during the time interval of their circulation in the patient was investigated by testing serum samples taken at different time points after IMAB362 administration in CDC assays on CHO-K positive CLDN18.2. 1 target cells. Serum samples were the source of patient-specific soluble effectors, including complementary, as well as for IMAB362. IMAB362 concentrations in serum samples were determined by ELISA (vivoScience) (Table 8) and adjusted to a final concentration of 0.5 IMAB362 µg / ml (mean EC50 of IMAB362) using the corresponding serum from each patient as pre-infusion diluents. As IMAB362 concentrations differ depending on treatment dose and blood collection time point, the dilution factor for samples differed considerably between patients ranging from 4.6fold to 688fold. A set of serum from healthy donors (HSC) was used as a control (Figure 4).
[00349] Compared to the positive control (pre-infusion of the respective patient's serum + fresh IMAB362)
162/217 death activity is retained within the first 24 hours, at which point the cytolytic activity of serum samples taken one week later is reduced, which is evolving in the following weeks (Figure 4). Even so, considerable cytotoxicity was performed by serum patients up to 2 weeks after IMAB362 administration. The loss of CDC activity after 28-32 days is significant and more pronounced in patients treated with low doses of IMAB362 (Figure 4). In patients treated with high doses (0204; 600 mg / m ² and 0205; 1000 mg / nr) CDC activity appeared to be better preserved during the investigated period of time. Based on currently available data, the underlying mechanism for this decline has not been understood until now.
Table 8: IMAB362 concentrations in the patient's serum at different time points used in the different ADCC and CDC assays, nd not available; ND: excluded, not determined; Concentration measured b vivoScience via ELISA
Subject No. IMAB362 treatment (mg / m ³ ) Time after IMAB362 treatment(days) IMAB362 concentration in undiluted patient serum ¹ [g / ml] Dilution factor for obtaining 0.5pg / ml IMAB362 in CDC assays(fold) IMAB362 concentrati on in cytotoxicity assays[Rg / ml] IMAB362 concentration in nonnormalized CDC assays[g / ml] 0201 33 1 11.17 22.34 2.79 2.2 7 6.63 13.27 1.66 1.4 14 5.30 10.60 1.32 1.1 30 2.31 4.62 0.58 0.5 0202 100 1 41.37 82.74 10.34 8.3 7 17.08 34.15 4.27 3.4 14 12.78 25.56 3.16 2.6 28 6.41 12.82 1.60 1.3 0203 100 1 36.58 73.17 9.15 7.3 7 18.24 36.48 4.56 3.6 14 12.63 25.26 3.16 2.5 28 6.34 12.69 1.59 1.3 0403 300 1 113.40 227.9 28.4 22.8
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1 9 7 43.91 87.81 10.98 8.7 14 20.97 41.95 5.24 4.2 28 11.41 22.81 2.85 2.3 1 340.38 680.76 85.10 68.1 7 at. at. at. at. 0204 600 14 87.53 175.06 21.88 17.5 32 52.86 105.72 13.22 10.6 1 343.93 687.86 85.60 68.8 0205 1000 7 155.28 310.56 38.82 31.1 14 105.91 211.81 26.48 21.2 30 38.92 77.84 9.73 7.8
[00350] Effect of serum components on induced cytotoxicity 1MAB362 [00351] The ADCC activity of mAbs can be impared in the presence of human serum. The effect of sera from patients with ADCC activity was investigated. For this purpose, the serum from each patient collected 7, 14 and 28-32 days after administration and 1MAB362 thus representing factors of the patient's most administered IV circulating IMAB362 complement was used. All serum samples were diluted to 25% (v / v) of final concentration serum and the remaining IMAB362 concentration in each patient test sample was calculated (Table 8). PBMCs from a healthy donor were used as effectors and NUGC-4 cells as target cells (E: T = 40: 1 ratio) in this ADCC assay. All trials for all patients were performed in parallel in a single experiment using the same conditions, target cells and donor PBMCs to ensure comparability. As a functional test to control the
164/217 set of healthy human serum was enriched with IMAB362 (200, 0 µg / ml). As an additional positive control, individual patient pre-infusion serum samples were increased with 200.0 μ / πι1 IMAB362 (representing in vitro ECso-ioo for IMAB362 in this system).
[00352] It was observed in all trials that IMAB362 antibodies present in the patient's serum after administration are highly active and induce cytotoxicity (Figure 5). Biological activity of IMAB362 was retained more than 28-32 days after administration with specific death was still above 48% in all dose groups. Global differences between dose groups were surprisingly modest, suggesting a saturation effect. In patients treated with low doses (33-300 mg / m 2) a moderate decrease in specific death
77.7-87.4% up to 48.3-66.8% was observed over time, correlating with the decrease in serum antibody concentrations (Figure 5 top panel). Greater activity maintained steadily over time was observed in patients treated with 600 or 1000 mg / m ² IMAB362 (Figure 5 bottom panel).
[00353] This assay was repeated with serum samples, in which complement factors were inactivated, incubating them at 56 ° C for 30 min. Cytotoxicity with samples of heat-inactivated patient serum was
lower than those obtained with serum samples
165/217 not treated in all cases. Similar decreases were also observed with the pool inactivated by heat from healthy donors (HSC, Figure 6).
[00354] In summary, these data indicate that the patient's serum does not inhibit the ADCC ability of soluble serum components but instead adds to the total cytolytic activity induced by IMAB362.
[00355] CDAB-mediated IMAB362 kinetics in the serum of patients [00356] In order to determine the kinetics of the IMAB362 CDC capacity in the serum of patients from different serum sample dosing groups were collected 1, 7, 14 and 28 days after the IMAB362 administration.
[00357] Once again, this serum served as a source of complement, as well as for IMAB362. Final serum concentrations were adjusted to 20% (v / v), final volume. Final IMAB362 concentrations in each CDC assay sample are listed in Table 7. As a positive control, pre-infusion patient samples were mixed with fresh IMAB362 antibody to a final concentration of 10 µg / ml (in vitro IM95362 EC95 in this CDC test system). In addition, for the functional control of the CDC assay, serial dilutions of IMAB362 (0.032-10 g / ml) were prepared in human pooled serum. A standardized assay with CHO-K.1 cells stably transfected with luciferase and
166/217
CLDN18.2 were used as target cells. All samples of
serum were thawed and tested in parallel in it experience.[00358] Activity in CDC correlates well with the concentration antibody in each sample of serum (Figure 7).
Most importantly, the data suggest that cytototxic-mediated CDC activity is maintained over 4 weeks. In particular, patients in the high dose groups did not show any gout CDC activity over this time.
[00359] Summary and conclusions [00360] Patients with GEC do not appear to be impaired in their ability to induce both ADCC and CDC of CLDN18.2 expressing target cells in conjunction with IMAB362. Notable, max. Specific lysis seen in ADCC and CDC and EC50 measured for ADCC were greater for patient 0204, who had the most prominent clinical response and tumor antigen serum.
[00361] Ex vivo analysis of CDC with IMAB362 circulating at different time points after its administration showed that still 2 weeks after administration there is sufficient active IMAB362 circulating in patients to induce deep ADCC and CDC.
[00362] Patients' CDC activity in conjunction with IMAB362 circulation is reduced over time for reasons hitherto unknown.
167/217 [00363] Example 3: Cytokines [00364] Cytokine levels can serve as indicators of a patient's immune status. In this clinical trial, the aim of analyzing cytokines was mainly to support safety monitoring. We reviewed cytokines within this accessory analysis from the point of view of defining potential candidate biomarkers.
[00365] Cytokine levels were determined on day 1 before the 1MAB362 infusion and on days 3 and 5 of the treatment cycle. Studied cytokines pro-inflammatory compound (IL-1, IL-6, IL-12, IFNy, TNFa) and anti-inflammatory (IL-4, IL-10) cytokines and cytokines necessary for the growth and function of T cells (IL -2) N and cell proliferation (IL-2, IL-15).
[00366] Cytokines were analyzed by ELISA and flow cytometry (Interlab). Cytokines were analyzed according to Interlab SOP-U-IMM.M.0144. 05 Flow Cytomix Cytokin- Check IL4, IL6, IL13, TNF-alpha, IFN-gamma, MCP-1, IL10 IL2, mal-β, IL12p70, IL8, IL17A, IL23 and SOP-MUIMM.M.0151.02 Interleukin Humanes 15.
[00367] The cytokine levels of IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-15, IFNy, and TNFa were analyzed for 14 of the 15 patients (Table 9 ). Without cytokine levels, patient 0403 (300 mg / m ^{2) was determined.} The serum cytokine level values only, which were
168/217 above the reference range, were analyzed for temporal changes. The reference range values have been defined by Interlab (see CSR GM-IMAB-001).
[00368] Table 9: serum levels of cytokines on the day
1, day 3 and day 5 [00369] The cytokine levels of all patients were measured on a dial, day 3 and day 5 are indicated. The reference interval for each cytokine. Values below or above the detection limit have been set for the respective detection limit for the calculations.
Patient 0103 0104 0201 0105 0202 0203 1101 1201 0204 0302 1202 0106 0112 0205 Dose [mg / m ² ] 33 100 300 600 1000 (reference range [g / mL]) Day Pro-inflammatory cytokines [g / mL]: IL-1(<5.2) 1 <4.2 <4.2 <4.2 <4.2 <4.2 11.7 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 3 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 5 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 <4.2 IL-6 (<4.5) 1 <1.2 <1.2 <1.2 10.7 <1.2 8.3 <1.2 2.6 6.4 4.2 2.8 <1.2 16 4.2 3 <1.2 25.7 <1.2 5.2 <1.2 5.9 <1.2 2.6 123 4.2 12.5 <1.2 <1.2 2.8 5 <1.2 11.6 <1.2 5.2 <1.2 7.5 5.3 <1.2 27.9 4.2 23.4 <1.2 <1.2 5.6 IL-12 (<11.6) 1 <1.5 <1.5 3.9 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 3 <1.5 <1.5 3.9 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 IFNy (<45.0) 1 <1.6 <1.6 182 <1.6 <1.6 26.2 30.7 <1.6 <1.6 <1.6 81.2 <1.6 <1.6 35.9 3 <1.6 <1.6 173 <1.6 <1.6 <1.6 <1.6 <1.6 <1.6 <1.6 <1.6 <1.6 <1.6 <1.6 5 <1.6 <1.6 71.8 <1.6 <1.6 <1.6 3.5 <1.6 <1.6 <1.6 <1.6 <1.6 <1.6 <1.6 TNFa(<17.5) 1 <3.2 <3.2 29.8 <3.2 <3.2 10.1 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 3 <3.2 <3.2 15.7 <3.2 <3.2 4.6 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 5 <3.2 <3.2 10.1 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 Anti-inflammatory cytokines [g / mL]: IL-4(20.8) 1 <20.8 <20.8 57.1 <20.8 28.1 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 3 <20.8 <20.8 <20.8 <20.8 21.2 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 5 <20.8 <20.8 <20.8 <20.8 28.1 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 <20.8 IL-10(<6.0) 1 20.7 27.3 38.1 <6.0 20.7 14.1 9.0 <6.0 <6.0 <6.0 <6.0 <6.0 <6.0 <6.0 3 19.4 23.3 28.6 <6.0 22.0 <6.0 <6.0 <6.0 <6.0 <6.0 <6.0 <6.0 <6.0 <6.0 5 20.7 27.3 23.3 <6.0 22.0 <6.0 13.6 <6.0 <6.0 <6.0 <6.0 <6.0 <6.0 <6.0 Cytokines for T cells and NK cell function and proliferation [g / mL]: IL-2 1 <16.4 <16.4 354 <16.4 55.3 45.4 <16.4 <16.4 <16.4 <16.4 42.8 <16.4 <16.4 42.8 (<20.0) 3 17.3 <16.4 259 <16.4 <16.4 <16.4 <16.4 <16.4 <16.4 <16.4 <16.4 <16.4 <16.4 <16.45 <16.4 <16.4 162 <16.4 <16.4 <16.4 71.4 <16.4 <16.4 <16.4 <16.4 <16.4 <16.4 <16.4 IL-15 (<3.0) 1 <3.0 698 <3.0 <3.0 51 <3.0 <3.0 18.3 <3.0 <3.0 23.1 <3.0 <3.0 <3.0 3 <3.0 646 <3.0 <3.0 43 <3.0 <3.0 18.3 <3.0 <3.0 14.7 <3.0 <3.0 <3.0 5 <3.0 582 <3.0 <3.0 47.7 <3.0 <3.0 11.7 6.0 <3.0 10 <3.0 <3.0 <3.0
[00370] levels of proinflammatory cytokines (IL-1,
IL-6
IL-12
IFNy, TNFa) were above their respective
169/217 reference values in 9 of the 14 patients (0104, 0105, 0201, 0203, 0204, 01 12, 1202, 01 12, 0205). IFNy levels were elevated in two patients (0201, 1202), the TNFa level was elevated in one of these two patients (0201). In both patients, IFNy and TNFa levels were elevated before IMAB362 administration and decreased in the following days. IL-6 levels were elevated in eight patients (0104, 0105, 0203, 1 101, 0204, 01 12, 1202, 0205). No clear pattern in IL-6 level changes with respect to the IMAB362 administration and dose-effect relationship becomes evident. Patient 0204's IL-6 (600 mg / m ² IMAB362) was not elevated before administration, but increased considerably 2 days after infusion, not a pattern exhibited by any other patient. The levels of IL-1 and IL-12 remained within the respective reference range for all patients.
[00371] The levels of anti-inflammatory cytokines (IL4, IL-10) were above the respective reference range in 6 of the 14 patients (0103, 0104, 0201, 0202, 0203, 1 101). IL-10 levels were elevated in six patients (0103, 0104, 0201, 0202, 0203, 1 101), IL-4 were high levels in two of these patients (0201, 0202). Fluctuations in levels of anti-inflammatory cytokines show no clear pattern with regard to IMAB362 administration and dose-effect relationship.
[00372] Cytokines for T cells and function of
170/217 NK cells and proliferation of IL-2 and IL-15 levels were above the respective reference range in 9 of the 14 patients (0104, 0201, 0202, 0203, 1 101, 1201, 0204, 1202, 0205). IL-2 levels were above the reference range in six patients (0201, 0202, 0203, 1 101, 1202, 0205), IL-15 levels were above the reference range in five patients (0104, 0202, 1201, 0204 , 1202). Seven of the nine patients (0104, 0201, 0202, 0203, 1201, 1202, 0205), with elevated levels of IL-2 / IL-15 pre-administration exhibited a decrease in the level of cytokines on subsequent days: IL-2 / IL -15 levels were above the respective reference range before IMAB362 administration and decreased on the second and fourth day after IMAB362 administration. The most pronounced decrease in this group was observed for serum IL-2 concentrations. In all five patients (0201, 0202, 0203, 1202, 0205) with the increase in IL-2 pre-infusion levels a reduction to less than 50% of the respective pre-infusion levels was observed on the fourth day after administration. This decrease can also be seen in one patient (0201) with a considerably high level of IL-2 (354 pg / ml) before the administration of IMAB362 of 33 mg / m ² .
[00373] IL-2 was different from the concentration profile shown by patient 1101 (300 mg / m ² IMAB362) with levels of IL-2 in the reference range before infusion and 2 days later, but high levels of IL -2 concentration
171/217 on the fourth day after the infusion.
[00374] The level of IL-15 was decreased on the fourth day after administration in all four patients (0104, 0202, 1201, 1202), with high levels of pre-infusion IL-15. This concentration profile is very similar to that of IL-2 observed in the concentration profile, although the decrease in the relative level is not as pronounced.
[00375] No dose-effect relationship can be discerned by any of the analyzed cytokines.
[00376] Summarizing the above, analysis of pre-treatment levels of patients showed that IL-6, IL-10, IL-2, IL-15 are elevated in a substantial fraction of patients with late gastroesophageal disease. In contrast, none or only the individual patients had elevated levels of IL-1, IL-12, IL-4, IFNy, and TNFα.
[00377] Analysis of changes in cytokine levels in the first 5 days after treatment led to IMAB362 following observations. In all five patients with high levels of IL-2, these levels were found to decrease profoundly, with four out of five patients who reached normal reference values. Likewise, a moderate decrease was observed in all four patients with high levels of IL-15 after IMAB362 administration. Decreases in elevated levels after treatment have also been observed for individual patients with elevated levels
172/217 of IFNy and TNFa, respectively. IL-6, in contrast, increased after IMAB362 administration, four pre-treatment patients and 7 out of 14 patients on day 5 post-treatment showing IL-6 above reference levels.
Example 4: International, multicentre, open, phase IIa multiple dose study to assess the efficacy and safety of multiple doses of IMAB362 in patients with advanced adenocarcinoma of the stomach or lower esophagus [00378] An international, multicentre, open-phase study Multiple dose Ila was performed to investigate the efficacy and safety of multiple doses of IMAB362 in patients with advanced adenocarcinoma of the stomach or lower esophagus. The main objective of this study was to study the remission rate (CR, PR) according to RECIST. The secondary objectives of this study were: frequency and severity of adverse events, according to CTCAE v3.0 and tolerability of multiple doses of IMAB362, time to progression-free survival (PFS): The time from the beginning of the first infusion to the date of first observed disease progression or death from any cause (whichever comes first), immunogenicity by analysis of human anti-chimeric antibodies, quality of life, clinical benefit (CR, PR and SD according to RECIST) and pharmacokinetics of IMAB362 by levels serum.
[00379] Patients underwent screening for
173/217 determination of the presence of target IMAB362 CLDN18.2 in your tumor. CLDN18.2 status was determined by immunohistochemistry with an anti-claudin-18 antibody, to be performed according to a standardized protocol. Patients with tumors with at least 50% of the cells were stained with at least 2 + (double intensity) staining intensity were enrolled in this trial. The inclusion and exclusion criteria were verified during the screening visit (VI). Patients were recruited from university hospitals specializing in the treatment of gastroesophageal cancer.
[00380] Patients had to fulfill all inclusion criteria:
• Metastatic, refractory or recurrent disease of advanced adenocarcinoma of the stomach or lower esophagus confirmed by histology • CLDN18.2 expression confirmed by immunohistochemistry in a tissue sample embedded in paraffin of the tumor in at least 50% of the tumor cells with an intensity of staining at least 2+ (on a scale of 0 to 3+) • At least one measurable site of disease, according to the RECIST criteria (CT scan or MRI no more than 2 weeks before the 2 visit) • Age > 18 years • Written informed consent
174/217 • ECOG performance status (PS) 0-1 or Kamofsky index of 70 - 100% • Life expectancy> 3 months • Platelet count> 100,000 / mm ³ • Hemoglobin> 10 g / dl • Bilirubin normal • AST and ALT <2.5 times the upper limit of normal (LSN) (5 times LSN if liver metastases are present) • Creatinine <1.5 x ULN • For women of childbearing age (last menstruation less than 2 years) before enrollment): Negative pregnancy test (β-HCG) at baseline and using two highly effective methods of contraception during the treatment phase and for 8 weeks after the last infusion of the study drug dying • Male patients whose sexual partners were women of childbearing potential and had to use an accepted method of contraception during the treatment phase and for 8 weeks after the last study drug infusion [00381] Patients who had any or more of the following criteria exclusion students were not eligible to enter the study:
• Pregnancy or breastfeeding • severe allergic reaction before or intolerance to a
175/217 monoclonal antibody, including chimeric or humanized antibodies • Less than 3 weeks since previous chemotherapy or radiation therapy • Other experimental agents or devices simultaneously or within 4 weeks before this study • Other concurrent anticancer therapies (not for indication under treatment in study) • Known HIV infection or known active hepatitis (A, B, C) • Concomitant anticoagulation with vitamin antagonists (eg Coumadin, Marcumar) • Therapeutic doses of heparin (prophylactic doses are accepted) • Uncontrolled disease, including , but not limited to any of the following:
> Course or active infection requiring parenteral antibiotics;
> Symptomatic congestive heart failure;
> Unstable angina pectoris;
> Uncontrolled hypertension;
> Clinically significant cardiac arrhythmia;
> Myocardial infarction in the last 6 months;
> Gastric bleeding within the last four weeks;
176/217> Symptomatic peptic ulcer;
> Clinical symptoms of brain metastasis; and> Psychiatric illness or social situations that would prevent study completion.
[00382] All patients in all groups received doses of IMAB362 every two weeks, repeated on visits 2, 5, 6, 7 and 8 (5 applications). The dose escalation procedure comprised the following cohorts with
two doses many different (antibody / area in surface body) of IMAB362:[00383] Cohort of 1: 300 mg / m 2 [00384] Cohort 2: 600 mg m ² [00385] Cohort 3: 600 mg m ² [00386] The solution antibody was given as a intravenous infusion of 2 h every two weeks. Was important
that the infusion time was not less than 2 hours. For the infusion, an infusion system (for example Infusomat ^® FMS) had to be used in order to control the infusion time. The infusion set delivered with the study drug, which was tested for compatibility with the manufacturer, had to be used for the application of the drug. The study drug infusion time had to be in the morning. A qualified doctor had to be available for the time during the infusion and 24 hours later.
[00387] Thirty-seven patients received at least
177/217 a treatment. Unfortunately, during 3 their documentation is not completely in database 34 so that patients will be included in the set of all treated patients (APT set) and will be used for security analysis. Four, six and 24 patients were allocated, respectively, to cohort 1 with 300 mg / m ² IMAB362, cohort 2 with 600 mg / m ² IMAB362 and cohort 3 with 600 mg / m ² IMAB362.
[00388] During the treatment phase of one patient in group 1, three patients in group 2, and 12 patients in group 3 discontinued the study before receiving 5 infusions of IMAB362 and completed visit 9 (incl second image. Tumor) two weeks after the fifth infusion. These patients were replaced.
[00389] Two patients in group 2 had no measurable disease at baseline and were excluded from the analysis of efficacy. Minor protocol deviations, such as baseline tumor assessment> 14 days earlier than Visit 2 (n = 3; 8.8%), hemoglobin <10 g / dL (n = 5; 14.7% ), abnormal bilirubin values (n = 3, 8.8%), ALT or AST> 2.5 ULN (> 5 ULN in case of liver metastases) (n = 2; 5.9%), a value of creatinine> 1.5 ULN (n = 1; 2.9%) and prolonged time windows (> 15 days) between the screening period and the start of treatment (n = 2; 5.9%) occurred, but not led to the exclusion of any analysis. a
178/217 patient had a myocardial infarction in the last 6 months. The waiver was granted.
[00390] Since in cohort 2 and 3, patients received the same dose of 600 mg / m ^{2, it was} decided to analyze these patients as a group. All patients (n = 34) in the APT set were Caucasian. The mean age was 62 (range 45-65 years) in the dose group of 300 mg / m ² and 61 in the dose group (range 42-77 years) of 600 mg / m ² .
[00391] An overview of the cancer location and the result of the histopathological classification is shown in Table 10. The average time between the first screening and diagnosis visit for this study was 16 weeks (2.7 min / max 56 ). The expression status of HER2 / neu was mostly unknown to patients, except in 5 patients treated with 600 mg / m ^2. One of these patients was 5 HER2 / neu positive.
[00392] TNM classification was specified for stomach cancer (n = 16) and the esophageal or gastroesophageal junction (n = 19). In the APT group, 25% of patients had primary tumors of the stomach classified as Tl or 2, 31% had T3, T4 25% with primary tumors and 19% were unknown. Sixty-nine (69)% of patients in the APT pool had at least one or two infiltrated lymph nodes indicated by classification n 1 and 56% of patients suffered from peripheral metastases (ml) in the
179/217 moment of diagnosis. Sixty-nine (69)% of patients with cancer of the esophagus or gastroesophageal junction were diagnosed with> T3. At least one or two infiltrated lymph nodes (NL) were reported by 84% of patients. In addition, 84% of patients had peripheral metastases.
[00393] Table 10: Overview of the site and type of tumor in the first diagnosis (One patient had esophageal cancer and stomach cancer; several patients had stomach cancer affecting different parts of the stomach).
300 mg / m ² N (%) 600 mg / m ² N (%) APT set N (%) Number of patients 4 30 34 Esophagus - 2 (6.7) 2 (6.7) Gastroesophageal Junction 1 (25.0) 16 (53.3) 17 (50.0) distal - 4 4 cardia - 8 8 subcardia 1 2 3 unspecific - 2 2 Stomach 3 (75.0) 13 (43.3) 16 (47.1) fundus - 2 2 corpus - 6 6 antrum - 3 3 pylorus - - - unspecific 3 6 9 Type of tumor intestinal - 8 8 diffuse 1 6 7 signet ring cell CA - 4 4 mixed - 1 1 unspecific 3 12 15 Histopathological classification G2 - 10 10 G2-3 1 2 3 G3 1 14 15 G3-4 - 1 1 Unknown 2 3 5
180/217 [00394] On a MedDRA SOC basis, the most frequent clinically relevant previous diseases were surgical procedures in 25 patients (73.5%), chemotherapy in 30 patients (88.2%) and radiation in 7 patients ( 79.4%). In most cases, surgery consisted of surgical removal of organs (such as gastrectomy (72%), esophagectomy (16%), lymphadenectomy (32%), cholecystectomy (20%)). All patients, with the exception of four, had at least one prior therapy for their study disease. On a WHO DD ATC basis, the most used drugs were pyrimidine analogues (tluorouracil and / or capecitabine), platinum compounds (cisplatin and / or oxaliplatin), and detoxifying agents for antineoplastic treatment (calcium folinate and / or folinic acid) . Other previous medical treatments (ending on the day of the infusion at the latest) have also been documented.
[00395] A total of 30 of the 34 patients (88.2%) had at least one concomitant disease, that is, a disease that was occurring on the day of the study medication infusion. On a MedDRA SOC basis, the most common diagnoses were gastrointestinal disorders' in 19 patients (56%), general disorders in 12 patients (35%), 'metabolism and nutrition' in 10 patients (29%) and Tissue disorders musculoskeletal and connective tissue 'in 8 patients (23.5%).
Concomitant therapies were
181/217 mainly drugs for acid-related disorders (17 patients; 50%), analgesics (12 patients; 35.3%) and drugs for GI diseases (10 patients; 29.4%).
A. SAFETY ASSESSMENT [00396] Since the study medication injections were administered by the investigators at the study centers and the patients had to remain in the hospital for observation for at least 24 hours and up to 72 hours, overall compliance was ensured according to the study protocol. The assignment of the themes eligible for the dose cohorts was performed exactly as specified by the study protocol (supervised by the DSMB). The duration of the study, defined as the time from the date of the screening visit part 1 to the last day of the study, varied from min. 18 to max. 355 days. The average duration was 106 study days. 16 patients finished the study prematurely before visiting target 9. Patients in all dosing groups had an average of 4.5 to 5 infusions of IMAB362. The average duration of an IMAB362 infusion in the APT set was 125 minutes. There was one patient with a duration of less than the 120 minutes specified in the protocol. This patient stopped the infusion due to vomiting and prematurely stopped the study.
[00397] The safety analysis was performed for the set of APT comprising all 34 patients who received at least a dose of 300 mg / m ² (N = 4) or 600
182/217 mg / m ² (N
30). Two hundred and forty-one (241) adverse events according to the physician's description were coded according to the MedDRA dictionary and translated into preferential terms.
Adverse events, according to preferential terms, were counted only once for each patient (also if the same adverse event occurred more than once for that patient during the study). The highest grade NCI-CTC occurring in each patient was recorded.
Thirty-two (32, 94%) patients had at least one adverse event (regardless of the relationship) during the study. No adverse events have been documented for patients. A total of 6 patients (18%) did not experience an adverse event, possibly related to the drug.
One hundred and four (104) of the drug adverse events related by preferential terms were reported by 28 patients. Eight (8) of possibly drugs related to serious adverse events were reported in 4 patients. The number of patients in the lowest dose group (300 mg / m ² ) was too small to allow a detailed comparison between the two dose groups. The incidence of patients with related adverse events in the 300 mg / m ² dose group and 600 mg / m ² dose group (group 2 and 3) is 75 and 83%, respectively.
[00398] In total, from AEs, gastrointestinal disorders were more frequently reported by SOCs (27/34
183/217 patient 79.4%), General disorders and changes in the administration site (26/34 patients, 76.5%). On an EDDRA PT basis, the most frequently documented AEs were nausea (57 events in 18 patients), 'vomiting' (52 events in 16 patients) and fatigue (20 events in 14 patients). In total, only 192 of the recorded AEs were assessed by the investigators as being related to the study medication. These treatment-related adverse events were classified into 104 different preferred terms and were observed in 28/34 patients.
[00399] Most related adverse events were mild to moderate. There were 8 (23.5%) patients with treatment of moderate drug-related events emerging and 12 (35.3%) patients with serious treatment-related emergent events.
[00400] Severe drug-related AEs have been reported for patients in the 300 mg / m ² dose group ² , vomiting and concomitant nausea in one patient. In the 600 mg / m ² dose group, 10 patients experienced severe drug-related adverse events, 6 patients with vomiting of whom 3 patients experienced nausea, one patient with hypersensitivity (allergic reaction), one patient with salivary hypersecretion a patient with dehydration, and a patient with hypoalbuminia. The last two patients also reported vomiting and nausea. Two
184/217 patients suffered a related hypersensitivity (allergic reaction) during a drug perfusion study, one of which was classified as moderate and one as severe. Both patients recovered after the infusion were stopped.
[00401] Throughout the study of drug action events emerging from the reported treatment it was necessary for 12/34 patients due to an AE. In 7 (21%) cases, an EA led to the interruption of a permanent study. The underlying adverse event was related to 3 drugs (hypersensitivity (allergic reaction) (n = 2), vomiting and abdominal pain) and unrelated to the other four drug patients (deterioration of general physical health (n = 3), pneumonia). In one patient dose it was reduced and in another administration a patient dose was postponed for 4 days, due to severe vomiting with nausea. In three patients, the infusion was interrupted for a prolonged period. Twenty-seven patients (79%) received concomitant therapy due to an AS. Eleven patients were hospitalized.
[00402] There were 13 patients with 31 S AEs documented. One patient died during the second screening phase of the study. Twelve patients had other serious adverse events, which were related study drugs in four patients. Vomiting, nausea and related adverse events, such as GI hemorrhage and exsiccosis were judged by the investigators to be related to the study medication. There were 4 SARs, and 2 SUSAR (vomiting and vomiting with GI hemorrhage)
185/217 in the present study. The end result was death in seven cases. None of the deaths were classified by the researchers as related to the study medication.
[00403] One patient was male, 45 years old, white, in good general condition (performance status ECOG grade 1, RNAofsky index 80%) with a fine dietary status (BMI 19.3).
[00404] The patient received infusions with 300 mg / m ² IMAB362 every two weeks on Nov 04, on November 22 and the third on December 06, 2011. Before the study, the patient had already suffered grade 1 nausea and vomiting On November 7, 2010 grade 3 vomiting was diagnosed. As it was assessed as severe, the patient had to be hospitalized. When vomiting turned into grade 1 on November 17, 2010 and finally stopped completely, the patient could be discharged from the hospital on the same day. Before the second and third IMAB362 infusion, the patient was treated with a potent premedication (alizapride, aprepitant, metoclopramide, dimehydrinate) as prophylaxis for nausea and vomiting, so that he does not suffer from nausea or vomiting again. The investigator assessed vomiting as related to the study drug. The report was received by the prosecutor on January 19, 2011 and the SAE judged it as not expected, but related to the study drug and therefore classified as SUSAR.
[00405] One patient was a 77-year-old male
186/217 male, white. He was in a very good general condition (ECOG Performance Status: grade 0, KRNAofsky Index: 100%), with normal nutritional status (BMI 24) at screening. Prior to the study, the patient already suffered from nausea and, therefore, he was treated as needed with metoclopramide. The patient received only one application of 600 mg / m ² IMAB362 on 09 Nov 201 1, as the study had to be stopped prematurely due to death. A pleural effusion in the left lung was diagnosed by X-ray before the infusion and reported as SAE. The following morning, hematemesis defined in. After administration of pantoprazole and 8 mg of IV ondansetron, vomiting and decreased hematemesis recovered on the same day. Vomiting decreased from grade 3 to grade 2 and finally stopped on November 12, 1 201, so that the patient can be discharged from the hospital on the 13th: November 201 1. The investigator evaluated the case in relation to the study drug . The report was received by the prosecutor on November 10, 201 1 and case dismissed as not expected, but related to the study drug and therefore classified as SUSAR. The patient's general condition worsened, he developed kidney failure and, unfortunately, died on December 6, 201 1.
[00406] One patient was a 42-year-old Caucasian male in very good general condition (ECOG performance grade 0; RNAofsky index: 100%), with a well-nourished nutritional status
187/217 (BMI 26). The patient received two injections of 600 mg / m ² IMAB362. On March 20, 2012, the patient received the first application of the study drug. As he suffered severe nausea and vomiting, the infusion rate had to be reduced after 35 minutes of infusion. The symptoms were treated with 40 mg of pantoprazole and 3 mg of granisetron and 2 vials of butylscopolamine and 80 mg of aprepitant. This serious adverse event led to prolonged hospitalization. The investigator assessed this event as related to the study drug. The SAE report was received by the prosecutor on March 21, 2012 and the event judged as expected and related to the study drug. A few days later, on March 24, 2012, the patient had to be hospitalized again due to severe dehydration, which was caused by nausea and vomiting. In addition, the patient suffered from pain in the epigastric region. He received 1 g metamizole iv, a buprenorphine patch and infusions for rehydration. On March 30, 2012, the symptoms were relieved and the patient rehydrated. The investigator rated this event as unrelated to the study drug. The SAE report was received by the prosecutor on March 26, 2012 and the event dismissed as unexpected and unrelated to the study drug. On April 3, 2012 the patient received the second infusion, which again led to nausea and vomiting AEs. He was treated with 30 drops of metoclopraminde powder and 1 iv bottle of dimenhydrinat. Like the
188/217 symptoms worsened on April 5, 2012, they were assessed as severe. In addition, the patient was uncomfortable with dysphagia and thus severely reduced food intake. On 15 Apr 2012, the symptoms had disappeared. The investigator assessed this event as related to the study drug. The SAE report was received by the prosecutor on April 19, 2012 and the event judged as expected and related to the study drug.
[00407] One patient was a 73-year-old Caucasian man in good general condition (ECOG performance status grade 1, KRNAofsky index: 90%) with a well-nourished dietary status (BMI 26). From November 8, 201 1 to January 3, 2012, the patient received the five planned applications of the 600 mg / m ² study drug IMAB362 every two weeks. On November 8, 2011, the patient received the first order for IMAB362. During and after the infusion, he suffered from nausea and vomiting. The symptoms became severe on Nov. 9, 2011. After treatment with metoclopramide the symptoms disappeared a day later. The investigator assessed the event as related to the study drug. The SAE report was received by the prosecutor on November 10, 2011 and was deemed expected and related to the study drug. On December 6, 2011 was the third infusion. The patient suffered from moderate vomiting and mild nausea treated with clemastine, ranitidine, and ondansetron. The vomiting lasted for a day.
189/217 [00408] Nausea continued for 7 days. The study ended on January 16, 2012, due to disease progression. No follow-up visit was carried out.
[00409] In conclusion, 1MAB362 has been found to be safe and well tolerated in a heavy pre-treated population of patients with advanced adenocarcinoma of the stomach, esophagus or gastroesophageal junction. In total, from AEs, gastrointestinal disorders' 'the SOCs (27/34 patient 79.4%), General disorders and changes in the administration site' (26/34 patients, 76.5%) were more frequently reported.
[00410] On a MedDRA PT basis, the most frequently documented AEs were nausea (57 events in 18 patients), 'vomiting' (52 events in 16 patients) and fatigue (20 events in 14 patients).
[00411] In total, 192 of the recorded AEs were assessed by the researchers as related to the study medication. These treatment-related adverse events were seen in 28 of 34 patients. Eighty-three (83) percent of these related adverse events were gastrointestinal diseases (68%, 130 AEs) recorded in 25 patients and general disorders (15%, 29 AEs) recorded in 16 patients.
[00412] On a MedDRA PT basis most related adverse events were mild to moderate with nausea (50%),
190/217 vomiting (47%), fatigue (27%), abdominal pain (15%), peripheral edema (15%), loss of appetite (12%) and diarrhea (12%.) Occurring in more than 10% of patients.
[00413] Two patients suffered a related hypersensitivity (allergic reaction), during study of drug perfusion, one of which was classified as moderate and one as severe. Both patients recovered after the infusion was stopped.
[00414] No study abnormal laboratory values related to drug grade 4 CTC (life-threatening) or 5 (death) have been reported.
[00415] There were 12 (35.3%) patients with serious treatment related emergent events. Severe drug-related AEs have been reported for patients in the 300 mg / m ² dose group ² , vomiting and concomitant nausea in one patient. In the 600 mg / m ² dose group, 10 patients experienced serious adverse drug-related events, 6 patients with vomiting, 3 of whom experienced nausea, 1 patient with hypersensitivity (allergic reaction), 1 patient with salivary hypersecretion, 1 patient with dehydration and one patient with hypoalbuminia The last two patients also reported vomiting and nausea.
[00416] At the time of analysis 13 patients have recovered from all related adverse events
191/217 with the drug, 2 patients were recovering, 11 patients did not recover from at least one of AE and 2 for the state was unknown. Of the 11 patients in a related adverse event, at least one drug was not recovered 9 had gastrointestinal disorders (4 nausea, 2 vomiting).
[00417] There were 13 patients with 31 documented SAEs, including 7 deaths. One patient died during the screening phase, that is, before the start of the infusion of the study drug, and was therefore classified as a screening event. In four patients treated for emergent gastrointestinal SAEs like vomiting (n = 4), nausea (n = 2), exsiccosis (n = l) and GI hemorrhage (n = l) were judged to be treatment related. One of these patients with vomiting was treated with 300 mg / m ^{2 and} the other three were treated with 600 mg / m ² IMAB362. Three of these four patients recovered except for one who died from unrelated kidney failure.
[00418] The incidence of drug-related adverse events was comparable between dose groups 300 and 600 mg / m ² with 75% and 83% of patients, respectively. The frequency and severity of nausea, vomiting and fatigue was also comparable between both groups. There was no clear relationship between the dose and the frequency and / or severity of adverse events.
[00419] The profile of adverse effects with the majority of
192/217 adverse events reported for the gastrointestinal tract coincide with the underlying disease and also the expression profile CLDN18.2. It is suggested that nausea and vomiting are an effect on the target, since CLDN18.2 is also expressed in gastric epithelial cells (in tight junctions).
[00420] Generally speaking, IMAB362 given in multiple doses of 300 and 600 mg / m ^{2 has} been found to be safe and well tolerated with vomiting and nausea being the most common related adverse event.
B. PHARMACOKINETIC AND IMMUNOGENICITY EVALUATION [00421] Preliminary drug concentration data for repeated dose application of IMAB362 is available for the four patients in the first and 34 patients in the second and third groups, who received 300 mg / m ² and 600 mg / m ² IMAB362, respectively.
Table 11: Cmax (maximum drug concentration in serum) after the first and fifth administration of IMAB362
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Cohort / dose Patient cmax after lst infusion [g / mL] cmax after 5th infusion [g / mL]1001-01 349.6 ± 179.2 ** 293.9 ± 26.9 Cohort 1 1001-07 341.6 ± 22.1 at. at. [300 mg / m ² ] 2002-02 253.3 ± 7.1 326.5 ± 3.61001-10 208.9 ± 3.2 259.1 ± 8.21005-03 343.6 ± 10.8 at. at.1005-04 256.9 ± 5.7 at. at. Cohort 2 1005-11 at. at. at. at. [600 mg / m ² ] 1001-08 325.1 ± 12.6 485.4 ± 6.72002-05 272.2 ± 3.6 642.6 ± 17.92002-07 325.3 ± 10.0 516.7 ± 1.91011-05 296.1 ± 13.7 385.8 ± 15.11013-02 310.9 ± 4.7 428.0 ± 17.94001-11 323.4 ± 8.9 at. at.1005-10 389.8 ± 0.7 457.6 ± 8.01001-24 390.8 ± 6.7 433.8 ± 6.61001-27 309.3 ± 10.7 at. at.1003-10 300.7 ± 2.1 at. at.1004-07 at. at. at. at.1004-10 448.3 ± 5.7 at. at.1004-11 at. at. at. at.1005-18 269.2 ± 6.7 at. at.1005-26 at. at. at. at.1005-27 473.6 ± 10.6 at. at. Cohort 3 1005-29 280.2 ± 16.7 at. at. [600mg / m2] 1005-34 385.5 ± 18.8 at. at.1006-03 317.0 ± 14.8 278.1 ± 8.91006-05 276.0 ± 18.2 at. at.1007-09 397.0 ± 12.5 at. at.1011-09 411.1 ± 12.1 535.9 ± 17.01011-16 322.5 ± 8.5 314.5 ± 15.61011-17 462.3 ± 10.6 at. at.1011-20 347.9 ± 14.4 570.3 ± 10.11012-01 433.7 ± 9.7 at. at.2003-08 575.1 ± 30.7 at. at.2003-10 421.8 ± 3.4 520.5 ± 6.52003-13 344.7 ± 21.6 at. at.2003-15 380.9 ± 3.1 at. at.2003-16 466.8 ± 6.8 at. at.
** high CV results from one outlier in the triplicate measurement [00422] Blood samples were taken before each infusion. After the infusion first additional samples were taken at the end of the infusion and 1, 1.5, 2, 3, 4, 6, 12, 24 hours, 3 and 6 days after the end of the infusion. After
194/217 last infusion samples were taken at the end of the infusion and 1, 1.5, 2 hours and 14 days, as well as 4 to 8 weeks after the end of the last infusion. No analyte can be detected in the pre-dosage samples from individual patients assigned to cohort 1-3.
[00423] After the first IMAB362 infusion, Cmax values varied between 208, 9 pg / ml and 349.6 pg / ml for the first group. For the second and third groups, taken together, the values varied between 269.1 pg / ml and 575, 1 pg / ml, after the first application.
[00424] In serum samples taken at subsequent time points (V3 to V5), a concentration-dependent reduction time of IMAB362 was observed (Figure 8). At visit 5 before the second infusion, minimum serum levels between 11.3 µg / ml and 36.8 µg / ml (mean value of 22.5 ± 10.5 µg / ml) were determined for the cohort of 1 and 17, pg / ml and 100.2 pg / ml (mean value 54.5 ± 29.0 pg / ml) for cohort 2 and 3 taken together.
[00425] At visit 8 (day 57) before the fifth minimum serum levels of infusion between 32.4 pg / ml and 67.1 pg / ml (mean value of 46.1 ± 18.5 ug / ml) were determined for the cohort of 1 and 28.3 pg / ml and 301.6 pg / ml (mean value of 147.2 ± 93.1 ug ml) per cohort 2 and 3 (Table 12).
[00426] After infusion at visit 8, Cmax values varied between 259.1 pg / ml and 326.5 pg / ml, for the first group and
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278.1 ng ml and 642.6 ng / ml for cohort 2 and 3 (Table 1 1).
[00427] For cohort 1, it means that Cmax values were determined for 90 min after the first IMAB362 infusion (270.6 ± 63.9 ug / ml) and 90 min after the fifth infusion (279.2 ± 27.7) . For cohort 2 and 3 together, mean C max, values were determined at the end of the first IMAB362 infusion (340.8 ± 80.2 ug / ml) and 60 min after the fifth infusion (443.3 ± 97.7) ( Table 12).
[00428] In summary, serum levels of IMAB362 measurements showed that in patients treated with 300 mg / m ² the serum concentration of IMAB362 falls below the desired level of 50-100 g ml between 2-week cycles. At a dose of 600 mg / m ² , in contrast, in the vast majority of patients IMAB362 serum levels were above 50 pg / ml, up to 2 weeks after the first application ^. Seven to 29 days (mean value of 15 days) after administration of 5, the dose level was above 50 pg / ml (mean value ± 151.3 90.1 ng / ml).
[00429] Table 12: Descriptive pharmacokinetic data from repeated administration of 300 and 600 mg / m ² of IMAB362 [00430] Mean ± SD concentration pg / ml) of IMAB362 in the serum of 4 patients treated with repeated doses of 300 mg / m ² (cohort 1) and up to 30 patients (first infusion of 30 patients, fifth infusion 12 patients), treated with repeated doses of 600 mg / m ² (cohort 2 and 3 together cohort).
[00431]
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Concentration (mean ± SD) IMAB362 [g / ml] Dose of IMAB362 300 mg / m ² 600 mg / m2 c _m x 1 after ^the infusion 270.6 ± 63.9 340.8 ± 80.2 Pre-dose level prior to the second infusion 22.5 ± 10.5 54.3 ± 29.0 Pre-dose level before the third infusion 34.9 ± 15.8 89.9 ± 62.3 Pre-dose level before the 4th infusion 44.9 ± 14.5 128.7 ± 80.6 Pre-dose level before the 5 infusion 46.1 ± 18.5 147.2 ± 93.1 c _m x 5 after ^the infusion 279.2 ± 27.7 443.3 ± 97.7 After administration 5 34.5 ± 12.2 151.3 ± 90.1
[00432] A slight accumulation of IMAB362 was observed from cycle to cycle. Accumulation factors ranged from 1.03 times to 3.52 times based on the first pre-dose value before the second infusion (mean value of 2.04).
Table 13: Accumulation of IMAB362 after repeated infusions [00433] To determine the accumulation factors, IMAB362 concentration ratios before visits 6, 7, 8 and 9.x (responder) and before the second infusion (visit 5) were calculated.
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Cohort 3 pre-infusion Accumulation factor Production clone V6 / V5 V7 / V5 V8 / V5 V9.1 / V5 V9.2 / V5 V9.3 / V5 V9.4 / V5 Patient 101105 F02 1.73 2.10 2.16 Patient 101302 F02 1.78 2.36 2.08 1.70 1.82 2.03 2.28 Patient 400111 F02 2.04 2.44Patient 100510 # 15 1.83 2.41 2.24 Patient 100124 # 15 2.33 2.69 2.42 1.98 3.17 2.54 1.49 Patient 100410 # 15 3.05 Patient 100529 F02 1.49 1.66Patient 100603 F02 1.21 1.39 1.66 1.92 3.52 Patient 100605 F02 1.12 1.83Patient 101109 # 15 2.12 2.70 2.29 Patient 101120 # 15 1.71 2.12 2.39 Patient 200310 # 15 1.03 1.35 1.30
[00434] In conclusion, the pharmacokinetics of IMAB362 were found to be dose dependent.
[00435] After the first ΙΜΆΒ362 infusion, Cmax values varied between 208, 9 pg / ml and 349.6 pg / ml for the first group. For the second and third groups, taken together, Cmax values varied between 269.1 pg / ml and 575, 1 pg / ml, after the first application.
[00436] In serum samples taken at the subsequent time points (V3 to V5), a reduction time dependent on the concentration of IMAB362 was observed. At visit 5 before the second infusion, minimum serum levels between 1 of 1.3 pg / ml and 36.8 pg / ml (mean value of 22.5 ± 10.5 g / ml) were determined for cohort 1 and 17 , 0 g / mL and 100.2 mg / mL (mean value of 54.5 ± 29.0 ng / mL) during cohort 2 and 3 taken together.
[00437] At visit 8 (day 57) before serum levels of minimum guinto infusion between 32.4 µg / ml and 67.1 µg / ml (mean value of 46.1 ± 18.5 µg / ml) were determined
198/217 for the cohort of 1 and 28.3 ug / mL and 301.6 μ ³ 4 / nil-, (mean value 147.2 ± 93.1 μ ³ 4 / nil.) For cohort 2 and 3.
[00438] After 5 infusion on business 8, Cmax values ranged from 259.1 g / ml and 326.5 g / ml for the first group and 278.1 ug / ml and 642.6 g / ml for the cohort 2 and 3.
[00439] For cohort 1, it means that the Cmax values were determined at 90 min after the first IMAB362 infusion (270.6 ± 63.9 μg / ml) and 90 min after the fifth infusion (279.2 ± 27.7 ). For cohort 2 and 3 together, they mean that the
values from C ax were determined in the end gives first infusion IMAB362 (340.8 ± 80.2 μg / ml) and 60 min after the fifth infusion (443.3 ± 97.7). [00440] In summary, the levels serum in measured
IMAB362 showed that in patients treated with 300 mg / m ² the serum concentration of IMAB362 falls below the desired level of 50-100 μ§ / ηύ between 2-weekly cycles. At a dose of 600 mg / m ^2, in contrast, in the vast majority of IMAB362 patients, serum levels were above 50 μg / ml, up to 2 weeks after the first application ^. Seven to 29 days (mean value of 15 days) after the 5th administration, the dose level was above 50 µg ml (mean value of 151.3 ± 90.1 µg / ml). C. EVALUATION OF ANTITUMORAL ACTIVITY
Full definition analysis (FAS):
[00441] Included all individuals who received study medication at least once and for whom data
199/217 effectiveness after treatment was available.
[00442] At the time of analysis, 50 patients were enrolled at a dose of 600 mg / m ² · Nine of them were included recently and no more data is currently available due to their recent enrollment. During ten patients no second imaging of tumors has been performed and, therefore, these patients are not included in the FAS set. The FAS set comprises 31 patients.
[00443] The average age was 57 years old with a range of 35 to 77. Patients in the FAS set had a median in the amofsky index of 90% (range 70-100%). The vast majority (81%) of patients have been pre-treated with at least one chemotherapy regimen. 6 (six) patients do not have a previous chemotherapy regimen.
Table 14: Details on previous chemotherapy regimens in the FAS set (n = 31).
5-FU / Capecitabi ne Platinum Compound Taxane Epirubicin Irinotec an mABs / other agents No. of patients 25 (81%) 23 (74%) 14 (45%) 8 (26%) 8 (26%) 6 (19%)
[00444] The median number of previous chemotherapy regimens was 2.0 (range 0 to 5). Chemotherapy regimens for gastroesophageal cancer mainly consist of various combinations of 5-FU derivative, composed of platinum, taxanes, epirubicin, irinotecan, trastuzumab for HER2 / neu positive patients and others
200/217 investigative agents. In the defined FAS 81% of patients had at least 5-FU or capecitabine at least 74% and were treated with a platinum compound at least once before inclusion. Six (6, 19%) patients were pretreated with trastuzumab or other investigative agents. Six (6, 19%) patients also had radiotherapy before the study started.
[00445] Due to the late stage of the disease, patients had an average of 2.0 metastatic sites (range 1 0.0-4.0). The most prominent were the lymph nodes (19 pts, 61%); liver (13 points, 42%); ascites (8 pts, 26%) and peritoneum (7 pts, 23%).
[00446] The overall disease control rate was 39% (Table 15). Four patients had a confirmed partial response, and 8 patients had a stabilization of the disease. The first reevaluation of these patients took place 8 to 1 a week after the first infusion, with the exception of two patients, for whom the first tumor reevaluation was done after 6 weeks, respectively.
[00447] Table 15: best evaluation of response of
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Total 31 100 [00448] In 6 out of 12 patients with clinical disease control, at least one tumor marker (CEA; CA19-9; CA125; CA15-3), which was increased at the beginning of the study, dropped from 35 to 76% over of the study. In three patients all tumor markers were below the cutoff value for one patient and no rumor marker results were available.
[00449] Interestingly, also 4 patients with progressive disease as the best response had a tumor marker decrease between 29 and 54% during the study.
[00450] Partial responses were achieved after 2.3 months of treatment (two patients), 6.5 months (one patient) and 4.8 months (one patient), respectively. The RP was confirmed for one patient, lasted 4.4 months more, which leads to a PFS of 9.2 months for this patient. For the other three patients, confirmations were made after 6 (one patient) and 12 weeks (two patients), respectively. More details can be found in Table 16.
Table 16: Detailed assessment of FAS defined on a per patient basis
at. - data are not yet available; n.d. - not detectable. * - Censored since the event did not occur until November 2012 or the exact date is unknown at the moment. Last follow-up date was used in all cases. # - Tumor marker is below cut. Not counted in the text
202/217 because of this.
Pat-No. No. of Inf. No. of provTx No. of met.locations IHC Status at entry Best answer PFS[week s] THE[months] Max. average no. cell s RECIST(exchange) Tumor marker 2002-05 17 0 2 3 87 at. SD (n.d.) CA 125 -59% ^# 401001-08 11 4 2 3 83 PD SD (± 0%) CA19-9 -35% 231007-02 16 1 2 2 70 SD PR (-51%) CA15_3 -65% ^# 404001-12 2 2 2 2 63 at. SD (+ 18%) at. 6 6.4 ¹ 1013-02 9 5 4 3 68 at SD (+5%) increase 181011-09 5 0 3 3 67 PD SD (-25%) CA 125 -25% 10 *1001-24 8 2 4 2 50 PD SD (0%) increase 16 * 5.6 ¹ 1006-03 17 3 3 3 70 SD PR (-34%) CA 125 -37% 34 *1007-09 11 1 2 3 73 SD SD (n.a.) CA15_3 -42% ^# 23 *2003-15 11 0 3 3 80 PD PR (-35%) CA 125 -35% 22 *2003-16 11 1 4 2 90 PD PR (-39%) CA19-9 -76%CA 125 -75% 22 *1005-34 5 4 3 2 50 SD SD (+ 17%) CEA -35% 11 *2002-07 5 0 1 2 50 PD PD (n.d.) n.d. 114001-09 5 0 1 2 40 PD PD (+40%) increase 101005-13 5 5 2 3 65 at. PD (+63%) increase 104001-01 5 2 3 2 60 PD PD (+60%) n.d. 91004-10 3 1 4 2 83 SD PD (+4%) increase 61005-10 5 4 2 3 53 PD PD (-2%) CA15_3-40%CA 125 -54% 101004-11 5 1 3 2 40 at. PD (+32%) CA19_9-32% 10 4.3 4001-11 4 4 3 3 80 at. PD (+6%) CA15_3-49%; 71011-05 5 1 2 2 58 at. PD (+72%) increase 101009-01 4 1 1 3 63 SD PD (+71%) increase 81011-16 5 2 1 3 75 PD PD (-26%) CEA -29% 9 3.8
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CA15_3 -28% 1012-01 3 2 1 2 50 at. PD (+12%) increase 71005-18 3 3 3 3 60 PD PD (+3%) increase 52003-10 5 0 4 2 60 at. PD (+8%) increase 101011-20 5 2 1 3 48 at. PD (+82%) increase 91003-10 2 2 2 3 50 at. PD (n.a.) at. 4 *1001-27 3 4 4 3 73 at. PD (n.a.) at. 5 * 1.6 1006-05 5 3 3 3 60 at. PD (+73%) increase 10 * 4.3 1005-29 3 3 2 3 75 SD PD (+3%) increase 7
[00451] The median survival progression for patients in the FAS set was 10 weeks (minimum 4 weeks; max 40 weeks). Due to the limited availability of events, median progression-free survival for patients with clinical benefit (PR + DP) shown in Figure 9, has limited value. Patients with no clinical benefit (PD) had a median progression-free survival of 9 weeks (min 4 weeks; max. 1.1 weeks) (Figure 9).
[00452] There were no differences between patients with clinical benefit (PR or SD as the best response) or progressive disease (PD as the best response) in relation to age (mean 56 vs. 59 years), not on previous chemotherapy regimen ( average of 1.9 vs 2.1), amofsky index (average of 89 vs 88%). Only the number of metastases was lower in the responder group with an average of 1.9 compared to 2.3 in the non-responder group. The difference is not statistically significant.
[00453] The intensity (medium and maximum) of coloring
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IHC was similar between patients with clinical benefit and progressive disease. The number of stained cells was different between both groups. The maximum number of stained cells and the average number of stained cells were higher in patients with clinical benefit with an average of 77% vs 67% and 71% vs. 60%, respectively. Differences were also noted on the location of metastases. In patients with clinical benefit, the frequency of pleural effusion (25% vs. 5%), peritoneal carcinomatosis (42% vs. 11%) and ascites (42% vs. 16%) was higher compared to patients with progressive disease such as best answer. The presence of liver metastases was much less (17% vs 58%) in patients with clinical benefit.
[00454] Per Protocol (PP):
[00455] The PP population included all patients who completed the treatment section (until visiting 9), without any major protocol deviation.
[00456] Of the 31 patients in the FAS set two had a major protocol violation (without target lesion) and nine patients did not complete the study protocol until the visit
9, and therefore they had any less of what at 5 infusions needed from IMAB362. O set PP comprises 20 patients. [00457] A age median was from 60 years with a range of
to 77. Patients in the PP set had an
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Kamofsky - median of 90% (range 70 - 100%). The vast majority (80%) of patients have been pre-treated with at least one chemotherapy regimen. Four (4) patients do not have a previous chemotherapy regimen.
[00458] Table 17: Details on previous chemotherapy regimens in the PP set (n = 20).
5-FU / Capecitabine Platinum compound Taxanos Epirubicin Irinotecan mABs / other agents No. of patients 16 (80%) 15 (75%) 11 (55%) 4 (20%) 5 (25%) 5 (25%)
[00459] The median number of previous chemotherapy regimens was 2.0 (range 0 to 5). Chemotherapy regimens for gastroesophageal cancer mainly consist of various combinations of 5-FU derivative, composed of platinum, taxanes, epirubicin, irinotecan, trastuzumab for HER2 / neu positive patients and other investigative agents. In the defined PP 80% of patients had at least once 5-FU or capecitabine and 75% were treated with a platinum compound at least once before inclusion. Five (5, 25%) patients were pretreated with trastuzumab or other investigative agents. More details can be found in Table 17. Five (5, 25%) patients also had radiotherapy before the study started.
[00460] Due to the late stage of the disease, patients had an average of 3.0 metastatic sites (range 1.0
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4.0). The most prominent were the lymph nodes (13 points, 65%); liver (9 pts, 45%); ascites (6 points, 30%) and lung (5 points, 25%).
[00461] The global disease control rate was 50%. Four patients had a confirmed partial response and 6 disease stabilization. The first reassessment of these patients took place 8 to 1 a week after the first infusion, with the exception of one patient, for whom the first re-evaluation of the tumor was done after 6 weeks, respectively (Table 18).
Table 18: evaluation of the best response according to RECIST, PP set.
Best answer N % PR 4 20 SD 6 30 PD 10 50
Total 20 100 [00462] In 6 out of 10 patients with clinical disease control, at least one tumor marker (CEA; CA19-9; CA125; CA15-3), which was increased at the beginning of the study, dropped from 35 to 76% over of the study. In two patients all tumor markers were below the cutoff value for one patient and no tumor marker results were available.
[00463] Interestingly also 3 patients with progressive disease as the best response had a tumor marker decrease between 29 and 54% during the study.
207/217 [00464] Partial responses were achieved after 2.3 months of treatment (two patients), 6.5 months (one patient) and 4.8 months (one patient), respectively. The RP was confirmed for one patient, lasted 4.4 months more, which leads to a PFS of 9.2 months for this patient. For the other three patients, confirmations were made after 6 (one patient) and 12 weeks (two patients), respectively. More details can be found in Table 19.
Table 19: Detailed assessment of the PP set on a per patient basis [00465] N.a. - data are not yet available; n.d. - not detectable. * - Censored since the event did not occur until November 2012 or the exact date is unknown at the moment. Last follow-up date was used in all cases. # - Tumor marker is below cut. They were not counted in the text because of this.
Pat-No. No. of Inf. No. of prev Tx No. of met.locations IHC Status at entry Best answer PFS[week s] THE[months] Max. Medium no.from cellulat RECIST(change) Tumor marker 1001-08 11 4 2 3 83 PD SD (± 0%) CA19-9 -35% 231007-02 16 1 2 2 70 SD PR (-51%) CA15_3 -65% ^# 401013-02 9 5 4 3 68 at SD (+ 5%) increase 181011-09 5 0 3 3 67 PD SD (-25%) CA125 -25% 10 *1001-24 8 2 4 2 50 PD SD (± 0%) increase 16 * 5.6 ¹
208/217
1006-03 17 3 3 3 70 SD PR (-34%) CA125 -37% 34 *1007-09 11 1 2 3 73 SD SD (n.a.) CA15_3 -42% #CA125 -27% # 23 *2003-15 11 0 3 3 80 PD PR (-35%) CA125 -35% 22 *2003-16 11 1 4 2 90 PD PR (-39%) CA19-9 -76%CA125 -75% 22 *1005-34 5 4 3 2 50 SD SD (+17%) CEA -35%CA 125 -11% 11 *4001-09 5 0 1 2 40 PD PD (+40% increase 101005-13 5 5 2 3 65 at. PD (+63%) increase 104001-01 5 2 3 2 60 PD PD (+60%) n.d. 91005-10 5 4 2 3 53 PD PD (-2%) CA15_3 -40%CA125 -54% 101004-11 5 1 3 2 40 at. PD (+32%) CA19_9 -32% 10 4.3 1011-05 5 1 2 2 58 at. PD (+72%) increase 101011-16 5 2 1 3 75 PD PD (-26%) CEA -29%CA15_3 -28% 9 3.8 2003-10 5 0 4 2 60 at. PD (+8%) increase 101011-20 5 2 1 3 48 at. PD (+82%) increase 91006-05 5 3 3 3 60 at. PD (+73%) increase 10 * 4.3
[00466] Measurements were made earlier, after 4 weeks and every 3-12 weeks thereafter throughout the study.
[00467] The average progression of free survival for patients in the PP set was 18 weeks (minimum 9 weeks; .. Max 40 weeks). Due to the restricted availability of events the median progression-free survival for patients with clinical benefit (PR + DP) shown in Figure
209/217 has a limited value. Patients with no clinical benefit (PD) had a median progression-free survival of 10 weeks (min 9 weeks; Max 10 weeks) (Figure 10).
[00468] There were no differences between patients with clinical benefit (PR or SD as the best response) or progressive disease (PD as the best response) in relation to age (mean 57 vs. 62 years), not on previous chemotherapy regimen ( average of 2.1 vs. 2.0), KRNAofsky- Index (average of 88 vs 88%). Only the number of metastases was higher in patients with clinical benefit with an average of 3.0 compared to 2.2 in the patient without benefit. The difference is not statistically significant. The intensity (average and maximum) of IHC staining was similar between patients with clinical benetitis and progressive disease. The number of stained cells was different between both groups. The maximum number of stained cells and the average number of stained cells were higher in patients with clinical benefit with a maximum of 76% vs. 66% and an average of 70% vs 66%, respectively.
[00469] Differences were also noted on the location of metastases. In patients with clinical benefit, the frequency of pleural effusion (30% vs. 10%), peritoneal carcinomatosis (40% vs. 0%) and ascites (40% vs. 20%) was higher compared to patients with progressive disease as better answer. The presence of liver metastases was much lower (20% vs 70%) in patients with clinical benefit.
210/217 [00470] In conclusion, the state of the tumor (according to RECIST) at 2 weeks after the 5 ^th IMAB362 infusion (V9) was compared with the baseline. For 31 patients (FAS) at least one staging after the start was available. Patients were enrolled in a terminal stage of the disease with a median of 2.0 previous chemotherapies and 2.0 metastatic sites.
[00471] A confirmed partial response was assessed in four patients leading to an overall response rate of 13%. Three of them are currently in progress and duration cannot be calculated. In addition, eight patients had a disease stabilization leading to a 39% disease control rate. At the time of analyzing the progression-free survival of these 12 patients with clinical benefit it ranged between 6 and 40 weeks. Median could not have calculated since event was not registered for 7 of those patients so tar. In 9 of the patients with clinical benefit, at least one tumor marker was elevated in the beginning and dropped by 35 to -76% in six of them concomitantly. Interestingly, 4 patients with progressive disease as the best response had a decrease between -29 and -54% of at least one of the elevated tumor markers during the course of the study. The overall average progression free survival was 10 weeks, with an interval of 4 to ~ 40 weeks.
[00472] In patients with clinical benefit (4 + 8 PR
211/217
SD = 39%) the incidence of peritoneal carcinomatosis, pleural effusion and ascites was higher, and the incidence of liver metastases was lower than in patients without the benefit. On the other hand, a patient with a confirmed partial response had frequent liver metastases.
[00473] With the current data set it appears that patients with a clinical benefit had a greater number of cells with positive IHC staining.
[00474] In addition, auxiliary data was collected in selected patients, which shows that patients and the components of patients' PBMC serum are fully functional and potent in mediating the main modes of action IMAB362 of CDC and ADCC, respectively.
[00475] In conclusion, anti-tumor activity (partial response, stable disease, decreased tumor marker) has been observed and IMAB362 ensures further investigation.
D. GLOBAL CONCLUSIONS [00476] This trial was designed as an ILA phase, multicenter, inter-patient multiple dose escalation, non-randomized, open clinical study with 3 cohorts. Patients eligible for this clinical study were asked to be refractory to standard treatment or to be without accepted therapy.
[00477] For this interim report 34 patients were evaluated by safety analysis (APT set) of
212/217 same 4 were included in group 1 (300 mg / m ²⁾ , 6 in group 2 (600 mg / m ²⁾ and 20 in cohort 3 (600 mg / m ²⁾ · IMAB362 given in a multiple dose schedule it was safe and well tolerated in heavy pretreated patients with gastro-oesophageal cancer, with nausea and vomiting being the most common related adverse event. Most adverse events were mild to moderate. There were only two patients with moderate-grade allergic reactions, one severe. There were no grade 4 and grade 5 adverse events (including laboratory parameters) in this phase IIa study and the previous phase I study I. This has meant that IMAB32 has so far not caused grade 4 related AEs is remarkable, since most of registered monoclonal antibodies are associated with life-threatening grade 4 and 5 side effects. The metastatic indication for breast cancer for bevacizumab was revoked by the FDA in November 201 1. After initial preliminary approval in 2008. Bevacizumab did not prolong life and caused severe blood pressure and hemorrhage, with intestinal perforation and perforation of the nasal septum. Cetuximab causes acne-like eruptions and grade 3-4 reactions to infusion, anaphylaxis and cardiac arrest, requiring antihistamine diphenhydramine prophylaxis before treatment. Trastuzumab is still widely used, while causing symptomatic cardiac dysfunction in 2 to 7% of patients, which has been known for more than 10 years.
[00478] The main measure for the evaluation of
213/217 potential antitumor activity was the tumor state according to RECIST. There were 31 patients who had at least one assessment after this baseline, so they were included in the FAS. Four PR and 8 SD in 31 (RR 13%, DCR 39%) pre-treated patients compare very well with response results in other phase II studies with target monotherapy approved as secondary or late stage treatment.
[00479] Cetuximab, an EGFR antagonist achieved an RR of 3% (with an additional 7% SD) in the late phase (most had 2 or more metastatic sites and previous therapies) GEC measured after 8 weeks in a phase II trial with thirty patients. In a second study with 55 end-stage patients cetuximab led to 5% RR and an additional 11% of SD measured after 8 weeks. Similar response rates were obtained in patients with EGFR cetuximab positive refractory CCRm, where it was later approved in this indication.
[00480] Sunitinib and erlotinib were tested in final phase GEC patients in different phase II studies with a total of about 150 patients. The DCR after 6-8 weeks ranged between 16 and 39% and the response rate was reported between 3 and 7%, respectively.
[00481] The objective phase II response rate for trastuzumab as secondary therapy for breast cancer was 1%, with a 9% increase in SD of> 6 months. For erlotinib
214/217 in pretreated lung cancer a 9% response rate has been reported. Sorafenib achieved an RR between 2% to 18% in two phase II trials in kidney cancer and for temsirolimus an RR of 7% was reported in a kidney cancer trial. These target therapy compounds were later developed in association with chemotherapy and became registered in these indications.
[00482] IMAB362 is a safe and effective antibody. As expected from the exquisite tissue-specificity of the target surface molecule and the high-precision antibody binding, the investigational drug is well tolerated compared to other commercially targeted therapy. In addition, in several patients evidence of clinical activity has been observed, comparable or better for the phase results of other targeted therapies already commercialized II.
Example 5: IMAB362 induced nausea / vomiting [00483] IMAB362 was observed to induce nausea / vomiting up to NCI-CTC grade 3. The symptoms can be described as follows: (i) non-dose dependent, (ii) acute occurrence mainly, within the first minutes of infusion, it can continue after completion of infusion, (iii) starts with epigastric pain, hypersalivation, (iv) vomiting can start without Prodromi, (v) rare reaction in patients with total gastrectomy, (vi) the first infusion
215/217 indicative against symptoms that increase from cycle to cycle.
[00484] The fact that these adverse reactions that rarely occur in patients who have undergone total gastrectomy, suggests that the underlying mechanism is an over-target effect. With IMAB362 vomiting is more frequent than nausea and is often reported to occur without prodromal nausea. Onset can be both acute and delayed. Our hypothesis is that small amounts of IMAB362 bind to the tightly accessible tight junction epitope. This results in a localized interruption of tight junctions and leakage of gastric to submucosal leaks. Resulting tissue reactions and cramps start a cascade nausea / vomiting.
[00485] Thus, the recommended countermeasures are efficient antiemetic prophylaxis and protection of the gastric mucosa.
[00486] For example, patients should receive antiemetic prophylaxis before starting medication. For both prophylaxis and curative intervention, a combination of an N.-1 receptor (eg, (aprepitant / Amendment) and a 5-HT3 receptor blocker (eg, ondansetron / Zofran) are recommended and can be extended with compounds Antiemetic medication is preferably given, at least in the first three days of each cycle. Prophylactic butylscopolamin / Buscopan
216/217 before each IMAB362 infusion can be considered.
[00487] Any measure designed to protect the mucosa can reduce gastric symptoms. In this regard, proton pump inhibitors and / or misoprostol can be used and can, for example, be administered on days 1, 2 or 3 of each cycle. Non-steroidal anti-inflammatory drugs (NSAIDs) should not be used, but acetaminophen is allowed. If paracetamol is not efficient for pain management NSAIDs it can be used if necessary for pain management to avoid opioid treatment. Patients receiving NSAIDs are preferably treated with proton pump inhibitors and / or Misoprostol.
[00488] Thus, antiemetic prophylaxis and protection of the gastric mucosa can be started immediately before the IMAB362 infusion. For example, the following combination can be administered, intravenous application being preferred:
• NK-1 RA: for example, Aprepitant / Emend (150 mg iv) • 5-HT3 RA: for example, Palonosetron (0.25 mg IV), Ondansetron / Zofran (8 mg IV), Granisetron (3 mg IV) • butylscopolamine / Buscopan • proton pump inhibitor: Pantoprazole / Pantozole [00489] Optionally, Metoclopramid / MCP, lorazepam, and / or Atropine can also be administered.
[00490] IMAB362 is an antibody, which is based on
217/217 predominantly on immunological modes of action, which can be compromised by immunosuppressive compounds. For this reason, steroids should be avoided in prophylaxis and antiemetics used only, if other compounds have failed.
[00491] In addition, exposure to IMAB362 must be aware. For example, close monitoring for the first 15-30 min is recommended. If necessary, the infusion rate should be delayed (for example, up to 4 h instead of 2 h) and infusion breaks should be included.
[00492] Antiemetic medication, as well as the protection of the gastric mucosa can be continued, for example, until the 3rd day of each cycle.

权利要求:
Claims (8)
[1]
1. In vitro method to determine the responsiveness of a cancer patient to treat a cancer disease with an antibody that has the ability to bind to CLDN 18.2 characterized by the fact that the referred method comprises the stage of determine the level in the blood of one or more markers in the patient, where the one or
more bookmarks are selected to from the group consisting of CA 125, CA 15-3, CA 19-9, CEA, IL-2, IL- 15, IL-6, IFNy, and TNFa.2. Method, in according to claim 1,
characterized by the fact that the level is determined in the blood, plasma or serum.
[2]
2/2
[3]
3. Method according to claim 1 or 2, characterized by the fact that the one or more markers are selected from the group consisting of CA 125, CA 15-3, CA 19-9, CEA, IL-2, IL-15, IFNy, and TNFa and a decrease in the level of at least one of the markers following treatment indicates that the patient responds to the treatment of a cancer disease.
[4]
4. Method according to claim 1 or 2, characterized by the fact that the marker is IL-6 and an increase in the level of the marker following treatment indicates that the patient responds to the treatment of a cancer disease.
Petition 870190048963, of 05/24/2019, p. 17/27
[5]
5. In vitro method to determine whether a cancer patient is in favor of treating a cancer disease with an antibody that has the ability to bind to CLDN18.2 characterized by the fact that said method comprises the step of determining the percentage of CLDN18.2 positive cancer cells.
[6]
6. Method according to claim 5, characterized by the fact that a level of at least 50% positive CLDN 18.2 cancer cells indicates that the patient is susceptible to the treatment of a cancer disease.
[7]
7. Method according to claim 5 or 6, characterized by the fact that a level of cancer cells of at least 50%, which are positive for the surface expression of CLDN18.2 indicates that the patient is susceptible to the treatment of a cancer disease.
[8]
8. Method, according with any an of claims 1 to 7, characterized by the fact in what CLDN18.2 It has the sequence in amino acids according defined by SEQ ID NO: 1.
Petition 870190048963, of 05/24/2019, p. 18/27

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同族专利:

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公开号 | 公开日

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SG10201707684WA|2017-11-29|

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AU2014234684A1|2015-08-20|

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AU2014234684B2|2018-12-06|

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ES2765949T8|2020-06-23|

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AU2019201513A1|2019-03-28|

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RU2015144666A|2017-04-21|

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US20190076525A1|2019-03-14|

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BR112015023910A2|2017-03-21|

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MX2015013355A|2016-06-24|

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KR20150130318A|2015-11-23|

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SG11201507764QA|2015-10-29|

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AU2019201513B2|2020-08-13|

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RU2015144666A3|2018-03-26|

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DK2976360T3|2020-01-27|

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JP2016517447A|2016-06-16|

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CN105189554A|2015-12-23|

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CA2899770A1|2014-09-25|

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JP2018184403A|2018-11-22|

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WO2014146778A1|2014-09-25|

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RU2019101923A|2019-02-22|

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UA119036C2|2019-04-25|

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AU2020210165A1|2020-08-13|

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NZ710648A|2021-03-26|

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WO2014146672A1|2014-09-25|

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PT2976360T|2020-01-22|

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IL240060D0|2015-09-24|

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ES2765949T3|2020-06-11|

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HK1219486A1|2017-04-07|

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US10137195B2|2018-11-27|

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法律状态:
2017-03-28| B65X| Notification of requirement for priority examination of patent application|

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2017-04-11| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|

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2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-11-21| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-02-26| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2019-07-16| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2019-09-10| 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 17/03/2014, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/03/2014, OBSERVADAS AS CONDICOES LEGAIS |

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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) |

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申请号 | 申请日 | 专利标题

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PCT/EP2013/000817|WO2014146672A1|2013-03-18|2013-03-18|Therapy involving antibodies against claudin 18.2 for treatment of cancer|

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PCT/EP2014/000719|WO2014146778A1|2013-03-18|2014-03-17|Therapy involving antibodies against claudin 18.2 for treatment of cancer|

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