HK1179521A - Neuropilin as a biomarker for bevacizumab combination therapies - Google Patents

Description

Neuropilin as biomarker for bevacizumab combination therapy

The present invention provides methods for improving the efficacy of treatment in patients with gastric cancer, particularly gastric or gastroesophageal junction ("GEJ") adenocarcinoma, by determining the level of expression of neuropilin relative to a control level determined in patients with gastric cancer, particularly gastric or gastroesophageal junction ("GEJ") adenocarcinoma by combining with a chemotherapy regimen bevacizumab (bevacizumab,) Treatment is performed. The improved therapeutic effect may be improved overall survival or improved progression-free survival. The invention further provides methods for evaluating patient pairs of bevacizumab in combination with a chemotherapy regimenBy determining the expression level of neuropilin relative to a control level determined in a patient having gastric cancer, particularly gastric or gastroesophageal junction ("GEJ") adenocarcinoma.

Thus, the present invention relates to the identification of one or more biomarkers of gastric cancer, in particular of gastric or gastroesophageal junction ("GEJ") adenocarcinomaIdentification and selection of the biomarkers in combination with angiogenesis inhibitors, e.g. bevacizumab, in combination with chemotherapeutic regimens, such as capecitabine (capecitabine) or 5-fluorouracil (5-fluorouracil) based chemotherapyIs correlated with sensitivity or responsiveness. In certain aspects, the invention relates to the use of tumor-specific expression of neuropilin determined relative to established controls in patients with gastric cancer, particularly gastric or GEJ adenocarcinoma, to identify the addition of an angiogenesis inhibitor, such as bevacizumab, to standard chemotherapySensitive or responsive patients. The invention also relates to a method for improving the therapeutic effect in patients with gastric cancer, in particular gastric or GEJ adenocarcinoma by determining the tumor-specific expression level of neuropilin relative to a control established in patients with gastric cancer, in particular gastric or GEJ adenocarcinoma by adding an angiogenesis inhibitor, e.g. bevacizumab, to a standard chemotherapy, e.g. capecitabine or 5-fluorouracil based chemotherapyTo proceed with. The therapeutic effect included the clinical parameters overall survival and progression free survival. The invention also provides methods for identifying inhibitors of angiogenesis, particularly bevacizumabKits and compositions for sensitive or responsive patients identified and defined according to the methods described herein.

Angiogenesis is essential for cancer formation, not only regulating primary tumor size and growth, but also affecting invasive and metastatic potential. Thus, mechanisms mediating the angiogenic process have been investigated as potential targets for targeted anti-cancer therapy. In previous studies of angiogenesis modulators, the Vascular Endothelial Growth Factor (VEGF) signaling pathway was found to preferentially modulate angiogenic activity in multiple cancer types, and various therapeutic agents have been developed to modulate this pathway at multiple points. These include bevacizumab, sunitinib (sunitinib), sorafenib (sorafenib), and vatalanib (vatalanib), among others. Although the use of angiogenesis inhibitors in the clinic has shown success, not all patients respond or fail to respond completely to angiogenesis inhibitor therapy. The underlying mechanism of such incomplete responses is unknown. Thus, there is a continuing need to identify subgroups of patients that are sensitive or responsive to anti-angiogenic cancer therapies.

Although many angiogenesis inhibitors are known, the most predominant angiogenesis inhibitor is bevacizumabBevacizumab is a recombinant humanized monoclonal IgG1 antibody that specifically binds VEGF (vascular endothelial growth factor) and blocks its biological effects. VEGF is a vital driver of tumor angiogenesis (tumor growth and metastasis, a necessary process required for tumor dissemination to other parts of the body).Four common cancer types approved in europe for the treatment of advanced stages: colorectal, breast, non-small cell lung (NSCLC) and renal cancers, which collectively cause over 250 million deaths each year. Has been used up to nowOver 50 million patients are treated, and a comprehensive clinical program with over 450 clinical trials is investigating the further use of Avastin in the treatment of a variety of cancer types (including colorectal, breast, non-small cell lung, brain, stomach, ovarian and prostate) in different contexts (e.g., advanced or early stage disease). Importantly, the amount of the active ingredients in the composition,has already shownShown promising as co-therapeutic agents that demonstrate efficacy when combined with a wide array of chemotherapy and other anti-cancer treatments. Several phase III studies have been published that demonstrate the beneficial effects of combining bevacizumab with standard chemotherapeutic regimens (see, e.g., Kang et al 2010, j.clin.oncol.,28:18s (supplement summary LBA4007); Saltz et al 2008, j.clin.oncol.,26: 2013-. However, as in previous studies of angiogenesis inhibitors, some phase III studies of these have shown that a fraction of patients underwent addition of bevacizumab to their chemotherapy regimenIncomplete response of (2).

Thus, there is a need for methods of determining those patients who respond or are likely to respond to patients comprising angiogenesis inhibitors, in particular bevacizumabThe combination therapy of (1). Thus, the technical problem underlying the present invention is to provide methods and means for identifying patients suffering from or susceptible to gastric cancer, in particular gastric or GEJ adenocarcinoma, who may benefit from the addition of angiogenesis inhibitors, in particular bevacizumab, to chemotherapeutic regimens, e.g. capecitabine or 5-fluorouracil based inhibitors

This technical problem is solved by providing the embodiments characterized in the claims.

Accordingly, the present invention provides a method for improving the therapeutic effect in a patient suffering from gastric cancer, in particular gastric or GEJ adenocarcinoma, by adding bevacizumab to a chemotherapy regimen, said method comprising:

(a) determining the expression level of neuropilin in a patient sample; and are

(b) Bevacizumab in combination with a chemotherapy regimen is administered to a patient having a reduced neuropilin level relative to a control level determined in a patient having gastric cancer, particularly gastric or GEJ adenocarcinoma.

The improved therapeutic effect may be a clinical parameter overall survival or may be progression free survival.

In other embodiments, the invention relates to an in vitro method for identifying a patient responsive or sensitive to the addition of bevacizumab to a chemotherapy regimen, said method comprising determining the expression level of neuropilin in a sample from a patient suspected of having or being susceptible to gastric cancer, particularly gastric or GEJ adenocarcinoma, wherein a decreased level of neuropilin relative to a control level determined in a patient having gastric cancer, particularly gastric or GEJ adenocarcinoma, is indicative of the sensitivity of the patient to the addition of bevacizumab to said regimen.

Thus, the present invention solves the identified technical problem, as it surprisingly shows that the tumor-specific expression level of neuropilin in a given patient is correlated with the therapeutic effect in those patients administered an angiogenesis inhibitor in combination with a chemotherapy regimen, relative to control levels determined in patients diagnosed with gastric cancer, in particular gastric or GEJ adenocarcinoma. Surprisingly, changes in tumor-specific neuropilin expression levels were identified as being responsive to the addition of bevacizumab to capecitabine-or 5-fluorouracil-based chemotherapeutic regimensFor a gastric cancer patient, improved progression-free survival and/or improved overall survival. In particular, the addition of bevacizumab to a chemotherapy regimen was demonstratedIs identified as having reduced neuropilin expression relative to a control level established in a sample obtained from a patient having or diagnosed with gastric cancer, particularly gastric or GEJ adenocarcinoma. The terms "marker" and "predictionThe term "may be used interchangeably and refers to the expression level of neuropilin, as described and defined herein.

In the context of the present invention, "neuropilin" refers to neuropilin-1 protein, a type I membrane protein also known as NRP-1, and is exemplified by the amino acid sequence shown in figure 3, SEQ ID NO:1 (NRP-1 precursor amino acid sequence is also available as UniProt accession No. O14786). As used herein, "neuropilin" may also refer to neuropilin-2 (also referred to as NRP-2), which shares about 44% homology with NRP-1, as is known in the art. Thus, the method of the present invention does not distinguish between NRP-1 and NRP-2. In the context of the present invention, the term "neuropilin" also encompasses homologues, variants and isoforms of NRP-1 and/or NRP-2, provided that said homologues, variants and isoforms are specifically recognized by one or more anti-neuropilin antibodies as described herein and/or as known in the art. The term "neuropilin" further encompasses peptides that are identical to the amino acid sequence of SEQ ID NO:1, or a sequence which is at least 85%, at least 90% or at least 95% homologous to one or more of NRP-1 and/or NRP-2 homologues, variants and isoforms (including splice isoforms) and fragments of said sequence, provided that the variant protein (including isoforms), homologous protein and/or fragment is recognized by one or more NRP-1 and/or NRP-2 specific antibodies, such as clone 446915 available from R & D Systems, Inc. (Minneapolis, Minnesota, U.S. A.), encoding sc-5307 in a catalog of products is available from Santa Cruz Biotechnology, Inc (Santa Cruz, California, u.s.a.) or otherwise known in the art.

Thus, the present invention encompasses determining the expression level of a protein, including but not limited to an amino acid sequence as described herein. In certain aspects, the invention encompasses detecting homologs, variants, and isoforms of neuropilin; the isoforms or variants include allelic variants or splice variants and the like. Also contemplated is the detection of proteins that are homologous to neuropilin or a fragment thereof as described herein, e.g., having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence SEQ id No. 1 or a fragment thereof. Alternatively, or in addition, the invention encompasses detecting the expression level of a protein encoded by a nucleic acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence encoding SEQ ID NO 1 or a fragment, variant or isoform thereof, or a fragment thereof. In this context, the term "variant" means that the neuropilin amino acid sequence, or a nucleic acid sequence encoding said amino acid sequence, differs from the unique sequence identified with SEQ ID NO:1 and/or available under the UniProt accession numbers identified above by mutations, e.g., deletions, additions, substitutions, inversions, and the like. In addition, the term "homologue" refers to a molecule having at least 60%, more preferably at least 80% and most preferably at least 90% sequence identity to one or more polypeptides or fragments thereof as shown in SEQ ID NO: 1.

To determine whether an amino acid or nucleic acid sequence has a certain degree of identity to an amino acid or nucleic acid sequence as described herein, the skilled person may use means and methods well known in the art, e.g. alignments, either manually or by using computer programs known in the art or described herein.

In accordance with the present invention, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably 70-95% identity, more preferably at least 95% identity to an amino acid sequence such as SEQ ID NO: 1) when compared and aligned for maximum correspondence over a comparison window, or within a designated region, as measured using sequence comparison algorithms as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such definitions also apply to the complement of the test sequence. Preferably, the identity described is present over a region of at least about 15 to 25 amino acids or nucleotides in length, more preferably over a region of about 50 to 100 amino acids or nucleotides in length. One skilled in the art would know how to determine percent identity between sequencing columns using, for example, algorithms such as those based on the CLUSTALW computer program (Thompson Nucl. acids Res.2(1994), 4673-.

Although the FASTDB algorithm does not generally consider internal mismatch deletions or additions, i.e., gaps, in the sequence in its calculations, this can be corrected manually to avoid over-assessment of% identity. CLUSTALW, however, does consider sequence gaps in its identity calculation. Also available to those skilled in the art are BLAST (Basic Local Alignment Search Tool) and BLAST2.0 algorithm (Altschul,1997, nucleic. acids Res.25: 3389-. The BLASTN program for nucleic acid sequences uses word lengths (W) of 11, expect (E) of 10, M =5, N =4, and a comparison of the two strands as a default. For amino acid sequences, the BLASTP program uses a word length (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS89:10915) uses as defaults comparisons of 50 for alignment (B), 10 for expected (E), M =5, N =4, and both strands.

The BLAST algorithm as discussed above produces an alignment of both amino acid and nucleotide sequences to determine sequence similarity. Because of the local nature of the alignment, BLAST is particularly useful for determining exact matches or identifying similar sequences. The basic unit of BLAST algorithm output is a High-scoring segment Pair (HSP). HSPs consist of two sequence segments of arbitrary but equal length, which are aligned to be the largest locally and for which the alignment score meets or exceeds a user-set threshold or cut-off score. The BLAST method looks for HSPs between query sequences and database sequences to evaluate the statistical significance of any matches found, and reports only those matches that meet a user-selected significance threshold. Parameter E establishes a statistical significance threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of occasional occurrences of HSPs (or groups of HSPs) within the context of an entire database search. Any database sequences whose matches satisfy E are reported in the program output.

Similar computer techniques using BLAST can be used to search for the same or related molecules in protein or nucleotide databases such as GenBank or EMBL. This assay is much faster than membrane-based multiplex hybridization. In addition, the sensitivity of a computer search can be improved to determine whether any particular match is classified as exact or similar. The basis for the search is a product score, which is defined as:

and consider both the degree of similarity between two sequences and the length of sequence match. For example, in the case of a product of 40, the match would be accurate within 1-2% error; and at 70 the match will be exact. Although lower scores may identify related molecules, similar molecules are generally identified by selecting those molecules that exhibit a product score between 15 and 40. Another example of a program that can produce sequence alignments is the CLUSTALW computer program (Thompson,1994, Nucl. acids Res.2: 4673-.

In accordance with the present invention, surprisingly, a greater therapeutic effect of bevacizumab in the AVAGAST population (see, e.g., Kang et al, 2010, j.clin.oncol.,28:18s (supplement summary LBA4007)) was found to be associated with lower tumor-specific neuropilin expression. In particular, relatively low tumor-specific neuropilin expression is associated with improved overall survival and/or improved progression-free survival in patients receiving bevacizumab in addition to a chemotherapy regimen.

The expression level of neuropilin (e.g., NRP-1, NRP-2, or a variant, homolog, truncate, or fragment thereof) can be assessed by any method known in the art suitable for determining the level of a particular protein in a patient sample, and preferably, by immunohistochemical ("IHC") methods employing antibodies specific for neuropilin. Such methods are well known in the art and are routinely performed, and corresponding commercial antibodies and/or kits are readily available. For example, commercial antibodies specific for neuropilin as described and defined herein can be obtained from R & D Systems, inc. (Minneapolis, Minnesota, u.s.a.) under clone 446915 and from Santa Cruz biotechnology, inc. (Santa Cruz, California, u.s.a.) under product catalog number sc-5307. Preferably, the expression level of the marker/indicator protein of the invention is assessed using the reagents and/or protocol recommendations of the antibody or kit manufacturer. The skilled person will also know other means of determining the expression level of neuropilin by IHC methods. Thus, the expression level of neuropilin and/or other markers/indicators as known in the art can be routinely and reproducibly determined by one skilled in the art without undue burden. However, to ensure accurate and reproducible results, the present invention also contemplates testing patient samples in specialized laboratories that can ensure the effectiveness of the testing protocol.

Preferably, the expression level of neuropilin is assessed in a biological sample containing or suspected of containing cancer cells and determined in a tumor-specific manner. The sample can comprise both cancerous cells (i.e., tumor cells) and non-cancerous cells (e.g., endothelial or non-malignant cells). In some aspects, determining tumor-specific expression of neuropilin refers to determining the expression level of only cancer cells, as opposed to other cell types that may be present in a tumor sample, such as endothelial or non-cancerous/non-malignant cells. In other aspects, determining tumor-specific expression of neuropilin refers to determining the expression level of cancer cells, as well as any other cell types (e.g., endothelial cells) that may be present in a tumor sample. A skilled artisan, such as a pathologist, can readily distinguish between cancerous and non-cancerous cells, such as endothelial cells. The sample may be a gastric tissue resection or gastric tissue biopsy obtained from a patient having, suspected of having, or diagnosed with gastric cancer, particularly gastric or GEJ adenocarcinoma. The sample may also be a resection or biopsy of a metastatic lesion obtained from a patient having, suspected of having, or diagnosed with gastric cancer, particularly gastric or GEJ adenocarcinoma. Preferably, the sample is a sample of stomach tissue or gastroesophageal junction tissue, or a resection or biopsy of stomach or gastroesophageal junction adenocarcinoma. The sample may also be a sample of a known or suspected metastatic gastric cancer lesion or section, or a blood sample, such as a peripheral blood sample, known or suspected to contain circulating cancer cells, such as gastric cancer cells. The analysis of the sample according to the method of the invention may be manual, as performed by a skilled technician, e.g. a pathologist, as known in the art, or may be automated using commercially available software designed for processing and analyzing pathological images, e.g. for analysis in tissue biopsy or resection (e.g. MIRAX SCAN, Carl Zeiss AG, Jena, Germany). Methods for obtaining biological samples, including tissue resections, biopsies and body fluids, such as blood samples containing cancer/tumor cells, are well known in the art.

In the context of the present invention, bevacizumab is to be administered in addition to or as co-therapy or co-treatment with one or more chemotherapeutic agents, which are administered as part of a standard chemotherapy regimen as known in the art. Examples of such chemotherapeutic agents include 5-fluorouracil, leucovorin (leucovorin), irinotecan (irinotecan), gemcitabine (gemcitabine) -erlotinib (erlotinib), capecitabine, and platinum-based chemotherapeutic agents such as paclitaxel (paclitaxel), carboplatin (carboplatin), cisplatin (cissplatin), and oxaliplatin (oxaliplatin). As demonstrated in the accompanying examples, the addition of bevacizumab to capecitabine-or 5-fluorouracil-based chemotherapy regimens achieves a progression-free prolongation of survival in gastric cancer patients and/or patient populations with lower neuropilin expression in tumor samples relative to established control levels in similarly situated patients, defined and selected according to the expression level of neuropilin, and correlated with overall survival.

The bevacizumab may be combined with a pharmaceutical composition based on capecitabine or 5-A combination of fluorouracil chemotherapy regimens. The choice between capecitabine and 5-fluorouracil is best determined by the treating physician based on well established criteria in the art. Examples of capecitabine-based chemotherapy regimens include the combination of capecitabine (or 5-fluorouracil) administered in combination with cisplatin. A typical cycle for capecitabine/cisplatin therapy may be 1000mg/m twice daily (bid) on days 1 to 14²The dose of capecitabine (b) is administered orally, followed by a 1 week rest, and a 2 hour infusion of 80mg/m at day 1 of the cycle in the presence of high hydration and premedication (steroids and antiemetics; 3 times/week)²I.v. administering cisplatin; the cisplatin and capecitabine cycles were continued until disease progression or unmanageable toxicity, with cisplatin administration limited to a maximum of 6 cycles. Thus, in certain aspects of the invention, a patient identified according to the methods herein is treated with bevacizumab in combination with capecitabine/cisplatin. Common modes of administration of bevacizumab include parenteral administration in bolus doses or as infusions over a fixed period of time, e.g., total daily doses administered over 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 3 hours, 4 hours, 5 hours or 6 hours. For example, bevacizumab at 7.5mg/kg may be administered to a gastric cancer patient by intravenous infusion over 15 to 30 minutes on day 1 of each capecitabine cycleAs described above. The skilled artisan will recognize that other modes of administration of bevacizumab are contemplated by the present invention, as determined by the particular patient and chemotherapy regimen, and that the particular mode of administration and therapeutic dose are best determined by the treating physician according to methods known in the art.

Patients selected according to the methods of the invention are treated with bevacizumab in combination with a chemotherapy regimen, and may be further treated with one or more additional anti-cancer therapies. In certain aspects, the one or more additional anti-cancer therapies are radiation.

In a preferred embodiment, a sample obtained from a patient is collected prior to the initiation of any other chemotherapeutic regimen or therapy, e.g., a therapy for treating cancer or managing or ameliorating a symptom thereof. Thus, in a preferred embodiment, the sample is collected prior to administration of the chemotherapeutic agent or initiation of the chemotherapy regimen.

The invention also relates to diagnostic compositions or kits comprising oligonucleotides or polypeptides suitable for determining the tumor specific expression level of neuropilin. As detailed herein, oligonucleotides such as DNA, RNA or a mixture of DNA and RNA probes can be used to detect mRNA levels of marker/indicator proteins, particularly neuropilin, while polypeptides can be used to directly detect protein levels of marker/indicator proteins via specific protein-protein interactions. In a preferred aspect of the invention, the polypeptide encompassed as a probe for the expression level of neuropilin and comprised in the kit or diagnostic composition described herein is an antibody specific for neuropilin, or specific for a homologue, variant and/or truncate thereof.

Thus, a further embodiment of the invention provides a kit useful for carrying out the methods described herein, comprising an oligonucleotide or polypeptide capable of determining the expression level of neuropilin. Preferably, the oligonucleotide comprises a primer and/or probe specific for mRNA encoding neuropilin as defined and described herein, and the polypeptide comprises a protein capable of specifically interacting with neuropilin, e.g. a marker/indicator specific antibody or antibody fragment.

In a further embodiment, the present invention provides the use of bevacizumab for improving the therapeutic effect in a patient suffering from gastric cancer, in particular gastric or GEJ adenocarcinoma, comprising the following steps:

(a) determining the expression level of neuropilin in a patient sample; and are

(b) Bevacizumab in combination with a chemotherapy regimen is administered to a patient having a reduced neuropilin level relative to a control level determined in a patient having gastric cancer, particularly gastric or GEJ adenocarcinoma.

The improved therapeutic effect may be improved overall survival or improved progression-free survival.

As demonstrated in the accompanying examples, the present invention solves the identified technical problem, since it can surprisingly be shown that the expression level of neuropilin in a given patient is correlated with the therapeutic effect in patients administered bevacizumab in combination with a capecitabine or 5-fluorouracil based chemotherapy regimen, relative to control levels determined in patients diagnosed with gastric cancer, in particular gastric or GEJ adenocarcinoma.

The phrase "responsive" in the context of the present invention refers to a subject/patient having, suspected of having, or susceptible to having, or diagnosed with gastric cancer, particularly gastric or GEJ adenocarcinoma, showing a response to a chemotherapy regimen comprising bevacizumab addition. The skilled person will be readily able to determine whether a human treated with bevacizumab according to the method of the invention shows a response. For example, the response may be reflected by a reduction in pain from gastric cancer, such as reduced and/or stopped tumor growth, reduction in tumor size, and/or improvement in one or more symptoms of gastric cancer (e.g., gastrointestinal bleeding, pain, anemia). Preferably, the response may be reflected by a decrease or reduction in the index of metastatic transformation of gastric cancer, e.g. preventing the formation of metastases or a decrease in the number or size of metastases.

The phrase "sensitive to …" in the context of the present invention refers to a subject/patient having, suspected of having, or susceptible to having, or diagnosed with gastric cancer, particularly gastric or GEJ adenocarcinoma, that in some way shows a positive response to treatment with bevacizumab in combination with a chemotherapy regimen. The patient's response may be less pronounced when compared to a patient who "responds" as described above. For example, the patient may experience less disease-related distress, although a reduction in tumor growth or metastatic indicators may not be measured, and/or the patient's response to bevacizumab in combination with a chemotherapy regimen may be only transient in nature, i.e., the growth of the tumor and/or metastasis may be only temporarily reduced or halted.

The phrase "patient with …" according to the present invention refers to a patient who shows clinical signs of gastric cancer, in particular gastric or GEJ adenocarcinoma. Gastric cancer can be metastatic, inoperable and/or adenocarcinoma of the locally advanced stomach or gastro-esophageal junction ("GEJ"). The phrase "susceptible to" or "to be susceptible to" in the context of gastric cancer refers to an indication disease in a patient based on, for example, possible genetic predisposition, prior or final exposure to a dangerous and/or carcinogenic compound, or exposure to an carcinogenic physical risk, such as radiation.

The phrase "therapeutic effect" encompasses in the context of the present invention the phrases "progression free survival" and "overall survival".

The phrase "progression free survival" in the context of the present invention refers to the length of time during and after treatment, the patient's disease does not deteriorate, i.e. does not progress, as assessed by the treating physician or investigator. As the skilled person will appreciate, progression free survival of a patient is improved or enhanced if the patient experiences a longer length of time for which the disease does not progress than the mean or mean progression free survival time of a control group of patients in similar circumstances.

The phrase "overall survival" in the context of the present invention refers to the average survival of patients within a patient group. As the skilled person will appreciate, the overall survival of a patient is improved or enhanced if the patient belongs to a subgroup of patients having a statistically significantly longer mean survival time compared to another subgroup of patients. Improved overall survival may be evident in one or more patient subgroups, but not when the patient population as a whole is analyzed.

As used herein, the term "administering" means administering an angiogenesis inhibitor, e.g., bevacizumab, to a patient in need of such treatment or medical intervention by any means known in the art suitable for administering a therapeutic antibodyAnd/or comprising angiogenesis inhibitors, e.g. bevacizumabPharmaceutical composition/treatment regimen of (a). Non-limiting routes of administration include by oral, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal, or intrabronchial administration (e.g., as achieved by inhalation). Particularly preferred in the context of the present invention is parenteral administration, e.g. intravenous administration. Bevacizumab for the treatment of colorectal cancerFor example, a preferred dose according to EMEA is 5mg/kg or 10mg/kg body weight administered once every 2 weeks or 7.5mg/kg or 15mg/kg body weight administered once every 3 weeks (for further details, see http:// www.emea.europa.eu/humandocs/PDFs/EPAR/avastin/EMEA-combined-h582en. pdf).

The term "antibody" is used herein in the broadest sense and includes, but is not limited to, monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, CDR-grafted antibodies, humanized antibodies, camelized (camelized) antibodies, single chain antibodies, and antibody fragments and fragment constructs, e.g., F (ab')₂Fragments, Fab fragments, Fv fragments, single chain Fv fragments (scFv), bispecific scFv, diabodies, single domain antibodies (dAb), and minibodies that exhibit a desired biological activity, particularly specific binding to one or more of VEGFA, HER2, neuropilin, and CD31, or to a homolog, variant, fragment, and/or isoform thereof.

As used herein, "chemotherapeutic agent" includes any active agent that can provide an anti-cancer therapeutic effect and can be a chemical or biological agent, particularly one that is capable of interfering with cancer or tumor cells. Preferred active agents are those that act as antineoplastic (chemotoxic or chemoinhibitory) agents that inhibit or prevent the formation, maturation or proliferation of malignant cells. Non-limiting examples of chemotherapeutic agents include alkylating agents (alkylating agents) such as nitrogen mustards (e.g., mechlorethamine (mechlorethamine), cyclophosphamide (cyclophosphamide), ifosfamide (ifosfamide), melphalan (melphalam) and chlorambucil (chlorembucil)), nitrosoureas (nitroureas) (e.g., carmustine (BCNU), lomustine (ccu), and semustine (semustine) (methyl-CCNU)), ethylenimines (ethylmelamines)/methylmelamines (melamines) (e.g., triethylmelamine (thienylmelamine) (TEM), triethyleneamine (triethylenethylamine) (TEM), triethyleneamine (thiophosphoramide) (e), hexamethylmelamine (e), and melaminee (e, e); antimetabolites such as folic acid analogs (e.g., methotrexate, trimetrexate), pyrimidine analogs (e.g., 5-fluorouracil, fluorodeoxyuracil (fluorodeoxyuridine), gemcitabine, cytarabine (cytosine arabine) (AraC, cytarabine), 5-azacytidine (azacytidine), 2 ' -difluorodeoxycytidine (2,2 ' -difluorodeoxycytidine), and pyrimidine analog prodrugs such as capecitabine), purine analogs (e.g., 6-mercaptopurine, 6-thioguanine, azathioprine (azathioprine), 2 ' -deoxyhelpfuligine (deoxyribomycin) (pentostatin), erythrohydroxynonyladenine (erythroxynyxyladine, EHNA), fludarabine (spinoflavine), and 2-chlorohexidine (2-A); antimitotic drugs developed from natural products (e.g., paclitaxel, vinca alkaloids (vinca alkaloids) (e.g., Vinblastine (VLB), vincristine (vinristine), and vinorelbine (vinorelbine)), taxotere (taxotere), estramustine (estramustine), and estramustine phosphate), epipodophyllotoxins (epedophylloxins) (e.g., etoposide (etoposide), teniposide (teniposide)), antibiotics (e.g., actinomycin (actimycin) D, daunomycin (daunomycin), doxorubicin (doxobicin), mitoxantrone (mitoxantrone), idarubicin (idarubicin), bleomycin (actinomycin)), mitomycin (streptomycin)), and interferons (e.g., mitomycin (mitomycin), and related enzymes (e.g., mitomycin (C)), and related enzymes (e.g., mitomycin (e), such as a, IL-2, G-CSF, GM-CSF); miscellaneous agents, including platinum coordination complexes (e.g., cisplatin, carboplatin), anthracenediones (e.g., mitoxantrone), substituted ureas (i.e., hydroxyurea), methylhydrazine derivatives (e.g., N-Methylhydrazine (MIH), procarbazine (procarbazine)), adrenocortical inhibitors (e.g., mitotane (o, p' -DDD), aminoglutethimide (aminoglutethimide)); hormones and antagonists, including adrenocortical steroid antagonists (e.g., prednisone and equivalents, dexamethasone, aminoglutethimide), progesterone (progestin) (e.g., hydroxyprogesterone hexanoate, medroxyprogesterone acetate, megestrol acetate), estrogen (e.g., diethylstilbestrol, ethinyl estradiol), and equivalents thereof); antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone propionate (testosterone), fluoxymesterone (fluoxymesterone), and equivalents thereof), antiandrogens (e.g., flutamide, gonadotropin-releasing analogs, leuprolide), and nonsteroidal antiandrogens (e.g., flutamide).

In the context of the present invention, reference to "homology" of an amino acid sequence is understood to mean at least 80% sequence identity, in particular at least 85%, preferably at least 90% and still more preferably at least 95% identity over the full length of the sequence as defined by SEQ ID NO as provided herein. In the context of the present invention, the skilled person will understand that homology further encompasses other allelic variations of marker/indicator proteins in different populations and ethnic groups.

As used herein, the term "polypeptide" refers to a peptide, protein, oligopeptide or polypeptide encompassing amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, the invention also encompasses peptide mimetics of such proteins/polypeptides in which an amino acid and/or peptide bond has been replaced by a functional analogue, for example an amino acid residue other than one of the 20 gene-encoded amino acids, for example selenocysteine. Peptides, oligopeptides, and proteins may be referred to as polypeptides. The terms polypeptide and protein are used interchangeably herein. The term polypeptide also refers to, and does not exclude, modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation, etc. Such modifications are well described in basic texts and are described in more detail in monographs and in multiple research literature.

As used herein, the terms "treatment" and "treating" refer to the remediation, amelioration, lessening of severity, or reduction in the course of time of a disease or any parameter or symptom thereof. Preferably, the patient is a human patient and the disease to be treated is gastric cancer, in particular gastric or GEJ adenocarcinoma. The term "assessing" such patients refers to determining the expression level of neuropilin and/or a method of selecting such patients based on the expression level of such marker/indicator proteins relative to established control levels in patients diagnosed with metastatic colorectal cancer.

In addition to the methods described above, the present invention also encompasses other immunohistochemical methods for assessing expression levels of neuropilin, such as by Western blotting and ELISA-based detection. The expression level of the marker/indicator protein of the invention can also be assessed at the mRNA level by any suitable method known in the art, such as Northern blotting, real-time PCR, and RT PCR, as understood in the art. Immunohistochemistry and mRNA based detection methods and systems are well known in the art and can be derived from standard textbooks such as Lottspeich (Bioanalytik, Spektrum Akademiesher Verlag,1998) or Sambrook and Russell (Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, U.S. A., 2001). The described methods are particularly useful for determining the expression level, e.g., tumor-specific expression level, of neuropilin in a patient or group of patients relative to established control levels in a population of similar conditions, e.g., having or diagnosed with gastric cancer, particularly gastric or GEJ adenocarcinoma.

The expression level of neuropilin can also be determined at the protein level by using immunoagglutination, immunoprecipitation (e.g., immunodiffusion, immunoelectrophoresis, immunosetting), Western blot techniques (e.g., (in situ) immunohistochemistry, (in situ) immunocytochemistry, affinity chromatography, enzyme immunoassay), and the like. The amount of purified polypeptide in solution can also be determined by physical means, such as photometry. Methods for quantifying a particular polypeptide in a mixture typically depend on, for example, the specific binding of an antibody. Specific detection and quantification methods that exploit antibody specificity include, for example, immunohistochemistry (in situ). For example, the concentration/amount of a marker/indicator protein of the invention (e.g., NRP-1, NRP-2 and/or variants, homologs or truncates thereof) in a cell or tissue can be determined by enzyme-linked immunosorbent assay (ELISA). Alternatively, Western blot analysis or immunohistochemical staining may be performed. Western blot combines separation of a mixture of proteins by electrophoresis with specific detection with antibodies. The electrophoresis may be multi-dimensional such as 2D electrophoresis. Typically, polypeptides are separated in 2D electrophoresis along one dimension according to their apparent molecular weight and along another direction according to their isoelectric point.

As mentioned above, the reduced expression of the marker/indicator protein according to the invention may also be reflected in a reduced expression of the corresponding gene of neuropilin (as described and defined herein). Thus, quantitative assessment of the pre-translational gene product (e.g., spliced, unspliced, or partially spliced mRNA) can be performed to assess the expression of the corresponding gene. Those skilled in the art will know standard methods to be used in this context or may derive these from standard textbooks (e.g. Sambrook,2001, supra). For example, quantitative data regarding the corresponding concentration/amount of neuropilin-encoding mRNA can be obtained by Northern blotting, real-time PCR, and the like.

In a further aspect of the invention, the method of the invention can be advantageously carried out using the kit of the invention and can be employed in a variety of applications, for example in the diagnostic field or as a research tool or the like. The parts of the kit of the invention may be packaged individually in vials or in combinations in containers or multi-container units. Preferably, the manufacture of the kit follows standard protocols, which are known to those skilled in the art. In accordance with the inventive methods described herein employing, for example, immunohistochemical techniques, kits or diagnostic compositions may be used to detect the expression levels of neuropilin (as defined and described herein).

Although exemplified by the use of bevacizumab, the present invention encompasses the use of other angiogenesis inhibitors known in the art for use in combination with standard chemotherapy regimens. As used herein, the term "angiogenesis inhibitor" refers to all agents that alter angiogenesis (e.g., the process of forming blood vessels), and includes agents that block blood vessel formation and/or stop or slow blood vessel growth. In addition to bevacizumab, non-limiting examples of angiogenesis inhibitors include pegaptanib (pegaptanib), sunitinib, sorafenib, and vatalanib. Preferably, the angiogenesis inhibitor used in accordance with the method of the invention is bevacizumab. As used herein, the term "bevacizumab" encompasses all corresponding anti-VEGF antibodies or anti-VEGF antibody fragments that meet the requirements necessary to obtain a marketing authorization as the same or a biologically similar product in a country or region selected from the united states, europe and japan.

For use in the detection methods described herein, the skilled artisan has the ability to label polypeptides or oligonucleotides encompassed by the invention. The hybridization probes used in detecting mRNA levels and/or the antibodies or antibody fragments used in IHC methods can be labeled and visualized according to standard methods known in the art, as routinely practiced in the art. Non-limiting examples of commonly used systems include the use of radioactive labels, enzymatic labels, fluorescent labels, biotin-avidin complexes, chemiluminescence, and the like.

One skilled in the art, e.g., the attending physician, is readily able to administer bevacizumab in combination with a chemotherapy regimen to a patient/patient group as selected and defined herein. In certain contexts, the attending physician will alter, change or modify the administration regimen of bevacizumab and chemotherapy regimen in accordance with his/her professional experience. Thus, in certain aspects of the invention, methods are provided for treating or improving the therapeutic effect (i.e. progression free or overall survival) with bevacizumab in combination with a chemotherapy regimen in a patient suffering from or suspected to suffer from gastric cancer, wherein said patient/patient group is characterized in the assessment of a biological sample (in particular gastric tissue resection, gastric tissue biopsy and/or metastatic lesions) exhibiting a reduced level of neuropilin expression relative to a control level established in a patient suffering from and/or diagnosed with gastric cancer, in particular gastric or GEJ adenocarcinoma. The present invention also provides the use of bevacizumab for the preparation of a pharmaceutical composition for the treatment of a patient having or suspected of having gastric cancer, in particular gastric or GEJ adenocarcinoma, wherein said patient is selected or characterized by a protein marker/indicator status as disclosed herein (i.e. a reduced level of neuropilin expression relative to a control level established in a patient having gastric cancer, in particular gastric or GEJ adenocarcinoma).

Brief Description of Drawings

The figure shows that:

FIG. 1: correlation of neuropilin expression with overall survival (median cut-off). Long dashed line, placebo, chemotherapy and neuropilin expression above median; short dashed line, bevacizumab therapy, chemotherapy, and biomarker expression above median; solid line, bevacizumab therapy, chemotherapy, and biomarker expression below or equal to median; medium/small dashed line, placebo, chemotherapy and biomarker expression below or equal to median.

FIG. 2: correlation of neuropilin expression with time to progression or death (median cut-off). Long dashed line, placebo, chemotherapy and neuropilin expression above median; short dashed line, bevacizumab therapy, chemotherapy, and biomarker expression above median; solid line, bevacizumab therapy, chemotherapy, and biomarker expression below or equal to median; medium/small dashed line, placebo, chemotherapy and biomarker expression below or equal to median.

FIG. 3: 1, representative amino acid sequence of neuropilin-1, SEQ ID NO.

FIG. 4: correlation between neuropilin expression and overall survival, time to progression or death, and Overall Response Rate (ORR).

Examples

Tissue samples were collected from patients participating in a randomized phase III study that compared the results of adding bevacizumab to first-line capecitabine (allowing for a 5-fluorouracil)/cisplatin combination chemotherapy regimen to treat metastatic or inoperable, locally advanced gastric or GEJ adenocarcinomas if capecitabine is contraindicated (AVAGAST study, see Kang et al, 2010, j.clin.oncol.,28:18s (supplement summary LBA4007) ("Kang")). A survey of biomarker states associated with angiogenesis and tumorigenesis revealed that decreased neuropilin expression levels relative to control levels determined in the entire patient population are indicative of improved overall survival and/or progression-free survival.

Patient and immunohistochemical method

A total of 774 patients participated in the avaast study and tumor samples from 629-. The processing arm is balanced. About 95% of patients are metastatic. Approximately 2/3 patients are male, 49% from the asia-pacific region, 32% from europe and 19% from america (see Kang).

Tissue samples are available as tissue blocks or as previously prepared slides. Immunohistochemical analysis was performed on 5 μm sections (for blocks) of formalin fixed paraffin embedded tissue samples or on previously prepared slides. After deparaffinization and rehydration, antigen retrieval was performed by citrate ph6.0 buffer in PT module at 95 ℃ for 30 minutes or CC1 buffer in Benchmark-XT (Ventana, Tucson, AZ, USA).

Based on known tumorigenic and angiogenic activity, initial biomarkers, including neuropilin, were selected for immunohistochemical analysis. Specifically, neuropilin was analyzed using an anti-human neuropilin murine monoclonal antibody available from Santa Cruz Biotechnology, Inc (Santa Cruz, California, u.s.a.) under product catalog number sc-5307.

Sections were stained on Autostainer or Benchmark-XT (for VEGFR-1) and the primary antibody was incubated for 1 hour. In particular for the san Cruz antibody, this anti-neuropilin antibody was used at 1/50 dilution. Binding of the primary antibody was visualized using the Envision system (DAKO, Glostrup, Denmark) or Ultraview (Ventana, Tucson, AZ USA). All sections were counterstained with Mayer's (Mayer) hematoxylin.

Confirmation reports showing correctness, specificity, linearity, and accuracy (reproducibility and repeatability) were generated for each IHC assay. Staining of the external control slide and the internal control element were documented.

Statistical analysis

H-scores were used for tumor markers to describe the overall distribution of biomarkers. The number of markers examined is limited and each marker is supported by biological basic principles; there was no formal correction for multiple tests. A priori cutoffs were used for protein expression levels: median (lower, upper) and quartile (25 or less, 25< x < 50,50< x < 75 > 75).

The effect of the treatment is assessed in a subset of patients defined by biomarker levels. Selecting overall survival ("OS") and/or progression free survival ("PFS") as a primary endpoint; a main descriptive analysis was performed using subgroup analysis. Testing of treatment for biomarker interactions (median cut-off) also provides a secondary analysis.

Results

Tumor marker

Results of the neuropilin analysis on tumor samples are provided in table 1.

Table 1: neuropilin H scores determined by IHC analysis of avaast samples

Median H-score for neuropilin expression for subsequent analysis was 90, and 25 th and 75 th percentile scores were 40 and 120, respectively.

Correlation of biomarkers with overall survival

Hazard ratios were determined for overall survival in patients separated by median or quartile neuropilin H scores.

Table 2: hazard ratio of overall survival in avaast patients separated by median neuropilin H score

Neuropilin H score	N	Confidence lower bound	Hazard ratio estimation	Upper confidence limit
					<= median value	350	0.59	0.75	0.97
>Median value	329	0.81	1.07	1.40

Table 3: hazard ratio of overall survival in AVAGAST patients separated by a quartile neuropilin H score

Neuropilin H score	N	Confidence lower bound	Hazard ratio estimation	Upper confidence limit
					<=P25	186	0.48	0.68	0.96
P25 to<=P50	164	0.57	0.83	1.19
					P50 to<=P75	184	0.67	0.97	1.42
>P75	145	0.79	1.19	1.78

Calculation of hazard ratios indicates that overall survival is improved in those patients exhibiting relatively reduced tumor-specific neuropilin expression when administered bevacizumab in combination with standard chemotherapy. In particular, in table 2, the upper limit of the 95% confidence interval for the therapeutic hazard ratio in the patient subgroup with lower tumor specific neuropilin expression (less than or equal to the median) is below 1. This supports a statistical correlation of the therapeutic effects (overall survival) observed in this patient subgroup.

A Kaplan-Meier curve (median cut-off) relating bevacizumab treatment to neuropilin expression in terms of overall survival is provided in fig. 1. It can also be seen in figure 1 that those patients with relatively low neuropilin expression had an improvement in overall survival (indicated by hazard ratio) when bevacizumab was added to chemotherapy. Median overall survival was improved for 1.8 months in patients with relatively low tumor-specific neuropilin expression (less than or equal to median) compared to only 0.8 months for patients with tumor-specific neuropilin expression above median. The results demonstrate that the therapeutic effect (overall survival) is improved in a subset of patients with relatively low neuropilin levels.

Raw materialAssociation of marker markers with progression free survival

Hazard ratios were determined for time to disease progression or death in patients separated by median or quartile neuropilin H scores.

Table 4: hazard ratio of time to disease progression or death in AVAGAST patients separated by median neuropilin H score

Neuropilin H score	N	Confidence lower bound	Hazard ratio estimation	Upper confidence limit
					<= median value	350	0.53	0.68	0.87
>Median value	329	0.62	0.80	1.05

Table 5: hazard ratio of time to disease progression or death in AVAGAST patients separated by a quartile neuropilin H score

Neuropilin H score	N	Confidence lower bound	Hazard ratio estimation	Upper confidence limit
					<=P25	178	0.51	0.71	0.98
P25 to<=P50	160	0.47	0.68	0.98
					P50 to<=P75	179	0.50	0.72	1.03
>P75	139	0.61	0.92	1.37

Calculation of hazard ratios indicates that progression-free survival improved with decreased tumor-specific neuropilin expression in those patients administered bevacizumab in combination with standard chemotherapy. In table 4, the upper limit of the 95% confidence interval for the therapeutic hazard ratio in the subset of patients with lower tumor specific neuropilin expression (less than or equal to median) is below 1. This supports a statistical correlation of the therapeutic effects (progression free survival) observed in this patient subgroup.

Table 6: hazard ratio of time to disease progression or death in AVAGAST patients separated by a quartile neuropilin H score (further analysis)

Neuropilin H score	N	Confidence lower bound	Hazard ratio estimation	Upper confidence limit
					<=P25	186	0.51	0.71	0.98

P25 to<=P50	164	0.47	0.68	0.98
					P50 to<=P75	184	0.51	0.73	1.05
＞P75	145	0.60	0.89	1.33

Table 5 was generated in a population by protocol that excluded patients with major protocol violations. Table 6 was generated in the intent-to-treat cohort, which included all randomized patients. Thus, table 6 provides a more accurate analysis.

A kaplan-meier curve (median cut-off) linking bevacizumab treatment and neuropilin expression in terms of progression-free survival is provided in fig. 2. It can also be seen in figure 2 that those patients with relatively low neuropilin expression had improved progression-free survival (indicated by hazard ratio) when bevacizumab was added to chemotherapy. Median progression-free survival was improved for 2.1 months in patients with relatively low tumor-specific neuropilin expression (less than or equal to median) compared to only 1.3 months for patients with tumor-specific neuropilin expression above median. The results demonstrate that the therapeutic effect (progression free survival) is improved in a subset of patients with relatively low neuropilin levels.

Claims (20)

1. A method of improving the treatment effect in a patient with gastric cancer by adding bevacizumab to a chemotherapy regimen, the method comprising:

(a) determining the expression level of neuropilin in a patient sample; and are

(b) Bevacizumab in combination with a chemotherapy regimen is administered to a patient having a reduced neuropilin level relative to a control level determined in a patient having gastric cancer.

2. An in vitro method for identifying a patient responsive or sensitive to treatment with bevacizumab to a chemotherapy regimen, said method comprising determining the expression level of neuropilin in a sample from a patient suspected of having or being susceptible to gastric cancer, wherein a decreased neuropilin level relative to a control level determined in a patient having gastric cancer is indicative of the patient's sensitivity to bevacizumab addition to said regimen.

3. The method of claim 1, wherein the therapeutic effect is overall survival.

4. The method of claim 1, wherein the therapeutic effect is progression free survival.

5. The method of any one of claims 1-4, wherein the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

6. The method of any one of claims 1-5, wherein the neuropilin expression levels are detected by immunohistochemical methods (IHC).

7. The method of any one of claims 1-6, wherein the sample is selected from the group consisting of: gastric tissue resection or gastric tissue biopsy.

8. The method of any one of claims 1-7, wherein the chemotherapy regimen is a capecitabine-based chemotherapy regimen or a 5-fluorouracil-based chemotherapy regimen.

9. The method of claim 8, wherein the capecitabine-based chemotherapy regimen is a regimen of capecitabine combined with cisplatin.

10. The method of claim 8, wherein the 5-fluorouracil-based chemotherapy regimen is a regimen of 5-fluorouracil in combination with cisplatin.

11. The method of any one of claims 1-10, wherein the patient is co-treated with one or more anti-cancer therapies.

12. The method of claim 11, wherein the anti-cancer therapy is radiation.

13. The method of any one of claims 1-12, wherein the expression level of neuropilin is determined prior to neoadjuvant therapy or adjuvant therapy.

14. A kit useful for carrying out the method of any one of claims 1-13 comprising an oligonucleotide or polypeptide capable of determining the expression level of neuropilin.

15. Use of an oligonucleotide or polypeptide for determining the expression level of neuropilin in any one of claims 1-13.

16. The kit of claim 14 or the use of claim 15, comprising a polypeptide capable of determining the expression level of neuropilin, wherein said polypeptide is suitable for use in an immunohistochemical method and/or is an antibody specific for neuropilin.

17. Use of bevacizumab for improving the therapeutic effect of a patient suffering from gastric cancer, comprising the following steps:

(a) determining the expression level of neuropilin in a patient sample; and are

(b) Bevacizumab in combination with a chemotherapy regimen is administered to a patient having a reduced neuropilin level relative to a control level determined in a patient having gastric cancer.

18. The use of claim 17, wherein the therapeutic effect is overall survival.

19. The use of claim 17, wherein the therapeutic effect is progression free survival.

20. The use of any one of claims 17-19, wherein the gastric cancer is gastric adenocarcinoma and/or gastroesophageal junction adenocarcinoma.