WO2016115149A1 - Neuropiline-1 utilisée en tant que biomarqueur sérique - Google Patents

Neuropiline-1 utilisée en tant que biomarqueur sérique Download PDF

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WO2016115149A1
WO2016115149A1 PCT/US2016/013068 US2016013068W WO2016115149A1 WO 2016115149 A1 WO2016115149 A1 WO 2016115149A1 US 2016013068 W US2016013068 W US 2016013068W WO 2016115149 A1 WO2016115149 A1 WO 2016115149A1
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cancer
neuropilin
tivozanib
subject
treatment
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PCT/US2016/013068
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English (en)
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Jie Lin
Bin Feng
Jeno Gyuris
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Aveo Pharmaceuticals, Inc.
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Publication of WO2016115149A1 publication Critical patent/WO2016115149A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • NEUROPILIN-1 AS A SERUM BASED BIOMARKER
  • the field of the invention is molecular biology, genetics, oncology, bioinformatics and clinical diagnostics.
  • biomarker is defined as "a characteristic that is objectively measured and evaluated as an indicator of normal biologic or pathogenic processes or pharmacological response to a therapeutic intervention.”
  • a prognostic biomarker is used to classify a cancer, e.g. , a solid tumor, according to aggressiveness, i.e. , rate of growth and/or metastasis, and refractiveness to treatment. This is sometimes called distinguishing "good outcome” tumors from “poor outcome” tumors.
  • a predictive biomarker is used to assess the probability that a particular subject will benefit from treatment with a particular drug.
  • HER2 HER2 or NEU
  • HERCEPTIN® trastuzumab
  • PD biomarkers are an indication of the effect(s) of a drug on a subject while the subject is taking the drug. Accordingly, PD biomarkers often are used to guide dosage level and dosing frequency, during the early stages of clinical development of a new drug. For a discussion of cancer biomarkers, see, e.g. , Sawyers, 2008, Nature 452:548-552.
  • Neuropilin-1 and neuropilin-2 are transmembrane glycoproteins of up to 923 and 926 amino acids respectively, sharing a similar domain structure and amino acid homology of 44%.
  • NRP-1 is known to bind to several members of the VEGF family, as well as class 3 semaphorins, an unrelated class of ligands with biological functions distinct from those of the VEGFs. NRP-1 is found in several isoforms, as a result of alternative splicing. Because NRP- 1 is highly expressed in diverse tumor cell lines and human neoplasms, it has been suggested as a potential therapeutic target in cancer, e.g. , through the use of blocking NRP1 antibodies, or small molecule antagonists of the ligand interaction with the NRP1 extracellular domain. See, Zachary, 2014, Chem Immunol Allergy 99:37-70.
  • Tivozanib (also known as AV-951) is a potent and selective small-molecule inhibitor of VEGF receptors 1, 2 and 3. Tivozanib exhibits picomolar inhibitory activity against all three receptors, and it exhibits antitumor activity in preclinical models (Nakamura et al , 2006, Cancer Res. 66:9134-9142). In a global, randomized Phase 3 superiority clinical trial evaluating the efficacy and safety of tivozanib compared to sorafenib in 517 subjects with advanced renal cell carcinoma ("TIVO-1 "), tivozanib yielded positive results (Motzer et al , 2012 ASCO Annual Meeting, Abstract No. 4501).
  • TIVO-1 advanced renal cell carcinoma
  • Bevacizumab (Avastin®) is an antibody that specifically targets VEGF A, but not other members of the VEGF family of growth factors (Los et al, 2007, The Oncologist,
  • the invention is based in part on the identification of lower circulating neuropilin-1 levels in a human subject's serum as a prognostic and/or predictive biomarker useful for identifying whether that human subject is likely to be highly responsive or less responsive to treatment with a VEGF inhibitor.
  • the invention provides a method of identifying a human subject likely to be responsive to treatment with a VEGF inhibitor.
  • the method includes comprise measuring the serum level of neuropilin-1 in the subject and determining whether said serum level of neuropilin-1 is below a defined threshold indicative that the subject is likely to be responsive to treatment with an inhibitor of VEGF - mediated angiogenesis.
  • the VEGF inhibitor is a VEGFR2 inhibitor or the VEGF-mediated angiogenesis if VEGFR2-mediated angiogenesis.
  • the invention provides a method of treating a human subject with a VEGF-associated condition in need thereof.
  • the method includes the step of administering an effective amount of a VEGF inhibitor to the human subject that has a serum level of neuropilin-1 below a defined threshold thereby to reduce VEGF-mediated angiogenesis associated with the condition in the subject.
  • the VEGF-mediated angiogenesis reduced is VEGFR2-mediated angiogenesis.
  • the condition is colorectal cancer and the threshold score is, for example, 300 pg/mL or about 300 pg/ml (298.5 pg/mL).
  • the condition is renal cell carcinoma and the threshold score is, for example, 185 pg/mL or about 185 pg/ml (185.5 pg/mL).
  • the invention involves determining whether a human subject with cancer or a tumor has a cancer or tumor type that is likely to respond to treatment with tivozanib and/or whether a particular subject is likely to benefit from treatment with tivozanib.
  • the inventors have surprisingly found that, while subjects with high serum levels of neuropilin-1 may benefit from treatment with tivozanib, subjects with low serum levels of neuropilin-1 are more likely to benefit from such treatment and may show superior response to treatment with tivozanib than those subjects with high levels of neuropilin- 1.
  • the invention provides a method of identifying a human cancer in a subject as responsive to treatment with tivozanib.
  • the method includes the steps of measuring a serum level of neuropilin-1 in the subject and determining whether the serum level of neuropilin-1 is below a defined threshold indicative that the cancer is likely to be responsive to tivozanib.
  • the method when the serum level of neuropilin-1 is below the defined threshold, it is indicative that the cancer is likely to be responsive to tivozanib, whereas when the serum level of neuropilin-1 is above the defined threshold, it is indicative that the cancer is likely to be resistant to tivozanib.
  • the method also includes a threshold determination analysis to generate a defined threshold.
  • the method includes a step of measuring the serum level of one or more additional biomarkers selected from Leptin, IGFBP2, Kallikrein5, endoglin, PSAT, IGFBP1 , IP-10 and MIG/CXCL9.
  • the method further comprises the step of performing a threshold determination analysis for neuropilin-1 and the one or more additional biomarkers, thereby generating a defined threshold neuropilin-1 score and a defined threshold score for the one or more additional biomarkers.
  • the human cancer is colorectal cancer while in another embodiment, the cancer is renal cell carcinoma.
  • the invention provides a method of determining whether to treat a cancer in a human subject with tivozanib rather than with bevacizumab (Avastin®).
  • the method involves measuring a serum level of neuropilin-1 in the subject and determining whether the serum level of neuropilin-1 is below a defined threshold indicative that the subject is more likely to benefit from treatment with tivozanib than with bevacizumb.
  • the cancer is colorectal cancer, while in another embodiment, the cancer is renal cell carcinoma.
  • the invention provides a method for treating a cancer in human subject in need thereof.
  • the method includes the step of administering an effective amount of tivozanib to the human subject, where the subject has been determined to have a serum level of neropilin-1 below a defined threshold thereby to treat the cancer.
  • a serum level of neuropilin-1 below the defined threshold indicates that the cancer is likely to be responsive to tivozanib
  • a serum level of neuropilin-1 above the defined threshold indicates that the cancer or tumor is likely to be resistant to tivozanib.
  • the cancer is colorectal cancer or renal cell carcinoma.
  • the serum level of neuropilin-1 below the defined threshold indicates that the cancer or the tumor is more likely to be responsive to treatment with tivozanib than with bevacizumab.
  • the invention provides a method of assessing the likelihood that a subject with cancer is likely to respond to treatment with tivozanib.
  • the subject may have renal cell carcinoma, or colorectal cancer.
  • the invention provides a method of assessing the likelihood that a subject with cancer is likely to respond more favorably to treatment with tivozanib than to treatment with bevacizumab.
  • the invention provides a method of identifying colorectal cancer as likely to be sensitive to treatment with tivozanib. In another embodiment, the invention provides a method of identifying colorectal cancer as likely to respond more favorably to tivozanib than it is likely to respond to treatment with bevacizumab .
  • the invention provides a method of assessing the likelihood that a subject with colorectal cancer is likely to respond to treatment with tivozanib. In a certain embodiment, the invention provides a method of assessing the likelihood that a subject with colorectal cancer is likely to respond more favorably to treatment with tivozanib than to treatment with bevacizumab. In certain embodiments, the invention provides a method for assessing a subject with colorectal cancer in order to decide whether to treat the subject with (1) bevacizumab + FOLFOX6; or (2) tivozanib + FOLFOX6.
  • the invention provides a method of assessing the likelihood that a subject with colorectal cancer is likely to respond to treatment with tivozanib. In a certain embodiment, the invention provides a method of assessing the likelihood that a subject with colorectal cancer is likely to respond more favorably to treatment with tivozanib than to treatment with bevacizumab. In certain embodiments, the invention provides a method for assessing a subject with colorectal cancer in order to decide whether to treat the subject with (1) bevacizumab + FOLFOX6; or (2) tivozanib + FOLFOX6.
  • the invention provides a method for determining whether to treat a human subject having a cancer with tivozanib in combination with FOLFOX6 rather than bevacizumab in combination with FOLFOX6.
  • the method includes the steps of measuring a serum level of neuropilin-1 in the subject and determining whether said serum level of neuropilin-1 is below a defined threshold indicative that the cancer is more likely to be responsive to treatment with tivozanib in combination with FOLFOX6 than treatment with bevacizumab in combination with FOLFOX6.
  • the cancer when a serum level of neuropilin-1 is below the defined threshold, it is indicative that the cancer is more likely to be responsive to treatment with tivozanib in combination with FOLFOX6 than to treatment with bevacizumab in combination with FOLFOX6.
  • the cancer is colorectal cancer, whereas in another embodiment, the cancer is renal cell carcinoma.
  • the invention provides a method of treating cancer in a human subject in need thereof.
  • the method includes the step of administering an effective amount of a combination of tivozanib and FOLFOX6 to the human subject where the human subject has been determined to have a serum level of neuropilin-1 below a defined threshold. Further, in this method, when the human subject has a serum level of neuropilin-1 below the defined threshold, it is indicative that the cancer is more likely to be responsive to treatment with the combination of tivozanib and FOLFOX6 than with a combination of bevacizumab and FOLFOX6.
  • the cancer is colorectal cancer while in another embodiment, the cancer is renal cell carcinoma.
  • the invention comprises measuring a subject's serum level of neuropilin-1, comparing the subject's serum level of NRP1 with a predetermined threshold serum level of NRP1; and, if the subject's serum level of NRP1 is below the predetermined threshold, treating the subject with the anti-cancer drug known as tivozanib. Accordingly, the invention provides a method for predicting quantitatively whether a human subject will be more likely to benefit from treatment with tivozanib, and may show superior response to treatment with tivozanib [i.e. , "NRP1 low” vs. "NRP1 high”].
  • Suitable methods of measurement include, for example, "capture-sandwich” assays like ELISA or a variation of it such as a Myriad RBM® assay.
  • Other platforms for measurement of circulating levels of protein may be used, such as, for example, a Luminex® microbead/microsphere assay.
  • measurements of circulating serum levels of NRP1 alone will be measured and used to determine whether to treat the subject with tivozanib.
  • the circulating levels of one or more additional serum factors related to angiogenesis may be measured in addition to NRP1, and a determination whether to treat the subject with tivozanib based thereon.
  • Suitable factors for measurement of circulating serum levels in combination with NRP1 include one or more of the following proteins: Leptin,
  • the method includes measuring the circulating serum levels of NR l and at least one other protein selected from the group consisting of Leptin, IGFBP2, Kallikrein5, endoglin, PS AT, IGFBPl, IP- 10 and MIG/CXCL9.
  • the method includes performing a threshold determination analysis, thereby generating a defined threshold.
  • the threshold determination analysis can include a receiver operator characteristic curve analysis.
  • the invention provides a method of treating a subject having cancer.
  • the method comprises: (a) determining whether the subject is likely to be responsive to tivozanib by measuring, in a sample from the serum of the subject, the relative circulating level of NR l; and (b) administering to the subject a therapeutically effective amount of tivozanib if the foregoing steps yield a result indicating that the cancer is likely to be responsive to tivozanib.
  • the method comprises (a) measuring in a serum sample from subject, the neuropilin-1 and at least one additional protein selected from the group consisting of: Leptin or IGFBP2 or Kallikrein5 or endoglin or PS AT or IGFBPl or IP- 10 or MIG/CXCL9 and determining a relative NRPl score; and (b) administering to the subject a therapeutically effective amount of tivozanib if the foregoing steps yield a result indicating that the cancer is likely to be responsive to tivozanib.
  • FIGURE 1 illustrates the difference in baseline pretreatment NRPl serum levels between Responders (Resp) and Non-Responders (Non-resp). See, Example 1 : Exploratory Serum Biomarker Analysis.
  • FIGURE 2 illustrates the difference in progression-free survival (PFS) observed between subjects with high levels of NRPl ("NRPl high”) and subjects with low levels of NRPl ("NRPl low”).
  • PFS progression-free survival
  • FIGURE 3 illustrates the difference in overall survival (OS) observed between subjects with high levels of NRPl ("NRPl high”) and subjects with low levels of NRPl ("NRPl low”).
  • OS overall survival
  • Subjects in the NRPl low group exhibited statistically significant improvement in median PFS over those subjects in NRPl high group; 17.9 months, v. 7.3 months, respectively.
  • Example 3 Neuropilin-1 Serum Levels in Colorectal Cancer: Treatment Of Subjects With Colorectal Cancer with Tivozanib and
  • Neuropilin-1 Serum Levels in Colorectal Cancer Treatment Of Subjects With Colorectal Cancer with Tivozanib and FOLFOX6 vs. Bevacizumab and FOLFOX6.
  • Subjects in the NRPl low group exhibited statistically significant improvement in median OS over those subjects in NRPl high group; >25 months vs. 16.2 months, respectively.
  • FIGURE 7 illustrates the lack of a statistically significant difference in
  • Example 3 Neuropilin-1 Serum Levels in Colorectal Cancer: Treatment Of Subjects With Colorectal Cancer with Tivozanib and FOLFOX6 vs. Bevacizumab and FOLFOX6. DETAILED DESCRIPTION OF THE INVENTION
  • the circulating serum level of neuropilin-1 in a subject can be used as a prognostic biomarker for classifying human tumors or cancers according to their likelihood of responding to treatment with the anti-tumor drug tivozanib, a VEGF tyrosine kinase inhibitor (TKI).
  • a VEGF tyrosine kinase inhibitor TKI
  • Such classification of tumors is useful for identifying human subjects who are suitable candidates for treatment with tivozanib in a clinical setting.
  • the methods of the present invention, in which quantifying serum levels of neuropilin-1 in a subject may surprisingly be used as a prognostic biomarker for treatment of such subject with angiogenesis inhibitors broadly, such as inhibitors of VEGF and inhibitors of VEGF -receptors.
  • Serum biomarker assays are advantageous over intratumoral tissue assays in that they are less invasive, drawing blood rather than requiring a tissue biopsy. Thus, when available, serum biomarkers are more easily tested multiple times, while the analysis of a biomarker found in or on the tumor itself is usually a one-time test result. Further, serum biomarker assays, when available, have the advantage of being more sensitive and accurate and have a wider dynamic range.
  • neuropilin-1 is surprisingly useful as a predictive biomarker in colorectal cancer as to whether the anti-tumor drug tivozanib is a better choice for treatment than bevacizumab (Avastin®).
  • serum expression levels of neuropilin-1 can be used to predict whether a particular subject is more likely to have a better response to treatment with tivozanib in combination with FOLFOX6, compared to treatment with bevacizumab (Avastin® in combination with FOLFOX6.
  • AV-951 and “tivozanib” mean N- ⁇ 2-chloro-4-[(6,7-dimethoxy-4- quinolyl)oxy]-phenyl ⁇ -N'-(5-methyl-3-isoxazolyl)urea, which has the following chemical structure, including salts and polymorphs thereof:
  • threshold neuropilin-1 score means the median neuropilin-1 score for a particular cancer at which the classifier gives the most desirable balance between the cost of false negative calls and false positive calls.
  • median neuropilin-1 score for a particular cancer means the numerical value calculated for a population of subjects, below which a subject is classified as being 'NRP1 LO' and therefore being likely to benefit from treatment with tivozanib.
  • the cancer or tumor of an 'NRP1 LO' subject is more likely to be responsive to treatment with tivozanib.
  • Neuropilin-1 scores higher than the threshold neuropilin-1 score level will be interpreted as indicating that the subject is 'NRP1 ⁇ and therefore less likely to benefit from treatment with tivozanib.
  • the cancer or tumor of an 'NRP1 ⁇ subject is less likely to be responsive (i.e. , may be resistant) to treatment with tivozanib.
  • the median neuropilin-1 score will vary depending upon known parameters. For example, as described herein, in a clinical trial of subjects with advanced colorectal cancer (CRC), the inventors determined the median NRPl level to be 300 pg/mL, more specifically, 298.5 pg/mL. Accordingly, for purposes of the present invention, a serum concentration of 300 pg/mL (298.5 pg/ml) may be considered appropriate for use as a 'median neuropilin-1 score' for subjects with CRC. A subject having CRC with serum NRPl below 300 pg/mL (298.5 pg/ml) may therefore be considered to have low levels of NRPl (i.e.
  • a subject having CRC with serum NRPl equal to or above 300 pg/mL may therefore be considered to have high levels of NRPl (i.e. , 'NRPl ⁇ ).
  • a serum concentration of 185 pg/mL (185.5 pg/mL) may be considered appropriate for use as a 'median neuropilin-1 score' for subjects with RCC.
  • a subject having RCC with serum NRPl below 185 pg/mL (185.5 pg/mL) may therefore be considered 'NRPl LO';
  • a subject having RCC with serum NRPl equal to or above 185 pg/mL (185.5 pg/mL) may therefore be considered 'NRPl HI.
  • ROC receiver operating characteristic
  • TPR true positive / (true positive + false negative)
  • response means, with regard to a treated subject, that the treated subject exhibits: (a) an increase in the period of progression-free survival ("PFS"); or (b) an increase in the period of overall survival, compared to untreated subjects.
  • PFS period of progression-free survival
  • responding an increase in the period of overall survival, compared to untreated subjects.
  • responsive means that the tumor of a treated subject displays: (a) slowing of growth, (b) cessation of growth, or (c) regression (i.e., decrease in size).
  • threshold determination analysis means analysis of a dataset representing a given cancer or tumor type, e.g., human renal cell carcinoma, to determine a threshold neuropilin-1 concentration, e.g. , an optimum threshold neuropilin-1 score, for that particular cancer or tumor type.
  • the dataset representing a given cancer or tumor type includes (a) actual response data (response or non-response), and (b) a threshold determination for each cancer or tumor from a group having such cancer or tumor type.
  • the group may be tumor-bearing mice or other non-human subject.
  • the group may be human subjects having such cancer type or tumor type.
  • Treatment in accordance with the methods and compositions described herein may improve or ameliorate one or more the characteristics or symptoms of a VEGF-associated condition.
  • "treat,” “treating” and “treatment” mean the treatment of a disease in a subject, e.g. , in a human. This includes: (a) inhibiting the disease, i.e. , arresting its development; and (b) relieving the disease, i.e. , causing regression of the disease state.
  • VEGF-associated condition means any disease or disorder associated with VEGF that can be treated with a VEGF inhibitor described herein.
  • exemplary VEGF-associated conditions include, but are not limited to, cancer, including colorectal cancer, kidney cancer, and breast cancer; rheumatoid arthritis; diabetic retinopathy; age-related macular degeneration (AMD); angiosarcoma; pulmonary emphysema; proteinuria; and preeclampsia.
  • Serum Samples A serum tissue sample collected from a human subject prior to treatment may be used as a baseline, and may be analyzed using any suitable assay for the measurement of serum proteins, so that a baseline neuropilin-1 serum level can be determined.
  • the tissue sample is optimally obtained from the subject and analyzed using conventional instruments and procedures.
  • NRP1 serum level may be determined using the Myriad RBM® platform (Myriad RBM, Inc., Salt Lake City, Utah). Other recognized medical procedures for obtaining and analyzing serum protein levels can be used by the skilled person to obtain and analyze serum NRP1 levels for practicing the methods described herein.
  • the serum tissue sample should be large enough to provide sufficient protein for measuring individual biomarker serum levels.
  • the sample is in the form of circulation tumor cells (CTCs) in a blood sample.
  • CTCs circulation tumor cells
  • Serum protein levels can be analyzed using an NRP1 ELISA kit.
  • kits are commercially available, for example, the Human Neuropilin-1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN); and Human Soluble Neuropilin-1 ELISA Kit (A viscera Science, Santa Clara, CA).
  • ELISA Performing a neuropilin-1 protein ELISA, requires at least one antibody against an NRP1 protein, i.e. , the detection antibody.
  • subject protein from a sample to be analyzed is immobilized on a solid support such as a polystyrene microtiter plate. This immobilization can be by non-specific binding of the subject, e.g. , through adsorption to the surface. Alternatively, immobilization can be by specific binding, e.g. , through binding of NRP1 protein from the sample by a capture antibody (anti-NRPl antibody different from the detection antibody), in a "sandwich" ELISA.
  • the detection antibody is added, and the detection antibody forms a complex with the bound NRP1.
  • the detection antibody is linked to an enzyme, either directly or indirectly, e.g. , through a secondary antibody that specifically recognizes the detection antibody.
  • Typical ELISA protocols also include one or more blocking steps, which involve use of a non-specifically-binding protein such as bovine serum albumin to block unwanted nonspecific binding of protein reagents to the plate.
  • the plate is developed by addition of an appropriate enzyme substrate, to produce a visible signal, which indicates the quantity of NRP1 in the sample.
  • the substrate can be, e.g., a chromogenic substrate or a fluorogenic substrate.
  • marker proteins e.g. , Leptin or IGFBP2 or Kallikrein5 or endoglin or PSAT or IGFBP1 or IP-10 or MIG/CXCL9
  • these marker proteins may be used in conjunction with NRP1.
  • biomarkers e.g. , Leptin or IGFBP2 or Kallikrein5 or endoglin or PSAT or IGFBP1 or IP-10 or MIG/CXCL9, as well as other serum-based protein biomarkers can be measured, for example, median biomarker serum protein levels can be determined and biomarker serum protein levels can be measured and compared to the median.
  • protein biomarkers may be measured, other than by measuring serum protein levels, using known methods of measuring protein levels, such as immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • one skilled in the art may identify non-protein biomarkers, such as DNA expression levels, RNA levels, etc. to be used in methods of the present invention.
  • the selection of additional biomarkers, if any, to be used in combination with serum levels of NRPl, and the method of measuring levels of such biomarkers, are design choices, and can be accomplished by a person of skill in the art, without undue
  • Neuropilin-1 score levels preferably are interpreted with respect to a threshold neuropilin-1 score.
  • a neuropilin-1 score lower than the threshold neuropilin-1 score will be interpreted as indicating that a cancer or tumor is likely to be responsive (sensitive) to tivozanib, and hence, a subject with such cancer or tumor may benefit from treatment with tivozanib.
  • Neuropilin-1 scores higher than the threshold neuropilin-1 score level will be interpreted as indicating that a cancer or tumor is likely to be non-responsive (resistant) to tivozanib, and hence, a subject with such cancer or tumor is not as likely to benefit from treatment with tivozanib.
  • cancer type or tumor type
  • cancer type or tumor type takes into account (a) species (mouse or human); and (b) organ or tissue of origin.
  • cancer type or tumor type further takes into account cancer
  • an optimum neuropilin-1 score can be determined (or at least approximated) empirically by performing a threshold determination analysis.
  • threshold determination analysis includes receiver operator characteristic (ROC) curve analysis.
  • ROC curve analysis is an established statistical technique, the application of which is within ordinary skill in the art.
  • ROC curve analysis see generally Zweig et at , 1993, "Receiver operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine,” Clin. Chem. 39:561-577; and Pepe, 2003, The statistical evaluation of medical tests for classification and prediction, Oxford Press, New York.
  • Neuropilin-1 scores and the optimum neuropilin-1 score may vary from cancer type to cancer type (or tumor type to tumor type). Therefore, a threshold determination analysis preferably is performed on one or more datasets representing any given cancer type or tumor type to be tested using the present invention.
  • the dataset used for threshold determination analysis includes: (a) actual response data (response or non-response), and (b) a neuropilin-1 score for each serum sample from a group of human cancers or tumors or mouse cancers or tumors. Once a neuropilin-1 score threshold is determined with respect to a given cancer type or tumor type, that threshold can be applied to interpret neuropilin-1 scores from cancers of that cancer type, or tumors of that tumor type.
  • the ROC curve analysis is performed essentially as follows. A sample with a neuropilin-1 score below threshold is classified as a responder, and a sample with a neuropilin- 1 score above the threshold is classified as a non-responder. Whether the neuropilin-1 score is higher or lower will depend on the type of assay and the units of measure employed. For every neuropilin-1 score from a tested set of samples, "responders" and “non-responders"
  • ROC curve is constructed by making a dot plot, using the TPR vector, and FPR vector. If the ROC curve is above the diagonal from (0, 0) point to (1.0, 0.5) point, it shows that the neuropilin-1 score test result is a better test than random.
  • the ROC curve can be used to identify the best operating point.
  • the best operating point is the one that yields the best balance between the cost of false positives weighed against the cost of false negatives. These costs need not be equal.
  • the average expected cost (C) of classification at point x,y in the ROC space is denoted by the expression
  • the smallest C can be calculated after using all pairs of false positive and false negative (x, y).
  • the optimum neuropilin-1 score threshold is calculated as the score of the (x, y) at C.
  • a neuropilin-1 score provides an approximate, but useful, indication of how likely a cancer or tumor is to be responsive or non-responsive. In general, the lower the neuropilin-1 score, the more likely a cancer or tumor is to be responsive to tivozanib, and the higher the lower the neuropilin-1 score, the more likely a cancer or tumor is to be resistant to tivozanib.
  • serum samples may include measurement of levels of one or more proteins used as internal standards.
  • the choice of proteins to be used as internal standards can be affected by the type of tissue sample to be tested. For any given type of tissue sample, there is wide latitude in the number and identity of internal standards. Choice of internal standards is within skill in the art.
  • the set of materials for analysis of serum samples form part of a diagnostic test kit that contains other components such as buffers, reagents and detailed instructions for using the set, e.g., in the methods described herein.
  • a diagnostic test kit can enhance the convenience and reproducibility in the performance of the methods described herein.
  • the kit may take advantage of availability of platform technologies that are well developed and currently available from commercial vendors, such as the Myriad RBM® platform described above.
  • a basic diagnostic test kit includes an ELISA assay for neuropilin-1.
  • the kit includes antibodies for ELISA assays for one or more additional proteins, such as Leptin, IGFBP2, Kallikrein5, endoglin, PS AT, IGFBP1, IP- 10 and/or MIG/CXCL9 for development of the score used in the methods described herein.
  • a more elaborate test kit contains not only antibodies for ELISA assays, but also buffers, reagents and detailed instructions for measuring the serum levels of the members of a neuropilin-1 score, using ELISA-based technology.
  • the kit includes a test protocol and all the consumables, i.e., reagents and single- use components (except test samples), needed to perform the methods disclosed herein.
  • the invention includes a method of treating a human subject with a VEGF- associated condition in need thereof.
  • the method includes administering an effective amount of a VEGF inhibitor to the human subject that has a serum level of neuropilin-1 below a defined threshold thereby to reduce VEGF-mediated angiogenesis associated with the condition in the subject.
  • the VEGF-mediated angiogenesis reduced is VEGFR2- mediated angiogenesis.
  • a therapeutically effective amount of active component e.g. , a VEGF- inhibitor
  • a therapeutically effective amount of active component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg.
  • the amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration.
  • the initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood- level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment.
  • Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg.
  • Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the antibody, and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks.
  • the optimal effective amount of the compositions can be determined empirically and will depend on the type and severity of the disease, route of administration, disease progression and health, mass and body area of the subject. Such determinations are within the skill of one in the art.
  • Examples of dosages of VEGF inhibitors which can be used for methods described herein include, but are not limited to, an effective amount within the dosage range of any of about 0.01 ⁇ g/kg to about 300 mg/kg, or within about 0.1 ⁇ g/kg to about 40 mg/kg, or with about 1 ⁇ g/kg to about 20 mg/kg, or within about 1 ⁇ g/kg to about 10 mg/kg.
  • the composition when administered subcutaneously, the composition may be administered at low microgram ranges, including for example about 0.1 ⁇ g/kg or less, about 0.05 ⁇ g/kg or less, or 0.01 ⁇ g/kg or less.
  • the invention includes a method of treating a cancer subject, comprising:
  • determining whether the subject is likely to be responsive to tivozanib by measuring, in a serum sample from the subject, the serum protein level of neuropilin-1 and optionally one or more of the following genes: Leptin, IGFBP2, Kallikrein5, endoglin, PSAT, IGFBP1, IP-10 and/or MIG/CXCL9; and
  • Tivozanib may be administered in combination or in sequence with any approved or known therapy regimen.
  • subjects with colorectal cancer are frequently treated with a combination of chemotherapy agents known as "FOLFOX.”
  • FOLFOX comprises a combination of folinic acid (or leucovorin) ("FOL"), fluorouracil (or 5-FU) ("F") and oxaliplatin (or Eloxatin® Sanofi S.A.) ("OX").
  • FOLFOX6 constitutes the sixth variation of the FOLFOX treatment regimen.
  • FOLFIRI which is a combination of folinic acid, fluorouracil and irinotecan hydrochloride (or Camptosar® Pfizer, Inc.)
  • XELOX which is a combination of capecitabine (or Xeloda® Genentech, Inc.)
  • mFOLFOX6 modified FOLFOX6
  • chemotherapy is administered in conjunction with tivozanib according to a 28 day cycle.
  • Tivozanib is administered orally at a dose of 1.5 mg daily beginning on Day 1 of each Cycle for 21 days, followed by 7 days off treatment, subject to dose reduction to 1.0 mg daily for Grade 3 drug-related adverse events. Hypertension events must be treated with anti-hypertensive drugs prior to dose reduction.
  • oxaliplatin is administered at 85 mg/m 2 via intravenous bolus in 500 mL of D5W over 2 hours on days 1 and 15 of the 28 day cycle.
  • Folinic acid is administered at 400 mg/m 2 via intravenous bolus in 500 mL of D5W over 2 hours on days 1 and 15 of the 28 day cycle, which may be concurrently with oxaliplatin via a separate IV line.
  • Fluorouracil bolus is administered at 400 mg/m 2 via intravenous over 5-15 minutes, or infused per institutional guidelines on days 1 and 15. Fluorouracil is additionally administered at 2400 mg/m 2 via infusion pump over 46-48 hours or infusion per institutional guidelines on days 1-3 and 15-17 of the 28 day cycle.
  • Non-responders' were subjects who exhibited both: (1) MPR varying from a reduction in tumor size of -7.8% to an increase in tumor size of 174.2%; and (2) PFS, measured by independent assessment, from 49 to 144 days. [0071] All 50 serum samples were profiled on Rules Based Medicine (RBM) Human Oncology Map 1.0 platform, which measures the serum levels of 99 proteins. Table 1 shows the results of this analysis for 10 serum factors that were found to have the most significant differences in Responders v. Non-Responders.
  • RBM Rules Based Medicine
  • the difference in NRPl serum levels between Responders and Non-Responders is shown in Figure 1.
  • TIVO-1 was a global, randomized Phase 3 superiority clinical trial evaluating the efficacy and safety of investigational drug tivozanib compared to sorafenib in 517 subjects with advanced RCC (Motzer et al, 2012 ASCO Annual Meeting, Abstract No. 4501). All subjects in TIVO-1 had clear cell RCC, had undergone a prior nephrectomy, and had not been treated previously with either a VEGF or mTOR therapy. TIVO-1 demonstrated a significant improvement in progression-free survival (PFS) in subjects receiving tivozanib vs. sorafenib.
  • PFS progression-free survival
  • Example 3 Neuropilin-1 Serum Levels in Colorectal Cancer: Treatment Of Subjects
  • modified FOLFOX6 mFOLFOX6
  • chemotherapy is administered in conjunction with tivozanib according to a 28 day cycle.
  • Tivozanib is administered orally at a dose of 1.5 mg daily beginning on Day 1 of each Cycle for 21 days, followed by 7 days off treatment, subject to dose reduction to 1.0 mg daily for Grade 3 drug- related adverse events. Hypertension events must be treated with anti-hypertensive drugs prior to dose reduction.
  • oxaliplatin In the mFOLFOX regimen, oxaliplatin is administered at 85 mg/m 2 via intravenous bolus in 500 mL of D5W over 2 hours on days 1 and 15 of the 28 day cycle.
  • Folinic acid is administered at 400 mg/m 2 via intravenous bolus in 500 mL of D5W over 2 hours on days 1 and 15 of the 28 day cycle, which may be concurrently with oxaliplatin via a separate IV line.
  • Fluorouracil bolus is administered at 400 mg/m 2 via intravenous over 5-15 minutes, or infused per institutional guidelines on days 1 and 15.
  • Fluorouracil is additionally administered at 2400 mg/m 2 via infusion pump over 46-48 hours or infusion per institutional guidelines on days 1-3 and 15-17 of the 28 day cycle.
  • bevacizumab is administered as an intravenous infusion at a dose of 5 mg/kg every 2 weeks beginning on day 1 of cycle 1. Subjects receive 2 treatments of bevacizumab in each treatment cycle, on day 1 and day 15. Bevacizumab will be administered prior to the start of the mFOLFOX6 chemotherapy regimen or per institutional guidelines.
  • NRPl low is a predictive biomarker for better efficacy of tivozanib over bevacizumab in subjects with low levels of NRPl.

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Abstract

L'invention concerne des procédés de pronostic et de diagnostic prédictif permettant de déterminer si un sujet humain répondra plus ou moins à un traitement par des inhibiteurs du VEGF, tels que le tivozanib (AV-951). Ces procédés sont basés sur la mesure des niveaux circulants de neuropiline-1 dans le sérum.
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