Classifications

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  • 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
  • G01N33/57488—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 involving compounds identifable in body fluids
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  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
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  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
• • A—HUMAN NECESSITIES
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  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
• • A—HUMAN NECESSITIES
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  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • 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
  • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
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  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• • 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

• the present invention relates to the field of biomarkers, and particularly concerns the use of pre-treatment levels of p2-glycoprotein 1 ( ⁇ 2 ⁇ ), particularly functional 2GPI, as an indicator to predict successful responses to therapies using PS-targeting antibodies such as bavituximab.
• IO immuno-oncology
• IO agents that target programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) have already received approval for the treatment of some advanced malignancies, while compounds that interact with other IO targets are in development.
• PD-1 programmed cell death protein 1
• PD-L1 programmed death-ligand 1
• PS membrane phospholipid, phosphatidyl serine
• 3G4 mouse-human chimeric monoclonal antibody
• bavituximab are part of a family of murine, chimeric and fully human antibodies that target PS in a p2-glycoprotein 1 (P2GPI)-dependent manner. That is, bavituximab and related PS-targeting antibodies bind to PS in the presence of P2GPI, such that they form a high affinity antibody- 2GPI-PS complex (Luster et al, 2006).
• P2GPI p2-glycoprotein 1
• these 2GPI-dependent PS-targeting antibodies are specific for PS in vivo, as most particularly shown by numerous imaging studies (Jennewein et al., 2008; Marconescu & Thorpe, 2008; Saha et al, 2010; Stafford & Thorpe, 201 1 ; Zhao et al, 201 1 ; Zhang et al, 2014; and Zhou, et al, 2014; U.S. Patent No. 7,790,860), including measuring and predicting response to therapy (Gong et al, 2013; Stafford et al, 2013).
• Bavituximab has demonstrated activity against a wide range of diseases in which PS is a marker, most particularly cancer and viral infections, but also infections of intracellular parasites, such as the parasitic protozoan, Leishmania amazonensis (Wanderley et al, 2013) and intracellular bacterial pathogens, such as Yersinia pestis and Francisella tularensis, which cause plague and tularemia, respectively (Lonsdale et al, 201 1).
• PS-targeting antibodies such as bavituximab have been shown to inhibit viral replication, decrease viral load in organs and increase survival (Soares et al , 2008; Moody et al, 2010; U.S. Patent No. 7,906,1 1 ).
• bavituximab and related PS-targeting antibodies have been demonstrated in an extensive number of pre-clinical studies and clinical trials, in which effects are mediated against tumor blood vessels as well as by blocking the immunosuppressive signaling of PS (Ran et al, 2005; U.S. Patent No. 7,572,448; DeRose et al, 201 1).
• the anti-tumor effects of PS-targeting antibodies such as bavituximab are enhanced when the antibodies are used in conjunction with agents or conditions that increase the exposure of PS in the tumor micro environment, such as by the use of radiation and/or the co- administration of chemotherapy (U.S. Patent No. 7,422,738; U.S. Patent No. 8,486,391 ; U.S.
• Patent No. 7,572,448 For example, improved anti-tumor effects have been demonstrated pre- clinically when using the bavituximab family of PS-targeting antibodies in combination with docetaxel to treat breast tumors (Huang et al., 2005); gemcitabine to treat pancreatic tumors (Beck et ai, 2006); irradiation to treat lung cancer (He et ai, 2007) and the brain cancer, glioblastoma (He et ai, 2009); docetaxel to treat prostate cancer and reactivate antitumor immunity (Yin et ai, 2013); and sorafenib to treat hepatocellular carcinoma (Cheng et al., 2016).
• PS-targeting antibodies such as bavituximab are used in combination therapies with other IO agents, as shown pre-clinically for the treatment of melanoma (Freimark et al., 2016) and triple -negative breast cancer (Gray et al., 2016a) in combination with checkpoint inhibitors in the form of antibodies to CTLA-4 or PD-1.
• HCV chronic hepatitis C virus
• HCC hepatocellular carcinoma
• RCC renal cell carcinoma
• the present invention addresses the foregoing and other needs of the prior art by providing new biomarker methods, compositions, kits and assays for optimizing treatment with PS-targeting antibodies such as bavituximab and like antibodies, e.g., 1N1 1.
• the invention particularly concerns the use of pre-treatment levels of p2-glycoprotein 1 ( ⁇ 2 ⁇ ), most preferably functional ⁇ 2 ⁇ , as an indicator or "biomarker" to predict successful responses to therapies using bavituximab.
• compositions, kits and assays provide for identifying and treating patients treatable with p2GPI-dependent PS-targeting antibodies, particularly cancer patients for treatment with a bavituximab-containing therapeutic regimen or combination therapy, most preferably, for identifying and treating patients with cancer using bavituximab (a first anti-cancer agent) and at least a second or third anti-cancer agent, all by selecting patients with defined ranges of pre-treatment blood concentrations of functional p2GPI.
• "Functional" p2GPI is p2GPI that binds to both PS and to a p2GPI-dependent PS-targeting antibody, preferably bavituximab.
• the invention concerns the selection, identification, diagnosis and preferably treatment of patients based on pre-treatment levels of functional P2GPI of equal to or greater than 200 ⁇ g/ml.
• the invention provides a method for treating a disease in which PS is a marker, particularly cancer, in a human patient, comprising administering a PS-targeting antibody, preferably bavituximab, to the patient, wherein the patient has a pre-treatment blood concentration of functional P2GPI of equal to or greater than 200 ⁇ g/ml.
• a PS-targeting antibody such as bavituximab, which is herein termed a "first therapeutic agent” and "first anti-cancer agent”
• first therapeutic agent and “first anti-cancer agent
• the invention also provides a method for treating a disease in which PS is a marker, particularly cancer, in a human patient, comprising (the steps of):
• a PS-targeting antibody preferably bavituximab
• a pre-treatment blood concentration of functional P2GPI of equal to or greater than 200 ⁇ g/ml.
• the PS-targeting antibody such as bavituximab is administered to the patient with at least a second therapeutic or anti-cancer agent.
• the invention further provides a method for treating a disease in which PS is a marker, particularly cancer, in a human patient, comprising (the steps of):
• a PS-targeting antibody preferably bavituximab
• a pre-treatment blood concentration of functional P2GPI of equal to or greater than 200 ⁇ g/ml.
• the PS-targeting antibody such as bavituximab is administered to the patient with at least a second therapeutic or anti-cancer agent.
• Also provided by the invention is a method for identifying a human patient, preferably a human cancer patient, treatable with a PS-targeting antibody, preferably bavituximab, and treating the patient, comprising (the steps of): (a) measuring the concentration of functional P2GPI in a pre-treatment blood sample obtained from the patient;
• a PS-targeting antibody preferably bavituximab
• a pre-treatment blood concentration of functional P2GPI of equal to or greater than 200 ⁇ g/ml.
• the patient is identified as treatable with bavituximab and at least a second therapeutic or anti-cancer agent and
• bavituximab and at least a second therapeutic or anti-cancer agent is administered to the patient.
• a method for identifying a human patient preferably a human cancer patient, treatable with a PS-targeting antibody, preferably bavituximab, and treating the patient, comprising (the steps of):
• a PS-targeting antibody preferably bavituximab
• a pre-treatment blood concentration of functional P2GPI of equal to or greater than 200 ⁇ g/ml.
• the patient is identified as treatable with bavituximab and at least a second therapeutic or anti-cancer agent and
• bavituximab and at least a second therapeutic or anti-cancer agent is administered to the patient.
• Another embodiment of the invention is a method of diagnosing a patient, preferably a cancer patient, treatable with a PS-targeting antibody, preferably bavituximab, wherein the method comprises measuring the concentration of functional P2GPI in a blood sample from the patient, wherein the patient is determined to be treatable with a PS-targeting antibody, preferably bavituximab, if the blood concentration of functional ⁇ 2 ⁇ is equal to or greater than 200 ⁇ g/ml.
• the patient is diagnosed as treatable with bavituximab and at least a second therapeutic or anti-cancer agent if the blood concentration of functional ⁇ 2 ⁇ is equal to or greater than 200 ⁇ g/ml.
• a further embodiment of the invention is a PS-targeting antibody, preferably bavituximab, for use in a method of treating a patient, preferably of treating cancer in a patient, wherein functional P2GPI is present at a concentration of equal to or greater than 200 ⁇ g/ml in a blood sample from the patient.
• this concerns bavituximab for use in such a method of treating cancer, further comprising administering at least a second anti-cancer agent.
• Yet another embodiment of the invention is a PS-targeting antibody, preferably bavituximab, for use in a method of treating a disease in which PS is a marker, preferably cancer, wherein the method comprises (the steps of):
• bavituximab and at least a second therapeutic or anti-cancer agent for use in a method of treating cancer.
• the invention concerns the selection of patients based on pre-treatment functional ⁇ 2 ⁇ in the range of 200-290 ⁇ g/ml.
• All numbers and ranges within these levels are included in each of the foregoing methods and uses, such as the selection, identification, diagnosis and preferably treatment of patients based on pre-treatment levels of functional P2GPI of equal to or greater than 200 or 210 ⁇ g/ml; and P2GPI in the ranges of from any one of 200 or 210 ⁇ g/ml as the low number, to any one of 270, 280, 290, 300, 310 or 320 ⁇ as the high number, including 200-270, 200-280, 200-290, 200-300, 200-310, 200- 320, 210-270, 210-280, 210-290, 210-300, 210-310 and 210-320 ⁇ and such like, with the ranges 210-270, 210-280, 210-290, 200-280 and 200-290 being currently preferred.
• a PS-targeting antibody preferably bavituximab
• the antibody is given at a dose of between about 1-10, 1 -6, 3-6 or 1-3 mg/kg, most preferably, of about 3 mg/kg.
• the invention is applicable to the use of a PS-targeting antibody, preferably bavituximab with a second or third anti-cancer agent that is a chemotherapeutic agent, such as sorafenib, paclitaxel, carboplatin, gemcitabine or docetaxel, for the treatment of solid rum, such as ovarian, gastric, liver, colorectal, breast, esophageal, brain (e.g., glioma, glioblastoma), prostate, skin (melanoma), head and neck, kidney, bladder, pancreatic or lung cancer, preferably non-small cell lung cancer (NSCLC), including non- squamous NSCLC.
• a chemotherapeutic agent such as sorafenib, paclitaxel, carboplatin, gemcitabine or docetaxel
• solid rumors such as ovarian, gastric, liver, colorectal, breast, esophageal, brain (e.g.
• bavituximab binding in a complex with functional ⁇ 2 ⁇ and PS and the immune activating mechanisms of bavituximab overall, are common to all bavituximab therapies. Therefore, the invention applies to the selection of patients for any therapies using PS-targeting antibodies such as bavituximab or like antibodies, e.g., 1N1 1 , particularly in combination therapies, such as with chemotherapy, and preferably with immuno-oncology (IO) agents.
• PS-targeting antibodies such as bavituximab or like antibodies, e.g., 1N1 1
• combination therapies such as with chemotherapy, and preferably with immuno-oncology (IO) agents.
• IO immuno-oncology
• Suitable IO agents are immune checkpoint antibodies, including agonistic (activating) antibodies that bind to an activating immune checkpoint, receptor or molecule, such as CD28, OX40 and/or GITR, and preferably antagonistic (blocking) antibodies that bind to an inhibitory immune checkpoint, receptor or molecule, such PD-1 , PD-L1 , CTLA-4, TIM-3 and/or LAG-3.
• Antagonistic (blocking) antibodies that bind to an inhibitory immune checkpoint, receptor or molecule are also herein termed "immune checkpoint inhibitors" or "ICIs”.
• immune checkpoint antibodies are blocking antibodies to CTLA-4, PD-1 or PD-L1 , such as ipilimumab, tremelimumab, nivolumab, pembrolizumab, durvalumab and atezolizumab.
• the invention particularly contemplates the selection, diagnosis and treatment of patients with a PS-targeting antibody, preferably baviruximab, and with a second and third therapeutic or anti-cancer agent.
• a chemotherapeutic agent and an immune checkpoint antibody or with two immune checkpoint antibodies, including treatment with a PS-targeting antibody, preferably baviruximab, in which treatment with a chemotherapeutic agent is followed by treatment with an immune checkpoint antibody.
• the present invention also provides new assay methods, compositions and kits particularly adapted for the detection and quantification of functional ⁇ 2 ⁇ .
• the invention therefore further provides methods of measuring functional ⁇ 2 ⁇ comprising (the steps of): optionally, coating a solid support such as an ELISA plate with PS to prepare a PS-coated solid support or PS-coated ELISA plate (or a pre-prepared PS-coated solid support or ELISA plate may be used); adding a PS-targeting antibody, preferably baviruximab, and a biological sample suspected of containing ⁇ 2 ⁇ to a PS-coated solid support, thereby co-incubating the antibody and sample under conditions effective to allow binding of ⁇ 2 ⁇ in the sample to both the PS-targeting antibody, preferably baviruximab, and the PS-coated solid support; and detecting the binding of the PS-targeting
• the biological sample suspected of containing ⁇ 2 ⁇ may be a blood sample, such as a plasma sample or a serum sample.
• Other biological fluid samples suspected of containing ⁇ 2 ⁇ may be used, including cell supernatants and the like.
• the PS-targeting antibody preferably bavituximab
• the PS-targeting antibody will itself be attached to a detectable agent that produces a detectable signal, such that the binding of the antibody and ⁇ 2 ⁇ to the PS-coated solid support is detected and measured by detecting and measuring the detectable signal.
• the PS-targeting antibody preferably bavituximab
• the PS-targeting antibody is added to the PS-coated solid support prior to adding the sample suspected of containing ⁇ 2 ⁇ , such as a blood sample.
• FIG. 1A, FIG. IB, FIG. 1C and FIG. ID Inhibition of tumor growth in mice treated with the 3G4 antibody, purified to apparent homogeneity.
• FIG. 1A established (0.6-0.7 cm diameter, 140 mm 3 volume) human MDA-MB-435 breast carcinomas growing in the mammary fat pads of SCID mice;
• FIG. IB established (0.5-0.7 cm diameter, 1 10 mm 3 volume) human MDAMB-231 breast carcinomas growing in the mammary fat pads of SCID mice;
• FIG. 1 C small Meth A fibrosarcomas growing subcutaneously in syngeneic BALB/c mice; and
• FIG. 1A, FIG. IB, FIG. 1C and FIG. ID Inhibition of tumor growth in mice treated with the 3G4 antibody, purified to apparent homogeneity.
• FIG. 1A established (0.6-0.7 cm diameter, 140 mm 3 volume) human MDA-MB-435 breast carcinomas growing in the mammary fat pads of SCID mice
• mice large (0.8-1 cm diameter, 370 mm 3 volume) human L540 Hodgkin's tumors growing subcutaneously in SCID mice.
• Groups of 8 to 10 mice were injected i.p. with 100 ⁇ g of the 3G4 antibody (O, open circles) or the control, BBG3 antibody ( ⁇ , closed circles) starting on the day indicated by the arrows. Treatments were continued thrice a week thereafter.
• FIG. 2A and FIG. 2B 3G4 antibody binding to PS-coated microtiter plates is serum- dependent.
• FIG. 2A the 3G4 antibody was purified from cells grown in bovine serum- containing media (A , SCM) or serum-free media ( ⁇ , SFM).
• a microtiter plate was coated with PS and blocked in 1 % ovalbumin from chicken egg white (OVA).
• Serial dilutions of 3G4 were performed in 10% fetal bovine serum (solid lines, FBS) or 1 % ovalbumin from chicken egg white (dashed lines, OVA).
• FIG. 2B the microtiter plate was coated with PS and blocked in 1% OVA.
• FIG. 3G4 antibody binds the plasma protein, ⁇ 2 ⁇ .
• a microtiter plate was coated with human ⁇ 2 ⁇ (1 ⁇ 20 ⁇ ) purified from human plasma and blocked in 1% OVA.
• Serial dilutions of a commercial mouse anti-human 2GPI ( ⁇ , ⁇ - ⁇ 2 ⁇ ), the 3G4 antibody from SFM ( ⁇ , 3G4) and a control mouse IgG (A , control mlgG) were performed in 1% OVA.
• FIG. 4 The 3G4 antibody binds to 2GPI at domain II.
• the wells of a microtiter plate were coated with recombinant full-length hp2GPI ( ⁇ , domain I-V) or h 2GPI peptides without domain I (-X-, domain II-V), without domains I and II ( ⁇ , domain III-V), without domains I, II and III ( A , domain IV-V) or without domains I, II, III and IV ( ⁇ , domain V).
• the plate was blocked in 1% OVA and serial dilutions of the 3G4 antibody from SFM were performed in l% OVA.
• FIG. 5 The ch3G4 antibody and ⁇ 2 ⁇ together bind cells with exposed PS.
• Adult bovine aortic endothelial (ABAE) cells were incubated for 30 min with 200 ⁇ lysophosphatidylcholine (LPC) in DMEM + 10% normal mouse serum (MS), plus (i) ch3G4 only, (ii) ch3G4 + hp2GPI simultaneously, or (iii) purified hp2GPI only. Cells were then washed and incubated for 30 min with (i) buffer only, (ii) buffer only, or (iii) ch3G4, respectively. Finally, cells were washed, fixed, and stained with fluorescent markers to detect binding of ch3G4.
• ch3G4 and hp2GPI were used at a concentration of 2 ⁇ g/ml.
• the pixel area of ch3G4 binding was quantified using MetaVue software. Values are relative to the binding of ch3G4 under condition (i), which was set to one.
• FIG. 6A and FIG. 6B The lipid binding region of P2GPI mediates binding of the ch3G4 antibody to cells with exposed PS.
• FIG. 6A ABAE cells were incubated with the ch3G4 antibody plus (i) a non-lipid binding form of ⁇ 2 ⁇ (nicked 1 ⁇ 20 ⁇ ) or (ii) intact b ⁇ 2GPI ("hp2GPI"). The incubations were performed in the presence or absence of 200 ⁇ LPC in DMEM + 10% MS for 30 min. Cells were then washed, fixed, and stained with fluorescent markers to detect binding of ch3G4. The ch3G4 antibody, intact hp2GPI and nicked hp2GPI were used at a concentration of 2 ⁇ / ⁇ 1.
• the pixel area of ch3G4 binding was quantified using MetaVue software. Values are relative to the binding of ch3G4 under condition (i) no LPC, which was set to one.
• FIG. 6B the wells of a microtiter plate were coated with intact h 2GPI (triangles, h 2GPI) or nicked h 2GPI (squares, nicked) and blocked in 1 % OVA. Serial dilutions of the ch3G4 antibody (closed) or a control mlgG (open) were performed in 1 % OVA.
• FIG. 7A and FIG. 7B Divalency is required for 2GPI-mediated 3G4 antibody binding to cells with exposed PS.
• FIG. 7A ABAE cells were incubated for 30 min with 20 nM 3G4, 3G4 F(ab') 2 or 3G4 Fab' monomer in the presence of 200 ⁇ LPC in DMEM + 10% FBS. Cells were then washed, fixed, and stained with fluorescent markers to detect binding of the 3G4 antibody or antibody fragments (3G4 F(ab') 2 and 3G4 Fab' are shown in FIG. 7A). The pixel area of antibody binding was quantified using MetaVue software. Values are relative to the binding of 3G4 in the absence of LPC, which was set to one.
• FIG. 7A ABAE cells were incubated for 30 min with 20 nM 3G4, 3G4 F(ab') 2 or 3G4 Fab' monomer in the presence of 200 ⁇ LPC in DMEM + 10% FBS. Cells were then was
• ABAE cells were incubated for 30 min with 200 ⁇ LPC, 40 nM purified hp2GPI, 20 nM ch3G4 and a titer of 3G4 Fab' monomer in DMEM + 10% MS. Cells were then washed, fixed, and stained with fluorescent markers to detect binding of ch3G4. The pixel area of ch3G4 binding was quantified using MetaVue software. Values are relative to the binding of ch3G4 without competing 3G4 Fab', which was set to 100.
• FIG. 8. The 3G4 and bavituximab family of antibodies bind to PS in a ⁇ 2 ⁇ - dependent manner.
• bavituximab and related therapeutic PS-targeting antibodies bind to 2GPI (at domain II) and 2GPI, in turn, binds to PS via domain V.
• ⁇ 2 ⁇ is monomelic, binds to PS only weakly and rapidly dissociates.
• PS-targeting antibodies such as 3G4 or bavituximab
• 3G4 or bavituximab when there is a surface with exposed PS, two molecules of ⁇ 2 ⁇ are able to bind to PS and become cross-linked by divalent antibody, such that a stable complex is formed.
• the antibody ⁇ 2GPI complex dissociates from cells with exposed PS more than 1 ,000-times more slowly than does monomelic ⁇ 2 ⁇ (in the absence of a PS-targeting antibody).
• FIG. 9A assuming a purity of the 3G4 antibody of 90% when produced from hybridoma cells, mice treated with the 3G4 antibody contain 2 ⁇ g/ml of bovine ⁇ 2 ⁇ at a molar ratio of ⁇ 2 ⁇ to antibody of 0.12.
• FIG. 9B at a starting point of 80% purity, mice treated with the 3G4 antibody contain 4 ⁇ / ⁇ 1 of bovine ⁇ 2 ⁇ at a molar ratio of 2GPI to antibody of 0.25.
• FIG. 10 Low levels of ⁇ 2 ⁇ support bavituximab binding to cells with exposed PS in vitro.
• ABAE cells were incubated for 30 min with 200 ⁇ LPC to induce PS exposure, 40 nM purified hp2GPI, and increasing concentrations of the ch3G4 antibody (bavituximab) in DMEM + 10% mouse serum. Cells were then washed, fixed and stained with fluorescent markers to detect binding of ch3G4. The pixel area of ch3G4 binding was quantified using MetaVue software. Values are relative to the binding of 320 pM ch3G4, which was set to one.
• FIG. 11 Low levels of ⁇ 2 ⁇ support 2aG4 antibody binding to PS on plates in vitro. Microtiter plates were coated with PS. Solutions containing a fixed amount of the 2aG4 antibody were tested for binding to PS in the presence of increasing amounts of human ⁇ 2 ⁇ (in ovalbumin). Bound antibodies were detected using HRP-conjugated anti-human IgG as a secondary antibody, with TMB as a substrate and absorbances were read at a wavelength of 450nm.
• FIG. 12 Bavituximab binding to PS in vitro in a range of human sera.
• Microtiter plates were coated with PS.
• Six different individual human serum samples were obtained, nominally termed sera # 3 ( ⁇ , dark blue line), # 4 ( ⁇ , pink line), # 9 ( A , yellow line), # 11 (-x-, light blue line), # 13 (-x-, purple line ) and # 19 ( ⁇ , brown line), and diluted in PBS to provide a range of % human serum down to 0.1 %.
• Bavituximab-HRP at 2 ⁇ g/ml was added to each % human serum solution. Those mixtures of bavituximab-HRP in human sera were added to the PS plates and allowed to bind. Plates were washed, TMB substrate was added and absorbances were read at a wavelength of 450nm.
• FIG. 13 Mean serum bavituximab concentrations following administration to patients with refractory advanced cancer.
• Bavituximab was administered intravenously at 0.1 mg/kg (O, open circles) or 0.3 mg/kg ( ⁇ , open squares) on days 0, 28, 35 and 42; and bavituximab was administered at 1 mg/kg ( ⁇ , closed circles) or 3 mg/kg ( ⁇ , closed squares) on days 0, 7, 14 and 28.
• the mean serum bavituximab concentrations were determined on the indicated days post-administration. The lower limit of quantitation was 0.1 ⁇ g/ml.
• FIG. 14. ⁇ 2 ⁇ levels in patients following bavituximab administration.
• bavituximab Repeat doses of bavituximab were administered (on the days indicated by the bold, black arrows, j) to patients infected with HCV in amounts of 1 mg/kg, 3 mg/kg and 6 mg/kg. Serum ⁇ 2 ⁇ levels were determined at the indicated days post-treatment for each dose (1 mg/kg, yellow, top line; 3 mg/kg, pink, middle line; 6 mg/kg, blue, bottom line) and compared to pre- treatment P2GPI levels.
• FIG. 15 The 1N1 1 (PGN635) antibody binds to PS in a serum-dependent manner. Binding of the scFv form of 1N1 1 was tested by ELISA against plated PS, and a mix of phosphatidylcholine (PC) and sphingomyelin (SM), (PC/SM). Polystyrene plates were coated with 10 ⁇ g/ml PS or the same amount of a mix of PC/SM (each dissolved in hexane). After the hexane had evaporated, 10% human serum (+ 10% serum) or 1% ovalbumin (+ 1 % OV) in PBS was added and incubated for one hour.
• PC phosphatidylcholine
• SM sphingomyelin
• FIG. 16 A standard curve for functional ⁇ 2 ⁇ . Samples of known amounts of functional ⁇ 2 ⁇ were tested in the functional ⁇ 2 ⁇ assay as described in Example XVI and plotted to provide a standard curve. From the standard curve, the amount of functional P2GPI in test samples, particularly diluted plasma or serum test samples, can be determined.
• FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D Distribution of pre-treatment functional P2GPI levels ⁇ g/ml) in patients participating in a Phase III trial of bavituximab and docetaxel to treat non-small cell lung cancer (NSCLC).
• FIG. 17A distribution of functional P2GPI for all 592 evaluable patients
• FIG. 17B Box Plot of P2GPI from the same patients
• FIG. 17C distribution of functional P2GPI for patients treated with bavituximab and docetaxel (294 evaluable patients)
• FIG. 17D distribution of functional P2GPI for patients treated with placebo and docetaxel (298 evaluable patients).
• FIG. 18A and FIG. 18B Kaplan-Meier survival curves from the Phase III trial showing that NSCLC patients having functional P2GPI levels of equal to or greater than 200 ⁇ g/ml have a trend for prolonged survival (mOS) when treated with bavituximab.
• FIG. 19 Comparison of the ⁇ 2 ⁇ levels that support the PS-binding, functional and anti-tumor activity of the 3G4 antibody in preclinical studies to the ⁇ 2 ⁇ levels in patients in the Phase III and other clinical trials.
• the distribution of pre-treatment functional ⁇ 2 ⁇ levels in 592 evaluable patients in the Phase III trial is the same as set forth in FIG. 17A.
• Functional ⁇ 2 ⁇ levels of equal to or greater than 200 ⁇ g/ml dark orange and light orange bars together
• FIG. 20A, FIG. 20B and FIG. 20C Distribution of pre-treatment functional ⁇ 2 ⁇ levels ⁇ g/ml) in patients in the Phase II trial of bavituximab and docetaxel to treat NSCLC of Example XIII, as reported in Example XVIII, A.
• FIG. 20A distribution of functional ⁇ 2 ⁇ for all 1 19 evaluable patients
• FIG. 20B distribution of functional ⁇ 2 ⁇ for patients treated with 3 mg/kg bavituximab and docetaxel (40 evaluable patients)
• FIG. 20C distribution of functional ⁇ 2 ⁇ for patients in the pooled control arm (placebo or 1 mg/kg bavituximab) (79 evaluable patients).
• FIG. 21 A, FIG. 2 IB and FIG. 21C Kaplan-Meier survival curves from the Phase II trial of Example XIII showing that NSCLC patients having functional ⁇ 2 ⁇ levels of equal to or greater than 200 ⁇ g/ml have a trend for prolonged survival when treated with bavituximab.
• FIG. 21 A, FIG. 2 IB and FIG. 21C Kaplan-Meier survival curves from the Phase II trial of Example XIII showing that NSCLC patients having functional ⁇ 2 ⁇ levels of equal to or greater than 200 ⁇ g/ml have a trend for prolonged survival when treated with bavituximab.
• FIG. 21 A in patients treated
• FIG. 21B in patients having functional ⁇ 2 ⁇ > 200 ⁇ g/ml, those treated with bavituximab 3 mg/kg (“Bavi"; blue, top line) have a trend for prolonged survival as opposed to patients in the combined control arm ("Placebo"; green, bottom line).
• FIG. 22 Distribution of pre-treatment functional ⁇ 2 ⁇ levels ⁇ g/ml) in patients in the Phase II trial of gemcitabine and bavituximab to treat pancreatic cancer of Example XII, as reported in Example XVIII, B. The distribution of functional ⁇ 2 ⁇ for all 31 evaluable patients is shown.
• FIG. 24A, FIG. 24B and FIG. 24C Distribution of pre-treatment functional ⁇ 2 ⁇ levels ⁇ g/ml) in patients in the Phase II trial of bavituximab and paclitaxel/carboplatin to treat NSCLC, as reported in Example XVIII, C.
• FIG. 24A distribution of functional ⁇ 2 ⁇ for all 84 evaluable patients
• FIG. 24B distribution of functional ⁇ 2 ⁇ for patients treated with bavituximab and paclitaxel/carboplatin (44 evaluable patients)
• FIG. 24C distribution of functional ⁇ 2 ⁇ for patients in the paclitaxel/carboplatin control arm (40 evaluable patients).
• FIG. 25A, FIG. 25B and FIG. 25C Kaplan-Meier survival curves from the Phase II trial reported in Example XVIII, C showing that NSCLC patients having functional ⁇ 2 ⁇ levels of equal to or greater than 200 ⁇ g/ml have a trend for prolonged survival when treated with bavituximab.
• FIG. 25B in patients having functional ⁇ 2 ⁇ > 200 ⁇ g/ml, those treated with bavituximab ("C/P+Bavi"; blue, top line) have a trend for prolonged survival as opposed to patients in the control arm ("C/P"; green, bottom line).
• FIG. 26 Kaplan-Meier survival curves showing that patients treated with bavituximab and docetaxel followed by subsequent immunotherapy (“SACT-IO”) (blue, top line) have a statistically significant better mOS as opposed to patients treated with docetaxel alone followed by subsequent immunotherapy (green, bottom line).
• SACT-IO Kaplan-Meier survival curves showing that patients treated with bavituximab and docetaxel followed by subsequent immunotherapy
• SACT-IO blue, top line
• the treatment groups, mOS and statistical analyses are tabulated for these survival curves in Table 16 (Example XIX).
• FIG. 27 Kaplan-Meier survival curve showing that NSCLC patients having functional p2GPI levels of equal to or greater than 200 ⁇ g/ml have a statistically significant better mOS when treated with bavituximab followed by subsequent immunotherapy (“SACT-IO").
• PS Phosphatidylserine
• PS-targeting antibodies therefore provide new treatment options for those diseases, including cancer.
• PS in normal cells, PS is segregated to the inner leaflet of the plasma membrane, but becomes externalized to the outer leaflet of the plasma membrane in diseased and aberrant cells in various disease states, particularly in cancer and viral infections.
• some of the environmental stressors that cause PS extemalization are hypoxia/reoxygenation, oxidative stress and exposure to certain cytokines. PS extemalization also occurs under conditions of cell death and immune phagocytic cell clearance (Birge et al., 2016).
• PS is recognized and bound by PS receptors ⁇ e.g., TIM 3 and TIM 4, BAI 1 , stabilin 2 and RAGE) on immune cells, optionally via one or more of a number of bridging proteins, such that PS induces and maintains immune suppression.
• PS receptors e.g., TIM 3 and TIM 4, BAI 1 , stabilin 2 and RAGE
• PS induces and maintains immune suppression.
• PS is exposed on the surface of tumor vascular endothelial cells, tumor cells and tumor-derived exosomes, and the process of immune suppression is duplicated, thus preventing antitumor and inflammatory reactions from occurring.
• Exposed PS is a phagocytic signal that facilitates the recognition and clearance of dying cells, triggers the release of immunosuppressive cytokines (e.g. , TGF- ⁇ and IL-10) and inhibits the production of proinflammatory cytokines ⁇ e.g., TNF-a and IL-12).
• PS also polarizes macrophages towards the immunosuppressive M2 phenotype, inhibits dendritic cell (DC) maturation and the ability of DCs to present antigen, while stimulating DCs to secrete immunosuppressive mediators that promote T cell tolerance.
• DC dendritic cell
• PS is a central factor in the induction and maintenance of an immunosuppressed tumor microenvironment.
• PS-targeting antibodies can be used to block the binding of PS to specific receptors on immune cells, and thus provide an effective cancer therapy (Yin et al., 2013).
• a number of such PS-targeting antibodies have been developed as therapeutics, as exemplified below.
• the group of "PS-targeting antibodies” includes all antibodies that operatively bind to PS in vitro and specifically localize and bind to PS exposed in disease states in vivo, particularly to PS on tumor cells and tumor blood vessels, irrespective of whether the antibodies bind directly to PS or require a serum protein to form a tight binding complex with PS.
• Such "direct” and “indirect” PS-targeting antibodies are described in more detail below.
• 3G4 An early monoclonal antibody generated to evaluate the preclinical potential of PS-targeting antibodies is the antibody termed 3G4, a mouse IgG 3 mAb (Example I; Ran et ai, 2005; Huang et ah, 2005).
• Samples of the hybridoma cell line secreting the 3G4 antibody were deposited with the American Type Culture Collection (ATCC) and given ATCC Accession number PTA 4545. Availability of the deposited hybridoma is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
• Bavituximab is a human chimeric version of the 3G4 mouse antibody, in which the murine variable (antigen binding) regions are operatively attached to a human antibody constant region (Example III, C).
• the bavituximab family of antibodies is described in detail in numerous U.S. Patents, e.g., U.S. Patent No. 7,247,303 and U.S. Patent No. 7,572,448, and bavituximab can be re-created by preparing a human chimeric form of the antibody deposited as ATCC PTA 4545.
• Bavituximab is less immunogenic when given to patients, because significant portions of the antibody are from human origin.
• the 3G4 and bavituximab antibodies bind strongly to anionic phospholipids, particularly PS, but also to phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG) and cardiolipin (CL), in the presence of serum (Ran et al, 2005).
• PI phosphatidylinositol
• PA phosphatidic acid
• PG phosphatidylglycerol
• CL cardiolipin
• PS is the most relevant, physiologically and pathologically.
• 3G4 and bavituximab exhibit no detectable binding to the neutral phospholipids, phosphatidylcholine (PC), sphingomyelin (SM) or phosphatidylethanolamine (PE), irrespective of the presence of serum.
• PC phosphatidylcholine
• SM sphingomyelin
• PE phosphatidylethanolamine
• 3G4 and bavituximab antibodies bound to PS directly, it was later determined that the PS-binding is mediated by a serum protein, which was identified as P2-glycoprotein 1 (P2GPI) (Example IV; Luster et al., 2006).
• P2GPI P2-glycoprotein 1
• 3G4 and bavituximab bind strongly to PS in enzyme-linked immunosorbent assays (ELISAs) conducted in the presence of ⁇ 2 ⁇ , which includes purified ⁇ 2 ⁇ as well as ⁇ 2 ⁇ provided simply by being present in the 10% serum typically used in ELISAs.
• ELISAs enzyme-linked immunosorbent assays
• P2GPI also known as apolipoprotein H, has five domains, I, II, III, IV and V (1 , 2, 3, 4 and 5), and the domain structure is conserved across mammals. ⁇ 2 ⁇ folds as a tertiary structure into those five discernable domains, and may have a closed, circular structure or an open, J-shape or hook structure.
• ⁇ 2 ⁇ binds to anionic phospholipids, particularly PS, through positively-charged regions in its C terminal domain, domain V, so long as domain V is not "nicked", such as by cleavage with the enzyme plasmin, at the Lys317/Thr318 cleavage site, which destroys PS binding (Hunt et al., 1993; Hunt & Krilis, 1994).
• the 3G4 and bavituximab antibodies bind to domain II of ⁇ 2 ⁇ . This reinforces the safety of 3G4 and bavituximab as therapeutic antibodies, because certain other antibodies that bind to p2GPI have been associated with pathologies, but those antibodies bind to domain I of ⁇ 2 ⁇ .
• bavituximab The binding of the 3G4 and bavituximab antibodies to PS is depicted in FIG. 8.
• the bavituximab family of antibodies binds to domain II of ⁇ 2 ⁇ .
• bavituximab binds to domain II of ⁇ 2 ⁇ , it is not linked with side-effects such as those associated with anti-phospholipid syndrome, in which antibodies are present that bind to domain I of p2GPI (de Laat et al., 2005; de Laat et al., 2006; loannou et al., 2007).
• the high affinity bivalent interaction of the antibody with ⁇ 2 ⁇ coordinates the resultant high-affinity binding to PS, including when PS is externalized on cell surfaces and membranes.
• the 3G4 and bavituximab antibodies bind to P2GPI, they are referred to as "PS-targeting antibodies" because they specifically localize and bind to PS exposed in disease states in vivo.
• PS-targeting antibodies As PS is maintained on the inside of healthy, normal cells, and only becomes exposed on the cell surface in disease states, antibody localization in vivo is not only specific to PS, but is specific for diseases in which PS is a marker, particularly cancer, but also viral infections and certain other pathologies.
• FIG. 8 also shows that 2GPI-dependent antibody binding to PS is the same in vitro as in vivo, such that an ELISA is an accurate model for therapy.
• the 3G4, bavituximab and like antibodies are able to form a stable binding complex with PS.
• the ELISA assay therefore mimics the in vivo situation during therapy, in which PS is uniquely exposed only on cells in the disease environment, such as cells in the tumor microenvironment or virally-infected cells.
• the 3G4 and bavituximab antibodies encounter exposed PS, they are able to form a stable binding complex with the ⁇ 2 ⁇ present in the blood.
• the antibody-P2GPI complex has more than 1 ,000-times higher affinity for PS than does monomelic ⁇ 2 ⁇ , i.e., ⁇ 2 ⁇ without a PS -targeting antibody.
• the entire family of PS-targeting antibodies includes antibodies that do bind directly to PS, i.e., direct PS-binding or PS-targeting antibodies.
• direct PS-binding antibody or “direct PS-targeting antibody” is an antibody that is not only functionally specific for PS, and targets and binds to PS in vitro and in vivo (as do the indirect binding antibodies), but that does not require a serum protein, such as ⁇ 2 ⁇ , to form a tight binding complex with PS, even in in vitro binding assays.
• a direct PS-binding antibody is the mouse monoclonal antibody termed 9D2 (Ran et al., 2002).
• the 9D2 antibody has been shown to localize to tumor blood vessels and to exert anti-tumor effects in vivo (Ran et al., 2002).
• Another example of a direct PS-binding antibody is the fully-human antibody termed 1 1.31 (PGN632).
• the 1 1.31 antibody has also been shown to exert anti-tumor effects in vivo ⁇ e.g., in mice bearing MDA-MB-435 mammary carcinoma xenografts) and shows impressive anti-viral effects (Moody et al, 2010; U.S. Patent No. 7,455,833).
• the direct PS-binding antibodies are therefore of use in treating the various diseases in which PS is a marker, most particularly cancer and viral infections.
• biomarkers for optimizing treatment with such direct binding, PS-targeting antibodies will typically not rely on serum proteins, such as 2GPI, as in the present invention, but on other factors.
• Useful biomarkers for the direct binding antibodies include immune biomarkers for PS-targeting antibodies.
• PS-targeting antibody family the indirect PS-binding antibodies.
• An "indirect PS-binding antibody” or “indirect PS-targeting antibody”, as used herein, is an antibody that is functionally specific for PS, operatively binds to PS in vitro and targets and binds to PS in vivo, but that requires a serum protein to form a tight binding complex with PS.
• the present invention is particularly concerned with a sub-set of the indirect PS-binding or PS-targeting antibodies, namely the 2GPI-dependent PS-binding or PS-targeting antibodies.
• a " ⁇ 2 ⁇ -dependent PS-binding antibody” or " 2GPI-dependent PS-targeting antibody”, as used herein, is an antibody that is functionally specific for PS, operatively binds to PS in vitro, i.e., binds to PS in vitro in assays conducted in the presence of serum containing ⁇ 2 ⁇ or purified serum, and targets and binds to PS in vivo, but that requires the serum protein, ⁇ 2 ⁇ to form a tight binding complex with PS.
• examples of such antibodies include the mouse antibody, 3G4 and the chimeric antibody, bavituximab.
• p2GPI-dependent PS-targeting antibodies are the fully-human antibodies termed 1N1 1 (PGN635) and 1G15, preferably the 1N1 1 antibody.
• 1N1 1 The PS binding properties of the 1N1 1 antibody are shown in FIG. 15.
• 1N1 1 was generated by phage display and selected using assays for binding to PS only in the presence of serum (or only in the presence of P2GP1). As shown by FIG. 15, those of skill in the art can routinely perform such studies to prepare and isolate further PS-targeting antibodies and 2GPI-dependent PS-targeting antibodies.
• PS-targeting antibodies have thus been shown to shift the functions of immune cells in tumors, resulting in multiple signs of immune activation and anti-tumor immune responses.
• PS-targeting antibodies such as bavituximab achieve this blocking of PS-mediated immunosuppression by multifocal reprograming of the immune cells in the tumor microenvironment to support immune activation (Yin et al., 2013). Bavituximab and related antibodies thus break immune tolerance in the tumor microenvironment.
• Antibody-mediated PS blockade reduces the levels of myeloid-derived suppressor cells (MDSCs), transforming growth factor-beta (TGF ) and interleukin-10 (IL-10), and increases the levels of pro- inflammatory cytokines such as interferon gamma (IFNy), tumor necrosis factor-alpha (TNFa) and interleukin-12 (IL-12).
• MDSCs myeloid-derived suppressor cells
• TGF transforming growth factor-beta
• IL-10 interleukin-10
• IFNy interferon gamma
• TNFa tumor necrosis factor-alpha
• IL-12 interleukin-12
• This PS blockade also repolarizes MDSCs and tumor-associated macrophages (TAMs) from predominant M2 to predominant Ml phenotype, promotes the maturation of dendritic cells (DCs), activates cytotoxic T-cells and induces potent adaptive antitumor T-cell immunity (Yin et al., 2013).
• TAMs tumor-associated macrophages
• Bavituximab and related antibodies also activate innate immunity, i.e., NK cells as well as Ml macrophages. Importantly, these antibodies also cause the selective shutdown of pre-existing tumor blood vessels, which uniquely expose PS (Ran et al., 2005; U.S. Patent No. 7,572,448), and this activity includes antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by tumor infiltrating Ml macrophages and NK cells. Destroying the tumor blood vessels in this way leads to tumor cell destruction.
• ADCC antibody-dependent cell-mediated cytotoxicity
• agents for use with bavituximab and related antibodies are agents and/or conditions that increase the exposure of PS in the tumor microenvironment, such as radiation and/or chemotherapeutics (U.S. Patent No. 7,422,738; U.S. Patent No. 8,486,391 ; U.S. Patent No. 7,572,448).
• Enhanced anti-tumor effects have thus been demonstrated pre-clinically in combination with docetaxel to treat breast tumors (Huang et al, 2005) and prostate cancer (Yin et al, 2013); gemcitabine to treat pancreatic tumors (Beck et al, 2006); irradiation to treat lung cancer (He et al, 2007) and glioblastoma (He et al., 2009); PRIMA-1 , which reactivates the mutant tumor suppressor, p53, for advanced breast tumors (Liang et al., 201 1); an adenoviral vector to re-target the adenovirus to tumor vasculature (Hogg et al., 201 1); cisplatin to treat lung cancer relapse after surgery (Judy et al., 2012); and sorafenib to treat hepatocellular carcinoma (Cheng et al., 2016).
• bavituximab Another group of agents for use with PS-targeting antibodies such as bavituximab are other IO agents.
• the mechanism of action of bavituximab is complementary to the available therapeutic agents, as PS is an upstream immune checkpoint.
• Impressive combination therapies have thus been shown pre-clinically for the bavituximab family of antibodies in combination with other checkpoint inhibitors in the form of antibodies to CTLA-4, PD-1 and PD-Ll (Freimark et al., 2016; Gray et al., 2016a).
• Such anti-tumor activity which included increased survival, was associated with increases in intratumoral activated CD8 T cells, a reduction of M2 macrophages and MDSCs coupled with PD-Ll expression, and increased tumor reactive T cells in the spleen when compared to PD-1 blockade alone.
• bavituximab family of PS-targeting antibodies reverses PS-mediated immunosuppression and initiates therapeutically effective adaptive antitumor immunity.
• treatment with bavituximab in combination with blockade of downstream immune checkpoints results in robust and long-lasting antitumor immunity that significantly improves outcomes, duration and levels of response (Freimark et al, 2016; Gray et al, 2016a).
• these antibodies may also be effectively combined in triple combination therapies, including triple combinations with radiation, chemotherapeutics ("chemoradiation”) and/or immunotherapeutics, and triple combinations with two immunotherapeutic agents. Impressive results were recently shown for a triple combination using antibodies that target PS, PD-1 and LAG-3 (Gray et al, 2016b).
• bavituximab has been evaluated in clinical studies in over 800 patients, mostly in combination with other indication-approved therapeutics.
• a range of anti-viral and anti-tumor studies have shown therapeutic activity.
• a dose of 3 mg/kg bavituximab given intravenously (IV) was determined and selected for most clinical studies in oncology, including in patients with lung, breast, liver, pancreatic, colorectal and kidney cancers.
• results from the Phase I and Phase II clinical studies demonstrated a clinically meaningful treatment effect of bavituximab.
• PS-targeting antibodies Although there is now a significant body of work showing successful treatment of a range of diseases using PS-targeting antibodies, to date, there are no known biomarkers for such therapies.
• the clinical experience with PS-targeting antibodies is largely based on the 2GPI-dependent PS-targeting antibodies such as bavituximab. It is for those antibodies that a biomarker to optimize treatment is most needed. If one or more circulating biomarkers for bavituximab treatment could be identified, along with sensitive and rapid methods to quantify such biomarker(s), this would be an important development, providing minimally invasive test(s) to facilitate patient selection for improved treatment outcomes.
• biomarkers play an increasingly important role in identifying specific patient characteristics that impact responses to treatment. This has been seen historically with targeted cancer treatments, as well as more recently with checkpoint inhibitors, including PD-1 and PD-L1 inhibitors. Biomarkers of importance to treatment with PS-targeting antibodies such as bavituximab are being analyzed.
• a "bavituximab biomarker” is a biomarker for use, either alone or as one of two or more, or multiple biomarkers, in selecting patients or patient populations for improved clinical benefit from treatment with therapies that comprise a PS-targeting antibody, preferably bavituximab, as at least part of the therapy.
• bavituximab biomarkers including ⁇ 2 ⁇ , may thus be used in methods to predict, in advance of treatment, whether a patient, patient population or sub-population is likely to benefit from a treatment comprising a PS-targeting antibody, preferably bavituximab, including a combination therapy that comprises a PS-targeting antibody, preferably bavituximab.
• Multi-marker signatures for identifying the most appropriate patient populations for improved clinical benefit from bavituximab-containing therapeutic regimens are also being considered.
• the first biomarker identified in these analysis is ⁇ 2 ⁇ (Section E; Section F).
• the pattern of biomarkers identified is a bavituximab "signature" to guide clinical development and treatment.
• bavituximab immune biomarkers are being analyzed. Such data support the use of bavituximab to "prime” the immune system, i.e. , to amplify anti-tumor immune responses.
• tumors can be placed on a scale from “hot” to “cold”, depending on how deeply they have been invaded by T cells and other immune cells.
• the level of immune infiltration (“heat") reflects whether the immune system is recognizing and engaging the tumor. Patients with a tumor that is "hot” have a better prognosis; with a "cold” tumor, the probability of relapsing is much higher.
• bavituximab is able to make a positive impact on the cold tumors, making them more amenable to therapy, including with other checkpoint inhibitors.
• the bavituximab immune biomarkers therefore have additional uses in not only selecting patients for bavituximab therapy, but in identifying patients for treatment with bavituximab and intelligently selected agents for combination therapies. Dl. Samples
• the invention may be used to test any biological sample that contains or is suspected to contain one or more of the biomarkers, including any tissue sample or biopsy from an animal, subject or patient, including fecal matter. Clarified lysates from biological tissue samples may be used.
• the invention is preferably used with natural body fluids, thus providing tests that can be performed on biological samples obtained using minimally- or non-invasive techniques, also termed "liquid biopsies". This is an advantage over more rigorous techniques like biopsies, which typically take longer to provide results and may pose health risks in themselves.
• biological fluids that contain or are suspected to contain one or more biomarkers
• biological fluids include blood, urine, ascites, cerebral and cerebrospinal fluid (CSF), sputum, saliva, nasal secretions, bone marrow aspirate, joint or synovial fluid, aqueous humor, amniotic fluid, follicular fluid, cerumen, breast milk (including colostrum), broncheoalveolar lavage fluid, semen, seminal fluid (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid or lavage, pericardial fluid, lymph, chyme, chyle, bile, liver perfusate, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, faecal fluid, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates
• biological fluids for testing are blood, urine and ascites fluid, particularly ascites fluid from an animal, subject or patient having or suspected of having ovarian cancer.
• a urine sample it will preferably be used in connection with cancers of the urinary, genitourinary and reproductive systems, such as, e.g., ovarian, prostate, renal, bladder, testicular, urethral and penile cancer.
• detecting and quantifying one or more of the other biomarkers is preferably performed from peripheral blood samples, preferably plasma, and most preferably serum.
• Exosomes have recently gained attention in connection with cancer. Exosomes are 40-50 to 100 nanometer (nm) size membrane-derived vesicles that are constitutively released by all cells in vivo and in vitro. Exosomes are biologically active molecular shuttles that play important roles in intercellular communication and influence many physiological and pathological processes. In cancer, exosome functions include the transfer of oncogenes between cancer cells and the tumor stroma that primes the so-called "metastatic niche" for metastatic spread (An et al., 2015).
• tumor exosomes Due to the multiple intracellular fusion events involved in exosome formation, the luminal contents and proteomic profile of the extracellularly released exosomes mirror those of the originating cell. Thus, tumor-derived exosomes (“tumor exosomes”) have profiles that reflect the cancer cell from which they arose. Indeed, the presence of cytosolic (particularly nucleic acids) and plasma membrane components from the originating cell means that circulating exosomes are readily accessible surrogates that reflect the properties of the parent cell for biomarker analysis.
• PS-positive tumor exosomes are characterized by having PS on their surface.
• PS-positive tumor exosomes can thus be used in the diagnosis of cancer.
• New and improved methods, compositions and kits for diagnosing cancer by detecting and quantifying PS-positive tumor exosomes in biological fluid samples using solid phase assays were recently reported. Such techniques are described in U.S. patent application Serial No. 15/177,747 and PCT patent application No. PCT/US 16/036629, each filed June 09, 2016.
• PS-targeting antibodies need to bind to PS in the disease microenvironment. Therefore, measuring the level of pre- treatment PS-positive tumor exosomes is particularly compelling for use as a predictive biomarker for response to therapy using PS-targeting antibodies such as bavituximab
• p2GPI is an abundant plasma (serum) glycoprotein found both free and associated with lipoprotein.
• the DNA and amino acid sequences of P2GPI from various mammalian species are known, including mouse, rat, dog, cow, chimp and human (Steinkasserer et al, 1991).
• the human p2GPI amino acid sequence is provided as Accession number 1C1ZA.
• p2GPI is glycosylated and is routinely reported as a 50 kDa protein (Example IV, A4,B3; see also, McNeil et al, 1990 at FIG. 3; Balasubramanian et al, 1998 at Fig. 1 ; Luster et al., 2006 at Figure ID).
• Example IV A4,B3; see also, McNeil et al, 1990 at FIG. 3; Balasubramanian et al, 1998 at Fig. 1 ; Luster et al., 2006 at Figure ID).
• P2GPI has been studied for decades, a precise physiological role for p2GPI remains unknown (Prakasam & Thiagarajan, 2012). Indeed, the apparently healthy life of knockout mice deficient in P2GPI indicates that its role is not critical (Sheng et al, 201 1).
• pre-treatment blood concentrations of p2GPI are effective as a biomarker to predict successful responses to therapies using PS-targeting antibodies such as bavituximab.
• levels of "functional" P2GPI meaning p2GPI that binds to both PS and to PS-targeting antibodies such as bavituximab, are useful alone as a biomarker for response to bavituximab.
• pre-treatment p2GPI levels are used alone as a biomarker for response to PS-targeting antibodies such as bavituximab
• those p2GPI levels are both numerically defined and measured in assays that are capable of detecting "functional" p2GPI, as defined herein.
• pre-treatment P2GPI levels are used as one of two or more, or a plurality, of biomarkers for response to PS-targeting antibodies such as bavituximab
• the P2GPI levels need not be so tightly numerically defined, nor solely measured in assays for functional p2GPI.
• the P2GPI levels as part of a dual or multi-marker signature for bavituximab-containing therapies can be "P2GPI high” vs. "p2GPI low", akin to descriptions such as VeriStrat Good (VS Good) and VS Poor, and rum that are "hot” or “cold”. Patients with " 2GPI high” should be selected for treatment with PS-targeting antibodies such as bavituximab.
• the levels of ⁇ 2 ⁇ that are " ⁇ 2 ⁇ high” are pre-treatment levels of ⁇ 2 ⁇ , either total ⁇ 2 ⁇ , or preferably functional ⁇ 2 ⁇ , of equal to or greater than about 180, 190, 200, 210, 220, 230, 240, 250 or 260 ⁇ g/ml, preferably of equal to or greater than about 200 ⁇ .
• “ 2GPI high” thus includes pre-treatment levels of ⁇ 2 ⁇ , either total ⁇ 2 ⁇ , or preferably functional ⁇ 2 ⁇ , of equal to about 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310 or 320 ⁇ g/ml.
• the invention also provides biomarkers in terms of certain numerically defined amounts and ranges of functional ⁇ 2 ⁇ , measured in assays that are capable of detecting functional ⁇ 2 ⁇ .
• the invention concerns the selection and treatment of patients based on pre-treatment levels of functional ⁇ 2 ⁇ of equal to or greater than 200 ⁇ g/ml (Example XVII; FIG. 18A and FIG. 18B; Example XVIII; Example XX). This may include pre-treatment levels of functional ⁇ 2 ⁇ of equal to about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310 or 320 ⁇ g/ml.
• bavituximab improved overall survival in patients with functional ⁇ 2 ⁇ of equal to or greater than 200 ⁇ g/ml in all clinical trials from which ⁇ 2 ⁇ data are available.
• the results are summarized in Table A, in which the Treatment column refers to bavituximab treatment of the listed indication in combination with the recited agent.
• the Treatment column refers to bavituximab treatment of the listed indication in combination with the recited agent.
• functional ⁇ 2 ⁇ of equal to or higher than 200 ⁇ g/ml was shown to provide a survival advantage for bavituximab over placebo (mOS not reached for bavituximab vs.
• certain currently preferred embodiments of the invention concern the selection and treatment of patients based on pre-treatment levels of functional ⁇ 2 ⁇ in the range of 200-290 ⁇ g/ml (Example XVII; compare Table 14A and Table 14B), particularly for treating NSCLC.
• This also includes pre-treatment levels of functional P2GPI in the ranges of 200-270, 200-280, 200-290, 200-300, 200-310, 200-320, 210-270, 210-280, 210-290, 210-300, 210-3 10 and 210- 320 ⁇ and such like, with the ranges 210-270, 210-280, 210-290, 200-280 and 200-290 being currently preferred.
• the invention concerns the selection and treatment of patients based on pre-treatment levels of functional ⁇ 2 ⁇ in the ranges of from any one of about 190, 200, 210 or 220 ⁇ g/ml as the low number, to any one of about 260, 270, 280, 290, 300, 310 or 320 ⁇ g/ml as the high number.
• ranges include all the following, from within which, the ranges of about 210-270, 210-280, 210-290, 200-280 and 200-290 ⁇ g/ml are preferred: about 190-260, 190-270, 190-280, 190-290, 190-300, 190-310 and 190-320; about 200-260, 200-270, 200-280, 200-290, 200-300, 200-310 and 200-320; about 210-260, 210-270, 210-280, 210-290, 210-300, 210-310 and 210-320; and about 220-260, 220-270, 220-280, 220-290, 220-320, 220-310 and 220-320.
• pre-treatment levels of functional ⁇ 2 ⁇ of equal to or greater than 200 ⁇ g/ml whichever one or more of the above numbers or ranges are chosen for any particular treatment or combination treatment
• the use of pre-treatment levels of P2GPI, preferably functional P2GPI, as a biomarker, or as part of a panel of biomarkers applies to the selection of patients with a wide range of diseases in which PS is a marker, most particularly cancer and viral infections, but also infections of intracellular parasites, and their treatment using any PS-targeting antibody, such as bavituximab, either alone, or preferably in any combination therapy.
• P2GPI As pre-treatment levels of P2GPI are a biomarker for bavituximab and related antibodies, the following guidance is provided concerning assays for ⁇ 2 ⁇ .
• the present invention also provides certain preferred assays for quantifying functional ⁇ 2 ⁇ (Section G).
• ⁇ 2 ⁇ As a serum protein, ⁇ 2 ⁇ is ideal for detection in peripheral blood (plasma, serum) samples, as described below.
• peripheral blood plasma, serum
• ⁇ 2 ⁇ localizes to endothelial cells in vivo
• the full range of biological samples can potentially be used for ⁇ 2 ⁇ detection.
• peripheral blood, plasma and serum samples are particularly preferred for detecting and quantifying ⁇ 2 ⁇ , whether total ⁇ 2 ⁇ or functional ⁇ 2 ⁇ (Section G).
• Whole blood may be used (red blood cells, white blood cells, platelets, proteins and plasma).
• plasma is used, which is the liquid remaining after the precipitation of red cells and white cells.
• Plasma contains fibrinogen and other clotting factors, so tends to clot on standing. Less clot-prone plasma is available, which is preferred; platelet-free plasma may also be used.
• serum is used for detecting and quantifying ⁇ 2 ⁇ . Serum is plasma without the clotting factors, mainly without fibrinogen, so serum does not clot on standing. Animal and human sera are routinely used for diagnostic purposes, and preparative techniques are widely known. Exemplary methods for preparing serum samples for P2GPI testing are shown herein (Example XV, A).
• the tests may be carried out directly on the biological sample, preferably blood, plasma or serum. Due to the sensitivity, ⁇ 2 ⁇ can readily be detected without any prior enrichment or concentration (although this is not excluded).
• the test samples preferably serum samples, may be fresh or previously frozen and then thawed.
• Example XV, Example XVI, Example XVII and Example XVIII show that ⁇ 2 ⁇ is stable to long-term storage at -70°C. Industry-standard techniques of freezing, storage and/or thawing should preferably be used, such as using cryogenic tubes or vials and/or protease inhibitors to limit proteolysis overall.
• assays for measuring P2GPI without reference to whether it is "functional" ⁇ 2 ⁇ i.e., assays for "total" ⁇ 2 ⁇
• assays for "total" ⁇ 2 ⁇ are applicable for use with those embodiments of the invention in which the pre-treatment ⁇ 2 ⁇ levels are used as only one of two or more biomarkers for bavituximab.
• a "functional" ⁇ 2 ⁇ assay should be used, such as described in Section G.
• Total ⁇ 2 ⁇ levels may be detected and preferably quantified using any one or more of the many in vitro binding assays and kits known in the art. Suitable binding assays include, for example, immunoblots, Western blots, dot blots, RIAs, immunohistochemistry, fluorescent activated cell sorting (FACS), immunoprecipitation, affinity chromatography, and the like. Although solid phase binding assays are typically preferred, various other methods for detecting ⁇ 2 ⁇ have been described in the literature, any of which may be used. For example, ⁇ 2 ⁇ levels may be accurately determined by radial immunodiffusion.
• ELISA enzyme linked immunosorbent assay
• modified capture ELISAs e.g., Mehdi et al., 1999
• competitive ELISAs e.g., Balasubramanian et al., 1998
• kits for assaying total ⁇ 2 ⁇ are available, as are commercially available anti ⁇ 2GPI antibodies, including those attached to diagnostic labels. Any such kits or antibodies may be used to detect and quantify total ⁇ 2 ⁇ .
• anti ⁇ 2GPI antibodies from US Biological are used herein in comparative assays (Example XVI, A10.B2).
• ELISAs for total ⁇ 2 ⁇ use one or more anti ⁇ 2GPI antibodies. Even though antibody technology is very advanced, the commercial kits and commercial anti ⁇ 2GPI antibodies often use polyclonal anti ⁇ 2GPI antibodies, which are completely suitable for use in such embodiments.
• anti ⁇ 2GPI antibodies are adsorbed to a solid surface, such as a 96 well plastic plate, and incubated with the biological sample suspected of containing ⁇ 2 ⁇ (in this case, the "antigen").
• Bound ⁇ 2 ⁇ (antigen) is detected using a secondary binding agent that is directly or indirectly labeled with a detectable agent, i.e. , an agent that produces a detectable signal, such as color or fluorescence, which can be detected and quantified.
• the secondary binding agent is an anti ⁇ 2GPI antibody that is labeled with a detectable agent.
• the present application discloses such an advantageous assay, which is uniquely adapted for the purpose of detecting and quantifying the amount of functional (active) ⁇ 2 ⁇ in human blood samples, such as plasma and serum, which assay can determine the level of ⁇ 2 ⁇ that is able to bind to both PS and to PS-targeting antibodies such as bavituximab.
• the present invention provides defined levels of pre-treatment ⁇ 2 ⁇ for use as a single biomarker for response to treatment with bavituximab and related PS-targeting antibodies.
• functional p2GPI of equal to or greater than 200 ⁇ g/ml (Example XVII; FIG. 18A and FIG. 18B; Example XVIII; Example XX) is broadly predictive of response to treatment with bavituximab, as exemplified by functional p2GPI in the range of 200-290 ⁇ g/ml (Example XVII; compare Table 14A and Table 14B).
• the preferred ELISAs for functional p2GPI provided by the invention are exemplified by the detailed teaching in Example XVI and are also described more fully below. Gl. Assay Methods
• solid phase assays such as ELISAs for functional ⁇ 2 ⁇ use both PS and a PS-targeting antibody such as bavituximab, at least one of which is operatively associated with a solid support and/or at least one of which is directly or indirectly labeled with a detectable agent.
• a PS-targeting antibody such as bavituximab
• the PS-targeting antibody could be adsorbed to the solid support and the PS labeled with a detectable agent.
• Many lipids such as PS labeled with detectable agents are known in the art, any of which could be used.
• the currently preferred embodiments are those in which PS is adsorbed to a solid surface, such as a 96 well plastic plate.
• the PS-targeting antibody such as bavituximab or 1N1 1
• the PS-targeting antibody can be labeled with a detectable agent, which is preferably a direct label attached to the antibody.
• the PS-coated solid support such as an ELISA plate (or wells thereof) is incubated with the PS-targeting antibody, such as bavituximab or 1N1 1 , and the biological sample suspected of containing ⁇ 2 ⁇ .
• the PS-targeting antibody preferably bavituximab
• the ⁇ 2 ⁇ samples are "co-incubated" on the PS-coated solid support or plate.
• the ⁇ 2 ⁇ can either bind to PS via (intact) domain V, followed by bavituximab binding to domain II of the ⁇ 2 ⁇ bound to the plate; or bavituximab can bind to domain II of the ⁇ 2 ⁇ in solution, followed by the complexed bavituximab ⁇ 2GPI (with an intact domain V) binding to the PS on the plate. Both binding events occur during the incubation time, so co-incubating encompasses all such mechanisms of binding.
• Co-incubating the PS-targeting antibody, preferably bavituximab, and the P2GPI samples on the PS-coated support thus means incubating together under "effective binding conditions", i.e., "under conditions and for a time effective to allow binding of ⁇ 2 ⁇ in the sample to both the PS-targeting antibody and the PS-coated support".
• "Binding” means under conditions and for a time effective to allow "specific binding”, i.e., binding that is not removed by routine washing. In using bavituximab, the effective binding conditions thus permit binding of PS to intact (non-nicked) domain V of ⁇ 2 ⁇ and binding of bavituximab to domain II of ⁇ 2 ⁇ .
• the effective binding conditions permit binding of PS to intact (non-nicked) domain V of ⁇ 2 ⁇ and binding of the PS-targeting antibody to ⁇ 2 ⁇ at a domain of ⁇ 2 ⁇ other than domain V, preferably to the hinge region joining domains I and II, and most preferably to domain II of P2GPI.
• the assays of the invention include a number of different formats.
• the PS-targeting antibody and the sample suspected of containing P2GPI may be added to the PS-coated support substantially simultaneously.
• the PS-targeting antibody and the sample suspected of containing P2GPI are added to the PS-coated support sequentially, i.e. , at times spaced apart.
• the PS-coated solid support such as an ELISA well, may first be incubated with the biological sample suspected of containing ⁇ 2 ⁇ and then co-incubated with the PS-targeting antibody, preferably bavituximab.
• the above assay may utilize a pre-prepared PS-coated support, in which case the assay comprises (or comprises the steps of):
• the PS-targeting antibody preferably bavituximab
• the PS-targeting antibody preferably bavituximab
• the PS-targeting antibody is applied to the PS-coated solid support first, followed by co-incubating with the biological sample suspected of containing P2GPI.
• Such an assay, or sequence of steps is preferred for technical reasons, e.g., to avoid cross-contamination during pipetting.
• Such a preferred assay comprises (or comprises the steps of):
• This assay may also utilize a pre-prepared PS-coated support, in which case the assay comprises (or comprises the steps of):
• the preferred assay with sequential binding steps in which the PS-targeting antibody, preferably bavituximab, and most preferably detectably-labeled bavituximab, is applied to the PS-coated solid support first, followed by co-incubating with the biological sample suspected of containing P2GPI, succinctly comprises (or comprises the steps of):
• a PS-targeting antibody preferably bavituximab, and most preferably detectably-labeled bavituximab
• a biological sample suspected of containing ⁇ 2 ⁇ to the PS-coated support under conditions effective to allow binding of ⁇ 2 ⁇ in the sample to both the PS-targeting antibody, preferably bavituximab, and the PS-coated support; preferably, wherein the PS-targeting antibody is added to the PS-coated support prior to adding the sample containing ⁇ 2 ⁇ and wherein they are co-incubated together;
• the assay may utilize a pre -prepared PS-coated support, in which case the assay comprises (or comprises the steps of): (a) adding a PS-targeting antibody, preferably bavituximab, and most preferably detectably-labeled bavituximab, and a biological sample suspected of containing ⁇ 2 ⁇ to a PS-coated support under conditions effective to allow binding of ⁇ 2 ⁇ in the sample to both the PS-targeting antibody, preferably bavituximab, and the PS-coated support; preferably, wherein the PS-targeting antibody is added to the PS-coated support prior to adding the sample containing ⁇ 2 ⁇ and wherein they are co-incubated together; and
• Bound PS-targeting antibody and ⁇ 2 ⁇ (antigen) is detected using at least a secondary binding agent in the form of at least a PS-targeting antibody, preferably bavituximab or 1N1 1 , which is directly or indirectly labeled with a detectable agent.
• An unlabeled PS-targeting antibody can be used in connection with a tertiary binding agent, preferably another antibody, which binds to the PS-targeting antibody and that is directly labeled with a detectable agent.
• tertiary binding antibodies are well-known in the art and, e.g., specifically bind to the Fc portion of the PS-targeting antibody.
• the currently preferred embodiments are those in which the PS-targeting antibody, preferably bavituximab, is itself directly attached to the detectable agent.
• the detectable agent is an agent that produces a detectable signal, such as color or fluorescence, which can be detected and measured or quantified.
• An exemplary detectable agent is the enzyme horseradish peroxidase (HRP), wherein the HRP cleaves the substrate 3,3'5,5' tetramethylbenzidine (TMB) to produce a colored signal that is detected and measured at 450 nm.
• HRP horseradish peroxidase
• TMB 3,3'5,5' tetramethylbenzidine
• the quantity of bound material measured from the signal is compared to the level of a "reference signal", such as a standard curve. If desired, a standard curve can be replicated in every assay.
• ⁇ 2 ⁇ capable of binding to both PS and to the PS-targeting antibody, i.e., ⁇ 2 ⁇ that is not removed overall by routine washing.
• These assays are therefore uniquely suited for detecting pre- treatment ⁇ 2 ⁇ in the form most relevant to clinical treatment, i.e., ⁇ 2 ⁇ that "functions" to form a binding complex with the administered antibody, preferably bavituximab, and the PS exposed in the disease site, preferably in the tumor microenvironment.
• the use of these assays is therefore advantageous in the selection of patients for improved treatment outcomes on bavituximab therapy.
• the functional ⁇ 2 ⁇ assays provided by the invention are also simple, reproducible, sensitive, cost-effective and ideal for use with biological samples obtained by minimally invasive techniques, particularly blood (serum and plasma) samples.
• the rapid nature of the assays provides the important advantage that the biomarker test can be performed quickly and treatment decisions made and implemented in a timely manner.
• the new assays for quantifying functional ⁇ 2 ⁇ provided by the invention are not limited solely to use in measuring ⁇ 2 ⁇ for use as a biomarker in a therapy using a PS-targeting antibody such as bavituximab.
• these assays may be used to measure functional ⁇ 2 ⁇ in any one or more such embodiments, e.g. , such as in connection with knock-out mice or antiphospho lipid syndrome (APS) in humans.
• APS antiphospho lipid syndrome
• binding constructs include constructs that bind to components useful in the detection of biomarkers.
• binding constructs include anti ⁇ 2GPI antibodies, PS and PS-targeting antibodies such as bavituximab.
• Such solid supports or substrates include, e.g. , plates, beads and fibers.
• the solid support or substrate is a multi-well plate, such as a standard 96-well plate.
• the solid support or substrate may be fabricated from any suitable material, such as sepharose, latex, glass, polystyrene, polyvinyl, nitrocellulose, silicon, silica, polydimethylsiloxane (PDMS) and the like.
• the binding construct is operatively associated with the solid support or substrate by effectively contacting at least one surface of the support or substrate with the binding construct.
• the binding construct is immobilized on at least one surface of the solid support or substrate.
• the binding constructs can also be printed onto coated glass slides and used in biomarker arrays or microarrays. Both non-contact and contact printing can be used to prepare such microarrays, with contact printing being preferred.
• Suitable detectable agents include, e.g., enzymes, such as horseradish peroxidase
• HRP horseradish-peroxidase detection system
• AP alkaline phosphatase
• AP alkaline phosphatase
• urease ⁇ -galactosidase and urease.
• a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
• TMB tetramethylbenzidine
• Other convenient enzyme-linked systems include, for example, the alkaline phosphatase detection system, which can be used with the chromogenic substrate p-nitrophenyl phosphate to yield a soluble product readily detectable at 405 nm.
• a ⁇ -galactosidase detection system can be used with the chromogenic substrate O-nitrophenyl-P-D-galactopyranoside (ONPG) to yield a soluble product detectable at 410 nm, or a urease detection system can be used with a substrate such as urea-bromocresol purple.
• O-nitrophenyl-P-D-galactopyranoside O-nitrophenyl-P-D-galactopyranoside
• a urease detection system can be used with a substrate such as urea-bromocresol purple.
• detectable agents include chemiluminescent labels and labels for fluorescent detection.
• Useful fluorochromes include DAPI (4',6-diamidino-2-phenylindole), fluorescein, Hoechst 3325S, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lissamine.
• Fluorescein or rhodamine labeled antibodies or annexins, and/or fluorescein- or rhodamine-labeled secondary antibodies can be used. Isotopes can also be useful in the detection methods, which moieties and assays are well known in the art.
• the detectable agent produces a detectable signal, which is then detected and preferably quantified.
• a detectable signal can be analyzed, e.g., using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation, such as a gamma counter for detection of ,25 I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. Where an enzyme-linked assay is used, quantitative analysis of the detectable signal can be performed using a spectrophotometer.
• the invention also provides a series of biomarker-based kits, including diagnostic, prognostic and predictive therapy kits.
• the biomarker kits will typically comprise one or more of the binding constructs useful in the detection of the biomarkers taught herein. Kits in connection with the P2GPI biomarker will generally comprise at least a first P2GPI binding construct, such as anti-p2GPI antibodies, PS and PS-targeting antibodies such as bavituximab.
• kits will comprise both binding constructs for biomarker detection and at least a first therapeutic agent for use in treating a selected patient, e.g., a PS-targeting antibody such as bavituximab or 1N1 1, or an immunoconjugate thereof.
• a PS-targeting antibody such as bavituximab or 1N1 1, or an immunoconjugate thereof.
• kits may further comprise at least a second or third distinct therapeutic agent for use in combination treatment with the PS-targeting antibody.
• chemotherapeutic, radiotherapeutic, anti- angiogenic, immunotherapeutic and/or anti-viral agents are examples of one or more chemotherapeutic, radiotherapeutic, anti- angiogenic, immunotherapeutic and/or anti-viral agents.
• kits will contain the stated components in at least a first suitable container (or container means).
• the containers will generally include at least one vial, test tube, flask, bottle, syringe or other container or container means, into which the desired agents are placed and, preferably, suitably aliquoted.
• the kits will also typically include a means for containing the individual vials, or such like, in close confinement for delivery, such as, e.g., injection or blow-molded plastic containers into which the desired vials and other apparatus are placed and retained.
• kits may be contained either in aqueous media or in lyophilized form.
• the powder can be reconstituted by the addition of a suitable solvent.
• the solvent may also be provided in another container within the kit.
• Any therapeutic components will preferably be in a pharmaceutically acceptable formulation, or ready for reconstitution as such.
• the kits may also contain a means by which to administer the therapeutic agents to an animal or patient, e.g., one or more needles or syringes, or an eye dropper, pipette, or other such like apparatus, from which the formulations may be injected into the animal or applied to a diseased area of the body.
• kits will preferably have distinct containers for each desired component or agent, particularly the biomarker detection and diagnostic components.
• the kits may comprise one container that contains two or more therapeutics, pre-mixed; either in a molar equivalent combination, or with one component in excess of the other.
• the kits may include pre-labeled antibodies in fully conjugated form, or separate label moieties to be conjugated by the user of the kit, preferably with instructions for attachment.
• one or more of the components, such as PS may already be bound to a solid support, such as a well of a micro titre plate.
• kits will preferably also include written or electronic instructions for use, e.g. in quantification, pre-clinical, clinical and/or veterinary embodiments, including for use in combined therapy.
• the kits will preferably further comprise control agents, such as suitably aliquoted biological compositions, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.
• the solid phase and ELISA-type biomarker assays can be automated or performed robotically, if desired, and the signal from multiple samples can be detected simultaneously.
• Various such assay formats have been used to detect and quantify biomarkers in general, although not in the context of the present invention.
• nano-plasmonic sensors and microfluidic devices termed "Chips" have been described and used for on-chip isolation, quantification and characterization of circulating biomarkers from cancer patients.
• the present assays can thus be accomplished using such microfluidic, chip, nano-tech and other streamlined and automated assays, whilst still retaining the specificities of the invention.
• the present invention also provides computer-based hardware and tests.
• Such computer-based embodiments of the invention include an interface configured for reading one or more laboratory biomarker tests, including for total p2GPI and/or functional p2GPI, and a computer programmed to analyze data from such biomarker tests and, preferably, to compare the analyzed data to established data sets, including test data sets and control data sets.
• the computer-implemented embodiments of the invention will preferably include memory storage, output functions and instructions configured to guide therapy based upon the output.
• the present invention provides biomarker methods, compositions and kits for selecting animals and humans and optimizing treatment with PS-targeting antibodies such as bavituximab, the following guidance concerning animals, subjects ind patients, including human patients, applies to both the biomarker detection and the treatment of the selected population.
• PS-targeting antibodies such as bavituximab
• the invention is most directly applicable to human subjects anl patients, such that the selection and treatment of humans are the most preferred embodirmnts. Nonetheless, the commonality and conservation of the biomarkers across species neans that invention is applicable to animals other than humans.
• mammals are preferred, most preferably, valued and valuable animals such as domestic pets, race hoises and animals used to directly produce (e.g., meat) or indirectly produce (e.g., milk) food for human consumption, although experimental animals are also included.
• the invention theiefore includes clinical, veterinary and research uses.
• the invention th;refore applies to dogs, cats, horses, cows, pigs, boar, sheep, goat, buffalo, bison, llama, desr, elk and other large animals, as well as their young, including calves and lambs, and to race, rats, rabbits, guinea pigs, primates such as monkeys and other experimental animals.
• a "therapeutically effective" amount or dose of a PS-targeting antibody such as bavituximab is an amount or dose that exerts a beneficial therapeutic effect when administered to an animal, preferably a human patient, in need of such a thirapy, including when administered as part of a combination therapy.
• a therapeutically effective anticancer dose is an amount or dose that exerts a beneficial anti-cancer effect when administered to an animal, preferably a human patient, with cancer, including when administered as part of a combination cancer therapy.
• a therapeutically effective anti-viral dose is an amount or dose that exerts a beneficial anti-viral effect when administered to an aninal, preferably a human patient, with a viral infection or disease, including when administered is part of a combination viral therapy.
• “Beneficial anti-cancer effects” include any consistently detectable anti-tumor and anticancer effect, including tumor vasculature thrombosis and/or destruction, tumor necrosis, tumor regression and tumor remission, up to and including cures.
• Clinical measures of beneficial anti-cancer effects include, for example, improvements ii overall response rate (ORR), including complete response (CR), partial response (PR), and CR + PR; time to tumor progression (TTP); duration of response (DOR or DR); and improvements or extensions in progression-free survival (PFS), disease-free survival (DFS) and overall survival (OS), including median overall survival (mOS), in individual patients, patient populations and sub- populations, as applicable.
• “Beneficial anti-viral effects” include any consistently detectable anti-viral effect, including inhibiting viral infection, replication, maturation, reproduction and egress and/or ongoing infection of, or spread to, additional cells (host cells) or tissues.
• Clinical measures of beneficial anti-viral effects include, for example, early virological response, reductions in viral load and clearance of virus, as well as improvements in the symptoms caused by the viral infection.
• beneficial therapeutic effects may not be curative, particularly in the intermediate or long term, but that does not negate the usefulness of the therapies.
• beneficial therapeutic, anticancer and anti-viral effects also include comparative and/or modest treatment effects, but with improvements in any one or more measures of safety.
• Another consideration for "beneficial" therapeutic effects is the fact that the PS-targeting antibodies such as bavituximab may predispose the disease or tumor to further therapeutic treatment, such that a subsequent treatment can result in an overall improved effect.
• the effective dose ranges of PS-targeting antibodies such as bavituximab given intravenously (IV) and quoted in mg/kg, will be between about 0.1 and about 13- 15, preferably between about 0.1 and about 6- 10; preferably, between about 0.3 and about 6; more preferably, between about 0.5 and about 6; more preferably, between about 1 and about 6; more preferably, between about 0.5 and about 3 or between about 3 and about 6; more preferably, between about 1 and about 3.
• Exemplary effective doses of PS-targeting antibodies such as bavituximab, given IV and quoted in mg/kg, will be about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 and about 15; preferably about 0.1 , 0.3, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 and about 6; more preferably, about 2 or 3; and most preferably, of about 3 mg kg.
• bavituximab administered intravenously (IV) for clinical treatment, particularly for all oncology indications, is recommended based on extensive pre-clinical and clinical data, and particularly on the pharmacokinetic profile in humans (Example VI), along with the extensive safety data. Nonetheless, a range of doses have been shown to be effective, including clinical anti-viral activity at 0.3 mg/kg (Example VI).
• bavituximab has been safely administered to rats and monkeys at doses above 10 mg/kg, up to 100 mg/kg. At the 100 mg/kg dose level in monkeys, bavituximab transiently decreased P2GPI in the systemic circulation, so such ultra-high doses are not recommended.
• the dose of 3 mg/kg although preferred, is not limiting on the invention. Accordingly, it will be understood that, given the parameters and detailed guidance presented herein, further variations in the active or optimal dose ranges and doses will be encompassed within the present invention. It will thus be understood that lower doses may be more appropriate in combination with certain agents, and that high doses can still be tolerated, particularly when treating a usually fatal disease.
• a pharmaceutically acceptable composition (according to FDA standards of sterility, pyrogenicity, purity and general safety) is administered to the animal or patient systemically. Intravenous injection is generally preferred, and a continuous infusion over a period of several hours is most preferred.
• the administration regimen can also be adapted to optimize the treatment strategy, as is well known to those of skill in the art. Some variation in dosage and treatment regimen may be necessary depending on the condition of the subject being treated. The physician(s) responsible will, in light of the present disclosure, be able to determine the appropriate treatment for the individual subject. Such optimization and adjustment is routinely carried out in the art, and by no means reflects an undue amount of experimentation. H3. Supplementing Treatment with P2GPI
• p2GPI levels as a biomarker for response to PS-targeting antibodies such as bavituximab, either alone, or as part of a multi-biomarker selection, and irrespective of whether total ⁇ 2 ⁇ or functional ⁇ 2 ⁇ is measured, the methods will select only a sub-set of patients for treatment.
• Another embodiment of the present application is thus to restore any non-selected patients to treatment eligibility by co-administering P2GPI to those patients along with the PS-targeting antibody such as bavituximab.
• the entire population becomes treatable with PS-targeting antibodies.
• a patient with a pre-treatment level of functional ⁇ 2 ⁇ of 150 g/ml could be returned to the treatable group by co-administering bavituximab in conjunction with sufficient functional ⁇ 2 ⁇ to restore the ⁇ 2 ⁇ levels to at least about 200 ⁇ g ml.
• the sample applies to whichever pre- treatment level of ⁇ 2 ⁇ is used in the biomarker analyses.
• PS-targeting antibodies such as bavituximab specifically target PS
• the first, and most important, indication for treatment is cancer (Section K), particular solid tumors and their metastases, but also liquid tumors, such as leukemias, and preferably, Hodgkin's Lymphoma.
• PS is maintained on the inside of the cell membrane and not accessible to binding. Only cells in diseases have PS exposed on the outside of the cell membrane, most particularly, cells in the tumor microenvironment, but also dying cells, aberrant cells, inappropriately activated cells, infected cells and pathogenic organisms themselves.
• PS exposure in the tumor microenvironment is "immunosuppressive", meaning that the body cannot adequately fight the cancer. By blocking PS, bavituximab overrides the PS-mediated immunosuppression, helping the body attack the tumor.
• PS is a relatively stable marker, meaning that it is an ideal target for therapy.
• PS is also exposed on the outside of cells, which means that bavituximab can be used in diagnosis and particularly for "imaging", i.e., in vivo diagnostics, of a variety of diseases in which increased or inappropriate cell death occurs, including such conditions as, e.g., cancer and heart attacks, (see below for imaging).
• Prominent pathogens that cause the host cell to externalize PS are viruses (Section J).
• PS and PS receptors as enhancers of enveloped virus entry and infection are now well-documented and applies to a wide range of viruses.
• connection between PS and viruses is not limited to enveloped viruses, but extends to non-enveloped viruses.
• PS lipid vesicles released from virally-infected cells enable efficient en bloc transmission of enteroviruses (Chen et al., 2015).
• PS-targeting antibodies such as bavituximab can localize to those cells and pathogens and exert beneficial effects.
• PS is a marker
• diseases in which aberrant vasculature (blood vessels) is involved including diseases and disorders having prothrombotic blood vessels (prone to clotting) and those involving aberrant angiogenesis.
• Angiogenesis is the process through which new blood vessels form from pre-existing vessels; the development of new blood vessels begins with the formation of endothelial cell sprouts, which requires PS (Weihua et al, 2005).
• Aberrant angiogenesis is involved in many diseases, most notably in cancer.
• PS-targeting antibodies such as bavituximab can treat benign (as opposed to malignant) tumors, such as benign prostatic hyperplasia (BPH), acoustic neuroma, neurofibroma, trachoma, granulomas including pyogenic granulomas and sarcoidosis (sarcoid), meningioma, angiofibroma, angioma, hemangiomas and systemic forms of hemangiomas, the hemangiomatoses.
• benign prostatic hyperplasia BPH
• acoustic neuroma acoustic neuroma
• neurofibroma neurofibroma
• trachoma granulomas including pyogenic granulomas and sarcoidosis (sarcoid)
• granulomas including pyogenic granulomas and sarcoidosis (sarcoid)
• meningioma angiofibroma
• angioma
• Conditions directly associated with aberrant vasculature that can be treated with PS-targeting antibodies include vascular restenosis (narrowing of blood vessels), including restenosis following angioplasty, vein occlusion, artery occlusion and carotid obstructive or occlusive disease; vasculitis (disorders that destroy blood vessels by inflammation), including Behcet's disease (also an eye disease), polyarteritis nodosa (panarteritis nodosa or PAN) and Wegener's granulomatosis (WG) or sarcoidosis (granulomatosis with polyangiitis, GPA); arteriovenous malformations (AVM) and arteriovenous fistula; epistaxis (nosebleeds); vascular adhesions; and hyperviscosity syndromes.
• vascular restenosis narrowing of blood vessels
• vasculitis disorders that destroy blood vessels by inflammation
• Behcet's disease also an eye disease
• PS-targeting antibodies such as bavituximab can treat clinically important diseases including joint diseases such as arthritis, including rheumatoid arthritis and osteoarthritis, synovitis, hemophilic joints and Paget's disease; skin diseases such as psoriasis, dermatitis, scleroderma (systemic sclerosis or CREST syndrome), pseudoxanthoma elasticum (PXE, known as Gronblad-Strandberg syndrome), rosacea, Stevens-Johnson syndrome or disease (PXE, rosacea and Stevens- Johnson syndrome are also eye diseases), pemphigoid, hypertrophic scars and keloids; Grave's disease; endometriosis; and Osier-Weber (or Osler-Weber-Rendu) syndrome or disease (also known as hereditary hemorrhagic telangiectasia, HHT).
• joint diseases such as arthritis, including rheumatoid arthritis and osteoarthritis, synovitis
• ocular neovascular diseases ocular neovascular diseases. These diseases are characterized by invasion of new blood vessels into the structures of the eye, such as the retina, choroid and/or cornea. They are the most common cause of blindness and are involved in approximately twenty eye diseases.
• ocular neovascular diseases are (proliferative) diabetic retinopathy, macular degeneration, including age-related macular degeneration (AMD), retinopathy of prematurity (ROP or Terry syndrome, previously known as retrolental fibroplasia, RLF), neovascular glaucoma, corneal graft neovascularization and corneal graft rejection.
• AMD age-related macular degeneration
• ROP or Terry syndrome previously known as retrolental fibroplasia
• neovascular glaucoma corneal graft neovascularization
• corneal graft neovascularization corneal graft rejection
• corneal graft rejection corneal graft rejection.
• CNV Choroidal neovascularization
• Other diseases associated with retinal/choroidal neovascularization that can be treated with PS-targeting antibodies such as bavituximab include syphilitic, mycobacterial and/or other eye infections causing retinitis or choroiditis; uveitis (iridocyclitis), including vitritis and pars planitis; Eales disease, presumed ocular histoplasmosis syndrome (POHS), Best's disease (vitelliform macular dystrophy), Stargardt disease, eye trauma and post-laser complications.
• POHS presumed ocular histoplasmosis syndrome
• Best's disease vitelliform macular dystrophy
• Stargardt disease eye trauma and post-laser complications.
• keratoconjunctivitis including keratitis (only the cornea is inflamed) and conjunctivitis (only the conjunctiva is inflamed), such as atopic keratitis, superior limbic keratitis, pterygium keratitis sicca and marginal keratolysis; phylectenulosis; Mooren ulcer; chemical burns, bacterial ulcers, fungal ulcers, Herpes infections and traumas of the eye and contact lens overwear.
• ocular diseases that can be treated with PS-targeting antibodies such as bavituximab include scleritis, rubeosis (neovascularization of the iris), neovascularization of the angle (NVA), and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy (PVR), whether or not associated with diabetes.
• PS-targeting antibodies such as bavituximab
• endothelial cell sprouts requires PS, so the development of new blood vessels also requires PS (Weihua et al, 2005).
• This process is also involved in certain normal physiological events, particularly wound healing and reproduction, and is important in ovulation and in the implantation of the blastula after fertilization.
• Prevention of this process using bavituximab can thus be used to induce amenorrhea (absence of a menstrual period in women of reproductive age), to block ovulation and/or to prevent implantation by the blastula, i.e. , as a contraceptive.
• Chronic inflammation also involves aberrant and pathological vasculature.
• chronic inflammatory disease states such as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels into the inflamed tissues.
• PS-targeting antibodies such as bavituximab.
• erythrocytes are prematurely senescent and PS-positive ("sickle erythrocytes"), as opposed to only about 1% in healthy people.
• the PS-positive sickle erythrocytes remain in circulation, adhere to the endothelium and their exposed PS acts as a platform for the initiation of the coagulation cascade that is responsible for clot propagation (Kennedy et al., 2015).
• PS is also expressed in atherosclerosis and PS-positive extracellular microvesicles are released from atherosclerotic plaques (Mallat et al., 1999).
• the plaques formed within the lumen of blood vessels, which are positive for PS, have also been shown to have angiogenic stimulatory activity.
• angiogenic markers such as VEGF
• PS-targeting antibodies such as bavituximab thus provide an effective treatment for atherosclerosis and obstructive coronary diseases.
• PS-positive extracellular microvesicles Both Type 1 and Type 2 diabetic patients have PS-positive extracellular microvesicles, as shown by being annexin V-positive (Sabatier et al, 2002).
• brain exosomes contain PS and amyloid ⁇ -peptide ( ⁇ ), the pathogenic agent of the disease (Yuyama et al., 2012).
• PS and PS-positive extracellular microvesicles are also involved in sepsis (septic shock), where they are markers and mediators of sepsis-induced microvascular dysfunction and immunosuppression (Souza et al., 2015).
• Antiphospholipid syndrome APS
• SLE or lupus systemic lupus erythematosis
• autoimmune disorders in which antibodies are produced against the body's own phospholipids, are associated with coagulation disorders, including miscarriages and thrombocytopenia (low platelet counts). Accordingly, the anti-phospholipid antibodies in these patients are pathogenic antibodies, which cause thrombosis.
• PS-targeting antibodies such as bavituximab, however, target PS without exhibiting any such side effects. Accordingly, bavituximab can also treat antiphospholipid syndrome, associated diseases and complications thereof.
• bavituximab can antagonize or compete with the pathogenic antibodies in APS patients, thus displacing the pathogenic antibodies from their phospholipid-protein targets in the body.
• pathogenic infections for example, intracellular parasites, such as the parasitic protozoan, Leishmania amazonensis, which causes leishmaniasis (Zandbergen et al., 2006; Wanderley et al., 2009; Wanderley et al, 2013); Plasmodium falciparum, which causes malaria (Eda & Sherman, 2002; Pattanapanyasat et al., 2010); and Trypanosoma cruzi, a parasitic protozoan that causes trypanosomiasis (DaMatta et al., 2007), all result in PS exposure.
• intracellular parasites such as the parasitic protozoan, Leishmania amazonensis, which causes leishmaniasis (Zandbergen et al., 2006; Wanderley et al., 2009;
• Schistosoma parasitic flatworms that cause schistosomiasis, also expose PS (van der Kleij et al., 2002), as does Toxoplasma gondii, which causes toxoplasmosis (Seabra et al, 2004).
• PS exposure has also been shown on the exterior cell surface following infection by intracellular bacterial pathogens, such as Yersinia pestis and Francisella tularensis, which cause plague and tularemia, respectively (Lonsdale et al., 201 1).
• Listeria monocytogenes which causes listeriosis, also promotes the release of membrane-derived vesicles with exofacial PS from infected host cells (Czuczman et al., 2014).
• endothelial cells infected with the meningitis-causing pathogen, Neisseria meningitidis exhibit PS translocation to the cell surface (Schubert-Unkmeir et al., 2007).
• the PS externalization common to the facultative intracellular parasites detailed above is likely to occur for other such pathogens, such as Brucella and Salmonella, which cause brucellosis and illnesses such as typhoid fever, paratyphoid fever and food poisoning, respectively.
• pathogens such as Brucella and Salmonella
• brucellosis and illnesses such as typhoid fever, paratyphoid fever and food poisoning, respectively.
• Chlamydia trachomatis can also be treated, which causes trachoma (also see above).
• PS externalization on host cells is now a generally recognized phenomenon in response to infection with a range of bacteria and pathogens (Wandler et al., 2010).
• H. pylori has direct contact with gastric epithelial cells, it induces externalization of PS to the outer leaflet of the host plasma membrane (Murata-Kamiya et al., 2010).
• PS is also present on Treponema pallidum, which causes syphilis.
• Bartonellosis a bacterial infection found in South America, can be treated with bavituximab, particularly because bartonellosis results in a chronic stage that is characterized by proliferation of vascular endothelial cells, and one of bavituximab's mechanisms of action, as clearly shown in cancer treatment, is to destroy vascular endothelial cells.
• PS-targeting antibodies such as bavituximab may be used for imaging any of the foregoing diseases, disorders and infections, most preferably, for imaging vascularized tumors (Jennewein et al., 2008; Marconescu & Thorpe, 2008; Saha et al, 2010; Stafford & Thorpe, 201 1 ; Gong et al, 2013; Stafford et al, 2013; U.S. Patent No. 7,790,860).
• Bavituximab may also be used for imaging vascular thromboses, particularly in or near the heart, such as in deep vein thrombosis, pulmonary embolism, myocardial infarction, atrial fibrillation, problems with prosthetic cardiovascular materials, stroke (cerebrovascular accident (CVA) or cerebrovascular insult (CVI)), and the like.
• PS-targeting antibodies such as bavituximab may also be used in imaging activated platelets, e.g. , in conditions such as abscesses, restenosis, inflammation of joints and in hemostatic disorders, such as arterial, coronary, venous and cerebral thrombosis and such like.
• PS-targeting antibodies such as bavituximab are thus suitable for treating and/or diagnosing all the above diseases and disorders, in which PS is a documented marker.
• Prominent pathogens that cause the host cell to externalize PS are viruses.
• the presence of PS has been demonstrated on the surface of viruses and virally-infected cells and/or shown to be important to infections from a wide range of viral families, including Arenaviridae, Bunyaviridae, Flaviviridae, Filoviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Poxviridae, Retroviridae and Rhabdoviridae (see also, Table 2A and Table 2B in U.S. patent application Serial No. 14/634,607 and PCT patent application No. PCT/US 15/18183, each filed February 27, 2015).
• connection between PS and viruses is not limited to enveloped viruses, but extends to non-enveloped viruses (Clayson et al, 1989; Chen et al, 2015).
• PS lipid vesicles essentially exosomes
• accompanies data showing that PS vesicles enable efficient en bloc transmission of enteroviruses.
• the following rationale explains that PS is involved in infections from both enveloped and non-enveloped viruses.
• Enveloped viruses utilize the host cell plasma membrane to embed viral proteins that mediate efficient entry of the progeny virions with the next host cell.
• PS is found on the exterior of virus infected cells prior to virus release and enveloped viruses incorporate PS into the viral envelope upon exiting the host cell.
• Non-enveloped viruses use to release new virions from the cell include lysis of the cell, which can be caused directly by the host immune response to the infected cells (T cells or macrophages), or due to the activity of virus directly on host cell protein synthesis or cellular structures.
• An example of a virus alters the cell structure to induce cell lysis is Adenovirus.
• Adenovirus expresses several proteins late during infection that alter the structural integrity of the cell by disrupting filament networks and protein synthesis.
• Some non-enveloped viruses are able to release their progeny viruses via a nondestructive mechanism without any cytopathic effect.
• Poliovirus While poliovirus induces cell lysis rapidly (about 8 hours), it is also released from cells in PS lipid vesicles that are capable of infecting new host cells. Poliovirus particles in PS-vesicles are more efficient in infecting HeLa cells and primary macrophages than virus particles removed from PS-vesicles and blocking the vesicles with Annexin V inhibited the vesicles from infected cells in a dose dependent manner, suggesting the PS lipids are cofactors for poliovirus infection.
• Coxsackievirus B3 and Rhinovirus particles are also released into PS lipid vesicles (Chen et ai, 2015), indicating a common mechanism utilized by enteroviruses to selectively release mature particles without lysis of the cell.
• SV40 is also released from cells in the above types of PS-lipid vesicles.
• SV40 particles can be found released from cells before induction of cytopathic effects (Clayson et ai, 1989).
• SV40 virions have been observed in cytoplasmic smooth vesicles at 48 hour post infection and the release of SV40 particles was inhibited by monensin, a sodium ionophore that blocks intracellular protein transport by blocking cation transport across lipid membranes.
• PS-targeting antibodies such as bavituximab thus include interference with proteins needed in cell activation or their ability to mediate viral egress, reversing the PS-mediated immunosuppression and clearance of infected cells or the virus by immune clearance mechanisms.
• PS-targeting antibodies such as bavituximab
• the potential mechanisms by which PS-targeting antibodies such as bavituximab have been shown to exert such anti-viral properties include: 1) binding to viral particles; 2) binding to infected cells; 3) inhibition of viral replication; and 4) enhancement of immune responses by blocking the immunosuppressive cell receptors that bind PS.
• Data in an HIV-1 model demonstrate that virions produced by virally infected macrophages have elevated levels of PS which serve as a cofactor for HIV-1 infection of macrophages. Blocking PS on HIV-1 with PS-targeting antibodies may prevent cell-cell interactions and block virus-target cell fusion. Results also indicate that bavituximab binds to pichinde viral particles and treatment of pichinde virus-infected guinea pigs enhances development of both of anti pichinde antibodies and cellular responses.
• PS-targeting antibodies such as bavituximab.
• the treatment of benign tumors is included, such as acoustic neuroma, neurofibroma, trachoma, pyogenic granulomas and BPH.
• the treatment of malignant tumors is preferred.
• tumor, tumors, cancer and cancers are intended to indicate malignancy, unless expressly stated otherwise.
• the tumors to be treated are solid or vascularized tumors, including tumors in which angiogenesis is active and tumors having prothrombotic blood vessels.
• Solid and “vascularized” tumors are tumors having a vascular component, i.e., which require tumor blood vessels for the provision of oxygen and nutrients to the tumor cells.
• All cancers are included, whether primary or metastatic, as exemplified by breast, ovarian, thoracic, lung, liver (hepatocellular carcinoma, HCC), colon, colorectal, rectal, prostate, pancreatic, brain (gliomas and glioblastomas), cervical, uterine, endometrial, head and neck, parotid, esophageal, larynx, thyroid, gastrointestinal, stomach, kidney (renal cell carcinoma, RCC), biliary tract, bladder, testicular and other cancers, including carcinomas (squamous and non-squamous, small cell and non-small cell), adenocarcinomas and neuroblastomas, as well as melanoma, merkel cell carcinoma and hematological malignancies.
• carcinomas squamous and non-squamous, small cell and non-small cell
• adenocarcinomas and neuroblastomas as well as melanoma, merkel cell carcinoma and hemat
• the invention particularly applies to non-small cell lung cancer (NSCLC) or to breast, pancreatic, liver, kidney, rectal or ovarian cancer or melanoma. Most particularly, the invention applies to NSCLC such as non-squamous NSCLC.
• NSCLC non-small cell lung cancer
• biomarker and treatment methods may thus be combined with any other methods generally employed in the treatment of the particular disease or disorder that the animal or patient exhibits, particularly cancer and viral infections and diseases. So long as a given therapeutic approach is not known to be detrimental to the patient's condition in itself, and does not significantly counteract the PS-targeting antibody therapy, its combination with the present invention is contemplated. Combination therapies for non-malignant diseases are also contemplated.
• the present invention may be used in combination with classical approaches, such as surgery, chemotherapy, radiotherapy, cytokine therapy, anti- angiogenesis and the like, and newer approaches such as immuno-oncology (10) agents.
• the invention therefore provides biomarker and combined therapies in which the PS-targeting antibodies such as bavituximab are used simultaneously with, before, or after surgery or radiation treatment; or are administered to patients with, before, or after conventional chemotherapeutic or radiotherapeutic agents, cytokines, anti-angiogenic agents, apoptosis- inducing agents, targeted therapies, IO agents or such like.
• any surgical intervention may be practiced in combination with the present invention.
• any mechanism for inducing DNA damage locally within tumor cells is contemplated, such as ⁇ -irradiation, X-rays, UV-irradiation, microwaves and even electronic emissions and the like.
• the directed delivery of radioisotopes to tumor cells is also contemplated, and this may be used in connection with a targeting antibody or other targeting means.
• the general use of combinations of substances in cancer treatment is well known.
• the "primary therapeutic agents" or “first anti-cancer agents” for use with the present invention, as used herein, are the PS-targeting antibodies such as bavituximab.
• the "secondary or tertiary therapeutic agents” or “at least a second or third anti-cancer agent”, as used herein, are second or third, distinct therapeutic agents, anti-cancer or anti-viral agents, i.e. , therapeutic agents, anti-cancer or anti-viral agents "other than” the primary therapeutic agent. Any secondary or tertiary therapeutic agent may be used in the combination therapies of the present invention.
• secondary or tertiary therapeutic agents may be selected with a view to achieving additive, greater than additive and potentially synergistic effects, according to the guidance in the present application and the knowledge of those of skill in the art.
• anti-tumor therapy or anti-viral therapy one would simply administer to an animal or patient a PS-targeting antibody such as bavituximab in combination with another, i.e. , a second or third, distinct therapeutic agent, anti-cancer or anti- viral agent, in a manner effective to result in their combined therapeutic, anti-tumor or antiviral actions within the animal or patient.
• the agents would therefore be provided in amounts effective and for periods of time effective to result in their combined presence within the disease site, e.g. , the tumor, tumor environment or microenvironment, and/or to exert their combined therapeutic actions in the animal or patient, preferably, to exert their combined therapeutic actions on the immune system of the animal or patient.
• the primary therapeutic agent and the second or third, distinct therapeutic agent may be administered substantially simultaneously, either in a single composition, or as two or three distinct compositions using different administration routes.
• the PS-targeting antibody such as bavituximab may precede, or follow, the second or third, distinct therapeutic agent, anti-cancer or anti-viral agent by, e.g. , intervals ranging from minutes to weeks or months.
• the primary therapeutic agent and the second or third, distinct therapeutic agent are applied separately to the animal or patient, one would ensure that an inoperative period of time did not exist between the time of each delivery, such that each agent would still be able to exert an advantageously combined effect. From standard practice, including the clinical experience to date with bavituximab, one or two weeks is not an inoperative period of time between administering bavituximab and a second or third, distinct therapeutic agent. Indeed, an interval of about one week may be preferred.
• one preferred combined tumor therapy concerns administering to an animal or patient a PS-targeting antibody such as bavituximab in combination with a second, distinct anti-cancer agent, either substantially simultaneously or preferably at intervals of weeks, followed by administering a third, distinct anti-cancer agent at a subsequent time and continuing the periodic administration of the third, distinct anti-cancer agent for a subsequently effective time, such as for a period of months.
• the secondary or tertiary therapeutic agents for separately timed combination therapies may be selected based upon certain criteria, including those discussed herein and known in the art. However, a preference for selecting one or more second or third, distinct therapeutic agents for prior or subsequent administration does not preclude their use in substantially simultaneous administration if desired.
• second or third, distinct anti-cancer agents selected for administration "prior to" the primary therapeutic agents, and designed to achieve increased and potentially synergistic effects include agents that induce the expression of PS in the tumor microenvironment.
• agents that stimulate localized calcium production activate membrane transporters that move PS to the outer surface of the plasma membrane, injure the tumor endothelium, cause preapoptotic changes and/or induce apoptosis in the tumor endothelium or tumor cells will generally result in increased PS expression.
• agents are docetaxel and paclitaxel.
• the PS can then be targeted using the PS-targeting antibody such as bavituximab, thus amplifying the overall therapeutic effect, and also giving increased attack via host effectors (complement, ADCC, antibody-mediated phagocytosis, CDC).
• Drugs that have selectivity for angiogenic, remodeling or activated endothelial cells, such as are present in tumor blood vessels, but not in normal resting blood vessels, can also be used to selectively causes exposure of PS in the tumor microenvironment.
• examples of such agents are combretastatins and docetaxel. This again would lead to increased antibody binding and enhanced initiation of host effector mechanisms.
• Second or third, distinct anti-cancer agents selected for administration "subsequent to" the primary therapeutic agents, and designed to achieve increased and potentially synergistic effects, include agents that benefit from the effects of the primary therapeutic agent.
• PS-targeting antibodies such as bavituximab cause tumor necrosis.
• effective second or third, distinct anti-cancer agents for subsequent administration include anti- angiogenic agents, which inhibit metastasis; agents targeting necrotic rumor cells, such as antibodies specific for intracellular antigens that become accessible from malignant cells in vivo (U.S. Patent No. 5,019,368; 5,882,626); and chemotherapeutic agents and anti-tumor cell immunoconjugates, which attack any tumor cells that may survive at the periphery.
• the currently most preferred second or third, distinct anti-cancer agents for administration subsequent to the PS-targeting antibody such as bavituximab are immune checkpoint inhibitors, as described below.
• the time period for treatment may be desirable to extend the time period for treatment significantly, where several days (2, 3, 4, 5, 6 or 7), several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
• Anti- angiogenics should be administered at a careful time after surgery, however, to allow effective wound healing. Anti-angiogenic agents may then be administered for the lifetime of the patient.
• the primary therapeutic agent or the second or third, distinct therapeutic agent may be utilized.
• the primary therapeutic agent and the second or third, distinct therapeutic may be administered interchangeably, on alternate days or weeks; or a sequence of one agent treatment may be given, followed by a sequence of the other treatment or treatments.
• all that is required is to deliver two or more agents in a combined amount effective to exert a therapeutic effect, irrespective of the times for administration.
• the PS-targeting antibodies such as bavituximab may be administered in combination with one or more chemotherapeutic agents or drugs.
• Chemotherapeutic drugs can kill proliferating tumor cells, enhancing the necrotic areas created by the overall treatment. The drugs can thus enhance the action of the primary therapeutic agents of the invention.
• chemotherapeutic drugs are selective for dividing, oxygenated cells. These have advantages in combined therapy as the chemotherapeutic drug acts on different targets from the primary therapeutic agents, leading to a more complete anti-tumor effect. For example, chemotherapeutic drugs are selectively active against the rapidly dividing, oxygenated tumor cells in the tumor periphery. Anti-angiogenic drugs that are selective for well-oxygenated, angiogenic vessels in the tumor periphery would also be effective in combination.
• the primary therapeutic agents of the present invention can also enhance the action of the chemotherapeutic drugs by retaining or trapping the drugs within the tumor.
• the chemotherapeutics are thus retained within the tumor, while the rest of the drug is cleared from the body. Tumor cells are thus exposed to a higher concentration of drug for a longer period of time. This entrapment of drug within the tumor makes it possible to reduce the dose of drug, making the treatment safer as well as more effective.
• Further drugs for combined use in the present invention are those that act on cells that are "sensitized" to the drug by the action of the primary therapeutic agent, such that reduced doses of the second drug are needed to achieve its anti-tumor effect.
• the primary therapeutic agent of the invention sensitizes tumor cells to a second drug, either directly or through stimulation of cytokine release.
• suitable second or third anti-cancer agents for combination therapy are those that enhance the activity of host effector cells, e.g. , by selectively inhibiting the activity of immunosuppressive components of the immune system.
• Such agents enable the primary therapeutic agents of the invention, which stimulate attack by effector cells as part of their mechanism, to work more aggressively.
• Examples of such gents are docetaxel and immune checkpoint inhibitors.
• Drugs that induce apoptosis may be used in the combination therapies.
• Docetaxel for example, induces apoptosis and therefore PS exposure by binding to microtubules and disrupting cell mitosis (Hotchkiss et al., 2002).
• Treatment of endothelial cells, which line tumor blood vessels, and tumor cells with docetaxel at subclinical concentrations is known to induce PS expression at the cell surface.
• the anti-tumor effects of PS-targeting antibodies such as bavituximab include Fc domain-mediated augmentation of immune effector functions, such as ADCC, CDC, stimulation of cytokine production, and such mechanisms in combination.
• This is also relevant to docetaxel, as other studies have shown that the treatment of breast cancer patients with docetaxel leads to increases in serum IFNy, IL-2, IL-6 and GM-CSF cytokine levels, augmenting the anti-rumor immune responses in these patients by enhancing the activity of natural killer (NK) and lymphokine activated killer (LAK) cells.
• NK natural killer
• LAK lymphokine activated killer
• docetaxel will both induce PS expression and binding of the administered antibody, and also enhance the activities of immune effectors, which mediate anti-tumor effects.
• combination of the antibodies with docetaxel is a preferred embodiment, particularly when also combined with or followed by treatment with an immune checkpoint inhibitor, as described below.
• docetaxel and other chemotherapeutic agents that induce apoptosis are certain preferred agents for use in the combination treatments of the present invention.
• Combinations with chemotherapeutics drugs that induce apoptosis, such as docetaxel should synergistically attack tumor vasculature endothelial cell and tumor cell compartments, leading to not only significantly enhanced treatment efficacy but also lower toxicity.
• These combinations are contemplated for use in breast cancer treatment, particularly the combination of metronomic chemotherapy using docetaxel with a PS-targeting antibody.
• Exemplary chemotherapeutic agents for combined therapy are described in U.S. Patent
• chemotherapeutic agents such as those chemotherapeutic agents, antibodies and other drugs known in the art are exemplary and not limiting. Variation in dosage can occur depending on the condition treated. The treating physician will be able to determine the appropriate dose for the individual subject.
• docetaxel is used, such as docetaxel administered at a starting dose of 60 mg/m 2 or docetaxel administered to a patient in an amount of 75 mg/m .
• a challenge to effective immunotherapy is to overcome multiple pathways that inhibit innate or adaptive immune activation.
• the PD-1 immune checkpoint has been identified as a major immunosuppressive pathway and has emerged as a promising target for cancer immunotherapy with less toxicity than chemotherapy. It functions to exhaust activated tumor- specific T cells and dampen their tumor-killing activity.
• PD-1 is absent on na ' ive T cells, B cells, macrophages, DCs, and monocytes but expressed highly on their activated counterparts.
• tumors and associated myeloid cells exploit the PD-1 pathway to generate innate and adaptive immune resistance through up-regulation of PD-Ll expression.
• Example XIX demonstrate that patients treated with bavituximab (and docetaxel) followed by subsequent immunotherapy (“SACT-IO”) have a statistically significant better overall survival in comparison to patients treated with placebo (docetaxel alone) followed by subsequent immunotherapy.
• SACT-IO subsequent immunotherapy
• bavituximab does, indeed, enhance the activity of immunotherapy agents in human patients.
• important embodiments of the present invention are the treatment of cancer patients with PS-targeting antibodies such as bavituximab in combination with immunotherapy or immuno-oncology (IO) agents.
• PS-targeting antibodies such as bavituximab
• IO immuno-oncology
• Exemplary immunotherapeutic agents for combined therapy are listed in Table C in provisional application Serial No. 62/406,727, filed October 1 1 , 2016; and in Table D in each of provisional application Serial No. 62/480,994, filed April 03, 2017 and provisional application Serial No. 62/507,580, filed May 17, 2017, of which combinations with NK cell and CAR-T therapies are currently preferred.
• IO agents are those approved for clinical treatment or in human clinical trials, preferably in late-stage clinical trials, such as those described in Table E in each of provisional application Serial No. 62/480,994, filed April 03, 2017 and provisional application Serial No. 62/507,580, filed May 17, 2017.
• the doses for use and indications for treatment are well-known to those of ordinary skill in the art, as exemplified by the details in Table E in each of provisional application Serial No. 62/480,994, filed April 03, 2017 and provisional application Serial No. 62/507,580, filed May 17, 2017.
• nivolumab at 240 mg or 3 mg/kg every 2 weeks to treat melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), classical Hodgkin (Hodgkin's) lymphoma, squamous cell carcinoma of the head and neck (head and neck cancer), bladder cancer, small cell lung cancer, brain cancers such as malignant glioma, anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM), hepatocellular cancer (HCC), esophageal cancer, gastric cancer, mesothelioma and multiple myeloma; pembrolizumab at 200 mg or 2 mg/kg every 3 weeks to treat melanoma, NSCLC, classical Hodgkin lymphoma, head and neck cancer, gastric cancer, breast cancer, bladder cancer, all solid tumors, colorectal cancer, RCC, multiple myeloma, esophageal cancer and
• IO agents are ipilimumab at 3 mg/kg or 10 mg/kg every 3 weeks to treat unresectable or metastatic melanoma; tremelimumab at 15 mg/kg every 3 months to treat melanoma; REGN2810 to treat NSCLC; PDROO l to treat melanoma; racotumomab to treat NSCLC; MEDI0562 and GSK3174998 to treat advanced solid tumors; urelumab to treat melanoma; utomilumab to treat DLBCL; and each of BMS-986016, LAG525, JNJ-61610588, TSR-022, MBG453, MEDI1873 and INCAGN01876 to treat solid tumors.
• IO agents for combination therapy with PS-targeting antibodies such as bavituximab, as directly supported by the data in Example XIX
• checkpoint inhibitors also termed herein “immune checkpoint antibodies”.
• Suitable “immune checkpoint antibodies” include agonistic (activating) antibodies that bind to an activating immune checkpoint, receptor or molecule, such as CD28, OX40 and/or GITR, and antagonistic (blocking) antibodies that bind to an inhibitory immune checkpoint, receptor or molecule, such PD-1 , PD-L1 , CTLA-4, TIM-3 and/or LAG-3.
• Such blocking antibodies are routinely termed "immune checkpoint inhibitors”, which is also used herein.
• immune checkpoint antibodies are "blocking antibodies that bind to CTLA-4, PD-1 or PD-L1 ".
• blocking antibodies that bind to CTLA-4, PD-1 or PD-L1 are well-known to those of ordinary skill in the art, as described in Table B.
• blocking antibodies to CTLA-4 such as ipilimumab and tremelimumab
• blocking antibodies to PD-1 such as nivolumab (Brahmer et al., 2015), REGN2810 and pembrolizumab (Garon et al., 2015)
• blocking antibodies to PD-L1 such as durvalumab (MEDI4736) and atezolizumab (Fehrenbacher et al., 2016); and combinations of any one or more of such antibodies, known as an "IO doublet”.
• tremelimumab, nivolumab, durvalumab and atezolizumab are currently preferred.
• patents for tremelimumab, nivolumab, durvalumab and atezolizumab are U.S. Patent No. 6,682,736, U.S. Patent No. 8,008,449, U.S. Patent No. 8,779,108 and U.S. Patent No. 8,217, 149, respectively.
• other suitable examples of anti-CTLA-4 antibodies are those described in U.S. Patent No. 6,207,156, which particularly concerns anti-CTLA-4 antibodies that comprise a CDR (CDR3, CDR2 or CDRl) selected from a defined antibody from a deposited hybridoma.
• CDR CDR3, CDR2 or CDRl
• anti-PD-Ll antibodies are those described in U.S. Patent No.
• antibodies to PD-L1 are those in U.S. Patent No. 7,943,743, No. 9,580,505 and No. 9,580,507, kits thereof (U.S. Patent No. 9,580,507) and nucleic acids encoding the antibodies (U.S. Patent No. 8,383,796).
• Such antibodies bind to PD-L1 and compete for binding with a reference antibody; are defined by VH and V genes; or are defined by heavy and light chain CDR3 (U.S. Patent No. 7,943,743), or heavy chain CDR3 (U.S. Patent No. 8,383,796), of defined sequences or conservative modifications thereof; or have 90% or 95% sequence identity to reference antibodies.
• anti-PD-Ll antibodies also include those with defined quantitative (including binding affinity) and qualitative properties, immunoconjugates and bispecific antibodies. Further included are methods of using such antibodies, and those with defined quantitative (including binding affinity) and qualitative properties, including antibodies in single chain format and those that are in the format of an isolated CDR, in enhancing an immune response (U.S. Patent No. 9,102,725). Enhancing an immune response, as in U.S. Patent No. 9,102,725, may be used to treat cancer or an infectious disease, such as a pathogenic infection by a virus, bacterium, fungus or parasite. These anti- PD-Ll antibody compositions and methods include the product, BMS936559.
• Suitable antibodies to PD-Ll are those in U.S. Patent Application No. 2016/0009805, which concerns antibodies to particular epitopes on PD-Ll, including antibodies of defined CDR sequences and competing antibodies; nucleic acids, vectors, host cells, immunoconjugates; detection, diagnostic, prognostic and biomarker methods; and treatment methods.
• the present example describes the immunization protocol, the generation and initial characterization of the murine PS-targeting antibody termed 3G4.
• PS anionic phospholipids
• the membrane-exposed PS surrounded by other membrane components, has a better conformation for raising antibodies.
• the intent was to immunize immunocompetent animals with autologous cells expressing PS, wherein the animals would not produce antibodies against all self, surface antigens, but would recognize the membrane-exposed PS as a foreign element.
• bEnd.3 mouse endothelioma cells, bEnd.3 (immortalized mouse (BALB/c strain) endothelial cells) were cultured in 10% DMEM with 9ml/500ml HEPES Buffer, in 10% C0 2 incubator. The bEnd.3 cells were expanded in T175 TC flasks until the desired number of cells was obtained. Typically, each flask at -70-80% confluency has about 3 x 10 6 cells, and each mouse should receive from 1 x 10 6 to 20 x 10 6 cells, up to 1 x 10 7 cells. bEnd.3 cells were treated with 50 ⁇ to 200 ⁇ of hydrogen peroxide for 1 or 2 hours at 37°C to expose anionic phospholipids, particularly PS, before immunization. The stock of
• the strategy to isolate monoclonal antibodies reactive with PS involved screening hybridoma supernatants on PS-coated plates using an anti-mouse IgG, Fc gamma specific secondary antibody. Screening was first conducted against four phospholipids (PS, phosphatidylserine; PE, phosphatidylethanolamine; CL, cardiolipin; and PC, phosphatidylcholine), as well as bEnd3 cells. Clones reactive with the neutral phospholipid, PC were discarded, as were clones non-reactive with bEnd3 cells. Clones with high binding to PS were selected. The wells that had PS only reactivity, or strong preference for PS were sub- cloned first, and wells that exhibited PS reactivity in combination with binding to other anionic phospholipids were sub-cloned second.
• PS phosphatidylserine
• PE phosphatidylethanolamine
• CL cardiolipin
• PC phosphatidylcholine
• the isotype of each selected hybridoma was determined.
• antibodies of IgG class have numerous advantages over IgM, including typically higher affinity, lower clearance rate in vivo and simplicity of purification, modification and handling, their generation was particularly desired.
• To focus on wells with homogeneous IgG isotype wells containing IgM or a mixture of different Igs were discarded or re-cloned. Sub-cloning of highly positive clones was repeated three to four times.
• the 3G4 antibody was tested for binding to PS in an ELISA in the presence and absence of serum, and was initially characterized as being "serum- independent", i.e., an antibody that binds to PS in the absence of serum.
• the 3G4 antibody was studied using the following "Standard ELISA", which was used to test binding to PS or other phospholipids.
• the phospholipid antigen PS antigen, P-6641 25mg lOmg/ml (solvent is Chloroform:MeOH 95:5) in 2.5ml bottle
• stock solution should be aliquoted and stored in an airtight container at -30°C.
• the preferred 96 well plates are Dynatech U bottom Immulon 1 (from Dynatech Labs, Cat# 01 1-010-3550).
• the standard blocking buffer used was 10% bovine serum dissolved in PBS.
• the primary antibody was the test sample.
• the secondary antibody was goat, anti-mouse IgG- HRP.
• the developing solutions were: 10 ml of 0.2M Na 2 P0 4 , 10 ml of 0.1M citric acid, one 10 mg tablet of OPD, and 10 ⁇ of hydrogen peroxide.
• the stop solution was 0.18 M H 2 S0 4 .
• the protocol entailed coating a 96-well plate with PS as follows: the PS stock solution was diluted in n-hexane to 10 ⁇ g/ml and mixed well. 50 ⁇ was added to each well and allowed to evaporate for one hour. 200 ⁇ of 10% serum (as a blocking buffer) was added to each well, covered and maintained at room temperature for 2 hours or overnight at 4°C. The plate was washed three times with PBS. The primary antibody (diluted in blocking buffer) was added and incubated for 2 hours at 37°C. The plate was washed three times with PBS. 100 ⁇ /well of secondary antibody (typically goat, anti-mouse IgG-HRP) was added and incubated for 1 hour at 37°C.
• secondary antibody typically goat, anti-mouse IgG-HRP
• the plate was washed three times with PBS.
• the ELISA was developed by adding 100 ⁇ of developing solution to each of the wells, developed for 10 minutes; then 100 ⁇ of stop solution was added to each plate and the O.D. read at 490 ran. It was determined that the 3G4 antibody had a relative affinity for PS that was improved compared to prior antibodies, and that the 3G4 antibody bound to PS, CL, PI (phosphatidylinositol), PA (phosphatidic acid) and PG (phosphatidylglycerol). In keeping with the model for targeting PS differentially expressed in tumors, the 3G4 antibody did not react with the neutral phospholipids, PC and SM.
• the 3G4 antibody was purified to apparent homogeneity from the supernatant of the cultured hybridoma using a standard Protein A procedure. Briefly, a sample containing the 3G4 antibody at physiological pH was applied to a Protein A column and allowed to slowly pass through, so that the IgG binds to immobilized Protein A. The column was washed with wash buffer to remove non-bound serum components. The 3G4 antibody was eluted from the column using an acidic elution buffer (about pH 2.8), and fractions containing the eluted antibody then neutralized or dialyzed to return to physiological pH. In testing this highly purified 3G4 antibody for binding to PS in an ELISA conducted in the absence of serum, it was still believed that 3G4 bound to PS directly, i.e. , without serum or serum proteins.
• mice were obtained from Charles Rivers Laboratories. The mice were 4-5 weeks, female, C.B-17 SCID or Fox Chase SCID mice. Mice were housed in autoclaved caging, sterile food and water, with sterile handling. All procedures were performed in laminar flow hoods. Mice were acclimated 1 week and then ear-tagged and a blood sample (approximately 75-100 ⁇ ) taken from the tail vein to check for leakiness by ELISA. Any mice that failed the leakiness ELISA test were not used for test procedures. Mice were injected orthotopically with tumor cells into mammary fat pad (MFP) or subcutaneously into the right flank 2-3 days post ear-tagging and blood sample removal.
• MFP mammary fat pad
• 1 x 10 7 cells in 0.1 ml DMEM were typically injected into MFP of anesthetized mice.
• Mice were anesthetized with 0.075 ml of mouse cocktail injected IP.
• the mouse cocktail is 5 ml Ketamine ( 100 mg/ml); 2.5 ml Xylazine (20 mg/ml); 1 ml Acepromazine (10 mg/ml); 1 1 ml sterile water. Dosage was 0.1 ml per 20-30 grams body weight via the IP route for a duration of 30 minutes.
• the mouse was laid on its left side and wiped with 70% ethanol just behind the head and around the right forearm/back area.
• a 2-3 mm incision was made just behind the right forearm (lateral thorax), which reveals a whitish fat pad when the skin flap is raised.
• 0.1 ml of cells were injected into the fat pad using a 1 ml syringe and a 27-gauge needle, producing a bleb in the fat pad.
• the incision was closed using a 9 mm sterile wound clip.
• the mouse was returned to its cage and observed until it had wakened from anesthesia and was mobile.
• mice were typically injected with 1 x 10 7 cells in 0.2 ml. Mice were not anesthetized, but were restrained using a steady grip of mouse skin exposing the right flank. A 1 ml syringe with a 23 gauge needle was used to inject 1 x 10 cells in 200 ⁇ , just under the skin of the mice and a bleb was seen. It was not unusual to observe a small amount of fluid leak from the injection site. A twisting motion was used when withdrawing the needle from the subcutaneous injection to reduce this leakage. Tumor volume was measured by LxWxH.
• mice were injected IV with 1000 U of heparin in 0.2 ml saline. Mice were then sedated by injecting the mouse IP with 0.1 ml mouse cocktail. Once the mouse was sedated enough, as measured by no reflex when toe/foot is pinched, the thoracic cavity is opened to expose the heart and lungs. A 30 gauge needle attached to tubing and perfusion pump was inserted into the left ventricle. The right ventricle was snipped so that blood can drip out. Saline was pumped through for 12 minutes at a speed of 1 ml per minute. At the end of the perfusion, the needle and tubing were removed. Tissues were removed for further studies, either immunohistochemistry or pathology.
• Meth A mouse fibrosarcoma tumor cells were used with
• mice BALB/c mice.
• human MDA-MB-231 breast tumor cells or MDA-MB-435 cells were seeded into the mammary fat pad of SCID mice.
• a large human Hodgkin's lymphoma L540 xenograft was established by injecting cells and allowing the tumor to grow to a size of over 500 mm 3 before treatment. Tumor-bearing mice (8-10 animals per group) were injected i.p.
• the 3G4 antibody thus caused tumor vascular injury, localized thrombosis, tumor necrosis and retarded tumor growth, with no evidence of toxicity.
• the present example provides the full sequences of the heavy and light chain variable regions of the 3G4 antibody, which together include the six complementarity determining regions (CDRs), and describes the generation of chimeric versions of the 3G4 antibody, including the mouse-human chimeric antibody (ch3G4), now called bavituximab.
• CDRs complementarity determining regions
• the original sequences of the 3G4 antibody variable regions were obtained by RACE from the hybridoma that produces the 3G4 antibody and the sequences verified.
• the nucleic acid and amino acid sequences of the variable region of the heavy chain (Vh) of the 3G4 antibody are shown in FIG. 18A in U.S. Patent No. 7,572,448.
• the heavy chain variable region sequence encompasses VH CDRl , VH CDR2 and VH CDR3, at locations predictable by Kabat (Kabat et al, 1991 ).
• the BstEII site in the nucleic acid sequence can be used as a convenient site to prepare a functional mouse variable region, e.g. , for use in grafting onto a human constant region.
• the 3G4-2BVH sequence has been grafted onto a human ⁇ constant region at the BstEII site using a Lonza pEE vector.
• the resultant product contains the mouse leader sequence and its VH is joined to the human CHI sequence in the manner shown in FIG. 18A in U.S. Patent No. 7,572,448.
• the nucleic acid and amino acid sequences of the variable region of the light chain have been grafted onto a human ⁇ constant region at the BstEII site using a Lonza pEE vector.
• the resultant product contains the mouse leader sequence and its VH is joined to the human CHI sequence in the manner shown in FIG. 18A in U.S. Patent No. 7,572,448.
• VK of the 3G4 antibody are shown in FIG. 18B in U.S. Patent No. 7,572,448.
• the light chain variable region sequence encompasses VL CDRl , VL CDR2 and VL CDR3, at locations predictable by Kabat (Kabat et al., 1991).
• the Bbsl site in the nucleic acid sequence can be used as a convenient site to prepare a functional mouse variable region, e.g., for use in grafting onto a human constant region.
• the 3G4-2BVL sequence has been grafted onto a human ⁇ constant region at the Bbsl site using a Lonza pEE vector.
• the resultant product contains the mouse leader sequence and its VL is joined within the human CL1 sequence in the manner shown in FIG. 18B in U.S. Patent No. 7,572,448.
• the human chimera of the murine 3G4 antibody is a human IgGj isotype (hlgGi ).
• the murine IgG homolog of ch3G4 corresponds to a mouse IgG 2a isotype (mIgG 2a )- This construct was made and tested, and shown to behave essentially the same as the original mouse IgG 3 antibody.
• the 3G4 light chain coding sequence was amplified by RT-PCR from total RNA isolated from the 3G4 hybridoma cell line.
• RT-PCR primers were designed such that the amplified fragment contained Xmal and EcoRI restriction enzyme sites on either end of the amplified product for cloning into the Lonza expression vector, pEE 12.4 vector.
• the variable region of the 3G4 heavy chain was amplified by RT-PCR from total RNA isolated from the 3G4 hybridoma cell line. Primers were designed such that the amplified fragment contained Hindlll and Xmal restriction enzyme sites on either end of the amplified product for cloning into the Lonza expression vector, pEE6.4 vector.
• the murine IgG2a constant region was amplified by PCR from a plasmid vector. PCR primers were designed with Bstll and EcoRI restriction enzyme sites at either end of the amplification product for cloning into the pEE6.4 + 3G4VH vector. The BstEII site was designed to be in- frame with the 3G4 VH variable region sequence upstream.
• the heavy and light chain constructs were combined into a single double gene vector (12.4 3G4 IgG2a) by cutting both vectors with Sail and Notl. The heavy and light chain coding regions were verified by sequencing.
• the 12.4 3G4 IgG2a vector was transfected into NSO cells by electroporation. Following transfection, the NSO cells were diluted and plated into 96-well plates in media lacking glutamine. Only cells transfected with the construct (which contains the glutamine synthease gene for positive selection) can grow in the absence of glutamine. Transfectants were identified and screened for antibody secretion using the Standard ELISA of Example I and those transfectants secreting the highest amounts of antibody were grown in large culture to generate purified antibody. The resultant 2aG4 antibodies were purified to apparent homogeneity and shown to have essentially the same affinity and binding profile as the 3G4 antibody.
• the chimeric construct containing the murine variable regions and the human constant regions has been produced (ch3G4) and shown to have essentially the same characteristics as the original murine antibody.
• the murine 3G4 antibody was converted into a human-mouse chimeric antibody.
• the murine VH was cloned and grafted onto the human ⁇ constant region at the BstEII site of the Lonza 2BVH vector.
• the murine V was cloned and grafted onto the human K constant region at the Bbsl site of the Lonza 2BVL vector.
• the sequences were verified.
• the entire construct was expressed in CHO (Chinese hamster ovary) cells and the antibody purified. This is the antibody now called bavituximab.
• the resultant ch3G4 bound at least as well as the murine 3G4 to phospholipid-coated ELISA plates using the Standard ELISA of Example I.
• the binding was antigen-specific, since no binding was observed with control antibodies of irrelevant specificity.
• ch3G4 was also shown to localize to tumor vascular endothelium and to exert anti-tumor effects and anti-viral effects in a wide range of studies.
• the chimeric 3G4 construct was expressed in CHO cells under serum- free conditions, and the purified antibody tested for binding to PS in an ELISA in the absence of serum, binding to PS was lost.
• This example provides data to resolve the apparent discrepancy in the PS binding profiles of the 3G4 antibody from the original hybridoma and the chimeric antibody expressed in CHO cells.
• the present example demonstrates that the interaction between the 3G4 antibody and PS is dependent on the plasma protein, 2-glycoprotein I ( 2GPI).
• Dulbecco's modified Eagle's medium (DMEM) and trypsin/EDTA were obtained from Mediatech, Inc. (Herndon, VA). Fetal bovine serum (FBS), normal human serum, normal rat serum and normal mouse serum were obtained from Biomeda (Foster City, CA). Fresh human plasma was obtained from Carter Blood Care (Dallas, TX). Serum-free Hybridoma Media, Synthechol NS0 supplement, L-a-phosphatidylserine (PS), bovine serum albumin (BSA) and ovalbumin from chicken egg white (OVA) and were obtained from Sigma Chemical Co. (St. Louis, MO).
• FBS Fetal bovine serum
• PS L-a-phosphatidylserine
• BSA bovine serum albumin
• OVA ovalbumin from chicken egg white
• Lipids (Alabaster, AL). Ninety six-well Immulon-I B and -2HB microtiter plates were obtained from Thermo Lab Systems (Franklin, MA). Tris-HCl gradient SDS-PAGE gels and an Opti-4CN Substrate kit were obtained from Biorad (Hercules, CA). Eight-well glass chamber slides were obtained from BD Biosciences (Bedford, MA).
• the 3G4 mouse monoclonal antibody which was generated to bind the anionic phospholipid PS, is the antibody described in Example I.
• 3G4 was produced originally in hybridoma supernatant (Example I; Example II).
• 3G4 was also converted to a mouse IgG2a isotype (Example III, B) and was produced in the NSO mouse myeloma cell line.
• NSO cells were cultured in DMEM supplemented with 10% FBS or serum-free Hybridoma Media with Synthechol NSO supplement.
• the human chimeric version of 3G4 (ch3G4; bavituximab) was generated (Example III, C) and the antibody produced from CHO cells under serum-free conditions.
• the mouse anti-human ⁇ 2 ⁇ (anti- 2GPI or ⁇ - ⁇ 2 ⁇ ) mAb was obtained from US Biological (Swampscott, MA).
• Rituximab human IgGl chimeric mAb
• All secondary antibodies were obtained from Jackson Immunoresearch Labs (West Grove, PA).
• 3G4 F(ab') 2 was generated by incubation with the protease pepsin.
• 3G4 Fab and control Fab 7H1 1 were generated by incubation with the protease papain. All antibody cleavage products were purified by FPLC, and verified by SDS-PAGE.
• Human ⁇ 2 ⁇ (hp2GPI) for use in the present example was purified from human plasma essentially as described previously (Polz et al, 1980; Wurm et al., 1984). Briefly, perchloric acid (70%) was added to pooled plasma to a final concentration of 1.57% (v/v). The precipitate was discarded and the supernatant was adjusted to pH 7.5 with saturated Na 2 C0 3 , followed by extensive dialysis against 50 mM Tris, pH 8.0. This material was applied to a DEAE cellulose column equilibrated with 50 mM Tris, pH 8.0 to remove contaminants.
• the DEAE column flow-through was then applied to a heparin-Sepharose affinity column equilibrated with 50 mM Tris, pH 8.0, and bound proteins were eluted using 1.0 M NaCl. Finally, the ⁇ 2 ⁇ preparation was dialyzed against PBS and purified further by protein A/G to remove contaminating IgG. The final preparation contained a homogeneous band at 50 kDa, as shown by non-reduced SDS/PAGE and Coomassie ® staining.
• the yeast shuttle expression vector pPIC6aA (Invitrogen) and host strain Mut + X-33 (Invitrogen) were used.
• the expression vector contains the 5' promoter and the 3' transcription termination sequences of the alcohol (methanol) oxidase gene (AOX1).
• the vector also has a yeast a mating factor signal sequence downstream of the AOX1 promoter to which foreign cDNA can be fused for secretion of recombinant heterologous protein into the culture medium.
• Expression in P. pastoris provides glycosylation and disulfide bond formation similar to that in mammalian cells.
• PCR amplified fragments were inserted in-frame between the EcoRl and Xbal restriction sites of pPICaA, directly downstream from the a mating factor signal sequence.
• a Stop codon was introduced at the end of each fragment to prevent fusion of the recombinant proteins to a c-myc epitope or a His tag at the C-terminus.
• Plasmid constructs were propagated in E. coli in presence of 100 ⁇ g/ml blasticidin and verified by restriction analysis and nucleotide sequencing. Recombinant proteins expressed by the above five constructs encoded proteins of approximately 36, 29, 24, 16 and 9 kDa, respectively, before glycosylation.
• the recombinant plasmid constructs were linearized with restriction enzyme Sacl, purified and 10 ⁇ g was used to transform host strain X-33 by the spheroplasts method (Invitrogen). Transformants for each of these constructs were selected on YPD (Yeast extract Peptone Dextrose Medium) plates containing 400 ⁇ g/ml blasticidin for 4 days. Several clones for each of these constructs were restreaked on YPD plates with 400 ⁇ g/ml blasticidin to determine the true integrants. Ten clones of each construct were then streaked on Minimal Dextrose (MD) and Minimal Methanol (MM) plates.
• MD Minimal Dextrose
• MM Minimal Methanol
• recombinant proteins were produced using culture conditions recommended by Invitrogen.
• a starter culture of each clone was cultured in 5 ml of buffered minimal glycerol-complex medium (BMGY) at 30°C with vigorous shaking overnight. Cells were collected, used to inoculate 25 ml of BMGY and grown for 2 days. Cells from the 25 ml culture were then used to inoculate 1 L of buffered minimal methanol-complex (BMMY) medium (1.0% methanol). Culture was continued for 4 days at 30°C with vigorous shaking and 100% methanol was added every 24 hours (final concentration of 1.0%) to maintain protein expression.
• BMGY buffered minimal glycerol-complex medium
• Culture medium was clarified by centrifugation (4000 x g, 15 min) and supernatant was dialyzed for 2 days at 4°C in 50 mM Tris buffer before being applied to a DEAE-sephacel column equilibrated with 50 mM Tris buffer. Flow through solution was collected and applied to a heparin-sepharose column.
• P2GPI was eluted from heparin-sepharose column with 1 M NaCl, dialyzed against 50 mM Tris buffer, concentrated using Amicon concentrator and analyzed by Western blot. The N-terminus of each protein was sequenced to confirm cleavage of the a-factor leader sequence. Protein yields varied from l O mg/L (full-length P2GPI) to 25 mg/L (P2GPI domain V).
• hp2GPI was prepared from intact hp2GPI purified from human plasma as described above in the present example. hp2GPI was incubated with plasmin-coated beads at 37°C for 17 hrs. The beads were removed by centrifugation and the supernatant containing the cleaved protein was recovered. Western blotting of the purified product indicated the nicked 2GPI preparations were plasmin-free and did not contain plasmin autoproteolytic products (no reactivity with anti-plasmin or anti-angiostatin antibodies). N-terminal sequence analysis revealed two N-termini that corresponded to the N-terminus of ⁇ 2 ⁇ and a new sequence generated at the Lys317/Thr318 cleavage site.
• Example I The Standard ELISA of Example I was adapted with the following modifications. PS-coated Immunlon IB microtiter plates were blocked overnight in 1 % OVA (w/v). The following day, serial 2-fold dilutions of 3G4 purified from serum-containing or serum-free supernatant were prepared from an initial concentration of 13.33 nM. Dilutions were performed in 1% OVA or 10% non-heat inactivated sera from cow, human, rat or mouse. Plates were incubated for 1 hr. at 37°C and binding of 3G4 was detected. All ELISA studies were performed at least three times. 8. Anti-hp2GPI ELISA
• the assay was performed as described above with the following modifications. h 2GPI, nicked h 2GPI, or recombinant h 2GPI peptides were coated on 96-well Immunlon 2HB microtiter plates overnight at a concentration of 10 ⁇ g/ml. Plates were then blocked in 1% OVA for 1 hr. at room temperature. 3G4, ch3G4, or anti- 2GPI were diluted in 1% OVA to an initial concentration of 13.33 nM and serial 2-fold dilutions were prepared. Plates were incubated for 1 hr. at 37°C and antibody binding was detected. All ELISA studies were performed at least three times.
• Protein samples were heated to 95°C for 5 min in non-reducing SDS sample buffer.
• ABAE aortic endothelial
• DMEM fetal bovine aortic endothelial
• ABAE cells were removed from subconfluent cultures by brief exposure to 0.25% trypsin/0.02% EDTA and 8-well chamber slides were seeded with 2xl0 4 cells/well. Following overnight culture, cells were washed gently with PBS and treated with 200 ⁇ lysophosphatidylcholine (LPC) to induce PS exposure.
• LPC-treatment was performed in the presence of 3G4, ch3G4, or control IgG for 30 min at 37°C in either 10% FBS or 10% normal mouse serum (MS). If LPC-treatment was performed in 10% MS, 1 ⁇ 2 ⁇ was added as a co-factor because 3G4/ch3G4 cannot bind PS in MS (see Results below in the present example).
• PS exposure was determined by immunofluorescence staining.
• Cells were washed thoroughly in PBS, fixed in 4% paraformaldehyde (w/v) and incubated with a biotin- conjugated anti-mouse secondary antibody. Next, cells were incubated with FITC-conjugated streptavidin (Jackson Immunoresearch) to detect antibody binding. Cells were then permeabilized with 0.1% Triton-XlOO in PBS and count erstained with Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR) and 4', 6-diamidino-2-phenylindole (DAPI; Molecular Probes). Images were captured using a Coolsnap digital camera (Photometries, Arlington, AZ) mounted on a Nikon microscope and processed with MetaVue software (Universal Imaging Corporation, Downingtown, PA).
• the area of antibody binding was determined using MetaVue image analysis software, which is able to quantify the number of illuminated pixels in an image. Images of FITC fluorescence were used to quantify antibody binding. Corresponding images of DAPI fluorescence were used to normalize the FITC images for the number of cells present in the field. A small FITC/DAPI ratio indicates a small antibody binding area, whereas a large FITC/DAPI ratio indicates a large binding area. The FITC/DAPI ratios were used to determine increases or decreases in antibody binding area relative to a basal amount of antibody binding under the selected conditions. Five images at 200x magnification were used for each analysis. Data are analyzed as average relative FITC/DAPI ratios with standard deviation. B. Results
• 3G4 and bavituximab antibodies and PS is dependent on the plasma protein, P2GPI.
• 3G4 is shown to bind to P2GPI at domain II, which is not linked to pathogenic antibodies isolated from patients with Anti-Phospholipid Syndrome (APS), which commonly recognize P2GPI domain I.
• APS Anti-Phospholipid Syndrome
• the data show that divalent 3G4/ 2GPI complexes are required for enhanced PS binding, including to PS-positive cells, since 3G4 Fab' fragments do not have this activity.
• the 3G4 antibody purified from serum-containing media (SCM) or serum-free media
• SFM serum-coated microtiter plates
• FBS FBS
• OVA which lacks bovine serum proteins
• 3G4 purified from SFM no longer binds PS (FIG. 2A, dashed line, ⁇ ). This finding indicates that a factor present in bovine serum mediates the interaction between 3 G4 and PS.
• aPL anti-phospholipid
• human p2GPI human p2GPI (hp2GPI) was coated on a microtiter plate and incubated with mouse anti -human P2GPI antibody (anti-p2GPI), the 3G4 antibody, or a control mouse IgG2a of irrelevant specificity (control mlgG).
• anti-p2GPI mouse anti -human P2GPI antibody
• 3G4 antibody mouse IgG2a of irrelevant specificity
• anti-p2GPI bound to 1 ⁇ 2 ⁇
• control mlgG did not (FIG. 3).
• the 3G4 antibody also bound strongly to the hp2GPI coated plate (FIG. 3).
• purified hp2GPI and 10% human serum were run on an SDS-PAGE gel and transferred to a membrane support for immunoblot.
• 3G4 detected the 50 kDa purified h 2GPI and a single band of similar size in human serum. Importantly, the 3G4 immunoblot was virtually identical to a blot generated using the anti-P2GPI antibody. The control mlgG antibody did not detect any protein.
• P2GPI has five domains, of which the fifth domain is responsible for binding to anionic phospholipids such as PS.
• anionic phospholipids such as PS.
• recombinant human ⁇ 2 ⁇ constructs were generated with different domain structures and tested alongside recombinant full-length hp2GPI.
• domain constructs were made by serial truncations from the N-terminus, and so lack each of the N-terminal domains in turn, as follows: recombinant full-length 1 ⁇ 2 ⁇ contains domains I-V; 1 ⁇ 20 ⁇ from which domain I has been deleted contains domains II-V; 1 ⁇ 2 ⁇ from which domains I and II have been deleted contains domains III-V; h 2GPI from which domains I, II and III have been deleted contains domains IV-V; and hp2GPI from which domains I, II, III and IV have been deleted contains domain V only.
• Pathogenic anti ⁇ 2GPI antibodies isolated from patients with APS commonly recognize domain I of P2GPI (de Laat et al, 2005). Anti-P2GPI antibodies from APS patients that recognize domain II are not often pathogenic. This likely explains the lack of toxicity associated with 3G4 following toxicological studies performed in a variety of animal models, and in extensive clinical experience, such as described herein.
• a live-cell binding assay was developed. This assay detects and measures antibody binding to cell membrane surfaces following treatment with the membrane disrupting agent, lysophosphatidylcholine (LPC) to induce PS exposure.
• LPC lysophosphatidylcholine
• ABAE cells were incubated with the 3G4 antibody or control mlgG in DMEM + 10% FBS in the presence or absence of 200 ⁇ LPC for 30 min. Cells were then washed, fixed and stained with fluorescent markers to visualize binding of antibody to the cell surface. The pixel area of 3G4 or mlgG binding was quantified using MetaVue software. All values were relative to the binding of 3G4 to non-LPC treated cells, which was set to one.
• the live-cell binding assay was performed in media containing 10% mouse serum rather than 10% FBS to prevent interference from bovine ⁇ 2 ⁇ .
• the 3G4 antibody does not bind PS in the presence of mouse serum.
• the 3G4 antibody did not detect any protein in 10% mouse serum by immunoblot, indicating that 3G4 does not recognize murine ⁇ 2 ⁇ .
• ch3G4 human chimeric 3G4 antibody
• ABAE cells were incubated with ch3G4 in the presence of 10% mouse serum and LPC, no antibody binding was detected (FIG. 5).
• addition of purified hp2GPI to the binding reaction supported widespread binding of ch3G4 (FIG. 5), demonstrating that the ch3G4 antibody binding is dependent upon ⁇ 2 ⁇ .
• ch3G4 binding was dependent upon LPC treatment and no binding was detected using a control human IgG of irrelevant specificity.
• the live-cell binding assay was performed using "nicked" hp2GPI.
• Nicked 1 ⁇ 2 ⁇ is unable to bind PS due to plasmin- mediated cleavage within the lipid binding region of domain V (Hunt et al. , 1993 ; Hunt & Krilis, 1994).
• 3G4 antibody detects PS by enhancing the avidity of ⁇ 2 ⁇ for anionic phospholipids.
• 3G4 F(ab') 2 and 3G4 Fab' monomers were generated and used in live-cell binding assays with the intact 3G4 antibody.
• the intact 3G4 antibody bound to LPC-treated ABAE cells, but not to untreated cells.
• An equivalent concentration of 3G4 F(ab') 2 also bound to LPC-treated ABAE cells (FIG. 7A), but binding of 3G4 Fab' was negligible (FIG. 7A).
• 3G4 Fab' The ability of 3G4 Fab' to inhibit ch3G4 binding confirms that 3G4 Fab' is able to bind 2GPI and that monomelic 3G4 Fab'/p2GPI complexes cannot bind cells with exposed PS. These data show that divalent complexes are required to bind to PS exposed on cell surfaces.
• the 3G4 antibody binds to ⁇ 2 ⁇ at domain II, and as shown in FIG. 6A and FIG. 6B, the lipid binding region of ⁇ 2 ⁇ domain V is required for co-binding of 3G4 (and ch3G4) and ⁇ 2 ⁇ to PS exposed on cells.
• antibody divalency is required for such co-binding of 3G4 (and ch3G4) and ⁇ 2 ⁇ to exposed PS.
• the present example provides pre-clinical data concerning interactions between the bavituximab family of antibodies, ⁇ 2 ⁇ and PS. Overall, the data show that relatively low levels of ⁇ 2 ⁇ , markedly below the typical amounts in the human population, are sufficient for effective binding of bavituximab to PS.
• the antibody was purified to apparent homogeneity from the supernatant of the cultured hybridoma using a standard Protein A procedure (Example I). In early studies in mice, this purified antibody was shown to exert anti-tumor effects in several models (Example II).
• Example IV After it was determined that the 3G4 antibody requires ⁇ 2 ⁇ for PS binding (Example IV), and that all species of ⁇ 2 ⁇ support PS binding except mouse (FIG. 2B), it was deduced that the anti-tumor effects of Example II are under-estimates of the potency of the 3G4 antibody. That is, the anti-tumor effects of the 3G4 antibody in FIG. 1A, FIG. IB, FIG. 1C and FIG. ID are supported by only the low level of bovine ⁇ 2 ⁇ that could have co-purified with the 3G4 antibody through the Protein A column.
• the 3G4 antibody was purified to apparent homogeneity using a Protein A column, which purifies antibodies based on the affinity for IgG.
• affinity of P2GPI for the 3G4 antibody is low (and less than the affinity between Protein A and 3G4), such that P2GPI would have separated from 3G4 during loading and washing.
• the low pH elution step to separate the 3G4 antibody from Protein A
• the smaller P2GPI protein would not have been collected. Nonetheless, as shown in FIG. 9A and FIG. 9B, even taking the hypothetical position that 10-20% of the apparently pure 3G4 antibody delivered to the mice was actually in form of 3G4 ⁇ 2GPI complexes, such levels of bovine ⁇ 2 ⁇ are still very low in absolute terms and in comparison to the antibody.
• the 3G4 antibody was produced from hybridoma cells in 10% FBS.
• a one liter volume would typically yield 10 mg/L of 3G4 antibody.
• FBS contains 200 ⁇ g/ml of bovine P2GPI (similar to the level of human P2GPI in human sera)
• 10% FBS would contain 20 ⁇ g/ml of bovine ⁇ 2 ⁇ .
• the resulting material Upon purification with Protein A, the resulting material would contain 10 mg of protein.
• the remaining 10-20% is a 3G4 ⁇ 2GPI "complex", in which one antibody is attached to two p2GPI (3G4-2x 2GPI).
• mice were administered 100 ⁇ g of such protein. At 90% purity, 90 ⁇ g of the 100 ⁇ g of administered protein is pure 3G4, with 10 ⁇ g of 3G4-2xP2GPI complex. Taking the molecular weight (MW) of the antibody to be 145 kD, and the MW of p2GPI to be 50 kD (Example IV, A4,B3; McNeil et al, 1990; Luster et ah, 2006), and with two ⁇ 2 ⁇ for each antibody in the complex, the ratio by weight is approximately 3 :2 (precisely, 59.2% antibody and 40.8% antibody).
• 10 ⁇ g of complex approximately 6 ⁇ g is 3G4, and 4 ⁇ g is p2GPI (precisely, 5.92 g 3G4, and 4.08 ⁇ g is P2GPI).
• the volume of blood of a mouse is 2 ml.
• the pure 3G4 antibody is present at 90 ⁇ g per mouse, or 45 ⁇ .
• 10 ⁇ g of complex per mouse contains about 6 ⁇ g of 3G4 and about 4 ⁇ g of p2GPI (3 ⁇ of 3 G4 and 2 ⁇ of P2GPI).
• the 2 ⁇ of ⁇ 2 ⁇ in the mouse corresponds to 0.04 ⁇ .
• 3G4 adding the 3 ⁇ of 3G4 from the administered 3G4-2xp2GPI complex to the 45 ⁇ g/ml pure 3G4, there is 48 ⁇ ⁇ of the 3G4 antibody. This corresponds to antibody at 0.33 ⁇ .
• this is a molar ratio of ⁇ 2 ⁇ to antibody of 0.12 (FIG. 9A).
• ABAE cells were treated with LPC to induce PS exposure (Example IV) and then incubated with 40 nM purified human P2GPI and varying concentrations of the ch3G4 antibody (bavituximab). It was determined that relative ch3G4 binding increased in a concentration-dependent manner from 320 pM to an apparent peak of 80 nM ch3G4, which is an antibody to 2GPI ratio of 2: 1 (FIG. 10). As shown in Table 1 (antibody MW, 145 kD; human P2GPI MW, 50 kD), the apparent peak binding in this study corresponds to a molar ratio of p2GPI to antibody of 0.5.
• Solutions containing a fixed amount of the 2aG4 antibody and increasing amounts of human P2GPI were prepared. Briefly 62.5 ng/ml (0.4 nM) of 2aG4 was added to 0.0032, 0.016, 0.08, 0.4, 2, 10 or 50 nM of human P2GPI in ovalbumin. The different 2aG4-p2GPI mixtures were added to PS micro titer plates and the molecules were allowed to bind for 1-2 h at 37°C. Unbound molecules were removed by washing with PBS. The secondary antibody was HRP-conjugated anti-human IgG in binding buffer. Plates were left for 1 hour at 37°C before unbound antibody was removed by washing five times with PBS.
• TMB substrate was added to each well at a volume of 100 ⁇ and left for 15 minutes at room temperature to allow the colorimetric reaction to occur. The reaction was stopped by addition of 100 ⁇ of 2M H 2 S0 4 . Absorbances (optical density, OD) were read by a plate spectrometer at a wavelength of 450nm within 30 minutes of adding the stop solution and analyzed using SoftMax Pro software (including subtracting the average OD of the control without P2GPI from the average OD of the test samples). This study showed that 2aG4 antibody binding to PS started to plateau at a molar ratio of P2GPI to antibody of about 1 , where both molecules were present at approximately 0.4 nM (FIG. 1 1 ). More precisely, as shown in Table 2 (antibody MW, 145 kD; human P2GPI MW, 2017/053370
• a molar ratio of p2GPI to antibody of 0.93 is effective in supporting antibody binding to plates coated with PS.
• HT 1080 cells were treated with 50 ⁇ etoposide for 18 h. Cells were then incubated with the different bavituximab-percentage FBS solutions, followed by secondary antibody against bavituximab to allow visualization of antibody bound to cells via flow cytometry. Negative controls included cells not treated with etoposide, which will not expose PS on the surface, and PBS solutions lacking any FBS.
• NFAT Nuclear factor of activated T-cells
• the NFAT signal transduction pathway and NFAT response elements have been used in the development of assays, and commercially available kits, to monitor NFAT signal transduction pathways in cultured cells.
• An NFAT bioassay has been developed for use with the bavituximab family of antibodies (Larson et al., 2013).
• Jurkat cells engineered to express the FcYRIIIa-V158 receptor on the cell surface (Promega) were also transfected with a genetic element containing the luciferase gene under the control of a minimal TATA promoter containing multiple NFAT-RE.
• NFAT effector cells which are co-cultured with PS-positive target cells.
• PS- targeting antibodies such as bavituximab bind to PS on the surface of target cells.
• the PS- targeting antibody's Fc region then binds to the FcYRIIIa-V158 receptor on the NFAT effector cells and signaling through the NFAT pathway is triggered.
• NFAT binds to the NFAT-RE and activates luciferase expression, which can be quantified.
• This NFAT assay is therefore a surrogate ADCC bioassay for bavituximab and other PS-targeting antibodies.
• FIG. 12 Exemplary results from the ELISA assay described above are presented in FIG. 12, which again show that dilute human sera, with low levels of P2GPI, effectively support antibody binding.
• PS ELISA using purified P2GPI FIG. 11
• there was some loss of PS binding at 50% and 100% human sera which is related to typical saturating effects commonly detected in ELISA assay formats, particularly when using undiluted sera.
• Such effects are not observed in FACS assays, and bavituximab binding to cells with exposed PS was shown to be essentially the same in 50%, 75% and 100% fetal bovine serum.
• bavituximab binding to PS in the ELISA started to plateau at about 1 % human sera.
• normal human sera contains, on average, 200 ⁇ g/ml P2GPI (Steinkasserer et al, 1991 ; Mehdi et al, 1999; Miyakis et al, 2004), 1 % human sera contains about 2 ⁇ or 0.04 ⁇ of p2GPI.
• FIG. 12 shows that bavituximab binding to PS was already at saturation at this concentration of P2GPI.
• the 1 % human sera in the ELISA corresponds to a molar ratio of P2GPI to antibody of 2.86. This generally accords with the rationale that each molecule of bavituximab needs to bind to two molecules of P2GPI to form a stable complex with PS on the cell surface (FIG. 8).
• Table 3 shows that each molecule of bavituximab needs to bind to two molecules of P2GPI to form a stable complex with PS on the cell surface (FIG. 8).
• the present example shows that molar ratios of ⁇ 2 ⁇ to antibody from as low as 0.12 to 2.86 support antibody binding to PS and PS-positive cells, facilitate activity in functional assays and permit effective treatment of mice with tumors.
• a molar ratio of ⁇ 2 ⁇ to antibody should be about 2.86 (Table 3), but that it does not need to be higher than about 3.
• a Phase I, multicenter, open-label, dose escalation study was conducted to evaluate the safety, tolerability and pharmacokinetics (PK) of bavituximab when administered intravenously (bavituximab monotherapy) to 26 patients with refractory advanced solid tumors.
• Patients were enrolled into four sequential dose-escalation cohorts (0.1, 0.3, 1 or 3 mg/kg bavituximab weekly) with two dosing schedules. In the 0.1 mg/kg and 0.3 mg/kg cohorts, patients received bavituximab on days 0, 28, 35 and 42; and in the 1 mg/kg and 3 mg/kg cohorts, patients were administered bavituximab on days 0, 7, 14 and 21.
• the upper dose of 3 mg/kg weekly was selected based on preclinical modeling
• Example V and experience in other patient populations.
• maximal efficacy was achieved at antibody doses of 0.5 mg/kg 3 times weekly, yielding a C max of 5.5 ⁇ g/ml with a half-life of 48 hours and a simulated average blood concentration of 2 ⁇ g/ml over the course of treatment.
• PS binding by bavituximab was presumably saturated, based on observations of the concentration at which binding of bavituximab to PS-positive cells becomes saturated in vitro (Example V).
• Samples were collected from patients in the 0.1 and 0.3 mg/kg dose cohorts before the study, on days 0, 1, 2, 4, 7, 10, 14, and every 7 days from days 21 to 70. Samples were collected from patients in the 1 and 3 mg/kg dose cohorts before the study, on days 0, 1, 2, 4, 7, 14, 21 , 22, 23, 25, and every 7 days from days 28 to 56. Bavituximab blood levels were determined by a validated ELISA.
• Table 4 presents a summary of the mean (coefficient of variation, CV) PK parameters of bavituximab following single-dose administration (day 0) and multiple-dose administration (day 21) in this Phase I trial, including maximum concentration (C ma x), clearance (CL), half- life (/i /2 ) and area under plasma concentration-time curve from time zero to infinity (AUCj nf )- Table 4
• the mean half-life of bavituximab ranged from 37.2 to 43.9 hours.
• the mean maximum serum concentration (C max ) ranged from 2.1 1 to 56.4 ⁇ g/ml (depending on dose) at the median time after administration when the maximum serum concentration was reached (T max ) (values ranging from 2.04 to 3.73 hours).
• T max median time after administration when the maximum serum concentration was reached
• the maximum serum concentration was 56.4 ⁇ g/ml.
• the bavituximab half-life ranged from 37 to 47 hours. No maximum tolerated dose was reached in this study.
• Bavituximab exhibited linear single-dose (day 0) and multiple-dose (days 21 or 42) PK characteristics (FIG. 13). Bavituximab did not exhibit appreciable accumulation or time- dependent PK differences following multiple-dose administration. In summary, this study showed that bavituximab was well tolerated at doses ranging up to 3 mg/kg weekly and the pharmacokinetics support a weekly dosing regimen.
• bavituximab exhibits linear single-dose and multiple-dose PK characteristics at doses ranging from 0.1 to 6 mg/kg, with no evidence of appreciable accumulation of bavituximab or time-dependent PK differences.
• the median T max was shown to occur within the first 2 to 3 hours following the end of the infusion. Serum bavituximab concentrations decline in an apparent mono-exponential or bi-exponential first-order manner. The more rapid distribution phase, where observed, is essentially complete within 6 hours and the terminal elimination half-life is approximately 1 to 2 days (21.9 to 46.8 hours).
• Bavituximab PK characteristics are generally similar in patients with cancer and chronic viral infections, as tested in patients chronically infected with HCV, with and without HIV.
• a Phase I, open-label, single center, dose escalation study evaluated a single intravenous infusion of bavituximab in patients chronically infected with HCV (Example VII, A). As shown in Table 5, it was found that the observed concentrations of bavituximab were very consistent with the predictions from the PK modeling data.
• bavituximab In the Phase lb study in patients co-infected with chronic HCV and HIV (Example VII, C), bavituximab exhibited linear single-dose PK characteristics on day 0 and linear multiple-dose PK characteristics on day 49 following once weekly administration at doses ranging from 0.3 to 6 mg/kg. Bavituximab did not exhibit time-dependent PK differences or accumulation following multiple-dose administration once weekly for 8 weeks. 2. PK in Combination Therapies
• bavituximab and other drugs were given in combination, there did not appear to be any clinically relevant pharmacokinetic interactions for either of the drugs. This includes when bavituximab and docetaxel were given in combination.
• a Phase lb, multi-center, open-label, non-randomized study first evaluated the safety, tolerability and PK of weekly intravenous administration of 3 mg/kg bavituximab when used in combination with gemcitabine, paclitaxel plus carboplatin or docetaxel in patients with refractory advanced solid tumors. It was determined that there were no significant differences in any measurable parameter among the three treatment groups following a single-dose (day O) or multiple-dose bavituximab administration (day 21). Evaluation of C max and AUC indicated that no accumulation of bavituximab following multiple-dose administration once weekly for eight weeks.
• data are presented to exemplify some of the clinical experience in treating viral infections in patients using bavituximab, including bavituximab in combination with ribavirin.
• Data are also presented to show that, at the selected clinical dose, administration of bavituximab does not appreciably reduce P2GPI levels in human subjects.
• Bavituximab was first evaluated in Phase I, open-label, dose escalation studies and Phase lb, open-label, escalating repeat-dose studies in patients chronically infected with hepatitis C virus (HCV). These studies concerned the safety, tolerability, PK profile, viral kinetics, maximum tolerated dose (MTD) and maximum effective dose (MED) of bavituximab. Doses of 0.1 , 0.3, 1 , 3 and 6 mg/kg were administered in Phase I (30 patients; successive cohorts of 6 patients), and doses of 0.3, 1 , 3 and 6 mg/kg were administered in Phase lb (24 patients; four cohorts of 6 patients).
• bavituximab 3 mg/kg dose of bavituximab for use in humans.
• This dose was determined to be the maximal dose at which bavituximab and ⁇ 2 ⁇ were present together at concentrations effective to allow the bavituximab ⁇ 2GPI complex to form and bind to PS exposed on cells in the disease site without depleting plasma p2GPI levels.
• the data also show that any reductions in P2GPI during bavituximab treatment are only temporary and that P2GPI levels are restored within 3 days.
• a separate Phase lb, multi-center, open-label, non-randomized, dose-escalating, repeat- dose study was conducted to evaluate bavituximab in patients co-infected with chronic HCV (majority of HCV genotype 1) and human immunodeficiency virus (HIV).
• the primary objectives were to determine the safety, tolerability, PK profile, viral kinetics, MTD and/or MED.
• the study involved 16 scheduled visits over approximately 16 weeks.
• Bavituximab was administered to successive cohorts of patients at the following doses: 0.3 mg/kg, six patients; 1 mg/kg, six patients; 3 mg/kg, nine patients; and 6 mg/kg, six patients.
• SAEs serious adverse events
• HCV The median baseline HCV viral load was 6.76 logio and the median baseline for HIV was 3.99 logio- Plasma viral loads of HCV and HIV were measured at specific time points during the study. When treated with bavituximab at all dose levels, several patients in each treatment group exhibited transient antiviral activity (maximum reduction in HCV and/or HIV viral load of > 0.5 logio from baseline). D. Phase II Study in HCV Patients
• the primary endpoint was the proportion of patients who showed an early virological response (EVR) at Study Week 12, with an EVR being defined as equal to or greater than a 2-logio international unit (IU) reduction in HCV RNA level.
• EVR early virological response
• IU 2-logio international unit
• the present example provides data from the treatment of patients with HER2-negative metastatic breast cancer using bavituximab in combination with the taxane, paclitaxel.
• Bavituximab (3 mg/kg) was given weekly until progression, in combination with docetaxel (35 mg/m 2 ), given on days 1, 8, and 15 of planned 4-week cycles for up to 6 cycles. All patients received one prior chemotherapy regimen. Of the most common TEAEs reported, only fatigue, headache, back pain and hypertension were Grade >3.
• HCC hepatocellular carcinoma
• HCC hepatocellular carcinoma
• data are presented from the treatment of patients with previously untreated stage IV pancreatic cancer using gemcitabine in combination with bavituximab.
• This study was a Phase II, randomized, open-label study to evaluate gemcitabine when administered with or without bavituximab in patients with previously untreated stage IV pancreatic cancer.
• the primary objective was to compare the OS of patients among the treatment arms. Secondary objectives included comparing PFS, OR , DR and safety.
• Enrolled patients were randomized in a 1 : 1 ratio to receive study treatment of gemcitabine alone or gemcitabine with weekly 3 mg/kg bavituximab.
• Gemcitabine 1000 mg/m 2
• a total of 70 patients were enrolled to the study. In general, the patient population had very extensive disease burden, which may have reduced the response in both arms.
• the most common TEAEs for the bavituximab plus gemcitabine treatment group were nausea (44.1%), anemia (35.3%), and fatigue, constipation and anorexia (each occurring in 32.4% of patients).
• Example XIV After the Phase III trial of Example XIV, and the functional p2GPI analyses of Example XVII, showing that functional p2GPI levels correlate with treatment outcomes, stored samples from the present Phase II trial were also tested for functional 2GPI. Results from these analyses, as reported in Example XVIII, strengthen the finding that levels of functional p2GPI are a biomarker for successful bavituximab treatment.
• the present example concerns a Phase II trial testing bavituximab plus docetaxel in patients with previously-treated Stage Illb/IV non-squamous non-small cell lung cancer (NSCLC).
• NSCLC non-squamous non-small cell lung cancer
• This study was a Phase II, randomized, double-blind, placebo-controlled trial evaluating bavituximab plus docetaxel in patients with previously treated locally advanced or metastatic non-squamous NSCLC.
• the primary objective of this study was to compare the ORR (CR + PR) among the treatment arms. Secondary objectives included comparing PFS, DR, OS, safety and PK.
• the combined control patients with fatal events included 1 patient with sepsis, 1 patient with a cerebrovascular accident, and 1 patient experiencing both pneumonia and pseudomonal sepsis.
• Example XIV Subsequent to the Phase III trial of Example XIV, and the analyses of functional ⁇ 2 ⁇ in Example XVII, which showed that functional P2GPI levels correlate with treatment outcomes, stored samples from the present Phase II trial were also tested for functional P2GPI. Results from these analyses, which are described in Example XVIII, further validate that levels of functional P2GPI are a biomarker for successful bavituximab treatment.
• Phase III trial was a randomized, double-blind, placebo-controlled multicenter trial of bavituximab plus docetaxel in patients with previously-treated Stage Illb/IV non-squamous non-small cell lung cancer (NSCLC). This global, double-blind Phase III trial was initiated in 2012.
• Selection criteria were for patients with Stage Illb IV non- squamous NSCLC who progressed on platinum-doublet chemotherapy (should have progressed on appropriate targeted therapy if known EGFR or ALK mutation), with ECOG PS 0-1 and prior immunotherapy allowed.
• the trial accrued 597 such patients in a 1 : 1 ratio to receive up to six 21 -day cycles of docetaxel (at 75 mg m ) in combination with either weekly 3 mg/kg bavituximab (bavituximab plus docetaxel) or placebo (docetaxel alone) until progression or toxicity.
• the primary endpoint was overall survival (OS) and secondary endpoints included objective response rate (Independent Central Review, ICR), progression- free survival (ICR), safety, PK, Quality of Life (LCSS) and exploratory biomarkers, including immune correlates.
• ICR Independent Central Review
• ICR progression- free survival
• PK PK
• LCSS Quality of Life
• exploratory biomarkers including immune correlates.
• the baseline characteristics of the selected patients are shown in Table 7, in which the 'Placebo' column refers to patients treated with docetaxel alone and the 'Bavituximab' column refers to patients treated with bavituximab plus docetaxel.
• treatment-related Grade 3/4 febrile neutropenia was slightly higher for bavituximab plus docetaxel (8%) than for docetaxel alone (5%).
• the mOS was 10.7 months (95% confidence interval [CI], 8.6-1 1.5) among 297 patients in the bavituximab plus docetaxel group and 10.8 months (95% CI, 9.2-12.6) among 300 patients in the docetaxel alone group (hazard ratio (HR) for death, 1.10 (0.89, 1.37)).
• Progression-free survival (PFS) was also similar in the two arms when 70% of the targeted OS events were reached, with a median PFS of 4.1 months for the bavituximab plus docetaxel group and 3.9 months for the docetaxel alone group.
• Subsequent immunotherapy was received by about 15% of the patients in the study, evenly distributed between the bavituximab plus docetaxel arm and the docetaxel alone arm (see Example XIX).
• the efficacy analysis (ITT) at this stage is listed in Table 8, in which the P-value is based on the two-sided stratified Cochran-Mantel-Haenszel exact method.
• Stratification factors include disease stage (IIIB vs. IV), geographic region (North America, Europe, Rest of World), previous maintenance and/or targeted therapy (Yes vs. No).
• biomarker analyses were conducted with a view to identifying one or more biomarkers, or a pattern of biomarkers (a bavituximab "signature"), for patients who receive the most benefit from a bavituximab- containing therapeutic regimen.
• the present example concerns the sample collection techniques that apply to later studies and describes the initial proteomic signature analyses.
• the Phase III trial was designed, and informed consent was obtained, for the collection of patient blood samples.
• Patient blood specimens were obtained using proper phlebotomy techniques.
• a tourniquet was placed 7 to 10 cm above the venipuncture site, but tourniquet application for preliminary vein selection was not permitted to exceed one minute.
• the patients were requested to close, but not pump, their first and the venipuncture site was cleaned with a 70% isopropyl alcohol pad using a circular motion from the center to the periphery and allow to air dry.
• Serum Separator Tube Using a 21 gauge needle, patient blood was collected in a 5.0 ml gold top Serum Separator Tube (SST). The tourniquet was released as soon as possible after the blood began to flow and the tube permitted to fill completely. The tube was immediately inverted 5 times after collection and allowed to clot for at least 30 minutes. To separate the serum, the tube was centrifuged within 30 to 60 minutes of collection at 1 ,000 to 1,300 g for 15 minutes. A pipette was used to transfer approximately 1.25 ml of serum into 3.6 ml cryovial tubes x2 and those samples were frozen.
• SST Serum Separator Tube
• the frozen vials were placed into a specimen bag and sealed tightly.
• the bottom of a dry ice shipper was layered with dry ice and the specimen bag placed in the box. Dry ice was added until the box was full, the lid was secured in place, and the samples were shipped to Central Lab for storage at -70° degrees Celsius.
• the Central Lab prepared the vials for sub-aliquotting by thawing the samples. Using a pipette at least 250 ⁇ of serum was transferred into 2 ml natural cap cryovial tubes x4 and re frozen at -70° Celsius. Repeating the same shipping directions, the sub-aliquoted samples were shipped frozen on dry ice to the testing labs for biomarker for testing.
• VeriStrat is a commercially available, blood-based predictive and prognostic proteomic test for patients with advanced NSCLC. In addition to being prognostic, VeriStrat is predictive of differential treatment benefit when selecting between single-agent treatment options. VeriStrat was retrospectively performed on patient samples from the Phase III trial.
• Pre-treatment serum samples from patients in the Phase III trial were tested for protein expression using mass spectrometry, classifying patients as VeriStrat (VS) Poor (VS-P), which correlates with a more aggressive disease, or VS Good (VS-G), which correlates with a more favorable prognosis.
• OS was analyzed by VeriStrat subgroups using Kaplan-Meier statistical methods. VeriStrat classification was available for 569 patients of the 597 randomized patients.
• VeriStrat results in the Phase III trial are overall prognostic for PFS and OS, but not predictive for bavituximab treatment response.
• the unexpected OS result in the docetaxel arm may have been impacted by the relatively high overall proportion of VeriStrat Good patients.
• the percentage of VeriStrat Good patients in this Phase III trial (greater than 80%) is higher than previously reported (approximately 67%), indicating that patients had better prognosis overall, thus partially explaining the better than expected performance of the docetaxel arm.
• the present example concerns the development of a ⁇ 2 ⁇ assay explicitly designed for the detection of functional (active) P2GPI in fluid samples.
• This test method is uniquely adapted to detect and quantify functional P2GPI, meaning ⁇ 2 ⁇ that is able to bind to both PS and to bavituximab.
• the present example thus provides a previously unavailable tool required for further meaningful biomarker analyses in connection with bavituximab treatment.
• the Wash Buffer is I X phosphate-buffered saline (PBS) and the Blocking Buffer is 2% Ovalbumin in lx PBS.
• bavituximab antibody was conjugated to horseradish peroxidase (HRP) to prepare a bavituximab-HRP detection agent for use in the assay.
• HRP horseradish peroxidase
• the conjugation was performed using EZ-Link® Plus Activated Peroxidase (Thermo Scientific, Cat# 31487) following the procedure for conjugating activated peroxidase to an antibody at pH 7.2 provided by the manufacturer. Briefly, 1 mg of bavituximab was diluted to 1 mg/ml in PBS, pH 7.2. This was added to 1 mg of lyophilized EZ-Link Plus activated peroxidase to reconstitute.
• the ELISA plates were coated with the PS antigen as follows: 5 ⁇ g/ml PS antigen was prepared and diluted into 6 ml of hexane in a fume hood with the blower off. 50 ⁇ of PS solution was added to each well using a 12-channel pipette. The fume hood blower was turned back on and the hexane allowed to evaporate for 30-45 minutes, typically 30 minutes.
• Blocking Buffer (2% Ovalbumin in IX PBS) was prepared. 200 ⁇ 1 of Blocking Buffer was added to each well using a 12-channel pipette. The blocked ELISA plates were incubated at 37°C for 120 minutes ( ⁇ 10 minutes, which did not alter the performance of the assay). 6. Sample Preparations
• the P2GPI standards for the positive control were obtained from Haematologic Technologies, Inc. (HTI; cat# B2G1-0001-C; 1.0 mg/ml) in a buffer of 0.2 M Glycine, 0.15 M NaCl, pH 7.4. A vial of 2GPI was thawed and the standard and positive control preparation was performed as follows:
• the unknown samples were prepared for testing as follows: unknown samples were prepared in Blocking Buffer with a final dilution of 1 :4000 and 1 : 8000; a 1 : 100 dilution of the unknown sample was prepared first; a 1 :40 dilution was prepared from the 1 : 100 dilution to achieve a 1 :4000 dilution; and a 1 :80 dilution was prepared from the 1 : 100 dilution to achieve a 1 :8000 dilution.
• the non-binding plate preparation was performed as follows: 75 ⁇ of Blocking Buffer was added to columns 1 -3 of rows B-H; 150 ⁇ of 250 ng/ml standard was added to columns 1 -3, row A; using a multichannel pipette, 75 ⁇ from columns 1-3 was serially diluted from row A through row G; 75 ⁇ of positive controls and samples was added to the designated wells; and 75 ⁇ of Blocking Buffer was added to any blank wells.
• the plate setup is shown in Table 10.
• bavituximab-HRP Prior to finish of the block, 6 ml of 300 ng/ml bavituximab-HRP was prepared in Blocking Buffer.
• the assay plate was washed with IX PBS by pipetting 250 ⁇ into each well and this was repeated 2 more times.
• a plate washer may be used, in which case, the plate is washed once with IX PBS. It was ensured that the plate was as dry as possible.
• bavituximab-HRP 50 ⁇ of 300 ng/ml bavituximab-HRP was added to all wells of the assay plate. 50 ⁇ was added from each corresponding well of the non-binding plate.
• Using an assay plate and a non-binding plate in this way means that the detectably-labeled bavituximab is added to the PS-coated assay plate first, prior to adding the samples containing ⁇ 2 ⁇ from the non-binding plate. This sequence avoids cross-contamination during pipetting. Bavituximab-HRP and the samples containing ⁇ 2 ⁇ are incubated together on the plate and incubation was conducted at 37°C for 90 minutes. 8. Development
• the TMB peroxidase substrate and TMB peroxidase Solution B was removed from the refrigerator at least 1 hour before use.
• the assay plate was washed with IX PBS by pipetting 250 ⁇ into each well and this was repeated 2 more times.
• a plate washer may be used, in which case, the plate is washed once with IX PBS. It was ensured that the plate was as dry as possible.
• TMB mixture 12 ml was prepared by mixing 6 ml of TMB peroxidase substrate with 6 ml of TMB solution B. 100 ⁇ of TMB solution was added to each well of the assay plate and allowed to develop for 5-6 minutes. Development was stopped by adding 100 ⁇ of 2M H 2 S0 4 to each well of the assay plate. The assay plate was read and optical density (OD) determined at 450nm within 30 minutes of stopping the reaction. The microplate reader was used in conjunction with the SoftMaxPro plate data and analysis template, which provides a printout of assay data. 9. Preparation of Nicked p2GPI
• An assay was designed that should detect total P2GPI, based on the manufacturer's specifications for the antibodies used. This is an assay using commercially available antibodies from US biological, in which plates are coated with a capture antibody against P2GPI and any bound P2GPI is detected using an anti-P2GPI-HRP conjugate as a detection antibody.
• the antibody catalog numbers are: Capture Antibody, US Biological # A2299-81 A, affinity-purified anti-P2GPI and Detecting Antibody, US Biological #A2299-81B, peroxidase- conjugated anti-p2GPI.
• a 1 100 dilution of the capture antibody was prepared in carbonate buffer (50 mM Sodium Bicarbonate) at pH 9.6. 100 ⁇ was added to each well of the ELISA plate and incubated at room temperature. The plate was washed with lxPBS buffer containing Tween-20, then blocked with 200 ⁇ /well of assay diluent containing 1% BSA and incubated at 37°C. Purified P2GPI was used to prepare a two-fold dilution standard curve starting at 500 ng/ml in assay diluent. Samples were diluted in assay diluent to achieve a concentration within the linear region of the standard curve.
• the plate was washed, followed by the addition of 100 ⁇ /well of the standard curve and samples in either duplicate or triplicate. After the addition of the standard curve and samples, the plate was incubated at 37°C.
• the detection antibody was diluted 1 :400 in assay diluent. After incubating the samples and standard curve, the plate was washed, followed by the addition of 100 ⁇ /well of the detection antibody. The plate was incubated at 37°C. After the secondary antibody incubation, the plate was washed, then developed with TMB. The plate was read on a plate reader at 450nm and the sample concentrations determined from the standard curve.
• total ⁇ 2 ⁇ assay using commercially available antibodies (Table 1 1 A), and the present, “functional ⁇ 2 ⁇ assay” (Table 11B), both read out similar concentrations of ⁇ 2 ⁇ (approximately 141 ng/ml and 136 ng/ml).
• concentrations of ⁇ 2 ⁇ approximately 141 ng/ml and 136 ng/ml.
• the assay is able to successfully determine the amount of functional ⁇ 2 ⁇ in fluid samples, which is p2GPI that binds to both PS and to bavituximab.
• This assay has now been routinely performed to prepare reproducible ⁇ 2 ⁇ standard curves.
• a Four- Parameter Logistic Fit is used, which is a statistical equation used for non-linear regression analysis.
• the Four-Parameter Fit Equation is:
• A is the Y-value corresponding to the asymptote (i.e. the flat part of the curve) for the low values of the X-axis;
• B is the coefficient that describes how rapidly the curve makes its transition from asymptotes in the center of the curve, and is commonly called as the slope factor
• C is the X value corresponding to the midpoint between A and D; commonly called the EC50;
• D is the Y-value corresponding to the asymptote for the high values of the X-axis.
• FIG. 16 A representative example of a standard curve for functional ⁇ 2 ⁇ is shown in FIG. 16. From such a standard curve, the concentrations of functional ⁇ 2 ⁇ in human blood samples, such as plasma or serum samples, can be determined. Mainly for accuracy, but also for economy of sample preparation, the standard curve is prepared in ng/ml (nanogram/ml). As the average levels of ⁇ 2 ⁇ in the normal human population are about 200 ⁇ g/ml (microgram/ml) (Mehdi et ai, 1999; Miyakis et ah, 2004), the standard curve is prepared in expectation that the test samples will be diluted before analysis in the assay. Diluted plasma or serum test samples are run in the assay and the concentration of ⁇ 2 ⁇ in the patient then calculated by adjusting for the dilution factor.
• ELISA plates are those optimized for lipid adsorption, which may be used to replace the ELISA plates in Section Al, above.
• ELISA plates are known that are optimized for lipid adsorption, which have surface chemistries providing better lipid (PS) binding.
• PS lipid binding
• One such ELISA plate is the ThermoFisher PolySorp plate, which has been used in a new assay format.
• the hexane-based PS coating method in Section A4, above, may preferably be replaced with an isopropanol-based PS coating method, which can provide certain safety benefits to the user (by avoiding the use of hexane).
• isopropanol as a coating buffer in a new assay format
• the ELISA plates are coated with PS antigen using 10 ⁇ g/ml PS antigen diluted in isopropanol and the incubation time is 90 min.
• any known method of obtaining P2GPI may be employed.
• P2GPI as purchased from a commercial vendor, such as HTI (Section A6, above).
• Alternative ⁇ 2 ⁇ preparations may also be developed for defined, reproducible calibration control.
• One such preferred method is to express ⁇ 2 ⁇ in CHO cells and purify the expressed ⁇ 2 ⁇ .
• a preferred purification of ⁇ 2 ⁇ from CHO cells includes: a harvest clarification, chromatin extraction step, which removes contaminants and allows the clarified harvest to pass through a 0.2 um filter; use of a tangential flow filtration (TFF) system, to buffer-exchange the clarified harvest and decrease its conductivity without increasing the volume; a capto adhere step in anion flow through mode, to remove further contaminants; a strong cation step using NuviaTM S to remove aggregates and other contaminants, concentrate the eluate and facilitate any buffer exchange step; and, optionally, use of a TFF system to buffer exchange and concentrate the purified ⁇ 2 ⁇ .
• ⁇ 2 ⁇ has been expressed and purified in this way and used in a new assay format.
• certain preferred bavituximab-HRP detection agents are conjugates crosslinked using either of two commonly-used, non-proprietary crosslinkers, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate) or SATA (N-succinimidyl S-acetylthioacetate).
• SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate
• SATA N-succinimidyl S-acetylthioacetate
• Other preferred bavituximab-HRP detection agents are conjugates in which the number of HRP is in excess to the bavituximab antibody, particularly those resulting in an HRP:bavituximab ratio of 2: 1 or 3: 1 , with essentially no free (unconjugated) antibody.
• Such conjugates are purified by an S-300 sizing column to remove unreacted reaction components.
• Bavituximab-HRP detection agents with each of these constituents and properties have been obtained from Columbia Biosciences, 4985 Winchester Blvd., Frederick, Maryland, 21703, and used at 600ng/ml in a new assay format.
• the present example reports the levels of pre-treatment functional P2GPI in the patients of the Phase III trial of Example XIV.
• the present example also concerns functional 2GPI as a biomarker for successful bavituximab treatment, such as in NSCLC patients treated with bavituximab and docetaxel and other combination therapies.
• the Phase III trial described above accrued 597 patients.
• the collection of blood samples from the patients in the Phase III trial is described in Example XV, A.
• Sub- aliquots of those 592 patient blood samples were tested for functional ⁇ 2 ⁇ , using the assay described in the example immediately above.
• the levels of pre-treatment functional ⁇ 2 ⁇ ranged from 0.5 to 402 ⁇ g/ml and the distribution of functional ⁇ 2 ⁇ for all patients is shown in FIG. 17A and FIG. 17B.
• functional P2GPI ranged from 22 to 365 g/ml, as shown FIG. 17C.
• the distribution of functional ⁇ 2 ⁇ in the patients treated with docetaxel alone is shown in FIG. 17D, which covers the full range for the study (0.5 to 402 ⁇ g/ml).
• step 1 is to search for OS separation or significant OS separation of a High ⁇ 2 ⁇ vs. Low ⁇ 2 ⁇ group for patients in the bavituximab plus docetaxel group
• step 2 is to search for OS separation or significant OS separation of the bavituximab plus docetaxel group vs. the docetaxel alone group (placebo) for those High P2GPI patients.
• FIG. 18A shows that for patients treated with bavituximab, those with functional P2GPI of equal to or higher than 200 ⁇ g/ml (167 patients) had a mOS of 11.4 months, vs.
• Step 1 is to search for significant OS separation of "Within Range” vs. "Outside of Range” for patients treated with bavituximab (plus docetaxel)
• Step 2 is to search for significant OS separation of the bavituximab vs. placebo arms for patients "Within Range”.
• the two cutoff method showed that pre-treatment levels of functional ⁇ 2 ⁇ within each of the ranges of 210-270, 210-280, 210-290, 200-280 and 200-290 ⁇ g/ml are statistically significant predictors of benefit in overall survival in patients treated with bavituximab plus docetaxel vs. those treated with docetaxel alone.
• These results for the functional ⁇ 2 ⁇ ranges of 210-270, 210-280, 210-290, 200-280 and 200-290 ⁇ are shown in Table 14A and Table 14B.
• each of the ranges of functional ⁇ 2 ⁇ of 210-270, 210-280, 210-290, 200-280 and 200-290 ⁇ have a hazard ratio of less than one, and a statistically significant P value, representing the improvement in survival.
• Table 14B naturally shows the opposite, in that patients with functional ⁇ 2 ⁇ outside of those stated ranges have a hazard ratio of more than one, and a statistically significant P value, representing a worsening of survival (or increased chance of death).
• Example V shows that molar ratios of ⁇ 2 ⁇ to antibody of 0.12 to 0.25 (FIG. 9A and FIG. 9B, with FIG. 1A, FIG. IB, FIG. 1C and FIG. ID); 0.125, 0.5 to 2 (FIG. 10); 0.93 (FIG. 1 1); and 1.43 to 2.86 (FIG. 12) are effective in supporting binding of bavituximab to PS.
• molar ratios of ⁇ 2 ⁇ to antibody of 0.12 to 0.25 FIG. 9A and FIG. 9B, with FIG. 1A, FIG. IB, FIG. 1C and FIG. ID
• 0.125, 0.5 to 2 FIG. 10
• 0.93 FIG. 1 1
• 1.43 to 2.86 FIG. 12
• Example XVII Following the identification of functional ⁇ 2 ⁇ as a biomarker for successful bavituximab treatment in Example XVII, the present example extends the use of the functional ⁇ 2 ⁇ assay to samples from earlier bavituximab clinical trials.
• the following results show that the same levels of functional ⁇ 2 ⁇ also correlate with successful treatment outcomes for bavituximab, thus confirming functional ⁇ 2 ⁇ as a biomarker for bavituximab.
• Example XIII Samples from the NSCLC Phase II trial of Example XIII (PPHM 0902) were tested using the functional ⁇ 2 ⁇ assay of Example XVI.
• the levels of pre-treatment functional P2GPI ranged from 0.5 to 266 ⁇ g/ml for all patients (FIG. 20A). Within the patients treated with bavituximab 3 mg/kg plus docetaxel, functional P2GPI ranged from 0.5 to 266 ⁇ g/ml (FIG. 20B).
• the distribution of functional P2GPI in the patients in the combined control arm was 0.5 to 257.4 g/ml (FIG. 20C).
• the levels of pre-treatment functional 2GPI are consistent with the average reported in the literature.
• Example XII Samples from the Phase II pancreatic cancer trial of Example XII (PPHM 1002) were tested using the functional P2GPI assay of Example XVI. There were 31 patient samples in which levels of pre-treatment functional ⁇ 2 ⁇ were evaluable. The levels of pre-treatment functional ⁇ 2 ⁇ ranged from 82.5 to 343.2 ⁇ g/ml for all patients (FIG. 22). For these 31 patients, the mean level of pre-treatment functional 2GPI (219.2 ⁇ / ⁇ was consistent with the average reported in the literature. Although the sample size is small, and the disease is very aggressive, using a cut-off of
• a randomized, open-label, Phase II trial (PPHM 1001 ) of paclitaxel/carboplatin with or without bavituximab was conducted in patients with previously untreated locally advanced or metastatic non-squamous NSCLC. Samples from this trial were tested using the functional 2GPI assay of Example XVI. There were 84 patient samples in which levels of pre-treatment functional P2GPI were evaluable, of which 44 patients were in the bavituximab arm and 40 patients were in the paclitaxel/carboplatin arm.
• the levels of pre-treatment functional P2GPI ranged from 0.5 to 326 ⁇ g/ml for all patients (FIG. 24A). Within the patients treated with bavituximab, functional P2GPI ranged from 0.5 to 326 ⁇ g/ml (FIG. 24B). Functional 2GPI in the patients in the paclitaxel/ carboplatin arm ranged from 88.8 to 292.7 ⁇ g/ml (FIG. 24C).
• Example XVII and Example XVIII consistently show that functional P2GPI levels correlate with treatment outcomes, thus validating functional P2GPI levels as a biomarker for successful bavituximab treatment.
• Example XIV Although the initial analyses of the Phase III trial of Example XIV did not show superior OS in the bavituximab plus docetaxel arm as compared to the docetaxel alone group, ongoing studies were conducted with a view to identifying other possible indicators of a therapeutic benefit to bavituximab treatment.
• the present example shows that patients treated with bavituximab and docetaxel followed by subsequent immunotherapy (SACT-IO) have a statistically significant better mOS as opposed to patients treated with docetaxel alone followed by subsequent immunotherapy.
• the particular immunotherapy agents of the "first subsequent IO” were also identified.
• the immunotherapy agents are shown in Table 17, all of which are checkpoint inhibitor antibodies (immune checkpoint inhibitors) in the form of a blocking antibody that binds to CTLA-4, PD-1 or PD-L1.
• the blocking antibodies used were tremelimumab, a blocking antibody that binds to CTLA-4; nivolumab, a blocking antibody that binds to PD-1 ; and durvalumab (MEDI4736), a blocking antibody that binds to PD-L1.
• 42/46 bavituximab patients subsequently received nivolumab; two received durvalumab monotherapy and two received tremelimumab + durvalumab (Table 17).
• the first subsequent IO in the 47 patients in the placebo arm was: tremelimumab (3), nivolumab (40), durvalumab (3), pembrolizumab (2) and REGN2810 (1), which is a total of 49 agents in 47 patients, with two patients receiving an IO doublet of durvalumab (MEDI4736)-tremelimumab. That is, 40/47 patients in the control arm subsequently received nivolumab; one received durvalumab monotherapy; one received tremelimumab monotherapy; two received tremelimumab + durvalumab; two received pembrolizumab and one received REGN2810.
• FIG. 26 shows the survival benefit of initial treatment with bavituximab prior to subsequent IO in terms of time since randomization.
• the survival benefit of initial bavituximab treatment prior to subsequent IO is even more pronounced when measured as the time since the first subsequent IO treatment.
• Example XIX patients treated with bavituximab (plus docetaxel) and subsequent IO have a markedly better mOS than patients treated with docetaxel alone and subsequent 10.
• the present example further validates the use of functional p2GPI as a bavituximab biomarker, showing that the same levels of functional ⁇ 2 ⁇ also correlate with successful treatment by bavituximab in combination with immunotherapy.
• Example XVII functional p2GPI levels of 200 ⁇ g/ml or higher are shown to correlate with successful bavituximab treatment, including in the Phase III trial (Example XVII).
• high p2GPI as being pre-treatment levels of functional p2GPI of equal to or higher than 200 ⁇ g/ml (> 200 ⁇ g/ml)
• p2GPI > 200 ⁇ g/ml correlated with increased overall survival in patients treated with bavituximab and subsequent IO, but not in control patients who received subsequent IO (FIG. 27).
• Mycobacterium tuberculosis ESAT-6 is a leukocidin causing Ca2+ influx, necrosis and neutrophil extracellular trap formation", Cell Death and Disease, 5:el474; doi: 10.1038/cddis.2014.394, 2014.
• Gerber et al. "Phase I Safety and Pharmacokinetic Study of Bavituximab, a Chimeric Phosphatidylserine-Targeting Monoclonal Antibody, in Patients with Advanced Solid Tumors", Clin. Cancer Res., 17(21): l-9, 201 1.
• Gerber et al "Docetaxel Combined with Bavituximab in Previously Treated, Advanced Nonsquamous Non-Small-Cell Lung Cancer", Clinical Lung Cancer, 17(3): 169-176, 2016.
• LAG3 is an immunotherapeutic target in murine triple negative breast cancers whose activity is significantly enhanced in combination with phosphatidylserine targeting antibodies
• Poster B019 CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference, New York, NY, September 25-28, 2016b.
• Hotchkiss et al. "Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (Taxotere): association with impaired repositioning of the microtubule organizing center", Mol. Cancer Titer., 1 (13): 1 191 -200, 2002.
• Meckes and Raab-Traub "Microvesicles and Viral Infection", J. Virology, 85(24): 12844- 12854, 201 1.
• Mehdi et al. "Genetic variation in the apolipoprotein H (p2-glycoprotein I) gene affects plasma apolipoprotein H concentrations", Hum. Genet., 105:6371, 1999.
• Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells", Science, 320:531-535, 2008.
• Pattanapanyasat et al. "Febrile temperature but not proinflammatory cytokines promotes phosphatidylserine expression on Plasmodium falciparum malaria-infected red blood cells during parasite maturation", Cytometry, Part A, 77A:515-523, 2010.

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